摘要
目的 通过应用重组逆转录病毒介导小鼠过氧化物酶体增殖体激活受体γ２（ｍＰＰＡＲγ２）基因整合入ＮＩＨ３Ｔ３细胞基因组中进行表达，并研究其功能。方法 从经测序证实含正确序列ｍＰＰＡＲγ２的重组质粒ｐｃＤＮＡ３／ｍＰＰＡＲγ２中，双酶切下ｍＰＰＡＲγ２全长ｃＤＮＡ序列，亚克隆入逆转录病毒载体ｐＧＣＥＮ中构建重组逆转录病毒载体ｐＧＣＥＮ／ｍＰＰＡＲγ２。对ｐＧＣＥＮ／ｍＰＰＡＲγ２及ｐＧＣＥＮ进行包装及Ｇ４１８抗性筛选，收集病毒上清，感染靶细胞ＮＩＨ３Ｔ３细胞，进行Ｇ４１８抗性筛选并进行扩增。在含过氧化物酶体增殖体激活受体γ（ＰＰＡＲγ）激活物５，８，１１，１４二十碳四烯酸等分化介质中，诱导ＰＰＡγ２表达增加，使ＮＩＨ３Ｔ３细胞向脂肪细胞分化。结果 构建了含ｍＰＰＡＲγ２全长ｃＤＮＡ重组逆转录病毒载体，获得了滴度分别为５ ｘ １０４ ＣＦＵ／ｍｌ和６ｘ１０５ ＣＦＵ／ｍｌ的ｐＧＣＥＮ／ｍＰＰＡＲγ２及ｐＧＣＥＮ的病毒上清。经油红Ｏ染色证实分化１０ｄ的表达ｍＰＰＡＲγ２的ＮＩＨ３Ｔ３细胞中明显积聚了较多的中性脂肪，其细胞形态也与体内成熟脂肪细胞相似。结论 本研究在体外建立了由ＰＰＡＲγ２诱导ＮＩＨ３Ｔ３细胞向脂肪细胞分?
Objective:To express the mouse peroxisome proliferator activated receptor γ2(mPPARγ2) in NIH3T3 cells mediated by the recombinant retrovirus and study its function. Methods mPPARγ２ gene digested from the recombinant plasmid pcDNA3/mPPARγ2 and confirmed to contain the target gent segment with fluorescence-sequencing was subcloned into retrovirus vector pGCEN to generate the recombinant retrovirus pGCEN/mPPARγ２.The recombinant retrovirus pGCEN/mPPARγ2 and pGCEN were packaged with PA3l7 cells and anti-G418 clones of PA317 cells were selected. Viral supernatants were collected and ed to infect NIH3T3 cells.Peroliferator activated receptor γ2 (PPARγ2)-expressing NIH3T3 cells cultured in the differentiation media containing PPARγ activator ETYA were induced into adipocytes.Results The recombinant retrovirus pGCEN/mPPARγ2 was constructed, 5 x 104 CFU/ml of the viral supernatants containing pGCEN/ mPPARγ2 and 6 x 105 f CFU/ml of the viral supernatants containing pGCEN were obtained. mPPARγ was expressed in NIH3T3 cells mediated by the recombinant retrovirus.Lipid accumulation obviously existed in PPARγ2- expressing NIH3T3 cells at 10 days postdifferentiation and the lipid-containing cells morphologically resembled the mature adipocytes in vivo. Conclusion An adipocyte differentiation model in vitro was established. The work is the basis for further researches on the molecular mechanism of adipocyte differentiation induced by PPARγ2.
出处
《中华内科杂志》
CAS
CSCD
北大核心
2001年第4期247-250,T002,共5页
Chinese Journal of Internal Medicine
基金
上海联合利华研究与发展基金资助(9807)
