【作者】 高旭辉；

【导师】 王武军；

【作者基本信息】 第一军医大学 ， 胸心血管外科， 2007， 博士

【摘要】 本课题来源：广东省自然科学基金，项目编号990386；广东省医学科研基金，项目编号A1999326；广州军区武汉总医院科研基金，2005年度。免疫豁免概念的提出已经有一百多年的历史，机体的某些器官，如脑、眼、角膜、卵巢、胎盘等，不受免疫系统的干扰和排斥，称为免疫豁免器官。对免疫豁免机制的研究，有可能找到使移植的细胞、组织和器官突破同种甚至异种移植免疫屏障的新方法，具有广阔的临床应用前景和潜在的巨大经济效益；加强对免疫豁免机制的研究，将使器官移植摆脱免疫抑制剂的束缚，而使病人长期存活。国内外目前对睾丸支持细胞（Sertoli cells，Sc）免疫豁免机制的研究才刚刚起步。初步研究发现睾丸Sertoli细胞能产生细胞因子FasL（Fas的配体，属肿瘤坏死因子家族）和TGF-β1（Transforming Growth Factor-β1，转化生长因子β1），这两种细胞因子可能与睾丸Sertoli细胞产生免疫豁免功能有关。因此我们开展了对睾丸Seaoli细胞免疫豁免机制的进一步研究，并探讨Seaoli细胞对同种心脏移植免疫排斥反应及细胞凋亡的影响。本研究目的：1、探讨睾丸Sertoli细胞分离纯化方法，检测睾丸Sertoli细胞FasL、TGF-β1的表达率；2、将Sertoli细胞与异基因T淋巴细胞共同培养，观察Sertoli细胞对异基因T淋巴细胞的毒性作用；3、观察Sertoli细胞对异基因T淋巴细胞凋亡的影响；4、制作同种异体大鼠腹腔异位心脏移植动物模型，在移植心脏种植与供心同基因Sertoli细胞，掌握制作大鼠腹腔异位心脏移植动物模型的外科技术；5、对种植Sertoli细胞的移植心脏与未种植Sertoli细胞的移植心脏，比较两组急性排斥反应（Acute Rejection，AR）、心肌细胞凋亡指数（ApoptosisIndex，AI）、心肌内Fas（Factor associated suicide，自杀相关因子，又称CD95或APO-1）mRNA、FasL mRNA、TGF-β1 mRNA的表达，并进行相关分析，明确Sertoli细胞对心脏移植免疫学影响，探讨Sertoli细胞对同种异体移植心脏是否具有免疫豁免作用。实验一、Sertoli细胞的分离纯化及其免疫豁免机制的研究一、目的1、探讨大鼠睾丸Sertoli细胞分离提纯方法，检测Sertoli细胞FasL、TGF-β-1的表达百分率；2、将大鼠睾丸Sertoli细胞与异基因T淋巴细胞共同培养，观察Sertoli细胞对异基因T淋巴细胞的毒性作用；3、观察大鼠Sertoli细胞对共同培养的异基因T淋巴细胞凋亡的影响。二、方法1、Wistar大鼠Sertoli细胞的制备取2～3周龄雄性Wistar大鼠的睾丸（生精细胞小于5％），消化酶消化，500um筛网过滤后，39℃（在此温度去除生精细胞）5％CO₂条件下孵育48小时后，除去损伤的细胞，获得高纯度的Sertoli细胞。2、SD大鼠T淋巴细胞制备取SD大鼠脾脏，于200目金属网研压，收集细胞悬液，用大鼠淋巴细胞分离液和尼龙毛柱法分离出高纯度T淋巴细胞。3、免疫组化SABC法检测Wistar大鼠Sertoli细胞FasL、TGF-β1的表达。4、不同数量的Wistar大鼠Sertoli细胞对SD大鼠T细胞的毒性作用取96孔培养板，每孔加入靶细胞----SD大鼠T细胞1x10⁵个（100ul），每孔加植物血凝素（Phytohemagglutinin，PHA，具有促进T细胞增殖的功能）20ul（20mg／L），再加经15戈瑞（Gray，Gy）照射1h的效应细胞----Wistar大鼠Sertoli细胞（15 Gy辐射使细胞失活）。根据效应细胞和靶细胞的比值分成T₀～T₅6组，效靶比依次为0／1、0.001／1、0.01／1、0.1／1、1／1、10／1；T₀组：T细胞+Sc细胞0个（100μL）+DMEM／F-12培养基100μL；T₁组：T细胞+Sc细胞1×10³／ml（100μL）+DMEM／F-12培养基100μL；T₂组：T细胞+Sc细胞1×10⁴／ml（100μL）+DMEM／F-12培养基100μL；T₃组：T细胞+Sc细胞1×10⁵／ml（100μL）+DMEM／F-12培养基100μL；T₄组：T细胞+Sc细胞1×10⁶／ml（100μL）+DMEM／F-12培养基100μL；T₅组：T细胞+Sc细胞1×10⁷／ml（100μL）+DMEM／F-12培养基100μL；T0组为空白对照组，每组6复孔，培养72小时，噻唑蓝比色法（MTT法）检测Sertoli细胞对T细胞的毒性作用。5、不同共培养时间Wistar大鼠Sertoli细胞对SD大鼠T细胞的毒性作用取96孔培养板，每孔加入靶细胞----SD大鼠T淋巴细胞1×10⁵个（100u1），每孔加植物血凝素20ul（20 mg／L），再加经15 Gy照射1h的效应细胞----Wistar大鼠Sertoli细胞1×10⁵个（100ul），效靶比为1／1；实验按培养天数进行分组：T0天组：T细胞+Sc细胞0个+DMEM／F-12培养基100μL；T1天组：T细胞+Sc细胞1×10⁵+DMEM／F-12培养基100μL；T2天组：T细胞+Sc细胞1×10⁵+DMEM／F-12培养基100μL；T3天组：T细胞+Sc细胞1×10⁵+DMEM／F-12培养基100μL；T4天组：T细胞+Sc细胞1×10⁵+DMEM／F-12培养基100μL；T5天组：T细胞+Sc细胞1×10⁵+DMEM／F-12培养基100μL；T6天组：T细胞+Sc细胞1×10⁵+DMEM／F-12培养基100μLT7天组：T细胞+Sc细胞1×10⁵+DMEM／F-12培养基100μL；T0天组为空白对照组，共8组，每组6复孔，按培养时间点，用MTT法检测Wistar大鼠Sertoli细胞对SD大鼠T淋巴细胞的毒性作用。6、Wistar大鼠Sertoli细胞对SD大鼠T淋巴细胞毒性作用机制的研究取96孔培养板，每孔加入靶细胞----SD大鼠T淋巴细胞1×10⁵个（100ul），每孔加植物血凝素20ul（20mg／L），再加经15Gy照射1小时的效应细胞----Wistar大鼠Sertoli细胞1x10⁵（100μl），效靶比为1／1；根据对Sertoli细胞施加的干扰因素不同进行分组：G0组：T细胞+Sc细胞0个（100μl）+DMEM／F-12培养基100μL；G1组：T细胞+经抗FasL和抗TGF-β1单抗孵育30min后的Sc细胞1x10⁵（100μl）+DMEM／F-12培养基100μL；G2组：T细胞+经抗FasL单抗孵育30min后的Sc细胞1x10⁵（100μl）+DMEM／F-12培养基100μL；G3组：T细胞+经抗TGF-β1单抗孵睜育30min后的Sc细胞1×10⁵（100μl）+DMEM／F-12培养基100μL；G4组：T细胞+孵育1x10⁵Sc细胞48小时后的培养基上清液（100μl）+DMEM／F-12培养基100μL；G5组：T细胞+Sc细胞1×10⁵（100μl）+DMEM／F-12培养基100μL；G0组为空白对照组，每组6复孔，培养72小时后，用MTT法检测Wistar大鼠Sertoli细胞对SD大鼠T淋巴细胞的毒性作用。