[1]段洪超,王庆超,张杰△.丹参注射液对兔软骨细胞增殖与凋亡的影响[J].西部中医药,2018,31(07):22-26.
　DUAN Hongchao,WANG Qingchao,ZHANG Jie.Effects of DanShen Injection on Apoptosis and Proliferation of Rabbit Chondrocytes[J].Western Journal of Traditional Chinese Medicine,2018,31(07):22-26.
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丹参注射液对兔软骨细胞增殖与凋亡的影响()
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《西部中医药》[ISSN:2096-9600/CN:62-1204/R]

卷:
31
期数:
2018年07期
页码:
22-26
栏目:
出版日期:
2018-07-15

文章信息/Info

Title:
Effects of DanShen Injection on Apoptosis and Proliferation of Rabbit Chondrocytes
文章编号:
1004-6852(2018)07-0022-05
作者:
段洪超，王庆超，张杰△
黑龙江中医药大学附属第二医院，黑龙江 哈尔滨 150009
Author(s):
DUAN Hongchao, WANG Qingchao, ZHANG Jie△
The Second Affiliated Hospital of Heilongjiang University of Chinese Medicine, Harbin 150009, China
关键词:
骨性关节炎; 兔软骨细胞; Bcl-2; Bax; 丹参注射液
Keywords:
osteoarthritis;  rabbit chondrocyte;  Bcl-2;  Bax;  DanShen injection
分类号:
R684.3
文献标志码:
A
摘要:
目的：观察丹参注射液对兔软骨细胞增殖与凋亡的影响。方法：取健康4周龄新西兰大白兔24只，取膝关节软骨组织并建立兔膝软骨细胞体外培养体系，采用四甲基偶氮噻唑蓝比色法(MTT)检测软骨细胞2-5代的增长曲线；采用梯度时间法优化丹参注射液干预软骨细胞活性的时效及量效关系；采用RT-PCR法检测各组软骨细胞B淋巴细胞瘤-2(Bcl-2)、Bax的表达。结果：第2-5代细胞的增殖曲线趋势：第1天各代细胞光密度值(OD值)没有显著差异(P＞0.05)，第2天增殖缓慢，第3天增殖逐渐加快，第4天进入生长期，第5、6天增殖变慢，第7、8天增殖进入平台期，第9、10天增殖抑制。OD值随细胞传代，呈减低趋势，第1、3代OD值高于第4、5代，但没有显著差异(P＞0.05)。时效关系：2组干预24小时OD值没有显著差异(P＞0.05)，干预48小时OD值中剂量组大于对照组(P＜0.05)，干预72、96小时OD值中剂量组大于对照组(P＜0.01)，中剂量组干预72小时OD值显著大于24、48、96小时(P＜0.05)，故选择细胞干预72小时作为以后实验检测时间点。量效关系：第3代软骨细胞用不同丹参注射液终浓度的10%FBS DMEM溶液干预，干预前细胞OD值没有明显差异(P＞0.05)；干预72小时后，400、200、100、50、25 μg/L 浓度细胞的OD值显著大于0 μg/L浓度的OD值(P＜0.01)；50 μg/L浓度的OD值显著大于400、200、100、25 μg/L浓度的OD值(P＜0.01)；100 μg/L浓度的OD值显著大于400、200、25 μg/L浓度的OD值(P＜0.01)；100 μg/L与50 μg/L比较细胞OD值没有显著差异(P＞0.05)。Bcl-2、Bax表达：诱导凋亡的第3代软骨细胞，加丹参注射液干预24小时后，细胞Bax表达中、高剂量组小于模型组(P＜0.05)，高剂量组小于低剂量组(P＜0.05)；细胞Bcl-2表达中、高剂量组大于模型组(P＜0.05)，高剂量组大于低剂量组(P＜0.05)。结论：丹参注射液能抑制软骨细胞调亡；可能通过下调软骨细胞促凋亡因子Bax的基因表达，上调软骨细胞促凋亡因子Bcl-2的基因表达，抑制软骨细胞线粒体调亡通路的信号转导实现。
Abstract:
Objective: To observe the effects of DanShen injection on apoptosis and proliferation of rabbit chondrocyte. Methods: All 24 healthy rabbits aging four weeks were chosen, and cartilaginous tissue of knee joint was incised and rabbit knee chondrocyte culture system in vitro was established, building-up curve of chondrocyte of generations two to five was detected by MTT method; time-effects and dose-effect relations of DanShen injection intervening chondrocyte active were optimized by using gradient time method; the expressions of Bcl-2 and Bax of chondrocyte in different groups were detected by using RT-PCR method. Results: Multiplication curvilinear trend of generations two to five: there was no significant difference in OD values of different generations cells at the first day (P＞0.05), the cells reproduced slowly at the second day, they gradually increased to reproduce at the third day, stepped into the growth period at the forth day, multiplication became slow at the fifth and sixth day, it entered into plateau at the seventh and eighth day, it was inhibited at the ninth and tenth day. OD value showed the downward tendency as the passage of the cells, OD values of the first and third generations were higher than these of the forth and fifth generations, while there was no significant difference(P＞0.05). Time-effects relation optimization: there was no significant difference in the comparisons of OD values when both groups were intervened for 24 hours (P＞0.05), moderate dose groups was larger than the control group in OD values after intervened for 48 hours (P＜0.05), moderate dose group was larger than the control group in OD values after intervened for 72 and 96 hours (P＜0.01), OD value of moderate dose group after intervened for 72 hours was larger than that in 24 hours, 48 hours and 96 hours notably(P＜0.05), therefore, cellular intervention for 72 hours was chosen as the detection time point of the experiment in future. Dose-effects relation: the chondrocyte of generation three was intervened by 10% FBS DMEM solution when DanShen injections were in different concentrations, cellular OD value had no obvious difference before the intervention(P＞0.05); after intervened for 72 hours, cellular OD values after dealt with DanShen injection in final concentrations of 400, 200, 100, 50 and 25 μg/L were larger than these in 0 μg/L significantly (P＜0.01); OD values in 50 μg/L were bigger than these in 400, 200, 100 and 25 μg/L notably (P＜0.01); OD values in 100 μg/L were bigger than these in 400, 200 and 25 μg/L remarkably (P＜0.01); the comparisons of cellular OD values had no significant difference after dealt with DanShen injection in the concentrations of 100 μg/L and 50 μg/L(P＞0.05). The expressions of Bcl-2 and Bax: apoptosis-induced chondrocyte of generation three was intervened by DanShen injection for 24 hours, the expressions of Bax in the cells in moderate and high dose groups were less than these of the model group (P＜0.05), high dose group was less than low dose group(P＜0.05); the expressions of Bcl-2 in the cells in moderate and high dose groups were bigger than these of the model group(P＜0.05), high dose group was larger than low dose group (P＜0.05). Conclusion: DanShen injection could inhibit the apoptosis of chondrocyte,realized by down regulating the gene expressions of apoptosis promoting factor Bax, up regulating gene expression of apoptosis promoting factor Bcl-2 and inhibiting signal transduction of mitochondria apoptosis pathway in chondrocyte.

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备注/Memo

备注/Memo:
收稿日期：2017-06-01 *基金项目：哈尔滨市科技创新人才研究专项资金(优秀学科带头人)项目(编号RC2014XK004078)。 作者简介：段洪超(1978—)，男，硕士学位，主治医师。研究方向：骨伤科疾病的中西医结合治疗。 △通讯作者：张杰(1968—)，女，博士学位，教授，主任医师。研究方向：骨伤科疾病的中西医结合治疗。
更新日期/Last Update: 2018-07-15