摘要
目的研究原纤毛在3T3-L1前体脂肪细胞分化中的表达及对脂肪分化的影响。方法诱导3T3-L1前体脂肪细胞向脂肪细胞分化,在分化第0天和第4天分别孵育水合氯醛以化学方式抑制原纤毛。Western blot法检测原纤毛关键蛋白驱动蛋白3a(Kif3a)、鞭毛内转运蛋白88(IFT88)和乙酰化α微管蛋白(acAT)以及脂肪分化关键蛋白过氧化物酶体增殖物激活受体γ(PPARγ)的表达;免疫荧光染色检测ac AT反映原纤毛数目;双萤光素酶报告基因系统检测PPARγ基因启动子活性;油红染色检测脂滴沉积程度。结果原纤毛关键蛋白Kif3a、IFT88以及ac AT在分化第0天呈高表达,分化第2天表达降低,分化第4天表达恢复至第0天水平,分化第8天又明显减少。在分化第0天给予水合氯醛,可明显抑制原纤毛关键蛋白Kif3a、IFT88以及ac AT的表达及数目,显著增加PPARγ的启动子活性和蛋白表达及脂滴沉积。而在分化第4天给予水合氯醛,与溶媒对照组相比,虽也显著抑制原纤毛生成,但PPARγ启动子活性和蛋白表达及脂滴沉积无明显变化。结论原纤毛在3T3-L1前体脂肪细胞分化过程中呈动态变化;分化早期原纤毛减少可明显增加脂滴生成能力,然而一旦分化过程被启动,原纤毛减少不影响脂滴生成。
Objective To investigate the dynamic changes and effect of primary cilia on adipogenesis during 3T3-L1 cell differentiation into adipocyte. Methods Chloral hydrate was applied to inhibit primary cilia on day 0 and 4 of 3T3-L1 cell differentiation into adipocyte. The expression levels of primary cilia key proteins like kinesin family member 3a （ Kif3a）, intraflageUar transport protein 88 （IFT88）, acetylated α-tubulin （acAT） and adipogenic key protein peroxisome proliferator activated receptor γ （PPARγ） were detected by Westem blotting. The number of primary cilia was analyzed by immunofluorescence assay. The promoter activity of PPARy gene was measured with dual-luciferase reporter assay system. The lipid accumulation was observed by oil red O staining. Results The levels of primary cilia key proteins, including Kif3a, IFT88, acAT, and the number of the primary cilia were higher on day 0 of cell differentiation, decreased on day 2, and then increased to the beginning levels on day 4, and significantly decreased again on day 8. Incubation with chloral hydrate on day 0 of differentiation significantly reduced the levels of Kif3a, IF188, acAT proteins and the number of primary cilia, dramatically enhanced the promoter activity of PPARγ gene, level of PPAR y protein and lipid accumulation. However, although primary cilia was significantly inhibited by the incubation with chloral hydrate on day 4 of differentiation, the promoter activity and protein expression of PPARy, lipid accumulation had no obvious changes compared with vehicle groups. Conclusion Pdmary cilia presented a dynamic change during 3T3-L1 cell differentiation into adipocytes. Few primary cilia in the early differentiation led to significantly enhanced adipogenesis, but once the differentiation was under way, the decrease of primary cilia would not affect adipogenesis.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2015年第11期1473-1478,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
中国博士后基金(2012M521590,2013T60792)
广东省自然科学基金(s2013040014350)