7、Wistar大鼠Sertoli细胞促进SD大鼠T淋巴细胞凋亡机制的研究取48孔培养板，每孔加入靶细胞----SD大鼠T淋巴细胞1×10⁶／ml（0.5ml），每孔加植物血凝素100ul（20mg／L），再加经15Gy照射1小时的效应细胞----Wistar大鼠Sertoli细胞1×10⁶／ml（0.5ml），效靶比为1／1；根据对Sertoli细胞施加的干扰因素不同进行分组：G0组：T细胞+SC细胞0个（0.5ml）+DMEM／F-12培养基0.5ml；G1组：T细胞+经抗FasL和抗TGF-B1单抗孵育30min后的SC细胞1x10⁶／ml（0.5ml）+DMEM／F-12培养基0.5ml；G2组：T细胞+经抗FasL单抗孵育30min后的Sc细胞1x10⁶／ml（0.5ml）+DMEM／F-12培养基0.5ml；G3组：T细胞+经抗TGF-β1单抗孵育30min后的Sc细胞1x10⁶／ml（0.5ml）+DMEM／F-12培养基0.5ml；G4组：T细胞+孵育1x10⁶／ml（0.5ml）SC细胞48小时后的培养基上清液（0.5ml）+DMEM／F-12培养基0.5ml；G5组：T细胞+SC细胞1x10⁶／ml（0.5ml）+DMEM／F-12培养基0.5ml；G0组为空白对照组，每组6复孔，培养72小时后，用DNA末端标记技术----TUNEL法检测T细胞的凋亡。三、结果1、Wistar大鼠每个睾丸可以分离产生睾丸细胞（2.68±0.29）×10⁷个：细胞的存活率达（94.15±2.23）％；睾丸细胞FasL表达率为（91.65±1.63）％；睾丸细胞TGF-β1表达率为（89.20±2.16）％。只有Sertoli细胞表达FasL和TGF-β1，表达FasL和TGF-β1的百分率即可认定是Sertoli细胞纯度。2、随Wistar大鼠Sertoli细胞数量的增加，对SD大鼠T淋巴细胞的毒性作用增大，当Sertoli细胞与T细胞之比（效靶比）为1／1时，毒性作用达到高峰，效靶比超过1／1时，其毒性作用不再增加，而维持在高水平。3、Wistar大鼠Sertoli细胞对SD大鼠T淋巴细胞毒性作用，随着培养时间延长其毒性作用增大，但超过3天后，其毒性作用不再增大，而维持在高水平。4、抗FasL单克隆抗体和抗TGF-β1单克隆抗体，分别能减弱Wistar大鼠Sertoli细胞对SD大鼠T细胞的毒性作用，但不能完全阻断其毒性作用；而同时使用抗FasL和抗TGF-β1单克隆抗体时，Wistar大鼠Sertoli细胞对SD大鼠T细胞的毒性作用消失；表明Wistar大鼠Sertoli细胞通过表达的细胞因子TGF-β1和FasL对SD大鼠T细胞产生毒性作用。而Wistar大鼠Sertoli细胞的培养基上清液对SD大鼠T细胞也产生毒性作用，可能与Sertoli细胞产生的可溶性细胞因子，如可溶性FasL（sFasL）有关。5、抗FasL单克隆抗体和抗TGF-β1单克隆抗体，分别能减弱Wistar大鼠Sertoli细胞诱导SD大鼠T细胞凋亡的作用，但不能完全阻断T细胞凋亡；而同时使用抗FasL和抗TGF-β1单克隆抗体时，Sertoli细胞促进T细胞的凋亡作用消失；表明Sertoli细胞通过表达的细胞因子TGF-β1和FasL诱导T细胞凋亡。而培养Sertoli细胞的培养基上清液也能诱导T细胞凋亡，可能与Sertoli细胞产生的可溶性细胞因子，如sFasL有关。四结论1、2～3周龄Wistar大鼠睾丸含有丰富的Sertoli细胞；2、Wistar大鼠睾丸Sertoli细胞高度表达FasL和TGF—β1两种细胞因子；3、Wistar大鼠Sertoli细胞对SD大鼠T细胞的毒性作用，随着数量的增加其毒性作用增大；当Sertoli细胞与T细胞之比（效靶比）为1／1时，毒性作用达到高峰，效靶比超过1／1时，其毒性作用不再增加，而维持在高水平。4、Wistar大鼠Sertoli细胞对SD大鼠T细胞的毒性作用，共培养3天时效应最显著，超过3天后，其毒性作用不再增加，而保持在高水平。5、Wistar大鼠Sertoli细胞对SD大鼠T细胞的毒性作用，是Sertoli细胞表达的FasL和TGF-β1两种细胞因子产生的生物学效应；Wistar大鼠Sertoli细胞的培养基上清液对SD大鼠T细胞具有细胞毒性，可能与Wistar大鼠Sertoli细胞产生的可溶性细胞因子，如sFasL有关。6、Wistar大鼠Sertoli细胞能够诱导SD大鼠T细胞凋亡，是Sertoli细胞表达的FasL和TGF-β1两种细胞因子产生的生物学效应；Wistar大鼠seftoli细胞的培养基上清液能诱导SD大鼠T细胞凋亡，可能与Wistar大鼠Sertoli细胞产生的可溶性细胞因子，如sFasL有关。实验二、供心种植Sertoli细胞的大鼠心脏移植动物模型的制作一、目的制作Wistar至SD大鼠腹腔异位心脏移植动物模型，并在移植心脏种植与供心同基因Sertoli细胞，掌握制作大鼠腹腔异位心脏移植动物模型的外科技术。二、方法制作Wistar至SD大鼠腹腔异位心脏移植动物模型40例，随机分成T1组和T2组，每组20例。T1组：制作Wistar→SD大鼠腹腔异位心脏移植动物模型20例，心脏移植完成后，供体心脏的心肌内注射DMEM／F-12培养基100ul；T2组：制作Wistar→SD大鼠腹腔异位心脏移植动物模型20例，心脏移植完成后，供体心脏的心肌内注射CFSE荧光标记的Wistar大鼠的Sertoli细胞1×10⁷个（100ul）；72小时内如移植心脏停跳或受体鼠死亡，视为移植手术失败，予以剔除；心脏移植手术成功的大鼠，纳入实验三。记录供心摘取时间、心脏灌注及修剪时间、主动脉吻合时间、肺动脉吻合时间、供心冷缺血时间、手术总时间和术中出血量。三、结果1、心脏移植例存活34例，T1组成功18例，失败2例；T2组成功16例，失败4例；总体移植成功率85.0％。T1组1例因麻醉过深致受体大鼠死亡，1例因主动脉吻合口出血死亡；T2组2例主动脉吻合口出血死亡，1例因麻醉过深致受体大鼠死亡，1例因弥漫性腹膜炎致受体大鼠死亡。34例大鼠移植心脏存活均超过7天，并进入实验三。2、T1组与T2组比较，经t检验分析显示，供心摘取时间、心脏灌注及修剪时间、主动脉吻合时间、肺动脉吻合时间、供心冷缺血时间、术中出血量、手术总时间的差异无统计学意义（P＞0.05）；表明T1组和T2组在手术创伤、供心的保护等条件上是一致的，没有显著性差异。四、结论1、必要的显微外科训练是大鼠心脏移植动物模型制作成功的基础；模型制作过程中适当深度的麻醉、良好的心脏灌注、尽可能短的缺血时间、彻底的止血、适量液体的补充和良好的肝素化是大鼠心脏移植动物模型制作成功的关键。2、T1组和T2组供心摘取时间、心脏灌注及修剪时间、主动脉吻合时间、肺动脉吻合时间、供心冷缺血时间、术中出血量、手术总时间的差异不显著，表明制作大鼠心脏移植物模型的条件是一致的，排除了人为因素对实验条件稳定性的影响。实验三供心种植Sertoli细胞对大鼠心脏移植急性排斥反应和心肌细胞凋亡的影响一、目的1、检测供心种植Sertoli细胞的大鼠组，移植心脏急性排斥反应（AcuteRejection，AR）、心肌细胞凋亡指数（Apoptosis Index，AI）、心肌FasmRNA、FasL mRNA、TGF-β1 mRNA的表达变化；与供心未种植Sertoli细胞的大鼠组进行比较；探讨Sertoli细胞对移植心脏的免疫学影响。2、比较供心种植Sertoli细胞的大鼠组和供心未种植Sertoli细胞的大鼠组移植心脏存活时间，并观察移植心脏排斥反应。探讨Sertoli细胞对同种异体移植心脏是否具有免疫豁免作用。二、方法1、实验分组心脏移植动物模型来源于实验二，实验二T1组手术成功18例，T2组手术成功16例。