日本血吸虫细胞培养条件的筛选和抗原含量动态变化的研究

发布时间：2018-01-14 16:42

本文关键词：日本血吸虫细胞培养条件的筛选和抗原含量动态变化的研究　出处：《武汉大学》2005年硕士论文　论文类型：学位论文

【摘要】：本文以琥珀酸脱氢酶(SDH)、乳酸脱氢酶(LDH)和葡萄糖-6-磷酸脱氢酶(G-6-PDH)为评价指标,运用正交试验法筛选日本血吸虫细胞的培养条件,探讨这3个评价指标的可靠性;并用筛选出的培养条件培养日本血吸虫细胞,研究日本血吸虫培养细胞抗原含量的动态变化。所获结果简述如下: 1.日本血吸虫细胞培养条件的选择以SDH、LDH和G-6-PDH为评价指标,运用正交试验法研究合成培养基(TC-199、RPMI-1640和DMEM)、细胞外基质(鼠尾胶、肝基质与肺基质)和血清浓度(5%、10%及20%)3个因素在3个不同水平对虫龄为21d的日本血吸虫培养细胞的影响。培养第5d,对日本血吸虫培养细胞进行SDH染色;培养第14d,分别进行LDH、G-6-PDH染色;培养第28d,进行LDH染色。将染色后的培养细胞置于Olympus-BH_2显微镜下观察并拍照,HPIAS-2000图像分析仪测量它们的含量,用SPSS10.0作统计分析。结果显示:培养早期,不论以SDH或LDH,还是G-6-PDH为指标,RPMI-1640含20%小牛血清组成的培养基培养接种于肝基质上的日本血吸虫细胞,培养细胞的SDH、LDH、G-6-PDH活性最强。血清浓度对培养细胞酶活性的影响最大,其次是细胞外基质,影响最小的是合成培养基。其中,血清浓度对3种酶活性的影响差异均显著(P0.01或P0.05);基质对SDH、LDH活性的影响差异显著(P0.01或P0.05),而对G-6-PDH活性的影响差异不显著(P0.05);合成培养基的影响均无差异(P0.05)。培养28d以后,DMEM含5%小牛血清组成的培养基培养接种于肝基质上的日本血吸虫细胞,培养细胞的LDH活性最强。血清浓度对培养细胞LDH活性的影响最大,其次是细胞外基质,影响最小的是合成培养基。血清浓度和细胞外基质对LDH活性的影响差异均显著(P0.05),合成培养基的影响均无显著性差异(P0.05)。因此,选择3种酶中任一,均可用来评价日本血吸虫细胞培养条件的优劣。在培养早期,选择三种合成培养基中的任一种与20%小牛血清组成的培养基培养接种于肝基质上的日本血吸虫细胞,培养细胞的SDH、LDH、G-6-PDH活性最强。培养28d以后,三种培
[Abstract]:In this paper, succinate dehydrogenase (SDH), lactate dehydrogenase (LDH) and glucose -6- phosphate dehydrogenase (G-6-PDH) as the evaluation index, screening of Schistosoma japonicum cell culture conditions by using orthogonal test method, the reliability of the 3 indexes; and screened cultured cells of Schistosoma japonicum and Schistosoma japonicum dynamic changes of cell culture antigen content. The results are summarized as follows:
Selection of cell culture conditions for Schistosoma japonicum 1.
In SDH, LDH and G-6-PDH as the evaluation index, using orthogonal test method based on Synthesis of culture (TC-199, RPMI-1640 and DMEM), extracellular matrix (rat tail collagen, liver and lung stromal matrix) and serum concentration (5%, 10% and 20%) 3 factors at 3 different levels of instar 21d the schistosome effects in cultured cells cultured in 5D cells. SDH staining, cultivation of Schistosoma japonicum; cultured in 14d, were analyzed by LDH and G-6-PDH staining; cultured 28d, LDH staining. After staining the cells cultured in Olympus-BH_2 observed and photographed under the microscope, measuring their content of HPIAS-2000 image analyzer, statistical analysis by using SPSS10.0. The results showed that early training, either at SDH or LDH, or G-6-PDH as the index, RPMI-1640 containing 20% calf serum. The cultured in culture medium on liver stromal cells of Schistosoma japonicum, the culture cells of SDH, LDH, G-6-PDH activity was the strongest. Effect of serum concentration on enzyme activity in cultured cells, followed by extracellular matrix, the impact is minimal synthetic medium. The difference of serum concentration of 3 enzymes were significantly (P0.01 or P0.05); matrix of SDH, influence the activity of LDH significantly (P0.01 or P0.05), but not significant effect on the activity of G-6-PDH difference (P0.05); there was no significant difference in effects of synthetic medium (P0.05). After 28d culture, DMEM containing 5% calf serum. The cultured in culture medium on liver stromal cells of Schistosoma japonicum, cultured cells LDH activity. The effects of serum concentration on LDH activity in cultured cells the largest, followed by the extracellular matrix, the impact is minimal synthetic medium. The serum concentration of extracellular matrix and the influence on the activity of LDH were significantly different (P0.05), there were no significant differences between the effects of synthetic medium (P0.05). Therefore, 3 kinds of enzyme in any, Can be used to evaluate the culture conditions of cells from Schistosoma japonicum. In the early stage of culture, culture medium were inoculated in the liver matrix choose three synthetic medium one and 20% calf serum. The cultured cells of Schistosoma japonicum, SDH, LDH, the strong G-6-PDH activity. 28d culture after three.

【学位授予单位】：武汉大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R383

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