【作者】 刘俐君；

【导师】 张海彬；

【作者基本信息】 南京农业大学 ， 临床兽医学， 2008， 硕士

【摘要】 脱氧雪腐镰刀菌烯醇（DON）广泛存在于世界范围内的谷物及其制品中,并可引起人和动物中毒,但其作用机制至今仍不十分清楚。本实验以不同组织来源的细胞为研究对象,从细胞毒性、凋亡形态学和早期凋亡检测三方面来比较细胞对DON的敏感性,为进一步研究DON诱导细胞凋亡的机制奠定基础。实验一:本试验采用不同组织来源的细胞（BHK-21、PIEC、HT-29、CHO-K1、BGC-823、BT-325和HL-7702）为研究对象,用不同浓度的DON（DON终浓度分别为125 ng/mL,250 ng/mL,500 ng/mL,1000 ng/mL,2000 ng/mL）处理不同时间（24 h、48 h和72 h）后,通过MTT分析法测定细胞增殖的变化,对比各细胞的敏感性。结果显示:不同浓度的DON均可抑制细胞的增殖,剂量越大抑制率越高,且与对照组相比差异极显著（P＜0.01）。500～2000 ng/mL浓度组,除CHO-K1 48 h和72 h段结果相近外,其它细胞三个时间段的抑制率呈显著的时间效应关系（P＜0.05）。各时间段1000和2000 ng/mL组抑制率相比较,除了BGC-823和BT-325外,其它细胞的差异不显著。由IC₅₀结果得出BHK-21是最敏感的细胞,HL-7702敏感性最低。DON处理24 h时BHK-21的IC₅₀为333ng/mL,其它六种细胞均大于2000 ng/mL。48 h测定结果从高到低依次为BHK-21＞PIEC＞HT-29＞CHO-K1＞BGC-823＞BT-325＞HL-7702。IC₅₀分别为320,361,598,620,1048,1485,＞2000 ng/mL。72 h的检测结果从高到低依次为:BHK-21＞PIEC＞HT-29＞CHO-K1＞BT-325＞BGC-823＞HL-7702,IC₅₀分别为228,238,438,678,832,908,＞2000 ng/mL。结论:DON对七种细胞均有毒性。根据细胞来源不同,毒性大小也有差异。DON的细胞毒性存在时间和剂量效应关系,并且与细胞的生长速度有关。BHK-21是所选细胞系中对DON最敏感的的细胞,HL-7702敏感性最低。实验二:本实验采用不同组织来源的细胞（BHK-21、PIEC、HT-29、CHO-K1、BGC-823、BT-325和HL-7702）为模型,用不同的浓度DON（DON的终浓度分别为125 ng/mL,250 ng/mL,500 ng/mL,1000 ng/mL,2000 ng/mL）处理24 h。染色后作形态学观察和流式细胞仪测定凋亡率,对比细胞对DON的敏感性。并用彗星实验验证DON对敏感细胞的DNA损伤情况。结果显示:不同浓度DON对七种细胞均有诱导凋亡的作用。细胞的形态变化和凋亡率与DON呈显著的剂量效应关系。除BGC-823的125 ng/mL组外,每种细胞各浓度组的凋亡率都显著高于对照组（P＜0.05）。细胞坏死率所占百分比均小于5%,且各细胞的凋亡率均在2000 ng/mL剂量组达到最高。结论:DON对所选七种细胞均有诱导凋亡的作用,DON对细胞形态学变化和诱导凋亡作用与毒素剂量、细胞来源细胞生长速度和细胞毒性有关。DON诱导细胞凋亡的敏感性顺序从高到低依次为HT-29＞BHK-21＞BT-325＞PIEC＞BGC-823,CHO-K1,HL-7702。DON对HT-29的DNA损伤与凋亡检测结果一致。HT-29细胞可用于深入研究DON诱导细胞凋亡的机制。更多还原

