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Abstract：
BACKGROUND: The transcription factor Sox9 is pivotal in the morphogenesis of hair follicles, but its role in sebocytes is poorly understood.
OBJECTIVE: We investigated the effect of Sox9 on human sebocyte proliferation, differentiation and lipogenesis.
METHODS: Sox9 expression was detected by immunohistochemistry in normal skin and acne lesion. Primary cultured human sebocytes were transfected with adenovirus expressing GFP-Sox9 or Sox9 microRNA. Sox9 and peroxisome proliferator-activated receptor (PPAR)γ expression in sebocytes was detected by quantitative real-time PCR, Western blot and immunocytofluorescence; cell proliferation was measured by MTS and [3H]-thymidine incorporation assays; cell cycle distribution and apoptosis were evaluated by propidium iodide staining-based flow cytometry; and intracellular lipid levels were assessed by Oil Red O stain.
RESULTS: Sox9 immunostaining was increased in mature sebocytes of acne lesion compared with normal skin. Expression of Sox9 mRNA and protein and PPARγ protein was elevated with cell confluent levels in sebocytes. Sox9 overexpression enhanced proliferation, differentiation, proportion of S and G2/M cells, lipogenesis and PPARγ expression in sebocytes, while Sox9 silencing caused inhibition of differentiation, lipogenesis and PPARγ expression, and increase of G1 and sub-G1 (apoptotic) cell fraction. The suppression of Sox9 knockdown on sebocyte growth was observed using [3H]-thymidine incorporation but not MTS assay.
CONCLUSIONS: These results demonstrate that Sox9 can reinforce sebocyte proliferation, differentiation and lipogenesis. The G1/S transition arrest and apoptotic induction might contribute to inhibitory effect of Sox9 silencing on sebocyte proliferation.
OBJECTIVE: We investigated the effect of Sox9 on human sebocyte proliferation, differentiation and lipogenesis.
METHODS: Sox9 expression was detected by immunohistochemistry in normal skin and acne lesion. Primary cultured human sebocytes were transfected with adenovirus expressing GFP-Sox9 or Sox9 microRNA. Sox9 and peroxisome proliferator-activated receptor (PPAR)γ expression in sebocytes was detected by quantitative real-time PCR, Western blot and immunocytofluorescence; cell proliferation was measured by MTS and [3H]-thymidine incorporation assays; cell cycle distribution and apoptosis were evaluated by propidium iodide staining-based flow cytometry; and intracellular lipid levels were assessed by Oil Red O stain.
RESULTS: Sox9 immunostaining was increased in mature sebocytes of acne lesion compared with normal skin. Expression of Sox9 mRNA and protein and PPARγ protein was elevated with cell confluent levels in sebocytes. Sox9 overexpression enhanced proliferation, differentiation, proportion of S and G2/M cells, lipogenesis and PPARγ expression in sebocytes, while Sox9 silencing caused inhibition of differentiation, lipogenesis and PPARγ expression, and increase of G1 and sub-G1 (apoptotic) cell fraction. The suppression of Sox9 knockdown on sebocyte growth was observed using [3H]-thymidine incorporation but not MTS assay.
CONCLUSIONS: These results demonstrate that Sox9 can reinforce sebocyte proliferation, differentiation and lipogenesis. The G1/S transition arrest and apoptotic induction might contribute to inhibitory effect of Sox9 silencing on sebocyte proliferation.
摘要：
背景：转录因子Sox9在毛囊的形态发生中起关键作用，但它在皮脂腺细胞中的作用却知之甚少。
目的：我们研究了Sox9对人皮脂腺细胞增殖的影响，分化和脂肪生成。
方法：用免疫组织化学方法检测正常皮肤和痤疮皮损中Sox9的表达。用表达GFP-Sox9或Sox9微小RNA的腺病毒转染原代培养的人皮脂细胞。实时定量PCR检测sox9和过氧化物酶体增殖物激活受体(PPAR)γ在皮脂腺细胞中的表达,蛋白质印迹和免疫荧光;通过MTS和[3H]-胸苷掺入测定测量细胞增殖;通过基于碘化丙啶染色的流式细胞术评估细胞周期分布和细胞凋亡;通过油红O染色评估细胞内脂质水平。
结果：与正常皮肤相比，痤疮病变的成熟皮脂细胞中Sox9免疫染色增加。在皮脂腺细胞中，Sox9mRNA和蛋白以及PPARγ蛋白的表达随着细胞融合水平的升高而升高。Sox9过表达增强增殖，分化,S和G2/M细胞的比例，脂肪生成和PPARγ在皮脂腺细胞中的表达，而Sox9沉默导致分化抑制，脂肪生成和PPARγ表达，以及G1和亚G1（凋亡）细胞分数的增加。使用[3H]-胸苷掺入而不是MTS测定观察到Sox9敲低对皮脂腺细胞生长的抑制。
结论：这些结果表明Sox9可以增强皮脂腺细胞的增殖，分化和脂肪生成。G1/S转换阻滞和凋亡诱导可能有助于Sox9沉默对皮脂腺细胞增殖的抑制作用。
目的：我们研究了Sox9对人皮脂腺细胞增殖的影响，分化和脂肪生成。
方法：用免疫组织化学方法检测正常皮肤和痤疮皮损中Sox9的表达。用表达GFP-Sox9或Sox9微小RNA的腺病毒转染原代培养的人皮脂细胞。实时定量PCR检测sox9和过氧化物酶体增殖物激活受体(PPAR)γ在皮脂腺细胞中的表达,蛋白质印迹和免疫荧光;通过MTS和[3H]-胸苷掺入测定测量细胞增殖;通过基于碘化丙啶染色的流式细胞术评估细胞周期分布和细胞凋亡;通过油红O染色评估细胞内脂质水平。
结果：与正常皮肤相比，痤疮病变的成熟皮脂细胞中Sox9免疫染色增加。在皮脂腺细胞中，Sox9mRNA和蛋白以及PPARγ蛋白的表达随着细胞融合水平的升高而升高。Sox9过表达增强增殖，分化,S和G2/M细胞的比例，脂肪生成和PPARγ在皮脂腺细胞中的表达，而Sox9沉默导致分化抑制，脂肪生成和PPARγ表达，以及G1和亚G1（凋亡）细胞分数的增加。使用[3H]-胸苷掺入而不是MTS测定观察到Sox9敲低对皮脂腺细胞生长的抑制。
结论：这些结果表明Sox9可以增强皮脂腺细胞的增殖，分化和脂肪生成。G1/S转换阻滞和凋亡诱导可能有助于Sox9沉默对皮脂腺细胞增殖的抑制作用。
