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目的:探讨间充质干细胞(MSCs)延缓睾丸衰老的机制。方法:通过皮下注射D-半乳糖建立衰老大鼠模型的同时,大鼠尾静脉注射MSCs进行生物治疗;应用终浓度为20μmol/L荧光染料CFSE标记的MSCs回输大鼠体内,制作睾丸冰冻切片,在荧光显微镜下观察MSCs是否定位于睾丸;制作睾丸组织切片,观察睾丸组织微细结构变化并计数萎缩曲细精管的比例;采用硫代巴比妥酸和黄嘌呤氧化法分别检测血清和睾丸中丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性;应用Real-time PCR和Western blotting检测睾丸组织中P16的表达。结果:MSCs能够归巢到衰老大鼠的睾丸并存活;MSCs治疗组与模型组相比,睾丸曲细精管管壁增厚,各级生精细胞层增多,曲细精管内成熟精子增多,间质细胞胞浆浓稠,染色质清晰;治疗组萎缩的曲细精管比例高于模型组(P<0.05);血清与睾丸中SOD的活性均明显增高、MDA含量也显著降低(P<0.05);与模型组相比,MSCs治疗组能够使衰老大鼠睾丸内的P16 mRNA和P16蛋白表达降低(P<0.05)。结论:MSCs下调P16的表达,可能是其改善衰老睾丸功能的机制之一。
Objective: To investigate the mechanism of mesenchymal stem cells (MSCs) delaying the testicular aging. Methods: MSCs were injected into the caudal vein of rats by subcutaneous injection of D-galactose to establish the aging rat model. The MSCs were transfused into the body of rats with 20 μmol / L fluorescent dye CFSE and frozen in testes , Observed under a fluorescence microscope MSCs located in the testis; testis tissue sections were made to observe the testicular microstructure changes and count the number of atrophy seminiferous tubules; using thiobarbituric acid and xanthine oxidation were detected in serum and testis (MDA) content and superoxide dismutase (SOD) activity in the testis tissue were detected by Real-time PCR and Western blotting. Results: MSCs could homing to the testes of aged rats and survived. Compared with the model group, the MSCs treated group had thicker seminiferous tubules, more layers of germ cells and more mature spermatozoa in the seminiferous tubules, The interstitial cytoplasm was dense and the chromatin was clear. The proportion of seminiferous tubules in atrophic group was higher than that in model group (P <0.05). The activity of SOD in serum and testis were significantly increased and the content of MDA was also decreased significantly (P < 0.05). Compared with the model group, MSCs treatment group decreased the expression of P16 mRNA and P16 protein in the testis of aging rats (P <0.05). Conclusion: The down-regulation of P16 expression by MSCs may be one of the mechanisms of improving the testicular function of aging testes.