發(fā)布時(shí)間：2018-04-23 09:25

  本文選題：SLC5A8 + 大腸癌　； 參考：《河北大學(xué)》2017年碩士論文

【摘要】：背景大腸癌(colorectal cancer,CRC)包括結(jié)腸癌和直腸癌,是最常見的消化道惡性腫瘤之一,全世界每年約有120萬新發(fā)病例,約60萬人死于該疾病。中國(guó)醫(yī)學(xué)科學(xué)院腫瘤醫(yī)院/國(guó)家癌癥中心在CA Cancer J Clin(臨床腫瘤)雜志發(fā)表的2015年中國(guó)癌癥統(tǒng)計(jì)數(shù)據(jù)顯示我國(guó)大腸癌發(fā)病率、死亡率在全部惡性腫瘤中均位居前5位,其中新發(fā)病例37.6萬,死亡病例19.1萬,已成為嚴(yán)重危害中國(guó)人民健康的疾病。隨著我國(guó)經(jīng)濟(jì)的發(fā)展,人民生活水平的不斷提高及飲食結(jié)構(gòu)的改變,進(jìn)食過多脂肪、肉類,而少膳食纖維,體育鍛煉較少,精神壓力過大,導(dǎo)致大腸癌的發(fā)病率、病死率逐年上升,同時(shí)基因的改變也導(dǎo)致了個(gè)體對(duì)大腸腫瘤的易感性。腫瘤的發(fā)生發(fā)展是一個(gè)復(fù)雜的病理、生理過程,涉及到多種基因改變的復(fù)雜的異質(zhì)性疾病。通常,基因組在復(fù)制、轉(zhuǎn)錄、翻譯等各個(gè)水平的改變均會(huì)影響其生物學(xué)行為的改變。因此對(duì)大腸癌相關(guān)分子機(jī)制的深入研究,將對(duì)大腸癌的預(yù)防和治療具有十分重要的意義。我們前期的研究發(fā)現(xiàn),SLC5A8基因在大腸癌組織中表達(dá)下降或缺失,提示該基因的沉默與大腸癌的發(fā)生存在一定關(guān)系。但是對(duì)SLC5A8基因在大腸癌中沉默的分子機(jī)制、作用底物、信號(hào)通路、與其他蛋白的相互作用等還不是很了解,仍需我們進(jìn)一步的深入研究。溶質(zhì)載體家族第5家族第8員(solute carrier family 5 member 8,SLC5A8)是2002年Rodriguez等人發(fā)現(xiàn)的新的候選抑癌基因,位于人類染色體12q13,屬于Na+/葡萄糖共轉(zhuǎn)運(yùn)蛋白家族成員,是短鏈脂肪酸(SCFA)如,醋酸、丙酸、丁酸、丙酮酸和乳酸的Na+-偶聯(lián)轉(zhuǎn)運(yùn)蛋白。國(guó)內(nèi)外學(xué)者發(fā)現(xiàn)SLC5A8在人類的食管癌、賁門癌、乳腺癌、甲狀腺癌、肺癌、胰腺癌、腦膠質(zhì)瘤、前列腺癌、急性髓系白血病等多種腫瘤組織中表達(dá)降低或缺失,說明其可能參與了腫瘤的發(fā)生、轉(zhuǎn)移、侵襲過程。研究發(fā)現(xiàn),SLC5A8基因在腫瘤組織中表達(dá)降低或缺失的原因是其GPG島的甲基化及組蛋白的去乙�；６∷嶙鳛镾LC5A8基因的轉(zhuǎn)運(yùn)底物之一,是由細(xì)菌發(fā)酵膳食纖維產(chǎn)生的。丁酸除了作為β-氧化和檸檬酸循環(huán)產(chǎn)生代謝能量的底物,同樣也是組蛋白去乙酰化酶抑制劑(HDAC),尤其是HDAC1和HDAC3亞型。這些內(nèi)源性HDAC抑制劑存在于正常結(jié)腸腔中,SLC5A8介導(dǎo)它們進(jìn)入結(jié)腸上皮細(xì)胞,并抑制HDACs。由于SLC5A8基因的表達(dá)減少或缺失,使丁酸不能進(jìn)入結(jié)腸腔中,這可能成為大腸癌發(fā)生的原因之一。目的探討SLC5A8基因轉(zhuǎn)染對(duì)大腸癌細(xì)胞株SW480增殖和侵襲力的影響。方法實(shí)驗(yàn)分三組,實(shí)驗(yàn)組:將含SLC5A8基因的p EX-4質(zhì)粒轉(zhuǎn)染到大腸癌細(xì)胞株SW480;空載對(duì)照組:將空載質(zhì)粒p EX-4轉(zhuǎn)染到大腸癌細(xì)胞株SW480;空白對(duì)照組:正常大腸癌細(xì)胞株SW480。(1)運(yùn)用q RT-PCR法對(duì)細(xì)胞中SLC5A8 m RNA表達(dá)水平進(jìn)行分析;(2)采用MTS法檢測(cè)細(xì)胞增值抑制率;(3)Transwell細(xì)胞侵襲實(shí)驗(yàn)檢測(cè)SLC5A8基因轉(zhuǎn)染SW480細(xì)胞后侵襲力的變化;(4)通過細(xì)胞劃痕實(shí)驗(yàn)觀察轉(zhuǎn)染SLC5A8基因?qū)W480細(xì)胞遷移能力的影響。統(tǒng)計(jì)方法:所有數(shù)據(jù)應(yīng)用SPSS19.0統(tǒng)計(jì)軟件分析;計(jì)量數(shù)據(jù)采用均數(shù)±標(biāo)準(zhǔn)差(sx±)表示;組間比較采用t檢驗(yàn)和ANOVA方差分析,檢驗(yàn)水準(zhǔn)P0.05為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果(1)熒光顯微鏡顯示:轉(zhuǎn)染p EX-4-SLC5A8、p EX-4空載質(zhì)粒的SW480細(xì)胞均顯示高強(qiáng)度的綠色熒光。SLC5A8擴(kuò)增產(chǎn)物溶解曲線呈現(xiàn)單峰,引物特異性良好,擴(kuò)增產(chǎn)物及擴(kuò)增循環(huán)數(shù)(CT值)均可用。q RT-PCR結(jié)果顯示,轉(zhuǎn)染后細(xì)胞中SLC5A8 m RNA表達(dá)水平顯著增高,表明轉(zhuǎn)染效率較高,實(shí)驗(yàn)方法及條件可靠。(2)SLC5A8基因轉(zhuǎn)染后,通過MTS法檢測(cè)發(fā)現(xiàn),當(dāng)培養(yǎng)基中加入丁酸時(shí),實(shí)驗(yàn)組細(xì)胞SW480的增殖抑制率高于對(duì)照組,差異具有統(tǒng)計(jì)學(xué)意義(P=0.013)。當(dāng)培養(yǎng)基中沒有丁酸,實(shí)驗(yàn)組細(xì)胞SW480的增殖抑制率與對(duì)照組之間差異無統(tǒng)計(jì)學(xué)意義(P=0.957)。實(shí)驗(yàn)組在培養(yǎng)基中有丁酸時(shí)對(duì)細(xì)胞SW480的增殖抑制率高于培養(yǎng)基中無丁酸,差異具有統(tǒng)計(jì)學(xué)意義(P=0.017)。無論培養(yǎng)基中有、無丁酸,對(duì)照組對(duì)細(xì)胞SW480的增值抑制率差異不具有統(tǒng)計(jì)學(xué)意義(P=0.930)。MTS實(shí)驗(yàn)結(jié)果說明實(shí)驗(yàn)組SW480細(xì)胞在有丁酸時(shí)增殖明顯受到抑制。(3)Transwell體外侵襲實(shí)驗(yàn)結(jié)果顯示,實(shí)驗(yàn)組的透膜細(xì)胞數(shù)低于空載對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(t=14.072,P=0.000)。