沉默DKK1、Sost重组腺病毒载体的构建及其对MG63细胞增殖、ALP活性和钙离子浓度影响研究

Construction of DKK1 and Sost recombinant adenovirus silencing vector and their effect on MG63 cell viability,ALP activity and Ca^(2+) concentration

目的构建Wnt信号通路抑制因子Dickkopf1(DKK1)、骨硬化蛋白Sclerostin(Sost)基因沉默重组腺病毒载体,观察其对MG63细胞增殖、碱性磷酸酶(ALP)活性和钙离子浓度影响作用。方法设计出特异性针对DKK1、Sost和无关对照序列(Scr),构建DKK1、Sost沉默重组腺病毒载体,用q PCR法和蛋白印记(Western blot)法选出沉默效率最好的扩增DKK1、Sost沉默重组腺病毒载体,并用荧光显微镜观察计算病毒滴度。MG63细胞被分成空白对照组、沉默DKK1(Ad-sh DKK1)组、沉默Sost(Ad-sh Sost)组、沉默DKK1+Sost(Ad-sh DKK1+Ad-sh Sost)组4组,分别用优选出的沉默效率最好的sh DKK1和sh Sost腺病毒载体转染MG63细胞,采用四甲基偶氮唑蓝微量酶反应比色法(MTT)检测观察细胞增殖、考马斯亮蓝蛋白定量法测定ALP活性、流式分析法测定钙离子浓度。结果 1DKK1 p Ad-sh DKK1-1和Sost p Ad-GFP-shRNA-2沉默效率最高;2与空白对照组对比,沉默DKK1组、沉默Sost组、沉默DKK1+Sost组的细胞光吸收值(OD)、ALP活性测定值均升高,而钙离子浓度均降低,以沉默DKK1+Sost组变化最明显,组间比较具有统计学差异(P<0.01和P<0.05)。结论 DKK1、Sost沉默可促进MG63细胞增殖、提高ALP活性,降低钙离子浓度,尤以二者同时沉默时的作用最强。

Objective To construct silent recombinant adenovirus vector targeting DKK1 and Sost gene and investigate their effect on MG63 cells viability,ALP activity and Ca2 ＋concentration. Me thods Designed specific sequences of DKK1,Sost and controlled sequence（ Scr）,constructed recombinant adenovirus vector of silencing about DKK1 and Sost,selected the best silencing genes to amplify recombinant adenovirus vector of silent DKK1 and Sost by q PCR and protein imprinting（ Western blot）,calculated virus degree with fluorescence microscopy. Mature MG63 cells were divided into blank control group,DKK1 silencing group,Sost silencing group and DKK1 ＋ Sost silencing group. According to different groups,MG63 cells were infected by adenovirus vector of sh DKK1 and sh Sost of the best efficiency,then we observed the cell viability through Tetramethyl azo salt azole trace enzyme reaction colorimetry（ MTT）,ALP activity through Coomassie brilliant blue protein quantitative method,Ca2 ＋concentration through flowanalysis. Results ① DKK1 p Ad-sh DKK1-1 and Sost p Ad-GFP-shRNA-2 had the highest efficiency; ② Compared with the blank control group,DKK1 silencing group,Sost silencing group and DKK1 ＋ Sost silencing group had increased cell viability and ALP activity,but decreased Ca2 ＋concentration,especially in the DKK1 ＋ Sost silencing group,and the differences among groups were statistically significant（ P〈0. 01 and P〈0. 05）. Conclusion DKK1 and Sost silencing can promote MG63 cell viability,ALP activity and reduce Ca2 ＋concentration,and silencing both of them have the strongest effects.