人釉原蛋白基因转导对人骨髓基质细胞增殖和碱性磷酸酶合成的影响

Effects of human amelogenin gene transduction on proliferation and synthesis of alkaline phosphatase of human bone marrow stromal cells

目的观察慢病毒介导的人釉原蛋白(hAm)基因转导对人骨髓基质细胞(hBMSCs)增殖及细胞成骨分化标志物碱性磷酸酶(ALP)合成的影响。方法采用RT-PCR技术获取hAm编码基因,扩增产物与携带绿色荧光蛋白(GFP)的慢病毒载体质粒(FUGW)连接构建重组慢病毒载体质粒FUAmW,以聚乙烯亚胺(PEI)法三质粒共转染293T细胞组装获取慢病毒,并感染hBMSCs(目的基因转导组);以感染FUGW和未感染病毒的hBMSCs分别作为对照基因转导组和空白对照组。流式细胞仪测定慢病毒感染效率,RT-PCR鉴定细胞hAm表达;MTT比色法检测细胞增殖;慢病毒感染后第7天,倒置相差显微镜观察各组细胞ALP染色情况;感染后第4、7、10天,采用RT-PCR技术检测各组hBMSCs内ALP mRNA表达。结果目的基因转导组细胞FUAmW的感染效率达40.29%,RT-PCR检测到540 bp的目的基因产物条带;细胞增殖水平显著高于对照基因转导组和空白对照组(P<0.05);慢病毒感染后第7天,目的基因转导组ALP染色阳性细胞数量明显少于对照基因转导组和空白对照组;感染后第4、7、10天,目的基因转导组细胞ALP mRNA表达显著低于对照基因转导组和空白对照组。结论导入外源性Am基因能刺激hBMSCs增殖,但细胞内ALP mRNA表达下调。

Objective To investigate the effects of lentivirus-mediated human amelogenin(hAm) gene transduction on proliferation and synthesis of alkaline phosphatase(ALP) of human bone marrow stromal cells(hBMSCs). MethodsRT-PCR was adopted to obtain hAm encoding gene,and recombinant lentivirus vector plasmid FUAmW was constructed by connection of amplified products with lentivirus vector plasmid(FUGW) carrying green fluorescent protein(GFP).Recombinant lentivirus was prepared from 293T cells by polyethylenimine(PEI)-mediated transient cotransfection,and hBMSCs were infected with generated lentivirus(target gene transduction group).hBMSCs with FUGW infection and those without virus infection were served as control gene transduction group and blank control group,respectively.The infection efficiency of lentivirus was analysed by flow cytometry,and the expression of hAm in cells was detected by RT-PCR.Cell proliferation was determined by MTT colorimetric assay.ALP staining was observed with inverted phase contrast microscopy 7 d after lentivirus infection.The expression of ALP mRNA in hBMSCs was detected by RT-PCR 4 d,7 d and 10 d after infection. Results In target gene transduction group,the infection efficiency of FUAmW was 40.29%,540 bp strap of target gene was detected by RT-PCR,and cell proliferation was significantly higher than that in control gene transduction group and blank control group(P<0.05).The number of cells with positive ALP staining in target gene transduction group was significantly smaller than those in control gene transduction group and blank control group 7 d after lentivirus infection,and the expression of ALP mRNA in target gene transduction group was significantly lower than that in control gene transduction group and blank control group 4 d,7 d and 10 d after infection. Conclusion Lentivirus-mediated transduction with Am gene may enhance the proliferation of hBMSCs,while reduce the expression of ALP mRNA in cells.