【作者】 赵亮；

【导师】 张学美；

【作者基本信息】 昆明医学院 ， 内科学， 2011， 硕士

【摘要】 目的：通过检测不同浓度三氧化二砷(ATO)对人皮肤T细胞淋巴瘤Hut-78细胞株的诱导凋亡作用、细胞周期及hTERT、PML、PML-Ⅰ、PML-Ⅳ和PML-V基因表达的影响,探讨上述基因在ATO诱导人皮肽T细胞淋巴瘤凋亡过程中的作用及可能机制。方法：本课题采用PI单标记流式细胞术检测不同浓度的ATO,作用不同时间后,对人皮肤T细胞淋巴瘤Hut-78细胞株的诱导凋亡作用及对细胞周期的影响;采用RT-PCR方法检测不同浓度的ATO,作用不同时间后,对人皮肤T细胞淋巴瘤Hut-78细胞株hTERT、PML、PML-Ⅰ、PML-Ⅳ及PML-Ⅴ基因表达变化的影响。结果：1、以2-10μmol/L浓度的ATO分别干预Hut-78细胞24、48、72小时,流式细胞仪分析各期细胞占细胞周期的比例及细胞凋亡(坏死)比率,结果示ATO干预后被阻滞于G2/M期的细胞随着ATO作用时间及浓度的增加比例逐渐增加,10μmol/L作用72小时时G2/M期比例最高,随药物浓度的增加及作用时间延长,Hut-78细胞的凋亡(坏死)比率逐渐增加；2、在ATO 2、5、10μmol/L三组浓度的作用下,用RT-PCR方法检测不同作用时间下Hut-78细胞hTERT、PML、PML-Ⅰ、PML-Ⅳ及PML-Ⅴ基因表达水平,结果示：(1)随着ATO浓度和作用时间的增加,hTERT mRNA的表达水平逐渐降低；(2)在不同浓度ATO作用48 h后PML的表达增加至最高,从48h至72h则逐渐降低；(3)在2umol/L 10umol/L和的ATO作用下,随着作用时间的延长,PML-I的表达逐渐增加；在对照组和5umol/L的ATO作用下,从24h至48h之间,随着时间延长,表达逐渐减少,而从48h至72h之间,则随着时间延长,表达逐渐增加；(4)在不同浓度ATO作用后,在24h至48h之间,随着时间延长,PML-IV的表达逐渐增加,在48至72h之间,随着时间延长,表达逐渐减低；(5)在对照组和2umol/L的ATO作用后,随着时间延长,PML-V的表达无明显变化；在5umol/L和10umol/L的ATO作用后,随着时间延长,表达逐渐增加,其中10umol/L的ATO作用后,PML-V表达量的增加趋势最大；3、PML-Ⅳ表达与hTERT基因的表达之间无相关性。结论：1、在一定浓度范围内,ATO对Hut-78细胞存在增殖抑制作用,其增殖抑制作用在一定程度上可能与下调hTERT的表达有关；2、ATO可诱导Hut-78细胞发生凋亡,凋亡主要发生在G2/M期,其凋亡作用可能与PML、PML-I及PML-IV的表达增高及下调端粒酶活性有关；3、ATO对Hut-78细胞的诱导凋亡作用呈显著的时间一剂量依赖性；4、不同浓度范围的ATO对Hut-78细胞PML-V基因的表达影响不同,推测]PML-V只有在药物强应激情况下,表达才会激增。更多还原

【Abstract】 Objective:To investigate the possible function and mechanism of the genes, such as Promyelocytic Leukemia, Promyelocytic Leukemia isoform I, Promyelocytic Leukemia isoformⅣ, Promyelocytic Leukemia isoform V and human telomerase reverse transcriptase, in Arsenic Trioxide on cutaneous T cell lymphoma cell line Hut-78 cells by detecting the apoptosis, effects of cell cycles and expression of these genes in cutaneous T-cell lymphoma cell line Hut-78 which was treated with Arsenic Trioxide of different concentrations.Methods:The effect of Arsenic Trioxide in different concentrations and for different duration of time on apoptosis and cell cycles of hut-78 cells were analyzed by Flow cytometry with PI staining. The transcription level changes of hTERT, PML, PML-I, PML-Ⅳand PML-Ⅴgenes were detected by the reverse transcriptase-polymerase chain reaction (RT-PCR)Results:1. Hut-78 cells were treated with the concentration range of 2-10μmol/L of ATO respectively for 24 h,48h and 72h. Then cell cycle, apoptosis and necrosis were measured by flow cytometry with PI staining. The results showed that, the ratio of G2/M phase of Hut-78 cells that was treated with Arsenic Trioxide was increased. When the Hut-78 cells were treated with 10μmol/L Arsenic Trioxide for 72 hours, the highest percentage of G2/M phase was detected. With the increasing of concentration and extending of action time, the rate of apoptosis and necrosis of Hut-78 cells was gradually increased; 2.The expressions of hTERT, PML, PML-Ⅰ, PML-Ⅳand PML-Ⅴgenes in Hut-78 cells treated with Arsenic Trioxide in different concentrations for different action time were detected by RT-PCR.The results show that:(1) The expression of hTERT mRNA gradually decreased with the concentration of ATO increasing and acting time extending; (2) After treated with ATO in different concentrations after 48h, the transcription level of PML reached a maximum value, which was gradually decreased from 48h to 72h; (3) With prolonging treatment with 2umol/L and 10umol/L ATO respectively,the transcription level of PML-Ⅰwas gradually increased,When the concentration of ATO is 0 umol/L and 5 umol/L respectively,with prolonging treatment,the transcription level was gradually decreased from 24h to 48h,while that was gradually increased from 48h to 72h; (4) With ATO in different concentration,the transcription level of PML-Ⅳwas gradually increased from 24h to 48h with the acting time extending,while that was gradually decreased from 48h to 72h; (5) When the concentration of ATO is 0 umol/L and 2 umol/L respectively,with prolonging treatment,The expression of PML-Ⅴdid not change significantly.When the concentration of ATO was 5 umol/L and 10 umol/L respectively, with prolonging treatment, the transcription level was gradually increased; And at the dosage of 10 umol/L the increased trend of the transcription level was the most significant; 3. There was no correlation between the expression level of PML-IVand hTERT.Conclusion:1.Arsenic trioxide can inhibit the proliferation of Hut-78 cells in a certain range of concentrations, which may be related to the decreasement in the transcription level of hTERT to some extent; 2.ATO can induce apoptosis of Hut-78 cells. And apoptosis occurs mainly in G2/M phase, which may be related to PML, PML-Ⅰand PML-Ⅳincreased and decreased the expression of telomerase activity; 3.ATO can induce apoptosis of Hut-78 cells, which is dependent on time and dose; 4.The expression of PML-Ⅴwas different with ATO in different concentrations, we speculated that the expression level of PML-Ⅴcould remarkably increase only in the strong stress caused by drug.更多还原