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目的:探讨ICAM1反义寡核苷酸对小鼠肾小管上皮细胞表达ICAM1的影响,为利用ICAM1反义寡核苷酸类药物治疗肾小管间质病变奠定基础。方法:小鼠ICAM1反义寡核苷酸及其对照物参照文献合成并行全硫代磷酸化修饰。部分ICAM1反义寡核苷酸在其5′端作FITC荧光标记。将ICAM1反义寡核苷酸单独或脂质体转染试剂DOTAP混合后转染至培养的肾小管上皮细胞之中,转染成功后加入IL1β诱导细胞产生ICAM1。采用对照寡核苷酸及不含寡核苷酸的细胞转染液作为实验对照及空白对照。利用免疫组化染色的方法检测细胞ICAM1表达变化并提取细胞总RNA行逆转录PCR及Northern杂交观察ICAM1mRNA的表达的变化。结果:两个不同浓度的ICAM1反义寡核苷酸均可明显阻断IL1β诱导的肾小管上皮细胞ICAM1及ICAM1mRNA的表达;对照的寡核苷酸不能减少IL1β诱导的肾小管上皮细胞ICAM1及ICAM1mRNA的表达。结论:小鼠ICAM1反义寡核苷酸可明显抑制IL1β诱导的小鼠肾小管上皮细胞ICAM1及ICAM1mRNA的表达,提示ICAM1的反义寡核苷酸有可能应用于肾小管间质病变的实验治疗之中。
Objective: To investigate the effect of ICAM1 antisense oligonucleotide on the expression of ICAM1 in mouse renal tubular epithelial cells, and lay the foundation for the treatment of tubulointerstitial lesions with ICAM1 antisense oligonucleotide. METHODS: Mouse ICAM1 antisense oligonucleotides and their controls were synthesized in parallel with per-thiophosphorylation. Some ICAM1 antisense oligonucleotides are FITC fluorescently labeled at their 5 ’end. ICAM1 antisense oligonucleotides alone or liposome transfection reagent DOTAP mixed transfected into cultured renal tubular epithelial cells were added after the successful transfer of IL1β induced cells to produce ICAM1. The control oligonucleotide and oligonucleotide-free cell transfection solution were used as experimental control and blank control. The changes of ICAM1 expression were detected by immunohistochemical staining and the total RNA was extracted. The expression of ICAM1 mRNA was observed by reverse transcription PCR and Northern blotting. Results: Both ICAM1 antisense oligonucleotides at different concentrations could obviously block the expression of ICAM1 and ICAM1 mRNA in renal tubular epithelial cells induced by IL1β. The control oligonucleotide did not decrease IL1β-induced ICAM1 and ICAM1 mRNA in renal tubular epithelial cells expression. CONCLUSION: Antisense oligonucleotide of ICAM1 can significantly inhibit the expression of ICAM1 and ICAM1 mRNA in renal tubular epithelial cells induced by IL1β, suggesting that ICAM1 antisense oligonucleotide may be used in the experimental treatment of tubulointerstitial lesions Among them.