發(fā)布時(shí)間：2018-05-09 04:15

  本文選題：結(jié)直腸癌 + 腫瘤轉(zhuǎn)移　； 參考：《南方醫(yī)科大學(xué)》2014年碩士論文

【摘要】：研究背景 結(jié)直腸癌已成為我國(guó)發(fā)病率增長(zhǎng)最快的惡性腫瘤之一,然而在結(jié)直腸癌相關(guān)性死亡中,大部分為轉(zhuǎn)移癌所致。近年來越來越多的研究聚焦于腫瘤轉(zhuǎn)移,并不斷探索腫瘤轉(zhuǎn)移的機(jī)制,旨在減少腫瘤轉(zhuǎn)移的發(fā)生,降低轉(zhuǎn)移癌的死亡率。腫瘤血管生成是腫瘤生長(zhǎng)以及腫瘤轉(zhuǎn)移過程中非常重要的環(huán)節(jié),VEGF是促進(jìn)血管生成的重要因子,在腫瘤血管生成中也具有重要作用。目前有實(shí)驗(yàn)通過抑制VEGF的生成來抑制腫瘤血管形成,從而抑制腫瘤,但是對(duì)于結(jié)直腸癌的轉(zhuǎn)移抑制作用不明顯。因此,有必要進(jìn)一步研究VEGF在結(jié)直腸癌形成以及轉(zhuǎn)移過程中的機(jī)制。 近年來,研究發(fā)現(xiàn)Lgr5為小腸、結(jié)腸以及毛囊等組織的干細(xì)胞特異性分子標(biāo)記物,并且在肝細(xì)胞癌、結(jié)腸癌、卵巢癌、基底細(xì)胞癌以及胃癌等實(shí)體腫瘤中高表達(dá)。Lgr5是Wnt信號(hào)通路的靶基因,并且可以反饋性地調(diào)節(jié)Wnt信號(hào)通路的活性,從而調(diào)控細(xì)胞的生長(zhǎng)、運(yùn)動(dòng)和分化。越來越多的研究證實(shí),Lgr5可能也為結(jié)直腸癌的腫瘤干細(xì)胞標(biāo)記物,可以與其他腫瘤干細(xì)胞標(biāo)記物一同用于結(jié)直腸癌腫瘤干細(xì)胞的篩選。根據(jù)“腫瘤干細(xì)胞學(xué)說”,有數(shù)據(jù)表明,結(jié)直腸癌可能來源于隱窩基底部Lgr5陽性的干細(xì)胞。這就證明Lgr5可能對(duì)于結(jié)直腸癌的發(fā)生發(fā)展具有重要作用。然而,目前關(guān)于Lgr5在腫瘤形成、發(fā)展以及轉(zhuǎn)移過程中的作用及其機(jī)制的研究較少。我們課題組前期證實(shí)在結(jié)直腸癌細(xì)胞中敲低Lgr5的表達(dá)后可以抑制腫瘤的血管生成。 研究目的 為了避免單方面敲低Lgr5表達(dá)引起實(shí)驗(yàn)結(jié)果的偶然性以及進(jìn)一步探討Lgr5在結(jié)直腸癌及其轉(zhuǎn)移癌中的作用,Lgr5對(duì)VEGF的影響及其機(jī)制,為以Lgr5為靶點(diǎn)的結(jié)直腸癌治療提供理論依據(jù)。 研究方法 1.構(gòu)建Lgr5過表達(dá)質(zhì)粒并建立穩(wěn)定細(xì)胞株 根據(jù)完整的人Lgr5(NM_003667)基因序列信息設(shè)計(jì)其編碼區(qū)擴(kuò)增引物,引物序列見表一,引物擴(kuò)增總長(zhǎng)度為2745bps。將其克隆到pcDNA3.1(+)真核表達(dá)載體中,經(jīng)檢測(cè)序列正確。利用Lipofectamine2000將pcDNA3.1(+)-Lgr5質(zhì)粒、pcDNA3.1(+)空載體質(zhì)粒瞬時(shí)轉(zhuǎn)染入SW480細(xì)胞,48小時(shí)后,以1000ug/mlG418進(jìn)行連續(xù)篩選,約2-3周挑克隆并擴(kuò)大培養(yǎng),以600ug/mlG418繼續(xù)維持。 2. Western blotting 提取細(xì)胞的總蛋白,用BCA蛋白質(zhì)定量試劑盒檢測(cè)蛋白濃度,蛋白質(zhì)樣品上樣量為20μg。電泳后將蛋白質(zhì)電轉(zhuǎn)至PVDF膜,5%脫脂牛奶封閉,4℃孵育一抗過夜,一抗稀釋比例為(Lgr51:500, VEGF1:200, GAPDH1:1000,β-actin 1：1000),次日二抗孵育1小時(shí),ECL化學(xué)發(fā)光法顯影。 