[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.
点击复制

p38MAPK基因RNAi慢病毒载体的构建及在MC3T3-E1细胞的表达(/HTML )
分享到:

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.

相似文献/References:

[1]陈莉丽,黄玫,雷利红,等.OPG/RANKL/RANK系统参与牙槽骨吸收及重建过程作用初探[J].陆军军医大学学报(原第三军医大学学报),2013,35(04):288.
 Chen Lili,Huang Mei,Lei Lihong,et al.Role of OPG/RANKL/RANK system during alveolar bone resorption and remodeling[J].J Amry Med Univ (J Third Mil Med Univ),2013,35(15):288.
[2]张纲,亓连军,谭颖徽,等.bFGF-PLA-Ns对体外培养成骨细胞增殖、分化及矿化的影响[J].陆军军医大学学报(原第三军医大学学报),2007,29(21):2073.
 ZHANG Gang,QI Lian-jun,TAN Ying-hui,et al.Effect of basic fibroblast growth factor-polylactide sustained release nanospheres on proliferation, differentiation and mineralization of osteoblasts in vitro[J].J Amry Med Univ (J Third Mil Med Univ),2007,29(15):2073.
[3]黄宏,朱方强,孙宏振,等.脂肪来源干细胞体外成骨诱导和成脂诱导分化[J].陆军军医大学学报(原第三军医大学学报),2008,30(13):1219.
 HUANG Hong,ZHU Fang-qiang,SUN Hong-zhen,et al.Differentiation of swine adipose tissue-derived stromal cells into adipocytes and osteoblasts in vitro[J].J Amry Med Univ (J Third Mil Med Univ),2008,30(15):1219.
[4]张恒,周占松,刘丽梅,等.P物质刺激下脊髓星形胶质细胞活化中磷酸化P38丝裂素活化蛋白激酶和c-fos的表达及作用[J].陆军军医大学学报(原第三军医大学学报),2006,28(21):2163.
[5]陈允嘉,王豫蓉,邹林洪,等.放线共生放线杆菌培养上清对成骨细胞分化及间隙连接通讯的影响[J].陆军军医大学学报(原第三军医大学学报),2010,32(10):1059.
 Chen Yunjia,Wang Yurong,Zou Linhong,et al.Actinobacillus actinomycetemcomitans induces differentiation and gap junctional intercellular communication in cultured osteoblasts[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(15):1059.
[6]叶平,杨波,唐文,等.p38MAPK信号通路对乙醛刺激的大鼠肝星状细胞增殖、凋亡及细胞周期的影响[J].陆军军医大学学报(原第三军医大学学报),2010,32(20):2197.
 Ye Ping,Yang Bo,Tang Wen,et al.p38MAPK signal pathway modulates proliferation, apoptosis and cell cycle of rat hepatic stellate cells stimulated by acetaldehyde[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(15):2197.
[7]谢会霞,郝进.雷公藤内酯醇通过p38MAPK信号通路诱导Hut102细胞凋亡[J].陆军军医大学学报(原第三军医大学学报),2010,32(19):2109.
 Xie Huixia,Hao Jin.Triptolide induces apoptosis of Hut102 cells through p38MAPK pathway[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(15):2109.
[8]王胜国,周力,陈扬熙,等.不同强度静磁场对成骨细胞细胞内钙离子浓度的影响[J].陆军军医大学学报(原第三军医大学学报),2010,32(23):2515.
 Wang Shengguo,Zhou Li,Chen Yangxi,et al.Effect of static magnetic field at different strengths on Ca2+ concentration in rat primarily cultured osteoblasts[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(15):2515.
[9]段小军,王富友,杨柳,等.应用凝胶接种技术体外分层构建工程化骨软骨复合组织的初步研究[J].陆军军医大学学报(原第三军医大学学报),2009,31(20):1969.
 DUAN Xiao-jun,WANG Fu-you,YANG Liu,et al.Preparation of laminated construction of engineered osteochondral tissue with proteic-gel technology in vitro[J].J Amry Med Univ (J Third Mil Med Univ),2009,31(15):1969.
[10]金鑫,易龙,陈春烨,等.膜电位及MAPK磷酸化在飞燕草素抑制Ox-LDL诱导的血管内皮细胞氧化损伤中的作用[J].陆军军医大学学报(原第三军医大学学报),2009,31(19):1854.
 JIN Xin,YI Long,CHEN Chun-ye,et al.Delphinidin-3-glucoside inhibits Ox-LDL-induced injury in vascular endothelial cells: roles of membrane potential and MAPK phosphorylation[J].J Amry Med Univ (J Third Mil Med Univ),2009,31(15):1854.

更新日期/Last Update: 2010-08-03