IGF-Ⅰ、GLP-Ⅰ、乳酸对体外培养新生牛脂肪细胞HSL mRNA丰

1、IGF-、GLP-、乳酸对体外培养新生牛脂肪细胞HSL mRNA丰度和酶活性的影响摘 要实验选取临床检查无异常的新生荷斯坦犊牛颈动脉放血致死，无菌开腹切取腹腔内小肠网膜约60g，D-Hanks液洗涤，分离去除脂肪组织中肉眼可见的纤维成份及血管，按照本实验室建立的犊牛前脂肪细胞培养方法进行脂肪细胞的原代单层培养，整个培养期为14d。培养至第8、12d，用油红O工作液和台盼蓝工作液对脂肪细胞分别进行染色，以便观察其形态；第14d，选取体外培养单层生长良好的脂肪细胞，在培养介质中分别添加0、10、20、30、40、50g/L胰岛素样生长因子（IGF-），0、100、250、500、750、1000n

2、mol/L胰高血糖素样肽(GLP-)，0、10、20、30、40、50mg/L乳酸（每浓度梯度设三个重复），再分别进行培养24h后提取细胞总RNA，20倍稀释后用Pharmacia Biotech公司的RNA/DNA calculator测定总RNA的浓度和纯度，并用DEPC-H2O将所有样品的总RNA浓度调节一致（以消除提取RNA过程中总RNA含量差异，确保定量精确性）。将常规RT-PCR 扩增HSL mRNA模板的纯化产物进行质粒的重组、克隆和序列测定。用灭菌纯水将以上重组质粒按梯度稀释后作为荧光定量PCR 反应的阳性标准模板，按已建立的PCR反应体系和条件在ABI PRISM 7000型

3、荧光定量PCR扩增仪上扩增，机器自动绘制出标准曲线。将IGF-、GLP-、乳酸处理的脂肪细胞总RNA的反转录产物以标准曲线为参照在ABI PRISM 7000型荧光定量PCR扩增仪上扩增，数据用SPSS10.0软件统计分析，观察IGF-、GLP-、乳酸处理的脂肪细胞HSL mRNA丰度的变化。IGF-、GLP-、乳酸处理的脂肪细胞在低温操作室参照南京凯基总蛋白提取试剂盒提取细胞总蛋白，采用华特生蛋白定量试剂盒测定所提细胞总蛋白浓度，最后用南京建成脂肪酶测定试剂盒测定IGF-、GLP-、乳酸处理的脂肪细胞 HSL活性变化，数据同样用SPSS10.0软件统计分析。实验结果表明：1. IGF-、GL

4、P-、乳酸对HSL mRNA表达的抑制作用存在剂量依赖性。IGF-浓度20g/L、GLP-浓度100nmol/L、乳酸浓度20mg/L时对HSL mRNA的表达抑制作用显著（P。2. IGF-、GLP-、乳酸对HSL活性的抑制作用同样存在剂量依赖性。IGF-浓度30g/L、GLP-浓度100nmol/L、乳酸浓度40mg/L时对HSL活性的抑制作用明显（P。3. IGF-、GLP-、乳酸可通过抑制奶牛脂肪细胞内HSL mRNA表达及HSL活性而抑制脂肪的分解，从而促进脂肪沉积。关键词：IGF-；GLP-；乳酸；体外培养；脂肪细胞；HSL；mRNA丰度；酶活性Effects of IGF-、GL

5、P-and Lactic acid on Abundance of HSL mRNA and Activity of HSL in Vitro Culture Bovine AdipocyteQian hui(Clinical Veterinary Medicine)Directed by Deng JunliangAbstractIn this experiment, the healthy and neoformtive Holstan calves were caused to death through cutting arteria carotis. About 60g epiplo

6、on of intestina parva was taken from abdominal cavity without contaminate.After being washed with D-Hanks,the fiber and blood vessel were rejected from the intestina parva.Following the method of cultivanting of adipose cell established by this laboratory, the adipose cells were cultivanted to the f

7、ourteen day.In the eighteenth、twelveth day,the adipose cells were observed after being stained with rathonum red and trypan blue respectively.Untill the fourteen day, IGF-、GLP-and lactic acid were added to the media with 0,10,20,30,40,50g/L、0,100,250,500,750,1000nmol/L、0,10,20,30,40,50mg/L respectiv

8、ely.Every concentration gradient was three dulplated.After 24 hours ,the total RNA and total protein were extracted from the adipose cells. The total RNA was 20 times diluted ,and then the concentration and purity were determined in the machine of RNA/DNA calculator.Finally,erery sample of the total

9、 RNA was adjusted to the same concentration and purity with DEPC-H2O to avoid the difference of contents in the extraction of it and to ensure truth of the quantitation.The depurant products of RT-PCR of HSLmRNA which were positive and normally quantitative were recombinated and cloned and sequence

10、determined.The above plasmids were diluted according to the specified concentration grad with the degerming water to be the positive and normal template of fluorescence PCR.The amplicification was done in the ABI PRISM 7000 PCR machine according to the institutional PCR system and qualification.The

11、standard curve was motily drawed by the machine.The total RNA of adipose cells dealed with IGF-、GLP-and the lactic acid respectively were reversly transcripted.The products of reverse transcription were amplicificated in ABI PRISM 7000 PCR machine according to the above standard curve.The numerical

12、datas were statistically analyzed with the software of SPSS10.0 to ensure the variations of abundances of HSL mRNA of adipose cells dealed with IGF-、GLP-and the lactic acid respectively.The total protein of adipose cells dealed with IGF-、GLP-and the lactic acid respectively were extracted in the hyp

13、othermal operating room according to kit of total protein extraction of Nanjing KaiJi corporation.The concentration of the total protein was determined with the kit of HuaTeSheng corporation.Finally,the activity of HSL of adipose cells dealed with IGF-、GLP-and the lactic acid respectively was determ

14、ined with the kit of Lipase Activity Assay. The numerical datas were also statistically analyzed with the software of SPSS10.0 to ensure the variations of the activity of HSL of adipose cells dealed with IGF-、GLP-and the lactic acid respectively.The results showed that the expression of HSL mRNA and

15、 the activity of HSL were suppressed in adipose cells treated with IGF-,lactic acid respectively.This suppression was dose dependent.Firstly,while the concentration of IGF-20g/L、the concentration of GLP-100nmol/L、 the concentration of the lactic acid20mg/L ,the suppressiones towards the abundance of HSL mRNA were all notable（P0.05 or 0.01）.Secondly, while the concentration of IGF-30g/L、the concentration of GLP-100nmol/L 、the concentration of the lactic acid40mg/L ,the suppressiones towards the activity of HSL were all notable（P0.05 or 0.01）. Finally,we can conclude that IGF-、GL