砷通过抑制BCAT1基因增加顺铂对肝癌及肝内胆管细胞癌的杀伤作用

发布时间：2018-01-09 14:28

本文关键词：砷通过抑制BCAT1基因增加顺铂对肝癌及肝内胆管细胞癌的杀伤作用　出处：《南京医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:肝细胞癌(Hepatocellularcarcinoma,HCC)是世界上第三大癌症,大多数患者需要几种化疗药物的治疗,然而HCC对化疗药物易产生耐药性,是患者化疗失败的主要原因之一。本研究通过检测BCAT1在HCC组织和HCC细胞株(HepG2和97H)中的表达,并进一步探讨BCAT1在HCC顺铂(cisplatin,CDDP)耐药中的作用及其调控机制,为ATO联合CDDP治疗HCC和药物靶向治疗HCC提供新的理论依据。方法:(1)运用western blot法和qRT-PCR法分别检测HCC组织和癌旁组织、HCC细胞株(HepG2和97H)和正常肝细胞(L02)中BCAT1的mRNA水平和蛋白水平。采用T检验方法比较组间有无差异。(2)HCC细胞对CDDP治疗常出现耐药现象,为了验证高表达的BCAT1是否在HCC的CDDP耐药中起到作用,首先运用小干扰RNA对HCC细胞株(HepG2和97H)中的BCAT1进行沉默,并用MTT法检测BCAT1沉默对CDDP处理过的HCC细胞活力的影响。同时,用流式细胞仪检测BCAT1沉默对CDDP处理过的HCC细胞凋亡的影响。(3)ATO在肿瘤治疗中有良好的效果,我们用western blot法检测ATO处理是否可以抑制HCC细胞中BCAT1的表达,并检测ATO和CDDP联合处理后HCC细胞中BCAT1的蛋白水平。(4)用MTT法检测ATO联合CDDP处理对HCC细胞(HepG2和97H)细胞活力的影响,同时检测BCAT1过表达对ATO联合CDDP处理过的HCC细胞(HepG2和97H)的细胞活力的影响。用流式细胞仪检测检测ATO联合CDDP处理对HCC细胞凋亡的影响,并检测BCAT1过表达对ATO联合CDDP处理过的HCC细胞凋亡的影响。结果:(1)实时荧光定量PCR结果表明HCC组织中BCAT1的mRNA水平显著高于癌旁组织,western blot法结果也表明HCC组织中BCAT1蛋白水平显着高于癌旁组织。然后我们用实时荧光定量PCR和蛋白免疫印迹法检测HCC细胞(HepG2和97H)和正常肝细胞(LO2)中BCAT1的mRNA水平和蛋白水平。与LO2细胞相比,HepG2和97H细胞中BCAT1的mRNA水平和蛋白水平显著更高。(2)HepG2细胞用不同浓度的CDDP处理48小时,BCAT1siRNA处理的HepG2细胞对CDDP表现出更大的敏感性,并且比对照细胞具有较低的增殖率和较高的凋亡率。类似地,在97H细胞中BCAT1的降低可以增强细胞对CDDP的化学敏感性,表现为较低的增殖率和较高的凋亡率。(3)我们用2.5、5和10μM的ATO处理HCC细胞,并通过western blot法测定48小时BCAT1的蛋白表达。结果表明,ATO以剂量依赖性方式显著降低HCC细胞中BCAT1的蛋白表达。此外,5 μMATO处理以时间依赖性方式抑制BCAT1表达。之前已经描述了 ATO处理降低了 c-Myc表达,c-Myc其是BCAT1基因的反式作用因子。因此,HepG2细胞用5μM ATO或/和20μM CDDP处理48小时后,进行western blot法测定c-Myc和BCAT1表达。结果显示5 μM的ATO抑制c-Myc和BCAT1表达。(4)当HepG2和97H细胞用5 μM的ATO和20μM CDDP处理48小时。ATO处理的细胞显示出比正常细胞更低的增殖和较高的凋亡速率。然而,外源性BCAT1过表达细胞显示出比ATO处理的细胞更高的增殖和较低的凋亡。结论:(1)HCC组织和HCC细胞株(HepG2和97H)中BCAT1的mRNA和蛋白水平显著升高。(2)BCAT1沉默可以导致HCC细胞对CDDP更敏感,表明BCAT1表达降低可以抑制HCC的CDDP耐药。(3)ATO通过c-Myc来调节BCAT1蛋白的表达降低。(4)ATO处理可以降低HCC细胞的CDDP耐药性,并且这种降低可以被BCAT1过表达部分逆转,提示BCAT1可以在HCC细胞CDDP耐药中起到一定作用。目的:肝内胆管癌(intrahepatic cholangiocarcinoma,ICC)是发病率仅次于肝细胞肝癌(HCC)的原发性肝癌,约占原发性肝癌的10-15%。本研究通过研究ATO联合CDDP化疗对ICC细胞的影响,并探讨BCAT1在ICC耐药CDDP中的潜在调控机制,为ATO联合CDDP治疗ICC提供一定的临床参考。方法:(1)为了评估化疗药物敏感性,我们先用20μM CDDP处理或和5μM ATO联合处理ICC细胞(RBE和HUCCT1细胞)48小时,然后用MTT法检测ATO联合CDDP处理对ICC细胞活力的影响。(2)为了验证BCAT1表达是否参与ICC的CDDP耐药,我们用western blot法检测BCAT1分子和H2AX的表达,而H2AX分子用来检测药物处理后ICC细胞的DNA损伤。(3)为了进一步验证BCAT1在ATO联合CDDP化疗中的作用,首先我们通过彗星实验检测ATO联合CDDP对HUCCT1细胞凋亡的影响,然后运用小干扰RNA对HUCCT1细胞中的BCAT1进行沉默,最后再用彗星实验检测BCAT1沉默对ATO联合CDDP所诱导的细胞凋亡的影响。结果:(1)CDDP单独处理RBE细胞时,RBE细胞活力未出现显著的降低;但当ATO联合CDDP处理RBE细胞时,RBE细胞对CDDP敏感性增加,细胞增殖功能发生障碍,细胞活力明显降低。此外,在另一株HUCCT1细胞中也发现相似现象,ATO联合CDDP处理显著降低HUCCT1的细胞活力。(2)单独CDDP处理RBE细胞时对BCAT1和H2AX的蛋白水平没有明显的影响,统计学没有意义。然而,ATO联合CDDP处理既能明显诱导H2AX蛋白水平的升高,又能诱导BCAT1蛋白水平的降低。我们又用另一株ICC细胞株HUCCT1来验证ATO联合CDDP的药物效果,实验结果和RBE细胞很相似,ATO处联合CDDP处理既能明显诱导H2AX蛋白水平的升高,又能诱导BCAT1蛋白水平的降低。(3)在彗星实验中,DNA损伤越严重,彗星的尾部的DNA碎片越多,彗星尾部的面积长度以及荧光强度就越大,通过测量这些指标可以对DNA损伤和细胞凋亡程度进行定量分析。实验结果显示单独ATO和单独CDDP处理后,HUCCT1细胞的DNA损伤比对照组有微弱的增加,但没有显著性差异;而ATO联合CDDP处理可以诱导HUCCT1细胞明显的DNA损伤。接着我们用小干扰对BCAT1进行沉默,验证BCAT1在ATO联合CDDP所诱导的DNA损伤中的作用,结果显示BCAT1沉默后,ATO联合CDDP处理的ICC细胞株出现明显的彗星拖尾现象,细胞凋亡增加。结论:(1)ATO处理使ICC细胞对CDDP更敏感,RBE和HUCCT1细胞增殖被抑制。(2)ATO联合CDDP处理不仅抑制BCAT1的蛋白表达,还可以增加H2AX的蛋白表达。(3)BCAT1沉默可以增强ATO联合CDDP化疗所诱导的HUCCT1细胞DNA损伤,提示BCAT1可能参与ATO联合CDDP诱导的ICC细胞DNA损伤。
