【摘要】：低密度脂蛋白受體相關(guān)蛋白5(LRP-5)是低密度脂蛋白受體家族成員之一,該蛋白質(zhì)屬于單跨膜受體,由1615個(gè)氨基酸殘基組成,分為胞內(nèi)區(qū)、跨膜區(qū)、胞外區(qū)三個(gè)區(qū)。LRP-5與其家族中另一個(gè)成員LRP-6高度同源。研究表明,LRP-5/LRP-6與七跨膜受體蛋白質(zhì)FZD共同構(gòu)成了WNT/β-catenin信號(hào)轉(zhuǎn)導(dǎo)通路的受體系統(tǒng)。LRP-5/LRP-6是配體WNTs蛋白質(zhì)結(jié)合的輔受體,當(dāng)配體WNTs與FZD和LRP-5/LRP-6同時(shí)結(jié)合時(shí),該信號(hào)通路才能被激活。而WNT/β-catenin信號(hào)轉(zhuǎn)導(dǎo)通路異常調(diào)控與癌癥的關(guān)系是目前研究的一個(gè)熱點(diǎn)問(wèn)題。本研究以WNT/β-catenin信號(hào)通路中輔受體LRP-5為切入點(diǎn),應(yīng)用RNAi技術(shù)沉默LRP-5基因表達(dá),探討WNT/β-catenin信號(hào)轉(zhuǎn)導(dǎo)通路改變對(duì)胃癌細(xì)胞增殖、凋亡、侵襲、遷移等生物學(xué)行為及差異蛋白質(zhì)組學(xué)的影響,以期探討胃癌發(fā)病機(jī)制,為胃癌治療的可能藥物靶點(diǎn)提供實(shí)驗(yàn)依據(jù)。首先,用qRT-PCR和免疫組化的方法檢測(cè)了不同分化程度的胃癌組織中LRP-5基因的表達(dá)狀態(tài),為下一步開展RNAi實(shí)驗(yàn)奠定可行性基礎(chǔ)研究。結(jié)果顯示,中分化、低分化的胃癌組織中LRP-5高表達(dá),與正常胃組織比較差異有統(tǒng)計(jì)學(xué)意義(P㩳0.05),而且伴有淋巴細(xì)胞轉(zhuǎn)移的胃癌組織中也呈現(xiàn)LRP-5高表達(dá)。這提示LRP-5與胃癌的發(fā)生、發(fā)展、轉(zhuǎn)移和惡性程度相關(guān)。其次,構(gòu)建了4個(gè)針對(duì)LRP-5基因不同位點(diǎn)的pGPU6/GFP/Neo-LRP-5干擾質(zhì)粒,進(jìn)行轉(zhuǎn)染效率和干擾效率的評(píng)價(jià)后,成功篩選出了pGPU6/GFP/Neo-LRP-5-Homo-4868(L68)干擾質(zhì)粒。經(jīng)瞬時(shí)轉(zhuǎn)染48h沉默MGC-803中LRP-5基因表達(dá)后,觀察其對(duì)細(xì)胞增殖、粘附、凋亡、周期、侵襲、遷移的影響。結(jié)果顯示:1、CCK8法檢測(cè),LRP-5基因干擾組轉(zhuǎn)染MGC-803細(xì)胞48h,相對(duì)生長(zhǎng)速度顯著低于空白對(duì)照組和陰性對(duì)照組(P0.05);2、LRP-5基因干擾組MGC-803細(xì)胞的粘附細(xì)胞數(shù)為155.00±18.33,較空白對(duì)照組和陰性對(duì)照組升高,差異有統(tǒng)計(jì)學(xué)意義(P0.05);3、流式細(xì)胞儀雙染法檢測(cè)細(xì)胞凋亡,在LRP-5干擾質(zhì)粒轉(zhuǎn)染MGC-803細(xì)胞48h后,活細(xì)胞數(shù)量減少、凋亡細(xì)胞增多,與陰性對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義(P㩳0.05);4、流式細(xì)胞儀PI單染檢測(cè)細(xì)胞周期,在LRP-5干擾質(zhì)粒轉(zhuǎn)染MGC-803細(xì)胞48h后,LRP-5干擾組的S期比例升高、G2期比例降低,發(fā)生了S期阻滯現(xiàn)象;5、Transwell小室基底膜實(shí)驗(yàn)表明,LRP-5干擾組侵襲遷移到Transwell基底膜下層的細(xì)胞計(jì)數(shù)為(38.67±3.51),與空白對(duì)照組(52.67±4.51)、Mock組(50.67±3.79)、陰性對(duì)照組(47.00±4.36)比較,細(xì)胞的侵襲能力有所下降,差異有統(tǒng)計(jì)學(xué)意義(P㩳0.05);6、劃痕實(shí)驗(yàn)顯示,LRP-5干擾組MGC-803細(xì)胞的遷移愈合率為0.7509±0.0108,與空白對(duì)照組和陰性對(duì)照組比較,愈合率下降,遷移能力下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。最后,本研究采用RNAi技術(shù)沉默人胃癌MGC-803細(xì)胞中LRP-5基因表達(dá),在轉(zhuǎn)染MGC-803細(xì)胞48h后,抽提干擾組和陰性對(duì)照組細(xì)胞總蛋白。應(yīng)用2D電泳分離純化蛋白質(zhì)、PDQuest軟件分析2D電泳膠圖,結(jié)果顯示,兩組之間共有29個(gè)差異蛋白質(zhì)點(diǎn)。選擇其中15個(gè)點(diǎn)進(jìn)行質(zhì)譜鑒定,成功12個(gè)。與陰性對(duì)照組比較,L68干擾組中有10個(gè)點(diǎn)上調(diào),2個(gè)點(diǎn)下調(diào)。這些蛋白質(zhì)包括:細(xì)胞骨架、分子伴侶、細(xì)胞周期、細(xì)胞增殖、糖代謝相關(guān)的蛋白質(zhì)。本研究首次應(yīng)用RNAi技術(shù)沉默LRP-5基因,對(duì)胃癌MGC-803細(xì)胞的生物學(xué)行為及差異蛋白質(zhì)的影響進(jìn)行深入研究,初步發(fā)現(xiàn)了LRP-5的低表達(dá)可抑制胃癌MGC-803細(xì)胞的增殖、遷移和侵襲,為探討胃癌發(fā)病機(jī)制和開發(fā)胃癌治療的可能藥物靶點(diǎn)提供了實(shí)驗(yàn)依據(jù)。
[Abstract]:The low-density lipoprotein receptor-related protein 5 (LRP-5) is one of the members of the low-density lipoprotein receptor family, which belongs to a single-transmembrane receptor and consists of 1615 amino acid residues, and is divided into three regions of an intracellular region, a transmembrane region and an extracellular region. The LRP-5 is highly homologous to the other member LRP-6 in the family. The study shows that the LRP-5/ LRP-6 and the seven-transmembrane receptor protein FZD form the receptor system of the WNT/ HCO3-catenin signal transduction pathway. LRP-5/ LRP-6 is an auxiliary receptor for ligand WNTs protein binding, which can be activated when ligand WNTs is combined with FZD and LRP-5/ LRP-6. The relationship between the abnormal regulation and the cancer of the WNT/ P-catenin signal transduction pathway is a hot issue in the current research. In this study, the LRP-5 gene expression in WNT/ HCO3-cattenin signal pathway was used to silence the expression of LRP-5 gene, and the effects of the change of WNT/ P-catenin signal transduction pathway on the proliferation, apoptosis, invasion and migration of gastric cancer cells and the differential proteomics were discussed. Objective To study the mechanism of gastric cancer and provide experimental basis for possible