【作者】 李鹏；

【导师】 雪原；

【作者基本信息】 天津医科大学 ， 外科学， 2011， 硕士

【摘要】 目的：用Wnt3a和smad4联合刺激纯化的MSCs,观察其miRNA谱的变化,为寻找成骨相关的miRNA提供实验基础。方法：选用出生后2周的SD大鼠,无菌下其股骨的骨髓细胞,通过分离、培养原代骨髓间充质干细胞；绘制其生长曲线；流氏细胞仪检测培养第6代的、已纯化的骨髓间充质干细胞。通过pcDNA3.1-smad4与穿梭质粒pGStrack-HB组装,依靠同源性将穿梭质粒pGStrack-smad4转移至pGSadeno腺病毒表达载体上；HEK 293包装重组腺病毒及线性化,完成pGSadeno-smad4的构建；PCR验证重组腺病毒的产生。以rAd5-smad4转染和Wnt3a联合刺激培养MSCs,激光共聚焦观察Wnt3a+smad4组β-cat/Smad4在MSCs内的聚合现象。Trizol法提取细胞中的总RNA,制作芯片,用LuxScan 10K/A双通道激光扫描仪扫描芯片。采用LuxScan3.0图像分析软件对芯片图像进行分析。用Significance Analysis of Microarrays检测Wnt3a+smad4对MSCs的miRNA普表达的影响,挑选出差异表达基因。并观察MSCs的存活时间及凋亡结果：经过分离、培养的原代骨髓间充质干细胞,经多次传代后,细胞逐渐纯化,并且细胞初期多以集落形式增长,第6代后细胞呈形态均一的纺锤形,分布均匀。流式细胞仪检测,结果显示为体积均一的细胞群,CD31、CD34、CD45的阳性率分别为5.67%、4.31%、4.42%,CD90、CD44、CD71的阳性率分别为97.17%、98.63%、95.86%。结果说明本试验培养分离的细胞具备了所有MSCs的抗原特性,细胞生长速率及形态正常。通过穿梭质粒、线性切割和包装构建rAd5-smad4; rAd5-smad4中smad4PCR片段大小约为1.8kb,结果均符合预期,说明重组腺病毒rAd5-smad4包装成功。以rAd5-smad4转染和Wnt3a联合刺激培养MSCs, microarry检测发现hsa-miR-122a、638、494、450表达上调；激光共聚焦显示MSCs内的β-cat/Smad4聚合。]Ad5-smad4转染和Wnt3a联合刺激后MSCs可存活2周/代；凋亡率0.02%。MSCs未经传代长期存活。并具有正常细胞的凋亡率结论：本试验分离、培养的细胞具备了所有MSCs的抗原特性,细胞生长速率及形态正常。通过穿梭质粒、线性切割和包装细胞系完成了rAd-smad4的构建表达；Wnt3a和转染rAd-smad4实现了联合对MSCs进行基因修饰目的,MSCs可存活2周/代；凋亡率0.02%。联合刺激后的MSCs高表达hsa-miR-122a、638、494、450。这为寻找成骨相关的miRNA提供了实验基础。更多还原

【Abstract】 Objictive:Combined stimulation of purified MSCs with smad4 and Wnt3a, observed changes in miRNA spectrum, to provide experimental basis for searching for related miRNA of osteogenesis.Methods:Two weeks old SD rats were choosed, gain the bone marrow cells of femoral. Then isolated and coltured the bone marrow mesenchyal stem cells. Drawed the curve of growth. Detected the purified MSCs with sixthed generation by Flow cytometry. Assembled pcDNA3.1-smad4 to pGStrack-HB, then transferred pGStrack-smad4 to pGSadeno which was adenovirus carrier. Recombined adenovirus by transfecting HEK293 cell, and here completed the construction of adenovirus of pGSadeno-smad4. Then validated the recombinant adenovirus by polymerase chain reaction. Combined the stimulation with Wnt3a and transfection of rAd-smad4 to cultured the MSCs, confocal observation of phenomenon aggregate ofβ-cat/Smad4 in MSCs, which were stimulated Wnt3a and smad4. Extracted the total RNA by using Trizol method, producted and scanned chips with LuxScan 10K/A of dual channel. Images were analyzed by Image analysis software LuxScan3.0. Used Significance Analysis of Microarrays to detect miRNA expression profile of MSCs, which were stimulated Wnt3a and smad4, selected the different expression of miRNAs. And observed the survival time and apoptosis of MSCs.Results:The bone marrow mesenchymal stem cells gradually purified after isolated and cultured primary MSCs were passaged for many generations, in the early stage of growth the cells formed colonies, the cells were uniform shape and distribution after the sixth generation. And flow cytometry showed that cell populations had homogeneous volume, the positive rates of CD31, CD34, CD45 were 5.67%,4.31%, 4.42%, and of CD90, CD44, CD71 were 97.17%,98.63%,95.86%. These results show that the isolated and cultured cells had the same antigenic characteristics of MSCs, growth rate and morphology were normal.Constructed rAD5-smad4 through the shuttle plasmid and packaging. PCR fragments size of smad4 rAd5-smad4 were about 1.8kb, the results are in line with expectation, which indicated that adenovirus rAd5-smad4 packaged successfully. Combined the stimulation with Wnt3a and transfection of rAd-smad4 to cultured the MSCs, detected hsa-miR-122a,638,494 and 450 upregulation by microarry. Confocal observed the phenomenon aggregate ofβ-cat/Smad4 in MSCs. Combined the stimulation with Wnt3a and transfection of rAd-smad4 to the MSCs, MSCs could survive for 2 weeks of generation with 0.02% of apoptosis rate. MSCs had normal rate of apoptosis.Conclusion:isolated and cultured primary MSCs had the same antigenic characteristics of MSCs, growth rate and morphology were normal. rAD5-smad4 was constructed successfully. Combined the stimulation with Wnt3a and transfection of rAd-smad4 to the MSCs, and achieved the purpose of genetically modified to MSCs. We found hsa-miR-122a,638,494 and 450 were upregulation, which provided experimental basis for searching for related miRNA of osteogenesis.更多还原