FasL基因转染Hela细胞体外诱导淋巴细胞凋亡的作用

Effect of Fas Ligand gene transfection in Hela cell on the induction of lymphocyte apoptosis

目的 应用FasL基因转染具有上皮细胞特性的Hela细胞,探讨FasL基因的表达对诱导同种异体淋巴细胞凋亡的作用,为解决同种异体组织工程化皮肤的免疫排斥问题提供一条研究途径。方法 PLNCX2-FasL质粒转化大肠杆菌,抽提纯化的PLNCX2-FasL质粒用限制性内切酶Xho Ⅰ,Not Ⅰ酶切,琼脂糖凝胶电泳得到755 bp大小的目的条带。紫外灯下切割回收目的条带并抽提纯化得到FasL基因,用Xho Ⅰ,Not Ⅰ酶切PCDNA3.1质粒载体并在相同位点插入FasL基因,T4酶连接,构建PCDNA3.1-FasL重组质粒。脂质体转染法将PCDNA3.1-FasL基因转染Hela细胞,G418筛选;经过免疫细胞化学法,RT-PCR鉴定后,进行单项淋巴细胞混合试验,检测FasL基因对诱导淋巴细胞凋亡的作用。结果 表达FasL基因的Hela细胞的淋巴细胞刺激指数较对照组转染空白质粒的Hela细胞下调,仅达到对照组刺激指数的17.01％。结论 FasL基因转染上皮细胞可诱导淋巴细胞凋亡,为研究避免同种异体组织工程化皮肤的免疫排斥提供了一种可行的方法和设想。

Objective To determine the effect of FasL gene expression of Hela cell on the induction of lymphocyte apoptosis. Methods Plasmid DNA (PLNCX2-FasL) was prepared from the transformed E. Coli cells. The XhoI-NotI fragment of PLNCX2-FasL containing the targeted gene of Fas L was inserted between XhoI and Not I sites of PCDNA3. 1. The resulting plasmid PCDNA3. 1-FasL was used for the liposome-mediated transfection of Hela cells, a cell line with the characteristics of epithelial cell. The transfected cell clones were selected by G418 and confirmed for their expression of Fas L with immunochemistry and RT-PCR. The effect of the epithelial expression of FasL on lymphocyte apoptosis was evaluated using mixed lymphocyte reaction. Results A reduction of simulating index (17. 01% ) was observed in the mixed lymphocyte reaction of FasL expressing Hela cells as compared to those of un-transfected cell. Conclusion Our result demonstrates that expression of FasL on epithelial cells can induce apoptosis of lymphocyte. Expression of FasL on epithelial cells might be a promising way to overcome immune rejection of allogeneic engineer skin.