drug target for gastric cancer treatment. First, the expression status of LRP-5 gene in gastric cancer tissues with different degree of differentiation was detected by qRT-PCR and immunohistochemistry, and the feasibility of the next step was studied. The results showed that the high expression of LRP-5 in the middle and low-differentiated gastric cancer tissues was significantly different from that of the normal gastric tissues (P <0.05), and the LRP-5 high expression was also present in the gastric cancer tissues with the lymphocyte metastasis. This suggests that LRP-5 is associated with the occurrence, development, metastasis and malignancy of gastric cancer. The pGPU6/ GFP/ Neo-LRP-5 interference plasmid was constructed and the pGU6/ GFP/ Neo-LRP-5-Homo-4868 (L68) interference plasmid was successfully screened. After transient transfection of the LRP-5 gene in MGC-803, the effects of the expression of LRP-5 on cell proliferation, adhesion, apoptosis, cycle, invasion and migration were observed. The results showed that: 1, CCK8 method, LRP-5 gene interference group was transfected into MGC-803 cells for 48h, and the relative growth rate was significantly lower than that of blank control group and negative control group (P0.05). The number of adherent cells of MGC-803 cells in LRP-5 gene interference group was 155. 00-18.33, and the control group and negative control group increased. The difference was significant (P0.05); 3, the cell apoptosis was detected by flow cytometry double-staining method, and after the LRP-5 interference plasmid was transfected into the MGC-803 cells for 48h, the number of living cells was reduced, the number of apoptotic cells increased, and the difference with the negative control group was statistically significant (P 0.05); and 4, The cell cycle of the cell cycle was detected by flow cytometry PI. The S-phase ratio of LRP-5 interference group was increased and the proportion of G2 phase was decreased after 48 h of the LRP-5 interference plasmid. The cell count of LRP-5 interference group invasion and migration to the lower layer of Transwell basement membrane was (38. 67-3.51), compared with the blank control group (52. 67-4.51), the Mock group (50. 67-3.79) and the negative control group (47. 00-4.36), the invasion ability of the cells decreased and the difference was statistically significant (P-0.05); The results showed that the rate of migration and healing of MGC-803 cells in LRP-5 interference group was 0. 7509-0. 0108, compared with the blank control group and the negative control group, the healing rate decreased, the migration ability decreased, and the difference was statistically significant (P0.05). Finally, the LRP-5 gene expression in the MGC-803 cell of human gastric cancer was silenced by RNAi, and the total protein of the interfering group and the negative control group was extracted after the MGC-803 cell was transfected for 48h. The protein and PDQuest software were separated and purified by 2D electrophoresis. The results showed that there were 29 different protein points between the two groups. 15 of them were selected for mass spectrometry and 12 were successful. Compared with the negative control group, 10 of the L68 interference groups were up-regulated and 2 points were down-regulated. These proteins include a cytoskeleton, a molecular chaperone, a cell cycle, cell proliferation, and sugar metabolism-related proteins. In this study, the LRP-5 gene was used to silence the effects of LRP-5 gene on the biological behavior and differential protein of MGC-803 cells in gastric cancer. The low expression of LRP-5 was found to inhibit the proliferation, migration and invasion of MGC-803 cells in gastric cancer. In order to study the mechanism of gastric cancer and the possible drug target for the development of gastric cancer, the experimental basis is provided.
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.2