[Abstract]:Objective: hepatocellular carcinoma (Hepatocellularcarcinoma, HCC) is the third largest in the world of cancer, most patients need treatment of some chemotherapy drugs, however HCC to chemotherapy drug resistance, is one of the main reasons for the failure of chemotherapy patients. This study through the detection of BCAT1 in HCC and HCC cell lines (HepG2 and 97H) expression in BCAT1 in HCC, and to further explore the role of cisplatin (cisplatin, CDDP) resistance and its regulation mechanism, provide a new theoretical basis for ATO combined with CDDP in the treatment of HCC and drug targeting HCC. Methods: (1) using Western blot method and qRT-PCR method were used to detect HCC tissues and adjacent tissues, cell lines (HCC HepG2 and 97H) and normal liver cells (L02) in BCAT1 mRNA and protein level. The T tests to compare the difference between groups. (2) HCC cells to CDDP treatment often appear resistance phenomenon, in order to verify whether the high expression of BCAT1 in HC Play the role of the CDDP resistant C, using small interfering RNA on HCC cell lines (HepG2 and 97H) BCAT1 in the silence effect of HCC cell activity and detection of BCAT1 silencing by MTT method to CDDP treated. At the same time, the effect of HCC cell apoptosis by flow cytometry BCAT1 silencing of CDDP the. (3) ATO has a good effect in the treatment of cancer, we used Western blot method to detect whether ATO can inhibit BCAT1 expression in HCC cells, BCAT1 protein level in HCC cells and the detection of ATO and CDDP after the treatment. (4) using MTT method combined with CDDP detection on HCC ATO processing cells (HepG2 and 97H) affect cell viability, simultaneous detection of BCAT1 overexpression on ATO combined with CDDP treated HCC cells (HepG2 and 97H) affected cell viability. Effect of combined treatment of ATO detection CDDP detection on the apoptosis of HCC cells by flow cytometry, and the detection of BCAT1 over expression on AT Effect of apoptosis of HCC cells combined with CDDP treated O. Results: (1) real time fluorescence quantitative PCR results showed that BCAT1 HCC in the level of mRNA was significantly higher than that in adjacent tissues, Western blot results also showed that BCAT1 protein level in HCC tissues was significantly higher than that in the adjacent tissues. Then we used real-time fluorescence quantitative PCR and protein Western blot detection of HCC cells (HepG2 and 97H) and normal