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目的探讨六味地黄丸对兔退变椎间盘髓核及纤维环细胞的保护作用。方法选择8月龄成年新西兰兔6只利用异常应力负荷及椎间失稳法建立新西兰兔腰椎椎间盘退变模型,病理切片确认模型成功后,取椎间盘分离软骨终板,将髓核及纤维环组织进行细胞培养,将生长良好的细胞随机分为对照组、损伤组与含药组。对照组细胞培养用胎牛血清体积分数为0.01的DMEM培养基;损伤组给予体积分数为0.01的胎牛血清与浓度为5μg/L的TNF-a混合的DMEM培养基;含药组为体积分数为0.01的六味地黄丸含药血清加体积分数为0.01的胎牛血清与浓度为5μg/L的TNF-a混合的DMEM培养基。使用细胞增殖—毒性检测试剂盒(CCK-8)检测各组兔退变椎间盘细胞增殖的影响。实时荧光定量PCR检测聚集蛋白聚糖、Ⅱ型胶原、基质金属蛋白酶-13基因的表达差别。结果对照组椎间盘细胞初期正常生长,增殖良好,随着时间延长,细胞数量减少,凋亡细胞数量增多,细胞外基质胶原减少,细胞形态变化快。损伤组能够明显抑制椎间盘软细胞的增殖,细胞及细胞外基质胶原数量减少明显,同时间点比较,细胞凋亡数量增多,与正常组比较差异有统计学意义(P<0.05),而含药组细胞增殖缓慢,细胞数量下降缓慢,细胞凋亡数量减少,细胞外基质胶原成分下降不明显,3组之间比较差异有统计学意义(P<0.05)。与对照组比较,损伤组椎间盘细胞蛋白聚糖、Ⅱ型胶原mRNA表达下调增加,基质金属蛋白酶-13基因表达上调明显。与损伤组比较,而六味地黄丸含药血清组椎间盘细胞蛋白聚糖、Ⅱ型胶原mRNA表达上调,基质金属蛋白酶-13基因表达下调,3组比较差异有统计学意义(P<0.05)。结论六味地黄丸含药血清能够抑制TNF-a诱导的兔退变椎间盘细胞损伤,起到延缓椎间盘损伤的作用。其机制可能与上调蛋白聚糖及Ⅱ型胶原表达及下调基质金属蛋白酶表达有关。
Objective To investigate the protective effect of Liuweidihuangwan on degenerative disc nucleus pulposus and fibrocystic ring cells in rabbits. Methods Six New Zealand white rabbits (8 months old) were selected to establish a New Zealand rabbit model of lumbar disc degeneration by means of abnormal stress load and intervertebral instability method. After the model was confirmed by pathological section, the cartilage endplate was separated from the disc and the nucleus pulposus and annulus fibrosus tissue The cells were cultured and the well-growing cells were randomly divided into control group, injury group and drug-containing group. The control group was cultured in DMEM with a volume fraction of fetal bovine serum of 0.01. The injured group was given DMEM medium containing fetal bovine serum with a volume fraction of 0.01 and TNF-a mixed with a concentration of 5 μg / L. The drug- 0.01 of Liu Wei Di Huang Wan containing serum plus volume fraction of 0.01 fetal bovine serum and concentration of 5μg / L of TNF-a mixed DMEM medium. The proliferation of degenerated intervertebral disc cells in each group was detected by CCK-8. Real-time fluorescence quantitative PCR was used to detect the difference of aggrecan, type II collagen and matrix metalloproteinase-13 gene expression. Results In the control group, the cells of the intervertebral discs grew normally initially and proliferated well. As the time prolonged, the number of the cells decreased, the number of apoptotic cells increased, the extracellular matrix collagen decreased and the morphology of the cells changed rapidly. Compared with the normal group, the number of apoptotic cells in the injured group was significantly decreased (P <0.05), and the content of the collagen in the cells and the extracellular matrix was significantly decreased The proliferation of the cells was slow, the number of cells decreased slowly, the number of apoptotic cells decreased and the composition of extracellular matrix collagen did not decrease obviously. The difference between the three groups was statistically significant (P <0.05). Compared with the control group, the expression of proteoglycan and type Ⅱ collagen in the intervertebral disc cells in injury group was increased and the expression of MMP-13 was up-regulated. Compared with the injury group, the expression of proteoglycan and collagen Ⅱ mRNA in the intervertebral disc cells of the Liuwei Dihuang pill-containing serum group was up-regulated and the expression of the matrix metalloproteinase-13 gene was down-regulated in the three groups (P <0.05). Conclusion Liuweidihuangwan serum can inhibit TNF-a-induced degeneration of intervertebral disc cells in rabbits and play a role in delaying disc injury. The mechanism may be related to up-regulation of proteoglycan and type II collagen expression and down-regulation of matrix metalloproteinase.