一、The Characteristics of Acute Rejection after Limb Allotransplantation in Rats─An Experimental Study（论文文献综述）
Christophe Caneparo,Luis Sorroza-Martinez,Stéphane Chabaud,Julie Fradette,Stéphane Bolduc[1]（2021）在《Considerations for the clinical use of stem cells in genitourinary regenerative medicine》文中研究说明The genitourinary tract can be affected by several pathologies which require repair or replacement to recover biological functions.Current therapeutic strategies are challenged by a growing shortage of adequate tissues.Therefore,new options must be considered for the treatment of patients,with the use of stem cells （SCs） being attractive.Two different strategies can be derived from stem cell use:Cell therapy and tissue therapy,mainly through tissue engineering.The recent advances using these approaches are described in this review,with a focus on stromal/mesenchymal cells found in adipose tissue.Indeed,the accessibility,high yield at harvest as well as anti-fibrotic,immunomodulatory and proangiogenic properties make adipose-derived stromal/SCs promising alternatives to the therapies currently offered to patients.Finally,an innovative technique allowing tissue reconstruction without exogenous material,the self-assembly approach,will be presented.Despite advances,more studies are needed to translate such approaches from the bench to clinics in urology.For the 21st century,cell and tissue therapies based on SCs are certainly the future of genitourinary regenerative medicine.
李钟奇[2]（2020）在《椎间盘退变关键标志物筛选及携载TGF-β3支架对椎间盘修复的实验研究》文中研究指明研究背景腰痛（Low back pain,LBP）是工业化国家的主要健康问题之一,是人致残的主要原因。LBP 与椎间盘退变（Intervertebral disc degeneration,IDD）有关。椎间盘（intervertebraldisk,IVD）高度因退变而降低,改变了受影响脊髓节段的力学特性。这个过程加速了邻近节段和其他脊柱结构的退变,如小关节、韧带和肌肉等。IDD不仅影响IVD,还影响其周围组织,如肌肉和韧带,并影响脊柱应对日常生活中正常生理负荷的能力。从长远来看,IDD会导致腰椎管狭窄和随之而来的神经组织压迫。腰椎管狭窄是引起下腰部疼痛和神经性跛行的主要原因,尤其是老年人。考虑到现今社会生活水平提高和医疗卫生事业的发展,老年人口在逐年增加。老年患者往往还合并有骨质疏松,心脑血管等慢性疾病,因此这类患者LBP和IDD相关疾病的治疗问题变得更加困难。目前对IDD的治疗包括药物治疗、物理治疗等保守治疗和椎间盘摘除术、脊柱融合术、椎间盘置换术等侵入性治疗,但这些方法都不能恢复IVD的原有结构和功能。近些年,生物信息学的快速发展为研究IDD带来了机遇,它将统计学、数学、信息学和计算机科学的方法恰当的整合在一起来解决问题。生物信息学应用的主要领域包括:发现基因、预测基因表达、序列比对、蛋白结构比对、预测蛋白结构、预测蛋白间相互作用等。通过公共数据库检索方式,可以挖掘并验证与IDD相关的潜在基因与信号通路,以此从基因层面了解IDD的发病机制,并为IDD的预防和治疗研究提供新的思路和研究方向。近年来,以细胞为基础的组织工程对IDD的治疗已被证明是一种很有前景的治疗方法,应用组织工程学方法构建与IVD结构和功能相符的组织工程植入体,可以替代退变IVD并保留其力学运动范围,从而对退变的IVD结构进行重建和修复,使IVD功能得到恢复,达到治疗IDD的目的。纤维环组织工程就是以纤维环的再生和修复为目的的一种修复方法。通过筛选,应用新型的复合支架,即脱细胞纤维环基质/壳聚糖水凝胶作为纤维环损伤修复的支架材料,因其具有较好的生物相容性、可降解性及力学性能,并能携载和连续释放合适的生长因子,满足新生纤维环组织在不同阶段对生长因子的需求,最终达到修复受损的IVD的目的。第一部分:椎间盘退变关键标志物的筛选研究目的:本研究同时应用GSE56081和GSE124272这两个数据集去挖掘和验证与IDD相关的潜在基因与信号通路,为IDD的发病机制的研究提供新思路。研究方法:首先,对GEO数据库下载的GSE56081和GSE124272这两个数据集中的数据进行预处理,然后利用limma包提供的经典贝叶斯方法对IDD和Healthy两组的差异表达基因进行分析。随后,在毒性与基因比较数据库（CTD）中搜索疾病相关基因,并将搜索到的疾病相关基因与差异表达基因取交集,得到的交集基因被判断为疾病相关的差异表达基因。接着根据得到的疾病相关的差异表达基因,利用 DAVID 软件进行 Gene Ontology（GO）和 Kyoto Encyclopedia of Genes and Genomes（KEGG）富集分析。并且,利用STRING数据库对疾病相关的差异表达基因进行蛋白互作（protein-protein interaction,PPI）网络分析,筛选PPI网络中的hub蛋白,通过Cytoscape软件的MCODE插件筛查重要模块,并利用DAVID对重要模块进行KEGG通路富集分析。接着将PPI网络中筛选得到的hub基因与模块基因取交集作为关键基因,并将在GSE124272数据集中验证成功的基因作为marker基因。随后使用ROC曲线来证明得到的marker基因的诊断效能,最后使用基因集合富集分析（GSEA）对marker基因进行分析。研究结果:本研究共筛选出1184个差异表达基因,随后与CTD数据库中得到的2295个疾病相关基因取交集,共得到142个疾病相关的差异表达基因。富集分析结果显示,这142个疾病相关的差异表达基因共富集了 130个GO-biological process（BP）,18 个 GO-cellular component（CC）,28 个 GO-molecular function（MF）以及27条KEGG信号通路（最显着富集的为肿瘤坏死因子信号通路和HTLV-I感染）。随后,还对这142个疾病相关的差异表达基因进行PPI网络分析,结果共得到了 223个PPI关系对,包括83个编码蛋白,其中金属蛋白酶组织抑制因子（TIMP1）连接度最高,并挖掘出1个关键的子模块,模块中包含的基因有基质金属蛋白酶2（MMP2）、I型胶原α2链（COL1A2）、SMAD家庭成员3（SMAD3）、SMAD家庭成员2（SMAD2）、转化生长因子β1（TGFB1）、Ⅳ型胶原α1链（COL4A1）、丝裂原活化蛋白激酶1（MAPK1）、转录因子snail 1（SNAI1）、TIMP1、聚集蛋白聚糖（ACAN）。KEGG信号通路富集分析结果显示,模块共富集到16条信号通路,其中最显着富集到结肠直肠癌信号通路。接着,将模块基因与top20 hub基因取交集,共得到9个交集基因,其中ACAN基因在GSE56081和GSE124272这两个数据集中的相对表达水平上下调一致。因此,将ACAN作为marker基因。ACAN的ROC面积在两个数据集中均达到了 0.8以上,具有良好的诊断效能。最后,GSEA富集分析显示,ACAN富集到10条正相关的信号通路和9条负相关的信号通路,其中最显着的正相关信号通路是帕金森病通路,最显着的负相关信号通路是嗅觉传导信号通路。研究结论:HTLV-I感染和肿瘤坏死因子信号通路可能在IDD过程中发挥着重要作用;TIMP1基因可能通过HIF-1α信号通路调节髓核细胞凋亡在IDD过程中扮演重要角色;COL1A2可能通过血小板激活、黏着斑、PI3K-Akt信号通路在IDD过程中起作用;ACAN基因可能是诊断IDD的一个潜在治疗靶点,并通过帕金森病通路和嗅觉传导信号通路发挥重要作用。本研究为IDD的潜在标志物进行分析,并对疾病相关的差异表达基因进行富集分析,为探索IDD侵袭、增殖、凋亡等生物学行为的调控机制提供了新的观点,并可能为探索IDD的研究提供了新的线索。第二部分:携载TGF-β3的脱细胞纤维环基质/壳聚糖水凝胶修复退变椎间盘的实验研究研究目的:运用脱细胞纤维环基质/壳聚糖水凝胶作为纤维环损伤修复的支架材料,通过包裹转化生长因子β3（Transforming Growth Factor-β3,TGF-β3）达到缓释效果,观察纤维环源干细胞在携载TGF-β3脱细胞纤维环基质/壳聚糖水凝胶的生长情况以细胞-基材复合体在体内外对退变纤维环修复情况,为构建IVD纤维环组织工程支架材料和细胞因子的选择提供一定的理论基础和实践依据。研究方法:首先从纤维环组织中分离并纯化纤维环源干细胞,检测其自我更新多向分化能力。接着制备携载TGF-β3脱细胞纤维环基质/壳聚糖水凝胶,并检测T GF-β3、细胞增殖、I型胶原蛋白（Collagen Ⅰ）、Ⅱ型胶原蛋白（Collagen Ⅱ）和聚集蛋白聚糖（Aggrecan,ACAN）的基因表达情况。最后,采用磁共振成像（MRI）方法和做苏木精-伊红染色（HE染色）观察纤维环修复情况,以检测携载TGF-β3的脱细胞纤维环基质/壳聚糖水凝胶在体内对IDD的修复作用。研究结果:从电镜图可以明显看到支架上孔结构比较大,孔与孔相连构成通孔,形成三维立体网状结构,并且。TGF-β3含量随时间的延长而增加,到第7天时达到峰值。在第1、3、5天,3组之间细胞增殖情况没有明显差异,但在第7天,携载TGF-β3的脱细胞纤维环基质/壳聚糖水凝胶组的细胞增殖情况明显高于其他两组。Collagen Ⅰ、Collagen Ⅱ和Aggrecan基因mRNA及蛋白表达水平在携载TGF-β3的脱细胞纤维环基质/壳聚糖水凝胶组明显高于不携载TGF-β3的脱细胞纤维环基质/壳聚糖水凝胶组。通过大鼠的体内实验,将所构建的携载TGF-β3的脱细胞纤维环基质/壳聚糖水凝胶植入受损的IVD内,通过4周、8周MRI结果和4周HE染色结果分析实验组和对照组,表明携载TGF-β3的脱细胞纤维环基质/壳聚糖水凝胶在体内对IDD有明显的修复作用。研究结论:所构建的携载TGF-β3的脱细胞纤维环基质/壳聚糖水凝胶具有相互沟通的空隙,初步显示出携载TGF-β3的脱细胞纤维环基质/壳聚糖水凝胶具有良好的结构性能;所构建的携载TGF-β3的脱细胞纤维环基质/壳聚糖水凝胶对体外分离培养的纤维环源干细胞的生长、增殖无明显的抑制作用,并且纤维环源干细胞能在其表面及内部黏附、生长和增殖,初步显示出携载TGF-β3的脱细胞纤维环基质/壳聚糖水凝胶具有良好的细胞相容性;所构建的携载TGF-β3的脱细胞纤维环基质/壳聚糖水凝胶植入动物体内,4周及8周以后能够缓解椎间隙的狭窄和生物力学性能的降低,4周HE染色提示结构接近正常,初步显示出携载TGF-β3的脱细胞纤维环基质/壳聚糖水凝胶对IDD具有良好的修复作用。
Ye Shen,He Shen,Dongyu Guo,Xinghuai Sun,Yuan Sun,Nan Hong,Xiawei Wang,Chen Xie,Yuan Zhao,Qin He,Le Jin,Yingying Wen,Bo Jiang,Chenying Yu,Miaomiao Zhu,Feng Cai,Jianwu Dai[3]（2020）在《Recent developments in regenerative ophthalmology》文中进行了进一步梳理Regenerative medicine（RM） is one of the most promising disciplines for advancements in modern medicine, and regenerative ophthalmology（RO） is one of the most active fields of regenerative medicine. This review aims to provide an overview of regenerative ophthalmology, including the range of tools and materials being used, and to describe its application in ophthalmologic subspecialties, with the exception of surgical implantation of artificial tissues or organs（e.g., contact lens, artificial cornea, intraocular lens, artificial retina, and bionic eyes） due to space limitations. In addition, current challenges and limitations of regenerative ophthalmology are discussed and future directions are highlighted.
