中药对犬骨髓干细胞成骨分化增殖的影响

目的 探讨活血化淤补肾壮骨中药对骨髓间充质干细胞(mesenchymal stem cells,MSC)向成骨细胞分化增殖的影响.方法 比格犬6只,雄性,体重10～15 kg.实验分为含药血清组(A组)、无药血清组(B组)和胎牛血清组(C组).A组的血清来自活血化淤补肾壮骨中药经煎制后,按犬体表面积折算等效剂量,2只比格犬连续喂服7 d后经股动脉采血制备而成;B组的血清采用等剂量生理盐水,2只比格犬喂服7 d后经股动脉采血制备而成;C组的血清直接购买.另外2只比格犬由胫骨获取骨髓,经Ficoll分离液进行梯度离心,MSC经含胎牛血清的DMEM培养,传代后在培养液加入矿化诱导液(β-甘油磷酸钠、维生素C和地塞米松)促使其向成骨细胞分化,I型胶原蛋白免疫组化鉴定成骨细胞,银染色和茜素红染色鉴定钙结节.分别将诱导后的细胞在A组、B组和C组DMEM中培养.通过甲基噻唑基四唑法(MTT)和碱性磷酸酶(ALP)活性的检测分别观察MSC分化生长的情况.结果 原代培养MSC细胞形态以梭形为主,诱导培养后细胞呈多边形,细胞有突起,I型胶原蛋白表达呈阳性,继续培养至10～14 d,细胞量达到高峰,出现钙化结节,银染色和茜素红染色呈强阳性.MTT法检测的生长曲线显示,3组细胞数量逐渐增加,培养6 d后吸光度值A组(0.696±0.188)、B组(0.374±0.093)及C组(0.296±0.106)比较,差异均有统计学意义(P<0.05).ALP活性随着培养时间延长而增加,A组的增加更为显著.培养5 d后ALP活性A组(36.72±2.02)、B组(26.90±2.46)及C组(24.50±1.56)比较,差异均有统计学意义(P<0.05).结论 补肾壮骨中药能明显促进骨髓间充质干细胞向成骨细胞分化增殖,促进骨形成.

Effects of traditional Chinese medicine on the differentiation of mesenchymal stem cells to osteoblasts

Objective To investigate the effects of traditional Chinese medicine on the differentiation of mesenchymal stem cells(MSC)to osteoblasts.Methods Six male Beagle dogs weighed 10-15 kg each were divided into three groups,group A:medicine serum group,group B:non-medicine serum group and group C:bovine serum group.The serum of group A was obtained from the femoral artery of 2 Beagle dogs drinking equivalent dose of traditional Chinese medicine according to body surface area for 7 continuous days.The serum of group B was collected from the femoral artery of 2 Beagle dogs fed with equal volume of normal saline for 7 days.The serum of group C was fetal bovine serum.The tibia marrow was harvested from another 2 Beagle dogs and MSC were isolated and purified by density gradient centrifugation.MSC were cultured in DMEM solution with fetal bovine serum. After MSC were digested by trypsin.MSC were cultured in DMEM solution wuth the osteogeneic inducer,which contained dexamethasone,antiscorbutic and β-glycerophosphate.Morphological and histological changes of the MSC were observed under an inverted microscope.Alizarin monosulfonate and nitric acid argentum staining was performed to observe the calcium deposition.MSC were curtured in DMED solution with medicine serum(group A),non-medicine serum(group B)and bovine serum(group C)respectively.The growth curve was detected by the methyl thiazoly tetrazolium (MTT). The alkaline phosphatase(ALP) activities were detected to observe the differentiation of MSC. Results The original MSC were observed as fibroblast-like cell shapes. After the osteogeneic inducer was added, MSC were polygon cells with a few polyprocess. Calcium deposition appeared during 10-14 days and alizarin monosulfonate and Von Kossa staining presented positive. MTT results showed that the number of adherent cells of group A was more than that of group B and that of group C significantly after 6 days (P <0. 05). ALP detection proved that ALP activity of group A was more than that of group B and that of group C significantly after 5 days ( P < 0. 05 ). Conclusions The traditional Chinese medicine promotes the differentiation of MSC to osteoblasts and osteogenesis.