目的：探究补肾活血中药对滑膜间充质干细胞（SMSCs） 成软骨分化的影响。方法：选择1 例膝骨关节炎并行全膝关节置换术患者，将手术中分离得到的滑膜组织进行消化培养，经流式细胞仪检测贴壁细胞的表面标志物表达。同时选取20 只SPF 级雄性SD 大鼠，随机分到中药血清组和空白血清组各10 只，中药血清组予以补肾活血中药汤剂灌胃，空白血清组予以等量0.9%氯化钠溶液灌胃，7 d 后经心脏取血获得含中药血清和空白血清，将SMSCs 置于不同浓度的中药血清及空白血清环境下培养24 h、48 h、72 h，CCK-8 检测对其细胞活性及毒性作用，获得合适培养浓度血清。通过不同诱导处理分为以下3 组：空白组予以含合适浓度的空白血清的完全培养基培养，诱导剂组予以含合适浓度的空白血清的成软骨诱导分化培养基培养，中药诱导剂组予以含合适浓度的中药血清的成软骨诱导分化培养基培养，培养21 d 后，观察各组阿利新蓝染色成软骨分化情况，统计并分析成软骨分化指标Ⅱ型胶原（Col Ⅱ）、蛋白聚糖（AGG） 和SOX9 的蛋白印迹检测及实时定量基因扩增荧光检测系统（qPCR） 检测结果数据。结果：流式细胞仪鉴定获得细胞的表面分子CD90、CD105 阳性率分别为99.08%、92.88%，CD31、CD34 阳性率0.07%、0.03%，经鉴定为SMSCs。72 h 内，10%浓度干预组随时间延长吸光度值逐渐增减；且在72 h 时，10%浓度含中药血清干预SMSCs 较0%浓度组的吸光度值有明显增长（P＜0.05），且30%浓度含中药血清干预SMSCs 较10%浓度组的吸光度值有所降低（P＜0.05），因此，选用10%的血清浓度作为合适浓度用作后续实验。分组诱导培养21 d 后，阿利新蓝染色显示中药诱导组蓝色广泛，呈聚集状态，阳性染色较其空白组及诱导剂组显著；经qPCR 检测，中药诱导剂组的成软骨分化指标Col Ⅱ、AGG、SOX9 的基因表达较诱导剂组有明显升高（P＜0.05），且均高于空白组（P＜0.05）。蛋白印迹检测中药诱导剂组的Col Ⅱ、SOX9 蛋白水平较诱导剂组有明显升高（P＜0.05），中药诱导剂组与诱导剂组的AGG 蛋白水平相当（P＞0.05），且高于空白组（P＜0.05）。结论：补肾活血中药可有效促进SMSCs 成软骨分化，其机制与上调Col Ⅱ、AGG、SOX9 表达相关。
Abstract： Objective： To explore the effect of traditional Chinese medicine of tonifying kidney and activating blood on chondrogenic differentiation of synovial mesenchymal stem cells(SMSCs). Methods：One patient with knee osteoarthritis and total knee arthroplasty was selected. The synovial tissue isolated during the operation was digested and cultured， and the expression of surface markers of adherent cells was detected by flow cytometry. At the same time，20 SPF male SD rats were randomly divided into traditional Chinese medicine serum group and blank serum group. The traditional Chinese medicine serum group was perfused with traditional Chinese medicine decoction of tonifying kidney and activating blood，and the blank serum group was perfused with the same amount of 0.9% spdoium chloride solution. After 7 days，blood was taken from the heart to obtain traditional Chinese medicine serum and blank serum. SMSCs were cultured in different concentrations of traditional Chinese medicine serum and blank serum for 24， 48 and 72 hours， CCK- 8 was tested for its cell activity and toxicity，and the appropriate culture concentration serum was obtained. Through different induction treatments，they were divided into the following three groups： the blank group was cultured in complete medium containing appropriate concentration of blank serum， the inducer group was cultured in chondrogenic induction differentiation medium containing appropriate concentration of blank serum，and the traditional Chinese medicine inducer group was cultured in chondrogenic induction differentiation medium containing appropriate concentration of traditional Chinese medicine serum for 21 days.The chondrogenic differentiation was observed by alcian blue staining in each group， and the Western blot detection and realtime quantitative gene amplification fluorescence detection system(qPCR) data of chondrogenic differentiation indexes typeⅡ collagen(ColⅡ)，aggrecan(AGG) and SOX9 were counted and analyzed. Results：The positive rates of CD90 and CD105 were 99.08% and 92.88% respectively， and the positive rates of CD31 and CD34 were 0.07% and 0.03% respectively. Within 72 hours， the absorbance value of 10% concentration intervention group increased and decreased gradually with the extension of time. At 72 h，the absorbance value of 10% serum containing traditional Chinese medicine intervention SMSCs was significantly higher than that of 0% concentration group(P＜0.05)，and the absorbance value of 30% serum containing traditional Chinese medicine intervention SMSCs was lower than that of 10% concentration group(P＜0.05). Therefore，10% serum concentration was selected as the appropriate concentration for follow- up experiment. After 21 days of grouping induction culture，alixin blue staining showed that the blue of the traditional Chinese medicine induction group was extensive and aggregated，and the positive staining was significantly higher than that of the blank group and the inducer group. qPCR showed that the gene expression of Col Ⅱ ， AGG and SOX9 in the traditional Chinese medicine inducer group was significantly higher than that in the inducer group(P＜0.05)， and they were higher than those in the blank group(P＜0.05). Western blot showed that the levels of Col Ⅱ and SOX9 protein in the traditional Chinese medicine inducer group were significantly higher than those in the inducer group(P＜0.05). The levels of AGG protein in the traditional Chinese medicine inducer group and the inducer group were the same(P＞0.05)， and higher than those in the blank group(P＜0.05). Conclusion：Traditional Chinese medicine for tonifying kidney and activating blood can effectively promote the chondrogenic differentiation of SMSCs，and its mechanism is related to the up regulation of the expression of Col Ⅱ，AGG and SOX9.