山奈酚对激素诱导的股骨头坏死中内皮细胞的保护作用研究  

Protective effect of Kaempferol on endothelial cell injury in glucocorticoid induced osteonecrosis of the femoral head

作　　者：徐鑫 范骁宇 吴鑫杰 时利军 王培旭 高福强[3] 孙伟 李子荣[3] XU Xin;FAN Xiaoyu;WU Xinjie;SHI Lijun;WANG Peixu;GAO Fuqiang;SUN Wei;LI Zirong(Department of Orthopedics,China-Japan Friendship Hospital,Graduate School of Peking Union Medical College,Chinese Academy of Medical Sciences,Beijing,100029,P.R.China;Department of Orthopedics,Peking University China-Japan Friendship School of Clinical Medicine,Beijing,100029,P.R.China;Center forOsteonecrosis and Joint Preserving&Reconstruction,China-Japan FriendshipHospital,Beijing,100029,P.R.China)

机构地区：[1]中日友好医院骨科北京协和医学院研究生院中国医学科学院,北京100029 [2]北京大学中日友好临床医学院骨科,北京100029 [3]中日友好医院骨坏死与关节保留重建中心,北京100029

出　　处：《中国修复重建外科杂志》2022年第10期1277-1287,共11页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(82972524)。

摘　　要：目的通过体外实验探讨山奈酚对糖皮质激素诱导的股骨头坏死(glucocorticoids-induced osteonecrosis of the femoral head,GIONFH)中骨微血管内皮细胞(bone microvascular endothelial cells,BMECs)的作用。方法从股骨颈骨折患者自愿捐赠的股骨头或股骨颈松质骨中分离BMECs,并行CD31、血管性血友病因子免疫荧光染色和体外成管实验鉴定。采用细胞计数试剂盒8(cell counting kit 8,CCK-8)法筛选地塞米松(dexamethasone,Dex)抑制细胞活性的最佳浓度及时间点和改善Dex抑制活性的最佳山奈酚浓度;然后将实验分为4组,分别为单纯细胞组(A组)、加入最佳浓度Dex组(B组)、加入最佳浓度Dex+1μmol/L山奈酚组(C组)和加入最佳浓度Dex+5μmol/L山奈酚组(D组)。采用EdU实验、体外成管实验、TUNEL实验、AnnexinⅤ/碘化丙啶(propidium iodide,PI)实验、Transwell迁移实验、划痕实验和Western blot检测山奈酚对Dex干预后BMECs增殖、成血管、凋亡、迁移和蛋白表达的影响。结果经鉴定所培养细胞为BMECs。CCK-8法检测示Dex抑制BMECs活性的最佳浓度及时间点为300μmol/L作用24 h,改善Dex抑制活性的最佳山奈酚浓度为1μmol/L。EdU和成管实验结果示B~D组细胞增殖率、成管长度和分支点数目显著低于A组,B、D组显著低于C组,差异均有统计学意义(P<0.05)。TUNEL和Annexin V/PI凋亡检测结果显示,B~D组TUNEL阳性细胞率和细胞凋亡率显著高于A组,B、D组显著高于C组,差异均有统计学意义(P<0.05)。划痕实验和Transwell迁移实验结果显示,B~D组划痕愈合率和细胞迁移数显著低于A组,B、D组显著低于C组,差异均有统计学意义(P<0.05)。Western blot检测示,B~D组Cleaved Caspase-3和Bax蛋白相对表达量显著高于A组,B、D组显著高于C组;B~D组基质金属蛋白酶2、Cyclin D1、Cyclin E1、VEGFA和Bcl2蛋白相对表达量显著低于A组,B、D组显著低于C组;差异均有统计学意义(P<0.05)。结论山奈酚可减轻GIONFH中BMECObjective To explore the effect of Kaempferol on bone microvascular endothelial cells(BMECs)in glucocorticoid induced osteonecrosis of the femoral head(GIONFH)in vitro.Methods BMECs were isolated from cancellous bone of femoral head or femoral neck donated voluntarily by patients with femoral neck fracture.BMECs were identified by von Willebrand factor and CD31 immunofluorescence staining and tube formation assay.The cell counting kit 8(CCK-8)assay was used to screen the optimal concentration and the time point of dexamethasone(Dex)to inhibit the cell activity and the optimal concentration of Kaempferol to improve the inhibition of Dex.Then the BMECs were divided into 4 groups,namely,the cell group(group A),the cells treated with optimal concentration of Dex group(group B),the cells treated with optimal concentration of Dex+1μmol/L Kaempferol group(group C),and the cells treated with optimal concentration of Dex+5μmol/L Kaempferol group(group D).EdU assay,in vitro tube formation assay,TUNEL staining assay,AnnexinⅤ/propidium iodide(PI)staining assay,Transwell migration assay,scratch healing assay,and Western blot assay were used to detect the effect of Kaempferol on the proliferation,tube formation,apoptosis,migration,and protein expression of BMECs treated with Dex.Results The cultured cells were identified as BMECs.CCK-8 assay showed that the optimal concentration and the time point of Dex to inhibit cell activity was 300μmol/L for 24 hours,and the optimal concentration of Kaempferol to improve the inhibitory activity of Dex was 1μmol/L.EdU and tube formation assays showed that the cell proliferation rate,tube length,and number of branch points were significantly lower in groups B-D than in group A,and in groups B and D than in group C(P<0.05).TUNEL and Annexin V/PI staining assays showed that the rates of TUNEL positive cells and apoptotic cells were significantly higher in groups B-D than in group A,and in groups B and D than in group C(P<0.05).Scratch healing assay and Transwell migration assay showed t

关 键 词：山奈酚 地塞米松 股骨头坏死 骨微血管内皮细胞 

分 类 号：R285[医药卫生—中药学]