LNK基因表达改变对THP-1细胞增殖及凋亡的影响

Effects of LNK gene expression on proliferation and apoptosis of THP-1 cells

目的初步探讨沉默及过表达LNK基因后对人急性单核细胞白血病细胞(THP-1)增殖及凋亡的影响,为研究白血病靶向治疗提供新方向。方法实验分为4组:沉默LNK组(Si-LNK)、过表达LNK组(VP-LNK)、空转组(Empty virus group)及对照组(Control group)。慢病毒转染THP-1细胞,建立稳定沉默及过表达LNK基因细胞株;转染72 h后,观察沉默LNK组、过表达LNK组及空转组绿色荧光蛋白(GFP)的表达量,流式细胞仪检测慢病毒感染效率;RT-PCR及Western blot检测LNK基因mRNA和蛋白表达水平;CCK-8法检测THP-1细胞在24、48、72 h的增殖情况,流式细胞术检测THP-1细胞凋亡率。结果经慢病毒感染72 h,沉默LNK组、过表达LNK组及空转组THP-1细胞的感染效率在99%以上。与对照组相比,沉默组LNK基因的mRNA水平及蛋白表达水平均显著降低,而过表达组均显著升高,说明LNK基因沉默或过表达成功。CCK-8法及流式检测结果显示沉默LNK组较对照组细胞增殖活性降低,且细胞凋亡率明显增高,差异均有统计学意义(P<0.05);与对照组及空转组相比,过表达LNK组THP-1细胞的增殖活性及细胞凋亡率无明显升高或降低,差异无统计学意义(P>0.05)。结论沉默LNK基因可显著抑制THP-1细胞增殖,促进其凋亡,但过表达LNK对细胞增殖及凋亡无明显影响。

Objective To investigate the effects of silencing and overexpression of LNK gene on proliferation and apoptosis of human acute monocyte leukemia(THP-1)cells and further provide a new direction for targeted therapy of leukemia.Methods This study was divided into four groups:silenced LNK group,overexpressed LNK group,idling group and control group.THP-1 cells were transfected with lentivirus to establish stable silencing LNK and overexpressing LNK gene cell lines.The expression of green fluorescent protein(GFP)in each group was observed under fluorescence microscope 72 h after transfection,and the efficiency of lentivirus infection was detected by flow cytometry.The expression of LNK gene was detected by RT-PCR and western blotting to understand the silencing and overexpression of LNK.The proliferation of THP-1 cells was detected by CCK-8 at 24,48 and 72 h,and the apoptosis rate of THP-1 cells was also measured by flow cytometry.Results After 72 h of lentivirus infection,the GFP expression of THP-1 cells reached more than 85%,and the cell infection efficiency was more than 99%.Compared with the control group,mRNA and protein expression levels of LNK gene in the silenced group were decreased,while those in the overexpressed group were increased,indicating the successful silencing or overexpression of LNK gene.The results of CCK-8 and flow cytometry showed that the proliferation activity of cells was decreased and the apoptosis rate was increased.Compared with the control group and idleness group,the proliferation activity and apoptosis rate in overexpressed LNK group were not significantly changed.Conclusion Silencing of LNK gene could inhibit the proliferation of THP-1 cells and promote their apoptosis,but overexpression of LNK has no significant effects on cell proliferation and apoptosis.