本文選題：LRG1 + 結直腸癌��； 參考：《中國人民解放軍醫(yī)學院》2017年博士論文

【摘要】：結直腸癌(Colorectal Cancer, CRC )是世界范圍內常見的實體腫瘤之一,嚴重威脅人類的健康。我國2000年至2011年結直腸癌的發(fā)病率和死亡率均逐年上升,據(jù)國家癌癥登記中心公布的最新數(shù)據(jù)表明,2015年我國結直腸癌共發(fā)病37.63萬人,死亡19.10萬人。目前手術切除仍然是根治結直腸癌的主要手段,但大部分晚期結直腸癌患者或術后復發(fā)轉移的結直腸癌患者,失去手術治療的機會,積極地內科治療是抑制腫瘤生長和延長生命的首選方法。結直腸癌的抗血管生成治療在內科治療當中具有舉足輕重的地位。因此進一步探索結直腸癌血管生成的機制,尋找抗血管生成的有效靶點具有重要的理論意義和潛在的臨床價值。富亮氨酸 α-2 糖蛋白 1 ( Leucine-rich-alpha-2-glycoprotein 1,LRG1 )是 1977 年發(fā)現(xiàn)的,在血液中和組織中表達的蛋白。近年來,研究發(fā)現(xiàn)LRG1在呼吸系統(tǒng)、消化系統(tǒng)和婦科腫瘤中的表達升高,且與腫瘤的惡性程度相關;也有研究發(fā)現(xiàn)LRG1促進腫瘤和其他疾病中的新生血管形成。但LRG1在結直腸癌發(fā)生發(fā)展和血管生成中的研究較少�；谝陨涎芯勘尘�,本課題通過臨床大樣本驗證,探索LRG1在結直腸癌發(fā)生發(fā)展和血管生成中的作用,并通過體外細胞實驗和體內成瘤實驗進一步驗證LRG1的促血管生成能力。最后通過信號通路研究,嘗試揭示LRG1促進血管生成功能的相關作用機制。實驗方法1.臨床樣本驗證:收集中國人民解放軍總醫(yī)院2005年至2009年接受根治性手術治療的III期結直腸癌的癌和癌旁標本,建立組織芯片和臨床信息數(shù)據(jù)庫。應用免疫組織化學染色技術檢測LRG1、CD34-微血管密度(Micro-vessel Density, MVD ),分析LRG1與臨床病理特征和患者預后的相關性,分析LRG1是否是影響患者生存的獨立預后因素。分析LRGI與MVD-CD34的關系,判斷LRG1對血管生成的影響。2. LRG1影響結直腸癌血管生成的體外研究:利用慢病毒構建基因過表達基因敲降穩(wěn)轉細胞株。通過Transwell侵襲實驗和劃痕試驗檢測LRG1對各穩(wěn)轉癌細胞株的侵襲和遷移能力的影響;并通過MTT實驗、Transwell遷移實驗和小管形成實驗判斷LRG1對人臍靜脈血管內皮細胞(HUVEC )的增殖、遷移和成管能力的影響。3. LRG1影響結直腸癌血管生成的體內研究:利用過表達和對照細胞株建立裸鼠皮下成瘤模型。測量瘤體體積變化,第30天取出瘤體稱重,并制作組織切片進行HE染色、CD34和VEGFA檢測,判斷LRG1對體內血管生成的影響。4. LRG1促進結直腸癌血管生成機制的初步研究:檢測LRG1過表達和敲降細胞株VEGFA的表達和分泌VEGFA的水平;檢測PI3K/AKT和MEK/ERK通路相關分子蛋白水平;檢測HIF-1α細胞質和細胞核內的分布情況,判斷LRG1是否介導HIF-1a移位入核發(fā)揮轉錄因子作用;檢測在信號通路抑制劑作用下,細胞中VEGFA表達水平是否改變,以及HUVECs成管能力是否下降,進一步確認LRG1通過AKT、ERK雙通路激活VEGFA誘導血管生成。實驗結果:1.共312例Ⅲ期結直腸癌患者納入本課題。免疫組化染色結果顯示60.9%病例癌組織內LRG1高表達,只有11.9%患者的癌旁正常組織內LRG1高表達,LRG1在癌組織內表達水平高于癌旁正常組織(X2=35.412, P 0.001 )。單因素分析顯示LRG1與腫瘤分化較差、T4分期和血管浸潤明顯相關(P=0.035,0.028,0.007)。多因素分析顯示LRG1是影響312例Ⅲ期結直腸癌患者總生存OS和無疾病生存的獨立預后因素(HR=1.517, P=0.020;HR=1.754,P=0.013)。并且 LRG1 水平與腫瘤 MVD 正相關(t=-8.603,P0.001)。2.選擇LRG1表達最低的DLD1和表達量最高的HT29細胞系,分別建立LRG1過表達和敲降細胞株。Real-Time PCR證實過表達組LRG1的轉錄水平升高6.7倍,敲降組與對照組相比LRG1轉錄水平下降了 35%～96%。體外實驗證明LRG1促進癌細胞的侵襲和遷移。進一步利用共培養(yǎng)HUVECs細胞進行體外功能實驗,LRG1基因過表達表達促進血管內皮細胞的細胞增殖,遷移和成管功能(P0.01)。3.裸鼠成瘤模型的腫瘤生長曲線顯示LRG1促進裸鼠體內移植瘤生長(P0.01 )。過表達組瘤體平均重量大于對照組(P0.05)。瘤體制成組織切片并進行HE染色,結果發(fā)現(xiàn)過表達組的移植瘤浸潤肌層,LRG1促進腫瘤的生長和浸潤。CD34-MVD和VEGFA熒光染色證明過表達組促進為血管形成和VEGFA表達。4.DLD1細胞中過表達組與對照組相比VEGFA表達和分泌水平均升高;HT29細胞中敲降組和對照組相比VEGFA表達和分泌水平下降。Western Blot結果顯示LRG1促進 PI3K/AKT/HIF-1α 和 Ras/MEK1/ERK1/2 通路的激活。并且 LRG1 誘導 HIF-1α從細胞質移位至細胞核。利用通路抑制劑分別抑制AKT和ERK通路后,DLD1-LRG1組的VEGFA表達量均有所下降,且LRG1誘導內皮細胞的小管形成能力被抑制。結論LRG1在結直腸癌腫瘤組織內高表達,且其表達水平明顯高于對應癌旁正常組織。并與結直腸癌的T分期、分化程度和血管浸潤明顯相關。LRG1是影響結直腸癌OS和DFS的獨立預后因素。LRG1與MVD成明顯的正相關。LRG1不僅能夠促進腫瘤細胞的侵襲和遷移能力,且能夠促進血管內皮細胞的增殖、遷移和成管能力。體內成瘤實驗證明LRG1促使移植瘤向肌層浸潤,也使CD34-MVD和VEGFA表達增加。腫瘤細胞中LRG1可通過PI3K/AKT/HIF1α和MEK/ERK雙通路上調VEGFA分泌介導的血管生成。
