兔髓核细胞原代培养及独活寄生汤对其细胞周期的影响

Primary Culture of Rabbit Nucleus Pulposus Cell and the Influence of Duhuo Jisheng Decoction(DHJSD) on Its Cell Cycle

[目的]1研究兔髓核细胞(nucleus pulposus cells,NPCs)的培养条件及生物学特性。2探讨独活寄生汤(duhuo jisheng decoction,DHJSD)对体外培养兔NPCs周期的影响。[方法]1无菌条件下分离兔腰椎髓核组织,酶消化法联合组织块法消化,含15%胎牛血清(fetal bovine serum,FBS)的DMEM/F12(1︰1)培养液培养,建立NPCs体外培养体系,细胞80%90%融合后进行传代培养。通过倒置相差显微镜观测细胞形态,通过II型胶原及集聚蛋白聚糖免疫组织化学进行细胞鉴定。2NPCs传第2代时进行实验。按照DHJSD药液不同浓度(100、200、400μg·m L-1)进行分组,0μg·m L-1为空白对照组。药物干预72h后,收集兔NPCs。MTT法检测细胞增殖能力;流式细胞仪检测细胞周期变化情况。[结果]兔NPCsⅡ型胶原和集聚蛋白聚糖免疫组织化学染色呈阳性,细胞质内有大量棕褐色颗粒状物质,核周尤为明显。与空白对照组比较,DHJSD干预后的兔NPCs OD值均增加(P<0.05);兔NPCs G1期比例降低(P<0.05),S期比例增加(P<0.05)。[结论]体外单层培养,成功培养兔NPCs。DHJSD通过促进NPCs周期关键限制点G1/S期的切换,促进NPCs增殖,抑制NPCs凋亡,从而减轻或延缓椎间盘退变。

[Objective]To research the annulus fibrosus cell culture conditions and biological charactefistics.To explore the influence of Duhuo Jisheng Decoction（DHJSD） on cell cycle of rabbit nucleus pulposus cells. [Methods] Lumbar nucleus pulposus was separated from ten New Zealand white rabbits, under aseptic conditions, used enzyme digestion method combined with tissue block method,cultivated with containing 15% FBS DMEM/F12（I：I） nutrient solution. When 90% cells fused,then using subculture. Inverted phase contrast microscope was used to observe cell morphology and identify through the detection of proteoglycan and type II collagen expression. The experiment started when the nucleus pulposus cells attained the second generation. Grouped according to the different doses of DHJSD（100μg· mL-1, 200μg· mL-1, 400μg· mL-1 ）, 0μg· mL-l was blank control group. After treated nucleus pulposus cells with the DHJSD for 72h ,cell proliferation was detected by MTT assay; and cell cycle analysis was performed with Flow Cytometry. [Result] The result of identification of the nucleus pulposus cells with immunohistochemical staining of proteoglycan and type II collagen was positive. It showed a large red-brown particulate matter significantly in the cytoplasm near the nuclear periphery. Compared with the control group, the OD values of DHJSD-treated groups were increased（P〈0.05）; the percentage of G1 phase cells was gradually decreased; however, the percentage of S phase cells and were increased（P〈0.05）. [Conclusion] Rabbit nucleus pulposus cells were successfully cultured by monolayer culture and enzyme digestion method combined with tissue block method in vitro. DHJSD could promote nucleus pulposus cells proliferation, inhibit nucleus pulposus cells apoptosis and delay intervertebral disc degeneration by promoting GI/S transition, in vitro.