【作者】 张勇；

【导师】 罗成基；

【作者基本信息】 第三军医大学 ， 防原医学， 2002， 博士

【摘要】 各种创伤、神经源性和肌源性肌病(如肌营养不良症)所致的肌损伤一直是影响人们生活的疾病之一，治疗上述肌损伤的手段有药物、物理疗法(包括各种理疗)、中医中药、介入等方法，但由于肌损伤的原因十分复杂，机理不清楚，且直接参与肌损伤修复的细胞——主要指卫星细胞数量十分有限，导致上述治疗效果不佳，故如何对肌损伤进行修复一直是人们研究的热点和难点。 间充质干细胞(mesenchymal stem cells，MSCs)基因疗法是近几年提出的修复肌损伤的新思路。早在1864年德国病理学家Conheim在研究创伤愈合时首先提出了骨髓中存在MSCs的观点，该观点在近十余年的研究中得到广泛的证实。MSCs是中胚层来源的具有多向分化能力的干细胞，主要存在于全身结缔组织和器官间质中，以骨髓中含量最丰富。该细胞具有分化成为成骨细胞、软骨细胞、脂肪细胞、肌腱细胞、成肌细胞、骨髓基质细胞和神经细胞等多种分化潜能，且易于分离和培养扩增，理论上讲可扩增40代而不丧失多向分化的间充质系的能力，是外源基因导入和表达的良好靶细胞，更是细胞基因治疗的首选载体。MyoD是骨骼肌表达的生肌调节因子(myogenic regulatory factors，MRFs)家族的重要组分，单一的MyoD即足以使肌干细胞(muscle stem cells)分化为成肌细胞(myoblasts)。由于转染MyoD可以启动肌分化过程，所以MyoD一直被当作最重要的成肌转录因子。MyoD转化的细胞移植后，参与再生的肌肉与卫星细胞来源的肌纤维形态学上完全一致。由于上述原因，我们利用MSCs有分化形成肌细胞的潜力，将带有增强型绿色荧光蛋白(enhance green fluorescent protein，EGFP)标记的真核表达的双顺反子质粒载体pIRES2-EGFP-MyoD转染进MSCs，使其分化为成肌细胞，并将MSCs和成肌细胞扩增后分别局部植入 Cardiotoxin造成的肌损伤组织中，观察MSCs和成肌细胞对肌损伤的修复 作用，从而为加速损伤部位肌肉组织再生和修复、减少伤残率提供一条新 的途径，主要结果如下： 1．成功地构建了真核表达的双顺质子质粒载体pIRESZ＊GFP－MyOD。一从原始质粒EMSV上用 ECORI酶切下 MyOD CDNA片段，进行琼脂凝胶电 泳，切胶回收纯化；ECORI酶切pIRESZ＊GFP，将线性化的载体与回收纯 化后的 MyOD CDNA 片段用 T。DNA 连接酶连接，形成 pIRESZEGFP－MyoD，将之转入工程菌 DHS a，铺板，挑选阳性克隆，摇 菌，提取质粒。Hind Jll酶切鉴定 MyOD cDNA插入方向，获得正向插入 MyoD的质粒载体。目的片段测序分析，与 Gene Bank报道序列完全一致。 2．运用脂质体转染的方法，将质粒载体pIRESZEGFP－MyOD成功地。转入 MSCS，转染效率为 10·3％，转染后的细胞经 G418筛选。转染后的 MSCs用 RTICR可检测出 MyoD的表达，扩增产物纯化测序与 Gene Bank 比较完全一致：荧光显微镜和激光共聚焦观察均可见胞体内有报告基因产 物的绿色荧光；免疫组化检坝MyoD、myogenln、myosln、myoglobln、desmln 表达均为阳性，而对照组为阴性；；未转染的MSCS荧光强度值很低，而转 染后的细胞具有很高的荧光强度值：与未转染的MSCS比较，转染后MSCS 其GO／GI期细胞的比例较高，而S、GZ、M期比例较低；转染后MSCS在＊电镜下观察表现为较为成熟细胞的形态学特点，且胞浆中有丝状物结构。 说明体外培养的MSCS在MyOD的调控下可以成功地分化为成肌细胞。 3．成功地制作了肌损伤模型。将 Cardiotoxin按 8 119电体重局部注入 SCID小鼠股四头肌中，24小时后可见肌细胞变性坏死，胞浆内有空泡形 成；7天后肌纤维发生明显断裂，变性坏死加重，并出现钙化；2周后变性 坏死钙化加重达到顶峰；4周后变性坏死钙化依然存在，但己可见部分肌 纤维修复；6周后变性坏死己不明显，钙化仍存在，肌细胞大部分修复。 4．将MSCS和成肌细胞扩增后分别局部植入肌损伤模型中，观察其对 肌损伤的修复效果，从不同时象点的病理切片和肌肉组织抗牵张力测定的 结果分析，MSCS和成肌细胞二者对肌损伤均有促进修复作用，肌肉组织 ·Vll· g抗牵张力的量化结果表明后者强于前者。 5．MSCS和成肌细胞对肌损伤的修复作用与损伤部位MyOD的表达密切相关，Western blot结果表明，二者呈正相关的关系；成肌细胞向肌细胞的分化过程主要受MAPK和PI。K信号通路调控，我们的研究表明，MSCS和成肌细胞对肌损伤修复的具体机制可能与MAPK家族中川KI、p38表达的增加，ERKZ的减少有关。 总之，本实验首先成功地构建了质粒载体pIRESZEGFP－MyOD，并将其成功地转染进了MSCS，使MSCS在MyOD的调控下分化为成肌细胞，并观察了MSCS和成肌细胞对肌损伤的修复作用，初步探讨了其修复的机制，旨更多还原

