FGF6基因高表达对鼠心肌细胞凋亡和增殖的影响

Effect of over-expressed FGF6 gene on myocardial cells apoptosis and proliferation

目的:克隆小鼠成纤维细胞生长因子6(FGF6)基因cDNA的读码框(CDS),构建携带FGF6基因的重组真核表达载体,并将重组质粒转染至心肌细胞系H9C2细胞中进行表达,测定转染后FGF6基因对心肌细胞增殖及凋亡的影响。方法:从小鼠心脏组织提取总RNA,通过逆转录得到总cDNA,PCR法扩增,产物连接pGEM-T Easy载体测序分析正确后,再以PCR方法扩增,将其连接入真核表达质粒PIRES2-DsRed2。以PCR、双酶切和测序鉴定正确后,将重组载体PIRES2-DsRed2-FGF6用脂质体包裹转染大鼠心肌细胞(H9C2),分组:空白对照(BC)组、转染空质粒(NC)组、转染重组FGF6-PIRES2-DsRed2质粒(P-FGF6)组,RT-PCR法检测转染后FGF6 mRNA表达水平,荧光显微镜观察转染效率。Cell Counting Kit-8(CCK-8)法检测转染后细胞的增殖能力;以血清饥饿诱导心肌细胞凋亡,同时进行FGF6质粒转染,流式细胞仪检测细胞凋亡指数,Western blot方法检测活化半胱氨酸蛋白酶3(caspase-3)表达。结果:成功克隆了小鼠FGF6基因cDNA,重组真核表达载体PIRES2-DsRed2-FGF6构建成功,且证实转染后大鼠心肌细胞的FGF6mRNA表达明显高于BC组(P<0.05)及NC组(P<0.05),转染FGF6基因能提高心肌细胞的增殖能力(P<0.05),FGF6基因能明显抑制血清饥饿引起的细胞凋亡(P<0.05),同时下调活化caspase-3表达(P<0.05)。结论:成功克隆了FGF6基因,并构建了真核表达载体PIRES2-DsRed2-FGF6,重组FGF6真核表达载体能够在H9C2细胞中获得有效过表达,并证实FGF6基因可促进心肌细胞的增殖及保护心肌细胞的凋亡。

Objective： To clone CDS of mouse FGF6 gene cDNA and construct its eukaryotic expression vector for its function study in ventricular septal defect. And then to construct and express PIRES2-DsRed2 vector of rat FGF6 full length gene in H9C2 myocardial cell lines. To investigate the effects of over-expressed FGF6 on proliferation and apoptosis in H9C2 cell lines. Methods： Total RNA was extracted from mouse heart tissue. FGF6 cDNA was prepared by RT-PCR, and inserted into pGEM-T Easy vector and confirmed by sequence analysis. Then the pGEM-T Easy vector which contained FGF6 cDNA was also amplified by PCR, and cloned into PIRES2-DsRed2 plasmid. And the recombinant plasmid was confirmed by PCR, double enzyme digestion analysis and DNA sequencing. Then the recombinant plasmid PIRES2-DsRed2-FGF6 was transiently transfected into H9C2 cells using Lipofectamin 2000. The level of expression of FGF6 mRNA was identified using RT-PCR after transfection. The transfection efficiency was observed directly under fluorescence microscope. The cell proliferation was measured with CCK-8. Cell apoptosis was induced by serum deprivation in H9c2 cells transfected with FGF6 gene. The apoptosis rate of the cells was determined by flow cytometry with annexin V- FITC and propidium iodide double staining. The protein expression of activated caspase-3 was measured by Western blotting. Results： The cDNA of FGF6 was cloned, and its recombinant eukaryotic expression vector was constructed successfully. Overexpression of FGF6 mRNA was observed in H9C2 cells transfected with FGF6 gene as compared to the blank control cells and the cells transfected with an empty vector （both P〈0.05）. Transfection of FGF6 gene promoted the proliferation of the cardiomyocytes （P〈0.05） and inhibited the apoptisis rates induced by serum deprivation （P〈0.05）.The expression of activated caspase-3 was increased by serum deprivation attenuated by FGF6 transfection （P〈0.05）. Conclusion： The PIRES2-DsRed2-FGF6 expression vector is successfully constructed and over-expression of FGF6 gene in H9C2 cells is obtained. In vitro, through overexpressing the FGF6 gene, we suppose that FGF6 is involved in the heart development by acting an anti- apoptosis factor in cardiomyocytes by promoting cell proliferation and inhibiting apoptisis.