补肾活血方联合富血小板血浆对干细胞移植延缓椎间盘退变的影响及分子机制研究

Chronic low back pain could be caused by extremely complex etiological factors, which makes it difficult to be diagnosed and cured. Biochemical changes in intervertebral discs were considered to be the most important pathological basis. Stem cell transplantation might be an ideal method, while the survival and transformation rates of cells still remains to be improved. The applications of platelet-rich plasma opened up a door of “autologous biological treatment”. Based on the previous work, we conjecture that the combination of Kidney-tonifying and Blood Circulation-promoting Recipes and platelet-rich plasma could improve the survival and transformation rates of stem cells and delay intervertebral disc degeneration through Wnt/beta-catenin signaling pathway.. A rabbit model of disc degeneration and cells co-culture system were developed for the in vivo and in vitro experiments in the current research. By using multiple advanced medical technologies of cell culture, immunohistochemistry, histopathology and molecular biology, we evaluated the effect of Kidney-tonifying and Blood Circulation-promoting Recipes and platelet-rich plasma on stem cell transplantation. In addition, the Wnt pathway was observed to find out the molecular mechanism. The final results were expected to provide some scientific evidence for the research of TCM on the remediation of chronic degenerative bone and joint disease.

慢性下腰痛机制不明给临床诊治造成诸多困扰，椎间盘的“生物学改变”是引发腰椎退变的重要病理基础，干细胞移植有望解决这一问题，然而移植后的存活率和转化率有待提高。富血小板血浆的应用开启了全新的“自体生物学”治疗模式，并有望解决干细胞移植中的诸多难点。本课题组前期研究发现，椎间盘退变可能与Wnt/β-catenin 通路的异常激活有关，而补肾活血方与富血小板血浆联合应用，很可能调节该通路而影响干细胞的活性和分化、提高其在椎间盘内移植后的存活率和转化率，最终实现延缓椎间盘退变的作用，此类研究尚未见报道。.研究采用体外和体内两种模型，分别以人脂肪干细胞/髓核细胞共培养体系和兔腰椎间盘退变模型为对象，应用细胞培养、免疫化学、病理组织学及分子生物学检测技术，观察补肾活血方、富血小板血浆对干细胞移植治疗椎间盘退变的影响以及作用机制，从分子水平、细胞水平和整体动物水平探讨中医药防治椎间盘退变的机理。

目的：本研究旨在观察补肾活血方及富血小板血浆对人脂肪间充质干细胞移植治疗椎间盘退变的影响，并探索其发挥作用的分子信号机制，以期解决椎间盘内干细胞移植存活率、转化率低的难题。方法：1.分离培养原代脂肪干细胞和髓核细胞，共培养后检测细胞COL2A1、ACAN和SOX9基因表达。2.制备富血小板血浆及补肾活血方含药血浆，观察干预后干细胞活性及COL2A1、ACAN、SOX9、COL1A1、MMP3等基因表达水平，并检测Wnt信号通路相关基因、蛋白表达水平。3.采用椎间盘穿刺法建立椎间盘退变大鼠模型，将干细胞移植入大鼠退变椎间盘内，观察补肾活血方及PRP对细胞移植活性的影响及Wnt/β-catenin信号通路变化情况。结果：1.采用胶原酶消化法可较好的分离培养出人脂肪间充质干细胞和髓核细胞。2.PRP组及M-PRP组细胞增殖率、蛋白聚糖表达显著上调（P<0.05），Ⅰ型胶原则显著下调。基因检测显示，PRP组和M-PRP组COL2A1、ACAN mRNA表达显著上调，MMP3、COL1A1 mRNA显著下调；PRP组WNT3A、CTNNB1 mRNA表达显著上调；而M-PRP组CTNNB1 mRNA显著下调。蛋白检测显示，M-PRP组β-catenin蛋白表达量显著下调。3.采用椎间盘穿刺法可成功建立椎间盘退变大鼠模型，ADSCs移植入退变大鼠椎间盘后可显著上调Collagen Ⅱ、Aggrecan，下调MMP9，补肾活血方和PRP的联合应用可增强其效果，并且下调Wnt通路相关蛋白表达。结论：1. ADSCs与退变NPCs共培养后表现出向类髓核细胞分化的趋势。2.补肾活血方联合富血小板血浆组在促进ADSCs成软骨分化及抑制细胞退变方面的功效较单纯富血小板血浆组更强。3. 补肾活血方及PRP的应用可增强干细胞移植的疗效，这种作用的发挥可能与其对Wnt/β-catenin信号通路的抑制有关。