本实验进一步分组如下：A组：由实验二T1组中随机抽出9例构成（移植心脏未种植Sc细胞）；B组：由实验二T2组中随机抽出9例构成（移植心脏种植SC细胞）；C组：由T1组剩余9例构成（移植心脏未种植Sc细胞）；D组：由T2剩余7例构成（移植心脏种植Sc细胞）。2、样本及数据采集①A组、B组大鼠在心脏移植术后第7天处死，无菌条件下切取移植心脏；即刻在心脏标本上取同一部位同等大小的心脏组织100mg，液氮速冻保存，为提取mRNA备用。剩余心脏标本10％中性甲醛浸泡，避光保存；避光条件下标本进行固定、包埋，石蜡切片，每标本切15张石蜡切片，分3组，每组5张；各组分别进行常规病理切片、荧光显微镜观察、细胞凋亡检测。②取液氮冷冻得心肌标本，用半定量RT-PCR方法检测移植心脏Fas mRNA、FasL mRNA、TGF-β1 mRNA的表达量。③观察C组、D组移植心脏存活时间及AR变化。三、结果1、荧光显微镜追踪观察移植的Sertoli细胞①Sertoli细胞标记效果良好，其标记效率达100％，细胞膜及细胞浆标记为黄绿色荧光。②心肌内种植Sertoli细胞组，术后1周Sertoli细胞由注射部位向周边迁移、分布，胞浆和细胞膜呈黄绿色荧光。2、A组和B组的急性排斥反应（AR）和心肌细胞凋亡指数（AI）的比较和相关分析①A组和B组AR差异有统计学意义，P=0.003；种植Sertoli细胞的B组移植心脏AR显著性降低。②A组和B组心肌细胞的AI差异有统计学意义，P=0.003；种植Sertoli细胞使移植心脏AI显著性降低。③Spearman相关分析显示，AR与AI成显著正相关，相关系数=0.811，相关显著，P＜0.001。3、A组和B组Fas mRNA、FasL mRNA、TGF-β1 mRNA表达量的比较①A组和B组FasL mRNA表达差异有统计学意义，P=0.022；B组FasL mRNA表达明显高于A组。②A组和B组Fas mRNA的表达差异无统计学意义，P=0.447。③A组和B组TGF-β1 mRNA表达差异有统计学意义，P＜0.001，B组TGF-β1 mRNA表达明显高于A1组。表明种植Sc细胞的B组FasL mRNA和TGF-β1 mRNA的表达显著增加。4、种植Sertoli细胞的B组内FasL mRNA、TGF-β1 mRNA与AR、心肌细胞AI的相关分析①AR与FasL mRNA的表达成显著负相关，相关系数-0.776，相关显著，P=P.014；②AR与TGF-β1 mRNA表达成显著负相关，相关系数-0.867，相关显著，P=0.002。③AI与FasL mRNA的表达成显著负相关，相关系数-0.800，相关显著，P=0.010；④AI与TGF-β1 mRNA的表达成显著负相关，相关系数-0.783，相关显著，P=0.013。5、C组和D组移植心脏存活时间及AR的比较①D组大鼠移植心存活时间显著大于C组大鼠移植心脏存活时间，P＜0.001。C组大鼠移植心脏存活时间为11.00±2.12天；D组大鼠移植心存活时间为17.29±1.80天；表明移植心脏种植Sertoli细胞显著延长心脏存活时间。②C组和D组移植心脏均死于急性排斥反应引起，两组AR无显著差别。四、结论1、CFSE荧光探针对Sertoli细胞的标记和示踪效果良好；2、种植Sertoli细胞显著减轻移植心脏急性排斥反应和心肌细胞凋亡；3、急性排斥反应与心肌细胞凋亡成显著正相关；4、移植心脏种植Sertoli细胞显著增加FasL mRNA表达和TGF-β1 mRNA表达，不能增加Fas mRNA的表达；5、移植心脏种植Sertoli细胞组，急性排斥反应、细胞凋亡分别与FasLmRNA、TGF-β1 mRNA的表达成显著负相关。6、移植心脏种植Sertoli细胞组与移植心脏未种植Sertoli细胞组比较，种植Sertoli细胞显著延长移植心脏存活时间；更多还原

【Abstract】 The study was supported by Guangdong Nature Science Foundation （No:990386） , by Guangdong Province Medical Research Foundation （No:A1999326） and by the Foundation of Wuhan General Hospital of Guangzhou Command（2005）.Immune privilege concept were proposed for more than 100 years.In vivo,brain,eyes, cronea, ovaries and placenta were the immune privilege organs,whiche were not interfered and rejected by the body. The research of immune privilege could make transplanted cells, tissure and organs break the immune barry of homogeneity and heteroplasty,which has wide perspective in clinical application and large economic benefit.Enhancing the research of immune privilege will make organ transplantation not need immunodepressant,but patients will get long-term surviving.In our country or abroad the research about immune privilege of sertoli cell started in recent years.The chief research was concentrated on the immune privilege of sertoli cells.Sertoli cells cound produe two kinds of cytokine ,FasL and TGF-β1,which was the chief reason of which sertoli cells had the function of immune privilege.So we carried the research on the assessment of sertoli cells immune privilege mechanisms and the effect to cardiac allograft of rats.Objective: 1.To investigate the purification method of sertoli cells and detect the expressing rate of FasL and TGF-β1 in sertoli cells.2.To obersve the toxic function of sertoli cells to T lymphocyte,when they were cocultured.3.To explore the apoptosis effect of sertoli cells to T lymphocyte.4.To make cardiac allograft animal models of myocardium transplanted with or without sertoli cells.5.To