liver cells (LO2) in BCAT1 mRNA and protein level in LO2 cells. Compared with BCAT1, HepG2 and 97H cells in mRNA and protein level were significantly higher. (2) HepG2 cells treated with different concentrations of CDDP for 48 hours, BCAT1siRNA the HepG2 cells of CDDP showed greater sensitivity to apoptosis and has lower proliferation rate and higher than the control cells. Similarly, BCAT1 decreased in 97H cells can enhance the chemosensitivity of cells to CDDP, is low The apoptosis rate and high proliferation rate. (3) we use ATO cells HCC 2.5,5 and 10 M, and the expression of Western by blot method for the determination of 48 hours of BCAT1 protein. The results showed that ATO significantly decreased the expression of BCAT1 protein in HCC cells in a dose-dependent manner. In addition, 5 MATO in time dependent inhibition of BCAT1 expression has been described before. ATO treatment reduced the expression of c-Myc, c-Myc is the BCAT1 gene trans acting factor. Therefore, HepG2 cells with 5 M and 20 M / ATO or CDDP after 48 hours of treatment, the Western blot method for the determination of c-Myc and BCAT1 expression. The results showed that 5 M ATO inhibited the expression of c-Myc and BCAT1. (4) when HepG2 and 97H cells with 5 M ATO and 20 M CDDP treatment for 48 h.ATO treated cells showed higher proliferation and apoptosis rate than normal cells lower. However, exogenous overexpression of BCAT1 cells than ATO treatment Apoptosis of higher proliferation and lower. Conclusion: (1) HCC and HCC cell lines (HepG2 and 97H) in BCAT1 mRNA and protein levels were significantly increased. (2) BCAT1 silencing can lead to HCC cells more sensitive to CDDP, indicating that BCAT1 expression decreased the resistance of CDDP can inhibit the HCC. (3) reduced the expression of ATO by c-Myc to regulate BCAT1 protein. (4) ATO treatment can reduce the CDDP resistance of HCC cells, and this decrease can be overexpression of BCAT1 partially reversed, suggesting that BCAT1 may play a certain role in CDDP resistance in HCC cells. Objective: intrahepatic cholangiocarcinoma (intrahepatic cholangiocarcinoma ICC) is a disease the rate of hepatocellular carcinoma (HCC) after the primary liver cancer, accounting for primary liver cancer 10-15%. the study of ATO combined with CDDP chemotherapy by ICC cells, and to explore the potential mechanism of regulation of BCAT1 in ICC resistant CDDP, ATO combined with CDDP in the treatment of ICC. For clinical reference. Methods: (1) in order to assess the sensitivity of chemotherapy drugs, we use 20 M CDDP or 5 M ATO treatment and combined treatment of ICC cells (RBE cells and HUCCT1) for 48 hours, then use MTT method to detect the effect of ATO combined with CDDP treatment on ICC cell viability (2) for. The validation