中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2014年
5期
1098-1100
,共3页
郭珈宜%崔宏勋%郭马珑%郭艳幸%郭艳锦
郭珈宜%崔宏勛%郭馬瓏%郭豔倖%郭豔錦
곽가의%최굉훈%곽마롱%곽염행%곽염금
软骨分化%生长分化因子5%油酸酰胺%缝隙连接蛋白43
軟骨分化%生長分化因子5%油痠酰胺%縫隙連接蛋白43
연골분화%생장분화인자5%유산선알%봉극련접단백43
Chondrogenic differentiation%Growth differentiation factor 5%Oleamide%Comexin 43
目的 观察缝隙连接细胞间通讯(GJIC)在生长分化因子5(GDF-5)诱导人骨髓间充质干细胞(hMSCs)成软骨分化中的作用.方法 体外培养hMSCs,分为3组,分别用含0、100 μg/LGDF-5、100μg/L GDF-5+2.5μmoL/L油酸酰胺的软骨诱导液干预.采用噻唑蓝(MTT)法检测各组细胞24、48、72 h增殖情况.以5×109/L的细胞密度进行高密度培养,干预2周后采用逆转录-聚合酶链反应(RT-PCR)检测各组Ⅱ型胶原、缝隙连接蛋白43(Cx43)和蛋白多糖mRNA的表达.采用Western blot检测各组Ⅱ型胶原、Cx43和蛋白多糖蛋白的表达.应用甲苯胺蓝染色观察各组细胞外基质着色情况.结果 MTT法结果显示油酸酰胺组与对照组的平均吸光度值比较,差异无统计学意义(P＞0.05).GDF-5组Ⅱ型胶原、蛋白多糖和Cx43在mRNA水平上较对照组表达显著增强(1.00±0.03比0.40±0.12;1.00±0.07比0.32±0.02;1.00±0.02比0.45±0.01,P＜0.01);在蛋白水平上较对照组表达亦显著增强(1.50±0.11比0.53 ±0.07;1.70 ±0.14比0.52 ±0.04;1.00±0.12比0.50±0.06,P＜0.01);油酸酰胺组Ⅱ型胶原和蛋白多糖在mRNA水平上表达较对照组无明显变化,而Cx43在mRNA水平上表达升高(0.94 ±0.12比0.45±0.01,P＜0.01);油酸酰胺组Ⅱ型胶原和蛋白多糖在蛋白水平上表达明显减弱(0.42 ±0.04比0.53 ±0.07;0.38 ±0.06比0.52±0.04,P＜0.01),但Cx43在蛋白水平上表达较对照组无明显变化.甲苯胺蓝染色结果显示GDF-5组细胞外基质明显蓝染并呈异染性着色,油酸酰胺及对照组蓝染稍弱,着色程度差异无统计学意义(P＞0.05).结论 Cx43介导的GJIC在GDF6诱导的软骨分化过程中起着重要作用.
目的 觀察縫隙連接細胞間通訊(GJIC)在生長分化因子5(GDF-5)誘導人骨髓間充質榦細胞(hMSCs)成軟骨分化中的作用.方法 體外培養hMSCs,分為3組,分彆用含0、100 μg/LGDF-5、100μg/L GDF-5+2.5μmoL/L油痠酰胺的軟骨誘導液榦預.採用噻唑藍(MTT)法檢測各組細胞24、48、72 h增殖情況.以5×109/L的細胞密度進行高密度培養,榦預2週後採用逆轉錄-聚閤酶鏈反應(RT-PCR)檢測各組Ⅱ型膠原、縫隙連接蛋白43(Cx43)和蛋白多糖mRNA的錶達.採用Western blot檢測各組Ⅱ型膠原、Cx43和蛋白多糖蛋白的錶達.應用甲苯胺藍染色觀察各組細胞外基質著色情況.結果 MTT法結果顯示油痠酰胺組與對照組的平均吸光度值比較,差異無統計學意義(P＞0.05).GDF-5組Ⅱ型膠原、蛋白多糖和Cx43在mRNA水平上較對照組錶達顯著增彊(1.00±0.03比0.40±0.12;1.00±0.07比0.32±0.02;1.00±0.02比0.45±0.01,P＜0.01);在蛋白水平上較對照組錶達亦顯著增彊(1.50±0.11比0.53 ±0.07;1.70 ±0.14比0.52 ±0.04;1.00±0.12比0.50±0.06,P＜0.01);油痠酰胺組Ⅱ型膠原和蛋白多糖在mRNA水平上錶達較對照組無明顯變化,而Cx43在mRNA水平上錶達升高(0.94 ±0.12比0.45±0.01,P＜0.01);油痠酰胺組Ⅱ型膠原和蛋白多糖在蛋白水平上錶達明顯減弱(0.42 ±0.04比0.53 ±0.07;0.38 ±0.06比0.52±0.04,P＜0.01),但Cx43在蛋白水平上錶達較對照組無明顯變化.甲苯胺藍染色結果顯示GDF-5組細胞外基質明顯藍染併呈異染性著色,油痠酰胺及對照組藍染稍弱,著色程度差異無統計學意義(P＞0.05).結論 Cx43介導的GJIC在GDF6誘導的軟骨分化過程中起著重要作用.
