基于衰老相关基因探讨补肾化瘀生新方延缓骨髓间充质干细胞衰老的机制

发布时间：2018-10-29 10:42

【摘要】：目的:基于衰老相关基因p16~(INK4a)、p53、p21,探讨补肾化瘀生新方延缓骨髓间充质干细胞(Bone mesenchymal stem cells,BMSCs)衰老的机制;方法:取60只SPF级SD大鼠,采用随机数字表法分为补肾化瘀生新方组和生理盐水组,用于含药血清的制备;SPF级SD大鼠,3周龄,进行原代BMSCs的提取,取状态良好的第3代BMSCs,用6g/L D-gal诱导建立BMSCs衰老模型;分组:正常组随机分为空白组、空白中药组、空白生理盐水组,衰老组随机分为模型组、模型中药组、模型生理盐水组。观察各组各代BMSCs的形态学变化;MTT法确定促进第3代BMSCs分化增殖的最佳含药血清的浓度;实时荧光定量PCR技术检测不同代次各组BMSCs p16~(INK4a)、p53、p21 mRNA的表达。结果:(1)镜下见年轻的BMSCs形态呈长梭形或纺锤形,漩涡样分布,生长迅速,折光性好;衰老的BMSCs多呈扁三角形、椭圆形,胞体扩散,生长缓慢,折光性差;(2)根据MTT法检测结果可知,促进BMSCs增殖的最佳浓度为10%;(3)实时荧光定量PCR结果显示:第3代BMSCs,空白组、模型组、模型中药组、模型生理盐水组之间相互比较,p16~(INK4a)、p53、p21 mRNA表达量均无统计学意义(P0.05);第6代BMSCs,与空白组相比,模型组、模型生理盐水组、空白生理盐水组的三个衰老关键基因p16~(INK4a)、p53、p21 mRNA的表达差异具统计学意义(P0.05),空白中药组、模型中药组的表达差异无显著性(P0.05);与模型组相比,模型中药组的表达差异具有显著性统计学意义(P0.01),模型生理盐水组的表达无统计学意义(P0.05);且模型中药组与模型生理盐水组、空白中药组与空白生理盐水组相比,p16~(INK4a)、p53、p21 mRNA的表达差异均具有统计学意义(P0.05)。第7代BMSCs,与空白组相比,空白中药组p16~(INK4a)、p53、p21 mRNA的表达差异无统计学意义(P0.05),余四个组的表达差异均具统计学意义(P0.05);与模型组相比,模型中药组的三个基因的表达具有统计学意义(P0.05),模型生理盐水组的表达无统计学意义(P0.05);模型中药组与模型生理盐水组、空白中药组与空白生理盐水组相比,差异均具有统计学意义(P0.05)。第9代BMSCs,与空白组相比,空白中药组p16~(INK4a)、p53、p21 mRNA的表达量差异无统计学意义(P0.05),余四个组的表达量差异均具有显著性(P0.01);与模型组相比,模型中药组p16~(INK4a) mRNA的表达量无统计学意义(P0.05),p53、p21 mRNA的表达量差异有统计学意义(P0.05),而模型生理盐水组三个基因mRNA的表达量差异均无统计学意义(P0.05);与空白中药组相比,空白生理盐水组三个基因mRNA的表达均具有统计学意义(P0.05);与模型中药组相比,模型生理盐水组p16~(INK4a) mRNA的表达不具有统计学意义(P0.05),模型生理盐水组p53、p21 mRNA的表达量差异均具有统计学意义(P0.05)。结论:(1)在自然衰老及成功诱导衰老模型的条件下,补肾化瘀生新方可延缓BMSCs的衰老,但对年轻的BMSCs作用不明显;(2)补肾化瘀生新方可通过下调衰老关键基因p16~(INK4a)、p53、p21的表达来延缓BMSCs的衰老,但随着衰老时间的延长,其作用力逐渐减弱,并且到一定程度,这种衰老具有不可逆性;(3)补肾化瘀生新方调控BMSCs的衰老与其抑制p16/pRb和p53/p21途径密切相关。
[Abstract]:Objective: To investigate the mechanism of delaying aging of bone marrow mesenchymal stem cells (BMSCs) based on senescence-related genes p16 ~ (INK4a), p53 and p21. Methods: 60 SPF SD rats were divided into two groups: Bushen Huayu Xinfang group and normal saline group. The method comprises the following steps of: preparing a drug-containing serum; SPF-grade SD rats; 3-week age; carrying out extraction of primary BMSCs; taking the third generation BMSCs with good condition; inducing the establishment of BMSCs aging model by using 6g/ L D-retinoic acid; grouping: the normal group is randomly divided into a blank group, a blank Chinese medicine group and a blank physiological saline group; The aging group was randomly divided into model group, model Chinese medicine group and model physiological saline group. Morphological changes of BMSCs were observed in each group; MTT method was used to determine the concentration of the best drug-containing serum for the differentiation and proliferation of the third generation BMSCs; and the expression of p16 ~ (INK4a), p53 and p21 mRNA in different generations of BMSCs was detected by real-time fluorescence quantitative polymerase chain reaction (PCR). Results: (1) The morphology of the young BMSCs under the scope of the mirror was in the form of a long shuttle shape or a crescent shape, the spiral pattern was distributed, the growth was rapid, the refractive index was good; the aging BMSCs were flat triangles, oval, cell diffusion, slow growth and poor refractive index; (2) According to the MTT method, it can be seen that, The optimal concentration of BMSCs was 10%; (3) Real-time fluorescence quantitative PCR showed that the expression of p16 ~ (INK4a), p53 and p21 mRNA was not statistically significant (P0.05). Compared with the blank group, the expression of p16 ~ (INK4a), p53 and p21 mRNA in the model group, normal saline group and blank saline group was statistically significant (P0.05). There was no significant difference in the expression of the model Chinese medicine group (P0.01), the expression of the model physiological saline group was not statistically significant (P0.05), and compared with the blank normal saline group, p16 ~ (INK4a) and p53 were found in the model Chinese medicine group. The expression of p21 mRNA was statistically significant (P0.05). Compared with the blank group, the expression of p16 ~ (INK4a), p53 and p21 mRNA in the blank Chinese medicine group was not statistically significant (P0.05). There was no significant difference in the expression of normal saline group (P0.05). There was no significant difference in the expression of p16 ~ (INK4a), p53 and p21 mRNA in the blank Chinese medicine group compared with the blank group (P <0.05). The expression of p16 ~ (INK4a) mRNA in the model Chinese medicine group was not statistically significant (P0.05). There was no significant difference in the expression of p21 mRNA in normal saline group (P0.05). Compared with the traditional Chinese medicine group, the expression of p16 ~ (INK4a) mRNA in normal saline group was not statistically significant (P0.05). Conclusion: (1) Under the condition of natural aging and successful induction of aging model, the aging of BMSCs can be delayed by tonifying kidney and blood stasis, but it is not obvious to young BMSCs; (2) BMSCs can be delayed by lowering the expression of p16 ~ (INK4a), p53 and p21. However, with the prolongation of aging time, its acting force is gradually weakened, and to some extent, this kind of aging has irreversibility; (3) The aging of BMSCs is closely related to the inhibition of p16/ pRb and p53/ p21.
【学位授予单位】：河南中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R285.5

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