体外条件下TGF-β3、BMP-2和DEX诱导兔滑膜间充质干细胞向软骨细胞谱系分化的研究

Induction of rabbit synovial-derived mesenchymal stem cells into the chondrocyte lineage with the synergic stimulation of TGF-β3,BMP-2 and DEX in vitro

目的本实验拟将分离纯化得到的滑膜间充质干细胞(synovial-derived mesenchymal stem cells,SMSCs)在体外培养条件下进行成软骨刺激诱导,并从转录和翻译两个水平寻找进入软骨细胞分化谱系的证据,进而判断转化生长因子β(transforming growth factor-β,TGF-β)、骨形态发生蛋白(bone morphogenetic protein-2,BMP-2)和地塞米松(dexamethasone,DEX)诱导SMSCs进入软骨细胞分化谱系。方法贴壁法分离纯化得到SMSCs,在体外培养条件下用含500 ng/ml BMP-2、10 ng/ml TGF-β3、10^(-7)M DEX的高糖DMEM培养基进行刺激诱导,并在倒置相差显微镜下观察分化过程中其形态学的变化,以RTPCR检测Ⅰ、Ⅱ型胶原及软骨特异性Aggrecan(AGN)的mRNA表达,细胞免疫荧光化学染色的方法检测细胞分化过程中Ⅰ、Ⅱ型胶原的表达,碱性甲苯胺蓝细胞化学染色检测软骨特异性GAG的表达,证实SMSCs的诱导成软骨作用。结果 SMSCs在前述诱导条件下,诱导后14天细胞逐渐由小梭形变为多角形、类软骨细胞样形态,RT-PCR可以检测到Ⅰ、Ⅱ型胶原及AGN基因的表达,细胞免疫荧光化学染色Ⅰ、Ⅱ型胶原、碱性甲苯胺蓝细胞化学染色结果呈阳性。而未经诱导的SMSCs形态基本保持梭形,基因表达和染色呈阴性,两组间差异显著。细胞免疫荧光化学染色分析SMSCs在软骨诱导培养基中诱导后14天表达Ⅰ、Ⅱ型胶原;未经诱导的SMSCs不表达Ⅰ、Ⅱ型胶原。说明SMSCs在软骨诱导培养基中诱导后14天进入软骨细胞分化谱系,可作为种子细胞在同样的诱导条件下向软骨分化。结论 SMSCs作为新的MSCs家族成员,显示出与BMSCs相似的多向分化潜能,500 ng/ml/BMP-2、10 ng/ml TGF-β、10^(-7)MDEX的高糖DMEM培养基中培养后14天,SMSCs已进入软骨细胞分化谱系。SMSCs可作为半月板组织工程的种子细胞。

Objective To explore that synovial-derived mesenchymal stem cells （ SMSCs ） differentiated into the chondrocyte lineage induced by transforming growth factor-β3 （ TGF-β3 ）, bone morphogenetic protein-2 （ BMP-2 ）, and dexamethasone （ DEX ） in vitro culture conditions. After isolation and purification, SMSCs were induced to chondrocytes, which was certiifed on the transcriptional and translational levels. Methods SMSCs were obtained after isolation and purification by the adherence method, which were induced in high glucose Dulbecco modiifed Eagle medium （ DMEM ） containing 500 ng/ml BMP-2, 10 ng/ml TGF-β3 and 10-7 M DEX in vitro culture conditions. The morphologic changes during the differentiation process were observed using the inverted phase contrast microscope, and collagen I, collagen II and messenger-Ribonucleic Acid （ m-RNA ） expressions of cartilage-specific Aggrecan （ AGN ） were detected by Reverse Transcription-Polymerase Chain Reaction （ RT-PCR ）. The immunolfuorescence staining was used to detect the expressions of collagen I and II during the differentiation process, and the alkaline toluidine blue staining was used to detect the expressions of cartilage-speciifc group-speciifc antigen gene （ GAG ） to conifrm the capability of SMSCs to differentiate into the chondrocyte lineage. Results After 14 days’ induction, the small spindle SMSCs were gradually changed into the polygonal morphology, just like the chondrocytes.Collagen I, collagen II and expressions of AGN could be detected by RT-PCR. The results of immunolfuorescence staining of collagen I and II and alkaline toluidine blue staining were positive. Without the induction, SMSCs basically maintained the spindle-shape morphology, and the gene expressions and staining results were negative. The differences between them were statistically signiifcant. Based on the results of immunolfuorescence staining, SMSCs expressed collagen I and II after 14 days’ induction in DMEM. Without the induction, SMSCs did not express collagen I or II. It was illustrated that SMSCs differentiated into the chondrocyte lineage after 14 days’ induction in DMEM, which could be used as the seed cells to differentiate into chondrocytes under the same condition. Conclusions As a new member in the family of mesenchymal stem cells （ MSCs ）, SMSCs show the multi-differentiation potential which is similar to that of bone marrow-derived mesenchymal stem cells （ BMSCs ）. After induced in the high glucose DMEM containing 500 ng/ml BMP-2, 10 ng/ml TGF-β3 and 10-7 M DEX, SMSCs enter into the chondrocyte lineage at the 14th day, which can be used as the seed cells for the meniscus tissue engineering.