3.實(shí)時(shí)熒光定量PCR (qRT-PCR) 提取細(xì)胞、組織總RNA并測(cè)定RNA濃度,用M-MLV逆轉(zhuǎn)錄酶合成cDNA第一鏈,使用Takara的SYBR Premix Ex Taq試劑盒進(jìn)行實(shí)時(shí)熒光定量PCR,引物序列見表一。 4.免疫組織化學(xué)(SP法) 收集南方醫(yī)院結(jié)直腸原發(fā)癌標(biāo)本58例,結(jié)直腸癌轉(zhuǎn)移癌標(biāo)本8例,組織經(jīng)10%福爾馬林固定,石蠟包埋,標(biāo)本切成4μm薄片,65℃烤箱烤片約3小時(shí),依次脫蠟水化,以0.01M枸櫞酸鈉緩沖液(pH6.0)高溫微波修復(fù)15mmin,利用邁新公司UltraSensitiveS-P超敏試劑盒,一抗4℃冰箱孵育過夜(Lgr51:50、CD311:50),經(jīng)DAB顯色劑顯色,蘇木素復(fù)染,脫水透明,封片風(fēng)干,顯微鏡下觀察并拍片,兩個(gè)病理科醫(yī)生分別獨(dú)立閱片并評(píng)分。 5.HE染色 組織經(jīng)10%福爾馬林固定,石蠟包埋,標(biāo)本切成4μ.m薄片,65℃烤箱烤片約30分鐘,依次脫蠟水化,蘇木素染色2-5分鐘,1%鹽酸酒精分化,流水沖洗返藍(lán)15分鐘,伊紅染色數(shù)秒至數(shù)分鐘,脫水透明,封片晾干,顯微鏡下觀察并拍片。 6. Transwell小室遷移與侵襲實(shí)驗(yàn) 將100u1濃度為以1x106/ml的細(xì)胞懸液加于上室,然后將500u1含10%FBS的培養(yǎng)基加于下室,培養(yǎng)24小時(shí)后,取出小室,4%多聚甲醛將細(xì)胞固定30分鐘,結(jié)晶紫中染色10分鐘,用棉簽擦掉上室殘留細(xì)胞,顯微鏡下隨機(jī)選取5個(gè)視野進(jìn)行細(xì)胞計(jì)數(shù)。侵襲實(shí)驗(yàn)在上室鋪有一層基質(zhì)膠,其余方法及操作均同遷移實(shí)驗(yàn)。 7.小管形成實(shí)驗(yàn) 質(zhì)粒瞬時(shí)轉(zhuǎn)染后48小時(shí)收集培養(yǎng)基,經(jīng)孔徑為0.22μmn的濾器分別過濾,收集為條件培養(yǎng)基�；|(zhì)膠加入96孔板中,每孔加入50ul,37℃溫度下交聯(lián)1小時(shí)使膠固化。每孔加入50u1濃度為2×105/ml的HMVEC細(xì)胞,并且加入相應(yīng)的條件培養(yǎng)基。于0h、2h、4h、8h在倒置顯微鏡下觀察HMVEC細(xì)胞形成小管樣結(jié)構(gòu)的情況,并拍照,計(jì)數(shù)視野中小管樣網(wǎng)狀結(jié)構(gòu)中的閉合環(huán)形小管結(jié)構(gòu)的個(gè)數(shù)。 8. Elisa實(shí)驗(yàn) 根據(jù)人血管內(nèi)皮生長(zhǎng)因子(VEGF) Elisa試劑盒(武漢華美生物工程有限公司)的操作說明,檢測(cè)重組細(xì)胞培養(yǎng)基中VEGF的濃度。 9. AOM/DSS模型的建立 選取5-6周balb/c小鼠,隨機(jī)分成2組,每組10只。實(shí)驗(yàn)組用終濃度為1.25mg/ml的AOM按照12.5mg/kg的劑量腹腔注射一次(10ul/g),對(duì)照組以腹腔注射等劑量生理鹽水。對(duì)照組不做特殊處理,實(shí)驗(yàn)組進(jìn)行如下處理：第1周正常飲水；第2周飲水含2.5%DSS,第3、4周連續(xù)2周正常飲水,此為一個(gè)循環(huán)；第5-10周,重復(fù)第2、3、4周處理2次,即總共三個(gè)循環(huán)。繼續(xù)飼養(yǎng)至17周,處死老鼠并取結(jié)腸組織,觀察其瘤體情況,將結(jié)腸以10%福爾馬林固定,石蠟包埋,行HE染色。