异槲皮苷联合大鼠骨髓间充质干细胞促进大鼠骨折愈合

Isoquercetin combined with rat bone marrow mesenchymal stem cells to promote fracture healing in rats

目的探讨异槲皮苷通过促进骨髓间充质干细胞迁移和成骨分化对大鼠骨折愈合的影响。方法体外实验:CCK-8检测骨髓间充质干细胞(BMSCs)增殖,Transwell实验检测BMSCs迁移,茜素红S染色检测BMSCs钙沉积,碱性磷酸酶(ALP)检测试剂盒检测BMSCs中ALP活性,Western blot检测runt相关转录因子2(Runx2)和骨钙素(OCN)蛋白表达;体内实验:40只骨折模型SD大鼠随机分为对照组、BMSC组、异槲皮苷组和BMSC+异槲皮苷组,每组10只。对照组大鼠尾静脉和腹腔注射PBS;BMSC组大鼠尾静脉注射2×106个BMSCs,腹腔注射PBS;异槲皮苷组大鼠腹腔注射40 mg/kg异槲皮苷,尾静脉注射PBS;BMSC+异槲皮苷组尾静脉注射2×106个BMSCs,腹腔注射40 mg/kg异槲皮苷。X线摄影检测大鼠骨折情况,免疫组织化学检测骨痂组织OCN和Ⅰ型胶原蛋白(ColⅠ)的表达。结果与对照组相比,异槲皮苷促进BMSC增殖,其中10μmol/L效果最明显(P<0.05);与对照组相比,异槲皮苷(5μmol/L和10μmol/L)促进BMSCs迁移、钙沉积和ALP活性,促进BMSCs中Runx2和OCN蛋白表达(均P<0.05)。体内实验中,与对照组及BMSC组相比,BMSC+异槲皮苷组大鼠在骨折2周后,骨折愈合评分、OCN和ColⅠ的表达升高(P<0.05);而BMSC组、异槲皮苷组与对照组相比,无明显差异(P>0.05)。与对照组相比,BMSC组、异槲皮苷组和BMSC+异槲皮苷组大鼠在骨折3周后,骨折愈合评分升高,OCN和ColⅠ的表达升高(P<0.01);与BMSC组相比,BMSC+异槲皮苷组大鼠在骨折3周后,骨折愈合评分升高,OCN和ColⅠ的表达升高(P<0.05)。结论异槲皮苷通过促进大鼠骨髓间充质干细胞迁移和成骨分化促进大鼠骨折愈合。

Objective To investigate the effect of isoquercetin on bone fracture healing in rats by promoting bone marrow mesenchymal stem cell migration and osteogenic differentiation.Methods CCK-8 was used to detect the proliferation of bone marrow mesenchymal stem cells(BMSCs).Transwell assay was used to detect the migration of BMSCs.Alizarin Red S staining was used to detect calcium deposition in BMSCs.Alkaline phosphatase(ALP)detection kit Detection of ALP activity in BMSCs.Western blotting was used to detect the protein expression of Runx2 and OCN.40 fracture SD rats were randomly divided into control group,BMSC group,isoquercetin group and BMSC+isoquercetin group,ten rats in each group.Rats in the control group were injected with PBS in the tail vein and intraperitoneal cavity,rats in the BMSC group were injected with 2×106 BMSCs in the tail vein,PBS was injected intraperitoneally,and rats in the isoquercetin group were injected intraperitoneally with 40 mg/kg isoquercetin and PBS in the tail vein.In the BMSC+isoquercetin group,2×106 BMSCs were injected into the tail vein,and 40 mg/kg isoquercetin was injected intraperitoneally.The fractures of rats were detected by X-ray photography;and the expression of OCN and ColⅠin the callus was detected by immunohistochemistry.Results Compared with the control group,isoquercetin(5μmol/L,10μmol/L,and 20μmol/L)promoted BMSCs proliferation,of which 10μmol/L had the most obvious effect(P<0.05).Compared with the control group,isoquercetin(5μmol/L and 10μmol/L)promoted BMSCs.Migration,calcium deposition and ALP activity promoted Runx2 and OCN protein expression in BMSCs(P<0.05).In vivo experiments,compared with the control group or the BMSC group,the rats in the BMSC+isoquercetin group had fracture healing scores,expressions of OCN,and ColⅠincreased after 2 weeks of fracture(P<0.05).There was no significant difference between the BMSC group and the isoquercetin group compared with the control group(P>0.05).Compared with the control group,rats in the BMSC group,the isoquercetin group,and the BMSC+isoquercetin group had a fracture healing score increased after 3 weeks of fracture,and the expressions of OCN and ColⅠincreased(P<0.01).Compared with the BMSC group,in the BMSC+isoquercetin group,the fracture healing score increased and the expressions of OCN and ColⅠincreased after 3 weeks(P<0.05).Conclusion Isoquercitrin promotes bone fracture healing in rats by promoting bone marrow mesenchymal stem cell migration and osteogenic differentiation in rats.