PKCI-1／HINT1表达质粒的构建及其对黑素瘤A375细胞凋亡及自噬影响的初步研究  

Construction of a eukaryotic expression plasmid carrying the PKCI-1/HINT1 gene and its effects on apoptosis and autophagy of A375 melanoma cells

作　　者：倪娜娜[1] 温斯健[1] 张韡[1] 姜祎群[1] 孙建方[1] 

机构地区：[1]中国医学科学院北京协和医学院皮肤病研究所病理科,南京210042

出　　处：《中华皮肤科杂志》2016年第5期348-352,共5页Chinese Journal of Dermatology

基　　金：国家自然科学基金（81272992）;江苏省自然科学基金（BK20131063）;高等学校博士学科点专项科研基金（20121106110040）

摘　　要：目的 构建人PKCI-1/HINT1基因的真核表达质粒,研究其在人黑素瘤A375细胞株中的表达并检测其对A375细胞凋亡及自噬的影响.方法 以人黑素瘤细胞A375的总RNA为模板,反转录-聚合酶链反应（RT-PCR）扩增PKCI-1/HINT1基因序列,将PKCI-1/HINT1基因克隆到真核表达载体PCDNA3.1（＋）中,构建PCDNA3.1（＋）-PKCI-1/HINT1重组体.将PCDNA3.1（＋）-PKCI-1/HINT1表达载体瞬时转染黑素瘤A375细胞,RT-PCR及Western印迹检测PKCI-1/HINT1在细胞内的表达,并以PCDNA3.1（＋）空载体转染细胞作为相应对照组.噻唑蓝（MTT）检测PKCI-1/HINT1转染后细胞的增殖变化,Hoechest 33258染色检测细胞凋亡,采用绿色荧光蛋白-微管相关蛋白轻链3（GFP-LC3）标记结合激光共聚焦显微镜法检测PKCI-1/HINT1对细胞自噬的的影响,并通过Western印迹检测PKCI-1/HINT1对细胞内caspase 3及自噬相关蛋白beclin1蛋白表达的影响.结果 PCDNA3.1 （＋）-PKCI-1/HINT1真核表达载体经酶切及测序鉴定构建成功,并能够在细胞内有效表达.MTT检测发现PKCI-1/HINT1能够明显抑制A375细胞的增殖,与PCDNA3.1（＋）对照组相比,在48 h,72 h及96 hPCDNA3.1 （＋）-PKCI-1/HINT1组活细胞数分别减少17.0％,25.6％、29.4％,差异有统计学意义（均P＜0.05）.Hoechest 33258染色显示PKCI-1/HINT1可促进A375细胞内凋亡小体形成.激光共聚焦显微镜发现PKCI-1/HINT1的过表达可使A375细胞内GFP-LC3B的点状聚集增加.Western印迹发现,PKCI-1/HINT1可促进细胞内caspase 3及beclin1蛋白表达.结论 成功构建真核表达载体PCDNA3.1 （＋）-PKCI-1/HINT1并在细胞内有效表达PKCI-1/HINT1.PKCI-1/HINT1的高表达可以抑制A375细胞增殖,促进其凋亡,同时可引发A375细胞的自噬过程.Objective To construct a eukaryotic expression plasmid carrying the PKCI-1/HINT1 gene,to investigate its expression in A375 melanoma cells after transfection,and to evaluate its effects on apoptosis and autophagy of A375 cells.Methods The PKCI-1/HINT1 gene sequence was amplified by reverse transcription PCR （RT-PCR） with total RNA extracted from A375 cells as the template,then inserted into the eukaryotic expression plasmid PCDNA3.1 （＋） to construct a recombinant plasmid,PCDNA3.1 （＋）-PKCI-1/HINT1.Some A375 cells were classified into two groups to be transiently transfected with the recombinant plasmid （PCDNA3.1 （＋）-PKCI-1/HINT1 group） or the empty plasmid PCDNA3.1 （＋） （control group）.After additional 48-hour culture,RT-PCR and Western blot analysis were performed to quantify the mRNA and protein expressions of PKCI-1/HINT1 respectively,Hoechst 33342 staining was conducted to detect apoptosis of A375 cells,Western blot analysis to detect the expressions of intracellular caspase-3 and autophagy-associated protein beclin1,and cell autophagy was observed by using the green fluorescent protein （GFP）-microtubule-associated protein 1 light chain 3 （LC3） labelling method combined with confocal laser scanning microscopy.Methyl thiazolyl tetrazolium （MTT） assay was performed to evaluate the proliferative activity of A375 cells at 24,48,72 and 96 hours after transfection.Results Enzyme digestion and sequencing analysis confirmed that the eukaryotic expression plasmid PCDNA3.1 （＋）-PKCI-1/HINT1 was successfully constructed and effectively expressed in the transfected A375 cells.MTT assay showed that PKCI-1/HINT1 could obviously inhibit the proliferation of A375 cells,and the number of live cells was decreased by 17.0%,25.6% and 29.4% in the PCDNA3.1 （＋）-PKCI-1/HINT1 group at 48,72 and 96 hours,respectively,compared with the control group （all P 〈 0.05）.Hoechest 33258 staining revealed that PKCI-1/HINT1 could promote the formation of apoptotic bodies in A375 cel

关 键 词：黑色素瘤 细胞系 肿瘤 细胞凋亡 自噬 PKCI-1/HINT1 

分 类 号：R739.5[医药卫生—肿瘤]