提取結(jié)腸組織RNA,并進(jìn)一步行實(shí)時(shí)熒光定量PCR檢測(cè)Lgr5以及VEGF的表達(dá)量。 10.裸鼠成瘤與肝轉(zhuǎn)移實(shí)驗(yàn) 裸鼠成瘤實(shí)驗(yàn) 選取5-6周Balb/c裸鼠,隨機(jī)分成2組,每組7只,飼養(yǎng)等級(jí)為SPF級(jí)。分別注射SW620Lgr5shRNA細(xì)胞、SW620NC細(xì)胞至裸鼠背側(cè),調(diào)整細(xì)胞濃度為1×107/ml,注射200ul,注射細(xì)胞數(shù)為2×106個(gè)。觀察裸鼠的成瘤情況,每隔3天稱量裸鼠的體重,測(cè)量腫瘤的長(zhǎng)徑(L)以及寬徑(W),以公式L×w2/2計(jì)算出瘤體的體積。4周后,裸鼠安樂死,并取下瘤體,經(jīng)10%福爾馬林固定石蠟包埋,供后續(xù)HE染色。 裸鼠結(jié)直腸癌肝轉(zhuǎn)移實(shí)驗(yàn) 前期細(xì)胞準(zhǔn)備與裸鼠同成瘤實(shí)驗(yàn),以1%戊巴比妥鈉6-8ul/g腹腔注射麻醉裸鼠,在裸鼠左側(cè)背部取長(zhǎng)約1cm小口,暴露脾臟,沿著脾門上緣從脾臟頭側(cè)進(jìn)針向脾臟尾部注射,將100ul濃度為1×107/ml的細(xì)胞緩慢注入脾被膜下,注射成功后,清理腹腔,逐層關(guān)腹并逐層縫合,并以紗布包扎。觀察裸鼠的生長(zhǎng)狀態(tài),每隔3天稱量裸鼠的體重。3周后,處死裸鼠,并取下脾臟、肝臟、肺臟等組織,經(jīng)10%福爾馬林固定石蠟包埋,供后續(xù)HE染色。 結(jié)果 1.免疫組化初步結(jié)果顯示,在結(jié)直腸原發(fā)癌以及轉(zhuǎn)移癌中,存在Lgr5表達(dá)越高,微血管密度(MVD)越高的趨勢(shì) 對(duì)結(jié)直腸原發(fā)癌、淋巴結(jié)轉(zhuǎn)移癌、肝轉(zhuǎn)移癌組織標(biāo)本進(jìn)行免疫組織化學(xué)染色,檢測(cè)Lgr5以及血管內(nèi)皮標(biāo)記物CD31的表達(dá),發(fā)現(xiàn)Lgr5的表達(dá)在淋巴結(jié)轉(zhuǎn)移癌與肝轉(zhuǎn)移癌中表達(dá)更高,并且CD31的表達(dá)同樣也升高,即微血管密度升高。免疫組化初步結(jié)果顯示,在結(jié)直腸原發(fā)癌以及轉(zhuǎn)移癌中,存在Lgr5表達(dá)越高,微血管密度(MVD)越高的趨勢(shì),然而這種趨勢(shì)以及二者的相關(guān)性還需要通過進(jìn)一步加大樣本量,獲得統(tǒng)計(jì)學(xué)數(shù)據(jù)加以證明。 2.成功構(gòu)建高表達(dá)Lgr5的穩(wěn)定細(xì)胞株 轉(zhuǎn)染Lgr5重組過表達(dá)質(zhì)粒pcDNA3.1(+)-Lgr5及pcDNA3.1(+)空載體質(zhì)粒進(jìn)入SW480并采用G418進(jìn)行篩選,分別采用western blotting與實(shí)時(shí)熒光定量PCR技術(shù)檢測(cè)Lgr5在SW480Lgr5與SW480vector中蛋白質(zhì)以及mRNA的表達(dá)水平。結(jié)果發(fā)現(xiàn),與SW480vector相比,Lgr5的蛋白質(zhì)表達(dá)水平在SW480Lgr5細(xì)胞中明顯升高；Lgr5的mRNA表達(dá)水平在SW480Lgr5細(xì)胞中同樣明顯升高(t=-17.499,p0.05)。 3.Lgr5對(duì)腫瘤細(xì)胞遷移與侵襲能力的影響 在遷移與侵襲實(shí)驗(yàn)中,與SW480vector細(xì)胞組相比,SW480Lgr5細(xì)胞組遷移與侵襲的細(xì)胞數(shù)明顯增多(遷移：t=4.291,p0.05；侵襲：t=3.127,p0.05)；與SW620NC細(xì)胞組相比,SW620Lgr5shRNA細(xì)胞組遷移與侵襲的細(xì)胞數(shù)明顯減少(遷移：t=6.557,p0.01；侵襲：t=2.297, p0.05)。