Micro-dystrophin基因修饰的骨髓间充质干细胞移植治疗后mdx鼠MyoD和devMHC的变化  

The changes of MyoD and devMHC in mdx mice after transplantation of bone marrow mesenchymal stem cells modified by Micro-dystrophin gene

作　　者：王淑辉[1] 尚延昌[2] 于美娟[3] 张雅妮[3] 张成[3] 

机构地区：[1]首都医科大学附属北京友谊医院神经内科,北京100050 [2]解放军总医院老年神经科,北京100853 [3]中山大学附属第一医院神经内科,广东广州510080

出　　处：《临床和实验医学杂志》2014年第12期953-957,共5页Journal of Clinical and Experimental Medicine

基　　金：国家自然科学基金资助项目(81100237);解放军总医院临床科研扶持基金资助项目(2012FC-TSYS-3057)

摘　　要：目的观察经人微小抗肌萎缩蛋白(Micro-dystrophin)基因修饰的骨髓间充质干细胞(mMSCs)移植治疗的mdx小鼠成肌调节因子(MRFs)的表达。方法采用脂质体介导Micro-dystrophin基因转染mdx小鼠的mMSCs通过尾静脉注射转染外源基因的mMSCs移植治疗mdx鼠,在移植后4周、8周、12周、16周分别应用免疫荧光、逆转录-聚合酶链反应(RT-PCR)、Western blot三种方法检测腓肠肌内MRFs肌分化因子(MyoD)和发育肌球蛋白重链(devMHC)的表达。结果免疫荧光结果显示对照组腓肠肌有少许MyoD表达,阳性率为(5.2±0.31)%,移植后4周组为(8.4±0.52)%,移植12周组为(19.8±1.63)%,移植16周组为(15.4±1.37)%。对照组腓肠肌可见少量devMHC表达,阳性率为(1.1±0.18)%,移植后4周组为(7.4±1.33)%,移植12周组为(15.8±2.95)%,移植16周组为(21.9±3.62)%。移植后各组MyoD及devMHC阳性率较对照组升高(P<0.05),移植后一组与前一组比较差异有统计学意义(P<0.05)。RT-PCR检测MyoD和devMHC的mRNA表达,Western blot检测MyoD和devMHC蛋白表达,移植后8周组MyoD表达灰度比值较对照组增高(P<0.05),12周时最高,16周组较12周组下降;移植后8周组devMHC表达较对照组及4周组增加(P<0.05);12周组较8周组升高(P<0.05);16周组较12周组升高,但差异无统计学意义(P>0.05)。结论以Micro-dystrophin基因修饰的MSC移植治疗mdx鼠可能通过激活MRFs的序列表达而促进成肌分化。Objective To study the changes of MyoD and developmental MHC （ dev MHC） in mdx mice after transplantation of bone marrow mesenchymal stem cells modified by Micro - dystrophin gene. Methods The plasmid PeDNA3.1 （ ＋ ）/Micro - dystrophin was transfected into mMSCs by lipofectamine. Then the microdys - mMSCs were transplanted into mdx through the tail vein. The gastrocnemius muscle from mice at each time point were collected. Expression of MyoD and devMHC was detected using the method of RT - PCR and Western blot. Results A small number of MyoD positive fibers （ 5.2 ± 0.31 ） % were detected in non - transplanted mice. The percent of MyoD positive fibers was （8.4 ±0.52）% at the time of 4 weeks after transplantation of genetically modified MSCs. At the time of 12 weeks after transplantation, the percent of MyoD positive fibers was （ 19.8 ± 1.63 ） %. 16 weeks after transplantation, the percent was （ 15.4 ± 1.37 ） %. A small number of devMHC positive fibers （1.1± 0.18 ）% were detected in non -transplanted mice. The percent of devMHC positive fibers at 4 weeks, 12 weeks, 16 weeks was respectively （7.4 ± 1.33 ） % , （ 15.8 ±2.95 ） %, （21.9 ± 3.62） % . MyoD and devMHC positive cells rate of every group after transplantation were higher than those of the control group. The difference was statistically significant （ P 〈 0. 05 ）. Comparison between two neighboring groups after transplantation, the difference was statistically significant （ P 〈 0.05 ）. The content of MyoD and devMHC was detected by RT - PCR and Western blot. The results showed that MyoD expression increased in 8 weeks group after transplantation than control group. The difference was statistically significant （ P 〈 0.05 ）. The expression of MyoD increased after transplantation, up to the maximum at 12 weeks, drop at 16 weeks. The difference was statistically significant （ P 〈0.05）. devMHC expression increased in 8 weeks group after transplantation than control group and 4 weeks group, the

关 键 词：MDX鼠 Micro—dystrophin基因 骨髓间充质干细胞 肌分化因子 发育肌球蛋白重链 

分 类 号：R746.2[医药卫生—神经病学与精神病学]