摘要
目的探讨3T3-L1脂肪前体细胞诱导分化过程中PRNP基因表达水平的变化及TNF-α对其调节作用。方法体外培养3T3-L1前体脂肪细胞,胰岛素加地塞米松加1-甲基-3-异丁基黄嘌呤（MDI）方案诱导3T3-L1细胞分化成熟,并收集分化前、分化0～10 d各时段细胞,采用RT-PCR技术检测诱导分化不同时段3T3-L1细胞PRNP基因表达水平;同时在成功诱导3T3-L1细胞分化成熟的基础上,应用不同水平TNF-α（0.1、1.0、10.0μg/L）干预分化后的成熟脂肪细胞（第10天）,收集TNF-α刺激前（0 h）及刺激后0.5、2.0、6.0、12.0、24.0 h的脂肪细胞,通过RT-PCR测定TNF-α干预前后不同时间点脂肪细胞中PRNP基因表达水平。采用Excel软件进行统计学分析。结果1.PRNP基因低表达于3T3-L1脂肪前体细胞中,随细胞分化成熟该基因表达水平逐渐上调,至分化第10天其表达水平最高。PRNP基因表达水平除在诱导分化前（第-1天）至第4天、第2～5天、第6～10天时段内无显著性差异外（Pa〉0.05）,其余各时段间表达水平均有显著性差异（Pa〈0.05）;2.在分化前的3T3-L1脂肪细胞和成熟脂肪细胞中,不同水平TNF-α（0.1、1.0、10.0μg/L）均能在短时间内明显抑制PRNP基因mRNA的表达,且呈时间依赖性。结论PRNP基因可能参与3T3-L1脂肪细胞分化及脂质积聚过程;不同水平重组TNF-α对成熟脂肪细胞的PRNP基因表达具有抑制作用,其抑制效应总体趋势上呈时间依赖性。
Objective To investigate the changes of PRNP gene expression during 3T3 - L1 preadipocyte differentiation and analyze the regulation role of tumor necrosis factor - alpha （ TNF - α） on PRNP gene expression in matured 3T3 - L1. Methods 3T3 - L1 preadipocytes were cultured in vitro and differentiated into the matured adipocytes with the standard insulin plus dexamethasone plus 3 - isobutyl - methyl xanthine （ MDI ） induction cocktail. Cells were collected before differentiate and at various time - points during differentiation. TNF - α with different concentrations （ 0.1, 1.0, 10.0 μg/L ） was added into the culture medium of fully differentiated adipocytes （Day 10） in various time - points （0,0.5, 2.0, 6.0, 12.0, 24.0 h）. The levels of 3T3 - L1 preadipocyte PRNP gene mRNA expression at different time were evaluated by reverse transcription - polymerase chain reaction （ RT - PCR）. Excel software was used to analyze the data. Results 1. In preadipocytes, the level of PRNP gene mRNA expression was low. With the 3T3 - L1 preadipocytes being differentiated into the matured adipocytes, the level of PRNP gene mRNA expression was upregulated and reached the higher level in fully differentiated adipocytes. There was a significant difference between any 2 detected phases in the levels of PRNP gene mRNA expression （ P 〈 0.05 ） , except that the levels of PRNP gene mRNA expression did not increase obviously in the stage of pre - differentiation to Day 4, Day 2 to Day 5, Day 6 to Day 10 （P 〉 0.05 ）. 2. Treatment of matured adipocytes with TNF - α of different concentrations resulted in a significant decrease in the level of PRNP gene mRNA expression. The suppressive effect of TNF - α on PRNP gene mRNA expression generally tended to be reinforced with the elongation of time course. Conclusions PRNP gene is related to the etiology of obesity. Up - regulation in the level of PRNP gent mRNA expression during 3T3 - L1 preadipocyte differentiation may be involved in the differentiation, mature and adipogenesis of adipocytes. The suppressive effect of TNF - α on PRNP gene mRNA expression is generally time - correlated.
出处
《实用儿科临床杂志》
CAS
CSCD
北大核心
2008年第7期489-491,共3页
Journal of Applied Clinical Pediatrics
基金
国家自然科学基金项目资助(39870362,30371502)
江苏省自然科学基金项目资助(BK99136,BK2001120)
江苏省教委自然科学基金项目资助(98KJB320002)
江苏省卫生厅项目资助(H200362)
