CAJ | 학술논문

目的：构建黑色素瘤抑制蛋白2（MIA2）慢病毒表达载体，并将其通过脂质体转染到HepG2细胞中，观察转染前后MIA2表达变化及其对细胞凋亡的影响。方法以pDONR223‐MIA2为模板，PCR扩增MIA2，构建pLVX‐IRES‐ZsGreen1‐MIA2慢病毒表达载体，体外瞬时转染肝癌细胞HepG2，荧光显微镜下观察MIA2的表达情况，用实时荧光定量PCR（RT‐PCR）检测HepG2细胞中MIA2mRNA的表达情况，噻唑蓝（MTT）法和克隆形成实验检测细胞增殖的情况。结果成功构建pLVX‐IRES‐ZsGreen1‐MIA2慢病毒表达载体；RT‐PCR结果显示，阴性对照组与实验组细胞MIA2mRNA表达水平差异有统计学意义（P＜0．05）；MTT检测发现，实验组细胞增殖数目较阴性对照组明显减少，差异有统计学意义（P＜0．05）；此外，实验组的克隆形成数和细胞迁移数均较阴性对照组明显减少，差异有统计学意义（P＜0．05）。结论pIRES2‐ZsGreen1‐MIA2可显著下调MIA2的表达水平进而抑制肝癌细胞HepG2的体外增殖及迁移能力。
목적：구건흑색소류억제단백2（MIA2）만병독표체재체，병장기통과지질체전염도HepG2세포중，관찰전염전후MIA2표체변화급기대세포조망적영향。방법이pDONR223‐MIA2위모판，PCR확증MIA2，구건pLVX‐IRES‐ZsGreen1‐MIA2만병독표체재체，체외순시전염간암세포HepG2，형광현미경하관찰MIA2적표체정황，용실시형광정량PCR（RT‐PCR）검측HepG2세포중MIA2mRNA적표체정황，새서람（MTT）법화극륭형성실험검측세포증식적정황。결과성공구건pLVX‐IRES‐ZsGreen1‐MIA2만병독표체재체；RT‐PCR결과현시，음성대조조여실험조세포MIA2mRNA표체수평차이유통계학의의（P＜0．05）；MTT검측발현，실험조세포증식수목교음성대조조명현감소，차이유통계학의의（P＜0．05）；차외，실험조적극륭형성수화세포천이수균교음성대조조명현감소，차이유통계학의의（P＜0．05）。결론pIRES2‐ZsGreen1‐MIA2가현저하조MIA2적표체수평진이억제간암세포HepG2적체외증식급천이능력。
Objective To construct an Lentiviral expression vector of pLVX‐IRES‐ZsGreen1‐MIA2 targeting to MIA2 and in‐vestigate its effect on the expression of MIA2 and growth of HCC cell line HepG2 in vitro ,observe MIA2 changes and the influence on apotheosis ,thus to provide preliminary experimental fundament for successive researching on the role of MIA2 in the pathogene‐sis of HCC .Methods The sequence of pLVX‐IRES‐ZsGreen1‐MIA2 was designed and synthesized .The pLVX‐IRES‐ZsGreen1‐MIA2 Lentiviral expression vector was constructed and then transiently transfected into HepG2 HCC cells in vitro .The proportion of pLVX‐IRES‐ZsGreen1‐MIA2 positive cells was observed under the fluorescence microscope .Then ,the expression level of MIA2 was detected by real time PCR .Moreover ,the proliferation of HepG2 cells was observed by MTT assay and colony formation as‐say .Finally ,the migration of HepG2 cells in vitro was also determined by Scratch assay .Results pLVX‐IRES‐ZsGreen1‐MIA2 Lentiviral expression vector was successfully constructed .Compared with control group (NC) ,the expression level of MIA2 was significantly decreased in transfected groups(P<0 .05);MTT assay showed that the proliferation of HepG2 cells was dramatically reduced in pIRES2‐ZsGreen1‐MIA2transfected groups(P< 0 .05);furthermore ,the number of both colony forming and migrating cells were also remarkably reduced in transfected groups(P<0 .05) .Conclusion The pIRES2‐ZsGreen1‐MIA2 can significantly re‐duce the expression level of MIA2 and inhibit the proliferation and migration of the HepG2 HCC cells in vitro .