發(fā)布時間：2018-03-11 12:16

  本文選題：TPX2　切入點：siRNA　出處：《青島大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的對于分期較晚不考慮外科治療的宮頸癌患者,主要的治療手段之一是放療。腫瘤細胞對射線殺傷是否敏感直接決定了療效和疾病預(yù)后。DNA修復(fù)蛋白與腫瘤的放射敏感性關(guān)系密切。TPX2主要在胞核中起作用,依賴細胞周期并受其調(diào)節(jié),與有絲分裂中紡錘體的準(zhǔn)確合成及結(jié)構(gòu)穩(wěn)定有直接關(guān)系。TPX2基因異常表達直接產(chǎn)生的結(jié)果是中心體出現(xiàn)反常擴增及異倍體形成,同時與腫瘤轉(zhuǎn)移和復(fù)發(fā)明顯相關(guān)。已有研究證實了射線依賴的?-H2AX形成量與DNA雙鏈斷裂點的數(shù)量之間有相關(guān)關(guān)系。最新的研究發(fā)現(xiàn)胞內(nèi)TPX2蛋白發(fā)生不同程度的消耗時,射線誘導(dǎo)的?-H2AX的數(shù)量會呈現(xiàn)短暫增加的現(xiàn)象。本實驗主要利用RNAi技術(shù),設(shè)計合成靶基因TPX2的小干擾RNA,觀察導(dǎo)入si RNA后HeLa細胞中TPX2 mRNA和蛋白表達的改變,以及HeLa細胞放射敏感性的變化,為宮頸癌的放射增敏提供了研究方向。方法選擇人宮頸癌HeLa細胞,根據(jù)小干擾RNA設(shè)計原則設(shè)計靶基因TPX2的si RNA,共設(shè)計3對分別為si RNA-1、si RNA-2和si RNA-3,同時設(shè)計陰性對照si RNA。利用ribo FECTTMCP轉(zhuǎn)染試劑,將si RNA轉(zhuǎn)染入HeLa細胞,對轉(zhuǎn)染后各組細胞內(nèi)TPX2 mRNA和蛋白質(zhì)的表達情況進行觀察。主要采用RT-PCR、Western Blotting法,于轉(zhuǎn)染后48 h進行,提取總RNA和總蛋白后檢測TPX2 mRNA和蛋白表達情況,并篩選出最佳轉(zhuǎn)染組及最佳轉(zhuǎn)染時間。實驗分組:目的基因組、陰性對照組、空白對照組。利用ribo FECTTMCP將si RNA轉(zhuǎn)染入宮頸癌HeLa細胞中進行4 Gy-X線照射,利用CCK8法和流式細胞術(shù)觀察TPX2表達下調(diào)的HeLa細胞對射線的敏感性的改變。結(jié)果轉(zhuǎn)染48 h后提取總RNA,利用RT-PCR觀察轉(zhuǎn)染后各組細胞TPX2 mRNA的相對表達量發(fā)現(xiàn),轉(zhuǎn)染針對靶基因TPX2的si RNA的各組細胞均表現(xiàn)出TPX2基因表達抑制效應(yīng),以si RNA-2效果最明顯,其TPX2的相對含量為(0.133±0.008)與空白對照組(1.000)及陰性對照組(0.981±0.814)相比有統(tǒng)計學(xué)意義(P0.05)。轉(zhuǎn)染后繼續(xù)培養(yǎng)48 h,用Western Blot法檢測HeLa細胞TPX2蛋白的表達。結(jié)果發(fā)現(xiàn)si RNA-2組TPX2蛋白表達明顯降低,蛋白相對含量為(0.518±0.685),空白對照組為(1.064±0.794),陰性對照組為(0.981±0.814),si RNA-2組與陰性對照組及空白對照組比較,有統(tǒng)計學(xué)意義(P0.05)。HeLa細胞放射敏感性的檢測:轉(zhuǎn)染后給予4 Gy X線照射,(1)利用CCK8檢測各組細胞OD值,并計算細胞增殖活力,沉默TPX2組為(0.147±0.003),陰性對照組為(0.224±0.009),空白對照組為(0.353±0.012),統(tǒng)計學(xué)分析得出F=9.243,P=0.0015,差異有統(tǒng)計學(xué)意義(P0.05);(2)用流式細胞術(shù)檢測各組細胞凋亡率發(fā)現(xiàn)各組細胞均出現(xiàn)凋亡增加,其中沉默TPX2組細胞凋亡率為(12.68±0.03%),陰性對照組為(9.37±0.11%),空白對照組為(5.81±0.21%),進行單因素方差分析,F=10.242,P0.0001,差異有統(tǒng)計學(xué)意義(P0.05)。結(jié)論針對靶基因TPX2的si RNA成功轉(zhuǎn)染HeLa細胞,并能發(fā)揮基因表達的特異性抑制作用,目的基因的mRNA和蛋白的表達都受到抑制,同時可以提高HeLa細胞的放射敏感性。
[Abstract]:Objective to treat cervical cancer patients whose stage is late and surgical treatment is not considered. One of the main therapeutic methods is radiotherapy. The sensitivity of tumor cells to radiation directly determines the effect and prognosis of disease. DNA repair protein is closely related to the radiosensitivity of tumor. TPX2 mainly plays a role in the nucleus. Depending on and regulated by cell cycle, the abnormal expression of TPX2 gene is directly related to the accurate synthesis and structural stability of spindle in mitosis. The abnormal amplification of centrosome and the formation of aneuploidy are the results of abnormal expression of TPX2 gene. There is also a clear correlation between tumor metastasis and recurrence. There is a correlation between the amount of H2AX formation and the number of double strand breaks in DNA. The number of H2AX increased briefly. In this experiment, we designed and synthesized the small interference RNAs of target gene TPX2 by RNAi technique, observed the changes of TPX2 mRNA and protein expression in HeLa cells after transfection of si RNA, and the radiosensitivity of HeLa