Transwell小室遷移與侵襲實(shí)驗(yàn)表明,升高Lgr5表達(dá)后腫瘤細(xì)胞的遷移與侵襲能力明顯增強(qiáng),敲除Lgr5表達(dá)后腫瘤細(xì)胞的遷移與侵襲能力明顯減弱。 4.Lgr5對(duì)內(nèi)皮細(xì)胞小管形成能力的影響 用內(nèi)皮細(xì)胞在Matrigel中形成閉合環(huán)形小管的數(shù)目表示內(nèi)皮細(xì)胞小管形成的能力。與SW480vector組相比,SW480Lgr5組小管樣結(jié)構(gòu)明顯增多(t=8.617,p0.05)；與SW620NC組相比,SW620Lgr5shRNA組小管樣結(jié)構(gòu)數(shù)明顯減少(t=5.670,p0.05)。 5.Lgr5對(duì)結(jié)直腸癌細(xì)胞內(nèi)血管內(nèi)皮生長(zhǎng)因子的影響 通過Western blotting發(fā)現(xiàn),與SW480vector細(xì)胞相比,SW480Lgr5細(xì)胞中VEGF的蛋白含量明顯增多,與SW620NC細(xì)胞相比,SW620Lgr5shRNA細(xì)胞VEGF的蛋白含量明顯減少。通過Elisa發(fā)現(xiàn),與SW480vector組相比,SW480Lgr5組中分泌VEGF的量明顯增多(t=12.58,p0.01),與SW620NC組相比,SW620Lgr5shRNA組分泌VEGF的蛋白含量明顯減少(t=8.104,p0.01)。 6. AOM/DSS模型中,腫瘤組織Lgr5與VEGF的mRNA表達(dá)明顯增高 成功建立AOM/DSS模型,提取建模成功后的小鼠結(jié)腸組織RNA,行qRT-PCR顯示,與對(duì)照組相比,實(shí)驗(yàn)組中Lgr5以及VEGF的mRNA的表達(dá)量明顯升高(Lgr5:t=14.30, p0.01; VEGF:t=9.213, p0.01)。 7.敲低Lgr5的表達(dá)后,降低結(jié)直腸癌細(xì)胞在裸鼠體內(nèi)的成瘤以及肝轉(zhuǎn)移能力 在成瘤實(shí)驗(yàn)中,NC組的瘤體體積明顯大于Lgr5shRNA組,并且這種顯著差異在第3周開始體現(xiàn)出來。在裸鼠結(jié)腸癌肝轉(zhuǎn)移實(shí)驗(yàn)中,與NC組相比,Lgr5shRNA組肝臟內(nèi)癌結(jié)節(jié)數(shù)目明顯較少(t=-4.386,p0.05)。 結(jié)論 通過以上實(shí)驗(yàn)結(jié)果,我們可以得出以下結(jié)論： 1.在體外,Lgr5可以促進(jìn)結(jié)直腸癌細(xì)胞的遷移與侵襲能力； 2.在體內(nèi),Lgr5可以促進(jìn)結(jié)直腸癌的成瘤以及肝轉(zhuǎn)移； 3.Lgr5可以促進(jìn)內(nèi)皮細(xì)胞的血管生成,可以促進(jìn)結(jié)直腸癌中VEGF的表達(dá)； 我們通過功能性獲得與失去實(shí)驗(yàn)、體內(nèi)外實(shí)驗(yàn),發(fā)現(xiàn)在結(jié)直腸癌中,Lgr5可能是通過促進(jìn)VEGF的表達(dá),從而促進(jìn)血管生成,最終促進(jìn)腫瘤形成與轉(zhuǎn)移。因而我們的研究可以為以Lgr5為靶點(diǎn)的治療策略提供理論依據(jù)。
[Abstract]:Research background