contrast the acute rejection,myocardium apoptosis,the expressing of Fas mRNA,FasL mRNA and TGF-β1 mRNA in the animal models’ transplanted hearts of myocardium with or without sertoli cells transplanted.To explore whether sertoli cells have immune privilege function to allografted hearts.Experiment 1: Isolation and purification of sertoli cells and identification immune privilege mechanisms of sertoli cells1. Objective1) To investigate isolation and the purification method of sertoli cells and detect the expressing rate of FasL and TGF-β1 in sertoli cells;2) To obersve the toxic function of sertoli cells to allogene T lymphocytes, when they were cocultured.3) To explore the apoptosis effect of sertoli cells to allogene T lymphocytes.2. Methods1) Preparing of Wistar rats’ sertoli cells Testes of 2-3 weeks Wistar rats were digested by digestive enzyme and then filtrated by 500um grid.The digested cells were incubated in 39℃for 48 hours, which temperature could remove the spermatogenic cells and remove the injured sertoli cells.2) Preparing of SD rats’ T lymphocytes To grind the SD rats’ spleen on 200 mesh wire gauze and collect the cells suspension.To separate high purity T lymphocytes by using lymphocyte isolation medium and nylon fiber column.3) Detection of the expression of FasL、TGF-β1 in sertoli cells by SABC method.4) Toxic function of different quantitive Wistar rats’sertoli cells to SD rats’ T lymphocytesAdding target cell 1^*10⁵ T cells and 20ul phytohemagglutinin （PHA, 20ug/ml） in each well of 96 wells cultivation plate, and adding effector cell sertoli cells which exposed to 15Gy ray for 1 hour. To separate 6 groups（T₀～T₅） according to the rate of effector cell target cell, and one by one the rate was 0/1、0.001/1、0.01/1、0.1/1、1/1、10/1 ; T0 group:T cells+Sc 0（100ul）+ DMEM/F-12 100ul; T1 group: T cells+Sc 1^*10³/ml （100ul）+ DMEM/F-12 100ul; T2 group: T cells+Sc 1^*10⁴/ml （100ul）+ DMEM/F-12 100ul; T3 group: T cells+Sc 1^*10⁵/ml （100ul）+ DMEM/F-12 100ul; T4 group: T cells+Sc 1^*10⁶/ml （100ul）+ DMEM/F-12 100ul; T5 group: T cells+Sc 1^*10⁷/ml （100ul）+ DMEM/F-12 100ul;TO group was blank group,and every group had 6 duplicated wells.To culture for 72 hours and detecte the toxic effect of sertoli cells to T lymphocytes by MTT method.5) Toxic function of Wistar rats’sertoli cells to SD rats’ T lymphocytes for different cultured timeAdding target cell 1^*10⁵ T cells and 20ul phytohemagglutinin （PHA, 20ug/ml）in each well of 96-wells cultivation plate and adding effector cell 1^*10⁵ sertoli cells （100ul） exposed by 15Gy ray.The rate of effector cell and target cell was 1/1. To group according to the culture time : T0d group:T cells+Sc 0（100ul）+ DMEM/F-12 100ul; T1d group: T cells+Sc 1^*10⁵ （100ul）+ DMEM/F-12 100ul; T2ds group: T cells+Sc 1^*10⁵ （100ul）+ DMEM/F-12 100ul; T3ds group: T cells+Sc 1^*10⁵ （100ul）+ DMEM/F-12 100ul; T4ds group: T cells+Sc 1^*10⁵ （100ul）+ DMEM/F-12 100ul; T5ds group: T cells+Sc 1^*10⁵ （100ul）+ DMEM/F-12 100ul; T6ds group: T cells+Sc 1^*10⁵ （100ul）+ DMEM/F-12 100ul; T7ds group: T cells+Sc 1^*10⁵ （100ul）+ DMEM/F-12 100ul;T0d group was blank group,and there were 8 groups,which every group had 6 duplicated wells.To culture and detecte the toxic effect of