實(shí)驗(yàn)組的透膜細(xì)胞數(shù)低于空白對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(t=17.710,P=0.000)�？蛰d對(duì)照組透膜細(xì)胞數(shù)與空白對(duì)照組之間的差異無統(tǒng)計(jì)學(xué)意義(t=1.540,P=0.162)。Transwell體外侵襲實(shí)驗(yàn)以透膜細(xì)胞數(shù)作為侵襲力的評(píng)價(jià)指標(biāo)。說明SLC5A8基因在丁酸的作用下可以抑制大腸癌細(xì)胞株SW480的侵襲能力。(4)應(yīng)用細(xì)胞劃痕實(shí)驗(yàn)觀察SLC5A8基因?qū)?xì)胞遷移的影響,劃痕后24小時(shí),實(shí)驗(yàn)組細(xì)胞遷移率低于空載對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(t=13.617,P=0.000)。實(shí)驗(yàn)組細(xì)胞遷移率低于空白對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(t=15.997,P=0.000)�？蛰d對(duì)照組細(xì)胞遷移率與空白對(duì)照組之間的差異無統(tǒng)計(jì)學(xué)意義(t=1.421,P=0.193)。同樣,劃痕后48小時(shí),實(shí)驗(yàn)組細(xì)胞遷移率低于空載對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(t=29.526,P=0.000)。實(shí)驗(yàn)組細(xì)胞遷移率低于空白對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(t=48.936,P=0.000)�？蛰d對(duì)照組細(xì)胞遷移率與空白對(duì)照組之間的差異無統(tǒng)計(jì)學(xué)意義(t=1.988,P=0.082)。說明SLC5A8基因在丁酸的作用下可以抑制大腸癌細(xì)胞株SW480的遷徙能力。結(jié)論1.SLC5A8基因在大腸癌細(xì)胞株SW480中表達(dá)下降或缺失。2.本實(shí)驗(yàn)所采用基因轉(zhuǎn)染技術(shù)成功將SLC5A8基因轉(zhuǎn)染到大腸癌細(xì)胞株SW480中。3.SLC5A8基因抑制大腸癌細(xì)胞株SW480的增殖、侵襲及遷徙是通過丁酸介導(dǎo)的,丁酸是其轉(zhuǎn)運(yùn)底物之一。4.本實(shí)驗(yàn)為大腸癌的基因治療提供新的方法。
[Abstract]:Background colorectal cancer (CRC), including colon and rectal cancer, is one of the most common digestive malignant tumors. There are about 120 million new cases in the world each year. About 600 thousand people die of the disease. The cancer hospital / National Cancer Center of the Chinese Academy of Medical Sciences in the 2015 magazine of the CA Cancer J Clin (clinical cancer), China Cancer system The data show the incidence of colorectal cancer in China, and the mortality rate is the top 5 in all malignant tumors, of which 376 thousand of the new cases and 191 thousand of the deaths have become a serious disease which seriously endangering the health of the Chinese people. With the development of China's economy, the people's living standards are constantly raised and the diet structure changes, eating too much fat and meat, With less dietary fiber, less physical exercise and excessive mental stress, the incidence of large intestine cancer and the mortality rate are increasing year by year. At the same time, the change of genes also leads to the susceptibility to colorectal cancer. The development of the tumor is a complex pathological process, involving complex heterogeneous diseases of various gene changes. It is of great significance for the prevention and treatment of colorectal cancer. Our previous studies have found that the expression of SLC5A8 gene in colorectal cancer is reduced or missing, which suggests that the gene in colorectal cancer can be reduced or missing. There is a certain relationship between the gene silencing and the occurrence of colorectal cancer. However, the molecular mechanism of the silence of the SLC5A8 gene in colorectal cancer, the role of the substrate, the signaling pathway, and the interaction with other proteins are not well understood. We still need further in-depth study. The eighth members of the fifth family of the solute carrier family (solute carrier family 5 member 8, SL C5A8) is a new candidate tumor suppressor gene found by Rodriguez