CAJ | 학술논문

目的探讨通过条件培养液(CM)和高浓度血清即20%的胎牛血清(FBS)联合作用下,myocardin在骨髓来源的间充质干细胞向平滑肌细胞(SMC)定向分化过程中的表达特点.方法采用伞骨髓贴壁法分离骨髓问充质干细胞,CM联合20%FBS诱导骨髓间充质干细胞,同时设立持续10%FBS、单20%FBS及单CM诱导下的骨髓间充质十细胞对照组和SMC阳性对照组,分别于诱导前及诱导3、7、10和14 d时观察细胞形态的变化,并在相应的时间点用免疫荧光法、逆转录聚合酶链反应法、Western blot半定量分析法榆测myocardin以及SMC表面各种标志基因的表达变化,用透射电镜检测诱导后细胞内肌丝存在以此来证实诱导分化成功.结果光镜观察显示诱导前至诱导3、7、10和14 d时,骨髓问允质十细胞南纺锤形、多角形、扁平形和小圆形等多种形态逐渐转变为单一的长梭形,在诱导21 d后显示出明显的峰-谷特征.免疫荧光结果显示表达myocardin的阳性细胞数量逐渐增多,表达部位由胞质逐渐移向胞核,而表达平滑肌标志基因Ot一平滑肌肌动蛋白(Ot.SMA)、平滑肌肌动蛋白重链(SM-MHC)的细胞也随之增加,同时联合诱导后3、7、lO和14 d表达myocardin和平滑肌标志基因(α-SMA或SM-MHC)的双阳性细胞数最逐渐增加.RT-PCR结果显示myocardin的mRNA表达逐渐上调,在诱导7 d达到高峰后表达基本稳定(P<0.05);平滑肌标志基因α-SMA及SM22α的表达随着myocardin表达上调而增加(r=O.81,P<0.05),诱导10 d后表达基本稳定.Western blot结果显示myocardin蛋白和平滑肌标志基因α-SMA蛋白表达量在诱导后明显增加(r=0.84,P<0.05).透射电镜结果显示诱导21 d后的分化细胞中有肌丝的存在.在不同时间点各组之间的差异有统计学意义(P<0.05).结论 CM联合高浓度的血清可有效诱导骨髓间充质干细胞向SMC分化,myocardin在此分化过程中起了重要作用.骨髓来源的间充质干细胞可以在诱导下定向分化成为SMC. 目的探討通過條件培養液(CM)和高濃度血清即20%的胎牛血清(FBS)聯閤作用下,myocardin在骨髓來源的間充質榦細胞嚮平滑肌細胞(SMC)定嚮分化過程中的錶達特點.方法採用傘骨髓貼壁法分離骨髓問充質榦細胞,CM聯閤20%FBS誘導骨髓間充質榦細胞,同時設立持續10%FBS、單20%FBS及單CM誘導下的骨髓間充質十細胞對照組和SMC暘性對照組,分彆于誘導前及誘導3、7、10和14 d時觀察細胞形態的變化,併在相應的時間點用免疫熒光法、逆轉錄聚閤酶鏈反應法、Western blot半定量分析法榆測myocardin以及SMC錶麵各種標誌基因的錶達變化,用透射電鏡檢測誘導後細胞內肌絲存在以此來證實誘導分化成功.結果光鏡觀察顯示誘導前至誘導3、7、10和14 d時,骨髓問允質十細胞南紡錘形、多角形、扁平形和小圓形等多種形態逐漸轉變為單一的長梭形,在誘導21 d後顯示齣明顯的峰-穀特徵.免疫熒光結果顯示錶達myocardin的暘性細胞數量逐漸增多,錶達部位由胞質逐漸移嚮胞覈,而錶達平滑肌標誌基因Ot一平滑肌肌動蛋白(Ot.SMA)、平滑肌肌動蛋白重鏈(SM-MHC)的細胞也隨之增加,同時聯閤誘導後3、7、lO和14 d錶達myocardin和平滑肌標誌基因(α-SMA或SM-MHC)的雙暘性細胞數最逐漸增加.RT-PCR結果顯示myocardin的mRNA錶達逐漸上調,在誘導7 d達到高峰後錶達基本穩定(P<0.05);平滑肌標誌基因α-SMA及SM22α的錶達隨著myocardin錶達上調而增加(r=O.81,P<0.05),誘導10 d後錶達基本穩定.Western blot結果顯示myocardin蛋白和平滑肌標誌基因α-SMA蛋白錶達量在誘導後明顯增加(r=0.84,P<0.05).透射電鏡結果顯示誘導21 d後的分化細胞中有肌絲的存在.在不同時間點各組之間的差異有統計學意義(P<0.05).結論 CM聯閤高濃度的血清可有效誘導骨髓間充質榦細胞嚮SMC分化,myocardin在此分化過程中起瞭重要作用.骨髓來源的間充質榦細胞可以在誘導下定嚮分化成為SMC.
