本文關鍵詞：砷通過抑制BCAT1基因增加順鉑對肝癌及肝內(nèi)膽管細胞癌的殺傷作用　出處：《南京醫(yī)科大學》2017年碩士論文　論文類型：學位論文

  更多相關文章： HCC BCAT1 三氧化二砷 順鉑 化療耐藥 ICC BCAT1 ATO CDDP 化療耐藥

【摘要】：目的:肝細胞癌(Hepatocellularcarcinoma,HCC)是世界上第三大癌癥,大多數(shù)患者需要幾種化療藥物的治療,然而HCC對化療藥物易產(chǎn)生耐藥性,是患者化療失敗的主要原因之一。本研究通過檢測BCAT1在HCC組織和HCC細胞株(HepG2和97H)中的表達,并進一步探討B(tài)CAT1在HCC順鉑(cisplatin,CDDP)耐藥中的作用及其調(diào)控機制,為ATO聯(lián)合CDDP治療HCC和藥物靶向治療HCC提供新的理論依據(jù)。方法:(1)運用western blot法和qRT-PCR法分別檢測HCC組織和癌旁組織、HCC細胞株(HepG2和97H)和正常肝細胞(L02)中BCAT1的mRNA水平和蛋白水平。采用T檢驗方法比較組間有無差異。(2)HCC細胞對CDDP治療常出現(xiàn)耐藥現(xiàn)象,為了驗證高表達的BCAT1是否在HCC的CDDP耐藥中起到作用,首先運用小干擾RNA對HCC細胞株(HepG2和97H)中的BCAT1進行沉默,并用MTT法檢測BCAT1沉默對CDDP處理過的HCC細胞活力的影響。同時,用流式細胞儀檢測BCAT1沉默對CDDP處理過的HCC細胞凋亡的影響。(3)ATO在腫瘤治療中有良好的效果,我們用western blot法檢測ATO處理是否可以抑制HCC細胞中BCAT1的表達,并檢測ATO和CDDP聯(lián)合處理后HCC細胞中BCAT1的蛋白水平。(4)用MTT法檢測ATO聯(lián)合CDDP處理對HCC細胞(HepG2和97H)細胞活力的影響,同時檢測BCAT1過表達對ATO聯(lián)合CDDP處理過的HCC細胞(HepG2和97H)的細胞活力的影響。用流式細胞儀檢測檢測ATO聯(lián)合CDDP處理對HCC細胞凋亡的影響,并檢測BCAT1過表達對ATO聯(lián)合CDDP處理過的HCC細胞凋亡的影響。結(jié)果:(1)實時熒光定量PCR結(jié)果表明HCC組織中BCAT1的mRNA水平顯著高于癌旁組織,western blot法結(jié)果也表明HCC組織中BCAT1蛋白水平顯著高于癌旁組織。然后我們用實時熒光定量PCR和蛋白免疫印跡法檢測HCC細胞(HepG2和97H)和正常肝細胞(LO2)中BCAT1的mRNA水平和蛋白水平。與LO2細胞相比,HepG2和97H細胞中BCAT1的mRNA水平和蛋白水平顯著更高。(2)HepG2細胞用不同濃度的CDDP處理48小時,BCAT1siRNA處理的HepG2細胞對CDDP表現(xiàn)出更大的敏感性,并且比對照細胞具有較低的增殖率和較高的凋亡率。類似地,在97H細胞中BCAT1的降低可以增強細胞對CDDP的化學敏感性,表現(xiàn)為較低的增殖率和較高的凋亡率。(3)我們用2.5、5和10μM的ATO處理HCC細胞,并通過western blot法測定48小時BCAT1的蛋白表達。結(jié)果表明,ATO以劑量依賴性方式顯著降低HCC細胞中BCAT1的蛋白表達。此外,5 μMATO處理以時間依賴性方式抑制BCAT1表達。之前已經(jīng)描述了 ATO處理降低了 c-Myc表達,c-Myc其是BCAT1基因的反式作用因子。因此,HepG2細胞用5μM ATO或/和20μM CDDP處理48小時后,進行western blot法測定c-Myc和BCAT1表達。結(jié)果顯示5 μM的ATO抑制c-Myc和BCAT1表達。(4)當HepG2和97H細胞用5 μM的ATO和20μM CDDP處理48小時。ATO處理的細胞顯示出比正常細胞更低的增殖和較高的凋亡速率。然而,外源性BCAT1過表達細胞顯示出比ATO處理的細胞更高的增殖和較低的凋亡。結(jié)論:(1)HCC組織和HCC細胞株(HepG2和97H)中BCAT1的mRNA和蛋白水平顯著升高。