Mohammad Zarif[4]（2020）在《辛伐他汀通过抑制TLR4的表达改善大鼠主动脉同种异体移植的动脉硬化》文中认为目的:探讨TLR4在大鼠同种异体血管病变中的病理作用以及辛伐他汀对其的影响。方法:以Wistar和SD大鼠腹主动脉移植为动物模型,观察移植动脉硬化的发展。实验分为同基因组（A组）,同种异体辛伐他汀组（B组）和同种异体对照组（C组）。在第70天,收获移植的动脉,测量移植的内膜的厚度,并进行IHC,HE染色以观察TLR4,NF-k B,VCAM,IVAM和PCM-1在移植的每个血管组织中的表达。血管组织,计算各组的积分光密度值。结果:移植后的动脉内膜在术后70天增厚,在C组最高。与A和B组相比,B组比A组最高,内膜厚度和积分光密度的差异显着（P<0.05）。C组中TLR4,NF-k B,VCAM,IVAM和PCM-1的表达也最强,而B组中TLR4,NF-k B,VCAM,IVAM和PCM-1的表达则明显弱于C组（p<0.05）。结论:1.采用改良的血管插管方法建立了大鼠胸主动脉腹膜移植的动物模型,可以更好地模拟心脏移植血管疾病的发生和发展。2.TLR4,NF-k B,VCAM,IVAM和PCM-1的表达增加可能与血管移植物疾病的发展有关,并且高度相关。3.辛伐他汀可以通过抑制TLR4,NF-k B,VCAM,ICAM和PCM-1的表达来改善血管移植疾病的进展。
Mahmoud Fathy Ahmed Aly Issa[5]（2019）在《Conversion of Mouse Embryonic Fibroblast and Bone Marrow Mesenchymal Stem Cells into Functional Osteoblast by Defined Factors》文中进行了进一步梳理Bone is a well specialized supporting framework of the body,characterized by its rigidity,hardness,regeneration power and repair.It shields the vital organs of the body,serves as an environment for marrow,and acts as a mineral stock for calcium homeostasis and a reservoir of growth factors and cytokines.It also contributes in acid-base balance.Understanding the role of bone cells is important,not only in the orthopedic field,but also in research field involving bone.Bone cells work in harmony to maintain the stability between bone formation and resorption;mean while remote bone structure and function.Bone has a natural ability to regenerate.However,bone grafts are required when complications are met such as excessive bone loss,which impede bone healing.Therefore,bone grafts are in growing demand worldwide.In such approach,autologous bone grafts are considered the "golden key" for bone repair.Even though,in some cases such as large bone loss,significant donor site morbidity and older patients,this procedure may not be practical or has poor outcomes.In these situations,allogeneic bone grafts may alternatively be used,but they have the potential risks of immune rejection,infection transmission,and the limited number of donors in comparison with the increased clinical needs.Tissue engineering is the use of chemical,biological,and engineering principles for regeneration,restoration or repair of living tissue by utilizing cells,biomaterials,and factors alone or in combination.The mechanism of bone tissue engineering is dynamic and complex.It includes migration and recruitment of osteoprogenitor cells,followed by their proliferation,differentiation,matrix formation,and finally remodeling of the generated tissue.The revolution of tissue engineering has raised the success of bone engineering in vitro.Over the past decades,important progress has been obtained in this field,particularly in cells,the biocompatible and biodegradable scaffolds,and the designs of bioreactors,which improve the in vitro osteogenic priming.In hand with identification and discover of many growth factors that can induce endogenous bone repair and vascular formation.Many pre-clinical trials with optimistic results have been conducted using various animal models.Despite this optimism,a poor solid clinical setting remains due to significant issues such as:optimization of cell sources,selection of biomaterial,in vitro preparation and the route of delivery.Based on the previous scientific background,our main goal was to find an available,biological applicable source for bone regeneration to overcome the limitations in Embryonic stem cells（ESCs）and Induced pluripotent stem cells（iPSCs）technology.Direct reprogramming technique was used to convert the mouse embryonic fibroblast（MEF）into functional osteoblast cells.For this purpose,the efficiency of using the human lim mineralization protein-3（hLMP-3）was examined with different combinations of Yamanaka factors.After selection of the suitable cocktail,an in vitro characterization of the osteoblast produced cells was performed.In addition,the selected cocktail was tested in an in vivo orthotopic animal model.Moreover,some natural inducers（Curcumin and All Trans Retinoic Acid（ATRA））were evaluated to reveal their ability to enhance the osteogenic differentiation of mouse bone marrow mesenchymal stem cells（BMSCs）in the culture medium.Finally,the osteogenic potency of the hLMP-3 reprogrammed MEF was tested,in an osteogenic differential medium supplemented with curcumin.To achieve these goals,firstly,the genes of interest were cloned inside a lentivirus expression vector.Secondly,these newly constructed lentivirus vectors containing the genes of interest were used for in vitro reprogramming of MEF to osteoblast.Different combinations of Yamanaka factors were tested with hLMP-3 to find out the best reprogramming cocktail.Afterwards,an in vitro characterization of these cells was performed all-over the reprogramming period.Later,this reprogramming cocktail was used to induce bone formation in an induced uni lateral cortical bone defect in an orthotopic animal model.Our results were obtained by specialized morphological,molecular and functional analyses.Finally,looking forward to improve the osteogenic culture medium,some natural inducers（Curcumin and ATRA）were tested during differentiation of BMSCs.Finally,the positive natural inducer was tested in the culture medium during reprogramming of MEF to osteoblast using hLMP-3 lentivirus vector.Our findings revealed the following points:1.Yamanaka transcription factors（Oct4,Sox2,c-Myc and Klf4）and hLMP-3 were successfully cloned inside the lentivirus expression vector（pGMLVPEl）.The transduction efficiency,and the multiplicity of infection（MOI）were tested by transducing sub-confluent MEFs at（P3）with Green Fluorescent Protein（GFP）lentivirus vector.Cells were transduced with varying amounts of Lentivirus GFP reporter with different concentration of polybrene.The best result was obtained at MOI=4,and polybrene concentration=4ng/μL.The Fluorescence-activated cell sorting