作者:张宇坤 杨述华 杨操 孙立 田洪涛作者单位:华中科技大学同济医学院附属协和医院骨科,武汉 430022 $ @" R y( Z' n' C6 I ; e8 C: J' h2 I/ P9 X" k5 L
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【摘要】 [目的]研究缝隙连接阻断剂1庚醇对小鼠骨髓基质干细胞体外软骨分化的影响。[方法]体外培养小鼠骨髓基质干细胞(MSCs),贴壁细胞传代,取第3代细胞,以生长分化因子5(GDF5)(100 ng/ml)和1庚醇(2.5 μmol/L)干预培养后,MTT法测定1庚醇对小鼠骨髓基质干细胞增殖的影响,RTPCR和免疫细胞化学法检测Ⅱ型胶原的表达,Western blotting检测Cx43蛋白的表达,阿尔辛蓝(Alcian)染色蛋白多糖。[结果]MTT结果显示2.5 μmol/L浓度1庚醇对小鼠MSCs的增殖不产生影响,对细胞没有毒性抑制作用;RTPCR和免疫细胞化学显示1庚醇能够抑制Ⅱ型胶原mRNA和蛋白的表达;Alcian染色结果显示1庚醇抑制分化细胞分泌蛋白多糖基质;Western blotting结果表明1庚醇对Cx43蛋白的表达没有作用。[结论]GDF5能够定向诱导小鼠骨髓基质干细胞向软骨方向分化,Cx43蛋白介导的缝隙连接细胞间通讯在GDF5诱导软骨分化中发挥着很重要的作用。 3 @7 k3 g3 x# O8 s3 c8 ? 【关键词】GDF-5 软骨 缝隙连接 小鼠 骨髓基质干细胞! Y4 @, E1 }! A) j% Z/ N: J
Effect of gap junctional blocker 1heptanol on chondrogenic differentiation of mouse bone marrow mesenchymal stem cells in vitro∥4 ~6 r8 Z7 l& g$ w/ k5 V% s7 P
. t, L& ]' d v6 T3 \ ZHANG Yukun,YANG Shuhua,YANG Cao,et al. % \, C, A" E) J8 k9 S4 U+ ~- z+ z% P& I8 j+ Z( F$ t! s6 I/ E4 [
Department of Orthopedics Affiliated to Union Hospital,Tongji Medical College of Huazhong Science and Technology University,Wuhan 430022,China% U1 K0 c, a4 }$ w+ v& c
* V1 Y6 i7 ` F1 `3 [- v 6 t' E; C1 h- T U * _# a/ T7 P! Y. _# @" Y) r( i( W Abstract:[Objective]To investigate the effect of gap junctional blocker 1heptanol on chondrogenic differentiation of bone marrow mesenchymal stem cells with the induction of GDF5 in vitro.[Method]The MSCs were isolated from mouse bone marrow and cultured in vitro.The cells in passage 3 were chosen to induce into chondrogenic differentiation with recombinant human GDF5(100 ng/ml)or 1 heptanol(2.5 μmol/L).The proliferation effect of 1heptanol on MSCs was investigated by MTT assay.Type Ⅱ collagen mRNA and protein were examined with RTPCR and immunocytochemistry respectively and the sulfate glycosaminoglycan was measured by Alcian blue.Connexin43 protein was examined with western blotting.[Result]MTT assay showed that 1heptanol had no effect on the proliferation of MSCs;RTDCR and immunocytochemistry all showed that 1heptanol could inhibit type Ⅱ collagen mRNA and protein alternatively expressed by MSCs with the intervene of GDF5 and 1 heptanol.Alcian blue staining revealed that 1heptanol could inhibit deposition of typical cartilage extracellular matrix promoted by recombinant GDF5.Western blotting demonstrated that 1 heptanol play no effect on the expression of connexin43.[Conclusion]These results suggest that mouse bone marrow mesencymal stem cells can be differentiated into chondrogenic phonotype with the induction of GDF5 in vitro,and that connexin43conaining gap junction cellular comunication plays an important role in chondrogenesis with the induction of GDF5.4 s" b4 g! x2 h( B0 P# [ p% A
" c1 _# j4 e2 \0 {6 V 总之,本研究初步探讨了GDF5与Cx43及其介导的缝隙连接细胞间通讯在体外软骨分化过程中的作用及联系,也说明GDF5在体内可以通过Cx43介导的缝隙连接细胞间通讯来实现对细胞聚集及软骨分化的调控,从而影响骨骼的发育过程。GDF5在骨骼发育和体外诱导软骨表型的机理还没有完全弄清楚,除了与Cx43介导的缝隙连接细胞间通讯功能的增强有关,细胞内信号Smad和MAP激酶途径也参与了软骨发生过程,相关的机理还需要进一步的研究。- ]) X& t# z4 E
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△基金项目:国家自然科学基金项目(No.30471753)作者: 科研人 时间: 2015-6-9 09:33