CAJ | 학술논문

目的探讨骨髓基质细胞(MSC)诱导分化为成骨细胞过程中各基因表达水平的变化,同时为合理选择及构建符合临床需求的组织细胞系在基因水平上寻求理论依据.方法无菌条件下分离SD大鼠MSC进行原代培养,根据培养体系不同,将其分为两组:①诱导分化组(6例),采用含浓度为1×10-8mol/L地塞米松,浓度为10 mmol/L β-甘油磷酸钠及浓度为50 mg/L维生素C的诱导性DMEM培养基进行诱导分化培养;②对照组(6例),采用DMEM培养基进行同步培养.通过倒置相差显微镜观察诱导分化组与对照组MSC的形态特征,并采用茜素红染色观察细胞的生长与分化情况.采用基因表达谱芯片杂交技术选出诱导分化组与对照组的表达水平存在差异的基因,并对差异基因进行生物学功能分析.结果 ①分离MSC进行原代培养.培养72 h后,MSC开始增殖,细胞形态多呈梭形、三角形或不规则细胞形态.继续培养6～7 d后,细胞逐渐形成散在的细胞集落,多呈成纤维细胞形态.培养10～12 d后,MSC集落间相互融合成单层细胞.②MSC传代培养,诱导分化组细胞与对照组相比细胞增殖速度缓慢,细胞形态多呈梭形或多角形,且逐渐向成骨细胞转变;培养10～12 d后,密集的细胞间出现较多散在的致密圆形透光性差的钙化结节;钙化结节经茜素红染色呈片状棕染,茜素红染色呈显著阳性.对照组细胞增殖迅速,多呈成纤维细胞形态生长,在常规培养14 d后未出现明显钙化结节.③诱导分化组细胞内表达水平发生改变的基因占总有效基因的27.7％,显著高于对照组MSC传代培养中表达水平发生改变的基因数,差异有统计学意义(P=0.01).诱导分化组细胞与原代细胞、对照组细胞基因表达水平的相关性较低(R2 =0.524 8,0.495 4),诱导分化组细胞表达水平发生改变的基因数目增多.诱导分化组与对照组MSC表达水平存在＞3倍差异的部分已知基因,包括参与基因表达或蛋白质合成、修饰、加工,跨膜运输与胞内膜泡运输、细胞外基质与黏附分子、细胞通讯与信号传导、细胞骨架及代谢功能相关的6大类基因,其中表达水平上调的基因为120个,下调的基因为17个.结论 MSC诱导分化为成骨细胞的过程中,细胞形态向成骨细胞转变,且与细胞生长,代谢及骨形成相关的基因表达水平发生改变,为后续构建符合临床需要的成骨细胞系在基因水平上提供理论依据. 目的探討骨髓基質細胞(MSC)誘導分化為成骨細胞過程中各基因錶達水平的變化,同時為閤理選擇及構建符閤臨床需求的組織細胞繫在基因水平上尋求理論依據.方法無菌條件下分離SD大鼠MSC進行原代培養,根據培養體繫不同,將其分為兩組:①誘導分化組(6例),採用含濃度為1×10-8mol/L地塞米鬆,濃度為10 mmol/L β-甘油燐痠鈉及濃度為50 mg/L維生素C的誘導性DMEM培養基進行誘導分化培養;②對照組(6例),採用DMEM培養基進行同步培養.通過倒置相差顯微鏡觀察誘導分化組與對照組MSC的形態特徵,併採用茜素紅染色觀察細胞的生長與分化情況.採用基因錶達譜芯片雜交技術選齣誘導分化組與對照組的錶達水平存在差異的基因,併對差異基因進行生物學功能分析.結果 ①分離MSC進行原代培養.培養72 h後,MSC開始增殖,細胞形態多呈梭形、三角形或不規則細胞形態.繼續培養6～7 d後,細胞逐漸形成散在的細胞集落,多呈成纖維細胞形態.培養10～12 d後,MSC集落間相互融閤成單層細胞.②MSC傳代培養,誘導分化組細胞與對照組相比細胞增殖速度緩慢,細胞形態多呈梭形或多角形,且逐漸嚮成骨細胞轉變;培養10～12 d後,密集的細胞間齣現較多散在的緻密圓形透光性差的鈣化結節;鈣化結節經茜素紅染色呈片狀棕染,茜素紅染色呈顯著暘性.對照組細胞增殖迅速,多呈成纖維細胞形態生長,在常規培養14 d後未齣現明顯鈣化結節.③誘導分化組細胞內錶達水平髮生改變的基因佔總有效基因的27.7％,顯著高于對照組MSC傳代培養中錶達水平髮生改變的基因數,差異有統計學意義(P=0.01).誘導分化組細胞與原代細胞、對照組細胞基因錶達水平的相關性較低(R2 =0.524 8,0.495 4),誘導分化組細胞錶達水平髮生改變的基因數目增多.誘導分化組與對照組MSC錶達水平存在＞3倍差異的部分已知基因,包括參與基因錶達或蛋白質閤成、脩飾、加工,跨膜運輸與胞內膜泡運輸、細胞外基質與黏附分子、細胞通訊與信號傳導、細胞骨架及代謝功能相關的6大類基因,其中錶達水平上調的基因為120箇,下調的基因為17箇.結論 MSC誘導分化為成骨細胞的過程中,細胞形態嚮成骨細胞轉變,且與細胞生長,代謝及骨形成相關的基因錶達水平髮生改變,為後續構建符閤臨床需要的成骨細胞繫在基因水平上提供理論依據.