analysis（FACS）confirmed this result,and revealed an increased fluorescence intensity（98.9%）compared with the non-transduced MEF cells.2.MEF cells were directly reprogramed with different combinations of Yamanaka factors with hLMP-3 to convert them into osteoblast cells.After transduction,cells were cultured in an osteogenic medium for 21 days,and examined for osteoblast formation.hLMP-3 gene expression was confirmed in the newly reprogrammed cells using the reverse transcription PCR（RT-PCR）reaction from the isolated RNA of the reprogrammed cells from different groups.The results demonstrated robust expression of the hLMP-3 all over the study period in comparison to the negative expression of the hLMP-3 in the control group（MEF cells）.The expression level changes of the bone gene markers during reprogramming revealed that the best reprogramming cocktail that induced the higher expression of bone markers was（c-Myc-Oct4）in addition to hLMP-3.This cocktail induced the highest level of the bone markers’ m-RNA expression all over the study period.Different assessment tools were used to compare the difference between using hLMP-3 overexpression alone（Transdifferentiation）and the overexpression of the selected cocktail（Direct reprogramming）.Both groups showed the morphological changes from the spindle-shaped cells of MEF to be flat,followed by relatively polygonal shape.After 14 days,MEF cultures which have been induced to osteoblast,displayed clusters with high cell density and large amounts of an extracellular matrix（ECM）,and finally the characteristic appearance of osteoblast cells（cuboidal shape）.The results of the in vitro mineralization assay were consistent with the RT-PCR results.We found that after 21 days post transduction,both Alizarin red staining（ALZ）,and Von Kossa staining（VK）showed calcium deposition,and bone nodules formation.The staining intensity of c-Myc,Oct4 with hLMP-3 combination was more significant than the staining intensity of hLMP-3 group at（P<0.05）&（P<0.01）for both ALZ&VK respectively.The last step through the characterization process was the evaluation of osteocalcin（OCN）expression using immunofluorescence technique（IF）,and on protein level using western blot analysis（WB）.The combination of c-Myc,Oct4 and hLMP-3 induced the expression of OCN more than hLMP-3 alone.Thus,this reprogramming cocktail revealed the strongest capability of inducing osteoblast-like cells among our study groups.3-The reprogramming of MEF with c-Myc,Oct4 and LMP-3 together didn’t pass through any intermediate pluripotency stage;by detection of the Nanog（pluripotency marker）expression on cells using IF technique,and RT-PCR at different time points in comparison with iPSCs.Our reprogramming cocktail revealed negative Nanog expression in contrast to the iPSCs that showed positive Nanog expression.Moreover,a significant decrease in c-Myc and Oct4 expression level was observed in comparison with iPSCs.4-The efficiency of the reprogramming cocktail was tested using an in vivo design to reveal the ability of the reprogrammed cells to induce bone formation in an induced unilateral cortical bone defect in the femur of an orthotopic rat animal model.The rat model was selected to proof that the reprogramming cocktail could induce bone formation in different animal models not only in mice.The results of the in vivo experiment proved the in vitro results and that the reprogramming cocktail could induce bone formation inside the bone defect after transplantation.The radiographic and histological examination revealed complete healing within one-month post-transplantation;while the control group didn’t show any signs of healing with many complications occurred as complete femoral fracture in some rats.5-To enrich the osteogenic culture medium,two natural inducers（curcumin&ATRA）were tested in BMSCs culture.First,BMSCs were isolated from five to six weeks old male BALB/c.mice.An almost homogeneous fibroblastic like cell population was observed after 15 days of culture,with little evidence of round or floating cells.These cells were induced to differentiate and proliferate into osteogenic lineage in vitro.The osteogenic medium was supplemented with 15μM curcumin,or 10"6 mol/L ATRA to study their effect on the osteogenic differentiation of the BMSCs.The morphology of BMSCs changed during the first week in an osteogenic medium from the characteristic spindle fibroblast to the characteristic primary osteoblast morphology（cuboidal appearance）in both control（BMSCs in osteogenic medium）,and curcumin groups.In contrast,cells in ATRA group displayed a more elongated shape,and dendrites appear clearly detectable.Both curcumin and control groups showed significant staining intensity in ALZ and VK after 28 days.The mineralization was greater in the curcumin group than the control group by measuring the staining intensity of both ALZ and VK（P<0.05）&（P<0.01）respectively.On the other hand,ATRA group did not show any signs of mineralization after staining.The same results were obtained after staining different groups with VK stain after 28 days.Curcumin group showed numerous areas of black deposits indicating sites of calcium deposition and mineralization.The black stained characteristic bone nodules were higher in the curcumin group than the control group;while the ATRA group did not demonstrate any positive staining results.On the same way,the curcumin group showed significant expression of m-RNA bone markers’ over the control and ATRA groups.In addition,curcumin supplemented osteogenic medium increased OCN expression as detected by IF and WB analysis.On the opposite side,ATRA inhibited the mineralization activity of the BMSCs during osteogenic differentiation.Finally,the curcumin supplementation effect on the osteogenic culture medium was examined on MEF cells after reprogramming with hLMP-3.The curcumin-supplemented group increased the hLMP-3 osteogenic potency in comparison to the non-supplemented hLMP-3 group.This was shown by a significance increase in the m-RNA expression of Runx2 at（P<0.01）,BMP-2 and Osx at（P<0.05）.
