發(fā)布時間：2018-01-30 11:16

  本文關鍵詞： 宮頸癌 SiHa細胞 CLCA2 侵襲 上皮-間質轉化(EMT)　出處：《蘭州大學》2017年碩士論文　論文類型：學位論文

【摘要】：背景與目的:宮頸癌是我國婦女最常見的惡性腫瘤,也是全世界導致婦女死亡的最主要惡性腫瘤之一,嚴重危害著婦女的健康。腫瘤的藥物耐藥、侵襲和轉移、術后復發(fā)是晚期宮頸癌患者常見的死亡原因,同時也是影響患者治療和臨床預后的重要因素。盡管晚期宮頸癌患者不能夠得到徹底的治愈,但有很多治療方式可以提高患者的生命長度和質量。近年來,精準醫(yī)學在的癌癥治療中的迅速發(fā)展以及對腫瘤生物學理解的逐漸加深為尋找治療癌癥的新方法提供了新的方向。CLCA2已被證實是p53基因的靶基因之一,其在調控細胞增殖、遷移,腫瘤細胞浸潤中起著重要的作用,目前發(fā)現(xiàn)其與多種腫瘤侵襲和轉移的發(fā)生關系密切,但其在宮頸癌中的作用尚不清楚,我們實驗組在前期的研究中通過應用基因芯片技術來研究異紫堇堿(Isocorydine)作用于宮頸癌SiHa細胞后基因譜的變化時發(fā)現(xiàn)了CLCA2基因在異紫堇堿作用48h后明顯上調。因此,本實驗旨在研究CLCA2基因在宮頸癌中的作用及其可能的機制,并初步探討CLCA2對臨床常用化療藥物紫杉醇的敏感性,以期對宮頸癌的診治及預防提供一定的實驗依據(jù)。方法:構建特異性CLCA2-shRNA,并借助慢病毒載體將其轉入宮頸癌Siha細胞中,實驗分為Siha組(空白對照組)、Siha-NC組(陰性對照組)、CLCA2-shRNA-1組(CLCA2-shRNA-1轉染的宮頸癌Siha細胞,下文以K1組表示)和CLCA2-shRNA-2組(CLCA2-shRNA-2轉染的宮頸癌Siha細胞,下文以K2表示),采用Western blot驗證CLCA2基因的敲除效率,隨后采用Transwell侵襲實驗觀察敲除CLCA2基因的宮頸癌Siha細胞侵襲力的變化,并用Western blot檢測各組細胞上皮-間質轉化標志物E-鈣黏蛋白(E-cadherin)及波形蛋白(Vimentin)的表達水平,并檢測基質金屬蛋白酶-2(matrix metalloproteinase2,MMP-2)和基質金屬蛋白酶-9(matrix metalloproteinase9,MMP-9)的表達。最后,利用MTS實驗初步探討敲除CLCA2基因的宮頸癌Siha細胞對紫杉醇的藥物敏感性。結果:1、.成功構建了慢病毒表達載體并且能夠高效轉入宮頸癌Siha細胞內。Western blot檢測顯示:與Siha組和Siha-NC組相比,K1組和K2組中CLCA2蛋白的表達水平明顯下降(P0.05);2、Transwell侵襲實驗顯示:與Siha組和Siha-NC組相比,K1組和K2組侵襲至Transwell孔下室的細胞數(shù)量明顯增多,敲除CLCA2基因的細胞的侵襲力明顯增加,差異具有統(tǒng)計學意義(P0.05);3、Western blot檢測顯K1和K2組中的E-cadherin蛋白的表達較Siha組和Siha-NC組均顯著下降,差異具有統(tǒng)計學意義(p0.05),而Vimentin蛋白的表達K1組僅與Siha組相比明顯上調,差異具有統(tǒng)計學意義(P0.05),而與Siha-NC組相比,差異無統(tǒng)計學意義(P0.05),K2組較Siha組和Siha-NC組均明顯升高,差異具有統(tǒng)計學意義(p0.05);4、Western blot的進一步檢測顯示K1組和K2組中MMP-2蛋白和MMP-9蛋白的表達較Siha組和Siha-NC組均顯著升高,差異具有統(tǒng)計學意義(p0.05);5.MTS實驗結果顯示:與Siha組和Siha-NC組相比,K1和K2組在不同濃度的紫杉醇(0.5,1,1.5,2,3,4,5nmol/l)作用下的抑制率明顯增高,差異具有統(tǒng)計學意義(P0.05)。結論:1、CLCA2基因的敲除能夠增強宮頸癌Siha細胞的侵襲能力;2、CLCA2基因的敲除能夠促進宮頸癌Siha細胞上皮-間質的轉化;3、CLCA2基因的敲除后能夠通過促進MMP-2和MMP-9的表達來增強腫瘤細胞的侵襲能力;4、CLCA2基因的敲除能夠增強紫杉醇對宮頸癌Siha細胞的敏感性。
[Abstract]:Background and objective: cervical cancer is the most common malignant tumors of women in our country, but also the whole world to one of the major malignant tumor death in women, seriously endanger the health of women. The drug resistance of tumor, invasion and metastasis, recurrence is common in patients with advanced cervical cancer deaths, but also an important factor affecting patients the treatment and clinical prognosis. Although patients with advanced cervical cancer can not be completely cured, but there are a lot of treatment can improve the patients' quality of life and length. In recent years, precision medicine in cancer treatment in the rapid development and gradually deepening understanding of tumor biology in order to find new method for the treatment of cancer provides a new the direction of.CLCA2 has been proven to be one of the target genes of p53 gene and its migration in the regulation of cell proliferation, tumor cells plays an important role in the invasion, and several There is a close relationship between tumor invasion and metastasis in cervical cancer, but its role is not clear, our experimental group in the previous study by using gene chip technology to study different Corydaline (Isocorydine) of CLCA2 gene was found in different Corydaline after 48h was significantly increased in the change of gene expression profile of human cervical cancer SiHa cells after the time. Therefore, the aim of this study is to investigate the role of CLCA2 gene in cervical cancer and its