et al. In 2002, located in the human chromosome 12q13, belonging to the Na+/ glucose co transporter family. It is the Na+- coupling transporter of short chain fatty acid (SCFA), acetic acid, propionic acid, butyric acid, pyruvic acid and lactic acid. Domestic and foreign scholars found SLC5A8 in human esophageal, cardiac and breast cancer. The reduction or deletion of the expression of the SLC5A8 gene in the tumor, the reduction or deletion of the gene in the tumor tissue is the methylation of the GPG island and the histone removal. Acetylation. As one of the transport substrates of SLC5A8 gene, butyric acid is produced by bacterial fermentation of dietary fiber. Butyric acid is also the substrate for producing metabolic energy as a beta oxidation and citric acid cycle. It is also a histone deacetylase inhibitor (HDAC), especially the HDAC1 and HDAC3 subtypes. These endogenous HDAC inhibitors exist in the normal colon. In the cavity, SLC5A8 mediates their entry into the colonic epithelial cells and inhibits the decrease or loss of the expression of HDACs. because of the SLC5A8 gene expression, which may not enter the colon cavity. This may be one of the reasons for the occurrence of colorectal cancer. Objective to investigate the effect of SLC5A8 gene transfection on the proliferation and invasiveness of colorectal cancer cell line SW480. Method experiments were divided into three groups. Test group: transfection of P EX-4 plasmid containing SLC5A8 gene into colorectal cancer cell line SW480; empty load control group: transfection of empty plasmid P EX-4 into colorectal cancer cell line SW480; blank control group: normal colorectal cancer cell strain SW480. (1) used Q RT-PCR method to analyze the expression level of SLC5A8 m in cells; (2) the inhibition of cell increment inhibition was detected by the method. Rate; (3) Transwell cell invasion test was used to detect the invasiveness of SLC5A8 gene transfected to SW480 cells; (4) the effect of transfection on the migration ability of SW480 cells by transfection of cells through cell scratch test. Statistical methods: all data were analyzed by SPSS19.0 statistical software; the count data were expressed by mean number + standard deviation (SX +); comparison was taken between groups. T test and ANOVA analysis of variance showed that the level of P0.05 was statistically significant. Results (1) the fluorescence microscope showed that the SW480 cells transfected with P EX-4-SLC5A8 and P EX-4 empty plasmid showed a single peak of high intensity green fluorescence.SLC5A8 product dissolution curve, and the primer specificity was good, the amplification products and the number of amplification cycles (CT values) were all The results of.Q RT-PCR showed that the expression level of SLC5A8 m RNA in the transfected cells was significantly higher, indicating that the transfection efficiency was higher and the experimental methods and conditions were reliable. (2) after the transfection of SLC5A8 gene, the MTS method was used to detect the proliferation and the inhibitory rate of SW480 in the experimental group was higher than that of the control group, and the difference was statistically