【Abstract】 Muscle injury resulting from various traumata, neurogenic and myogenic myopathy, such as muscular dystrophy, has been long affecting people’s life, though there are many therapeutic means including medicine, physical therapy, traditional Chinese medicine and herbs, intervention and so forth. However, because of the complicated causes resulting in muscle injury, the unclear mechanism of pathogenesis, and the limit amount of cells, mainly satellite cells, directly involved in the healing of muscle injury, the outcomes of above-mentioned treatment are not satisfying. Thus, how to repair muscle injury has been considered a heat and difficult issue for a long time.In recent years, gene therapy with mesenchymal stem cells (MSCs) has been studied in the treatment of injury of muscle. As early as 1864, German pathologist Conheim, for first time acclaimed that there exist MSCs in the bone marrow when he studied the healing of lesion, which has been proved in all round in the studies in later dozens of years. MSCs, stem cells derived from the mesoderm, are capable of multi-differentiating, mainly existing in connective tissue in all over the body and organ mesenchyma, especially abundant in the bone marrow. The cells, potential to differentiate into osteoblasts, chondrocytes, lipocytes, myoblasts, marrow stromal cells and neurocytes, are easy to be isolated and cultured, and theoretically, keep the differentiation to be mesenchymal system after 40 passages, so, the cells can be regarded as better target cells for extrogenic gene transference andexpression and optimal vector for cellular gene therapy. MyoD, one main member of myogenic regulatory factors Family because its transfection can initiate the process of muscle differentiation, merely make muscle stern cells differentiate into myoblasts. After the transportation of MyoD-transfected cells, the regenerated muscle has morphology totally in common with myofibers derived from satellite cells. Due to the reasons mentioned above, based on the potential of myoblast differentiation, we transfected an eukaryotic expression bicistron plasmid vector pIRES2-EGFP-MyoD labeled with enhance green fluorescent protein (EGFP) into MSCs and induced the cells differentiate into myoblasts. After proliferative culture, the differentiated MSCs and normal MSCs were respectively grafed into the focuses of muscle injury induced by cardiotoxin. Roles of MSCs and MSCs-differentiated myoblasts were investigated in the reparation of muscular damages in order to explore a novel method to accelerate the regeneration and repair of muscle injury and reduce the rate of disability. The main results are as follows.1. Bicistron plasmid vector pIRES2-EGFP-MyoD expressing in eukaryotic cells was constructed in success. After the MyoD cDNA fragment was cut form original plasmid EMSV with EcoR I and purified with electrophoresis, it was ligated with linearized EcoR I -cut vector pIRES2-EGFP in present of a ligase T4 DNA. The obtained pIRES2-EGFP-MyoD was then transferred into an engineered bacterium DH5a to replicate the target plasmid. After the indentification of insert direction of MyoD cDNA fragment with Hind III digestion, those plasmid vectors with positive insertion were harvested. The fragment sequence was confirmed to be consistent with the reported one in Genbank.2. The plasmid pIRES2-EGFP-MyoD was transfected into MScs by 10.3% with aid of lipotransfection method. After G418 screening, the expression of MyoD was detected in the transfected MSCs with RT-PCR andthe amplified product was in same in sequence with that from Genbank. Fluorescence was observed in the transfected cells under a fluorescent and laser cofocal microscopes, indicating the existence of reporter gene. Immunohistochemical methods indicated the expressions of MyoD, myogenin, myosin, myoglobin and desmin. But they are negative in control. Fluorescent intensity was stronger in the cells with MyoD transfection than those without. Compared with the later, the former had more in the phase of G0/G2更多还原