转化生长因子-β_1对大鼠脑膜间皮细胞结缔组织因子表达的影响

Effect of transforming growth factor beta-1 on expression of connective tissue growth factor in rat leptomeningeal mesothelial cells

目的观察转化生长因子-β1(TGF-β1)对大鼠软脑膜间皮细胞(RLMCs)结缔组织生长因子(CTGF)表达的影响。方法体外培养RLMCs,并将其分为4组:(1)0组(正常对照组):用无血清培养基(FSM)培养细胞;(2)1组:用含TGF-β11 ng/ml培养细胞;(3)2组:用含TGF-β12 ng/ml培养细胞;(4)3组:用含TGF-β14 ng/ml培养细胞;各组分别在TGF-β1刺激6、12及24 h后,采用逆转录-聚合酶链反应(RT-PCR)方法测定CTGF mRNA水平;蛋白免疫印迹(Western blot)测定CTGF蛋白表达。数据以平均值±标准差(x珋±s)表示,各个浓度点组间比较采用单因素方差分析(One-way ANOVA),两两比较采用最小显著法(LSD)检验,以P<0.05有统计学意义。结果在6、12及24 h分别以TGF-β11 ng/mL、2 ng/mL、4 ng/mL处理,CTGF mRNA表达水平与对照组比较,差异具有显著性(F6h=46.549、F12h=287.098、F24h=109.202,P均<0.001),呈剂量依赖性,以TGF-β1处理12 h组CTGF mRNA表达差异明显。western blotting:正常对照组RLMCs细胞和不同浓度的TGF-β1刺激后均表达CTGF蛋白,在TGF-β1刺激6、12及24 h后,各组均随着浓度的增加而增加,差异有显著性(F6h=52.988、F12h=95.331、F24h=157.107,P均<0.001),呈剂量依赖性,以TGF-β1处理6 h组CTGF蛋白表达差异明显。结论 TGF-β1能诱导RLMCs中CTGF通路激活。TGF-β1刺激RLMCs中CTGF mRNA和蛋白的表达,而且随浓度增加而显著。CTGF可能作为神经系统疾病中抑制脑膜纤维化的靶点而进一步研究。

Objective To investigate the role of transforming growth factor beta-1 （TGF-β1 ） on the expression of connective tissue growth factor （CTGF） in rat leptomeningeal mesothelial cells （RLMCs）. Methods RLMCs cultured in vitro were divided into 4 groups ： （ 1 ） control group ; （ 2 ） 1 ng/ml TGF-β1 group;（ 3 ）2 ng/ml TGF-β1 group; （4）4 ng/ml TGF-β1 group. Cultured RLMCs were incubated in TGF-β1 at different density for 6 h, 12 h and 24 h, the expression of CTGF mRNA was detected by reverse transcription-polymerase chain reaction （RT-PCR）, the expression of CTGF protein was examined by Western blot analysis. Results were expressed as mean ＋ SD. Statistical significances during doses were determined by one factor analysis of variance （one-way ANOVA） and followed by Fisher＇s least significant difference （LSD） test for multiple comparison. P 〈 0.05 was considered significant. Results RT-PCR： The dose-response of TGF-β1 elevated the CTGF mRNA： The expression of CTCF mRNA stimulated by incubation of TGF-β1 1 ng/ml group, 2 ng/ml group, 4 ng/ml group were significantly higher than that in control group （F6h = 46. 549, F12h = 287. 098, F24h = 109. 202,P 〈 0.001 ） , and TGF-β1 performed in a dose-dependent manner; TGF-[31 mostly increased CTGF mRNA after 12 h treatment. Western-blotting： Compare to control group, CTGF protein was obviously up-regulated by TGF-β1 at all the tree doses and time points （ F6h = 52. 988, F12h = 95.331,F24h = 157. 107,P 〈 0.00l ） . The regulation showed in a dosedependent manner and was extremely significant at 6 h. Conclusions TGF-β1 may activate CTGF pathway in RLMCs. TGF-β1 may up-regulate CTGF expression in RLMCs and the effect become more and more prominant with increased cell density. CTGF may be further investigated as the target of suppressing meninges fibrosis.