| 摘要: |
| 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。 |
| 关键词: 肝细胞 Bcl6 细胞增殖 细胞凋亡 |
| DOI：10.3724/SP.J.1008.2012.00349 |
| 投稿时间：2012-01-10修订日期：2012-03-19 |
| 基金项目:上海市科委重点基础项目(10JC1417400). |
|
| Up-regulation of B-cell lymphoma 6 expression suppresses apoptosis of rat liver cells |
| PAN Chuan-yong1,YANG Sheng-sheng1,FU Hai-long2,MIAO Ming-yong1*,JIAO Bing-hua1* |
(1. Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Second Military Medical University, Shanghai200433, China
2. Department of Anesthesiology, Changzheng Hospital, Second Military Medical University, Shanghai200003, China
*Corresponding authors.) |
| Abstract: |
| Objective To clone the gene of B-cell lymphoma 6 (Bcl6), construct a green fluorescent expression vector carrying rat Bcl6 gene, and to evaluate its roles in rat liver cells. Methods Total liver RNA was isolated from rat liver samples and cDNA was synthesized by reverse transcription; the coding sequence of Bcl6 gene was obtained by nested PCR. pEGFP-Bcl6 fusion expression vector was constructed by recombination technique and confirmed by sequencing. Then the recombinant vectors were transfected into rat liver cell line BRL-3A by lipofectamine; the expression levels of Bcl6 mRNA and protein were determined by real-time PCR and Western blotting analysis, respectively. Finally, the apoptosis of BRL-3A cells transfected with recombinant vector was analyzed by flow cytometry and cell proliferation was examined by MTT assay. Results The recombinant vector pEGFP-Bcl6 was successfully constructed as determined by PCR and sequencing, and was effectively delivered to BRL-3A cells by transient transfection as confirmed by PCR and Western blotting analysis. Result of flow cytometry proved that Bcl6 up-regulation suppressed apoptosis of rat liver cells and MTT assay found that Bcl6 up-regulation promoted proliferation of liver cells. Conclusion We have successfully cloned rat Bcl6 gene and constructed its green fluorescent expression vector. Our results initially prove that Bcl6 may inhibit cell apoptosis and promote cell proliferation in rat liver cells. |
| Key words: hepatocytes Bcl6 cell proliferation apoptosis |
