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目的检测胰岛素样生长因子-Ⅰ(insulin-like growth factor-Ⅰ,IGF-Ⅰ)蛋白及其m RNA在肥胖大鼠行Roux-en-Y胃旁路手术(RYGB)前后的表达水平变化,并探讨IGF-Ⅰ表达和脂肪细胞增殖、凋亡的关系。方法1选取SPF级SD雄性大鼠70只,随机分为空白对照组(NC组)10只和高脂饮食组60只。高脂饮食组大鼠给予特定配方高脂饲料,NC组大鼠给予特定配方维持饲料,6周后测量高脂饮食组大鼠的体质量,选择体质量增加在前20位的肥胖大鼠,随机分为胃旁路术组(GB组)10只和假手术组(SO组)10只。GB组大鼠行RYGB,SO组大鼠行假手术,NC组大鼠不接受任何手术。GB组和SO组大鼠于术中、3组大鼠于处理后12周取腹股沟脂肪组织〔代表皮下脂肪组织(subcutaneous adipose tissue,SAT)〕和附睾脂肪组织〔代表内脏脂肪组织(visceral adipose tissue,VAT)〕各0.5 g,行免疫印迹法和实时荧光定量PCR法分别检测脂肪组织中IGF-Ⅰ蛋白及其m RNA的表达。2转染实验。将SAT细胞分为空白对照组(BC组,未行转染)、IGF-Ⅰ(+)组(即基因过表达组)、IGF-Ⅰ(+)空载体组、IGF-Ⅰ(-)组(即基因沉默组)及IGF-Ⅰ(-)空载体组,转染相应载体,每组设3个复孔。转染48 h后进行细胞增殖与凋亡实验,并采用免疫印迹法检测蛋白激酶B(AKT)、磷酸化蛋白激酶B(p-AKT)、磷酸化磷脂酰肌醇3-激酶(p-PI3K)及磷脂酰肌醇3-激酶(PI3K)的表达水平。3 Wortmannin实验。将SAT细胞分为Wortmannin(+)IGF-Ⅰ(+)组、Wortmannin(+)IGF-Ⅰ(-)、Wortmannin(-)IGF-Ⅰ(+)及Wortmannin(-)IGF-Ⅰ(-)组,转染相应载体类型24 h后,加入0.1 mmol/L Wortmannin,24 h后采用免疫印迹法检测AKT、p-AKT、p-PI3K、PI3K及GAPDH的表达水平。结果 1 PCR结果表明,在SAT组织中,同GB术前组比较,GB术后组的IGF-Ⅰ蛋白及其m RNA的表达水平均降低(P<0.01),而SO术前组和SO术后组的IGF-Ⅰ蛋白及其m RNA的表达水平比较差异均无统计学意义(P>0.05);VAT中,5组大鼠的IGF-Ⅰ蛋白及其m RNA的表达水平比较差异均无统计学意义(P>0.05)。2四甲基偶氮唑盐(MTT)比色法结果表明,同对应的空载体组比较,IGF-Ⅰ(+)组的增殖能力升高(P=0.04),IGF-Ⅰ(-)组的细胞增殖缓慢(P=0.04);流式细胞仪检测结果表明,同对应的空载体组比较,IGF-Ⅰ(+)组的细胞凋亡率较低(P=0.04),IGF-Ⅰ(-)组的细胞凋亡率较高(P=0.04)。3与IGF-Ⅰ(+)空载体组比较,IGF-Ⅰ(+)组的p-PI3K/PI3K(P=0.03)和p-AKT/AKT(P=0.04)比值均升高;与IGF-Ⅰ(-)空载体组相比,IGF-Ⅰ(-)组的p-PI3K/PI3K(P=0.04)和p-AKT/AKT(P=0.04)比值均降低。与Wortmannin(+)IGF-Ⅰ(+)组比较,Wortmannin(-)IGF-Ⅰ(+)组的p-AKT/AKT比值较高(P<0.05);与Wortmannin(-)IGF-Ⅰ(+)组比较,Wortmannin(-)IGF-Ⅰ(-)组的p-AKT/AKT比值较低(P<0.05)。结论 IGF-Ⅰ参与大鼠皮下脂肪富集,RYGB能明显降低大鼠皮下脂肪中IGF-Ⅰ蛋白及其m RNA的表达水平,从而达到减重效果。
Objective To detect the expression of insulin-like growth factor-Ⅰ (IGF-Ⅰ) protein and m RNA before and after Roux-en-Y gastric bypass surgery (RYGB) in obese rats To investigate the relationship between IGF-Ⅰ expression and adipocyte proliferation and apoptosis. Method 1: Seventy SPF SD male rats were randomly divided into blank control group (n = 10) and high fat diet group (n = 60). The rats in high-fat diet group were given high-fat diet with specific formula. Rats in NC group were given special formula to maintain the feed. After 6 weeks, the body weight of rats in high-fat diet group was measured. In the top 20 obese rats, Randomly divided into gastric bypass group (GB group) 10 and sham operation group (SO group) 10. Rats in GB group were treated with RYGB, and rats in SO group were sham operated. Rats in NC group were not given any surgery. The rats in GB group and SO group were intraoperatively and the rats in three groups were treated with inguinal adipose tissue [representative of subcutaneous adipose tissue (SAT)] and epididymal adipose tissue [representative of visceral adipose tissue , VAT)] 0.5 g each. The expression of IGF-Ⅰ protein and m RNA in adipose tissue were detected by Western blotting and real-time fluorescence quantitative PCR respectively. 