of the BCAT1 expression of CDDP is involved in ICC resistance, we use Western blot method to detect the expression of BCAT1 molecules and H2AX H2AX molecules to DNA, ICC cell damage detection after treatment. (3) in order to further verify BCAT1 in ATO combined with CDDP chemotherapy in the first, we through the comet assay combined with CDDP on ATO HUCCT1 cell apoptosis, and then use the small interfering RNA silencing on HUCCT1 cells in BCAT1 cell apoptosis and comet assay of BCAT1 silencing induced by ATO and CDDP. Results: (1) CDDP alone and RBE cells, RBE cells The cell activity was not significantly reduced; but when ATO and CDDP treated RBE cells, increased sensitivity to CDDP RBE cells, cell proliferation disorders, cell viability decreased significantly. In addition, a similar phenomenon is also found in other HUCCT1 cells, the combined treatment of CDDP ATO significantly decreased the viability of HUCCT1 (2). CDDP treatment of RBE cells when the protein level of BCAT1 and H2AX had no obvious effect, no statistical significance. However, not only can induce increased H2AX protein levels of CDDP and ATO combined treatment, reduce the induced level of BCAT1 protein. We also use another ICC cell lines HUCCT1 to verify the effect of ATO combined with drugs CDDP, experimental results and RBE cells are very similar, ATO increased and CDDP treatment can induce H2AX protein level decreased, and can induce BCAT1 protein level. (3) in the comet assay, DNA damage more serious, comet The more DNA fragments of the tail of the comet tail length and the area of fluorescence intensity is greater, by measuring these indicators can be used for the quantitative analysis of DNA damage and cell apoptosis. Experimental results show that the single ATO and single CDDP treatment, DNA damage of HUCCT1 cells slightly increased than the control group, but no significant difference; ATO combined with CDDP treatment can significantly induce DNA damage in HUCCT1 cells. Then we used small interfering BCAT1 silencing, DNA damage induced by BCAT1 in ATO and verify the role of CDDP, the results showed that BCAT1 silencing, ICC cell line joint CDDP ATO appears obvious comet tail, increased apoptosis. Conclusion: (1) ATO treatment of ICC cells more sensitive to CDDP, RBE and HUCCT1 cell proliferation was inhibited. (2) the combined treatment of CDDP ATO not only inhibited the protein expression of BCAT1, but also can increase the expression of H2AX protein. (3) BCAT1 silencing can enhance the DNA damage induced by ATO combined with CDDP chemotherapy, suggesting that BCAT1 may be involved in ICC cell DNA injury induced by ATO combined with CDDP.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735

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