淫羊藿苷促进人牙周膜干细胞增殖及骨向分化的作用  

Effects of Icariin promotion on proliferation and osteogenic differentiation of human periodontal ligament stem cells

作　　者：秦子顺[1] 殷丽华[1] 王开娟[1] 刘琪[1] 程文晓[1] 高鹏[1] 孙可墨 钟梅[1] 余占海[1] 

机构地区：[1]兰州大学口腔医院修复科,兰州730000

出　　处：《华西口腔医学杂志》2015年第4期370-376,共7页West China Journal of Stomatology

基　　金：甘肃省自然科学基金资助项目(1208RJZA148);甘肃省中医药科研课题基金资助项目(GZK-2012-47)

摘　　要：目的采用体外和体内方法研究淫羊藿苷(ICA)对人牙周膜干细胞(h PDLSCs)增殖及骨向分化的影响。方法采用体外酶消化组织块法培养h PDLSCs,有限稀释克隆法分离纯化,检测细胞表面标志物以进行鉴定。体外实验:h PDLSCs与1×10-7 mol·L-1 ICA共同培养,用四甲基偶氮唑盐法检测其增殖情况,经碱性磷酸酶(ALP)染色后采用酶联免疫仪检测其骨向分化情况,逆转录聚合酶链反应(RT-PCR)检测其成骨基因的表达情况,茜素红染色检测其骨量表达。体内实验:将h PDLSCs和纳米羟磷灰石(n HAC)复合物经1×10-7 mol·L-1 ICA共同诱导培养后,将其植入免疫缺陷小鼠,术后30 d采用组织切片法观察成骨状况。结果体外实验:1×10-7 mol·L-1 ICA作用于h PDLSCs后,与不含ICA的对照组相比,实验组从培养的第2天开始,h PDLSCs增殖明显;培养3、5、7 d,实验组ALP活性明显升高;培养7 d,成骨基因的相对表达量明显增高;培养14、21、28 d,实验组钙化结节的面积明显大于对照组。体内实验:实验组骨量较对照组明显增加。结论 1×10-7 mol·L-1 ICA可以促进h PDLSCs的增殖及骨向分化,提示采用ICA代替常规生长因子应用于牙周组织工程具有一定可行性。Objective To evaluate the effects of Icariin（ICA） on the proliferation and osteogenic differentiation of human periodontal ligament stem cells（h PDLSCs） in vitro and in vivo. Methods An enzymatic digestion block was used in vitro to culture h PDLSCs, which were separated and purified by limited dilution cloning. The h PDLSCs were identified using cellsurface markers and cocultured with 1×10-7 mol·L-1 ICA solution. The proliferation ability of these cells was determined by thiazolyl blue tetrazolium bromide（MTT） assay. After staining with alkaline phosphatase（ALP）, osteogenesis was detected by enzyme-linked immunosorbent assay. Osteoblast-related genes were analyzed by reverse transcription-polymerase chain reaction. Alizarin red staining was performed to measure the level of calcium deposition. The h PDLSCs were cocultured with 1×10-7 mol·L-1 ICA and nano-hydroxyapatite scaffolds in vivo before transplantation into subcutaneous tissues of nude mice. Osteogenic abilities were histochemically analyzed after 30 days of induction. Results The h PDLSCs were affected by 1×10-7 mol·L-1 ICA, and MTT assay showed that the proliferation of the groups treated with ICA in vitro was better than that of the control groups on the second day. The ALP activity of the treated h PDLSCs was significantly enhanced after cell culture for 3, 5, and 7 days. The gene expression of osteoblastic markers was also significantly enhanced after 7 days. The deposition of mineralization after incubation with 1×10-7 mol·L-1 ICA increased compared with the control after cell culture for 14, 21, and 28 days. Furthermore, the bone expression of the treatment groups in vivo was significantly enhanced compared with that of the control groups. Conclusion Treatment with 1×10-7 mol·L-1 ICA can significantly promote proliferation and differentiation of h PDLSCs in vitro and in vivo. ICA can effectively function as a bioactive growth factor in periodontal tissue engineering to replace traditional growth factors.

关 键 词：人牙周膜干细胞 淫羊藿苷 增殖 分化 纳米羟磷灰石 

分 类 号：R781.4[医药卫生—口腔医学]