反义寡聚核苷酸治疗支气管哮喘的实验研究  

Experimental Study on Treatment of Bronchial Asthma With Antisense Oligonucleotid

作　　者：王美琴[1] 白春学[2] 钮善福[2] 方晓惠[2] 陈常庆[3] 陈波[3] 

机构地区：[1]华山医院内科，上海 200040 [2]上海医科大学中山医院呼吸病研究所，上海 200032 [3]中国科学院上海生物工程中心，上海 200233

出　　处：《上海医科大学学报》2000年第6期464-467,470,共5页Journal of Fudan University(Medical Science)

摘　　要：目的　探讨反义寡聚核苷酸治疗支气管哮喘的可能性及效果。方法　根据小鼠IL 5cDNA序列设计合成反义寡聚核苷酸片段 ,用T4 噬菌体多核苷酸激酶标记反义寡聚核苷酸的 5′端 ,观察硬脂胺 (SA)脂质体对反义寡聚核苷酸的转染效率的影响。用卵蛋白和氢氧化铝复制哮喘模型。用聚酰纤维分离小鼠脾T淋巴细胞进行体外培养并导入由阳离子脂质体携带的不同浓度的反义寡聚核苷酸 ,观察其对T淋巴细胞合成IL 5的影响。采用酶联免疫吸附 (ELISA)法测定细胞培养上清液中IL 5浓度。结果　SA脂质体能显著提高反义寡聚核苷酸的转染效率 ,1∶15m/m(反义寡聚核苷酸和SA脂质体质量比 )时转染效率最佳 ,提高反义寡聚核苷酸转染效率 12倍。未经卵蛋白刺激的正常鼠和哮喘鼠T淋巴细胞培养上清液中未测到IL 5 ,经卵蛋白刺激后 ,T淋巴细胞培养上清液中IL 5含量明显增高。导入不同浓度的反义寡聚核苷酸 (10、2 0及 30 μmol/L)后 ,脾T淋巴细胞合成IL 5明显降低 ,细胞培养上清液中IL 5含量由 (4 4.6 0± 6 .2 3)pg/ml分别降低到 (30 .70± 7.36 2 )、(17.2 0± 6 .181)和 (8.16± 2 .34) pg/ml,抑制IL 5合成百分率分别为 31.17%、6 1.43%和81.7%。结论　反义寡聚核苷酸可明显抑制IL 5的合成 ,且呈明显的量效关系。Purpose To explore the possibility and the effect of therapeutic bronchial asthma by antisense oligonucleotid. Methods Based on the IL?5 cDNA sequence of mouse,a segment of antisense oligonucleotid was designed and synthetized.5′ labeling of antisense oligonucleotid was signed by T 4 PNK in order that the efficiency of stearylamine liposome in transfe ting antisense oligonucleotid can be evaluated. Astham model was duplicated with ovalbumin (OVA) absorbed to aluminum hydroxide. T lymphocytes of mice were separated by nylon fiber method,then T lymphocytes transfected a different content of antisense oligonucleotid with stearylamine phys. positive liposome were cultured respectively in order to observe the effect of antisense oligonucleotid on IL?5 produced by T lymphocytes. IL?5 levels in the supernatants of T lymphocytes culture were determined by ELISA. Results Stearylamine liposome could markedly increase the efficiency of antisense oligonucleotid transfection. The efficiency of antisense oligonucleotid transfection was the best at 1∶15 m/m (antisense oligonucleotid and SA liposome) and it was increased approximately 12 times.In healthy and asthma Balb/c mice, IL?5 was not detected in the supernatants of T lymphocytes culture without challenge with OVA.However,IL?5 was increased markedly in the supernatants of T lymphocytes culture challenged with OVA. After transfecting a different concentration antisense oligonucleotid, IL?5 levels in the supernatants of T lymphocytes culture were significantly lower than those in control cells without antisense oligonucleotid transfection. IL?5 levels decreased from (44.60±6.23) to (30.70±7.362),(17.20±6.181) and(8.16±2.34)pg/ml respectively. And IL?5 synthesis was inhibited by 31.17% , 61.43% and 81.7% respectively. Conclutions IL?5 synthesis could be obviously inhibited by antisense oligonucleotid and showed a markedly relation between quantitative and effect. It is supported that the production of IL?5 be inhibited through preventing the transcript

关 键 词：反义寡聚核苷酸 支气管哮喘 基因治疗 IL-5 

分 类 号：R562.250.5[医药卫生—呼吸系统] R394[医药卫生—内科学]