Effects Of IGF-Ⅰ,GLP-Ⅰ And Lactic Acid On Abundance Of HSL MRNA And Activity Of HSL In Vitro Culture Bovine Adipocyte

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-â… ,GLP-â… and 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-â… ,GLP-â… and 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-â… ,GLP-â… and the lactic acid respectively. Thetotal protein of adipose cells dealed with IGF-â… ,GLP-â… and 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-â… ,GLP-â… and 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-â… ,GLP-â… and the lactic acid respectively. The results showed thatthe expression of HSL mRNA and the activity of HSL were suppressed in adipose cellstreated with IGF-â… , 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-â… ,GLP-â… ,thelactic acid can regulate the fat lipoclasis through regulating the abudance of HSL mRNAand the activity of it. And then, to promote adipo-synthesis.