cells. Methods Human cervical cancer HeLa cells were selected. Si RNAs of target gene TPX2 were designed according to the principle of small interfering RNA design. Three pairs of siRNA-1 siRNA-3 were designed respectively. Si RNA was transfected into HeLa cells by ribo FECTTMCP transfection reagent. After transfection, the expression of TPX2 mRNA and protein in the cells was observed. The expression of TPX2 mRNA and protein was detected by RT-PCR Western Blotting method at 48 hours after transfection. The total RNA and total protein were extracted, and the expression of TPX2 mRNA and protein were detected after transfection. The best transfection group and optimal transfection time were selected. The experimental groups were as follows: objective genome, negative control group and blank control group. Si RNA was transfected into HeLa cells of cervical cancer by ribo FECTTMCP for 4 Gy-X irradiation. The changes of radiosensitivity of HeLa cells with down-regulated TPX2 expression were observed by CCK8 assay and flow cytometry. Results Total RNAs were extracted 48 h after transfection, and the relative expression of TPX2 mRNA was observed by RT-PCR. Si RNA transfected with target gene TPX2 showed inhibitory effect on the expression of TPX2 gene, especially si RNA-2. The relative content of TPX2 was 0.133 鹵0.008), which was significantly higher than that of control group (0.133 鹵0.008) and negative control group (0.981 鹵0.814). The expression of TPX2 protein in HeLa cells was detected by Western Blot assay at 48 h after transfection. The results showed that the expression of TPX2 protein in si RNA-2 group was significantly lower than that in control group. The relative content of protein was 0.518 鹵0.685g, 1.064 鹵0.794 in the blank control group, 0.981 鹵0.814 RNA-2 in the negative control group, and compared with the negative control group and the blank control group. Detection of radiosensitivity of P0.05U. HeLa cells: after transfection, 4 Gy X ray irradiation was given. OD value of each group was measured by CCK8, and cell proliferation activity was calculated. The rate of apoptosis was 0.147 鹵0.003 in the silencing TPX2 group, 0.224 鹵0.009 in the negative control group and 0.353 鹵0.012 in the blank control group. The apoptosis rate of silencing TPX2 group was 12.68 鹵0.03, that of negative control group was 9.37 鹵0.11, and that of blank control group was 5.81 鹵0.21. The single factor ANOVA analysis showed that the cell apoptosis rate was significantly different (P 0.05). Conclusion the si RNA for target gene TPX2 was successfully transfected into HeLa cells. Both mRNA and protein expression of the target gene were inhibited, and the radiosensitivity of HeLa cells was enhanced.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.33

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2 Qingquan Liu;Pinghua Yang;Kangsheng Tu;Hongyong Zhang;Xin Zheng;Yingmin Yao;Qingguang Liu;;TPX2 knockdown suppressed hepatocellular carcinoma cell invasion via inactivating AKT signaling and inhibiting MMP2 and MMP9 expression[J];Chinese Journal of Cancer Research;2014年04期

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本文編號：1598124