significant. P=0.013). When there was no butyric acid in the medium, there was no significant difference between the proliferation inhibition rate of SW480 in the experimental group and the control group (P=0.957). The inhibition rate of the proliferation of SW480 in the experimental group was higher than that in the medium (P=0.017). The difference was statistically significant (P=0.017). The difference of the inhibitory rate of SW480 in the group was not statistically significant (P=0.930).MTS experimental results showed that the proliferation of SW480 cells in the experimental group was obviously inhibited when there was butyric acid. (3) the experimental results of Transwell invasion in vitro showed that the number of membrane cells in the experimental group was lower than that of the empty control group, the difference was statistically significant (t=14.072, P=0.000). The number of membrane cells in the group was lower than that in the blank control group. The difference was statistically significant (t=17.710, P=0.000). There was no significant difference between the number of membrane cells in the empty control group and the blank control group (t=1.540, P=0.162).Transwell in the invasion experiment in vitro using the number of transmembrane cells as the evaluation index of the emplacement force. The effect of SLC5A8 gene in butyric acid was explained. The invasion ability of SW480 was inhibited. (4) the effect of SLC5A8 gene on cell migration was observed with the cell scratch test. The cell migration rate of the experimental group was lower than that of the control group at 24 hours after scratch (t=13.617, P=0.000). The cell migration rate in the experimental group was lower than that in the blank control group. The difference was statistically significant T=15.997 (P=0.000). There was no significant difference between the cell migration rate and the blank control group in the control group (t=1.421, P=0.193). Also, the cell migration rate of the experimental group was lower than that of the control group at 48 hours after the scratch (t=29.526, P=0.000). The cell migration rate of the experimental group was lower than that of the blank control group, and the difference was statistically significant Significance (t=48.936, P=0.000). There was no significant difference in the cell migration rate between the blank control group and the blank control group (t=1.988, P=0.082). It indicated that the SLC5A8 gene could inhibit the migration of SW480 in the colorectal cancer cell line under the action of butyric acid. Conclusion the 1.SLC5A8 gene was expressed in the colon cancer cell line SW480 to decrease or lose the.2. based experiment. The gene transfection technique successfully transfected the SLC5A8 gene into the colorectal cancer cell line SW480.3.SLC5A8 gene to inhibit the proliferation of colorectal cancer cell line SW480. Invasion and migration are mediated by butyric acid, and butyric acid is one of its transport substrates. This experiment provides a new method for the gene therapy of colorectal cancer.

【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.34

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