Colorectal cancer has become one of the fastest growing malignant tumors in China. However, in colorectal cancer related deaths, most of them are caused by metastatic cancer. In recent years, more and more studies focus on tumor metastasis and explore the mechanism of tumor metastasis to reduce the occurrence of tumor metastasis and reduce the mortality of metastatic cancer. Guan Shengcheng is a very important link in the process of tumor growth and tumor metastasis. VEGF is an important factor promoting angiogenesis and also plays an important role in angiogenesis. At present, there are experiments to inhibit the formation of VEGF to inhibit tumor angiogenesis and inhibit tumor, but the inhibition of metastasis of colorectal cancer is unknown. Therefore, it is necessary to further study the mechanism of VEGF in the process of colorectal cancer formation and metastasis.
In recent years, Lgr5 has been found to be a specific marker of stem cell specific molecules in small intestine, colon, and hair follicle, and high expression of.Lgr5 is the target gene of Wnt signaling pathway in hepatocellular carcinoma, colon cancer, ovarian cancer, basal cell carcinoma and gastric cancer, and can regulate the activity of Wnt signaling pathway by feedback. More and more studies have shown that Lgr5 may also be a marker for cancer stem cells in colorectal cancer and can be used with other tumor stem cell markers for screening cancer stem cells in colorectal cancer. According to the "tumor stem cell theory", the data suggest that colorectal cancer may originate from the basement of the crypts of Lgr. 5 positive stem cells. This suggests that Lgr5 may play an important role in the development of colorectal cancer. However, there are few studies on the role and mechanism of Lgr5 in the process of tumor formation, development and metastasis. Our group has previously confirmed that the tumor's blood vessels could be suppressed after the low Lgr5 expression in colorectal cancer cells Generate.
