本文選題：肌衛(wèi)星細(xì)胞 + 肌再生��； 參考：《華南農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：運(yùn)動拉傷、撞傷等或肌肉代謝紊亂引起的肌肉損傷和缺失,嚴(yán)重影響我們的日常生活。而肌肉組織的可再生能力主要依靠肌衛(wèi)星細(xì)胞,大面積的肌肉損傷由于損傷部位肌衛(wèi)星細(xì)胞的缺乏而不能完全再生,產(chǎn)生沒有肌肉收縮功能的疤痕。已有研究表明體外分離的肌衛(wèi)星細(xì)胞移植到肌萎縮癥動物模型后,肌肉修復(fù)能力有所提高,但是仍然存在許多問題,使得最終移植效果不顯著。本實(shí)驗(yàn)室前期研究結(jié)果表明,CTX損傷小鼠腓腸肌后移植同源異體肌衛(wèi)星細(xì)胞,不同時期受損組織石蠟切片HE染色觀察到肌肉修復(fù)明顯加快。因此,本研究則通過RT-PCR和Western-blot檢測肌衛(wèi)星細(xì)胞移植后不同時間點(diǎn)肌生成相關(guān)基因,Notch信號部分基因,炎癥反應(yīng)相關(guān)基因的表達(dá)變化,從分子水平進(jìn)一步探討肌肉損傷修復(fù)加速的機(jī)理。研究結(jié)果如下:1.移植肌衛(wèi)星細(xì)胞組的小鼠受損部位的肌衛(wèi)星細(xì)胞活化、增殖、分化相關(guān)基因MyoD,Pax7,P21,MyoG(myogenin)的mRNA表達(dá)水平在第3天達(dá)到最高值,提前且高于注射PBS組。而這些基因的蛋白表達(dá)水平也在移植肌衛(wèi)星細(xì)胞組更早的達(dá)到較高水平。在移植肌衛(wèi)星細(xì)胞組分化后期表達(dá)基因MYH1的mRNA和蛋白水平也提前且高于對照組,說明移植肌衛(wèi)星細(xì)胞后受損部位肌衛(wèi)星細(xì)胞參與到肌再生過程的時間提前,肌再生能力也加強(qiáng)了。2.Notch1的mRNA和蛋白表達(dá)在移植肌衛(wèi)星細(xì)胞組中第7天達(dá)到最高峰高于對照組。檢測Notch信號中配體Jagged1的mRNA表達(dá)也是在第7天移植肌衛(wèi)星細(xì)胞組顯著高于注射PBS組,同時Notch1胞內(nèi)活化片段NICD的蛋白表達(dá)在移植肌衛(wèi)星細(xì)胞組第3,7天均高于PBS組。說明在CTX損傷的腓腸肌中移植肌衛(wèi)星細(xì)胞能更早的提高Notch信號通路中配體Jagged1,活化受體Notch1的表達(dá),以及它們之間的作用。而Notch信號靶基因Hes1的mRNA表達(dá)在第3天移植肌衛(wèi)星細(xì)胞組的表達(dá)降低,直到移植后的第14天Hes1的表達(dá)在肌衛(wèi)星細(xì)胞組顯著高于PBS組,表明Hes1可能還受到其他Notch受體的影響。3.移植肌衛(wèi)星細(xì)胞組TNFα和IL-10的mRNA表達(dá)達(dá)到高峰均提前于注射PBS組,而蛋白水平TNFα無明顯變化,IL-10的蛋白表達(dá)移植肌衛(wèi)星細(xì)胞組提前達(dá)到最大值。表明在肌衛(wèi)星細(xì)胞移植后炎癥相關(guān)反應(yīng)進(jìn)程加速了。本研究初步分析了肌衛(wèi)星細(xì)胞移植后肌衛(wèi)星細(xì)胞的活化能力加強(qiáng);炎癥因子的表達(dá)增加,且炎癥反應(yīng)過程縮短;Notch信號活化增強(qiáng)。因此,肌再生、炎癥反應(yīng)和Notch信號通路的共同加速可能促進(jìn)了受損肌肉的修復(fù)進(jìn)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 偽 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 偽 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.
【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R685

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本文編號：1866658