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目的 探讨表达c FLIPs重组腺病毒诱导淋巴细胞抗凋亡作用。方法 采用RT PCR方法,从人T淋巴细胞株的总RNA中克隆c FLIPs基因;通过pAdeasy系统,构建表达c FLIPs的重组腺病毒Ad c FLIPs;Hochest染色法及PI染色流式细胞仪检测anti Apo 1诱导感染Ad c FLIPs后的H9细胞凋亡。结果 采用RT PCR方法从人T淋巴细胞株中扩增出c FLIPs基因;构建重组腺病毒Ad c FLIPs ;经Hoechst染色,荧光显微镜下观察细胞核的形态,结果发现,经anti Apo 1诱导凋亡处理2 4h后,未经Ad c FLIPs感染的H9细胞有大量的细胞核呈浓染致密的固缩形态或颗粒状荧光的凋亡细胞;Ad c FLIPs感染的H9细胞中细胞核呈浓染致密的固缩形态或颗粒状荧光的细胞数明显减少,大部分细胞染色质呈弥漫均匀低强度荧光;流式细胞仪分析经PI染色后的H9细胞,未经Ad c FLIPs预处理的H9细胞在anti Apo 1作用2 4h后,细胞的凋亡率分别为4 8 33%±7 4 1% ;经Ad c FLIPs预处理1d的H9细胞,其细胞凋亡率分别下降到3 6 0 %±0 2 1%。结论 重组腺病毒Ad c FLIPs可有效地诱导淋巴细胞的抗凋亡作用。
Objective To investigate the anti-apoptotic effects of recombinant adenovirus expressing c FLIPs on lymphocytes. Methods The c FLIPs gene was cloned by RT-PCR from the total RNA of human T lymphocyte strain. Recombinant adenovirus Ad c FLIPs expressing c FLIPs were constructed by pAdeasy system. Hochest staining and flow cytometry were used to detect anti-Apo 1 induced apoptosis of H9 cells infected with Ad c FLIPs. Results The c FLIPs gene was amplified from human T lymphocyte cell line by RT-PCR and the recombinant adenovirus Ad c FLIPs was constructed. The morphology of the nucleus was observed under a fluorescence microscope by Hoechst staining. The results showed that anti-Apo 1 -induced apoptosis After 24 hours, a large number of apoptotic cells were found in H9 cells without Ad c FLIPs infection. The nuclei of H9 cells infected with Ad c FLIPs were condensed and condensed The morphological or granular fluorescent cells decreased significantly, most of the chromatin was diffusely uniform low intensity fluorescence; Flow cytometry analysis of PI-stained H9 cells without Ad c FLIPs pretreated H9 cells in the anti Apo 1 The apoptosis rates of H9 cells pretreated with Ad c FLIPs for 1 day were decreased to 36.0% ± 0 2 1% . Conclusion Recombinant adenovirus Ad c FLIPs can effectively induce lymphocyte anti-apoptotic effects.