【摘要】：宮頸癌是女性最多見的惡性腫瘤,嚴重威脅著女性的健康與生活。順鉑是治療宮頸癌的常用化療藥物,但癌細胞對順鉑的抗藥性使其治療效果大為減弱。在用順鉑處理的宮頸癌細胞SiHa,C33A中,CDC25B 與 CDC25C基因的mRNA的選擇性剪接發(fā)生了改變,由于CDC25B 與 CDC25C在正常生理條件下和應(yīng)對DNA損傷時對細胞周期的檢查點功能有重要的調(diào)控作用,因此推測CDC25B與CDC25C基因的mRNA的剪接變化很可能與細胞耐受順鉑有關(guān)。本研究擬探究CDC25B與CDC25C的各主要剪接isoforms具有抗凋亡還是促凋亡的作用?這一剪接變化是否增加了宮頸癌細胞對順鉑的抗性?得到的結(jié)果和信息將有助于闡明CDC25B 與 CDC25C在腫瘤發(fā)生與發(fā)展中的作用,解讀腫瘤細胞對放化療‘抗性的機制。如果CDC25B 與 CDC25C 的 mRNA的剪接變化會影響其checkpoint的功能,則對其的調(diào)節(jié)有可能會提高順鉑的療效,調(diào)節(jié)的途徑和基因?qū)⒊蔀樾碌闹委煱悬c,為癌癥的個體化治療提供重要的信息和依據(jù)。目的：探究CDC25B與CDC25C 的 mRNA的選擇性剪接變化對宮頸癌細胞SiHa和C33A的生長、增殖、凋亡以及順鉑敏感性的影響。方法：應(yīng)用MTT比色法確定宮頸癌細胞SiHa, C33A的順鉑半致死量濃度；RT-PCR檢測相同時間、不同濃度順鉑干預(yù)及不同時間、半致死量順鉑干預(yù)對各組宮頸癌細胞CDC25B, C的mRNA的選擇性剪接的影響；將CDC25B1,B3及CDC25C1.C5基因轉(zhuǎn)染宮頸癌SiHa, C33A細胞,RT-PCR 及 Western Blot分別檢測轉(zhuǎn)染細胞中CDC25B1, B3, C1, C5 的 mRNA和蛋白表達水平;MTT比色法測量CDC25B1, B3, C1, C5基因過表達的宮頸癌SiHa, C33A細胞的順鉑半致死量濃度,與之前比較得出結(jié)論。結(jié)果：SiHa細胞順鉑的半致死量濃度約為40μmol/L,C33A細胞順鉑的半致死量濃度約為22.5 μmol/L。選取差異最為明顯的CDC25B1,B3, C1, C5四種剪接體進行轉(zhuǎn)染、上調(diào),RT-PCR及Western Blot分別檢測到相應(yīng)的mRNA和蛋白表達均上調(diào)；MTT比色法測量順鉑半致死量濃度發(fā)現(xiàn),與之前所得到的順鉑半致死量濃度相比,分別上調(diào)了CDC25B1與CDC25C1的細胞組的順鉑半致死量濃度顯著升高,差異具有統(tǒng)計學意義(P0.01)；而分別上調(diào)了CDC25B3與CDC25C5的細胞組的順鉑半致死量濃度顯著降低,差異具有統(tǒng)計學意義(P0.01)。結(jié)論：CDC25B的mRNA的剪接體CDC25B1能夠增強宮頸癌細胞系SiHa, C33A對順鉑的抗性；剪接體CDC25B3能夠減弱宮頸癌細胞系SiHa,C33A對順鉑的抗性。CDC25C的mRN A的剪接體CDC25C1能夠增強宮頸癌細胞系SiHa,C33A對順鉑的抗性；剪接體CDC25C5能夠減弱宮頸癌細胞系SiHaC33A對順鉑的抗性。
[Abstract]:Cervical cancer is the most common malignant tumor in women, which seriously threatens the health and life of women. Cisplatin is a commonly used chemotherapeutic agent for cervical cancer, but the resistance of cancer cells to cisplatin weakens its therapeutic effect. The selective splicing of CDC25B and CDC25C mRNA in the cervical cancer cell line SiHaC33A treated with cisplatin has been changed, because CDC25B and CDC25C play an important role in regulating the checkpoint function of cell cycle under normal physiological conditions and in response to DNA damage. It is suggested that the splicing changes of CDC25B and CDC25C mRNA may be related to cell tolerance to cisplatin. The purpose of this study is to investigate whether major splicing isoforms of CDC25B and CDC25C have anti-apoptotic or pro-apoptotic effects. Does this splicing change increase the resistance of cervical cancer cells to cisplatin? The results and information obtained will be helpful to clarify the role of CDC25B and CDC25C in carcinogenesis and development, and to interpret the mechanism of radio-chemotherapeutic resistance of tumor cells. If the splicing changes of CDC25B and CDC25C mRNA affect the function of checkpoint, the regulation of CDC25B may improve the efficacy of cisplatin, and the pathway and gene of regulation will become a new therapeutic target. To provide important information and basis for individualized treatment of cancer. Aim: to investigate the effects of selective splicing of CDC25B and CDC25C mRNA on the growth, proliferation, apoptosis and cisplatin sensitivity of cervical cancer cells SIHA and C33A. Methods: MTT colorimetric assay was used to determine the semi-lethal concentration of cisplatin in SiHaand C33A cells. RT-PCR was used to detect the same time, different concentration of cisplatin and different time. Effects of semi-lethal cisplatin intervention on selective splicing of CDC25B, C mRNA in cervical cancer cells in each group; The mRNA and protein expression levels of CDC25B1, B3, C1, C5 gene were detected by RT-PCR and Western Blot. MTT colorimetric assay was used to measure the cisplatin semi-lethal concentration of CDC25B1, B3, C1, C5 gene overexpression in cervical cancer SiHaand C33A cells. Compare with the previous conclusion. Results the semi-lethal concentration of cisplatin was about 40 渭 mol 路L ~ (-1) 路L ~ (-1) and about 22.5 渭 mol 路L ~ (-1) 路L ~ (-1) of cisplatin. Four splices CDC25B1B3, C1, C5 were selected for transfection. The up-regulated mRNA and protein expression were detected by RT-PCR and Western Blot respectively. MTT colorimetric assay was used to measure the semi-lethal concentration of cisplatin. Compared with the previously obtained semi-lethal concentration of cisplatin, the CDC25B1 and CDC25C1 cell groups increased the CDC25B1 and CDC25C1 semi-lethal concentration significantly (P0.01). The semi-lethal concentration of cisplatin in CDC25B3 and CDC25C5 cells was significantly lower than that in CDC25B3 and CDC25C5 cells (P0.01). Conclusion the splicing of CDC25B mRNA can enhance the resistance of cervical cancer cell line SiHaC33A to cisplatin. CDC25B3 could attenuate the resistance of SiHaC33A to cisplatin. CDC25C1 of mRNA of CDC25C could enhance the resistance of cervical cancer cell line SiHaC33A to cisplatin, and CDC25C5 could weaken the resistance of cervical cancer cell line SiHaC33A to cisplatin.
【學位授予單位】：昆明理工大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R737.33

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