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3 周韻斕;應(yīng)激條件下胃癌細(xì)胞高表達(dá)線粒體活性氧和前梯度蛋白2促進(jìn)腫瘤進(jìn)展的研究[D];上海交通大學(xué);2014年

4 徐同鵬;SP1誘導(dǎo)的長(zhǎng)非編碼RNA TINCR通過(guò)調(diào)控KLF2 mRNA穩(wěn)定性影響胃癌細(xì)胞的增殖與凋亡[D];南京醫(yī)科大學(xué);2015年

5 王暢;miR-34a通過(guò)miR-34a-c-myc/Bmi-1負(fù)反饋通路負(fù)調(diào)控胃癌干細(xì)胞樣特性[D];復(fù)旦大學(xué);2014年

6 羅貫虹;MGr1-Ag/37LRP參與PrP~C介導(dǎo)的胃癌細(xì)胞多藥耐藥[D];第四軍醫(yī)大學(xué);2014年

7 默董亮;人類解旋酶RecQL4調(diào)控胃癌細(xì)胞耐藥性分子機(jī)制研究[D];中國(guó)科學(xué)院北京基因組研究所;2016年

8 劉皎婧;PI3K/Akt/GSK3β/p190ARhoGAP/RhoA信號(hào)通路在Wnt5a誘導(dǎo)胃癌細(xì)胞遷移中作用的研究[D];南京醫(yī)科大學(xué);2014年

9 Muhammad Kamran（卡姆拉）;[D];大連醫(yī)科大學(xué);2015年

10 王瑛;PKGⅡ?qū)PA誘導(dǎo)的胃癌細(xì)胞徖移及RhoA和Rac1激活的影響[D];江蘇大學(xué);2015年

相關(guān)碩士學(xué)位論文 前10條

1 林光錟;CyclinA/CDK2介導(dǎo)BAG3 Ser291位點(diǎn)的磷酸化并影響胃癌細(xì)胞的增殖和凋亡[D];福建醫(yī)科大學(xué);2015年

2 蔡洙哲;p12-LOX對(duì)胃癌細(xì)胞MKN-28的增殖和遷移的影響[D];延邊大學(xué);2015年

3 李龍;Her-2高表達(dá)對(duì)胃癌細(xì)胞侵襲、遷移的影響[D];長(zhǎng)江大學(xué);2015年

4 趙婷婷;GPIbα對(duì)胃癌細(xì)胞粘附、遷移和侵襲能力的影響及其機(jī)制的初步探討[D];蘭州大學(xué);2015年

5 謝瑞霞;盤狀結(jié)構(gòu)域受體1調(diào)控上皮—間質(zhì)轉(zhuǎn)化影響胃癌侵襲轉(zhuǎn)移分子機(jī)制的研究[D];蘭州大學(xué);2015年

6 王東倉(cāng);NM23-H2通過(guò)抑制Wnt/β-catenin信號(hào)通路抑制胃癌細(xì)胞的侵襲遷移[D];蘭州大學(xué);2015年

7 朱瑞雪;NDRG2基因?qū)ξ赴┘?xì)胞惡性表型的影響[D];鄭州大學(xué);2015年

8 董凱楠;內(nèi)質(zhì)網(wǎng)應(yīng)激對(duì)胃癌細(xì)胞侵襲遷移能力的影響[D];鄭州大學(xué);2015年

9 單爭(zhēng)爭(zhēng);二甲雙胍在IL-6誘導(dǎo)胃癌細(xì)胞EMT中的作用[D];鄭州大學(xué);2015年

10 田莉;NM23-H2通過(guò)誘導(dǎo)自噬抑制胃癌細(xì)胞EMT的發(fā)生[D];蘭州大學(xué);2015年

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