188Re-k-ras-AGPNA诱导胰腺癌细胞凋亡及在荷瘤裸鼠体内的生物分布

摘要

Objective To investigate the depressing effect of antigene peptide nucleic acid (AGPNA)on the k-ras gene expression of human pancreatic cancer Patu8988 cells, the inducing apoptosis effect on Patu8988 cells, and the biodistribution characteristics in nude mice bearing xenografts using 188Re-k-ras-AGPNA.Methods The expression level of k-ras mRNA and the expression ratio of k-ras protein in Patu8988 cells transfected with AGPNA was measured by RT-PCR and flow cytometry ,respectively. The degree of cellular apoptosis 3 to 5 d after treating Patu8988 cells with 188Re-k-ras-AGPNA or 188ReO4- was determined by flow cytometry. For biodistribution study, 58 nude mice bearing Patu8988 cell xenografts were divided into two groups: intratumoral injection of 188Re-k-ras-AGPNA (Group A) and 188ReO4- (Group B). At different time points, the mice were sacrificed and organs of interest were excised, weighted and counted by a gamma counter. The organ uptake was calculated as a % ID/g and the absorbed doses of organs were calculated. One-way analysis of variance was used. Results After transfected with 1 nmol/ml AGPNA, the k-ras mRNA gray scale ratio and the expression ratio of k-ras protein were 1.00 ± 0.39 and (15.05 ± 5.07)%, respectively. They were significantly lower than those of the control group with 1.86 ± 0.07 and (24. 38 ± 5.40) % (F = 2. 545, 5. 327, P<0. 05). At 4 and 5 d after treatment in Group A, float cells' apoptosis ratios were (26.30 ± 7.45) % and (27.90 ± 10. 38) %, respectively. Tumors were the major distribution site in Group A with uptake of (37.47 ±21.31), (35.96 ±7.80) and (15.46 ±4.93) %lD/g at 1 h, 1 d and 7 d after intra-tumor injection, respectively. The absorbed dose of tumor was 15 569 mGy/MBq. Condusions Transfection with k-ras-AGPNA on Patu8988 cells may inhibit k-ras expression at mRNA and protein expression level, and 188Re-k-ras-AGPNA can induce apoptosis of Patu8988 cells.Tumor is the major distribution site in nude mice bearing human pancreatic cancer xenografts after intratumoral injection of 188Re-k-ras-AGPNA.%目的 研究胰腺癌k-ras点突变基因的反基因肽核酸(AGPNA)生物活性和188Re-k-ras-AGPNA诱导人胰腺癌细胞凋亡及在荷瘤裸鼠体内的生物分布.方法 (1)用RT-PCR检测转染kras-AGPNA后胰腺癌细胞k-ras癌基因的mRNA表达水平.(2)用流式细胞仪检测转染后胰腺癌细胞的k-ras蛋白表达水平.(3)给予188Re-k-ras-AGPNA和188ReO4 3～5 d后,用流式细胞仪检测胰腺癌细胞凋亡率.(4)58只荷人胰腺癌裸鼠分为188Re-k-ras-AGPNA和188ReO4- 2组,采取瘤内注射给药方式,在不同时间点(2组分别为15 min,1 h,4 h,1 d,3 d,5 d,7 d与15 min,1 h,2 h,4 h,24 h)显像后处死裸鼠,计算各组织的%ID/g,并计算各组织的吸收剂量(mGy/MBq).对数据行单因素方差分析.结果 (1)转染1 nmol/ml k-ras-AGPNA组细胞的k-ras突变基因mRNA的灰度比和k-ras蛋白表达率分别为1.00±0.39和(15.05±5.07)%,比未给药的肿瘤细胞对照组[分别为1.86±0.007和(24.38±5.40)%]低(F=2.545,5.329,P均<0.05).(2)给药后4,5 d,188Re-k-ras-AGPNA组漂浮细胞的凋亡率分别为(26.30±7.45)%和(27.90±1 0.38)%,比188ReO4-组[分别为(8.75±3.11)%和(16.87±5.85)%]高(F=9.376,P均<0.05).(3)瘤内注射188Re-k-ras-AGPNA后1 h和1,7 d肿瘤内放射性摄取分别为(37.47±21.31)、(35.96±7.80)和(15.46±4.93)%ID/g.肿瘤内吸收剂量为15 569mG;y/MBq.结论 k-ras-AGPNA能抑制k-ras基因在mRNA和蛋白水平的表达.188Re-k-ras-AGPNA能诱导胰腺癌细胞凋亡且瘤内注射后肿瘤部位摄取高、滞留时间长.