Ghrelin对3T3-L1前脂肪细胞增殖和分化的影响及相关机制探讨

Effects of ghrelin on the proliferation and differentiation of 3T3-L1 preadipocytes and its possible mechanisms

摘要:

目的：探讨ghrelin对3T3-L1前脂肪细胞增殖和分化的影响及作用机制。方法：体外培养3T3-L1前脂肪细胞，MTT法检测不同浓度ghrelin对其增殖活力的影响，半定量RT-PCR检测ghrelin对c-myc和胸苷激酶mRNA表达水平的影响；同时在利用胰岛素或ghrelin诱导分化的不同时段，通过油红O染色测定细胞分化程度，半定量RT-PCR检测过氧化物体增殖剂活化受体γ（PPARγ）、CAAT/增强子结合蛋白α（C/EBPα）mRNA的表达。结果：10-7~10-15 mol/L ghrelin作用24 h明显促进前脂肪细胞增殖，ghrelin组c-myc和胸苷激酶mRNA表达水平明显升高，与对照组相比差异均有显著性意义（P<0.05）。Ghrelin干预后，可诱导前脂肪细胞向成熟脂肪细胞的形态转变，但诱导分化率仍少于胰岛素；同时其PPARγ和C/EBPα mRNA表达水平较对照组明显升高（P<0.05），ghrelin组和胰岛素组PPARγ和C/EBPα mRNA表达量随着分化时间的延长而增加，分化8 d与2 d相比，差异均有显著性意义（P<0.01）。结论：Ghrelin明显促进3T3-L1前脂肪细胞增殖与分化。Ghrelin可能通过增加c-myc的含量，进而引起胸苷激酶的活化，从而导致细胞周期的激活，促进细胞增殖；同时ghrelin可能通过增加PPAR-γ、C/EBP-α mRNA的表达，促进细胞分化，从而提高脂肪细胞对胰岛素的敏感性。［中国当代儿科杂志，2009，11（1）：69-73］

Abstract:

OBJECTIVE: To investigate the effects of ghrelin on the proliferation and differentiation of 3T3-L1 preadipocyte, and study the possible mechanisms. METHODS: 3T3-L1 preadipocytes were cultured in vitro. The proliferation potentials of 3T3-L1 preadipocytes that were treated with different concentrations of ghrelin were evaluated by MTT methods. The levels of c-myc and thymidine kinase mRNA were detected using RT-PCR. 3T3-L1 preadipocytes were differentiated into the matured adipocytes with insulin (INS) or ghrelin. The morphological changes of 3T3-L1 adipocytes were observed and the differentiation rate was assayed by oil-red O staining. Total RNA was extracted from adipocytes at various times, and the levels of peroxisome proliferation activated receptor γ (PPARγ) and CAAT/enhancer binding protein(C/EBPα) mRNA expressions were detected using RT-PCR. RESULTS: Ghrelin at concentrations of 10-7 to 10-15 mol/L significantly stimulated preadipocyte proliferation (P<0.05). The levels of c-myc and thymidine kinase mRNA significantly increased in 3T3-L1 preadipocytes with 10-9 mol/L and 10-11 mol/L ghrelin treatment (P<0.01). The 3T3-L1 preadipocytes treated with 10-11 mol/L ghrelin had lots of lipid droplets in the cytoplasma, but the differentiation rate was lower than those treated with INS. Ghrelin of 10-11 mol/L significantly increased the mRNA expression of PPARγ and C/EBPα in the course of 3T3-L1 preadipocyte differentiation, compared with the normal control group (P<0.05). The PPARγ and C/EBPα mRNA expression increased with the prolonged differentiation of preadipocytes induced by ghrelin or INS. There were significant differences in the levels of PPARγ and C/EBPα mRNA expression between the 2nd and 8th days of differentiation（P<0.01）. CONCLUSIONS: Ghrelin promotes the proliferation and differentiation of 3T3-L1 preadipocytes. The proliferation of 3T3-L1 preadipocytes induced by ghrelin may be associated with increased c-myc levels. Ghrelin may promote differentiation of 3T3-L1 preadipocytes by increasing mRNA expression of PPARγ and C/EBPα, thus enhances the sensitivity of adipocytes to INS.［Chin J Contemp Pediatr, 2009, 11 (1):69-73］