sertoli cells to T lymphocytes by MTT method.6) The mechanism of sertoli cells toxic function to T lymphocytes Adding target cell 1^*10⁵ T cells and 20ul phytohemagglutinin （PHA, 20ug/ml）in each well of 96-wells cultivation plate and adding effector cell 1^*10⁵ sertoli cells （100ul） exposed by 15Gy ray. The rate of effector cell and target cell was 1/1. To group according to the interfering factors: G0 group:T cells+Sc 0（100ul）+DMEM/F-12 100ul; G1 group: T cells+Sc 1^*10⁵cultured for 30min within FasL-mAb and TGF-β1 mAb（100ul）+DMEM/F-12 100ul; G2 group: T cells+Sc 1^*10⁵ cultured for 30min within FasL-mAb （100ul） +DMEM/F-12 100ul; G3 group: T cells+Sc 1^*10⁵ cultured for 30min within TGF-β1 mAb （100ul）+DMEM/F-12 100ul; G4 group: T cells+ supernatant solution of cultured Sc 1^*10⁵ for 48hours （100ul）+DMEM/F-12 100ul; G5 group: T cells+Sc 1^*10⁵ （100ul）+DMEM/F-12 100ul;G0 group was blank group, and there were 6 groups,which every group had 6 duplicated wells.To culture for 72 hours and detecte the toxic effect of sertoli cells to T lymphocytes by MTT method.7) The mechanism of sertoli cells apoptosis function to T lymphocytes Adding target cell 1^*10⁶/ml（0.5ml）T cells and 100ul phytohemaggluti -nin （PHA, 20ug /ml） in each well of 48-wells cultivation plate and adding effector cell 1^*10⁶/ml（0.5ml） exposed to 15Gy ray for 1 hour.The rate of effector cell and target cell was 1/1. To group according to the interfering factors: G0 group:T cells+Sc 0（0.5ml）+DMEM/F-12 0.5ml; G1 group: T cells+Sc 1^*10⁶/ml（0.5ml ）cultured for 30min within FasL-mAb and TGF-β1 -mAb+DMEM/F-12 0.5ml; G2 group: T cells+Sc 1^*10⁶/ml（0.5ml ）cultured for 30min within FasL-mAb +DMEM/F-12 0.5ml; G3 group: T cells+Scl^*10⁶ /ml（0.5ml ） cultured for 30min within TGF-β-mAb+DMEM/F-12 0.5ml; G4 group: T cells+ supernatant solution of cultured Sc 1^*10⁶/ml（0.5ml ） for 48hours +DMEM/F-12 0.5ml; G5 group: T cells+Sc 1^*10⁶/ml（0.5ml ）+DMEM/F-12 0.5ml;GO group was blank group, and there were 6 groups,which every group had 6 duplicated wells.To culture for 72 hours and to detecte the apoptosis effect of sertoli cells to T lymphocytes by TUNEL method.3. Results1) Every testis of Wistar rats can be separated （2.68±0.29）×10⁷ sertoli cells. Survival rate of sertoli cells attained （94.15±2.23）%.The sertoli cells’ expressing rate of FasL arrived （91.65±1.63） %. The sertoli cells’ expressing rate of TGF-β1 arrived （89.20±2.16） %.2) With increasing of sertoli cells, the toxic function to allogeneic T lymphocytes increased.When the rate of sertoli cells and T cells attained 1/1,the toxic function arrived at the peak effect.When the rate exceeded 1/1,the effect of sertoli cells could not increase,but still in high level.3) With the cocultivation time increasing, the toxic effect of sertoli cells to allogeneic T lymphocytes increased; but when cocultivation time passed 3 days,the toxic effect no longer increasd and still stayed in high level. 