补肾化瘀生新方延缓骨髓间充质干细胞衰老的作用及其抗衰老机制研究

With the deepening of researches into the stem cell, people realized that the senescence of organism is actually the senescence of stem cells. All the aging phenomena reflect the different aging levels of organism stem cells. BMSCs distribute in different tissues and organs, thus play an important role in maintaining the physiological stability and reproduction and recovery of tissue organs of the organism. Its senescence will decrease the tissue stability and renewing ability and hasten the aging process of organism. Therefore, studying the senescence process of BMSCs has great significance to researches of new methods of antisenility. On the basis of previous work, the author uses the formula of experienced and famous TCM doctors as intervention means and the models of cell senescence and rat senescence, RT-PCR, Immunohistochemically, explored the influence of Formula of reinforcing kidney and resolving stasis to reproduce new cells to telomerase activity, Wnt/β-catenin signal path and beta - galactosidase activity. The author also discovers the formula's protective effects to organs of rat models, antisenility effects of vein transplantation of BMSCs, revealing the antisenility effects of the formula in delaying the senescence of BMSCs and it's antisenility mechanism. He elaborated the scientificity of the theory of kidney deficiency and blood stasis delaying the reproduction and eventually promoted the TCM researches in the field of antisenility.

随着干细胞研究的深入，人们认识到生物体的衰老实际上是干细胞的衰老，所有衰老现象都反映出机体干细胞衰老水平的变化。骨髓间充质干细胞(BMSCs) 分布于多种组织和器官，在维持机体生理性的稳定和组织器官的再生修复中发挥重要作用，其衰老将降低组织的稳态性和去旧更新能力,加快机体的衰老进程，因此，明确BMSCs衰老机制对探索新的抗衰老方法有意义。 在前期工作基础上，以名老中医经验方-补肾化瘀生新方为干预手段，利用细胞衰老和大鼠衰老模型、RT-PCR、免疫组化等方法，探究补肾化瘀生新方对不同代次BMSCs的端拉酶活性、Wnt/β-catenin信号通路和β-半乳糖苷酶活性等影响，及其对模型大鼠各脏器中BMSCs衰老的保护作用、静脉移植BMSCs对模型大鼠的抗衰老作用，揭示了补肾化瘀生新方延缓BMSCs衰老的作用及其抗衰老机制，阐释"肾虚血瘀，生新乏力"致衰理论的科学性，推动中医药抗衰防衰研究深入。

目的：探讨补肾化瘀生新法延缓骨髓间充质干细胞衰老作用及其抗衰老机制。.方法：.体外研究：提取鉴定BMSCs，并纯化和培养；制备补肾化瘀生新方和生理盐水含药血清；6g/L D-gal诱导建立BMSCs衰老模型。自然衰老细胞随机分为正常组、治疗组、对照组:加入DMEM液、补肾化瘀生新和生理盐水含药血清。诱导衰老细胞随机分为正常组、模型组、治疗组、对照组:加入正常培养液、DMEM液、补肾化瘀生新和生理盐水含药血清。光镜观察各代细胞形态；MTT法检测细胞活性、绘制生长曲线；比色法检测β-半乳糖苷酶活性。流式细胞术检测增殖率和细胞周期；实时荧光PCR技术检测端粒酶活性、p16INK4a、p53、p21mRNA表达；Western blotting检测β-catenin、p53、β-actin蛋白表达。.在体研究：采用125mg•kg-1•d-1的D-半乳糖溶液皮下注射，1次/日，连续45d，建立大鼠衰老模型。随机分为补肾化瘀生新方高、中、低剂量组（按2.728g/ml、1.364g/ml、0.682g/ml灌胃），模型组生理盐水灌胃，正常组不给药，2次/日，连续45d。另补肾化瘀生新方协同静脉移植研究的造模、取材和灌胃方法同上，并尾静脉注射3.0×106／ml的BMSCs，1次/周，连续四次。流式细胞仪检测外周血和骨髓中CD105、CD117变化；实时荧光PCR和免疫荧光染色法测定脑、肝、肺、脾、肾、心组织中CD105、P53、P21、p16INK4amRNA和蛋白表达。.结果： 1、治疗组与模型组相比各代次β-半乳糖苷酶活性、倍增时间均降低；2、治疗组与模型组相比各代次细胞增殖率、端粒酶活性、G0/G1期和S期的比例均升高。3、治疗组与模型组相比各代次P53、P21、p16INK4a-mRNA和蛋白表达均升高，P＜0.05。4、高、中剂量组与模型组相比，外周血和骨髓中CD105、CD117明显增多；5、各剂量组与模型组比均能降低脑、肝、肺、脾、肾、心组织中P53、P21、p16INK4amRNA表达，P＜0.05。.结论：补肾化瘀生新方能够延缓BMSCs衰老，其机制可能与降低β-半乳糖苷酶活性、激活端粒酶活性、降低p16INK4a/Rb和P53/P21途径上的基因表达及下调Wnt/β-catenin信号通路有关，揭示“肾虚血瘀，生新乏力”致衰理论科学内涵。