FasL^+组织工程化猪软骨细胞的同种异体移植

Allogenic porcine transplantation of FasL^+ -transfected tissue engineered chondrocytes

目的:探讨表达FasL的猪组织工程化软骨细胞在同种异体内的生长状况和免疫排斥反应。方法:实验于2002-09/2004-03在上海交通大学医学院,上海市免疫学研究所进行。①分离制备猪软骨细胞。②制备FasL+软骨细胞,构建重组pGCEN-FasL反转录病毒载体,转染PA317细胞。经G418筛选获得分泌pGCEN-FasL病毒颗粒的PA317细胞克隆,选择高滴度的病毒液,感染猪软骨细胞。再经过G418筛选获得FasL+的软骨细胞克隆,扩增培养。③FACS检测FasL的表达,应用JAM试验测定FasL+的软骨细胞诱导Fas+的Jurkat细胞及活化的T细胞的凋亡率。同时制备转染pGCEN反转录病毒空载体的软骨细胞为对照。④取FasL+的软骨细胞与可注射性生物材料PluronicF127混合,注射于同种异体猪的腹壁皮下。在第4,5周取材,通过组织病理和免疫组织化学等方法,检测软骨细胞生长和免疫排斥反应。结果:①经转染的软骨细胞表面FasL的表达率为57%。②FasL+的软骨细胞具有明显诱导Fas+细胞和活化的同种异体T细胞的凋亡,最大的凋亡率分别为53.41%,30.38%(效/靶=10∶1),对照组分别为32.27%,13.16%(效/靶=10∶1)。③组织工程化猪软骨结节的结构与正常软骨组织基本一致,可见清晰的软骨凹陷和软骨膜,仅细胞排列较正常软骨略显混乱、不均匀现象。④免疫组织化学染色显示FasL+的组织工程化猪软骨结节的Ⅱ型胶原蛋白分布均匀,形状清楚,与正常软骨比较基本一致。⑤第5周的软骨细胞表面的FasL分子表达明显,周围的炎性细胞浸润相对较少。而对照组的软骨细胞周围可见到大量浸润的炎性细胞。结论:成功构建FasL+软骨细胞并有效表达,抑制免疫排斥反应,为建立同种异体软骨细胞移植的免疫耐受提供了实验依据。

AIM： To explore the cell growth features and immune rejection responses of FasL-transfected tissue engineered chondrocytes in allogenic porcine transplantation. METHODS： The experiment was done in Shanghai Immunology Institute, Medical College, Shanghai Jiao Tong University from September 2002 to March 2004. ①The chondrocytes were isolated from porcine cartilage tissue. ②FasL^＋ chondrocytes ware isolated, and pGCEN-FasL retrovirus vectors were established. PA317 cells were transfected. After G418 screening, PA317 cell clone excreted with pGCEN-FasL retrovirus particles. High-titer virus fluid was selected to infect chondrocytes in allogenic porcine. After G418 screening, FasL^＋ chondrocyte clone was obtained, amplified and cultured. ③FasL expression in transfected chondrocytes was analyzed by FACS. The apoptosis rate of Fas^＋ Jurkat cells and activated T cells with FasL^＋ chondrocytes induction were detected by JAM test. At the same time, chondrocytes with pGCEN retrovirus vector was as control. ④The FasL^＋ chondrocytes mixed with Pluronic F127 were injected subcutaneously into abdomen wall in allogenic porcine. Transplanted complexes in local tissue harvested from receipted porcine at weeks 4 or 5, the chondrocytes growth features and immune rejection reaction were detected by histopathological and immunohistochemical methods. RESULTS： ①The expression rate of FasL on chondrocytes was 57%. ②FasL^＋ chondrocytes could induce apoptosis of Fas^＋ cells and activated T cell. The largest rate of apoptosis of Fas^＋ cell and activated T cell were 53.41% and 30.38% （E/T=10：1）, respectively, while those in control group were 32.27% and 13.16% （E/T=10：1）. ③The structural feature of FasL^＋ chondrocytes in reformed cartilage tissue was identical with that of normal cartilage tissue, there were obvious cartilage introcession and membrane respectively. A little unregulated align was observed in reformed cartilage tissue. ④ Immunohistochemistrical staining showed that the type Ⅱ collagen in reformed cartilage tissue was even and with clear form, which was identical with that of normal cartilage tissue. ⑤FasL was highly expressed in chondrocytes at the 5^th week. Compared with tissue-engineered FasL^- chondrocytes （negative control）, the presence of infiltrating lymphocytes was obviously decreased around the FasL^＋ chondrocytes. A mass of infiltrating inflammatory cells appeared around chondrocytes in the control group. CONCLUSION： Tissue engineered chondrocytes are successfully constructed and FasL^＋ is highly expressed, which lays the experimental foundation of immune tolerance for allogenic chondrocyte transplantation.