【作者】 钱辉；

【导师】 邓俊良； 王哲；

【作者基本信息】 四川农业大学 ， 临床兽医学， 2007， 硕士

【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-Hank′s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、50μg／L胰岛素样生长因子Ⅰ（IGF-Ⅰ），0、100、250、500、750、1000nmol／L胰高血糖素样肽Ⅰ（GLP-Ⅰ），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PER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型荧光定量PCR扩增仪上扩增，机器自动绘制出标准曲线。将IGF-Ⅰ、GLP-Ⅰ、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-Ⅰ、GLP-Ⅰ、乳酸处理的脂肪细胞HSL mRNA丰度的变化。IGF-Ⅰ、GLP-Ⅰ、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-Ⅰ、GLP-Ⅰ、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-Ⅰ、GLP-Ⅰ、乳酸对HSL mRNA表达的抑制作用存在剂量依赖性。IGF-Ⅰ浓度≥20μg／L、GLP-Ⅰ浓度≥100nmol／L、乳酸浓度≥20mg／L时对HSL mRNA的表达抑制作用显著（P＜0.05或0.01）。2．IGF-Ⅰ、GLP-Ⅰ、乳酸对HSL活性的抑制作用同样存在剂量依赖性。IGF-I浓度≥30μg／L、GLP-Ⅰ浓度≥100nmol／L、乳酸浓度≥40mg／L时对HSL活性的抑制作用明显（P＜0.05或0.01）。3．IGF-Ⅰ、GLP-Ⅰ、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。更多还原

【Abstract】 In this experiment, the healthy and neoformtive Holstan calves were caused to deaththrough cutting arteria carotis. About 60g epiploon of intestina parva was taken fromabdominal cavity without contaminate. After being washed with D-Hank’s, the fiber andblood vessel were rejected from the intestina parva. Following the method of cultivantingof adipose cell established by this laboratory, the adipose cells were cultivanted to thefourteen day. In the eighteenth、twelveth day, the adipose cells were observed after beingstained with rathonum red and trypan blue respectively. Untill the fourteen day, IGF-Ⅰ、GLP-Ⅰand lactic acid were added to the media with 0,10,20,30,40,50μg/L、0,100,250,500,750,1000nmol/L、0,10,20,30,40,50mg/L respectively. Every concentrationgradient was three dulplated. After 24 hours, the total RNA and total protein were extractedfrom the adipose cells. The total RNA was 20 times diluted, and then the concentration andpurity were determined in the machine of RNA/DNA calculator. Finally, erery sample of thetotal RNA was adjusted to the same concentration and purity with DEPC-H₂O to avoid thedifference of contents in the extraction of it and to ensure truth of the quantitation. Thedepurant products of RT-PCR of HSLmRNA which were positive and normallyquantitative were recombinated and cloned and sequence determined. The above plasmidswere diluted according to the specified concentration grad with the degerming water to bethe positive and normal template of fluorescence PCR. The amplicification was done in theABI PRISM 7000 PCR machine according to the institutional PCR system andqualification. The standard curve was motily drawed by the machine. The total RNA ofadipose cells dealed with IGF-Ⅰ、GLP-Ⅰand the lactic acid respectively were reverslytranscripted. The products of reverse transcription were amplicificated in ABI PRISM 7000 PCR machine according to the above standard curve. The numerical datas were statisticallyanalyzed with the software of SPSS10.0 to ensure the variations of abundances of HSLmRNA of adipose cells dealed with IGF-Ⅰ、GLP-Ⅰand the lactic acid respectively. Thetotal protein of adipose cells dealed with IGF-Ⅰ、GLP-Ⅰand the lactic acid respectivelywere extracted in the hypothermal operating room according to kit of total proteinextraction of Nanjing KaiJi corporation. The concentration of the total protein wasdetermined with the kit of HuaTeSheng corporation. Finally, the activity of HSL of adiposecells dealed with IGF-Ⅰ、GLP-Ⅰand the lactic acid respectively was determined withthe kit of Lipase Activity Assay. The numerical datas were also statistically analyzedwith the software of SPSS 10.0 to ensure the variations of the activity of HSL of adiposecells dealed with IGF-Ⅰ、GLP-Ⅰand the lactic acid respectively. The results showed thatthe expression of HSL mRNA and the activity of HSL were suppressed in adipose cellstreated with IGF-Ⅰ, lactic acid respectively. This suppression was dosedependent. Firstly, while the concentration of IGF-Ⅰ≥20μg/L、the concentration ofGLP-Ⅰ≥100nmol/L、the concentration of the lactic acid≥20mg/L, the suppressionestowards the abundance of HSL mRNA were all notable （P＜0.05 or 0.01）. Secondly, whilethe concentration of IGF-Ⅰ≥30μg/L、the concentration of GLP-Ⅰ≥100nmol/L、theconcentration of the lactic acid≥40mg/L, the suppressiones towards the activity of HSLwere all notable （P＜0.05 or 0.01）. Finally, we can conclude that IGF-Ⅰ、GLP-Ⅰ、thelactic acid can regulate the fat lipoclasis through regulating the abudance of HSL mRNAand the activity of it. And then, to promote adipo-synthesis.更多还原