【作者】 陶梅梅；

【导师】 李太生； 黎明；

【作者基本信息】 中国协和医科大学 ， 感染内科学， 2008， 博士

【摘要】 目的:1、研究司他夫定(D4T)、齐多夫定(AZT)和去羟肌苷(DDI)对3T3-L1前脂肪细胞增殖和分化的影响。2、观察D4T、AZT和DDI对处于增殖和分化过程中的3T3-L1前脂肪细胞分泌胰岛素样生长因子—1(IGF-1),以及成熟脂肪细胞分泌脂联素(APN)的影响。通过上述研究,探讨HIV/AIDS患者接受高效抗逆转录病毒治疗(HAART)后出现脂肪萎缩的发生机制。方法:1、将不同浓度的D4T、AZT和DDI(0.1μg/ml、1μg/ml、10μg/ml)作用于CFSE标记的3T3-L1前脂肪细胞,应用流式细胞仪测定细胞增殖情况。2、选择D4T(1μg/ml、4μg/ml)、AZT(0.1μg/ml、1μg/ml)或DDI(1μg/ml、10μg/ml)作用于3T3-L1前脂肪细胞,使用MTT比色法测定细胞增殖情况。3、将不同浓度的D4T、AZT和DDI(0.1μg/ml、1μg/ml、10μg/ml)作用于3T3-L1前脂肪细胞,诱导分化至第8天,使用油红0脂肪染色法观察细胞分化情况。4、收集3T3-L1前脂肪细胞增殖和分化过程中的培养上清液,应用ELISA方法测定APN和IGF-1水平。结果:1、流式细胞仪CFSE标记法检测结果显示,与对照组相比,不同浓度D4T干预组(0.1μg/ml、1μg/ml、10μg/ml)的增殖指数降低程度分别为19.1%、39.8%和50.8%;AZT干预组(0.1μg/ml、1μg/ml、10μg/ml)的降低程度分别为6.7%、18.1%和30.9%。MTT比色法也证实D4T和AZT能够减弱3T3-L1前脂肪细胞的增殖能力。2、高浓度D4T(10μg/ml)和AZT(10μg/ml)能够抑制3T3-L1前脂肪细胞分化能力,造成成熟脂肪细胞数量减少。3、ELISA检测结果发现,在细胞增殖过程中,与对照组相比,不同浓度D4T干预组(0.1μg/ml、1μg/ml、10μg/ml)中的IGF-1含量下降程度分别为21.5%、31.7%和48.7%;D4T还能降低分化第8天的成熟脂肪细胞分泌IGF-1的能力,呈浓度依赖性,下降程度分别为10.5%、30.7%和42.6%;而AZT(0.1μg/ml、1μg/ml、10μg/ml)仅能降低增殖过程中前脂肪细胞分泌IGF-1的能力,与对照组相比下降程度分别为8.6%、21.7%和29.5%。4、与对照组相比,在10μg/ml的D4T干预组和AZT干预组中,成熟脂肪细胞分泌的APN水平下降。5、DDI对3T3-L1前脂肪细胞增殖、分化和IGF-1、APN的分泌未见明显影响。结论:1、本研究首次发现D4T能够降低3T3-L1前脂肪细胞的增殖能力,为浓度依赖性;同时证实AZT能够降低3T3-L1前脂肪细胞的增殖能力。2、本研究证实了D4T和AZT能够抑制3T3-L1前脂肪细胞分化能力,使成熟脂肪细胞数量下降。3、本研究首次在体外实验中发现,D4T和AZT均能够减弱增殖过程中的前脂肪细胞分泌IGF-1的能力,呈浓度依赖性;D4T还能够减弱成熟脂肪细胞分泌IGF-1的能力。4、本研究证实了D4T和AZT能够使成熟脂肪细胞分泌的APN含量下降。由此,D4T和AZT可能通过影响前脂肪细胞的增殖、分化及脂肪因子分泌等途径,导致脂肪萎缩的出现。更多还原

【Abstract】 Objective:1、To study the effects of D4T,AZT and DDI on proliferation and differentiation in 3T3-L1 preadipocytes.2、To study the effects of D4T,AZT and DDI on IGF-1 production by cultured proliferating preadipocytes and differentiating adipocytes.To study the effects of D4T,AZT and DDI on adiponectin secretion by differentiated adipocytes.To investigate the pathogenesis of highly active antiretroviral therapy（HAART） associated lipoatrophy based on above studies.Methods:1、3T3-L1 preadipocytes dying with CFSE were cultured in DMEM/F12 growth medium containing D4T,AZT or DDI at different concentrations（0.1μg/ml、1μg/ml、10μg/ml）.Flow cytometry was used to measure the proliferation index of preadipocytes in treated and control groups.2、3T3-L1 preadipocytes were cultured with D4T（1μg/ml、4μg/ml）, AZT（0.1μg/ml、1μg/ml） or DDI（1μg/ml、10μg/ml）.MTT assay was applied to evaluate growth of preadipocytes.3、3T3-L1 preadipocytes were induced to differentiate in both treated groups containing D4T、AZT or DDI at different concentrations（0.1μg/ml、1μg/ml、10μg/ml） and control groups.Oilred O staining was used to assess lipid accumulation.4、ELISA was designed to measure IGF-1 and adiponectin levels in cell culture supernatants during the course of proliferation and differentiation.Results:1、It was shown by CFSE with flow cytometry that D4T in different concentrations（0.1μg/ml、1μg/ml、10μg/ml） could make proliferation index decrease 19.1%,39.8%,50.8%respectively compared with control group.The decreased levels by AZT interfering groups（0.1μg/ml、1μg/ml、10μg/ml） were 6.7%、18.1%,30.9%respectively.The fact that D4T and AZT were able to attenuate the proliferation ability of 3T3-L1 preadipocyte was also confirmed by MTT method.2、D4T and AZT in high concentration could also inhibit the differentiation ability of 3T3-L1 preadipocyte,then make less mature adipocyte.3、It was also discovered by ELISA testing that D4T in different concentrations（0.1μg/ml、1μg/ml、10μg/ml） could make the concentration of IGF-1 decrease 21.5%,31.7%,48.7%respectively in proliferation phrase compared with control group.D4T could also low down the IGF-1 secretion by mature adipocyte in the 8^th day of differentiation.The secretion decrease, which was concentration-dependent,were 10.5%,30.7%,42.6%respectively. AZT in different concentrations（0.1μg/ml、1μg/ml、10μg/ml） could only make the concentration of IGF-1 decrease 8.6%,21.7%,29.5%respectively in proliferation phrase compared with control group.4、Adiponectin secreted by mature adipocyte was also decreased in D4T in 10μg/ml and AZT in 10μg/ml interfering groups compared with control group.5、DDI had no effect on proliferation,differentiation and production of IGF-1 and APN in 3T3-L1 preadipocytes.Conclusion:1、Our study discovers that D4T and AZT impair the proliferative activity of 3T3-L1 preadipocytes dose-dependently by flow cytometry and MTT assay.2、We confirm that D4T and AZT induce the less number of mature adipocytes by impairing the differentiation of 3T3-L1 preadipocytes.3、To our knowledge,in vitro experiments our study is the first to discover that D4T and AZT impair the production of IGF-1 in proliferating preadipocytes,and D4T impairs the production of IGF-1 in mature adipocytes.4、It is confirmed that D4T and AZT can lead to the reduction of adiponectin in supernanants of differentiated adipocytes.So,D4T and AZT might induce lipoatrophy by bothering the proliferation, differentiation of preadipocytes and the secretion of adipokines.更多还原