自体垂直悬吊牵引与靶向调控caspase3对兔退变椎间盘细胞的影响

发布时间：2018-01-21 01:56

本文关键词： 椎间盘退变细胞凋亡牵引　出处：《解放军医学杂志》2017年12期 　论文类型：期刊论文

【摘要】：目的探讨自体垂直悬吊牵引与靶向调控caspase 3对兔退变椎间盘细胞凋亡的影响。方法体外实验:分离培养兔椎间盘髓核细胞,进行caspase3 siRNA和阴性对照siRNA转染,分别置于血清饥饿培养基中培养6h和48h,设正常细胞和血清饥饿细胞对照,应用RT-q PCR法和流式细胞术Annexin V法分析caspase3 mRNA表达和细胞凋亡情况。体内实验:将35只新西兰大白兔随机分为模型组(n=10)、阴性siRNA对照组(n=10)、caspase3 siRNA组(n=10)和caspase3siRNA牵引组(n=5),采用16G针刺损伤腰椎椎体右前侧纤维环制备椎间盘退变模型;后3组按照造模入路用26G针头从造模对侧分别注入阴性对照siRNA或caspase3 siRNA转染复合体至兔髓核中心,模型组不予处理。48h后前3组每组随机选取5只实验兔,处死取髓核组织,提取总RNA,采用RT-q PCR和Western blotting分析退变椎间盘内caspase3 mRNA和蛋白表达情况;同时caspase3 siRNA牵引组再行自体垂直悬吊牵引,30min/d,连续2周后处死获取髓核组织,采用ELISA检测髓核内TNF-α和IL-1β表达情况,HE染色观察组织形态学变化。结果 Caspase 3 siRNA转染后兔髓核细胞caspase3 mRNA表达水平明显降低,在饥饿培养条件下细胞凋亡明显减少(P0.05)。椎间盘退变模型兔在纤维环穿刺注入caspase3 siRNA后,椎间盘髓核内caspase3 mRNA和蛋白表达明显下调;2周后caspase3 siRNA牵引组TNF-α和IL-1β表达与模型组和阴性siRNA对照组比较显著减少(P0.05),但与caspase3 siRNA组比较差异无统计学意义(P0.05)。HE染色显示caspase3 siRNA牵引组椎间盘内活细胞和细胞外基质明显增多,胶原纤维排列整齐。结论自体垂直悬吊牵引和靶向调控caspase3能有效阻止细胞凋亡的发生,延缓早期椎间盘退变。
[Abstract]:Objective to investigate the effect of autologous vertical suspension traction and targeted regulation of caspase _ 3 on apoptosis of rabbit degenerative disc cells. Caspase3 siRNA and negative control siRNA were transfected and cultured in serum starvation medium for 6 h and 48 h, respectively. Normal cells and serum hungry cells were used as control. The expression and apoptosis of caspase3 mRNA were analyzed by RT-q PCR assay and flow cytometry Annexin V method. 35 New Zealand white rabbits were randomly divided into model group (. 10). The control group with negative siRNA, caspase3 siRNA group (10%) and the caspase3siRNA traction group (5%). The model of disc degeneration was established by 16G acupuncture on the right anterior fibrous ring of lumbar vertebrae. In the latter three groups, negative control siRNA or caspase3 siRNA were injected into the nucleus pulposus center of rabbits with 26G needle from the contralateral side of the model respectively. After the model group was not treated for 48 hours, 5 rabbits in each group were randomly selected, and the tissues of nucleus pulposus were killed and the total RNA was extracted. The expression of caspase3 mRNA and protein in degenerative disc was analyzed by RT-q PCR and Western blotting. At the same time, the caspase3 siRNA traction group was treated with autologous vertical suspension traction for 30 min / d, and then killed for 2 weeks to obtain the nucleus pulposus tissue. The expression of TNF- 伪 and IL-1 尾 in nucleus pulposus was detected by ELISA. Results the expression of caspase3 mRNA in rabbit nucleus pulposus cells was significantly decreased after transfection of Caspase 3 siRNA. Apoptosis was significantly decreased in starved cultured rabbits after caspase3 siRNA was injected into the disc degeneration model. The expression of caspase3 mRNA and protein in nucleus pulposus of intervertebral disc was down-regulated. After 2 weeks, the expression of TNF- 伪 and IL-1 尾 in caspase3 siRNA traction group was significantly lower than that in model group and negative siRNA control group (P 0.05). But there was no significant difference between caspase3 siRNA group and caspase3 siRNA group (P0.05). HE staining showed that the number of living cells and extracellular matrix in intervertebral disc in caspase3 siRNA traction group was significantly increased. Conclusion Autologous vertical suspension traction and targeted regulation of caspase3 can effectively prevent apoptosis and delay early degeneration of intervertebral disc.
【作者单位】：广州医科大学附属第五医院中医科;广州中医药大学动物实验中心;
【基金】：广州医科大学博士启动项目(2014C41)~~
【分类号】：R681.53
【正文快照】： 椎间盘退变性疾病包括腰椎椎管狭窄、椎间盘突出症等,是骨科的常见病,也是导致肢体功能障碍的主要原因[1]。椎间盘退变的病理机制尚未完全阐明,但椎间盘内部异常的生物力学环境、椎间盘细胞凋亡及其相互作用可能是诱发和加速椎间盘退变从而出现腰腿痛的重要因素,因此早期改善

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