成纤维细胞生长因子-21表达变化对3T3-L1脂肪细胞成纤维细胞生长因子受体及脂肪细胞因子的影响

The effects of fibroblast growth factor-21(FGF-21) on FGF receptors and adipokines in 3T3-L1 adipocytes

目的探讨FGF-21表达变化对FGF相关受体(FGFRs)和脂肪细胞因子表达的影响。方法设计并构建2条针对小鼠FGF-21基因的短发夹状RNA(shRNA)序列,运用双荧光蛋白检测系统初步验证其有效性,筛选出抑制有效的pGenesil-FGF21重组质粒,分别用FGF-21过表达质粒pcDNA-FGF21和FGF-21 RNAi表达载体pGenesil-FGF21转染3T3-L1脂肪细胞,采用荧光定量PCR法检测FGF-21、FGFRs、脂联素、Visfatin以及瘦素(Leptin)的mRNA表达水平。结果成功构建2条FGF-21-shRNA重组质粒。转染上述重组载体的细胞内,两组pGenesil-FGF21 GFP平均荧光强度/RFP平均荧光强度均低于阴性对照组(均P<0.05),且pGenesil-FGF21-1组更低。转染pcDNA-FGF21的3T3-L1脂肪细胞内FGF-21 mRNA表达明显增加(P<0.01),转染pGenesil-FGF21-1的细胞内FGF-21 mRNA表达降低77.8%(P<0.05)。FGF-21过表达的脂肪细胞内以β-klotho和FG-FR1 mRNA表达明显升高,而Leptin mRNA表达明显降低;而FGF-21表达缺陷时则相反(均P<0.05)。结论 FGF-21可能主要通过β-klotho和FG-FR1途径激活受体后信号系统,并且可能通过影响脂肪细胞因子表达从而对代谢发挥其调控作用。

Objective To investigate the effects of fibroblast growth factor-21 （FGF-21） on fibroblast growth factor receptors （ FG- FR） and adipokines in 3T3-LI adipocytes. Methods Two shRNA motifs targeting on FGF-21 gene were designed and synthesized respectively. The effective shRNA-vector, pGenesil-FGF-21 were verified by double-fluorescence reporter assay system. The two vectors, pcDNA- FGF21 for over-expression and pGenesil-FGF21 for low-expression were transfeeted into 3T3-L1 adipocytes. The mRNA expressions of FGF- 21, FGFRs, adiponectin, visfatin and Leptin were detected by real-time quantitative PCR. Results The FGF21-shRNA vectors were constructed successfully. Fluorescence ratio of GFP/RFP in both groups were lower than that in HK group （ both P 〈0. 05 ）. And the lowest was in pGenesil-FGF21-1 group. The transfeetion of pcDNA-FGF21 significantly increased FGF-21 mRNA expression in adipocytes （ P 〈0. 01 ）. However, FGF-21 expression was down-regulated by 77.8% with transfection of pGenesil-FGF21 in adipocytes （ P 〈 0. 05 ）. The mRNA ex- pressions of β-klotho and FGFRI were markedly increased in the adipocytes of FGF-21 over-expression （ both P 〈 0. 05 ）. Meanwhile, the down-regulation of leptin was also observed in these adipocytes （P 〈 0. 05 ）. The reverse changes happened in adipocytes with FGF-21 expressions deficient （ both P 〈0. 05 ）. But the mRNA expressions of visfatin and adiponectin had no differences in these adipocytes. Conclusions In 3T3-L1 adipocytes, activating FGF-21 signaling downstream may be mainly through β-klotho and FGFR1, and FGF-21 to play an important role in metabolic homeostasis by accommodating some adipokines.