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研究融合蛋白scFvCD20:sTRAIL诱导B淋巴瘤细胞BJAB凋亡的发生以及凋亡相关蛋白的表达变化。采用PCR、重叠PCR、酶切、连接等方法构建pLentiR.scFvCD20:sTRAIL、pLentiR.ISZ-sTRAIL、pLentiR.scFvCD20及pLentiR.CopGFP慢病毒表达载体,瞬时转染293T细胞获得含有scFvCD20:sTRAIL融合蛋白的细胞培养上清,采用CCK8细胞增殖抑制实验检测scFvCD20:sTRAIL融合蛋白对CD20阳性BJAB细胞的生长抑制作用,流式细胞仪检测其对BJAB细胞诱导凋亡,Western blotting检测凋亡过程中内源性及外源性凋亡信号传导途径相关蛋白的变化。结果显示,成功构建了慢病毒表达载体pLentiR.scFvCD20:sTRAIL、pLentiR.ISZ-sTRAIL、pLentiR.scFvCD20及pLentiR.CopGFP,瞬时转染293T细胞后并可获得融合蛋白的表达。与ISZ-sTRAIL比较,融合蛋白scFvCD20:sTRAIL可不同程度地提高对CD20阳性BJAB细胞的生长抑制作用,凋亡细胞增多,其中Bcl-2表达下降、Bax表达升高,Caspase 8、Caspase 9、Caspase 3及PARP被特异性剪切活化。研究表明,融合蛋白scFvCD20:sTRAIL可诱导BJAB细胞凋亡,与bcl-2/bax的表达变化以及Caspase的剪切活化有关。
To investigate the apoptosis of BJAB induced by sTRAIL and the expression of apoptosis related proteins in the fusion protein scFvCD20: sTRAIL. The pLentiR.scFvCD20: sTRAIL, pLentiR.ISZ-sTRAIL, pLentiR.scFvCD20 and pLentiR.CopGFP lentiviral expression vectors were constructed by PCR, overlap PCR, restriction enzyme digestion and ligation and transiently transfected into 293T cells to obtain the recombinant lentiviral vector containing scFvCD20: sTRAIL fusion protein Cell culture supernatants were used to detect the growth inhibition effect of scFvCD20: sTRAIL fusion protein on CD20 positive BJAB cells by CCK8 cell proliferation inhibition assay. Flow cytometry was used to detect the apoptosis of BJAB cells induced by apoptosis. Western blotting was used to detect the endogenous And exogenous apoptotic signal transduction pathway related proteins. The results showed that the lentiviral vector pLentiR.scFvCD20: sTRAIL, pLentiR.ISZ-sTRAIL, pLentiR.scFvCD20 and pLentiR.CopGFP were successfully constructed and transiently transfected into 293T cells. Compared with ISZ-sTRAIL, the fusion protein scFvCD20: sTRAIL could enhance the growth inhibition of CD20-positive BJAB cells to varying degrees, and the apoptotic cells increased. The expression of Bcl-2, Bax and Caspase 8, Caspase 9, Caspase 3 and PARP are specifically cleaved and activated. Studies have shown that the fusion protein scFvCD20: sTRAIL can induce apoptosis in BJAB cells, which is related to the change of bcl-2 / bax and the cleavage of Caspase.