PAMM不影响人脂肪干细胞的成脂分化能力  

PAMM Does not Affect Adipogenic Differentiation of Human Adipose-derived Stem Cells

作　　者：张明萌 王颖 梁杰 吴洪福 史玉仓 吴志远 饶敏腊 彭健愉 蒋智文 刘新光 孙雪荣 ZHANG Ming-Meng;WANG Ying;LIANG Jie;WU Hong-Fu;SHI Yu-Cang;WU Zhi-Yuan;RAO Min-La;PENG Jian-Yu;JIANG Zhi-Wen;LIU Xin-Guang;SUN Xue-Rong(Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics,Institute of Aging Research,Guangdong Medical University,Dongguan 523808,China;Department of Blood Transfusion,Qingdao Municipal Hospital(Group),Qingdao 266011,China;The Second Clinical School,Guangdong Medical University,Dongguan 523808,China;Department of Plastic Surgery,Affiliated Hospital of Guangdong Medical University,Zhanjiang 524001,China;Key Laboratory of Stem Cell and Regenerative Tissue Engineering,Guangdong Medical University,Dongguan 523808,China;Institute of Biochemistry&Molecular Biology,Guangdong Medical University,Zhanjiang 524023,China)

机构地区：[1]广东医科大学广东省医学分子诊断重点实验室衰老研究所,东莞523808 [2]青岛市市立医院集团输血医学科,青岛266011 [3]广东医科大学第二临床医学院,东莞523808 [4]广东医科大学附属医院整形外科,湛江524001 [5]广东医科大学干细胞与再生组织工程重点实验室,东莞523808 [6]广东医科大学生物化学与分子生物学研究所,湛江524023

出　　处：《生物化学与生物物理进展》2022年第7期1305-1317,共13页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(81971329);广东省自然科学基金(2021A1515012236);广东省扬帆计划“引进紧缺拔尖人才”项目(4YF16001G);东莞市科技特派员项目(20201800500042);广东医科大学附属医院“临床医学+”科技合作项目(CLP2021B011);广东医科大学重点培育项目(GDMUZ2020007);湛江市科技发展专项资金竞争性分配项目(2020A01033)资助。

摘　　要：目的PAMM(peroxiredoxin-like 2 activated in M-CSF stimulated monocytes)是近年来发现的一种在脂肪组织中高表达的分泌因子,但其生物学功能尚不完全清楚。为了给PAMM功能研究提供新线索,本文探讨了PAMM在白色脂肪生成中的可能作用及PAMM调节的下游基因。方法利用成脂分化液或“成脂分化液+IL-1α”建立人脂肪干细胞(adipose-derived stem cells,ADSCs)的成脂分化和成脂抑制模型。用si RNA干扰和过表达质粒转染的方法抑制或上调PAMM表达水平。采用基因芯片、m RNA测序及定量RT-PCR检测基因的m RNA表达水平,采用蛋白质印迹法评价蛋白质表达水平,用油红O染色检测脂滴沉积。结果随着ADSCs向白色脂肪分化,PAMM表达水平明显上升,而随着成脂抑制,PAMM表达明显下降。在ADSCs中沉默或过表达PAMM后,再进行成脂分化。结果发现,上调或下调PAMM对脂滴形成及成脂相关基因的表达均无明显影响。类似地,在高度分化的脂肪细胞中敲低PAMM表达,对细胞形态及脂滴含量亦无明显影响。本文进一步利用si RNA干扰、m RNA测序及q RT-PCR,筛选和验证了一批受PAMM调节的下游基因及功能性基因集,包括SULF1、A2M基因及调节P53稳定性的基因集等。结论PAMM可作为白色成脂分化的标志基因,但其自身对白色脂肪细胞生成无明显影响,本研究筛选出来的PAMM下游基因及基因集,可为进一步研究PAMM的功能提供新线索。ObjectivePAMM(peroxiredoxin-like 2 activated in M-CSF stimulated monocytes) is a secreted protein which shows high expression in white adipose tissues, but the roles of PAMM in many biological processes are still unknown. To provide new clues for PAMM function research, this study is intended to investigate the possible role of PAMM in white adipogenesis as well as the downstream genes regulated by PAMM. Methods Adipogenic differentiation and adipogenic inhibition models of human ADSCs(adipose-derived stem cells) were established using adipogenic cocktail(AC) medium or AC plus IL-1α, respectively.Expression of PAMM in ADSCs was suppressed or overexpressed using si RNA interference or plasmid transfection. Gene array,m RNA sequencing and quantitative RT-PCR were employed to detect the m RNA expression level. Western blot was used to evaluate protein expression and Oil red O staining was adopted to assess lipid droplets accumulation. Results Expression of PAMM was increased following white adipogenic differentiation of ADSCs and decreased following adipogenic inhibition. However, when PAMM was knocked down or overexpressed before adipogenic differentiation of ADSCs, the downregulation and upregulation of PAMM expression generally did not exert evident influence on the formation of lipid droplets and the expression of adipogenesisrelated genes. Similarly, PAMM knockdown in highly differentiated adipocytes had no obvious effect on cellular morphology and the accumulation of lipid droplets. Finally, a bunch of functional genes and gene sets regulated by PAMM, such as SULF1, A2 M genes and P53 stability gene set, were screened out and confirmed through si RNA interference, m RNA sequencing and q RT-PCR.Conclusion This study suggests PAMM could serve as a useful marker of white adipogenic differentiation of ADSCs, but it exerts no evident effect on white adipogenic differentiation. The unveiled downstream genes and gene sets regulated by PAMM would provide new clues for the functional research of PAMM.

关 键 词：PAMM 脂肪细胞 成脂分化 干细胞 脂肪因子 

分 类 号：R34[医药卫生—基础医学]