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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
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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
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Journal Article
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
2017
Overview
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（P〈0.05）。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（P〈0.05）,但与caspase3 siRNA组比较差异无统计学意义（P〉0.05）。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。
