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益气化瘀中药结合BMSCs移植干预腰椎间盘退变的实验研究
【作者】 权学民；
【导师】 徐林；
【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2011， 博士
【摘要】 研究背景下腰痛是极为常见的临床症状之一,80%以上的人群一生中都会发生,已成为影响人类正常生活和工作的首要问题。下腰痛的原因多种多样,但主要原因与腰椎间盘退变(lumbar intervetebral disc degeneration, LIDD)密切相关。椎间盘退变是以细胞凋亡和基质重建为基础的病理过程,其过程为非自限性,还可继发椎间盘突出、椎管狭窄、脊柱畸形等严重的退行性改变,椎间盘退变是一切病理改变的中心环节。椎间盘退变的治疗一直是医学上的难题,以往临床上普遍认为其进程是不可逆的。近年来,随着细胞分子生物学、组织工程学的研究进展,提出治疗椎间盘退变的新思路,即细胞移植治疗椎间盘退变。间充质干细胞(mesenchymal stem cells, MSCs)因来源丰富、易于扩增、多向分化、免疫逃避等诸多优点成为细胞移植的最佳选择。目前MSCs移植治疗椎间盘退变的效果在动物实验中得到肯定,但改善影响MSCs存活、增殖、分化的椎间盘内环境,如营养和生物因子等仍是研究的热点。中药作用机理的研究认为益气化瘀中药可以增加椎间盘营养供应,减少基质降解及细胞凋亡,因此益气化瘀中药结合MSCs移植的中西医结合方法可能提高椎间盘退变的治疗效果。研究目的通过BMSCs联合益气化瘀中药干预SD大鼠腰椎间盘退变模型,观察BMSCs联合益气化瘀中药对腰椎间盘退变的影响,为腰椎间盘退变的防治提供新思路和方法；通过益气化瘀中药含药血清干预大鼠BMSCs培养,观察益气化瘀中药对BMSCs增殖的影响,探讨益气化瘀中药结合BMSCs治疗腰椎间盘退变的可能机理。研究方法1.雄性SD大鼠16只,显露L3-4、L4-5、L5-6及L6-S1椎间盘,L4-5和L6-S1用23G针穿刺纤维环全层制备椎间盘退变模型,L3-4和L5-6作为对照,2、4、6和8周(n=8)通过HE染色,免疫组化,Alcian Blue染色观察退变情况。2.用全骨髓贴壁法分离、培养SD大鼠BMSCs,分为6组,分别加入10%含药血清、10%大鼠血清、5%FBS+5%含药血清、5%FBS+5%大鼠血清、10%FBS、无血清,培养24h、48h、72h,MTT法检测不同血清对体外培养BMSCs增殖的影响。3.雄性SD大鼠128只,造模手术当时L4-5间盘注入BMSCs+PBS, L5-6间盘注入PBS,L6-S1间盘空针穿刺作为对照组。再随机分为两组,A组大鼠每日灌服益气化瘀中药0.85g/kg,B组大鼠每日灌服蒸馏水1ml/100g,取2、4、6和8周时间点(n=8)取材,通过HE染色,免疫组化,Alcian Blue染色和DMB分光光度法观察干预效果。研究结果1.23G针刺纤维环全层可以有效建立椎间盘退变模型,8周后组织学评分、Ⅱ型胶原和蛋白多糖含量与正常组比较差异有显著性意义。2.大鼠血清和含药血清培养组OD值各时间点高于FBS培养组,而且随着大鼠血清和含药血清浓度增加OD值增高；不同浓度含药血清培养组OD值各时间点高于大鼠血清培养组,72h时间点差异有显著性意义。3.组织学检查及生化检测显示,与对照组比较单纯BMSCs移植组和益气化瘀中药结合BMSCs移植组干预椎间盘退变模型组织学评分低,Ⅱ型胶原和蛋白多糖含量明显增多,其中益气化瘀中药结合BMSCs移植组效果更好；与对照组比较单纯益气化瘀中药组干预椎间盘退变模型Ⅱ型胶原和蛋白多糖含量增多,但差异无统计学意义。结论23G针刺纤维环全层可诱导SD大鼠椎间盘缓慢退变,为研究椎间盘退变的机理及治疗提供有效的手段；大鼠血清和含药血清环境都有利于BMSCs存活、增殖,其中益气化瘀中药含药血清效果更佳；益气化瘀中药结合BMSCs移植或单纯BMSCs移植都可以有效延缓椎间盘退变,益气化瘀中药结合BMSCs移植效果优于单纯BMSCs移植。
【Abstract】 ObjectivesTo observation the therapeutic effect of Yiqi Huayu Recipe combining BMSCs on the lumbar intervertebral disc degeneration model of rats, which can provides new ideas and methods for the prevention and control of degenerative lumbar intervertebral disc degeneration; To observation the influence of Yiqi Huayu Recipe medicated serum on proliferation of BMSCs, and analyze the mechanism of Yiqi Huayu Recipe combining BMSCs on the lumbar intervertebral disc degeneration.Methods1.16 Sprague-Dawley male rats were carried out transperitoneal ventral surgery and anular stab was performed using 23-gauge needle to make a full-thickness stab incision in the L4-5 and L6-S1 anterior-lateral disc. The L3-4 and L5-6 level was used as a control. Execute a certain number of rats to collect specimens after 2 weeks,4 weeks,6 weeks, and 8 weeks separately(n=8). Finally, we analyze disc degeneration states by hematoxylin-eosin staining, Alcian blue staining and immunohisto-chemical studies.2. whole bone marrow culture method was used to isolate and purify Sprague-Dawley rats BMSCs that were incubated