research objective
In order to avoid the incidental results of Lgr5 expression, and to further explore the role of Lgr5 in colorectal cancer and its metastatic carcinoma, the effect of Lgr5 on VEGF and its mechanism will provide a theoretical basis for the treatment of colorectal cancer with Lgr5 as the target.
research method
1. construction of Lgr5 overexpression plasmid and establishment of stable cell line
The amplified primers were designed according to the sequence information of the complete human Lgr5 (NM_003667) gene sequence. The primer sequence was shown in Table 1. The primer amplification length was 2745bps. and was cloned into the pcDNA3.1 (+) eukaryotic expression vector. The sequence was correct. PcDNA3.1 (+) -Lgr5 plasmid and pcDNA3.1 (+) empty body particles were transfected into SW4 instantaneously by Lipofectamine2000. 80 cells were continuously screened by 1000ug/mlG418 for 48 hours, and then cloned and expanded for about 2-3 weeks to maintain 600ug/mlG418.
2. Western blotting
The total protein of the cell was extracted and the protein concentration was detected by the BCA protein quantitative kit. After the sample sample was 20 u g. electrophoresis, the protein was electrically transferred to the PVDF membrane, the 5% skimmed milk was closed, and a night resistance was incubated at 4. The first anti dilution ratio was (Lgr51:500, VEGF1:200, GAPDH1:1000, beta -actin).
1:1000), two days after incubation for 1 hours, ECL was developed by chemiluminescence.
3. real time fluorescence quantitative PCR (qRT-PCR)
The cells were extracted, the total RNA was organized and the concentration of RNA was measured. The first chain of cDNA was synthesized by the M-MLV reverse transcriptase. The real-time fluorescent quantitative PCR was carried out by the SYBR Premix Ex Taq kit of Takara. The primer sequence was shown in Table 1.
4. immuno histochemistry (SP method)
58 specimens of primary colorectal cancer in the southern hospital and 8 cases of metastatic carcinoma of colorectal cancer were collected, the tissues were fixed by 10% formalin, paraffin embedded, the specimens were cut into 4 micron m slices, 65 Centigrade oven roasted slices for 3 hours, and then dewaxing and hydrating, and using 0.01M sodium citrate buffer (pH6.0) to repair 15mmin with high temperature microwave, and the UltraSensitiveS-P hypersensitivity test of Maoxin company was used. The agent box, one anti 4 C refrigerator incubated for night (Lgr51:50, CD311:50), was coloured by DAB coloring agent, restained with hematoxylin, dehydrated transparent, sealed by wind, observed under microscope and filming. Two pathologist independently read the film and score.
5.HE staining
The tissue was fixed by 10% formalin, paraffin was embedded, the specimens were cut into 4 mu.M slices, and the roast slices of 65 Centigrade oven were about 30 minutes, and then dewaxing and hydrating in turn. The hematoxylin was stained for 2-5 minutes, 1% hydrochloric acid alcohol was differentiated, the water was washed to blue for 15 minutes, the eosin was stained for several seconds to several minutes, the dehydration was transparent, the seal was dried, under microscope observation and flapping.
6. Transwell compartment migration and invasion experiment
The concentration of 100u1 was added in the cell suspension with 1x106/ml in the upper chamber, then the medium of 500u1 containing 10%FBS was added to the lower chamber. After 24 hours culture, the cell was removed and 4% polyformaldehyde was fixed for 30 minutes, and the crystal violet was stained for 10 minutes. The residual cells in the upper chamber were wiped out with the cotton swab. The cells were randomly selected under the microscope microscope. The cells were counted and the cells were counted. The invasion experiment was carried out. In the upper chamber there is a layer of matrix adhesive, the rest of the methods and operations are in the same migration test.
