中国组织工程研究
中國組織工程研究
중국조직공정연구
Journal of Clinical Rehabilitative Tissue Engineering Research
2013年
40期
7028-7033
,共6页
焦淑贤%胡彬%赵林%刘晓华%冯智慧
焦淑賢%鬍彬%趙林%劉曉華%馮智慧
초숙현%호빈%조림%류효화%풍지혜
干细胞%骨髓干细胞%骨髓间充质干细胞%Ca2+%肝细胞生长因子%细胞因子%细胞分化%ERK%干细胞图片文章
榦細胞%骨髓榦細胞%骨髓間充質榦細胞%Ca2+%肝細胞生長因子%細胞因子%細胞分化%ERK%榦細胞圖片文章
간세포%골수간세포%골수간충질간세포%Ca2+%간세포생장인자%세포인자%세포분화%ERK%간세포도편문장
背景：骨髓间充质干细胞增殖和分化中的具体机制仍不清楚，Ca2+信号、骨髓间充质干细胞增殖与分化信号如何协调和交叉成复杂信号网络等问题仍有待研究阐明。<br> 目的：探讨细胞内Ca2+在骨髓间充质干细胞向肝细胞定向诱导分化过程中的作用。<br> 方法：用全骨髓贴壁法从大鼠骨髓中分离骨髓间充质干细胞后进行纯化和扩增，并加入肝细胞生长因子诱导骨髓间充质干细胞向肝细胞分化，应用流式细胞技术分别检测肝细胞生长因子诱导分化骨髓间充质干细胞和对照骨髓间充质干细胞内游离[Ca2+]i。将不同浓度的尼莫地平加入肝细胞生长因子诱导的骨髓间充质干细胞(肝细胞生长因子组)培养液中进行干预后分为3组：肝细胞生长因子+尼莫地平10 mg/L组、肝细胞生长因子+尼莫地平50 mg/L组、肝细胞生长因子+尼莫地平100 mg/L组，在倒置相差显微镜下观察细胞生长情况并用免疫细胞化学法检测AAT的表达；并用RT-PCR检测对照组和尼莫地平干预组钙调蛋白mRNA的表达，免疫印迹法检测上述各组磷酸化ERK的表达。<br> 结果与结论：①加入肝细胞生长因子诱导分化的骨髓间充质干细胞内[Ca2+]i显著高于对照组(P<0.05)。②加入较大剂量的尼莫地平干预后，未见分化细胞且骨髓间充质干细胞的生长状态差；肝细胞生长因子+尼莫地平各组表达AAT的阳性细胞很少。③与对照组比较，肝细胞生长因子组和肝细胞生长因子+尼莫地平10 mg/L组钙调蛋白mRNA表达显著增加了(P<0.05)，肝细胞生长因子+尼莫地平50 mg/L组、肝细胞生长因子+尼莫地平100 mg/L组与对照组比较组间差异无显著性意义(P>0.05)。说明Ca2+不仅参与细胞因子诱导骨髓间充质干细胞向肝细胞的定向分化，而且也参与维持骨髓间充质干细胞的存活和增殖。
揹景：骨髓間充質榦細胞增殖和分化中的具體機製仍不清楚，Ca2+信號、骨髓間充質榦細胞增殖與分化信號如何協調和交扠成複雜信號網絡等問題仍有待研究闡明。<br> 目的：探討細胞內Ca2+在骨髓間充質榦細胞嚮肝細胞定嚮誘導分化過程中的作用。<br> 方法：用全骨髓貼壁法從大鼠骨髓中分離骨髓間充質榦細胞後進行純化和擴增，併加入肝細胞生長因子誘導骨髓間充質榦細胞嚮肝細胞分化，應用流式細胞技術分彆檢測肝細胞生長因子誘導分化骨髓間充質榦細胞和對照骨髓間充質榦細胞內遊離[Ca2+]i。將不同濃度的尼莫地平加入肝細胞生長因子誘導的骨髓間充質榦細胞(肝細胞生長因子組)培養液中進行榦預後分為3組：肝細胞生長因子+尼莫地平10 mg/L組、肝細胞生長因子+尼莫地平50 mg/L組、肝細胞生長因子+尼莫地平100 mg/L組，在倒置相差顯微鏡下觀察細胞生長情況併用免疫細胞化學法檢測AAT的錶達；併用RT-PCR檢測對照組和尼莫地平榦預組鈣調蛋白mRNA的錶達，免疫印跡法檢測上述各組燐痠化ERK的錶達。<br> 結果與結論：①加入肝細胞生長因子誘導分化的骨髓間充質榦細胞內[Ca2+]i顯著高于對照組(P<0.05)。②加入較大劑量的尼莫地平榦預後，未見分化細胞且骨髓間充質榦細胞的生長狀態差；肝細胞生長因子+尼莫地平各組錶達AAT的暘性細胞很少。③與對照組比較，肝細胞生長因子組和肝細胞生長因子+尼莫地平10 mg/L組鈣調蛋白mRNA錶達顯著增加瞭(P<0.05)，肝細胞生長因子+尼莫地平50 mg/L組、肝細胞生長因子+尼莫地平100 mg/L組與對照組比較組間差異無顯著性意義(P>0.05)。說明Ca2+不僅參與細胞因子誘導骨髓間充質榦細胞嚮肝細胞的定嚮分化，而且也參與維持骨髓間充質榦細胞的存活和增殖。
배경：골수간충질간세포증식화분화중적구체궤제잉불청초，Ca2+신호、골수간충질간세포증식여분화신호여하협조화교차성복잡신호망락등문제잉유대연구천명。<br> 목적：탐토세포내Ca2+재골수간충질간세포향간세포정향유도분화과정중적작용。<br> 방법：용전골수첩벽법종대서골수중분리골수간충질간세포후진행순화화확증，병가입간세포생장인자유도골수간충질간세포향간세포분화，응용류식세포기술분별검측간세포생장인자유도분화골수간충질간세포화대조골수간충질간세포내유리[Ca2+]i。장불동농도적니막지평가입간세포생장인자유도적골수간충질간세포(간세포생장인자조)배양액중진행간예후분위3조：간세포생장인자+니막지평10 mg/L조、간세포생장인자+니막지평50 mg/L조、간세포생장인자+니막지평100 mg/L조，재도치상차현미경하관찰세포생장정황병용면역세포화학법검측AAT적표체；병용RT-PCR검측대조조화니막지평간예조개조단백mRNA적표체，면역인적법검측상술각조린산화ERK적표체。<br> 결과여결론：①가입간세포생장인자유도분화적골수간충질간세포내[Ca2+]i현저고우대조조(P<0.05)。②가입교대제량적니막지평간예후，미견분화세포차골수간충질간세포적생장상태차；간세포생장인자+니막지평각조표체AAT적양성세포흔소。③여대조조비교，간세포생장인자조화간세포생장인자+니막지평10 mg/L조개조단백mRNA표체현저증가료(P<0.05)，간세포생장인자+니막지평50 mg/L조、간세포생장인자+니막지평100 mg/L조여대조조비교조간차이무현저성의의(P>0.05)。설명Ca2+불부삼여세포인자유도골수간충질간세포향간세포적정향분화，이차야삼여유지골수간충질간세포적존활화증식。