in L-DMEM containing 10% medicated serum,10% rat serum,5% FBS+5% medicated serum,5% FBS+5% rat serum, and 10% FBS and serum-free media. Morphology and proliferation of BMSCs were observed by MTT method at 24h,48h and 72h in vitro.3.128 Sprague-Dawley male rats, just at the time of establishing disc degeneration models, L4-5 level was injected with BMSCs-PBS complex, L5-6 level was injected with only PBS, L6-S1 level is a control, which just be treated with needling. The animals are randomly divided into two groups, group A ones were fed with Yiqi Huayu Recipe decoction by gastric perfusion 0.85 g/kg daily, and group B ones were fed with distilled water 1ml/100g. execute a certain number of rats to collect specimens after 2 weeks,4 weeks,6 weeks, and 8 weeks separately (n=8). Finally, we analyze disc degeneration states by hematoxylin-eosin staining, immunohisto-chemical studies, Alcian blue staining and DMB spectrophotometer method.Results1. Full-thickness anulus stab incision with 23-gauge needles defined gauges is effective to establish a rat model of intervertebral disc degeneration. After 8 weeks, histologic scores, collagen type II and proteoglycans analysis are significant difference.2. The OD values of the Rats serum and medicated serum training groups are higher than FBS training group at different time points, and OD values are increasing with rats serum and medicated serum concentrations increased; The OD values in different concentrations medicated serum training group higher than ratsserum training group at different time points, they have the remarkable significance difference at 72h point.3. Histological examination and biochemical tests showed that compare with the control group, pure BMSCs transplantation group and Yiqi Huayu Recipe decoction combined with BMSCs transplantation group have less histological score and more collagen typeⅡand proteoglycan content. Yiqi Huayu Recipe decoction with BMSCs transplantation group is better than the other. compared with the control group, pure Yiqi Huayu Recipe decoction group’s collagen typeⅡand proteoglycan content increased, but the difference was not statistically significant.Conclusions●Full-thickness anulus stab incision with 23-gauge needles approach results in a slowly progressive intervertebral disc degeneration in rat model. This model is available for studying the mechanism of intervertebral disc degeneration and treatment.●Rat serum group and medicated serum group are both suitable for BMSCs microenvironment to live and proliferation, medicated serum group is better than the other..●Both Yiqi Huayu Recipe decoction combined with BMSCs transplantation and pure BMSCs transplantation supporting its potential use as a treatment of intervertevral disc degeneration, Yiqi Huayu Recipe decoction with BMSCs transplantation group is better than the other.