益气化瘀补肾方对退变椎间盘细胞蛋白聚糖和Ⅹ型胶原mRNA表达的影响

目的：观察益气化瘀补肾方血清对脊髓型颈椎病患者退变椎间盘细胞蛋白聚糖和Ⅹ型胶原mRNA表达的影响。方法：取6例确诊为脊髓型颈椎病的住院患者行颈椎减压融合手术摘除的退变椎间盘组织块,采用组织块法培养原代细胞;SD大鼠10只,灌胃制备大鼠益气化瘀补肾方含药血清。取传代第1代的退变椎间盘细胞,分为益气化瘀补肾方含药血清高浓度组（20%）、中浓度组（10%）、低浓度组（5%）及相应体积比浓度氯化钠溶液血清对照组。MTT法检测不同浓度的益气化瘀补肾方血清对椎间盘细胞增殖的影响,实时荧光定量聚合酶链反应检测不同给药时间点各组椎间盘细胞蛋白聚糖和Ⅹ型胶原mRNA的表达。结果：第1代退变椎间盘细胞传代第6天时细胞增殖达到顶峰,8d后开始下降,故选择细胞传代第6天为给药干预时间点。与对照血清比较,不同浓度的益气化瘀补肾方血清可促进退变椎间盘细胞的增殖（P〈0.01）,提高退变椎间盘细胞蛋白聚糖mRNA的表达（P〈0.01）,并下调Ⅹ型胶原mRNA的表达（P〈0.01）,且以中浓度的作用最为明显（P〈0.05）。结论：益气化瘀补肾方通过调节退变椎间盘的胶原及蛋白多糖的表达而产生有效防治脊髓型颈椎病椎间盘退变的作用。

Effects of Chinese herbal medicine Yiqi Huayu Bushen Recipe on expressions of aggrecan and type X collagen mRNAs in cells from degenerated human intervertebral discs

Objective：To investigate the effects of serum containing Yiqi Huayu Bushen Recipe,a compound Chinese herbal medicine,on expressions of aggrecan and type Ⅹ collagen mRNAs of the degenerated intervertebral disc cells in patients with cervical spondylotic myelopathy.Methods：Six inpatients diagnosed as cervical spondylotic myelopathy and undergoing cervical decompression and fusion surgery to remove the degenerated disc tissue were enrolled in this study.Primary intervertebral disc cells were isolated using small tissue mass method.Serum containing Yiqi Huayu Bushen recipe were obtained after SD rats were fed with Yiqi Huayu Bushen recipe for 3 days.MTT method was used to detect proliferation of the first-passage cells isolated from degenerated intervertebral discs and growth curve was drawn.In the following tests,the first-passage cells were cultured with the sera containing drugs and sodium chloride（NaCl）,respectively.Proliferation of intervertebral disc cells was detected by MTT method and expressions of aggrecan and type Ⅹ collagen mRNAs were detected by real-time polymerase chain reaction.Results：The growth curve of the first-passage cells of the degenerated intervertebral discs showed that the time of cell proliferation peak was the sixth day.Compared with the same concentrations of NaCl solution,serum containing Yiqi Huayu Bushen Recipe at 5%,10% and 20% promoted cell proliferation and expression of aggrecan mRNA and decreased expression of type Ⅹ collagen mRNA（P0.01）.10% serum containing Yiqi Huayu Bushen recipe was more effective than 5% and 20%.Conclusion：Yiqi Huayu Bushen Recipe inhibits degeneration of the intervertebral disc cells by regulating collagen and proteoglycan expressions.