本文選題：失神經(jīng)肌萎縮 + 肌衛(wèi)星細(xì)胞��； 參考：《遵義醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的:建立家兔腓腸肌失神經(jīng)肌萎縮模型,分離提取肌衛(wèi)星細(xì)胞并移植,觀察移植后延緩骨骼肌萎縮的效果。方法:(1)家兔骨骼肌衛(wèi)星細(xì)胞分離提取與培養(yǎng):選取出生后1周內(nèi)的乳兔,取下四肢骨骼肌,經(jīng)膠原酶和胰酶混合液消化,再經(jīng)兩次差速貼壁得到原代肌衛(wèi)星細(xì)胞,繼續(xù)培養(yǎng)觀察以備移植。(2)動(dòng)物分組與建模:選用健康家兔35只,雌雄不限,體重2.0~2.5Kg,隨機(jī)分為正常組(normal)5只、模型組(model)10只、對(duì)照組(control)10只、移植組(transplant)10只;正常組不作任何處理,建模移植前完整取下腓腸肌,稱量肌濕重,于肌腹中部切取肌組織塊用于后續(xù)實(shí)驗(yàn),其余三組均進(jìn)一步分為4周和8周兩個(gè)亞組,無(wú)菌條件下在左后肢乆窩處顯露、切斷脛神經(jīng)并將兩斷端縫合固定于深筋膜。(3)肌衛(wèi)星細(xì)胞的標(biāo)記與移植:移植組左側(cè)腓腸肌用CM-Di L標(biāo)記的肌衛(wèi)星細(xì)胞0.5m L肌內(nèi)注射,對(duì)照組相同部位注射等體積細(xì)胞培養(yǎng)基。(4)腓腸肌的形態(tài)觀察與肌濕重測(cè)量:模型組、對(duì)照組、移植組各組分別于移植后第4、8周完整取下雙側(cè)腓腸肌稱量肌濕重,于肌腹中部切取肌組織塊用于后續(xù)實(shí)驗(yàn)。(5)石蠟切片HE染色測(cè)量腓腸肌的肌纖維橫截面積。(6)冰凍切片Kamovsky-roots法染色檢測(cè)腓腸肌運(yùn)動(dòng)終板。(7)Western blot檢測(cè)Bcl-2和Bax的蛋白表達(dá)。(8)統(tǒng)計(jì)學(xué)t檢驗(yàn)和單因素方差分析數(shù)據(jù)。結(jié)果:(1)通過(guò)混合酶消化法和兩次差速貼壁法分離提取得到圓形透亮的的原代肌衛(wèi)星細(xì)胞,繼續(xù)培養(yǎng)觀察到長(zhǎng)梭形貼壁細(xì)胞,最后觀察到細(xì)胞逐漸融合成多核細(xì)胞。(2)模型組與正常組比較,4周、8周后,模型組的腓腸肌肌濕重明顯減輕,肌纖維橫截面積明顯減小,P0.05,差異有統(tǒng)計(jì)學(xué)意義,但在4周時(shí),兩組的運(yùn)動(dòng)終板形態(tài)變化不明顯,均呈褐色分布,輪廓清晰可見,至8周時(shí),正常組的運(yùn)動(dòng)終板仍出現(xiàn)褐色分布,而模型組運(yùn)動(dòng)終板形態(tài)已經(jīng)基本消失,細(xì)胞溶解現(xiàn)象明顯。(3)對(duì)照組與模型組比較,兩組在4周、8周時(shí),肌濕重、肌纖維橫截面積以及Bcl-2和Bax的蛋白表達(dá)均未出現(xiàn)明顯差異(P0.05),同時(shí)兩組的運(yùn)動(dòng)終板的形態(tài)變化相同,4周時(shí),均呈褐色,邊緣清晰,8周時(shí),運(yùn)動(dòng)終板消失,細(xì)胞溶解現(xiàn)象明顯。(4)移植組與模型組比較,4周、8周后,移植組的肌濕重和肌纖維橫截面積下降較小,Bcl-2蛋白表達(dá)量升高和Bax表達(dá)量降低(P0.05);運(yùn)動(dòng)終板在4周時(shí),變化不明顯,均觀察到褐色分布,8周時(shí),模型組運(yùn)動(dòng)終板消失,移植組仍可觀察到運(yùn)動(dòng)終板,但邊緣已經(jīng)模糊。(5)4周、8周時(shí)熒光顯微鏡下可以清晰觀察到移植組有標(biāo)記的肌衛(wèi)星細(xì)胞,但隨著時(shí)間延長(zhǎng),熒光強(qiáng)度減弱,熒光數(shù)量減少。結(jié)論:(1)通過(guò)切斷家兔的脛神經(jīng)可以成功建立失神經(jīng)骨骼肌萎縮模型。(2)骨骼肌失神經(jīng)支配后,肌濕重明顯下降,肌纖維橫截面積明顯減小,運(yùn)動(dòng)終板數(shù)減少和結(jié)構(gòu)破壞。(3)肌衛(wèi)星細(xì)胞移植可延緩失神經(jīng)骨骼肌萎縮,并伴隨有受體肌的Bcl-2表達(dá)上調(diào)和Bax表達(dá)下調(diào)。
[Abstract]:Aim: to establish rabbit model of denervated gastrocnemius muscle atrophy, isolate and transplant muscle satellite cells, and observe the effect of delaying skeletal muscle atrophy after transplantation. Methods the isolated and cultured rabbit skeletal muscle satellite cells were isolated and cultured. The skeletal muscle of the limbs was removed and digested by collagenase and trypsin mixture, and then the primary muscle satellite cells were obtained by two differential adhesions. The animals were divided into normal group (n = 5), model group (n = 10), control group (n = 10), transplant group (n = 10), normal control group (n = 10) and control group (n = 10). The whole gastrocnemius muscle was removed before transplantation, the wet weight of the muscle was weighed, and the muscle tissue mass was removed from the middle of the muscle abdomen for further experiment. The other three groups were further divided into two subgroups: 4 weeks and 8 weeks, and exposed in the left hind limb under the condition of sterility. Labeling and transplantation of tibial nerve transection and suture of two ends to deep fascia. The left gastrocnemius muscle was injected intramuscularly with 0.5 mL of CM-Di L-labeled myosatellite cells in the left gastrocnemius transplantation group. Observation of the morphology and wet weight of gastrocnemius muscle in the control group: the wet weight of bilateral gastrocnemius muscle was completely removed from the model group, the control group and the transplantation group at the 4th week after transplantation. Measurement of muscle fiber cross section area of gastrocnemius muscle by HE staining in paraffin sections of gastrocnemius muscle. (6) Kamovsky-roots staining of gastrocnemius muscle motor end plate by Kamovsky-roots method. Detection of protein expression of Bcl-2 and Bax by Western blot in gastrocnemius muscle T test and ANOVA data. Results the circular and transparent primary myosatellite cells were isolated by mixed enzyme digestion and two differential adherence methods. The long fusiform adherent cells were further cultured and observed. Finally, it was observed that the wet weight of gastrocnemius muscle in the model group decreased significantly, and the cross sectional area of muscle fiber decreased significantly (P 0.05) in the model group compared with the control group after 4 weeks and 8 weeks, the difference was statistically significant, but at 4 weeks after the model group was compared with the normal group, the wet weight of gastrocnemius muscle was significantly reduced, and the cross-sectional area of the muscle fibers decreased significantly (P 0.05). The shape of the motor endplate in the two groups was not obvious, and the shape of the motor endplate was brown distribution, and the contour was clearly visible. At 8 weeks, the motor endplate of the normal group still appeared brown distribution, but the shape of the motor endplate in the model group had basically disappeared. Compared with the model group, the muscle wet weight of the two groups was found at 4 weeks and 8 weeks. The cross sectional area of muscle fiber and the protein expression of Bcl-2 and Bax were not significantly different between the two groups. At the same time, the morphological changes of the motor endplate in the two groups were brown at 4 weeks, and disappeared at 8 weeks with clear edges. After 4 weeks and 8 weeks compared with the model group, the muscle wet weight and muscle fiber cross-sectional area of the transplantation group decreased slightly, the expression of Bcl-2 protein increased and the expression of Bax decreased P0.05a, but the motor endplate did not change significantly at 4 weeks. After 8 weeks of brown distribution, the motor endplates disappeared in the model group, and the motor endplates were still observed in the transplantation group, but marked myosatellite cells were clearly observed in the transplanted group under fluorescence microscope after 4 weeks and 8 weeks. However, with the prolongation of time, the fluorescence intensity decreased and the amount of fluorescence decreased. Conclusion 1) the denervated skeletal muscle atrophy model can be successfully established by transection of the tibial nerve of the rabbit.) after denervated skeletal muscle, the wet weight of the muscle and the cross sectional area of the muscle fiber are obviously decreased. Reduced number of motor endplates and structural damage. 3) muscle satellite cell transplantation could delay denervated skeletal muscle atrophy, accompanied by up-regulation of Bcl-2 expression and down-regulation of Bax expression in recipient muscle.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R746.4

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