4) The toxic effect of sertoli cells to T lymphocytes was blocked by anti-FasL and anti-TGF-β1. The toxic effect could not be blocked by anti-FasL or anti -TGF-β1 respectivly.The culture solution had toxic effect to T lymphocytes, which showed that the culture solution had some cytokine as solubility FasL which had toxic effect.5) The apoptosis effect of sertoli cells to T lymphocytes was blocked by anti-FasL mAb and anti-TGF-β1. The apoptosis effect could not blocked by anti-FasL mAb or anti-TGF-β1 mAb respectivly.The culture solution had apoptosis effect to T lymphocytes,which showed that the culture solution had some cytokine as solubility FasL which had apoptosis effect.The apoptosis and toxic function of sertoli cells to allogeneic T lymphocuye were at the equal place.4. Conclusion1) The testes of 2 weeks rats contained plenty of sertoli cells.2) Sertoli cells expressed FasL and TGF-β1 in high level.3) With increasing of sertoli cells, the toxic function to allogeneic T lympho -cytes increased.When the rate of sertoli cells and T cells attained 1:1,the toxic function arrived at the peak effect.When the rate exceeded 1:1,the effect of sertoli cells could not increase,but still in high level.4) With the cocultivation time increasing,the toxic effect of sertoli cells to allogeneic T lymphocytes increased;but when cocultivation time passed 3 days,the toxic effect no longer increasd and still stayed in high level.5) The toxic effect of sertoli cells to T lymphocytes was blocked by anti-FasL and anti-TGF-β1. The toxic effect could not blocked by anti-FasL or anti -TGF-β1 alone.The culture solution had toxic effect to T lymphocytes, which showed that the culture solution had some cytokine as solubility FasL.6) The apoptosis effect of sertoli cells to T lymphocytes was blocked by anti-FasL and anti-TGF-β1. The apoptosis effect could not blocked by anti-FasL or anti -TGF-β1 alone.The culture solution had apoptosis effect to T lymphocytes,which showed that the culture solution had some cytokine as solubility FasL.The apoptosis and toxic function of sertoli cells to allogeneic T lymphocuye were at the epual place. Exeperiment 2.Making cardiac allograft animal models of rats with myocardium transplanted allogeneic sertoli cells1. ObjectiveMaking Wistar to SD rats allograft cardiac animal models with transplanting Wistar rats’ sertoli cells into donator’s myocardium.Contrast to the group without sertoli cells transplanted into donator myocardium,observing the survival time of transplanted hearts.2. MethodsMaking 40 cases of Wistar rats to SD rats allograft cardiac animal models. 40 cases were divided in two groups.Every group had 20 cases.Contrast group （T1） : When the heart transplantation finished, DMEM/F-12 culture medium（100ul） were injected into donators’ myocardium.Experiment group（T2）: When the heart transplantation finished, 1^*10⁷ Wistar rats’ sertoli cells（100ul） were injected into donators’ myocardium. If transplanted hearts stoped beating or recipient died,those cases were regarded as unsuccessful cases.The successful cases were sended into the experiment 3.Recorded the time of obtaining donator hearts、perfusing heart、clipping heart、aorta arterial anastomosis、pulmonary arterial anastomosis、cold ischemia、anesthesia and volume of bleeding in operation.3. Results1) Made allografted animal models 40 cases.Survival cases were 34 cases. In T1 group, survival cases were 18, and defeated cases were 2; In T2 group, survival cases were 16, and defeated cases were 