摘要
目的探讨Ki67基因放射性核素反义治疗对人肾癌细胞生长及凋亡的影响。方法人工合成Ki67基因反义核酸(ASODNs),氯胺-T法125I标记ASODNs(125I-ASODNs)。①体外实验:125I-ASODNs(10.0μmol/L、2.59MBq/L)转染人肾癌786-0细胞,采用免疫组化、Westernblot检测Ki67表达;免疫组化TUNEL法检测细胞凋亡。以ASODNs(10.0μmol/L)为对照。②动物实验:建立786-0细胞BALB/C-nu裸鼠模型,125I-ASODNs治疗组瘤体注射0.1ml125I-ASODNs(7.4MBq/L、10nmol),ASODNs治疗组瘤体注射0.1mlASODNs(10nmol),连续4天。药物处理后第6天取瘤组织检测肿瘤体积、Ki67表达、细胞凋亡。结果①体外实验:125I-ASODNs治疗组肾癌细胞Ki67表达阳性率[(21.5±1.2)%]降低,Ki67蛋白[(49.9±4.5)%]降低,细胞凋亡率[(28.9±1.3)%]升高,与ASODNs治疗组[(29.9±0.4)%、(82.1±1.9)%、(15.7±0.5)%]比较差异有显著性(P<0.01)。②动物实验:125I-ASODNs治疗组小鼠肿瘤体积(mm3)(76.1±22.9)减小,Ki-67表达率[(20.8±1.0)%]下降,Ki-67蛋白量[(58.6±2.9)%]降低,肿瘤细胞凋亡率[(24.9±2.0)%]升高,与ASODNs治疗组[(185.7±37.7)、(29.2±1)%、(79.7±3.4)%、(17.8±1.9)%]比较差异有显著性(P<0.01)。结论Ki67基因放射性核素反义治疗较单纯反义治疗具有更强的阻抑肾癌细胞Ki67基因表达、抑制增殖、促进凋亡作用。
Objective To evaluate the effects of ^125Ⅰ - labeled antisense oligonucleotide （ ^125Ⅰ - ASODNs） targeting Ki67 gene on the proliferation and apoptosis of human renal carcinoma （HRC） cell line in vitro and in vivo. Methods ASODNs targeting Ki67 gene was coupled with ^125Ⅰ by chloraseptine- T methods. HRC cell line 786-0 cells in vitro were treated with ^125Ⅰ - ASODNs （ 10.0 μmol/L, 2.59 MBq/L） and ASODNs （ 10.0 μmol/L） separately. For in vivo study, the 786 - 0 cells were implanted subcutaneously in BALB/C - nu nude mice to form the implanted tumor model of HRC. In ^125Ⅰ-ASODNs treatment group,0. 1 ml ^125Ⅰ -ASODNs（7.4 MBq/L, 10 nmol）was directly injected into the tumor in nude mouse qd for 4 days. In ASODNs treatment group, 0. 1 ml ASODNs（ 10 nmol）was injected into the tumor qd for 4 days. The nude mice were killed 6 days later to determine the Ki67 expression of 786 -0 cells by immunohistochemical technique and Western blot method, to examine the apoptosis of 786 -0 cells by TUNEL assay, and to calculate the volume of the tumor by measuring it sizes. Results I. In vitro ： When compared to that of the ASODN treated ceils, the Ki67 positive rate（ % ） of 786 - 0 cells treated with ^125Ⅰ - ASODN was lower （ 29.9 ± 0.4 vs. 21.5 ±1. 2, P 〈 0.01）. the expression rate of Ki67 protein was lower（82.1±1.9 vs. 49.9±4.5, P〈0.01） , while the apoptosis rate was higher （15.7±0.5 vs. 28.9 ±1. 3, P 〈0.01 ）. 2. In vivo： When compared to that in the ASODNs treatment group, the tumor volume （ram3） in the ^125Ⅰ -ASODNs treatment group was significantly smaller（ 185.7 ± 37.7 vs. 76.1 ± 22.9, P 〈 0.01 ）, the rate（ % ） of Ki67 expression was significantly lower （29.2 ± 1. 0 vs. 20, 8 ±1. 0, P 〈 0：01 ）, the Ki67 protein rate（ % ） was significantly lower （79.7 ± 3.4 vs. 58.6 ±2.9, P 〈 0.01 ）, and the apoptosis rate（ % ） was significantly higher （17.8 ±1.9 vs. 24.9 ±2.0, P〈0.01）. Conclusion The effects of ASODNs targeting Ki67 gene on the proliferation and apoptosis of human renal carcinoma cells will be augemented if the ASODNs are coupled with ^125Ⅰ. Radionuclide antisense therapy may serve as a novel treatment for renal carcinoma.
出处
《徐州医学院学报》
CAS
2007年第1期30-33,共4页
Acta Academiae Medicinae Xuzhou
基金
江苏省卫生厅医学科技发展基金(H2000153)
江苏省高校自然科学基金(02KJD320030)
