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  

Effects of gravitational traction and targeted regulation of caspase3 on the degenerative intervertebral disc cells in rabbits

作　　者：佟德民[1] 孙凤杰[1] 孙利群[1] 冯福盈 梁健宁 谭滴清[2] 

机构地区：[1]广州医科大学附属第五医院中医科,广州510700 [2]广州中医药大学动物实验中心,广州510405

出　　处：《解放军医学杂志》2017年第12期1072-1077,共6页Medical Journal of Chinese People's Liberation Army

基　　金：广州医科大学博士启动项目(2014C41)~~

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(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。Objective To explore the effects of gravitational traction and target regulation of caspase3 on the degenerative intervertebral disc cells in rabbits. Methods Rabbits nucleus pulposus cells transfected with caspase3 siRNA or negative control siRNA were incubated in serum-starved medium for 6 hours and 48 hours. The expression of caspase3 siRNA and cell apoptosis were analyzed by RT-qPCR and Annexin V-fluorescein staining. In order to create intervertebral disc degeneration model, the right anterior side of the annulus fibrosus of lumbar vertebrae of 35 rabbits were damaged by 16-gauge needle. After confirming the success of modeling, 35 animal models were randomized into 3 groups： model group （n--10）, negative siRNA group （n=10） and caspase3 siRNA group （n=5）. Either negative control siRNA or caspase3 siRNA was injected into the center of nucleus pulposus using a 26pgauge needle from the left anterior side, while the model group received no injection. Nucleus pulposus tissue of 5 rabbits selected randomly from every group after 48 hours were analyzed by PCR and Western blotting for caspase3 mRNA and protein expressions. Half of the casepase3 siRNA group were selected randomly and received a routine gravitational traction using a model of our own design, 30min per day for 2 weeks, while other groups received no treatment. TNF-α and IL-1β expression levels and histopathological observations were performed after intervention. Results Expression of caspase3 mRNA in rabbit nucleus pulposus cells transfected with caspase3 siRNA decreased obviously in serum-starved medium, and the apoptosis rate of cells cultured in serum-starved medium decreased significantly （P〈0.05）. Caspase3 mRNA and caspase3 protein expression of nucleus pulposus injected by caspase3 siRNA down-regulated in the caspase3 siRNA group. Compared with model group and negative control siRNA group, TNF-~x and IL-113 expression levels of caspase3 siRNA traction group decreased significantly （P〈0.05）, but there was no statistical

关 键 词：椎间盘退变 细胞凋亡 牵引 

分 类 号：R681.5[医药卫生—骨科学]