4. The achievement ratio were 85.0% in total. In T1 group,one cases was died of anesthesia and one case died of bleeding in arterial anastomosis position. In T2 group, 2 cases was died of anesthesia, one case died of bleeding in arterial anastomosis position,and one case died of diffused peritonitis.All transplanted hearts of 34 cases survived for more than 7days,and went into the experiment 3.2) In T1 and T2 group,with t-test analysis,the time of obtaining donator hearts、perfusing heart、clipping heart、aorta arterial anastomosis、pulmonary arterial anastomosis、cold ischemia、anesthesia and volume of bleeding in operation were not statistically significant（P＞0.05）. T1 group and T2 group had no statistically significant in operation wounded and myocardium protection.4. Conclusion1) The microsurgery practice was necessary,which was the basement to make cardiac allograft animal models. Good anesthesia,effective heart perfusion,short ischemic time, gracious surgery skill, good hemostasis andeffective heparinization;which were very importance in making cardiac allograft procedure.2) In T1 and T2 group, the time of obtaining donator hearts、perfusing heart、clipping heart、aorta arterial anastomosis、pulmonary arterial anastomosis、cold ischemia、anesthesia and volume of bleeding in operation were not statistically significant. The operation condition of T1 group and T2 group at was at the equal place in operation wounded and myocardium protection,which eliminated the interfering of human factor.Experiment 3. The effect to acute rejection and apoptosis in cardiac allograft animal models with myocardium being transplanted sertoli cells1. ObjectiveObserved the acute rejection（AR）,apoptosis index（AI） and expression of Fas mRNA ,FasL mRNA and TGF-β1 mRNA in the models with or without myocardium transplanted sertoli cells.With correlation analysis, to identify the effect of sertoli cells to transplanted hearts and explored the immune pretection of sertoli cells to cardiac allografts.2. Methods1) Experiment group:The heart transplanted animal models were came from experiment 2.T1 group had survival 18 cases and T2 group had 16 survival cases. A group: To choose 9 cases from T1 group; B group:To choose 9 cases from T2 group; C group: T1 group residued 9 cases; D group:T2 group residued 7 cases;2) Experiment samples gathering and processing of the data. At the 7^th day after operation,transplanted hearts were obtained in steriled condition.100mg tissue of every transplanted heart were obtained and preserved in liquid nitrogen for extracting mRNA.Cutting hearts were preserved in 10% neutral formalin away from light.In condition of away from light samples were carried out solidification, embedment and paraffin section.Every paraffin section were incised into 15 slides which were divided into 3 groups .Every group including 5 slides.By turn every group was carried out making pathological section, observing under fluorescence microscope,detecting apoptosis index of transplanted hearts..With QRT-PCR method ,expression of Fas,FasL and TGF-β1 mRNA was detected.Observed the survival time of transplanted hearts in C groups and D group.3. Results1) Observing sertoli cell under fluorescence microscope Sertoli