7. tubule formation experiment
The medium was collected 48 hours after the plasmid transfection. The medium was filtered by the filter with the pore size of 0.22 Mn respectively. The medium was collected as the conditioned medium. The matrix gum was added to the 96 hole plate, each hole was added to 50ul, and the glue was cured for 1 hours at 37 C. The HMVEC cells with the concentration of 2 x 105/ml were added to each hole, and the corresponding conditioned medium was added. 0h, 2h, 4h, 8h were at 0h. The microtubule like structure of HMVEC cells was observed under the inverted microscope, and the number of closed tubular structures in the meso tube reticular structure was counted.
8. Elisa experiment
According to the operation instructions of human vascular endothelial growth factor (VEGF) Elisa Kit (Wuhan Huamei Biological Engineering Co., Ltd.), the concentration of VEGF in the recombinant cell culture medium was detected.
The establishment of 9. AOM/DSS model
5-6 weeks of balb/c mice were randomly divided into 2 groups, each group had 10 rats in each group. The experimental group was injected with AOM of the final concentration of 1.25mg/ml according to the dose of 12.5mg/kg (10ul/g), and the control group was injected with equal dose of saline in the abdominal cavity. The control group did not do special treatment. The experimental group was treated as the following: first weeks of normal drinking water; second weeks of drinking water containing 2.5%DSS, third, 4 weeks of continuous 2 weeks of normal drinking water, this is a cycle, this is a cycle of 5-10 weeks, repeat week 2,3,4 treatment 2 times, that is, a total of three cycles. Continue to feed to 17 weeks, kill the mice and take the colon tissue, observe the tumor body condition, the colon is fixed with 10% formalin, paraffin embedded, HE color. Extract the colon tissue RNA, and walk in real-time real-time quantitative PC fluorescence quantitative PC R detected the expression of Lgr5 and VEGF.
10. experimental tumor and liver metastases in nude mice
Tumor formation experiment in nude mice
5-6 weeks of Balb/c nude mice were randomly divided into 2 groups, 7 rats in each group, and the feeding grade was SPF grade. SW620Lgr5shRNA cells were injected respectively, SW620NC cells to the dorsal side of nude mice. The cell concentration was 1 x 107/ml, 200ul was injected, and the number of injected cells was 2 x 106. The tumor formation of nude mice was observed, the length of the nude mice was weighed every 3 days and the length of tumor (L) was measured and measured. A wide diameter (W) was used to calculate the volume of the tumor by formula L * w2/2. After.4 weeks, the nude mice were euthanized and the tumor was removed. The tumor was then embedded in 10% formalin fixed paraffin for subsequent HE staining.
Experimental liver metastases in nude mice with colorectal cancer
The early cell preparation was prepared with the nude mice, with 1% pentobarbital sodium 6-8ul/g injected into the nude mice by intraperitoneal injection of nude mice. The spleen was taken on the left back of the nude mice and the spleen was exposed. The spleen was injected along the upper margin of the spleen from the spleen to the tail of the spleen. The cells with 100ul concentration of 1 x 107/ml were injected into the splenic membrane slowly, and the abdominal cavity was cleaned after the injection was successful. The growth state of nude mice was observed and the nude mice were weighed every 3 days after.3 weeks. The nude mice were killed and the spleen, liver, lung and other tissues were removed, and 10% formalin fixed paraffin was embedded for subsequent HE staining.
Result
1. immunohistochemical results showed that the higher the expression of Lgr5 and the higher the microvessel density (MVD) were in colorectal cancer and metastatic cancer.
The expression of Lgr5 and vascular endothelial marker CD31 was detected by immunohistochemical staining in the specimens of primary colorectal cancer, lymph node metastasis and liver metastasis. It was found that the expression of Lgr5 was higher in lymph node metastasis and liver metastasis, and the expression of CD31 was also elevated, that is, the increase of microvascular density. The higher the expression of Lgr5 and the higher the microvessel density (MVD) in colorectal primary and metastatic cancers, however, the trend and the correlation between the two also need to be obtained by further increasing the sample size to obtain statistical data.