목적 탐토골수기질세포(MSC)유도분화위성골세포과정중각기인표체수평적변화,동시위합리선택급구건부합림상수구적조직세포계재기인수평상심구이론의거.방법 무균조건하분리SD대서MSC진행원대배양,근거배양체계불동,장기분위량조:①유도분화조(6례),채용함농도위1×10-8mol/L지새미송,농도위10 mmol/L β-감유린산납급농도위50 mg/L유생소C적유도성DMEM배양기진행유도분화배양;②대조조(6례),채용DMEM배양기진행동보배양.통과도치상차현미경관찰유도분화조여대조조MSC적형태특정,병채용천소홍염색관찰세포적생장여분화정황.채용기인표체보심편잡교기술선출유도분화조여대조조적표체수평존재차이적기인,병대차이기인진행생물학공능분석.결과 ①분리MSC진행원대배양.배양72 h후,MSC개시증식,세포형태다정사형、삼각형혹불규칙세포형태.계속배양6～7 d후,세포축점형성산재적세포집락,다정성섬유세포형태.배양10～12 d후,MSC집락간상호융합성단층세포.②MSC전대배양,유도분화조세포여대조조상비세포증식속도완만,세포형태다정사형혹다각형,차축점향성골세포전변;배양10～12 d후,밀집적세포간출현교다산재적치밀원형투광성차적개화결절;개화결절경천소홍염색정편상종염,천소홍염색정현저양성.대조조세포증식신속,다정성섬유세포형태생장,재상규배양14 d후미출현명현개화결절.③유도분화조세포내표체수평발생개변적기인점총유효기인적27.7％,현저고우대조조MSC전대배양중표체수평발생개변적기인수,차이유통계학의의(P=0.01).유도분화조세포여원대세포、대조조세포기인표체수평적상관성교저(R2 =0.524 8,0.495 4),유도분화조세포표체수평발생개변적기인수목증다.유도분화조여대조조MSC표체수평존재＞3배차이적부분이지기인,포괄삼여기인표체혹단백질합성、수식、가공,과막운수여포내막포운수、세포외기질여점부분자、세포통신여신호전도、세포골가급대사공능상관적6대류기인,기중표체수평상조적기인위120개,하조적기인위17개.결론 MSC유도분화위성골세포적과정중,세포형태향성골세포전변,차여세포생장,대사급골형성상관적기인표체수평발생개변,위후속구건부합림상수요적성골세포계재기인수평상제공이론의거.
Objective To investigate gene expression levels in the process of marrow stromal cell (MSC) differentiation,and to seek theoretical basis for making cell lines which are more suitable for clinical use at genetic level.Methods MSC of SD rats were isolated and cultured.According to cell culture system,cultured MSC were divided into two groups.① Induced differentiation group (6 cases) were cultured in DMEM media adding 1 × 10 8 mol/L dexamethasone,10 mmol/L beta-glycerin sodium phosphate and 50 mg/L vitamin C.② Control group (6 cases) were cultured in DMEM media synchronously.Morphological characteristics of MSC in two groups were observed by inverted phase contrast microscope,and growth and differentiation of MSC were tested by alizarin red staining.Differential gene of two groups were screened by microarray,and the function of differential gene were analyzed.Results ① MSC were successfully isolated.After 72 h,MSC began to proliferate and cell morphology began changing to fusiform,triangular or irregular shape.After 6 to 7 d,cells gradually formatted dispersed cell colony.Cell colonies fused into a single layer of cells after 10 to 12 d.② MSC proliferation of induced differentiation group was slower than control group.After induction,MSC mainly shaped as spindle and polygon,and gradually transformed into osteoblasts.After 10 to 12 d,scattered dense circular calcified nodules with poor transparency were appeared in dense cells.The alizarin red staining shows calcified nodules appeared flaky brown dyed with a significant positive response.MSC proliferation of control group was rapid and no calcified nodule was appeared after 14 d.③ The number of genes with expression level differences in induced differentiation group were accounted for 27.7％ of total effective gene,and were far higher than those in control group,and the difference was statistically significant (P=0.01).The correlation of gene expression levels between induced differentiation group and primary MSC,control group were low(R2 =0.524 8,0.495 4).Genes with expression level difference were increased in induced differentiation group.Genes with expression level difference which were greater than 3 times between induced differentiation group and control group were involved in gene expression and protein synthesis,modification,processing,transmembrane transport and intracellular membrane vesicle transport,extracellular matrix and adhesion molecule,cell communication and signal transduction,cytoskeletal and metabolic function.Among those differential genes,expression levels of 120 genes were upregulated,and 17 genes were downregulated.Conclusions MSC transformed into osteoblasts in the process of induced differentiation.Expression levels of genes which were associated with cell growth,metabolism and bone formation were changed.And theoretical basis for making osteoblast cell lines which met clinical needs at genetic level was provided.