反义寡聚核苷酸治疗支气管哮喘的实验研究

反义寡聚核苷酸治疗支气管哮喘的实验研究
王美琴;白春学;钮善福;方晓惠;陈常庆;陈波
【期刊名称】《复旦学报（医学版）》
【年(卷),期】2000(027)006
【摘要】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 T4 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 transcription of IL-5 from T lymphocytes. The study provides foundation for antisense gene therapeutic asthma.%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-5 cDNA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∶15 m/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、20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(44.60±6.23)pg/ml分别降低到(30.70±7.362)、
(17.20±6.181)和(8.16±2.34)pg/ml，抑制IL-5合成百分率分别为31.17%、61.43%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。

支持其通过抑制致敏小鼠T淋巴细胞IL-5的转录而抑制其合成，为反义基因治疗哮喘提供了依据
【总页数】5页(P464-467，470)
【作者】王美琴;白春学;钮善福;方晓惠;陈常庆;陈波
【作者单位】华山医院内科上海 200040;上海医科大学中山医院呼吸病研究所上海 200032;上海医科大学中山医院呼吸病研究所上海 200032;上海医科大学中山医院呼吸病研究所上海 200032;中国科学院上海生物工程中心上海 200233;中国科学院上海生物工程中心上海 200233
【正文语种】中文
【中图分类】Q524+.3
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