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咪唑作为一种弱碱性物质,处理动物子宫内膜细胞后,可诱导其产生空泡化现象,进而影响其生殖生理功能。本研究使用人子宫内膜癌细胞HEC-1B作为模型,使用咪唑处理导致细胞空泡化后探讨空泡化产生的生理机制。通过基因芯片分析、荧光定量PCR检测、激光共聚焦显微镜观察等方法进行研究。结果表明:咪唑处理人子宫内膜癌细胞HEC-1B后,空泡型质子泵(V-ATPase)亚基V0D2基因表达量上调。使用荧光定量PCR验证芯片结果,V-ATPase亚基V0D2 mRNA表达量有所上调。对V-ATPase另外一个亚基V1E1基因进行荧光定量PCR,其mRNA表达量也有所上调。使用V-ATPase抑制剂巴弗洛霉素A1和咪唑共同处理人子宫内膜癌细胞HEC-1B后,通过吖啶橙染色,激光共聚焦显微镜观察,咪唑诱导的空泡被显著抑制。上述结果证明了V-ATPase在咪唑诱导的细胞空泡化中的重要作用。
Imidazole as a weakly basic substance, after treatment of animal endometrial cells, can induce its generation of vacuolization, thereby affecting its reproductive physiological functions. In this study, human endometrial cancer cell line HEC-1B was used as a model, and after imidazole treatment resulted in cellular vacuolization, the physiological mechanism of the vacuolization was explored. By gene chip analysis, fluorescence quantitative PCR detection, laser confocal microscopy and other methods to study. The results showed that the V0D2 gene expression of vacuolar proton pump (V-ATPase) subunit was up-regulated after imidazole treatment of human endometrial carcinoma cell HEC-1B. Using fluorescence quantitative PCR to verify the results of the chip, V-ATPase subunit V0D2 mRNA expression was increased. Fluorescent quantitative PCR of V1E1 gene, another subunit of V-ATPase, was also up-regulated in mRNA level. After co-treatment of human endometrial carcinoma cell line HEC-1B with the BAC and the imidacloprid inhibitor of V-ATPase, imidazole-induced vacuoles were significantly inhibited by acridine orange staining and laser confocal microscopy. The above results demonstrate the important role of V-ATPase in imidazole-induced cell vacuolization.