丹参注射液体外对原代人软骨细胞的生物效应实验研究

目的：通过对丹参注射液对体外培养的原代人软骨细胞的生物效应分析，探讨其治疗骨关节炎的作用机理。方法：以丹参注射液丹参生药（1.5 mg·mL^-1）作为计算药物浓度。药物分组：对照组、0.5、1.0、2.0、4.0、8.0、16.0 mg·mL^-1。用MTT法评价原代人关节软骨细胞（第3代）增殖活性；用定量PCR法测定细胞Ⅰ、Ⅱ型胶原的表达量，评价软骨细胞的分化；用酶联免疫方法检测细胞Ⅰ、Ⅱ型胶原的含量；用糖胺多糖试剂盒法检测细胞总糖胺多糖的含量，评价软骨细胞的功能蛋白水平。结果：MTT实验结果显示，各浓度丹参注射液组的细胞相对活性均低于对照组，且降低趋势有量-效关系，提示丹参注射液对原代人关节软骨细胞有生长抑制效应。定量PCR结果显示，各浓度组细胞Ⅰ型胶原mRNA表达量均低于对照组，且有一定的量-效关系。Ⅰ型胶原蛋白检测结果显示，低剂量（≤ 2.0 mg·mL^-1）组可显著抑制Ⅰ型胶原表达。结果提示低剂量的丹参注射液可能对原代人关节软骨细胞的脱分化有一定的抑制作用。同时，低剂量丹参注射液（≤ 4.0 mg·mL^-1）能促进糖胺多糖的含量增加，提示低剂量丹参注射液有促进软骨形成的生物效应。结论：低剂量的丹参注射液可轻微抑制人第3代关节软骨细胞的增殖，抑制Ⅰ型胶原（脱分化指标）的形成和促进糖胺多糖的合成，提示其具有促进软骨形成的生物效应。

Objective:This study was to in vitro investigate the biological effects of salvia miltiorrhiza injection(SMI)on the primary human articular chondrocytes. The mechanism of the treatment of osteoarthritis was discussed. Methods:The concentration of SMI used in experiment was calculated by Salvia miltiorrhiza Bge(1.5 mg·mL^-1).The control group was cultured with dulbecco's modified eagle medium/F12 medium containing 10% fetal bovine serum,while the experimental groups were administrated with 0.5,1.0,2.0,4.0,8.0,16.0 mg·mL^-1 of SMI. The methylthiazole tetrazolium(MTT)assay was used to evaluate cell proliferation activity of the third passage human articular chondrocytes.The mRNA expression level of collagen typeⅠ/Ⅱ was determined by real time-polymerase chain reaction(PCR),which was to evaluate cell differentiation. Enzyme-linked immunosorbent assay(ELISA)was used to assess the content of collagen typeⅠ/Ⅱ.The total content of glycosaminoglycan(GAG)in cells was determined using glycosaminoglycan(DMMB)kit,which was to evaluate the level of functional protein in chondrocytes.Results:The MTT assay showed that the cell proliferation in all of SMI administration groups were lower than that in the control group with a dose-dependent manner,indicating that SMI had inhibition effect on proliferation of the primary human articular chondrocytes.The mRNA expression level of collagen typeⅠin all of SMI administration groups were lower than that in the control group with a dose-dependent manner.The collagen typeⅠprotein content in the groups with lower concentration(≤ 2.0 mg·mL^-1)of SMI significantly decreased,being compared with that in the control group.The results suggested that lower concentration of SMI should have a role in preventing de-differentiation of chondrocytes. Meanwhile,lower concentration(≤ 4.0 mg·mL^-1)of SMI increased the content of GAG in the primary human chondrocytes,indicating that SMI had effect on chondroregeneration. Conclusion:Salvia miltiorrhiza injection could slightly inhibit the proliferation of the third passage primary human articular chondrocytes.It also inhibited the production of collagen typeⅠ(according to the de-differentiation assay)and promoted the synthesis of GAG.These results indicated that SMI could promote chondroregeneration.