陈建武[6]（2016）在《异体复合组织移植中血管化骨髓的致耐受研究》文中研究指明【背景】严重烧、创伤往往伴有深部软组织和骨组织缺损,由于缺损组织多、面积大,传统“拆东墙补西墙”的修复方式一般无法修复。异体复合组织（VCA）,由于取自尸体相同部位的组织,可避免以上不足,达到比较满意的修复外形,甚至恢复部分功能,有望成为此类缺损的理想修复手段。自1998年首例手移植和2005首例脸移植成功以来,全球已进行超过100例手移植和30多例脸移植手术,取得了较好的外观和功能修复结果,鼓舞着后来人对异体复合组织移植的探索。然而,复合组织移植面临的主要问题是排斥和终身服用免疫抑制药物,这对“life-saving”的器官移植可以接受,但对“life-changing”的复合组织移植无疑不可取,限制了其应用和推广。所以,如何减少或避免免疫抑制剂的使用成为异体复合组织移植能否进一步发展的关键。骨髓细胞移植具有免疫耐受诱导作用,相关机制也得到大量研究。目前已有成功诱导临床肾耐受的报道,先后在三个中心获得成功,术后无需服用免疫抑制剂即可长期存活,而且无排斥反应发生。某些复合组织,如脸移植和手移植,本身包含血管化的骨瓣,具有骨髓和骨髓微环境,有可能作为一种更加高效的骨髓移植方式。既然移植骨髓无需经历骨髓加工、分离和归巢等过程,在其原有微环境即可增殖分化,为受体持续供应供体细胞,从而促进耐受形成。业已证实,后肢在清髓性预处理条件下可自我诱导耐受,灵长类动物的脸移植也因附带骨瓣而使存活时间延长。所以,针对复合组织含血管化骨髓这一特性,本实验拟研究这一特殊骨髓移植方式的致耐受潜能,从而试图为复合组织致耐受,也为器官移植致耐受,开辟一个新的途径。为此,首先有必要构建一个合适的带血管骨髓移植模型。传统的模型多以大鼠后肢为移植模型,它模拟了临床手移植,既有利于研究移植物的免疫学特性,还可评估术后功能恢复情况。然而,该模型手术操作复杂,涉及截骨和髓内固定,受体创伤大、死亡率和并发症高。既然,我们的研究主要关注于复合组织的免疫学特性,即血管化骨髓,而对功能要求不高。因此,我们拟替代后肢,构建一个组织成分与之类似,但操作更为简便的模型,从而有利于血管化骨髓的研究。血管化骨髓不仅包含造血干细胞,还包括内皮细胞、成纤维细胞、脂肪细胞和成骨细胞等基质细胞,他们对移植骨髓细胞功能的发挥至关重要。大量研究证实,骨髓细胞联合成骨细胞、间充质干细胞甚至去骨髓的骨瓣等均可促进骨髓植入,有利于清髓受体的造血重建及耐受诱导。那么这种带血管的骨髓移植是否也像静脉骨髓移植一样具有造血能力?能否作为一种高效的骨髓移植方式?这都是需要研究回答的问题。研究血管化骨髓的最终目的是耐受,所以我们拟在非清髓性免疫诱导方案作用下,探索血管化骨髓的致耐受潜能。通常,骨髓诱导耐受有赖于受体去t细胞治疗、胸腺照射和清髓性预处理,三者缺一不可,其中前两者是为克服外周血和胸腺内的排斥反应,而后者是为移植骨髓“腾出空间”,提高骨髓细胞的植入率。这也是现有免疫诱导方案的基本组成部分,然而该方案涉及全身清髓,易导致骨髓抑制、三系细胞减少等并发症,不适于复合组织移植。血管化骨髓同时包含骨髓和微环境,即自带“生存空间”,可能无需“腾空间”的全身清髓即可诱导耐受。所以,我们拟设计了一个低毒、无清髓的诱导方案,并基于此方案来阐明血管化骨髓的致耐受作用。【目的】1)构建一个简便、可靠的带血管骨髓移植模型;2)探索带血管骨髓的造血重建潜能,并与传统的静脉骨髓移植进行比较,观察其可否作为一种有效的骨髓移植方式;3)探索复合组织移植中血管化骨髓对其存活的影响,并对相关的致耐受机制进行研究。【方法】1)模型构建:以股骨作为骨髓载体,在同基因的lewis大鼠间将含腹股沟皮瓣和部分股骨的骨肌皮瓣移植到受体腹股沟区,显微吻合供受体间的营养血管即股动静脉。术前通过解剖学研究,确定移植物的营养血管和基本手术步骤;术中通过美兰灌注,观察营养血管的供血分布情况;术后从组织病理学角度分析移植物的活性,验证该模型的可行性;最后改进实验模型,衍生多个不同的带血管骨髓移植模型,为后续不同实验的开展提供模型工具。2)带血管骨髓的造血潜能评估-1:以上部分实验构建的股骨移植模型为骨髓载体,将gfp大鼠股骨移植给经5gy放射清髓的lewis大鼠,术后给予环孢菌素a抑制排斥和gvhd（移植物抗宿主疾病）,然后通过血常规、脾指数测定、骨髓细胞计数和组织病理等方法评估受体的造血恢复情况,并用流式、荧光显微镜等方法分析细胞来源。所得结果与静脉骨髓移植组（静注与股骨等量的骨髓细胞,阳性对照）和假手术组（阴性对照）进行比较,评估血管化骨髓的造血潜能。3)带血管骨髓的造血潜能评估-2:为避免因排斥和供体基因不纯而引起的实验误差,类似实验在同基因lewis大鼠中进行重复,再次评估带血管骨髓、静脉骨髓细胞悬液和假手术组的造血重建情况。4)带血管骨髓致耐受性评估:以mhc完全不相符的近交系lewis和bn大鼠作为移植供受体,所有大鼠行腹股沟皮瓣移植,同时分别联合股骨移植（vbmt组）、静脉骨髓移植（i.v.bmt组）和生理盐水注射（control组）,并给予抗淋巴细胞血清、胸腺照射和环孢菌素a进行免疫诱导。术后对比各组皮瓣的存活时间和外周血、组织中供体特异性嵌合水平,并通过组织病理学方法评估各组皮瓣的排斥程度,组织切片和流式分析移植股骨的活性和组织来源,探索其致耐受的可能机制。【结果】1)（1）股骨移植平均手术时间（159.0±8.3）min,其中供体剥离时间（68.0±4.8）min,缺血时间（55.8±6.8）min;（2）美兰灌注显示股动静脉可以保证股骨和皮瓣的血供,模型具有一定的可行性;（3）所有10个移植物长期存活,无一例坏死,皮瓣外观红润,有毛发生长;（4）皮肤和股骨的he染色结果基本正常,证实了模型的可行性;（5）在此模型基础上进一步发展了含皮瓣的全股骨移植模型和不含皮瓣的全股骨移植模型。2)（1）与假手术对照组相比,带血管骨髓和静注的骨髓细胞悬液都显着加速了清髓大鼠白细胞和血小板的恢复（p<0.05）,且恢复细胞主要来自gfp供体而非受体;（2）带血管骨髓促进了脾集落的形成,脾脏指数高于假手术组（p<0.05）,脾脏he染色虽未见结构清晰的白髓,但可见大量淋巴小结和淋巴细胞,而假手术组脾内细胞成分明显减少;（3）同样,带血管骨髓移植组受体骨髓he染色也可见稀疏的骨髓细胞,而对照组骨髓基本被空泡性组织所取代;单根胫骨骨髓细胞数也较对照组高一个数量级（p<0.05）;且骨髓流式和骨髓涂片显示90%以上为gfp+,提示移植骨髓重建受体的造血系统。（4）移植后15d切除移植股骨,恢复的白细胞和血小板再次减少,进一步验证了vbm在早期受体造血重建中的关键作用;（5）然而,以上各项指标,除第5d时vbmt组血小板数高于i.v.bmt组外,其余指标vbmt和i.v.bmt均无明显差异,甚至在骨髓重建上vbmt还稍慢于i.v.bmt;（6）血管化骨髓的同基因移植所得结果与上述结果类似,再次验证了vbmt的造血潜能。3)（1）通过抗淋巴细胞血清、胸腺照射和环孢素a联合诱导,可以有效的清除受体外周血t细胞和淋巴细胞,为耐受诱导提供了条件;（2）在此诱导方案作用下,带血管骨髓显着延长皮瓣存活到78.8±13.0d,而静脉骨髓组和对照组分别在60.4±1.7d和58.6±1.3d发生排斥;（3）带血管骨髓诱导了稳定的外周血嵌合,约5.81%±1.98%,至75d时消失,而另两组整个观察期几乎无嵌合形成;（4）带血管骨髓促进供体细胞定植到受体脾、骨髓、淋巴结,但胸腺无供体定植;（5）移植股骨所含骨髓细胞逐渐被受体细胞所取代,不能持续为受体供应供体细胞;（6）移植股骨内骨髓可能与皮瓣同时发生排斥或先于皮瓣,且最终纤维化。【结论】我们的研究有几个重要发现:1)通过股骨移植,我们成功构建了多个简便、可靠的带血管骨髓移植模型,为复合组织移植中血管化骨髓的研究提供了模型基础;2)证实了血管化骨髓的造血作用,可以作为一种有效的骨髓移植方式,为血管化骨髓的致耐受研究提供了理论依据;3)在非清髓性免疫诱导方案下,发现复合组织中血管化骨髓可以延长复合组织的存活时间,并诱导稳定的嵌合;4)发现血管化骨髓不能源源不断为受体提供供体细胞,且最终被受体细胞所取代。总之,以上研究证实了异体复合组织中血管化骨髓的免疫保护作用,为复合组织免疫耐受的诱导提供了一个新的途径。
Tian Yu,Vijayalakshmi Rajendran,May Griffith,John V Forrester,Lucia Kuffová[7]（2016）在《High-risk corneal allografts: A therapeutic challenge》文中研究说明Corneal transplantation is the most common surgical procedure amongst solid organ transplants with a high survival rate of 86% at 1-year post-grafting. This high success rate has been attributed to the immune privilege of the eye. However, mechanisms originally thought to promote immune privilege, such as the lack of antigen presenting cells and vessels in the cornea, are challenged by recent studies. Nevertheless, the immunological and physiological features of the cornea promoting a relatively weak alloimmune response is likely responsible for the high survival rate in "low-risk" settings. Furthermore, although corneal graft survival in "lowrisk" recipients is favourable, the prognosis in "high-risk" recipients for corneal graft is poor. In "high-risk" grafts, the process of indirect allorecognition is accelerated by the enhanced innate and adaptive immune responses due to pre-existing inflammation and neovascularization of the host bed. This leads to the irreversible rejection of the allograft and ultimately graft failure. Many therapeutic measures are being tested in pre-clinical and clinical studies to counter the immunological challenge of "high-risk" recipients. Despite the prevailing dogma, recent data suggest that tissue matching together with use of systemic immunosuppression may increase the likelihood of graft acceptance in "high-risk" recipients. However, immunosuppressive drugs are accompanied with intolerance/side effects and toxicity, and therefore, novel cell-based therapies are in development which target host immune cells and restore immune homeostasis without significant side effect of treatment. In addition, developments in regenerative medicinemay be able to solve both important short comings of allotransplantation:（1） graft rejection and ultimate graft failure; and（2） the lack of suitable donor corneas. The advances in technology and research indicate that wider therapeutic choices for patients may be available to address the worldwide problem of corneal blindness in both "low-risk" and "high-risk" hosts.
