MNNG诱导对日本血吸虫成虫培养细胞核仁组织区相关嗜银蛋白的影响

Effects of N-methyl-N-nitro-N-nitrosoguanidine on argyrophilic nucleolar organizer region associated proteins in cultured cells from adult Schistosoma japonicum

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3g／ml MNNG的常规培养基处理48h，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 study the dynamic effects of N-methyl-N-nitro-N-nitrosoguanidine （MNNG） on the contents of argyrophilic nucleolar organizer region associated proteins （Ag-NORs） in the cultured cells and the proliferation of the cultured cells from adult Schistosoma japonicum. Methods The cultured ceils from 32-day old S. japonicum were inoculated on cover slips and cultured in routine medium which was made up of RPMI-1640, 20% calf serum and moderate amount of antibiotics. At the 4th day, the cultured cells were divided into a test group and control group at random. The cells in the test group were treated with MNNG which final concentration in the routine medium was 3 g/ml for 48 h, whereas those in the control group were treated with the routine medium free MNNG for the same time. After being washed thoroughly, they went on being cultured in the routine medium for 3 weeks; then were cultured in the low-serum medium which serum concentration was just 5%. The cells were stained weekly to show Ag-NORs in nuclear by the silver colloid technique from the 1st to 9th week. The number of granules and average optical density of Ag-NORs were measured by using photometerimage analytical instrument （HPIAS-2000） and analyzed by the statistic method. Results Along with the extending of the culture time, the staining of the cultured cells in the control group became weak, whereas those in the test group became strong except for the 2nd week from the 1 st to 6th week, especially at the 6th week （the 5th week after MNNG treatment）, cell nuclei were deeply stained into deep-brown, mass and gross Ag-NORs were observed and most nucleoli were black. At this stage, the dividing cells were observed also. The staining became weak gradually in the test group after the 7th week. The statistical analysis showed that the contents of Ag-NORs in the ceils of the control groups were the highest at the 1 st week, and then decreased with the cultured time. The contents of Ag-NORs in cells of test groups were increased with the cultured time except for the 2nd week,and at the 6th week, the contents of Ag-NORs in cells were highest, then decreased with the cultured time after the 7th week. Conclusion The rDNA transcription of the cultured cells from adult S. japonicum and its reproductive activity could be enhanced evidently after MNNG treatment, and the cell proliferation were most prominent at the 5th week after the MNNG treatment.