兔髓核细胞原代培养及独活寄生汤对Fas介导的凋亡通路的影响  

Primary culture of rabbit nucleus pulposus cell and the influence of Duhuo Jisheng Decoction on its Fas-mediated apoptosis pathway

作　　者：卢建华[1] 苏陈颖[2] 王靖[2] 俞冰[3] 张春丽[1] 钱志钢[4] 柯高峰[3] 

机构地区：[1]浙江中医药大学附属第一医院,杭州310006 [2]温州医科大学附属第一医院,温州325000 [3]浙江中医药大学,杭州310053 [4]浙江医院,杭州310013

出　　处：《中华中医药杂志》2016年第12期5341-5344,共4页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：浙江省自然科学基金资助项目(No.Y13H270012)~~

摘　　要：目的:研究兔髓核细胞的培养条件及生物学特性,并探讨独活寄生汤对体外培养兔髓核细胞凋亡的影响。方法:无菌环境下分离兔腰椎髓核组织,酶消化法消化,用DMEM/F12(1∶1)培养液培养。通过Ⅱ型胶原及集聚蛋白聚糖免疫组织化学检测进行细胞鉴定。髓核细胞传至第二代时开始药物干预。根据独活寄生汤药液不同浓度(100、200、400μg/m L)进行分组,0μg/m L为空白对照组。药物干预72h后,收集髓核细胞。RT-PCR法检测Fas、Fas L、Bcl-2、Bid、Bax、Caspase-8、Caspase-3 m RNA表达情况。结果:髓核细胞Ⅱ型胶原和集聚蛋白聚糖免疫组化染色为阳性。与空白对照组比较,独活寄生汤干预后能够下调Fas、Fasl、Bax、Bid、Caspase-3、Caspase-8 m RNA的表达(P<0.05),上调Bcl-2 m RNA的表达(P<0.05)。结论:体外环境下单层培养、酶消化法成功培养了兔髓核细胞。独活寄生汤通过抑制Fas介导的Ⅰ型和Ⅱ型凋亡通路的激活,从而抑制髓核细胞凋亡。Objective： To research the annulus fibrosus cell culture conditions and biological characteristics, and to explore the influence of Duhuo Jisheng Decoction（DHJSD） on apoptosis of rabbit nucleus pulposus cells. Methods： Lumbar nucleus pulposus was separated from rabbits, under aseptic conditions, cultivated with containing 15% FBS DMEM/F12（1：1） nutrient solution. It was identified 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（100, 200, 400μg/m L）, 0μg/m L was blank control group. After treated nucleus pulposus cells with the DHJSD for 72 h, the m RNA expression levels of Fas, Fas L, Bcl-2, Bid, Bax, Caspase-8 and Caspase-3 were measured by RT-PCR. Results： The result of identification of the nucleus pulposus cells with immunohistochemical staining of proteoglycan and type II collagen is positive. DHJSD could inhibit Fas, Fas L, Bax, Bid Caspase-3, Caspase-8 expression（P〈0.05） while promoting Bcl-2 expression（P〈0.05）. Conclusion： Rabbit nucleus pulposus cells are successfully cultured by monolayer culture and enzyme digestion method in vitro. DHJSD could inhibit the type I and II Fas-mediated apoptosis pathway in rabbit nucleus pulposus cells in vitro.

关 键 词：组织培养 髓核细胞 独活寄生汤 细胞凋亡 椎间盘退变 

分 类 号：R285[医药卫生—中药学]