possible mechanism, and to investigate the sensitivity of CLCA2 to clinical commonly used chemotherapy drug taxol, provide some experimental basis for the diagnosis and prevention of cervical cancer. Methods: to construct specific CLCA2-shRNA, and put it into the cervix cancer cell Siha by slow virus vector, the experiments were divided into Siha group (control group), Siha-NC group (negative control group), CLCA2-shRNA-1 group (CLCA2-shRNA-1 transfected Siha cell, below in the K1 group Said) and group CLCA2-shRNA-2 (cervical cancer Siha cells transfected with CLCA2-shRNA-2 below K2), using Western blot to verify CLCA2 gene knockdown efficiency, followed by Transwell invasion assay. Knockout invasion of cervical cancer Siha cells CLCA2 gene, and Western blot to detect the expression of epithelial mesenchymal transition mark E- cadherin (E-cadherin) and vimentin (Vimentin) expression and detection of matrix metalloproteinase -2 (matrix metalloproteinase2 MMP-2) and matrix metalloproteinase -9 (matrix Metalloproteinase9 MMP-9) expression of MTS. Finally, study the knockout drug of cervical cancer Siha cells CLCA2 gene to paclitaxel sensitivity use. Results: 1. Successfully constructed the lentiviral expression vector and can be efficiently transferred to the display of cervical cancer Siha cells in.Western blot detection with Siha group and Siha-NC group. Than, the expression level of CLCA2 protein in K1 group and K2 group decreased significantly (P0.05); 2, Transwell invasion assay showed that: compared with Siha group and Siha-NC group, the number of cells in K1 group and K2 group to the Transwell invasion chamber was significantly increased, CLCA2 knockout cell invasiveness has increased significantly statistically significant difference (P0.05); 3, the expression of Western blot and K2 K1 detection of E-cadherin protein in the group than in Siha group and Siha-NC group were significantly decreased, the difference was statistically significant (P0.05), and the expression of K1 protein in Vimentin group was significantly increased compared with Siha group, the difference was statistically significant (P0.05). Compared with Siha-NC group, the difference was not statistically significant (P0.05), K2 group than in Siha group and Siha-NC group were significantly higher, the difference was statistically significant (P0.05); 4, further detection of Western blot showed that the expression of MMP-2 protein and MMP-9 protein in K1 group and K2 group than in Siha group And the Siha-NC group were significantly increased, the difference was statistically significant (P0.05); 5.MTS results showed: compared with Siha group and Siha-NC group, K1 group and K2 in different concentrations of paclitaxel (0.5,1,1.5,2,3,4,5nmol/l) inhibited the rate increased significantly, the difference was statistically significant (P0.05). Conclusion: 1. CLCA2 gene knockout of cervical carcinoma can enhance the invasion ability of Siha cell; 2, CLCA2 gene knockout can promote cervical cancer Siha cell epithelial mesenchymal transformation; 3, CLCA2 gene knockout can promote the expression of MMP-2 and MMP-9 to enhance the ability of tumor cell invasion; 4, CLCA2 gene knockout to enhance paclitaxel on human cervical cancer Siha cell sensitivity.

【學位授予單位】：蘭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.33

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