cells were labeled in high quality by CFSE,which labeled rate attained at 100%. Under fluorescence microscope cytolymph and cells’ membrane of sertoli cells showed yellow green color.In experimental group,sertoli cells were dispersed from the injecting place to the periphery, which cytolymph and cells’ membrane of sertoli cells showed yellow green color.2) Analysis of acute rejection（AR） and apoptosis index（AI） of A group and B groupThe acute rejection of A group and B group displayed statistical significance, P=0.003.The acute rejection of B group was decreased significance than A group.AI of A and B group displayed statistical significance, P=0.003. The apoptosis of B group was decreased signifycance than A group.With Spearman correlation,the relation between the AR and AI displayed positive correlation,which correlation coefficient was 0.881, P＜0.001.3) Expression of cytokine in A group and B groupThe expression of FasL mRNA in A and B group had statistical significance, P=0.022;The expression of FasL mRNA in B group was high than the A group.The expression of Fas mRNA in A group and B group had not statistically significant.The expression of TGF-β1 mRNA in A and B group had statistical signifycance, P= 0.000. The expression of TGF-β1 mRNA in B group was higher than A group.Sertoli cells increased highly the expression of Fas mRNA and TGF-β1 mRNA.4) The corrrelation among acute rejection,apoptosis and cytokine in B group was analyzed with Spearman correlationAR had had inverse correlation with the expression of FasL mRNA, which correlation coefficient was -0.776, P=0.014.AR had inverse correlation with the expression of TGF-β1 mRNA, which correlation coefficient was -0.867, P=0.002.AI had inverse correlation with the expression of FasL mRNA, which correlation coefficient was -0.800, P=0.010. AI had inverse correlation with the expression of TGF-β1mRNA, which correlation coefficient was -0.783, P=0.013.5) To contrast the survival time of the cases of C group and D group In C group transplanted hearts survived 11.00±2.1days. In D group transplanted hearts survived 17.29±1.80 days.With t-test, survival time of C group and D group had statistically significant p＜0.001.The animal models of myocardium with transplanted sertoli cells in D group survived longer than the animal models without sertoli cells in C group.4. Conclusion1) Sertoli cells were labeled in high quality and in high tracing effective by CFSE.2) Transplanted hearts of which sertoli cells were transplanted decreased significance acute rejection and myocardium apoptosis.3) Acute rejection were positive correlation with Apoptosis idex.4) Transplanted hearts of which sertoli cells were transplanted into increased significance the expression of FasLmRNA and TGF-β1 mRNA,but did not increased the expression of Fas mRNA.5) Transplanted hearts of which sertoli cell were transplanted increased significance the expression of FasL mRNA and TGF-β1 mRNA, and the expression of FasL mRNA and TGF-β1 mRNA displayed significance negative correlation with acute rejection and myocardium apoptosis.6) The animal models of myocardium with transplanted sertoli cells survived longer than the animal models without sertoli cells.更多还原