【Abstract】 Deoxynivalenol （DON） occours worldwidly in cereal grains and their feeds, which have been associated with human and animal toxicoses, serious questions remain regarding how it should be regulate. We adopted cell lines from different origin, and compared their sensitivity about DON from cytotoxicity, morphology and early apoptosis. The aim of this study was to establish fundament for the further mechanism investigation of apoptosis induced by DON.TEST 1:In the present experiment, cell lines from different origins （BHK-21、PIEC、HT-29、CHO-K1、BGC-823、BT-325 and HL-7702） were exposed to five different concentration of DON （125 ng/mL, 250 ng/mL, 500 ng/mL, 1000 ng/mL, 2000 ng/mL） for 24 h, 48 h and 72 h, respectively. The MTT bioassay was performed to determine the inhibition ratio on proliferation of the seven cell lines, and compared their sensitivity. The results showed that the different concentrations of DON could inhibit the proliferation of these cell lines. High inhibition depended on the increased concentration. The inhibition on proliferation of each concentation was the extremely significant compared to the controls （P<0.01）. Significant time-dependent manner（P<0.05） was observerd in DON 500～2000 ng/mL groups of these cell lines from 24 h, 48 h and 72 h, except CHO-K1 in 48 h and 72 h groups. There was no significant change of inhibition in DON 1000 and 2000ng/mL groups in each time except BGC-823 and BT-325. The IC₅₀ value of BHK-21 was 333 ng/mL, after 24 h exposured to DON, and the others exceeded 2000ng/mL. The sensitivity of the cell lines were found in the decreasing order of BHK-21 > PIEC > HT-29 > CHO-K1> BGC-823 > BT-325 > HL-7702 after 48-h exposure, with IC₅₀ values of 320, 361, 598, 620, 1048, 1485, >2000ng/mL respectively. The sensitivities of the cell lines were found to be in the decreasing order of BHK-21 > PIEC > HT-29> CHO-K1> BT-325 > BGC-823> HL-7702 after 72-h exposure, with IC₅₀ values of 228, 238, 438, 678, 832, 908, >2000ng/mL, respectively. In conclusion, DON was toxical effect for these cell lines. The cytotoxicity was associated with does, duration time, cell origin and proliferation velocity. BHK-21 was the most sensitive about DON in these cells. However, HL-7702 was the less sensitive about DON than others.TEST 2:In the present experiment, cell lines from different origins （BHK-21、PIEC、HT-29、CHO-K1、BGC-823、BT-325 and HL-7702） were exposed to five different concentrations of DON （125 ng/mL, 250 ng/mL, 500 ng/mL, 1000 ng/mL, 2000ng/mL） for 24 h. The morphological changes of the cell lines were observed under microscope after staining. The apoptotic ratio was analyzed by flow cytometry to compare their sensitivity about DON. The DNA damage of sensitive cell was detected by comet assay. The result showed that Different concentration of DON induced apoptosis of all the cell lines The morphological changes and the apoptotic ratio of the cell lines was observed in a significant dose-dependent manner. A significant change of apoptotic ratio of these cell lines in defferent concentrations comparing with their control group（P<0.05）, except BGC-823 in DON 125 ng/mL. The necrosis cell were less than 5% of the total cell numbers. The apoptotic ratio reached the highest level at 2000 ng/mL in these cell lines. In conclusion, DON can induced apoptosis in these cell lines. The morphological changes and the apoptotic ratio of the cell lines were associated with does, cell origin and cytotoxicity. BHK-21 was the most sensitive for DON and HL-7702 is the less sensitive about DON than others. The sensitivities of the cell lines were found to be in the decreasing order of HT-29>BHK-21>BT-325>PIEC > BGC-823, CHO-K1, HL-7702. The effect of DON on DNA damage of HT-29 is associated with apoptosis. HT-29 could adopt for the further mechanism investigation of apoptosis induced by DON.更多还原