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

Inducing apoptosis effect by 188 Re-k-ras-AGPNA on pancreatic cancer cells and biodistribution characteristics in nude mice bearing xenografts

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”。Re—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k—ras．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188ReO4-3～5d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15min，1h，4h，1d，3d，5d，7d与15min，1h，2h，4h，24h）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1nmol／mlk-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±0．07和（24．38±5．40）％]低（F=2．545，5．329，P均〈0．05）。（2）给药后4，5d，188Re-k-ras-AGPNA组漂浮细胞的凋亡率分别为（26．30±7．45）％和（27．90±10．38）％，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—ra8-AGPNA后1h和1，7d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15569mGy／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—ra,s—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。

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 Pa- tu8988 cells, and the biodistribution characteristics in nude mice hearing xenografts using ISSRe-k-ras-AGPNA. Methods The expression level of k-ras mRNA and the expression ratio of k-ras protein in Patu8988 ceils trans- fected 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 biedistribution 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 ） % ID/g at 1 h, 1 d and 7 d after intra-tumor injection, respectively. The absorbed close of tumorwas 15 569 mGy/MBq. Conclusions Transfection with k-ras-AGPNA on Patu8988 cells may inhibit k-ras expression at mRNA and protein expression level, and 188 Re-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 lSSRe-k-ras-AGPNA.