溶血磷脂酸通过RhoA-YAP通路调控脂肪干细胞增殖的研究

Lysophosphatidic acid regulates the proliferation of adipose-derived stem cells via the Rho A/YAP signaling pathway

目的探讨溶血磷脂酸(lysophosphatidic acid,LPA)调控脂肪干细胞(adipose-derived stem cells,ASCs)增殖的作用及其分子机制。方法分离SD大鼠ASCs,利用LPA对其进行干预,干预时间为1 h,采用Western Blot检测YES相关蛋白(yes associated protein,YAP)、结缔组织生长因子(connective tissue growth factor,CTGF)蛋白表达水平。利用免疫荧光检测YAP亚细胞定位,逆转录聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)检测CTGF和Ankrd1的m RNA表达水平。慢病毒转染ASCs,Western Blot检测不同分组YAP蛋白表达。进一步利用流式细胞术和CCK-8法检测不同分组中ASCs增殖情况。最后采用Rho A抑制剂C3干预,免疫荧光检测不同分组中YAP亚细胞定位,Western Blot检测YAP、CTGF和GTP-Rho A的表达情况。结果 LPA能显著促进YAP的表达和在细胞核内的聚集,同时LPA也能够促进YAP靶基因CTGF在蛋白水平的表达。LPA能上调YAP靶基因CTGF和锚蛋白重复域1(ankyrin repeating domain 1,Ankrd1)的m RNA表达水平。慢病毒转染敲除YAP表达后,LPA对YAP的上调作用被明显抑制。细胞周期流式细胞术和CCK-8检测结果显示LPA可显著促进ASCs的增殖,但在sh YAP慢病毒转染特异性敲除YAP后,LPA对ASCs的促增殖能力被明显削弱。Rho A抑制剂C3处理后,LPA对YAP细胞核聚集的促进作用被削弱,同时LPA对YAP、CTGF和GTP-Rho A表达的促进作用也得到了明显抑制。结论 LPA能够通过Rho A-YAP通路调控ASCs的增殖。

Objective To study the role and molecular mechanism of lysophosphatidic acid （LPA） in regulating adipose-derived stem cells （ASCs） proliferation. Methods ASCs were isolated from SD rats and treated with LPA for 1 h. Western blotting was applied to detect the expression of YAP and CTGF proteins. Immunofluorescence staining was performed to detect YAP subcellular localization. RT-PCR was used to detect the expressions of YAP target genes CTGF and Ankrd1. Western blotting was done to detect YAP protein expression after lentivirus transfection. Flow cytometry and CCK-8 assay were carried out to detect the prolifera-tion of ASCs in different groups. Finally, RhoA inhibitor C3 was utilized and YAP was subcellularly localized by immunofluorescence staining. The expression of YAP, CTGF and GTP-RhoA was examined by Western blot-ting. Results LPA could significantly promote the expression and nuclear localization of YAP. LPA could also promote the expression of YAP target gene CTGF. The RT-PCR also showed that LPA promoted the expression of YAP target genes CTGF and Ankrd1. The promotion effect of LPA on YAP protein expression was abrogated after shYAP lentivirus transfection. LPA could also significantly promote proliferation of ASCs as demonstrated by the flow cytometry and CCK8 assay. However, the effect was abrogated by transfection of shYAP lentivirus. The effect of LPA on YAP nuclear localization was abrogated by RhoA inhibitor C3 treatment. C3 treatment also abrogated the effect of LPA on YAP, CTGF and GTP-RhoA protein expression. Conclusion LPA could promote proliferation of ASCs via the RhoA/YAP signaling pathway.