Toll样受体对大鼠骨髓间充质干细胞成骨的影响

Effect of toll like receptor to ossification of bone marrow mesenchymal stem cells of rat

目的 观察Toll样受体（TLR）不同亚型对大鼠骨髓间充质干细胞（BMSCs）成骨的影响。方法 分离纯化SD大鼠BMSCs,传代扩增。免疫荧光分析检测CD44、CD105、波动蛋白（Vimentin）,流式细胞术检测CD34、CD14、CD29、CD95、CD4和人白细胞抗原（HLA）-I等标志物。将第3代细胞,随机分为空白组、TLR-3激活组和TLR-4激活组。后两组分别以聚肌胞苷酸Poly（I：C）,1 mg/L）和脂多糖（LPS,10μg/L）激活。成骨诱导培养液培养。Western blot法检测I型胶原（Collagen-I）和骨钙素（Osteocalcin）的表达,并定量分析。在培养的第10、14天进行碱性磷酸酶（ALP）及茜素红染色。结果 全骨髓培养法提取到BMSCs,免疫荧光显示CD44、CD105、Vimentin阳性。流式细胞术显示CD29、CD95、CD44、HLA-I阳性,CD14、CD34阴性。Collagen-I和Osteocalcin的蛋白表达量依次为,TLR-4激活组〈TLR-3激活组〈空白组。3组ALP染色及茜素红染色均呈阳性,TLR-3激活组染色程度最强,空白组和TLR-4染色程度无明显差异。结论 TLR-3和TLR-4均能促进大鼠BMSC向成骨细胞（OB）分化。TLR-4作用更明显。在OB矿化过程中,TLR-3起促进作用,TLR-4则起抑制作用。

Objective To explore the effect of toll like receptor （TLR） on ossification of bone marrow mesenchymal stem cells of rat （BMSCs）. Methods After isolation and purification, rat-BMSCs were identified by detecting surface makers, such as CD44, CD105, CD34, CD14, CD29, CD95, HLA-I and Vimentin, using immunofluoreseence or flow cytometry （FCM）. BMSCs of 3rd passage were randomly divided into 3 groups ： control group, TLR3-Primed group （ Poly 〈 I ： C 〉 , 1 mg/L ） and TLR4-primed group （LPS, 10 μg/L）. Alkaline phosphatase （ALP） and alizarin red staining were performed in 10th day, and 14th day. Expression of collagen-I and osteocalcin were detected by Western blotting, then statis- tically analysed. Results CD44, CD105, Vimentin, CD29, CD95, human leukocyte antigens （HLA）-I were positive, CD14, while CD34 were negative. Collagen-I and Osteocalcin were all expressed in in three groups, strongest in TLR-4-primed group, weakest in Controlled group. ALP and alizarin red staining were all positive in all groups, stronger in TLR-3-primed group than other two groups, TLR-4-primed group and controlled group had no statistically difference. Conclusion The differentiation of BMSCs to osteoblasts （OB） can be promoted by TLR-3 and TLR-4. TLR-4 had stronger effect in this process. The ossification of OB to osteocytes was promoted by TLR-3, but inhibited by TLR-4.