摘要
目的:观察3T3-L1脂肪细胞分化过程中G蛋白亚单位、磷脂酰肌醇3激酶(PI3K)亚单位和蛋白激酶C(PKC)亚型蛋白表达的时序性变化规律,探讨Gβ/Gαq-PI3K-PKC信号通路在脂肪细胞分化过程中的作用。方法:体外培养3T3-L1前脂肪细胞,采用经典激素鸡尾酒诱导法(分化诱导剂1-甲基3-异丁基黄嘌呤+地塞米松+胰岛素)诱导分化,并以加入诱导分化液的时间为起点,于0 d、6 h、12 h、1 d、3 d、6 d、9 d收集细胞后立即进行蛋白抽提,用Western blotting分别检测细胞中Gβ、Gαq/11、PI3K-IA类和IB类部分亚单位磷酸化水平、磷酸化PKCα和ζ的表达变化。结果:诱导分化进程中:6 h时,各检测信号物质表达均不同程度升高;12 h时,磷酸化PI3K-p85、p110γ表达达高峰(P<0.05),Gβ明显升高(P<0.05),p101轻度升高,磷酸化PKCζ水平有所下降;1 d时,Gβ表达达高峰(P<0.01),PI3K-p85总水平及磷酸化PI3K-p85、p55、p110γ、p101开始不同程度下降,Gαq/11和磷酸化PKCα继续升高(P<0.01);3d时,Gαq/11和磷酸化PKCα表达达高峰(P<0.01);6d时,Gαq/11和磷酸化PKCα表达仍维持在较高水平,PKCζ明显降低(P<0.01);成熟脂肪细胞内(分化9 d)磷酸化p55、p85和PKCζ水平分别是前脂肪细胞的33.55%(P<0.01)、29.68%(P<0.05)和45.52%(P<0.05)。结论:Gβ/Gαq-PI3K-PKC信号通路检测信号分子于分化早期出现峰值,提示与细胞早期分化的克隆性增殖阶段密切相关,而磷酸化p55、p85和PKCζ亚单位在成熟脂肪细胞较前脂肪细胞水平显著下调提示其在分化后期对细胞分化起抑制性作用。
AIM： To investigate the role of individual protein isoforms in the regulation of 3T3 - L1 adipecyte development through detecting temporal patterns of Gβ,Gα/q, phosphorylated phosphoinositide 3 - kinase IA and IB isoforms and activated protein kinase C α and ξ subtypes involved in Gβ/Gαq - PI3K - PKC signaling pathway during 3T3 - LI preadipocyte differentiation. METHODS： The cells were induced by 1 - methyl - 3 - isobutylxanthine, dexamethasone and insulin in vitro, and harvested at indicated time points （0 d, 6 h,12 h, 1 d, 3 d, 6 d, 9 d）, then the total proteins of these cells were extracted. The expressions of Gβ,Gα/q, p101, phosphorylated p85, p55, p110γ, PKCα and ξ were assayed by Western blotting. RESULTS： （ 1 ） Gαq/11 and GIB increased after induction of differentiation, reaching maxima respectively at 3 d and 1 d when cellular level of GGβ was 1.97 ±0. 16 -fold higher and expression of Gαq/11 was 2. 34 ± 0. 22 - fold higher than that in just - confluent （0 d） ceils. Subsequently the expression declined. （2） Compared to 0 d, phosphorylated p110γ, p55 and p85 elevated slightly at 12 h, and decreased significantly by the end of the treatment period （9 d） which coincided with maximal differentiation. While the expression of p101 elevated slightly at 12 h, no statistical significance through differentiation was found. （ 3 ） Phosphorylated PKCα increased significantly, peaking at 3 d of differentiation. Expression of phosphorylated PKCα decreased during differentiation and its level 45. 52% lower in adipocyfes than that in preadipoeyte was observed. CONCLUSION： The protein levels elevating at the early stage of differentiation correlate with the time point at which clonal expansion of cells is observed. Phosphorylated p55 and PKCα decrease in adipocytes than that in preadipocyte, indicating an inhibitory influence upon the late differentiation process.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2009年第1期172-176,共5页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.30600213)