ALANI MOHANAD KHALID AHMED（李剑）[8]（2015）在《跟腱干细胞及骨髓间充质干细胞移植在大鼠跟腱再生中的研究》文中进行了进一步梳理目的：肌腱是一类由平行排列的纤维胶原束构成的致密结缔组织,连接肌肉-骨,支持身体的运动。随着人们生活水平的提高,运动成为一种流行的、健康的生活方式,肌腱损伤的病例也迅速增多,其中,有35%属于跟腱断裂。肌腱损伤后,由于组织内血供较少,组成的成纤维细胞代谢较低,修复缓慢,且恢复到损伤前水平较难。目前,常见的肌腱损伤治疗方法可分为：保守治疗和手术治疗。保守治疗仅仅能缓解症状,且耗时长,病人需要经历长期的康复过程,临床疗效不能令人满意；手术治疗除了常规的手术缝合外,还依赖于移植物,包括白体、异体、假体移植。但是这些移植治疗方法又有其特有缺陷,例如白体移植需要牺牲自身供区的组织,是一种“拆东墙补西墙”的方法,还可能会引发供区出现并发症,会导致病人额外的创伤；异体移植则存在组织来源困难、异体疾病传染风险、免疫排斥等弊端；假体移植存在移植物整合差、远期疗效不佳等缺点。因此,有必要探索新的、更加有效的跟腱损伤治疗途径。干细胞具有克隆形成、多向分化、自我更新的特性,是组织再生的必要因素。大量研究表明,骨髓间充质干细胞（bone marrow-derived mesenchymal stem cells, BMSCs）具有潜在分化为多种结缔组织的特性,包括：骨、软骨、肌腱、肌肉、骨髓、脂肪等。有报道表明,骨髓间充质干细胞能促进肌腱组织的再生修复。由肌腱、韧带组织分离的肌腱干细胞（tendon-derived stem cells, TDSCs）是一种成体干细胞,可参与肌腱损伤的修复过程。有报道表明,TDSCs较BMSCs具有更强的克隆形成能力,更快的增殖速率。在蛋白表达谱及基因表达水平上,TDSCs是较BMSCs更原始的一种干细胞种类。同时,TDSCs高表达肌腱组织相关标志物：比如转录因子scleraxis （Sex）和tenomodulin （Tnmd）,以及细胞外基质成分Collagen Ⅰ、 Collagen Ⅲ和decorin （Dcn）等。因此TDSCs可能是一种更加优良的可用于促进肌腱组织再生修复的种子细胞。但是,对于TDSCs及BMSCs用于肌腱断裂修复的作用差异未见系统的研究报道。因此,本文采用SD大鼠为研究对象,提取分离两种干细胞。在体外考察两者的基因转录、蛋白表达、细胞表型、增殖速率等差异；在此研究基础上,以大鼠跟腱断裂为疾病模型,考察细胞移植对于肌腱断裂修复的作用。方法：本研究中,我们首先成功的从大鼠骨髓与跟腱中分离得到了BMSCs和TDSCs,通过流式细胞检测、多向分化诱导实验测定其干细胞属性。此外,连续观察两种干细胞由第一代至第七代的细胞形态,考察两种干细胞的细胞形态差异及生长状态。在体外实验中,我们通过RT-PCR、免疫荧光染色实验、EdU细胞增殖实验、MTS光密度比色细胞生存活力检测实验分别比较了TDSCs和BMSCs的Oct-4,Nucleostemin, P75, Nanog, α-SMA, Biglycan, Vimentin, Decorin, Scleraxis, Tenascin-C, Collagen Ⅲ, Collagen Ⅱ, Collagen Ⅰ, SDF-1基因表达差异、CD29, CD44, CD90, CD 146, Oct-4, α-SMA及Nucleostemin蛋白表达谱差异、DNA合成速率以及细胞增殖的差异。在体内实验中,我们首先建立了SD大鼠跟腱断裂模型,在跟腱损伤后将两种干细胞分别移植到大鼠跟腱断裂处,取不同时间点（1周、2周、3周）,从外观检查、微观组织学评价、生物力学测定来考察两种干细胞对于断裂肌腱的修复作用。同时,采用定量PCR技术对于移植后的肌腱组织内胶原基因表达状况及表达差异进行分析,采用切片免疫荧光染色技术探讨移植后两种干细胞促进肌腱组织修复的可能作用机制。结果：由SD大鼠跟腱及骨髓中分离得到的TDSCs与BMSCs具有向骨、软骨、脂肪细胞分化的能力,并且均表达干细胞表明标志物：CD29, CD44, CD90,这两种细胞具有干细胞的基本特征,可认定两者为多潜能干细胞。由体外培养细胞形态学观察可知,两种细胞在第三代至第五代时细胞核较大,细胞体较为短小呈现了较好的细胞形态,其中TDSCs多数细胞呈现短梭型,具有较大的核质比；BMSCs多呈现为菱形或梭型,细胞形态均一。而细胞体外培养至第六代的时候,细胞开始出现老化,细胞形态变形。因此,由形态学观察可知,处于P3至P5代的细胞状态良好,可用于后续的实验研究。在体外比较实验中,我们发现TDSCs的Tenascin-C及Scleraxis基因表达水平较BMSCs高,这两个基因是肌腱组织修复、形成的关键因素之一。此外,TDSCs高表达Nucleostemin,而BMSCs的Nucleostemin表达量较低。此结果表明TDSCs表达更多的肌腱组织相关基因,并具有更强的干性特征。同时我们发现,TDSCs的DNA合成速率、细胞增殖速率均较BMSCs快。将两种细胞分别移植到大鼠跟腱断裂处,外观观察显示TDSCs处理组肌腱比BMSCs处理组肌腱具有更接近健康肌腱的外观形态,且修复速度更快。组织学检测显示,TDSCs处理组比BMSCs处理组更早出现较为规则的胶原纤维束排列的组织学形态,且细胞外基质更丰富。本研究还新研制了一种适合生理状态下测试生物组织力学性能的夹具,成功应用于肌腱的力学测试。结果表明,在修复早期（2周内）TDSCs处理组肌腱比BMSCs处理组肌腱能承受更大的力学拉伸。进一步的修复机制研究表明,两种细胞均能促进Tenascin-C在受损跟腱处的表达,且TDSCs的促进作用更加强。对修复后的肌腱进行基因和蛋白表达检测,结果也表明TDSCs处理组表达更为丰富的细胞外基质成分。此外,两种细胞在移植4周后,依然能在肌腱组织内检测到,说明移植的细胞已经整合进了宿主的肌腱组织中。因此,从微观及宏观水平上均表明,TDSCs较BMSCs更能促进肌腱组织的再生修复,其机制可能与TDSCs较好的促进了细胞外基质成分表达有关。结论：与BMSCs比较,TDSCs具有更高的增殖速率,表达更丰富的肌腱相关基因与蛋白。当移植入大鼠跟腱断裂处后,TDSCs和BMSCs都能促进肌腱组织的修复,但是TDSCs表现了更强的促进断裂肌腱再生能力。在断裂肌腱组织处,TDSCs高表达肌腱修复关键因子Tenascin-C可能是TDSCs较BMSCs修复作用更佳的原因之一。总之,本研究的结论表明TDSCs较BMSCs具有更优异的促进跟腱损伤修复的潜能,可作为跟腱损伤修复组织工程学理想的种子细胞。
Leandro Ryuchi Iuamoto,Alberto Meyer,Eleazar Chaib,Luiz Augusto Carneiro D’Albuquerque[9]（2014）在《Review of experimental attempts of islet allotransplantation in rodents:Parameters involved and viability of the procedure》文中提出The purpose of the present study was to organize the parameters involved in experimental allotransplantation in rodents to elaborate the most suitable model to supply the scarcity of islet donors. We used the PubMed database to systematically search for published articles containing the keywords "rodent islet transplantation" to review. We included studies that involved allotransplantation experiments with rodents’ islets, and we reviewed the reference lists from the eligible publications that were retrieved. We excluded articles related to isotransplantation, autotransplantation and xenotransplantation, i.e., transplantation in other species. A total of 25 studies related to allotransplantation were selected for systematic review based on their relevance and updated data. Allotransplantation in rodents is promising and continues to develop. Survival rates of allografts have increased with the discovery of new immunosuppressive drugs and the use of different graft sites. These successes suggest that islet transplantation is a promising method to overcome the scarcity of isletdonors and advance the treatment options for type 1 diabetes.