2. the successful construction of a stable cell line with high expression of Lgr5
The transfected Lgr5 recombinant overexpressed plasmid pcDNA3.1 (+) -Lgr5 and pcDNA3.1 (+) empty body particles entered SW480 and screened by G418. Western blotting and real-time fluorescent quantitative PCR technique were used to detect the protein and expression level of Lgr5 in SW480Lgr5 and SW480vector. The level of SW480Lgr5 increased significantly in Lgr5 cells, and the expression level of mRNA in SW480Lgr5 cells was also significantly increased (t=-17.499, P0.05).
Effects of 3.Lgr5 on migration and invasion of tumor cells
In the migration and invasion experiments, the number of migrating and invading cells in the SW480Lgr5 cell group increased significantly compared with the SW480vector cell group (migration: t=4.291, P0.05; invasion: t=3.127, P0.05); compared with the SW620NC cell group, the number of cells migrated and invaded by the SW620Lgr5shRNA cell group decreased significantly (migration: t=6.557, P0.01; invasion: t=2.297, P0.05).Transwell cell migration and invasion experiments showed that the migration and invasion ability of tumor cells increased obviously after the increase of Lgr5 expression, and the migration and invasion ability of tumor cells decreased significantly after knocking out Lgr5 expression.
Effect of 4.Lgr5 on endothelial cell tubule formation
The number of closed loop tubules formed by endothelial cells in Matrigel showed the ability to form endothelial cells. Compared with the SW480vector group, the tubule like structure in the SW480Lgr5 group increased significantly (t=8.617, P0.05). Compared with the SW620NC group, the number of tubules in the SW620Lgr5shRNA group decreased significantly (t=5.670, P0.05).
Effect of 5.Lgr5 on vascular endothelial growth factor in colorectal cancer cells
Compared with SW480vector cells, the protein content of VEGF increased significantly in SW480Lgr5 cells compared with SW480vector cells. Compared with SW620NC cells, the protein content of VEGF in SW620Lgr5shRNA cells decreased significantly. Compared with the SW480vector group, the amount of VEGF in SW480Lgr5 group increased significantly compared with the SW480vector group. Compared with group SW620Lgr5shRNA, the protein content of VEGF secreted significantly decreased (t=8.104, P0.01).
In the 6. AOM/DSS model, the expression of Lgr5 and VEGF mRNA in tumor tissues increased significantly.
The AOM/DSS model was successfully established to extract the RNA of mouse colon tissue after successful modeling, and qRT-PCR was shown. Compared with the control group, the expression of Lgr5 and VEGF in the experimental group increased significantly (Lgr5:t=14.30, P0.01; VEGF:t=9.213, P0.01).
7. knocking down the expression of Lgr5 decreases the tumorigenesis and liver metastasis of colorectal cancer cells in nude mice.
In the tumor formation experiment, the volume of the tumor body in the NC group was significantly larger than that in the Lgr5shRNA group, and the significant difference was found at third weeks. In the liver metastasis test of nude mice, the number of tumor nodules in the Lgr5shRNA group was significantly less than that in the NC group (t=-4.386, P0.05).
conclusion
From the above results, we can draw the following conclusions:
1. in vitro, Lgr5 can promote the migration and invasion of colorectal cancer cells.
2. in vivo, Lgr5 can promote tumor formation and liver metastasis of colorectal cancer.
3.Lgr5 can promote the angiogenesis of endothelial cells and promote the expression of VEGF in colorectal cancer.
Through functional acquisition and loss of experiments and in vitro and in vivo experiments, we found that in colorectal cancer, Lgr5 may promote angiogenesis by promoting the expression of VEGF, and ultimately promote tumor formation and metastasis. Therefore, our research can provide a theoretical basis for the therapeutic strategy targeting Lgr5.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R735.34

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