2 transfection experiments. The SAT cells were divided into blank control group (BC group, untransfected), IGF-Ⅰ (+) overexpression group, IGF-Ⅰ (Ie, gene silencing group) and IGF-Ⅰ (-) empty vector group were transfected with the corresponding vector, each set three wells. After 48 h of transfection, cell proliferation and apoptosis assays were performed. Protein kinase B (AKT), phosphorylated protein kinase B (p-AKT), phosphorylated phosphatidylinositol 3-kinase ) And phosphatidylinositol 3-kinase (PI3K) expression levels. 3 Wortmannin experiment. The SAT cells were divided into Wortmannin (+) IGF-Ⅰ group, Wortmannin (+) IGF-Ⅰ (-), Wortmannin (-) IGF-Ⅰ (+) and Wortmannin (-) IGF- After transfection with corresponding vector types for 24 h, 0.1 mmol / L Wortmannin was added and the expression of AKT, p-AKT, p-PI3K, PI3K and GAPDH was detected by Western blotting 24 h later. Results 1 PCR results showed that in the SAT tissue, compared with the pre-GB group, the expression of IGF-Ⅰprotein and m RNA in GB group decreased (P <0.01), while the preoperative SO and SO There was no significant difference in IGF-Ⅰprotein and m RNA expression between the two groups (P> 0.05). There was no significant difference in IGF-Ⅰprotein and m RNA between the five groups Statistical significance (P> 0.05). MTT assay showed that compared with the corresponding empty vector group, the proliferation of IGF-Ⅰ (+) group (P = 0.04) and IGF-Ⅰ (-) group (P = 0.04). The results of flow cytometry showed that the apoptosis rate of IGF-Ⅰ (+) group was lower than that of the corresponding empty vector group (P = 0.04) -) group had a higher rate of apoptosis (P = 0.04). Compared with the IGF-Ⅰ (+) empty vector group, the ratios of p-PI3K / PI3K (P = 0.03) and p-AKT / AKT The ratios of p-PI3K / PI3K (P = 0.04) and p-AKT / AKT (P = 0.04) in IGF-Ⅰ (-) group were all lower than those in I (-) The ratio of p-AKT / AKT in Wortmannin (-) IGF-Ⅰ (+) group was higher than that in Wortmannin (+) IGF-Ⅰ ) Group, the ratio of p-AKT / AKT in Wortmannin (-) IGF-Ⅰ (-) group was lower (P <0.05). Conclusion IGF-Ⅰ is involved in subcutaneous fat accumulation in rats. RYGB can significantly reduce the expression of IGF-Ⅰ protein and m RNA in rat subcutaneous fat to achieve weight loss.