化痰除湿祛瘀剂对人骨关节炎软骨细胞凋亡的影响

The Effects of Huatan Chushi Qiuyu Preparation on Cell Apoptosis of Human Osteoarthritis Chondrocytes

目的:研究化痰除湿祛瘀剂对人骨关节炎(osteoarthritis,OA)软骨细胞凋亡的影响。方法:(1)在苏州中医院骨伤科协助下,取因外伤急性截肢患者的软骨组织以及全膝关节置换手术OA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,观察显微镜观察其形态变化。(2)将软骨细胞分成OA软骨细胞组、OA软骨细胞组+化痰除湿祛瘀剂(0.016、0.08、0.4、2、10 mg/mL)组,根据分组情况分别培养24 h或48 h后,应用MTT法比较各组细胞的增殖情况。(3)根据增殖结果将软骨细胞分成OA软骨细胞组、OA软骨细胞组+化痰除湿祛瘀剂(0.05、0.1、2 mg/mL)组,用原位末端标记法(TUNEL)检测各组细胞凋亡情况。结果:(1)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;甲苯胺蓝证明所培养的细胞为来自中胚层的软骨细胞。(2)与对照组比较(24 h+),不同浓度的化痰除湿祛瘀剂对OA软骨细胞活力无影响。与对照组(OD值:0.393±0.010,λ=450 nm)比较(24 h+),化痰除湿祛瘀剂(1 mg/mL,OD值:0.441±0.009)和(2mg/mL,OD值:0.430±0.012)组可明显促进软骨细胞增殖(均P<0.001)。(3)OA软骨细胞经TUNEL标记后的结果显示,对照组的凋亡指数为(42.21±2.67)%,化痰除湿祛瘀剂(0.1 mg/mL,37.14%±4.01%;2 mg/mL,34.21%±5.00%)可明显抑制软骨细胞早期凋亡与对照组比较(P<0.05或P<0.01)。结论:化痰除湿祛瘀剂促进细胞增殖,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 explore the effect of Huatan Chushi Qiuyu preparation on cell apoptosis of human osteoarthritis chondrocytes. Methods：（ 1） The chondrocytes used in these researches were available with primary tissue culture by enzymatic digestion,cartilage tissue obtained during surgery for knee replacements from OA patients. toluidine blue stain were used to identify the cell source; observed its morphologic changesunder inverted microscope.（ 2） The chondrocytes were divided into OA chondrocyte group,OA chondrocyte ＋ Huatan Chushi Qiuyu preparation（ 0. 016,0. 08,0. 4,2,10mg/mL） groups,All the cells were treated according to the grouping for 24 h or 48 h,Then cell proliferation was detected by Methyl thiazolyl tetrazolium（ MTT） method.（ 3） According to the result of proliferation,The chondrocytes were divided into OA chondrocyte group,OA chondrocyte ＋ Huatan Chushi Qiuyu preparation（ 0. 05,0. 1,2 mg/mL） groups,apoptosis was detected by Td T-mediated d UTP nick end labeling（ TUNEL）. Results：（ 1） Successfully established a finite cell line of human osteoarthritis chondrocytes,which growth in good condition. Toluidine blue stain identified that the cells are chondrocytes from mesodermal.（ 2） compared with control group,Different concentrations of Huatan Chushi Qiuyu preparation had no effect on the viability of OA chondrocytes（ 24 h ＋）. Compared with the control group（ OD： 0. 393 ＋0. 010）,Huatan Chushi Qiuyu preparation（ 0. 1 mg/mL,OD： 0. 441 ± 0. 009） and（ 2 mg/mL,OD： 0. 430 ± 0. 012） group could promote the proliferation of cartilage cells（ 48 h ＋,P〈0. 001）.（ 3） The apoptosis index of control group was（ 42. 21 ± 2. 67） % were labeled by TUNEL,compared with control group,Huatan Chushi Qiuyu preparation（ 0. 1 mg/mL,37. 14% ± 4. 01%） and（ 2mg/mL,34. 21% ± 5. 00%） could obviously inhibit the early apoptosis of cartilage cells（ P〈0. 05 or P〈0. 01）. Conclusion： Huatan Chushi Qiuyu preparation can promote cell proliferation and inhibit cell apoptosis,which may be the important mechanism to inhibit OA cartilage damage.