[1]冯正平,邓华聪,姜蓉,等.p38MAPK基因RNAi慢病毒载体的构建及在MC3T3-E1细胞的表达[J].陆军军医大学学报(原第三军医大学学报),2010,32(15):1598-1601.
　Feng Zhengping,Deng Huacong,Jiang Rong,et al.Construction of recombinant lentiviral vector of siRNA for p38MAPK gene and its expression in MC3T3-E1 cells[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(15):1598-1601.
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p38MAPK基因RNAi慢病毒载体的构建及在MC3T3-E1细胞的表达(/HTML )
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陆军军医大学学报（原第三军医大学学报）[ISSN:1000-5404/CN:51-1095/R]

卷:
32卷
期数:
2010年第15期
页码:
1598-1601
栏目:
论著
出版日期:
2010-08-15

文章信息/Info

Title:
Construction of recombinant lentiviral vector of siRNA for p38MAPK gene and its expression in MC3T3-E1 cells
作者:
冯正平; 邓华聪; 姜蓉; 杜佳; 陈丹燕; 梁小燕
重庆医科大学：附属第一医院内分泌科,干细胞与组织工程实验室
Author(s):
Feng Zhengping;  Deng Huacong;  Jiang Rong;  Du Jia;  Chen Danyan;  Liang Xiaoyan
Department of Endocrinology, First Affiliated Hospital,  Laboratory of Stem Cells and Tissue Engineering, Chongqing Medical University, Chongqing, 400016, China
关键词:
p38MAPK; 成骨细胞; RNA干扰; 慢病毒属
Keywords:
MAP kinase;  osteoblast;  RNA interference;  lentivirus
分类号:
R322.71;R394-33;R394.2
文献标志码:
A
摘要:
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，Western blot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、2.5×108 TU/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。
Abstract:
Objective    To construct the lentiviral vector of mouse p38MAPK gene RNAi and study its effect on p38MAPK expression and apoptosis of MC3T3-E1 cells.     Methods    Three complementary oligoDNA fragments of mouse p38MAPK gene were designed and synthesized. After phosphorylation and annealing, double-strand DNA was subcloned into HpaⅠ sites of the siRNA expression vector PLL3.7. The inserted sequence was identified after screening and restriction enzyme digestion. 293T cells were co-transfected with lentiviral vector and recombinant plasmids. Viral titer was determined by flow cytometry (FCM). Recombinant lentivirus 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 containing shRNAs targeting p38MAPK gene was successfully constructed and transfected into MC3T3-E1 cells. Recombinant plasmids were named p38MAPK-shRNA1, p38MAPK-shRNA2, and p38MAPK-shRNA3, respectively. Their viral titer was 2.4×108, 2.8×108 and 2.5×108 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 non-p38MAPK-transfected group than in those of control group (P<0.01) and was significantly lower in MC3T3-E1 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.

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更新日期/Last Update: 2010-08-03