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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 study the expression of fibrillary cilia in 3T3-L1 preadipocyte differentiation and its effect on adipose differentiation. Methods 3T3-L1 preadipocytes were induced to differentiate into adipocytes, and chloral hydrate was separately incubated on day 0 and day 4 of differentiation to chemically inhibit fibrillation. Western blot was used to detect the expression of Kif3a, IFT88 and acAT and the expression of peroxisome proliferator-activated receptor gamma ( PPARγ). The number of acin was detected by immunofluorescence staining and the promoter activity of PPARγ gene was detected by dual luciferase reporter system. The extent of lipid droplet deposition was detected by oil red staining. Results The expression of Kif3a, IFT88 and ac AT were significantly increased on the 0th day of differentiation and decreased on the 2nd day of differentiation. The expression on the 4th day of differentiation recovered to the level of day 0 and remarkably decreased on the 8th day. Chloral hydrate on day 0 of differentiation significantly inhibited the expression and number of the key proteins of keloid cows, such as Kif3a, IFT88 and ac AT, significantly increased the promoter activity and protein expression of PPARγ and lipid droplets deposition. However, chloral hydrate on the 4th day of differentiation showed no significant change in PPARγ promoter activity, protein expression and lipid droplets deposition compared with vehicle control group. Conclusion Fibrillae showed a dynamic change during the differentiation of 3T3-L1 preadipocytes. The decrease of fibrils at early differentiation significantly increased lipid droplet production. However, once the differentiation process was initiated, the decrease of fibril did not affect the formation of lipid droplets.