목적 탐토통과조건배양액(CM)화고농도혈청즉20%적태우혈청(FBS)연합작용하,myocardin재골수래원적간충질간세포향평활기세포(SMC)정향분화과정중적표체특점.방법 채용산골수첩벽법분리골수문충질간세포,CM연합20%FBS유도골수간충질간세포,동시설립지속10%FBS、단20%FBS급단CM유도하적골수간충질십세포대조조화SMC양성대조조,분별우유도전급유도3、7、10화14 d시관찰세포형태적변화,병재상응적시간점용면역형광법、역전록취합매련반응법、Western blot반정량분석법유측myocardin이급SMC표면각충표지기인적표체변화,용투사전경검측유도후세포내기사존재이차래증실유도분화성공.결과 광경관찰현시유도전지유도3、7、10화14 d시,골수문윤질십세포남방추형、다각형、편평형화소원형등다충형태축점전변위단일적장사형,재유도21 d후현시출명현적봉-곡특정.면역형광결과현시표체myocardin적양성세포수량축점증다,표체부위유포질축점이향포핵,이표체평활기표지기인Ot일평활기기동단백(Ot.SMA)、평활기기동단백중련(SM-MHC)적세포야수지증가,동시연합유도후3、7、lO화14 d표체myocardin화평활기표지기인(α-SMA혹SM-MHC)적쌍양성세포수최축점증가.RT-PCR결과현시myocardin적mRNA표체축점상조,재유도7 d체도고봉후표체기본은정(P<0.05);평활기표지기인α-SMA급SM22α적표체수착myocardin표체상조이증가(r=O.81,P<0.05),유도10 d후표체기본은정.Western blot결과현시myocardin단백화평활기표지기인α-SMA단백표체량재유도후명현증가(r=0.84,P<0.05).투사전경결과현시유도21 d후적분화세포중유기사적존재.재불동시간점각조지간적차이유통계학의의(P<0.05).결론 CM연합고농도적혈청가유효유도골수간충질간세포향SMC분화,myocardin재차분화과정중기료중요작용.골수래원적간충질간세포가이재유도하정향분화성위SMC.
Objective To investigate the expression profiles of myoeardin gene during the differentiation of bone marrow-derived mesenchymal stem cell to smooth muscle cells in the conditional medium combiued with a high concentration of fetal bovine serum (FBS). Methods Marrow-derived mesenchymal stem cells were isolated and purified from mouse femoral bone and shinbones using differential adherent methods. Cells at the third passage were induced by 20% FBS in conditioned medium, conditioned medium alone, 20% FBS or 10% FBS alone respectively. Mouse aortic smooth muscle cells were cultured as the positive control. Levels of mRNA and protein expression of myoeardin and several smooth muscle cells marker genes were determined by immumofluorescenee, RT-PCR and Western blot before and 3, 7, 10, 14d after the induction. The presence of smooth muscle myofilaments was detected by using transmission electron microscope. Results Naive bone marrow-derivod mesenchymal stem ceils displayed multiple morphological forms including fusiform, polygon, oval, and micro-spherical, as compared to the single macro-spindle form after the induction. Typical appearance of peak valley was displayed on the 21st day after induction. At the same time, the expression of smooth muscle marker genes was reinforced along with an up-regulation of myocardin expression, lmmunofluoreecence study showed that the cells expressing myocardin and smooth muscle marker genes such as α-SMA and SM-MHC increased. Fluorescence domain of myocardin translocated from cytoplasm to nucleus and the amounts of double positive cells for myecardin with α-SMA or SM-MHC also increased. RT-PCR confirmed that the mRNA expression of myocardin increased gradually and remained stabilized after achieving its peak on the 7th day after induction. The expression of smooth muscle marker genes, α-SMA and SM22α, remained stable on the 10th day of induction. It was also confirmed by Western blot that the protein expression of both myocardin and α-SMA were markedly increased during the induction. Finally, transmission electron microscopy revealed the presence of mynfilament on the 21st day after induction. Conclusions Bone marrow-derived mesenchymal stem cells can be effectively induced into smooth muscle-like cells by conditioned medium combined with 20% FBS. Myocardin plays an important role in the differentiation process of bone marrow-derived mesenchymal stem cells to the peripheral smooth muscle cells.