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目的克隆大鼠B细胞淋巴瘤因子6(B-cell lymphoma 6,Bcl6)基因,构建绿色荧光蛋白融合表达载体pEGFP-Bcl6,初步探讨大鼠Bcl6转录因子在大鼠肝细胞中的生物学作用。方法首先提取大鼠肝脏总RNA,经反转录和巢式PCR扩增Bcl6编码区的CDS序列后,通过基因重组的方法构建pEGFP-Bcl6重组质粒并鉴定;然后利用脂质体转染法将重组质粒转入大鼠肝细胞BRL-3A中,利用实时定量PCR和蛋白质印迹法检测其表达;最后应用流式细胞术分析转染融合表达质粒后肝细胞的凋亡,采用MTT法检测肝细胞的增殖情况。结果 PCR法和测序法鉴定重组质粒pEGFP-Bcl6构建成功,实时定量PCR法和蛋白质印迹法鉴定重组质粒瞬时转染成功,流式细胞术证实Bcl6具有抑制肝细胞凋亡的作用,MTT法证实Bcl6具有促进肝细胞增殖的作用。结论成功克隆了大鼠Bcl6基因,构建了Bcl6绿色荧光蛋白表达载体,在大鼠肝细胞中初步证明Bcl6具有促进肝细胞增殖和抑制凋亡的作用。
Objective To clone the Bcl - 6 gene of rat and construct a green fluorescent protein fusion expression vector pEGFP - Bcl6 to investigate the biological role of Bcl6 transcription factor in rat hepatocytes. Methods The total RNA of rat liver was extracted firstly, and the CDS sequence of Bcl6 coding region was amplified by reverse transcription and nested PCR. The recombinant plasmid pEGFP-Bcl6 was constructed and identified by PCR. Then, The recombinant plasmids were transfected into rat hepatocytes BRL-3A. The expression of recombinant plasmids was detected by real-time PCR and Western blotting. Finally, the apoptosis of hepatocytes was analyzed by flow cytometry. The hepatocytes Proliferation situation. Results The recombinant plasmid pEGFP-Bcl6 was successfully constructed by PCR and sequencing. The recombinant plasmids were identified by real-time PCR and Western blotting. Transient transfection was confirmed by flow cytometry. Bcl6 could inhibit the apoptosis of hepatocytes. MTT assay confirmed that Bcl6 With the promotion of liver cell proliferation. Conclusion The Bcl6 gene was successfully cloned and the Bcl6 green fluorescent protein expression vector was constructed. It was preliminarily proved that Bcl6 could promote hepatocyte proliferation and inhibit apoptosis in rat hepatocytes.