曹姣[10]（2012）在《CTLA4-Ig重组慢病毒引流淋巴结定向表达的研究》文中认为新型免疫抑制药物的研制及发展，为整形外科在异体复合组织移植领域的稳步发展带来了新的机遇。然而，异体复合组织移植（CTA）的发展远远不似内脏器官移植一样迅速，究其根本，是因为异体复合组织移植物组分复杂，并且抗原性不均一。自1998年首次手移植被Dubernard成功实施以来，到目前为止,在临床上已完成超过60例手移植和23例异体颜面移植。第一例异体颜面移植在法国成功施行手术后，曾经发生2次急性排斥反应，第二例患者是在本科室成功实施后，术后也发生了3次急性排斥反应。加大免疫抑制剂用量是使排斥反应得到控制的有效方法，但是往往会出现各种棘手的并发症如急性肾衰、高血压症等。鉴于上述原因，寻找可以使免疫抑制剂用量最小化的方法，已成为CTA移植领域重点研究的项目。免疫应答的一般规律同样适用于异体复合组织移植排斥反应的发生，首先，外来的异体抗原被免疫识别之后激活机体免疫系统，继而产生对移植物的免疫排斥反应，也就是说，阻断免疫应答中的任何一个环节，都可以实现对移植免疫耐受的诱导。细胞毒性T淋巴细胞相关抗原4抗体融合蛋白（CTLA4-Ig）可以阻断CD28-B7途径，能有效诱导免疫耐受的发生。树突状细胞毋庸置疑是最主要的抗原提呈细胞，在其表面可以高水平表达MHC-II类分子以及CD80、CD86等共刺激分子，通过B7分子与CD28的结合，开启第二信号刺激途径，从而激活初始T细胞。成熟的树突状细胞具有淋巴归巢的特性，因此，CTLA4-Ig重组慢病毒转染的成熟树突状细胞有可能成为细胞回输治疗方案的备选细胞。目的：研究CTLA4-Ig直接淋巴结注射后其表达效率、CTLA4-Ig重组慢病毒转染成熟树突状细胞后相关基因的表达，局部注射转染后的成熟树突状细胞后观察CTLA4-Ig在引流淋巴结内的表达，探索淋巴结直接基因治疗方法的可行性；试图通过局部细胞治疗诱导免疫耐受，降低全身免疫抑制剂的用量，为同种异体复合组织移植探索新途径。方法：1．大鼠淋巴结内注射CTLA4-Ig重组慢病毒，观察CTLA4-Ig在淋巴结内的表达。2．贴壁培养法体外诱导成熟树突状细胞，进行形态学鉴定、活性及表型鉴定，体外鉴定其免疫学功能。3．体外试验研究CTLA4-Ig重组慢病毒转染大鼠成熟树突状细胞，观测CTLA4-Ig的表达时间及表达高峰。4．体内试验研究CTLA4-Ig重组慢病毒转染的成熟树突状细胞于大鼠引流淋巴结的定向表达。结果：1．实时荧光定量PCR及冰冻切片示，空白对照及阴性对照组淋巴结内均无CTLA4-Ig的表达。CTLA4-Ig重组慢病毒淋巴结内注射组有明显的CTLA4-Ig表达。2．光镜见培养的成熟树突状细胞集落分散，悬浮，分布较均匀。扫描电镜见细胞表面大量褶皱与粗细不等的突起。流式细胞术显示成熟树突状细胞表面的DC功能相关抗原MHC II、CD80、CD86均高表达。功能试验检测其分泌IL-12能力较未成熟树突状细胞强，混合淋巴细胞反应显示其有强的刺激T细胞增殖的能力。活细胞率为（80.34±1.25）%。3．慢病毒转染成熟树突状细胞76h后，于倒置荧光显微镜下可观察到荧光表达。随时间延长，荧光表达增多，细胞形态亦出现老化。转染后第7d细胞明显老化。Q-PCR示CTLA4-Ig重组慢病毒转染成熟树突状细胞第3天，于mRNA水平有CTLA4-Ig的表达，western blotting检测所示结果与Q-PCR一致。4．距大鼠腋固有淋巴结处2公分皮下缓慢注射所收获的CTLA4-Ig重组慢病毒转染后第3天的DCs，术后第4天取材，Q-PCR显示注射CTLA4-Ig重组慢病毒转染的DCs组于淋巴结内可检测到CTLA4-Ig的表达。结论：1．CTLA4-Ig重组慢病毒淋巴结内直接注射可行,但范围局限且损伤较大。2．细胞治疗初步显示可行，其效果需在异体复合组织移植模型中做进一步的验证。
二、The Characteristics of Acute Rejection after Limb Allotransplantation in Rats─An Experimental Study（论文开题报告）
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三、The Characteristics of Acute Rejection after Limb Allotransplantation in Rats─An Experimental Study（论文提纲范文）
（2）椎间盘退变关键标志物筛选及携载TGF-β3支架对椎间盘修复的实验研究（论文提纲范文）
| 摘要 |
| Abstract |
| 符号说明 |
| 前言 |
| 第一部分 椎间盘退变关键标志物的筛选 |
| 1 材料与方法 |
| 2 结果 |
| 3 讨论 |
| 4 结论 |
| 第二部分 携载TGF-β3的脱细胞纤维环基质/壳聚糖水凝胶对椎间盘退变修复的实验研究 |
| 1 材料与方法 |
| 2 结果 |
| 3 讨论 |
| 4 结论 |
| 参考文献 |
| 综述 |
| 参考文献 |
| 附图 |
| 附表 |
| 致谢 |
| 攻读硕士期间发表文章 |
| 学位论文评阅及答辩情况表 |
| 英文论文一 |
| 英文论文二 |
（4）辛伐他汀通过抑制TLR4的表达改善大鼠主动脉同种异体移植的动脉硬化（论文提纲范文）
| 中文摘要 |
| abstract |
| 1.Introduction: |
| 2.Methodology |
| 2.1 Experimental Animals |
| 2.2 Experimental Groups of Animals |
| 2.2.1 Isogenic group (group A): SD to SD |
| 2.2.2 Simvastatin intervention group(Group B) (Wistar to SD) |
| 2.2.3 Allogeneic Transplantation Group(Group C) (Wistar to SD) |
| 2.3 Experimental equipment |
| 2.4 Experimental drugs and reagents |
| 2.5 Surgery of Donor Rats |
| 2.6 Preparation or Fabrication of vascular cannula |
| 2.7 Recipient surgery: |
| 2.8 Extracting the graft artery |
| 2.9 Animal model intervention |
| 2.10 Specimen Collection |
| 2.11 Specimen HE Staining |
| 2.12 Immunohistochemistry(IHC)of specimen cells |
| 2.13 Pathological and Morphological observation of transplanted carotid artery |
| 2.14 Measurement of intimal thickness of transplanted arteries |
| 2.15 Light microscope observation |
| 2.16 Research Difficulties and Solutions |
| 2.17 Statistical analysis of data |
| 3.Results |
| 4.Discussion |
| 4.1 Current status of heart graft vascular disease |
| 4.2 Modeling Experience |
| 4.3 Treatment of heart graft vascular disease (CAV) |
| 4.4 The role of TLR4 in heart graft vascular disease |
| 4.5 Statin related research and its clinical application |
| 4.6 Simvastatin may help to delay the development of endometrial proliferation of heart grafts |
| 4.7 Simvastatin inhibits the activity of TLR4,NF-Kb,VCAM,ICAM and MCP-1 and delay the development of endometrial proliferation of heart grafts |
| 4.8 Limitations of the research |
| 4.9 Conclusion of my research |
| 5.References |
| Review Research progress of vascular graft disease |
| References |
| 8.Acknowledgement |
（5）Conversion of Mouse Embryonic Fibroblast and Bone Marrow Mesenchymal Stem Cells into Functional Osteoblast by Defined Factors（论文提纲范文）
| Abstract |
| List of abbreviations |
| Chapter 1. Review of literature |
| 1. Bone biology |
| 1.1 Bone function |
| 1.2 Bone anatomy |
| 1.3 Bone components |
| 1.3.1 Bone matrix (ECM) |
| 1.3.2 Cellular components |
| 1.3.3 Bone marrow |
| 1.4 Bone development (formation) |
| 1.4.1 Endochondral ossification |
| 1.4.2 Intramembranous ossification |
| 1.5 Bone remodeling |
| 1.6 Bone healing |
| 1.6.1 The inflammatory phase |
| 1.6.2 The reparative phase |
| 1.6.3 The remodeling phase |
| 2. The Need for Bone Repair and Current Therapies |
| 2.1 The need for bone repair |
| 2.2 The current treatments for bone repair |
| 3. Tissue engineering strategies for bone regeneration |
| 3.1 Cell based strategies |
| 3.1.1 Tissue specific cells (Osteoblasts) |
| 3.1.2 Stem cells |
| 3.1.2.1 Mesenchymal stem cells (MSCs) |
| 3.1.2.2 Embryonic stem cells (ESCs) |
| 3.1.2.3 Induced pluripotent stem cells (iPSCs) |
| 3.2 Osteoinductive factors |
| 3.2.1 Osteoinductive chemical factors |
| 3.2.2 Osteoinductive biological factors |
| 3.3 Biomaterials (Scaffold materials) |
| 4. Gene therapy strategies for bone regeneration |
| 4.1 Methods of Gene delivery |
| 4.1.1 The in vivo gene delivery |
| 4.1.2 The ex vivo gene delivery |
| 4.1.2.1 Cell-mediated gene therapy |
| 4.2 Vectors for gene delivery |
| 4.2.1 Gene delivery via non-viral vectors |
| 4.2.2 Gene delivery via viral vectors |
| 4.2.2.1 Retrovirus/Lentivirus |
| 4.2.2.2 Adenovirus |
| 4.2.2.3 AAV |
| 5. Lineage reprogramming |
