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 RNAi lentiviral vector of mouse p38MAPK gene and investigate its effect on the expression of p38MAPK and the apoptosis of MC3T3-E1 osteoblasts. Methods Three pairs of complementary oligo DNA fragments targeting mouse p38MAPK mRNA were designed and synthesized. The double-stranded DNA was annealed to form the double-stranded DNA. After digested with Hpa I, the positive clones were screened by PCR and identified by sequencing. 293T cells were co-transfected with the recombinant plasmid and the lentiviral packaging vector, and the lentivirus was packaged to produce the lentivirus. The virus titer was detected by flow cytometry. The MC3T3-E1 cells were transfected with p38MAPK-shRNA lentiviral vector and the MC3T3-E1 cells were detected by real- p38MAPK mRNA expression, p38MAPK interference effective target screening. The MC3T3-E1 cells were cultured with 22.2 mol / L glucose for 7 days. Western blot was used to detect the expression of p38MAPK in MC3T3-E1 cells. Flow cytometry was used to detect the apoptosis of MC3T3-E1 cells. Results The digestion and sequencing of the recombinant plasmids confirmed that the nucleotide sequences of the recombinant plasmids were correctly inserted. The resulting plasmids were named as p38MAPK-shRNA1, p38MAPK-shRNA2 and p38MAPK-shRNA3 respectively. The virus titers were 2.4 × 108 and 2.8 × 108, 2.5 × 10 8 TU / ml. The efficiency of p38MAPK-shRNA transfected MC3T3-E1 cells was over 74%. The results of RT-PCR showed that the expression of p38MAPK mRNA in MC3T3-E1 cells decreased by 78.8%, 84.3% and 60.2% compared with the control group % (P <0.01), among which p38MAPK-shRNA2 had the highest interference efficiency. Western blot results showed that p-p38MAPK protein expression was significantly increased in MC3T3-E1 cells transfected with high glucose and empty vector compared with normal control group (P <0.01). p38MAPK-shRNA lentiviral transfection group MC3T3-E1 cells p-p38MAPK protein expression was significantly lower (P <0.01). Flow cytometry results showed that compared with the normal control group, the apoptosis rate of MC3T3-E1 cells in high glucose group was significantly increased (P <0.01); p38MAPK-shRNA lentiviral transfection group and p38MAPK signal transduction blocker group The apoptosis rate of MC3T3-E1 cells in higher glucose group was significantly decreased (P <0.05, P <0.01). Conclusion The RNAi lentiviral vector targeting p38MAPK gene was successfully constructed, which can effectively inhibit the expression of p38MAPK gene in MC3T3-E1 cells and reduce the apoptosis of MC3T3-E1 cells induced by high glucose.