转染mFas配体的COS-7细胞诱导Fas+淋巴瘤细胞系(Yac-1)凋亡

本研究探讨通过Fas配体(Fas ligand,FasL)-Fas途径进行免疫治疗淋巴瘤的可能性。常规转化pBillneo-mFasL至大肠杆菌DH5α,经扩增、质粒抽提和纯化后,进行限制性内切酶酶切、mFasL基因PCR及其产物DNA测序,以鉴定pBillneo-mFasL内mFasL基因一级结构及插入方向;用脂质体法转染 pBillneo-mFasL至猴肾COS-7细胞,G418选择培养后,Western印迹分析外源性mFasL cDNA基因表达水平,将高表达mFasL的COS-7细胞与Fas~+小鼠淋巴瘤细胞系Yac-1以不同比例混合培养,5小时后收集悬浮的Yac-1细胞,用膜联蛋白(annexin)V/PI标记后,借助FCM观察细胞调亡率。结果表明,质粒pBillneo-mFasL的EcoRI酶切获得920 bp和7227 bp产物,Hind Ⅲ酶切获得1293 bp和6807 bp产物,初步证实插入mFasL cDNA片段与理论预计大小一致,并系正向插入;PCR扩增出自起始密码子(ATG)至终止密码子(TAA)后+36 bp的mFasL cDNA全长890 bp序列,DNA测序结果与基因库已知序列完全一致;pBillneo-mFasL转染COS-7细胞,并经G418选择培养后,Western印迹检测到明显mFasL蛋白质表达;当用这种高表达mFasL蛋白质的COS 7细胞与Fas~+ Yac-1细胞以1:1,5:1和10:1混合培养5小时后,用annexin V/PI标记悬浮Yac-1,结果后者调亡率分别为(22±4.8)％,(32.18±7.8)％和(51.8±5.4)％,与

Apoptosis of Fas+ Yac-1 Cells Induced with Fas Ligand-transfected COS-7 Cells

The possibility of immunotherapy for lymphoma by single FasI^Fas way was investigated.After pBillneo-mFasL was transformed into competent E.coli DH5o and amplified,the plasmid DNA was prepared and purified from the DH5a.To determine the primary structure and inserting direction of mFasL cDNA gene in pBillneo-mFasL,the plasmid DNA was cleaved by restriction enzyme,and the mFasL cDNA of pBillneo-mFasL was amplified by polymerase chain reaction(PCR),the DNA sequence of the PCR product was analysed by automatic DNA sequencing.After pBillneo-mFasL was transfected into COS-7 cells by liposome,the COS-7 cells were selected with G418 selective medium,and the expressing levels of mFasL cDNA on the COS-7 cell membrane was assayed by Western Blot.After the COS-7 cells higher expressing mFasL protein and mouse lymphoma cell line Yac-1 expressing Fas were cocultured for 5 hours,the suspending Yac-1 cells were collected and labeled by annexin V/PI kit.The apoptosis rate of the Yac-1 cells was tested by flow cytometry.The EcoRI cleaving products of pBillneo-mFasL included 920 bp and 7 227 bp fragments.Its Hind M cleaving products included 1 293 bp and 6 807 bp fragments.These results showed: (1) the length of DNA sequence containing mFasL cDNA within pBillneo-mFasL is the same as theoretical length; (2)the inserting of mFasL cDNA in pBillneo-mFasL was in positive orientation.The expected 890 bp DNA fragments of mFasL cDNA(from ATG to + 36 bp following TAA ) emerged in PCR product with pBillneo-mFasL as a template.The sequencing result of the PCR product equaled the known mFasL cDNA sequence in the gene bank.The COS-7 cells transfected by pBillneo-mFasL and selected withG418 culture medium expressed more mFasL membrane protein assayed by Western Blot.After the COS-7 cells were cocultured with Fas Yac-1 cells in different E'T ratios(l: 1,5-1 and 10-1) for 5 hours,the apoptosis rates of Yac-1 cells were (22 + 4.8)%,(32.18+7.8)%,and (51.8+5.4)%,respectively.These were obviously different from the control group( P < 0.01),in which the COS-7 cell was transfected by pBillneo( not carrying mFasL gene).It was concluded that lymphoma cells highly expressing Fas can be effectively killed through single Fas-FasL way in vitro.