| 5.1 Trans-differentiation (trans-determination or direct conversion) |
| 5.2 Direct reprogramming |
| 6. References |
| Chapter 2. Conversion of mouse embryonic fibroblasts (MEF) into functional osteoblasts by defined factor |
| 1. Introduction |
| 2. Material and methods |
| 2.1 Genetic materials |
| 2.1.1 Primer design and PCR amplification |
| 2.1.2 Gel purification of PCR Products |
| 2.1.3 Ligation of Genes into cloning vector |
| 2.1.4 Restriction Enzyme Digestion & Purification of the interested products |
| 2.1.5 Preparation of the competent cells for transformation |
| 2.1.6 Transformation of Ligated DNA into Bacterial Cells |
| 2.1.7 Colonies pick up, screening for positive colonies and plasmid extraction |
| 2.1.8 Screening for Positive Colonies |
| 2.1.9 DNA Sequencing to Confirm Correct DNA Identity |
| 2.1.10 Construction of the Lentivirus expression vectors |
| 2.2 Cells |
| 2.2.1 Isolation of MEF |
| 2.2.2 Cell Culture |
| 2.2.3 Cell Harvesting |
| 2.2.4 Cell freezing |
| 2.2.5 Cell Thawing and Recovery |
| 2.3 Study design |
| 2.4 In vitro osteogenic Induction |
| 2.4.1 Determination of the multiplicity of infection (MOI) |
| 2.4.2 Lentivirus mediated transduction |
| 2.5 Characterization of the osteogenic induction |
| 2.5.1 Real-Time RT-PCR |
| 2.5.1.1 RNA extraction |
| 2.5.1.2 cDNA synthesis |
| 2.5.1.3 RTPCR reaction |
| 2.5.2 In vitro mineralization assay |
| 2.5.2.1 ALP staining |
| 2.5.2.2 Alizarin Red staining |
| 2.5.2.3 Von Kossa staining |
| 2.5.3 Fluorescence Immunocytochemistry |
| 2.5.4 Western blot analysis |
| 2.6 Statistical analysis |
| 3. Results |
| 3.1 PCR amplification of the interested factors |
| 3.2 Confirmation of the successful cloning of the desired genes |
| 3.3 Confirmation of the correct DNA Identity |
| 3.4 Isolation and culture of MEF cells |
| 3.5 Lentivirus transduction efficiency and the multiplicity of infection |
| 3.6 Induction of osteoblasts from MEF by the combination of hLMP-3 and Yamanaka factors |
| 3.6.1 Morphological changes during reprogramming process |
| 3.6.2 Molecular characterization of the hLMP-3 in the transduced cells |
| 3.6.3 Expression level changes of the bone gene markers during reprogramming |
| 3.7 Conversion efficiency of induced osteoblasts from c-Myc, Oct4 and hLMP-3 combination |
| 3.7.1 Characterization of the generated osteoblasts using in vitro mineralization assay |
| 3.7.2 Evaluation of OCN expression as a late osteogenic marker |
| 3.8 MEF cells were directly reprogrammed into osteoblast like cells without passing through anintermediate pluripotency stage |
| 4. Discussion |
| 5. References |
| Chapter 3. In vivo study of the osteoblast formed cells after direct reprogramming of MEFs to form new bone inan induced bone defect |
| 1. Introduction |
| 2. Material and methods |
| 2.1 Animal model |
| 2.2 MEF cells isolation, transduction, and characterization |
| 2.3 Cell seeding and scaffold material |
| 2.4 Animal anesthesia and surgical procedures: |
| 2.5 Evaluation of the transplantation procedure |
| 2.5.1 Radiographic examination |
| 2.5.2 Histological examination |
| 3. Results |
| 3.1 Induction of a unilateral cortical femoral defect in SD rat |
| 3.2 Osteoblasts from the COL group induced bone repair after transplantation into a unilateralbone defect |
| 4. Discussion |
| 5. References |
| Chapter 4.Comparison between curcumin and all-trans retinoic acid in the osteogenic differentiation of mouse bonemarrow mesenchymal stem cells |
| 1. Introduction |
| 2. Material and methods |
| 2.1 Experimental animals |
| 2.2 Cells |
| 2.2.1 Isolation and culture of mouse BMSCs |
| 2.2.2 BMSCs freezing, thawing and recovery |
| 2.2.3 Characterization of mouse BMSCs |
| 2.3 Study design |
| 2.4 In vitro osteogenic Induction |
| 2.4.1 Experiment (Ⅰ) |
| 2.4.2 Experiment (Ⅱ) |
| 2.5 Characterization of the osteogenic differentiation |
| 2.5.1 Real-Time RT PCR |
| 2.5.2 In vitro mineralization assay |
| 2.5.3 Fluorescence Immunocytochemistry |
| 2.5.4 Western blot analysis |
| 2.6 Statistical analysis |
| 3. Results |
| 3.1 Morphological and immunotyping characterization of mouse BMSCs |
| 3.2 Morphological changes during the osteogenic differentiation of BMSCs |
| 3.3 Osteogenic differentiation capacity of BMSCs after induction with curcumin and ATRA |
| 3.4 The effect of curcumin and ATRA on the expression level of the bone associated gene markersduring BMSCs osteogenic differentiation |
| 3.5 Expression of the OCN during the osteogenic differentiation process |
| 3.6 Effect of curcumin supplemented OM on the osteogenic differentiation of lentivirustransduced MEFs with hLMP-3 |
| 4. Discussion |
| 5. References |
| Conclusion |
| Innovations |
| Acknowledgements |
| Publications |
（6）异体复合组织移植中血管化骨髓的致耐受研究（论文提纲范文）
| 缩略语表 |
| 中文摘要 |
| ABSTRACT |
| 前言 |
| 文献回顾 |
| 一、异体复合组织移植研究进展 |
| 二、骨髓细胞移植研究进展 |
| 三、带血管骨髓移植诱导异体复合组织免疫耐受的进展 |
| 四、小结 |
| 第一部分：构建大鼠带血管的骨髓移植模型 |
| 0 引言 |
| 1 材料 |
| 2 手术方法 |
| 3 实验方法 |
| 4 结果 |
| 5 讨论 |
| 6 小结 |
| 第二部分：带血管骨髓移植重建清髓受体的造血系统 |
| 0 引言 |
| 1 材料 |
| 2 方法 |
| 3 结果 |
| 4 讨论 |
| 5 小结 |
| 第三部分：带血管骨髓移植延长大鼠复合组织存活时间及可能机制探索 |
| 0 引言 |
| 1 材料 |
| 2 方法 |
| 3 结果 |
| 4 讨论 |
| 5 小结 |
| 小结 |
| 参考文献 |
| 附录 |
| 个人简历和研究成果 |
| 致谢 |
（8）跟腱干细胞及骨髓间充质干细胞移植在大鼠跟腱再生中的研究（论文提纲范文）
| 中文摘要 |
| ABSTRACT |
| 1 Introduction |
| 1.1 Issues Raised and Research Significance |
| 1.1.1 Issues Raised |
| 1.1.2 The research significance |
| 1.2 Research Status |
| 1.2.1 The structure and function of the tendon |
| 1.2.2 Achilles tendon injuries |
| 1.2.3 The strategies for healing the Achilles tendon injury |
| 1.2.4 Surgical and Non-Surgical Approach |
