【摘要】 目的:观察转化生长因子β对体外培养人成骨细胞结缔组织生长因子mRNA表达的影响。方法:实验在青岛海慈医疗集团检验科完成。选择2005-02/03在青岛海慈医疗集团骨科住院的无其他疾患的骨折患者3例,年龄30～50岁,经患者知情同意后,取其髂骨松质骨,剪碎,Ⅳ型胶原酶消化后,将骨片贴于25cm2培养瓶,加入MEM培养液约15d后,可见细胞游出,约25d达汇片,胰酶消化,按1×106个细胞接种培养后第2天,换含1g/LBSAMEM培养液继续培养24h后,加入10ng/L转化生长因子β干预培养0,3,6,12,24h,或在转化生长因子β作用峰值时,分别给予不同浓度(0,5,10,25ng/L)干预,抽提总RNA,采用半定量反转录-聚合酶链反应和Northernblot法检测干预后成骨细胞结缔组织生长因子mRNA表达水平。结果:①半定量反转录-聚合酶链反应检测转化生长因子β对体外培养成骨细胞结缔组织生长因子mRNA表达的影响:与0ng/L对照组相比,5,10和25ng/L转化生长因子β干预后结缔组织生长因子mRNA表达水平显著升高(P<0.01),呈明显剂量依赖关系;10ng/L转化生长因子β干预6h时转化生长因子β上调作用达高峰(P<0.01),然后下降,至24h时转化生长因子β对结缔组织生长因子的上调作用基本消失(P>0.05)。②Northernblot法检测转化生长因子β对体外培养成骨细胞结缔组织生长因子mRNA表达的影响:与0ng/L对照组相比,10和25ng/L转化生长因子β干预后结缔组织生长因子mRNA表达水平显著升高(P<0.01),但无明显剂量依赖关系(P>0.05);10ng/L转化生长因子β干预6h时转化生长因子β上调作用达高峰(P<0.01),然后下降,至24h时转化生长因子β对结缔组织生长因子的上调作用基本消失(P>0.05)。结论:转化生长因子β可呈剂量依赖性上调人成骨细胞结缔组织生长因子mRNA的表达。更多还原

【Abstract】 AIM: To observe the effect of transforming growth factor β (TGF-β) on the gene expression of connective tissue growth factor (CTGF) in human osteoblasts cultured in vitro. METHODS: The experiment was conducted in the Clinical Laboratory of Qingdao Haici Hospital. Three bone fracture inpatients without other disease aged 30-50 years and admitted in the Department of Orthopedics, Qingdao Haici Hospital were selected. After the agreement of patients, surgical specimens without surrounding connective tissues were isolated from adult iliac bones. The obtained trabecular bones were cut into small pieces, and cultured in a 25 cm2 flask in MEM after digested by Ⅳ collagenase. Cells migrated from the bone particles 15 days later, and reached confluence by 25 days. The cells were then digested with 0.25% trypsin-EDTA and subcultured at a density of 1×106 in a 25 cm2 flask. After 2 days, the cells were subsequently exposed to phenol red-free alpha-MEM containing 0.1% BSA for 24 hours and then treated with 10 ng/L TGF-β for 0, 3, 6, 12 and 24 hours, respectively or with various concentrations (0, 5, 10 and 25 ng/L) TGF-β. The expression of CTGF mRNA in human osteoblasts after treated with TGF-β was examined with semiquantitive RT-PCR and Northern blot. RESULTS: ①Results of RT-PCR: Compared with 0 ng/L control group, 5, 10 and 25 ng/L TGF-β could up-regulate the expression of CTGF mRNA (P < 0.01), which showed a dose-dependent pattern; the up-regulating effect of 10 ng/L TGF-β reached the peak after 6 hours (P < 0.01), and gradually decreased until 24 hours (P > 0.05). ②Results of Northern blot: Compared with 0 ng/L control group, 10 and 25 ng/L TGF-β could up-regulate the expression of CTGF mRNA (P < 0.01), but without dose-dependent relationship (P > 0.05); the up-regulating effect of 10 ng/L TGF-β reached the peak after 6 hours (P < 0.01), and gradually decreased until 24 hours (P > 0.05). CONCLUSION: The expression of CTGF mRNA is up-regulated by TGF-β with dose-dependent manner.更多还原