家兔肌卫星细胞移植治疗失神经骨骼肌萎缩的实验研究

发布时间：2018-05-19 22:43

本文选题：失神经肌萎缩 + 肌卫星细胞　；参考：《遵义医学院》2017年硕士论文

【摘要】：目的:建立家兔腓肠肌失神经肌萎缩模型,分离提取肌卫星细胞并移植,观察移植后延缓骨骼肌萎缩的效果。方法:(1)家兔骨骼肌卫星细胞分离提取与培养:选取出生后1周内的乳兔,取下四肢骨骼肌,经胶原酶和胰酶混合液消化,再经两次差速贴壁得到原代肌卫星细胞,继续培养观察以备移植。(2)动物分组与建模:选用健康家兔35只,雌雄不限,体重2.0~2.5Kg,随机分为正常组(normal)5只、模型组(model)10只、对照组(control)10只、移植组(transplant)10只;正常组不作任何处理,建模移植前完整取下腓肠肌,称量肌湿重,于肌腹中部切取肌组织块用于后续实验,其余三组均进一步分为4周和8周两个亚组,无菌条件下在左后肢乆窝处显露、切断胫神经并将两断端缝合固定于深筋膜。(3)肌卫星细胞的标记与移植:移植组左侧腓肠肌用CM-Di L标记的肌卫星细胞0.5m L肌内注射,对照组相同部位注射等体积细胞培养基。(4)腓肠肌的形态观察与肌湿重测量:模型组、对照组、移植组各组分别于移植后第4、8周完整取下双侧腓肠肌称量肌湿重,于肌腹中部切取肌组织块用于后续实验。(5)石蜡切片HE染色测量腓肠肌的肌纤维横截面积。(6)冰冻切片Kamovsky-roots法染色检测腓肠肌运动终板。(7)Western blot检测Bcl-2和Bax的蛋白表达。(8)统计学t检验和单因素方差分析数据。结果:(1)通过混合酶消化法和两次差速贴壁法分离提取得到圆形透亮的的原代肌卫星细胞,继续培养观察到长梭形贴壁细胞,最后观察到细胞逐渐融合成多核细胞。(2)模型组与正常组比较,4周、8周后,模型组的腓肠肌肌湿重明显减轻,肌纤维横截面积明显减小,P0.05,差异有统计学意义,但在4周时,两组的运动终板形态变化不明显,均呈褐色分布,轮廓清晰可见,至8周时,正常组的运动终板仍出现褐色分布,而模型组运动终板形态已经基本消失,细胞溶解现象明显。(3)对照组与模型组比较,两组在4周、8周时,肌湿重、肌纤维横截面积以及Bcl-2和Bax的蛋白表达均未出现明显差异(P0.05),同时两组的运动终板的形态变化相同,4周时,均呈褐色,边缘清晰,8周时,运动终板消失,细胞溶解现象明显。(4)移植组与模型组比较,4周、8周后,移植组的肌湿重和肌纤维横截面积下降较小,Bcl-2蛋白表达量升高和Bax表达量降低(P0.05);运动终板在4周时,变化不明显,均观察到褐色分布,8周时,模型组运动终板消失,移植组仍可观察到运动终板,但边缘已经模糊。(5)4周、8周时荧光显微镜下可以清晰观察到移植组有标记的肌卫星细胞,但随着时间延长,荧光强度减弱,荧光数量减少。结论:(1)通过切断家兔的胫神经可以成功建立失神经骨骼肌萎缩模型。(2)骨骼肌失神经支配后,肌湿重明显下降,肌纤维横截面积明显减小,运动终板数减少和结构破坏。(3)肌卫星细胞移植可延缓失神经骨骼肌萎缩,并伴随有受体肌的Bcl-2表达上调和Bax表达下调。
[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.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R746.4

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