| 1.3 Achilles tendon regeneration through tissue engineering |
| 1.3.1 Stem cells and their application in tendon injury repair |
| 1.3.2 Characteristics and usefulness of TDSCs as an alternative for tissue engineering |
| 1.3.3 Mesenchymal Stem Cells as a Candidate for Tendon and Ligament Repair |
| 1.3.4 Similarity and Immunohistochemically comparison of MSCs and TDSCs |
| 1.3.5 Advantages and disadvantages of TDSCs and BMSCs |
| 1.4 Objective and research content of this article |
| 1.4.1 Objectives |
| 2 Cell isolation and identification of TDSCs and BMSCs |
| 2.1 Introduction |
| 2.2 Materials and methods |
| 2.2.1 Experimental Animals |
| 2.2.2 The main experimental equipment and supplies |
| 2.2.3 The main laboratory reagents |
| 2.2.4 Preparation of some reagents |
| 2.2.5 Animals Experiments |
| 2.2.6 Observation of cell isolation and morphology |
| 2.2.7 Cell identification |
| 2.3 Results |
| 2.3.1 Cell morphology observation |
| 2.3.2 Cell multi-differentiation identification |
| 2.3.3 Cell surface markers |
| 2.4 Discussion |
| 2.5 Conclusion |
| 3 In vitro comparison among TDSCs and BMSCs through geneexpression, stem cell markers and cell proliferation |
| 3.1 Introduction |
| 3.2 Materials and methods |
| 3.2.1 The main experimental equipment and supplies |
| 3.2.2 The main laboratory reagents and supplies |
| 3.2.3 Preparation of common reagents |
| 3.2.4 RT-PCR assay (reverse transcription-polymerase chain reaction) |
| 3.2.5 Immunofluorescence staining (IF) |
| 3.2.6 MTS assay |
| 3.2.7 Ed U assay |
| 3.2.8 Data analyses |
| 3.3 Results |
| 3.3.1 Gene expression analyses of TDSCs and BMSCs |
| 3.3.2 Comparison of TDSCs and BMSCs Stem cells markers |
| 3.3.3 Cellular viability comparison among TDSCs and BMSCs |
| 3.3.4 Comparison for DNA synthesis in TDSCs and BMSCs |
| 3.4 Discussion |
| 3.5 Conclusion |
| 4 The Biomechanical testing comparison |
| 4.1 Introduction |
| 4.2 Materials and methods |
| 4.2.1 Material of the device |
| 4.2.2 Material used in the experiment |
| 4.2.3 Methods |
| 4.3 Results |
| 4.4 Discussion |
| 4.5 Conclusion |
| 5 The transplantation of TDSCs and BMSCs on rat ruptured Achillestendon |
| 5.1 Introduction |
| 5.2 Materials and methods |
| 5.2.1 Devices and matreial Manufacturer |
| 5.2.2 Reagent Manufacturer |
| 5.2.3 Preparation of some reagents |
| 5.2.4 Experimental animals |
| 5.2.5 Animal model and surgical procedure |
| 5.2.6 Macroscopic Assessment |
| 5.2.7 Histological evaluation |
| 5.2.8 Biomechanical Testing |
| 5.2.9 Quantitative Polymerase Chain Reaction (q PCR) |
| 5.2.10 Immunofluorescent testing |
| 5.2.11 Statistical analysis |
| 5.3 Results |
| 5.3.1 Macroscopic assessment of cell transplantation |
| 5.3.2 Findings of cell transplantation |
| 5.3.3 The ultimate failure load of biomechanical Testing |
| 5.3.4 Analyses of q RT-PCR |
| 5.3.5 Analyses of immunofluorescence |
| 5.4 Discussion |
| 5.5 Conclusion |
| 6 Conclusion and Future Perspective |
| 6.0 Conclusion |
| 6.1 Innovations Novel Findings |
| 6.2 Future perspective |
| 7 结论与未来展望 |
| 7.1 结论 |
| 7.2 创新点 |
| 7.3 未来展望 |
| ACKNOWLEDGEMENTS |
| REFERENCE |
| APPENDIX |
| A. Publications in the PH.D study |
| B. Scientific payoffs in the PH. D study |
| C. Scientific research projects in the PH.D study |
| D. AWARD |
（9）Review of experimental attempts of islet allotransplantation in rodents:Parameters involved and viability of the procedure（论文提纲范文）
| INTRODUCTION |
| SEARCH PROCESS |
| Search process |
| DATA ABSTRACTION |
| SEARCH RESULTS |
| DISCUSSION |
| CONCLUSION |
（10）CTLA4-Ig重组慢病毒引流淋巴结定向表达的研究（论文提纲范文）
| 缩略语表 |
| 中文摘要 |
| Abstract |
| 前言 |
| 文献回顾 |
| 1 同种异体复合组织移植临床进展概述 |
| 2 同种异基因移植排斥反应的防治进展 |
| 3 次级淋巴组织 |
| 4 CTLA4 -Ig |
| 5 成熟树突状细胞 |
| 实验一 大鼠淋巴结内注射 CTLA4-Ig 重组慢病毒 |
| 1 材料与方法 |
| 2 结果 |
| 3 讨论 |
| 实验二 大鼠成熟树突状细胞的培养及鉴定 |
| 1 材料与方法 |
| 2 结果 |
| 3 讨论 |
| 实验三 CTLA4-Ig 重组慢病毒转染大鼠成熟树突状细胞 |
| 1 材料和方法 |
| 2 结果 |
| 3 讨论 |
| 实验四 CTLA4-Ig 重组慢病毒转染 DCs 引流淋巴结定向表达 |
| 1 材料和方法 |
| 2 结果 |
| 3 讨论 |
| 小结 |
| 参考文献 |
| 个人简历和研究成果 |
| 致谢 |
四、The Characteristics of Acute Rejection after Limb Allotransplantation in Rats─An Experimental Study（论文参考文献）
- [1]Considerations for the clinical use of stem cells in genitourinary regenerative medicine[J]. Christophe Caneparo,Luis Sorroza-Martinez,Stéphane Chabaud,Julie Fradette,Stéphane Bolduc. World Journal of Stem Cells, 2021(10)
- [2]椎间盘退变关键标志物筛选及携载TGF-β3支架对椎间盘修复的实验研究[D]. 李钟奇. 山东大学, 2020(04)
- [3]Recent developments in regenerative ophthalmology[J]. Ye Shen,He Shen,Dongyu Guo,Xinghuai Sun,Yuan Sun,Nan Hong,Xiawei Wang,Chen Xie,Yuan Zhao,Qin He,Le Jin,Yingying Wen,Bo Jiang,Chenying Yu,Miaomiao Zhu,Feng Cai,Jianwu Dai. Science China（Life Sciences）, 2020(10)
- [4]辛伐他汀通过抑制TLR4的表达改善大鼠主动脉同种异体移植的动脉硬化[D]. Mohammad Zarif. 福建医科大学, 2020(07)
- [5]Conversion of Mouse Embryonic Fibroblast and Bone Marrow Mesenchymal Stem Cells into Functional Osteoblast by Defined Factors[D]. Mahmoud Fathy Ahmed Aly Issa. 扬州大学, 2019(02)
- [6]异体复合组织移植中血管化骨髓的致耐受研究[D]. 陈建武. 第四军医大学, 2016(02)
- [7]High-risk corneal allografts: A therapeutic challenge[J]. Tian Yu,Vijayalakshmi Rajendran,May Griffith,John V Forrester,Lucia Kuffová. World Journal of Transplantation, 2016(01)
- [8]跟腱干细胞及骨髓间充质干细胞移植在大鼠跟腱再生中的研究[D]. ALANI MOHANAD KHALID AHMED（李剑）. 重庆大学, 2015(07)
- [9]Review of experimental attempts of islet allotransplantation in rodents:Parameters involved and viability of the procedure[J]. Leandro Ryuchi Iuamoto,Alberto Meyer,Eleazar Chaib,Luiz Augusto Carneiro D’Albuquerque. World Journal of Gastroenterology, 2014(37)
- [10]CTLA4-Ig重组慢病毒引流淋巴结定向表达的研究[D]. 曹姣. 第四军医大学, 2012(01)
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