人胚胎干细胞体内向肌细胞分化的研究

发布时间：2018-05-09 21:37

本文选题：胚胎干细胞 + 骨骼肌细胞　；参考：《扬州大学》2008年硕士论文

【摘要】： 人胚胎干细胞(human embryonic stem cells, hES细胞)是能向多种细胞类型分化的全(多)能性干细胞,如何将这类细胞诱导分化成骨骼肌细胞,为治疗肌肉退行性疾病,如杜氏肌营养不良症(Duchenn’s Muscular Dystrophy,DMD),提供足够数量的细胞来源,一直是干细胞分化研究领域的热点。本研究针对这一问题进行探讨,以人ES细胞系SHES 39作为实验材料,成功的将SHES 39细胞在体外诱导成骨骼肌细胞的前体细胞。建立合适的小鼠肌肉损伤模型后,将这些前体细胞移植入小鼠肌肉中,能进一步整合分化为骨骼肌细胞和肌肉组织干细胞——卫星细胞。实验结果如下: 1.为了建立合适的肌肉损伤模型,对20 ug心肌毒素(cardiotoxin)单独处理以及处理24小时后辅以X-射线局部照射小鼠胫骨前肌这两种方案进行比较,并对18,25和50Gy的照射剂量进行比较。病理检查的结果证实:心肌毒素能在注射部位对肌肉造成严重破坏,大部分骨骼肌细胞变性坏死;辅以25Gy的X-射线局部照射能明显抑制肌细胞损伤后内源性细胞的修复增生,大大提高人源性细胞的整合率(28.65%±13.69%,P0.01)。另外,18Gy的照射剂量也可达到相似效果,但50Gy的照射剂量效果相对较差。 2.利用经典遗传学的杂交-互交方法,将8周龄的非糖尿病-严重联合免疫缺陷(NOD/scid)雄鼠(scid-/-)与mdx纯合雌鼠(mdx-/-)相杂交,再用其所生的8周龄F1代雄鼠(scid+/-/mdx-/-)和雌鼠(scid+/-/mdx+/-)互交,提取46只F2代杂合子小鼠的基因组DNA,对dystrophin以及scid基因进行PCR检测,共获得13只双基因缺陷的纯合小鼠( scid-/-/mdx-/-),作为研究杜氏肌营养不良症的免疫缺陷型动物模型。 3.人胚胎干细胞悬浮培养,诱导分化成类胚体,在合适培养液中贴壁后继续分化生长,多次传代,直至形成类似于成纤维细胞样的骨骼肌前体细胞后,将这些前体细胞植入经心肌毒素+25Gy X-射线局部照射联合处理的NOD-SCID小鼠胫骨前肌。为了确定最佳的细胞移植时间,分别在照射后0小时、24小时、48小时三个时点进行细胞移植的实验。免疫组化结果表明:照射后24小时或48小时进行移植的外源细胞整合率相对较高,分别为24.32%和23.48%。 4.心肌毒素+25Gy X-射线局部照射联合处理NOD-SCID小鼠24小时后,将hES来源的前体细胞移植到NOD-SCID小鼠的胫骨前肌中,2-16周后取材做冰冻切片,随后的免疫组化和原位杂交结果证实,约10%-20%的细胞能整合分化成骨骼肌细胞,并表达人特异性的dystrophin基因,部分细胞能分化成为卫星细胞,并表达卫星细胞的相关标志,如pax7等。将这些细胞以同样方式移植入免疫缺陷的杜氏肌营养不良症的动物模型-scid/mdx小鼠肌肉后,也能整合分化成骨骼肌细胞并表达dytrophin基因。 5.在hES细胞来源的前体细胞移植后14-128天的共12只小鼠(26处移植位点)中,都有人源性的细胞整合,而未见畸胎瘤的形成。说明hES细胞有潜力成为临床治疗杜氏肌营养不良症的细胞来源。
[Abstract]:Human embryonic stem cells ( hES cells ) are all ( many ) pluripotent stem cells capable of differentiating into various types of cells . How to differentiate these cells into skeletal muscle cells is a hot spot for the treatment of muscular degenerative diseases , such as Duchenn ' s sarcoma Dystrophy ( DMD ) .

1 . In order to establish a proper model of muscle damage , the two schemes were compared and the irradiation doses of 18 , 25 and 50 Gy were compared .

2 . The genomic DNA of 46 F2 - generation hybrid mice was obtained by cross - fertilization with mdx - / - ( mdx - / - ) in 8 - week - old non - diabetic - severe combined immunodeficient mice ( mdx - / - ) , and the genomic DNA of 46 F2 - generation hybrid mice was extracted by PCR , and 13 homozygous mice with double - gene defects were obtained as an animal model for studying the immune deficiency of Duchenne muscular dystrophy .

3 . Human embryonic stem cells were cultured in suspension culture to induce differentiation into the embryonic body . After the cells were inserted into the appropriate culture medium , the cells were continued to differentiate and grow , and the cells were passaged for many times until the precursor cells were formed similar to the fibroblasts . The results showed that the integration rate of exogenous cells was 24.32 % and 23.48 % , respectively , after 24 hours , 24 hours and 48 hours after irradiation .

4 . After 24 hours of combined treatment with myocardial toxin + 25Gy X - ray for 24 hours , hES - derived precursor cells were transplanted into the anterior tibialis anterior muscle , 2 - 16 weeks later , the cells could differentiate into skeletal muscle cells and express human - specific markers such as pax7 and so on .

5 . In total 12 mice ( 26 transplantation sites ) from 14 to 128 days after precursor cell transplantation from hES cells , there was a source cell integration without the formation of teratomas , suggesting that hES cells had the potential to be a source of clinical treatment for Duchenne muscular dystrophy .

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

【共引文献】