同源异体肌卫星细胞移植加速受损肌肉的修复

发布时间：2018-05-09 16:05

本文选题：肌卫星细胞 + 肌再生　；参考：《华南农业大学》2016年硕士论文

【摘要】：运动拉伤、撞伤等或肌肉代谢紊乱引起的肌肉损伤和缺失,严重影响我们的日常生活。而肌肉组织的可再生能力主要依靠肌卫星细胞,大面积的肌肉损伤由于损伤部位肌卫星细胞的缺乏而不能完全再生,产生没有肌肉收缩功能的疤痕。已有研究表明体外分离的肌卫星细胞移植到肌萎缩症动物模型后,肌肉修复能力有所提高,但是仍然存在许多问题,使得最终移植效果不显著。本实验室前期研究结果表明,CTX损伤小鼠腓肠肌后移植同源异体肌卫星细胞,不同时期受损组织石蜡切片HE染色观察到肌肉修复明显加快。因此,本研究则通过RT-PCR和Western-blot检测肌卫星细胞移植后不同时间点肌生成相关基因,Notch信号部分基因,炎症反应相关基因的表达变化,从分子水平进一步探讨肌肉损伤修复加速的机理。研究结果如下:1.移植肌卫星细胞组的小鼠受损部位的肌卫星细胞活化、增殖、分化相关基因MyoD,Pax7,P21,MyoG(myogenin)的mRNA表达水平在第3天达到最高值,提前且高于注射PBS组。而这些基因的蛋白表达水平也在移植肌卫星细胞组更早的达到较高水平。在移植肌卫星细胞组分化后期表达基因MYH1的mRNA和蛋白水平也提前且高于对照组,说明移植肌卫星细胞后受损部位肌卫星细胞参与到肌再生过程的时间提前,肌再生能力也加强了。2.Notch1的mRNA和蛋白表达在移植肌卫星细胞组中第7天达到最高峰高于对照组。检测Notch信号中配体Jagged1的mRNA表达也是在第7天移植肌卫星细胞组显著高于注射PBS组,同时Notch1胞内活化片段NICD的蛋白表达在移植肌卫星细胞组第3,7天均高于PBS组。说明在CTX损伤的腓肠肌中移植肌卫星细胞能更早的提高Notch信号通路中配体Jagged1,活化受体Notch1的表达,以及它们之间的作用。而Notch信号靶基因Hes1的mRNA表达在第3天移植肌卫星细胞组的表达降低,直到移植后的第14天Hes1的表达在肌卫星细胞组显著高于PBS组,表明Hes1可能还受到其他Notch受体的影响。3.移植肌卫星细胞组TNFα和IL-10的mRNA表达达到高峰均提前于注射PBS组,而蛋白水平TNFα无明显变化,IL-10的蛋白表达移植肌卫星细胞组提前达到最大值。表明在肌卫星细胞移植后炎症相关反应进程加速了。本研究初步分析了肌卫星细胞移植后肌卫星细胞的活化能力加强;炎症因子的表达增加,且炎症反应过程缩短;Notch信号活化增强。因此,肌再生、炎症反应和Notch信号通路的共同加速可能促进了受损肌肉的修复进程。
[Abstract]:Muscle damage and loss caused by sports injuries, collisions, etc., or muscle metabolic disorders seriously affect our daily life. The regenerative ability of muscle tissue mainly depends on muscle satellite cells. Due to the lack of muscle satellite cells in the injured site, large area of muscle injury can not be completely regenerated, resulting in no scar of muscle contraction function. Some studies have shown that the muscle repair ability of the isolated muscle satellite cells has been improved after transplantation to the animal model of muscular dystrophy in vitro, but there are still many problems, which make the final transplantation effect is not significant. The experimental results showed that homologous homologous muscle satellite cells were transplanted after CTX injury in mice. The repair of muscle was observed by HE staining in paraffin sections of damaged tissues at different stages. In this study, RT-PCR and Western-blot were used to detect the expression of Notch signaling genes and inflammatory response related genes at different time points after muscle satellite cell transplantation. The mechanism of accelerated repair of muscle injury was further discussed at the molecular level. The results are as follows: 1. The mRNA expression of myosatellous cells in the injured site of the transplanted muscle satellite cells reached the highest level on the 3rd day, and was earlier and higher than that in the injected PBS group. The protein expression level of these genes also reached a higher level earlier in the transplanted muscle satellite cell group. The mRNA and protein levels of MYH1 expression gene in the later stage of transplantation were also early and higher than those in the control group, which indicated that the time of muscle satellite cells participating in the process of muscle regeneration was earlier in the injured part of the transplanted muscle satellite cells. The ability of muscle regeneration also enhanced the expression of mRNA and protein of Notch1. The expression of mRNA and protein reached the peak on the 7th day in the transplanted satellite cells group, which was higher than that in the control group. The mRNA expression of ligand Jagged1 in the Notch signal was also significantly higher in the transplanted muscle satellite cells group than in the injected PBS group on the 7th day, and the expression of NICD in the Notch1 intracellular activation fragment was higher than that in the PBS group on the 7th day. These results suggest that the expression of ligand Jagged1, the activated receptor Notch1, and their roles in the Notch signaling pathway can be increased by the transplantation of muscle satellite cells in gastrocnemius muscle injured by CTX. However, the mRNA expression of Hes1, the target gene of Notch, decreased on the 3rd day, until the 14th day after transplantation, the expression of Hes1 was significantly higher in the myosatellar group than in the PBS group, suggesting that Hes1 might also be affected by other Notch receptors. The peak of mRNA expression of TNF 伪 and IL-10 in the transplanted muscle satellite cells group was earlier than that in the injected PBS group, while the protein level of TNF 伪 did not change significantly. The protein expression of IL-10 protein reached the maximum in the transplanted muscle satellite cell group. The results suggest that the inflammatory response is accelerated after the transplantation of myosatellite cells. In this study, we preliminarily analyzed the enhanced activation ability of myosatellous cells after transplantation, the increased expression of inflammatory factors and the increased activation of Notch signal in the process of inflammation. Therefore, muscle regeneration, inflammatory response and Notch signaling pathway may accelerate the repair process of damaged muscle.
【学位授予单位】：华南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R685

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