(2)BCAT1沉默可以導致HCC細胞對CDDP更敏感,表明BCAT1表達降低可以抑制HCC的CDDP耐藥。(3)ATO通過c-Myc來調(diào)節(jié)BCAT1蛋白的表達降低。(4)ATO處理可以降低HCC細胞的CDDP耐藥性,并且這種降低可以被BCAT1過表達部分逆轉(zhuǎn),提示BCAT1可以在HCC細胞CDDP耐藥中起到一定作用。目的:肝內(nèi)膽管癌(intrahepatic cholangiocarcinoma,ICC)是發(fā)病率僅次于肝細胞肝癌(HCC)的原發(fā)性肝癌,約占原發(fā)性肝癌的10-15%。本研究通過研究ATO聯(lián)合CDDP化療對ICC細胞的影響,并探討B(tài)CAT1在ICC耐藥CDDP中的潛在調(diào)控機制,為ATO聯(lián)合CDDP治療ICC提供一定的臨床參考。方法:(1)為了評估化療藥物敏感性,我們先用20μM CDDP處理或和5μM ATO聯(lián)合處理ICC細胞(RBE和HUCCT1細胞)48小時,然后用MTT法檢測ATO聯(lián)合CDDP處理對ICC細胞活力的影響。(2)為了驗證BCAT1表達是否參與ICC的CDDP耐藥,我們用western blot法檢測BCAT1分子和H2AX的表達,而H2AX分子用來檢測藥物處理后ICC細胞的DNA損傷。(3)為了進一步驗證BCAT1在ATO聯(lián)合CDDP化療中的作用,首先我們通過彗星實驗檢測ATO聯(lián)合CDDP對HUCCT1細胞凋亡的影響,然后運用小干擾RNA對HUCCT1細胞中的BCAT1進行沉默,最后再用彗星實驗檢測BCAT1沉默對ATO聯(lián)合CDDP所誘導的細胞凋亡的影響。結(jié)果:(1)CDDP單獨處理RBE細胞時,RBE細胞活力未出現(xiàn)顯著的降低;但當ATO聯(lián)合CDDP處理RBE細胞時,RBE細胞對CDDP敏感性增加,細胞增殖功能發(fā)生障礙,細胞活力明顯降低。此外,在另一株HUCCT1細胞中也發(fā)現(xiàn)相似現(xiàn)象,ATO聯(lián)合CDDP處理顯著降低HUCCT1的細胞活力。(2)單獨CDDP處理RBE細胞時對BCAT1和H2AX的蛋白水平?jīng)]有明顯的影響,統(tǒng)計學沒有意義。然而,ATO聯(lián)合CDDP處理既能明顯誘導H2AX蛋白水平的升高,又能誘導BCAT1蛋白水平的降低。我們又用另一株ICC細胞株HUCCT1來驗證ATO聯(lián)合CDDP的藥物效果,實驗結(jié)果和RBE細胞很相似,ATO處聯(lián)合CDDP處理既能明顯誘導H2AX蛋白水平的升高,又能誘導BCAT1蛋白水平的降低。(3)在彗星實驗中,DNA損傷越嚴重,彗星的尾部的DNA碎片越多,彗星尾部的面積長度以及熒光強度就越大,通過測量這些指標可以對DNA損傷和細胞凋亡程度進行定量分析。實驗結(jié)果顯示單獨ATO和單獨CDDP處理后,HUCCT1細胞的DNA損傷比對照組有微弱的增加,但沒有顯著性差異;而ATO聯(lián)合CDDP處理可以誘導HUCCT1細胞明顯的DNA損傷。接著我們用小干擾對BCAT1進行沉默,驗證BCAT1在ATO聯(lián)合CDDP所誘導的DNA損傷中的作用,結(jié)果顯示BCAT1沉默后,ATO聯(lián)合CDDP處理的ICC細胞株出現(xiàn)明顯的彗星拖尾現(xiàn)象,細胞凋亡增加。結(jié)論:(1)ATO處理使ICC細胞對CDDP更敏感,RBE和HUCCT1細胞增殖被抑制。(2)ATO聯(lián)合CDDP處理不僅抑制BCAT1的蛋白表達,還可以增加H2AX的蛋白表達。(3)BCAT1沉默可以增強ATO聯(lián)合CDDP化療所誘導的HUCCT1細胞DNA損傷,提示BCAT1可能參與ATO聯(lián)合CDDP誘導的ICC細胞DNA損傷。
[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.

【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735

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