IGF-Ⅰ、GLP-Ⅰ、乳酸對體外培養(yǎng)新生牛脂肪細(xì)胞HSL mRNA豐

1、IGF-、GLP-、乳酸對體外培養(yǎng)新生牛脂肪細(xì)胞HSL mRNA豐度和酶活性的影響摘 要實驗選取臨床檢查無異常的新生荷斯坦?fàn)倥ｎi動脈放血致死，無菌開腹切取腹腔內(nèi)小腸網(wǎng)膜約60g，D-Hanks液洗滌，分離去除脂肪組織中肉眼可見的纖維成份及血管，按照本實驗室建立的犢牛前脂肪細(xì)胞培養(yǎng)方法進行脂肪細(xì)胞的原代單層培養(yǎng)，整個培養(yǎng)期為14d。培養(yǎng)至第8、12d，用油紅O工作液和臺盼藍(lán)工作液對脂肪細(xì)胞分別進行染色，以便觀察其形態(tài)；第14d，選取體外培養(yǎng)單層生長良好的脂肪細(xì)胞，在培養(yǎng)介質(zhì)中分別添加0、10、20、30、40、50g/L胰島素樣生長因子（IGF-），0、100、250、500、750、1000n

2、mol/L胰高血糖素樣肽(GLP-)，0、10、20、30、40、50mg/L乳酸（每濃度梯度設(shè)三個重復(fù)），再分別進行培養(yǎng)24h后提取細(xì)胞總RNA，20倍稀釋后用Pharmacia Biotech公司的RNA/DNA calculator測定總RNA的濃度和純度，并用DEPC-H2O將所有樣品的總RNA濃度調(diào)節(jié)一致（以消除提取RNA過程中總RNA含量差異，確保定量精確性）。將常規(guī)RT-PCR 擴增HSL mRNA模板的純化產(chǎn)物進行質(zhì)粒的重組、克隆和序列測定。用滅菌純水將以上重組質(zhì)粒按梯度稀釋后作為熒光定量PCR 反應(yīng)的陽性標(biāo)準(zhǔn)模板，按已建立的PCR反應(yīng)體系和條件在ABI PRISM 7000型

3、熒光定量PCR擴增儀上擴增，機器自動繪制出標(biāo)準(zhǔn)曲線。將IGF-、GLP-、乳酸處理的脂肪細(xì)胞總RNA的反轉(zhuǎn)錄產(chǎn)物以標(biāo)準(zhǔn)曲線為參照在ABI PRISM 7000型熒光定量PCR擴增儀上擴增，數(shù)據(jù)用SPSS10.0軟件統(tǒng)計分析，觀察IGF-、GLP-、乳酸處理的脂肪細(xì)胞HSL mRNA豐度的變化。IGF-、GLP-、乳酸處理的脂肪細(xì)胞在低溫操作室參照南京凱基總蛋白提取試劑盒提取細(xì)胞總蛋白，采用華特生蛋白定量試劑盒測定所提細(xì)胞總蛋白濃度，最后用南京建成脂肪酶測定試劑盒測定IGF-、GLP-、乳酸處理的脂肪細(xì)胞 HSL活性變化，數(shù)據(jù)同樣用SPSS10.0軟件統(tǒng)計分析。實驗結(jié)果表明：1. IGF-、GL

4、P-、乳酸對HSL mRNA表達的抑制作用存在劑量依賴性。IGF-濃度20g/L、GLP-濃度100nmol/L、乳酸濃度20mg/L時對HSL mRNA的表達抑制作用顯著（P。2. IGF-、GLP-、乳酸對HSL活性的抑制作用同樣存在劑量依賴性。IGF-濃度30g/L、GLP-濃度100nmol/L、乳酸濃度40mg/L時對HSL活性的抑制作用明顯（P。3. IGF-、GLP-、乳酸可通過抑制奶牛脂肪細(xì)胞內(nèi)HSL mRNA表達及HSL活性而抑制脂肪的分解，從而促進脂肪沉積。關(guān)鍵詞：IGF-；GLP-；乳酸；體外培養(yǎng)；脂肪細(xì)胞；HSL；mRNA豐度；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