肝细胞生长因子诱导前软骨干细胞向软骨细胞的分化

Hepatocyte growth factor induces the differentiation of precartilaginous stem cells into chondrocytes

目的:探讨肝细胞生长因子诱导前软骨干细胞向软骨细胞分化的机制。方法:实验于2005-12/2006-03在河南省正骨研究院完成。①实验材料:普通级新生兔10只,平均体质量3kg。②实验干预:采用免疫磁珠分离技术分离新生兔长骨干骺端前软骨干细胞。利用肝细胞生长因子(浓度分别为0.5,1,2,4μg/L)作用前软骨干细胞;另以2μg/L肝细胞生长因子作用前软骨干细胞,对照用相同剂量的培养基,检测前软骨干细胞Ⅱ型胶原及Ⅱ型胶原mRNA的表达。③实验评估:采用光学显微镜观察前软骨干细胞形态及生长情况;四甲基偶氮唑盐比色法检测前软骨干细胞的增殖;免疫荧光检测Ⅱ型胶原表达;反转录-聚合酶链反应检测Ⅱ型胶原mRNA的表达。结果:①前软骨干细胞形态及生长情况:分离纯化的前软骨干细胞贴壁稳定,在光学显微镜下呈多角和梭形,生长旺盛,曲光度好。②前软骨干细胞增殖情况:细胞增殖率上升,在24,48,72h内呈时间浓度依赖。③Ⅱ型胶原表达:肝细胞生长因子作用第5天前软骨干细胞出现Ⅱ型胶原蛋白的表达。④Ⅱ型胶原mRNA的表达:在肝细胞生长因子刺激3d开始出现表达,并且5d,7d逐渐增加。结论:肝细胞生长因子作用前软骨干细胞后,出现软骨特征标志抗原Ⅱ型胶原的表达,细胞增殖活性上升,表明肝细胞生长因子能诱导前软骨干细胞向软骨细胞方向分化。其机制可能是前软骨干细胞也存在肝细胞生长因子受体。

AIM： To explore the mechanism of hepatocyte growth factor （HGF） to induce precartilaginous stem cells （PSCs） into chondrocytes. METHODS： The experiment was conducted in the Henan Research Institute of Orthopedics from December 2005 to March 2006. PSCs were isolated from the metaphysis of long bone of ten neonatal rabbits （average body mass of 3 kg） by immunomagnetic beads coasted with the second antibody. MTT assay was performed to observe the proliferation of PSCs cultured with HGF at concentrations of （0.5, 1, 2, and 4 μg/L）; As observing the effect of HGF on differentiation toward chondrocytes, 2 μg/L HGF was co-cultured with PSCs and the cells in control group was cultured with medium. The morphous and proliferation were observed by light microscope; the expression of type Ⅱ collagen was observed by immunofluorescence to observe, and expression of type Ⅱ collagen mRNA was determined by RT-PCR. RESULTS： （1）After isolated and purified, PSCs stably attached the culture flask, and presented polygon and fusiform shape, with good growth condition and luminosity by light microscope study. （2）The proliferation of PSCs was promoted by HGF in a dose- and time-dependent manner in 24, 48, and 72 hours. （3）Expression of type Ⅱ collagen protein appeared on the fifth day by HGF. （4）Expression of type Ⅱ collagen mRNA appeared on the third day induced by HGF, and was increasing in five and seven days. CONCLUSION： Expression of type Ⅱ collagen is found in PSCs induced by HGF, and the proliferation of PSCs is promoted. It indicates that HGF can induce PSCs to differentiate toward chondrocytes, which may be because there is HGF receptor in PSCs.