p38MAPK基因RNAi慢病毒载体的构建及在MC3T3-E1细胞的表达

Construction of recombinant lentiviral vector of siRNA for p38MAPK gene and its expression in MC3T3-E1 cells

目的构建小鼠p38MAPK基因RNAi慢病毒载体,观察其对MC3T3-E1成骨细胞p38MAPK表达及细胞凋亡的影响。方法设计并合成3对互补的针对小鼠p38MAPK mRNA的oligoDNA片段,退火形成双链DNA,与经HpaⅠ酶切后的载体连接,PCR筛选阳性克隆,测序鉴定。重组质粒与慢病毒包装载体共转染293T细胞,包装产生慢病毒,流式细胞仪检测病毒滴度,p38MAPK-shRNA慢病毒载体转染体外培养MC3T3-E1细胞,荧光定量PCR检测MC3T3-E1细胞p38MAPK mRNA表达,进行p38MAPK干扰有效靶点的筛选。22.2 mol/L葡萄糖刺激培养MC3T3-E1细胞7 d,Westernblot检测MC3T3-E1细胞p38MAPK蛋白的表达,流式细胞术检测MC3T3-E1细胞凋亡。结果酶切和测序均证实各重组质粒核苷酸序列插入正确,所得质粒分别命名为p38MAPK-shRNA1、p38MAPK-shRNA2、p38MAPK-shRNA3,流式细胞仪测定病毒滴度分别为2.4×108、2.8×108、2.5×108TU/ml。p38MAPK-shRNA转染MC3T3-E1细胞效率达到74%以上,RT-PCR检测结果显示,各p38MAPK-shRNA转染组MC3T3-E1细胞p38MAPK mRNA表达较正常对照组分别下降了78.8%、84.3%和60.2%(P<0.01),其中以p38MAPK-shRNA2的干扰效率最高。Western blot检测结果显示,与正常对照组相比,高糖组、空载体转染组MC3T3-E1细胞p-p38MAPK蛋白表达明显增加(P<0.01)。p38MAPK-shRNA慢病毒转染组MC3T3-E1细胞p-p38MAPK蛋白表达水平较高糖组明显下调(P<0.01)。流式细胞仪检测结果显示,与正常对照组相比,高糖组MC3T3-E1细胞凋亡率显著增加(P<0.01);p38MAPK-shRNA慢病毒转染组以及p38MAPK信号转导阻断剂组较高糖组MC3T3-E1细胞凋亡率明显减少(P<0.05,P<0.01)。结论成功构建了靶向p38MAPK基因RNAi慢病毒载体,其能有效抑制MC3T3-E1细胞p38MAPK基因表达,减少高糖诱导的MC3T3-E1细胞凋亡。

Objective To construct the lentiviral vector of mouse p38MAPK gene RNAi and study its effect on p38MAPK expression and apoptosis of MC3T3-EI cells. Methods Three complementary oligoDNA fragments of mouse p38MAPK gene were designed and synthesized. After phosphorylation and annealing, double- strand DNA was subcloned into Hpa I sites of the siRNA expression vector PLL3.7. The inserted sequence was identified after screening and restriction enzyme digestion. 293T cells were eo-transfected with lentiviral vector and recombinant plasmids. Viral titer was determined by flow cytometry （FCM）. Recombinant ]entivirus vector system was used to transfect the MC3T3-E1 cells, mRNA expression levels of p38MAPK were analyzed by real time PCR. MC3T3-E1 cells were collected in 7 d after culture in high glucose. Cell apoptosis was detected by FCM. Protein level in p-p38MAPK was measured by Western blotting. Results The lentiviral vector contai- ning shRNAs targeting p38MAPK gene was successfully constructed and transfected into MC3T3-E1 cells. Recombinant plasmids were named p38MAPK-shRNA1, p38MAPK-shRNA2, and p38MAPK-shRNA3, respec- tively. Their viral titer was 2.4 ×10^8, 2.8 ×10^8 and 2.5 ×10^8 TU/ml, respectively. The expression of p38MAPK mRNA was 78.8% , 84.3% and 60.2% , and was significantly lower in MC3T3-E1 cells of different p38MAPK-shRNA-transfected groups than in control group（P 〈 0.01 ）. Western blot analysis showed that the p-p38MAPK protein expression level was significantly higher in MC3T3-E1 cells of high glucose group and nonp38MAPK-transfected group than in those of control group （P 〈0.01 ） and was significantly lower in MC31B-EI cells of high glucose group and non-p38MAPK-transfected group than in those of control group after p38MAPK- shRNA transfection （ P 〈 0.01 ）. FCM showed that the apoptosis rate of MC3T3-E1 cells was significantly higher in high glucose group than in control group （P 〈 0.01 ） and significantly lower in high glucose group than in p38MAPK-shRNA transfection group （P 〈0.01 ）. Conclusion The recombinant lentivirus vector of shRNA targeting p38MAPK gene constructed by our group can effectively inhibit the expression of p38MAPK in MC3T3-E1 cells, thus decreasing their apoptosis induced by high glucose.