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目的观察甲基硝基亚硝基胍(MNNG)诱导后日本血吸虫成虫培养细胞核仁组织区相关嗜银蛋白(Ag-NORs)含量的动态变化,探讨MNNG诱导后培养细胞的增殖能力。方法将日本血吸虫成虫细胞接种于小盖玻片上,置于含20%小牛血清附加常量抗生素的RPMI-1640常规培养基中培养;培养第4天,细胞随机分为实验组和对照组两组,实验组细胞以含3 g/ml MNNG的常规培养基处理48 h,对照组细胞则用不含MNNG的常规培养基作相同处理。细胞经彻底清洗后继续以常规培养基培养3周,然后换用含5%小牛血清的低血清培养基培养。MNNG处理后第1~9周,每周取实验组和对照组细胞采用略作修改的胶银法进行Ag-NORs染色,光镜下观察与拍照,HPIAS-2000图像分析仪测定代表培养细胞内Ag-NORs含量的吸光度(A)值,并进行统计学分析。结果培养过程中,对照组细胞着色逐渐变浅,MNNG组细胞除在第2周时着色变浅外,其余均逐渐加深,尤其在第6周(MNNG诱导后第5周),细胞核呈深棕色,可见粗大的银染颗粒,核仁呈黑色,并观察到分裂细胞。第7~9周,两组细胞着色均逐渐变浅。定量分析发现,对照组细胞在培养初期Ag-NORs含量最高,随后逐渐降低;MNNG组的Ag-NORs含量在培养第2周时稍有降低,随后逐渐升高;在培养第6周即MNNG诱导后第5周时达到高峰,然后又逐渐下降。结论MNNG诱导可显著增强培养细胞的rDNA转录活性,提高细胞的分裂增殖能力,在MNNG诱导后第5周细胞增殖能力最强。
Objective To observe the dynamic changes of Ag-NORs in cultured nuclei of adult Schistosoma japonicum induced by MNNG, and to explore the proliferative ability of cultured cells after MNNG induction. Methods Adult Schistosoma japonicum cells were inoculated into small coverslips and cultured in RPMI-1640 medium supplemented with 20% fetal bovine serum with constant antibiotics. On the 4th day of culture, the cells were randomly divided into experimental group and control group , The experimental group treated with conventional medium containing 3 g / ml MNNG for 48 h, while the control group treated the same medium with conventional medium without MNNG. The cells were thoroughly washed and then cultured in a regular medium for 3 weeks, then switched to low serum medium containing 5% fetal bovine serum. MNNG treatment 1 to 9 weeks, the experimental group and the control group each week to take the cells modified with Ag-NORs slightly modified Ag-NORs staining, light microscopy and photographed, HPIAS-2000 image analyzer measured on behalf of cultured cells Ag-NORs content of the absorbance (A) values, and statistical analysis. Results During the culture, the cells in the control group gradually became lighter and the cells in the MNNG group became darker than those in the 2nd week, especially in the 6th week (the 5th week after the MNNG induction), the nucleus was dark brown , Visible thick silver-stained particles, black nucleolus, and observed dividing cells. From the 7th to the 9th week, the cell coloring of both groups gradually became lighter. Quantitative analysis showed that the content of Ag-NORs in the control group was the highest at the beginning of culture, and then gradually decreased. The content of Ag-NORs in MNNG group decreased slightly at the second week of culture and then increased gradually. At 6 weeks of culture, MNNG induced The fifth week after reaching its peak, and then gradually decline. Conclusion MNNG induction can significantly enhance the rDNA transcription activity of cultured cells and enhance the ability of cell division and proliferation. The cell proliferation ability of MNNG after 5 weeks of induction is the strongest.