목적 관찰봉극련접세포간통신(GJIC)재생장분화인자5(GDF-5)유도인골수간충질간세포(hMSCs)성연골분화중적작용.방법 체외배양hMSCs,분위3조,분별용함0、100 μg/LGDF-5、100μg/L GDF-5+2.5μmoL/L유산선알적연골유도액간예.채용새서람(MTT)법검측각조세포24、48、72 h증식정황.이5×109/L적세포밀도진행고밀도배양,간예2주후채용역전록-취합매련반응(RT-PCR)검측각조Ⅱ형효원、봉극련접단백43(Cx43)화단백다당mRNA적표체.채용Western blot검측각조Ⅱ형효원、Cx43화단백다당단백적표체.응용갑분알람염색관찰각조세포외기질착색정황.결과 MTT법결과현시유산선알조여대조조적평균흡광도치비교,차이무통계학의의(P＞0.05).GDF-5조Ⅱ형효원、단백다당화Cx43재mRNA수평상교대조조표체현저증강(1.00±0.03비0.40±0.12;1.00±0.07비0.32±0.02;1.00±0.02비0.45±0.01,P＜0.01);재단백수평상교대조조표체역현저증강(1.50±0.11비0.53 ±0.07;1.70 ±0.14비0.52 ±0.04;1.00±0.12비0.50±0.06,P＜0.01);유산선알조Ⅱ형효원화단백다당재mRNA수평상표체교대조조무명현변화,이Cx43재mRNA수평상표체승고(0.94 ±0.12비0.45±0.01,P＜0.01);유산선알조Ⅱ형효원화단백다당재단백수평상표체명현감약(0.42 ±0.04비0.53 ±0.07;0.38 ±0.06비0.52±0.04,P＜0.01),단Cx43재단백수평상표체교대조조무명현변화.갑분알람염색결과현시GDF-5조세포외기질명현람염병정이염성착색,유산선알급대조조람염초약,착색정도차이무통계학의의(P＞0.05).결론 Cx43개도적GJIC재GDF6유도적연골분화과정중기착중요작용.
Objective To investigate the effect of gap junctional blocker oleamide on growth differentiation factor 5 (GDF-5) induced chondrogenic differentiation.Methods hunan mesenchymal stem cells (hMSCs) were isolated and cultured in vitro,and the cells from Passage 3 were used in this study.hMSCs were randomly grouped into three according to the kinds of induction medium (0,100 μg/L GDF-5,100 μg/L GDF-5 + 2.5 μmol/L oleamide).The proliferation of hMSCs was investigated by methyl thiazol tetrazolium (MTT) assay.The cells were resuspended and the pellets were constructed by centrifugation at a density of 5 × 109/L,and then continued to be kept for two weeks.Reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect the expression of mRNA of type Ⅱ collagen,comexin 43 (Cx43)and aggrecan.Western blotting was performed to detect the expression of protein of type Ⅱ collagen,Cx43and aggrecan.Toluidine blue was performed to detect extracellular matrix staining.Results GDF-5 can promote the mRNA expression of type Ⅱ collagen,Cx43 and aggrecan (1.00 ± 0.03 vs.0.40 ± 0.12 ;1.00 ±0.07 vs.0.32 ±0.02; 1.00 ±0.02 vs.0.45 ±0.01,P ＜0.01),and the relative protein expression was also increased (1.50±0.11 vs.0.53 ±0.07; 1.70 ±0.14 vs.0.52 ±0.04; 1.00 ±0.12 vs.0.50±0.06,P＜0.01).While oleamide could up-regulate the expression of mRNA of Cx43 (0.94 ±0.12 vs.0.45 ±0.01,P ＜0.01),but had no effect on type Ⅱ collagen and aggrecan mRNA expression.Oleamide could down-regulate the expression of protein of type Ⅱ collagen and aggrecan,but had no effect onCx43 protein expression(0.42 ± 0.04 vs.0.53 ±0.07; 0.38 ±0.06 vs.0.52 ±0.04,P＜0.01).Toluidine blue staining showed that GDF-5 can promote the synthesis of aggrecan.Conclusion GDF-5 modulation of chondrogenesis involves gap junction-mediated intercellular communication.
