化痰除湿祛瘀剂膝痹康对膝骨关节炎患者软骨细胞凋亡调控基因表达的影响  

Effects of Xibikang preparation of eliminating dampness and phlegm and blood stasis on apoptosis regulated genes expression of chondrocytes in patients with knee osteoarthritis

作　　者：孟祥奇[1] 梁国强 尢君怡[1] 马奇翰 

机构地区：[1]江苏省苏州市中医医院骨伤科,江苏苏州215003 [2]吴门医派研究院,江苏苏州215003

出　　处：《河北中医》2017年第1期94-99,103,共7页Hebei Journal of Traditional Chinese Medicine

基　　金：江苏省苏州市科技局2014年度第十五批科技发展计划(应用基础研究.医疗卫生)项目(编号:SYS201420)

摘　　要：目的研究化痰除湿祛瘀剂膝痹康对人骨关节炎(osteoarthritis,OA)软骨细胞调控基因表达的影响。方法取全膝关节置换手术膝骨关节炎患者的软骨组织,采用酶消化培养法培养人软骨细胞,采用甲苯胺蓝染色鉴定细胞来源。将软骨细胞分为对照组、膝痹康0.016、0.08、0.4、2.0、10.0 mg/m L组,培养24 h后,应用甲基噻唑基四唑(MTT)法检测各组细胞的增殖情况。根据增殖结果将软骨细胞分为对照组、膝痹康低、中、高剂量组(0.05、0.1、2.0 mg/m L),用TUNEL法检测各组细胞凋亡情况,以实时荧光定量PCR(RT-PCR)法检测各组细胞Bcl-2、Bcl-2同源水溶性相关蛋白X(Bax)及p53 mRNA表达。结果本研究成功建立了人骨关节炎软骨细胞有限细胞系,细胞生长状态良好;甲苯胺蓝染色证明所培养的细胞为来自中胚层的软骨细胞。膝痹康在0.016~10.0 mg/m L范围内对OA软骨细胞波长=450 nm处24 h的光密度(OD)有影响。与对照组相比,膝痹康低剂量组细胞凋亡率无明显变化(P>0.05),膝痹康中、高剂量组下降(P<0.05,P<0.01)。膝痹康低、中、高剂量组间比较显示,两两比较差异均有统计学意义(P<0.05)。与对照组相比,膝痹康低剂量组Bcl-2、Bax及p53 mRNA相对表达量无明显变化(P>0.05),膝痹康中、高剂量组Bcl-2 mRNA表达量明显上调(P<0.05),Bax及p53 mRNA表达量明显下调(P<0.05,P<0.01);与膝痹康低剂量组相比,膝痹康中、高剂量组Bcl-2 mRNA表达量明显上调(P<0.05),Bax及p53 mRNA表达量明显下调(P<0.05,P<0.01);与膝痹康中剂量组相比,膝痹康高剂量组Bax mRNA表达量明显下调(P<0.05)。结论化痰除湿祛瘀剂膝痹康调控细胞凋亡基因的表达,抑制软骨细胞过度凋亡,这可能是抑制软骨破坏的重要机制之一。Objective To observe the effects of Xibikang preparation of eliminating dampness and phlegm and blood stasis the expression of apoptosis regulated genes in human knee osteoarthritis（OA） chondrocytes. Methods The chondrocytes originatef from cartilage tissue in knee OA patients after total knee replacement,which were cultured by enzyme digestion,cellular sources were identified by toluidine blue staining. The chondrocytes were divided into control group and Xibikang 0. 016,0. 08,0. 4,2. 0,10. 0 mg/m L groups,the cellular proliferation in each group was detected by methyl thiazolyl tetrazolium（MTT）after 24 h training. The chondrocytes were divided into control group and Xibikang low,medium and high dose groups（0. 05,0. 1,2. 0 mg/m L） according to cell proliferation,the cellular apoptosis was detected by Td T-mediated Dutp nick end labeling（TUNEL）,the expressions of B-cell lymphoma-2（Bcl-2） and Bcl-2-associated X protein（Bax） and p53 mRNA were detected by reverse transcription-polymerase chain reaction（RT-PCR）. Results This study successfully established a human osteoarthritis chondrocytes limited cell line,with normal growth condition,all cells originating from mesoderm chondrocytes which proved by toluidine blue. Xibi kang（range from 0. 016 to 10. 0 mg/m L） had effect of 24 h optical density in OA chondrocytes（ware length = 450 nm）. There was no statistical difference on apoptosis rate between low dose group and control group（P〈0. 05）,but that in medium and high dose groups were decreased（P〈0. 05）. There were significant differences on apoptosis rate through pairwise omparision among low,medium and high dose groups（P〈0. 05）. As compared with control group,there were no significantly changes on the relative transcript level of Bcl-2,Bax and p53 mRNA in low dose group（P〈0. 05）,the Bcl-2 mRNA was significantly up-regulated in medium and high dose group（P〈0. 05）,and the Bax and p53 mRNA were significantly down-regulated（P〈0. 05,P〈0. 01）. As comp

关 键 词：骨关节炎 化痰 细胞凋亡 BCL-2相关X蛋白质 基因 BCL-2 基因 p53 

分 类 号：R684.3[医药卫生—骨科学] R349.5[医药卫生—外科学]