BACKGROUND:The mechanism of differentiation and proliferation of bone marrow-derived mesenchymal stem cells remains unclear. In addition, issues such as how signal pathways such as Ca2+and bone marrow-derived mesenchymal stem cellproliferation and differentiation signals form complex signal network remain poorly understood. <br> OBJECTIVE:To investigate the effect of Ca2+in the induced differentiation of bone marrow-derived mesenchymal stem cells into hepatocytes. <br> METHODS:Bone marrow-derived mesenchymal stem cells were isolated from rat bone marrow using whone bone marrow adherence method, purified, amplified, and induced with hepatocyte growth factor. [Ca2+]i in the directional differentiated bone marrow-derived mesenchymal stem cells and control bone marrow-derived mesenchymal stem cells were detected with flow cytometry. Bone marrow-derived mesenchymal stem cells induced with hepatocyte growth factor were mixed with nimodipine of different concentration, and cells were divided into three groups:hepatocyte growth factor+nimodipine 10 mg/L, 50 or 100 mg/L groups. cellgrowth was observed with inverted phase contrast microscope and alpha 1-antitrypsin expression of the cells was confirmed by immunocytochemistry. The calcineurin M and the activation of extracellular signal regulated kinase pathway was detected by reverse transcription-PCR and western blotting, respectively. <br> RESULTS AND CONCLUSION:[Ca2+]i in the directional differentiated bone marrow-derived mesenchymal stem cells was higher than in the control group (P<0.05). After addition of a larger dose of nimodipine, no differentiation of cells was obeserved and growth of bone marrow-derived mesenchymal stem cells was getting worse. There were few alpha 1-antitrypsin positive cells in the nimodipine groups. Calcineurin Mexpression was significantly increased in directional differentiated bone marrow-derived mesenchymal stem cells and smal dose of nimodipine than the controls (P<0.05). However, no significant difference was found among middle, high dose nimodipine and control groups (P>0.05). These findings indicate that Ca2+could participate in the differentiation of bone marrow-derived mesenchymal stem cells into hepatocytes incuded with cytokines, and also maintain the survival and proliferation of bone marrow-derived mesenchymal stem cells.