[Abstract]:Colorectal Cancer (CRC) is one of the most common solid tumors in the world. It is a serious threat to human health. The incidence and mortality of colorectal cancer in China from 2000 to 2011 are increasing year by year. According to the latest data published by the National Cancer Registration Center, there are 376 thousand and 300 cases of colorectal cancer in China in 2015 and 19.10 deaths in 2015. Surgical excision is still the main cure for colorectal cancer, but most advanced colorectal cancer patients or recurrent colorectal cancer patients have lost the chance of surgical treatment. Active internal medicine is the first choice to suppress tumor growth and prolong life. Antiangiogenic treatment for colorectal cancer is treated in internal medicine. It is of great importance. Therefore, it is of great theoretical significance and potential clinical value to further explore the mechanism of angiogenesis in colorectal cancer and to find effective targets for anti angiogenesis. The leucine alpha -2 glycoprotein 1 (Leucine-rich-alpha-2-glycoprotein 1, LRG1) is discovered in 1977 and is in the blood and tissue. The protein expressed. In recent years, studies have found that LRG1 has increased in the respiratory system, digestive system and gynecologic tumors, and is associated with the malignancy of the tumor. There are also studies found that LRG1 promotes neovascularization in tumors and other diseases. But LRG1 is less studied in the development and angiogenesis of colorectal cancer. Background, this topic is to explore the role of LRG1 in the development and angiogenesis of colorectal cancer by large clinical samples, and to further verify the angiogenesis ability of LRG1 through in vitro cell experiment and in vivo tumorigenesis experiment. Finally, through the study of signal pathway, we try to reveal the relevant mechanism of LRG1 promoting angiogenesis. Test method 1. clinical sample validation: collect cancer and paracancerous specimens of III stage colorectal cancer in General Hospital of PLA from 2005 to 2009, establish tissue chip and clinical information database. Use immunohistochemical staining technique to detect LRG1, CD34- microvascular density (Micro-vessel Density, MVD), and analyze L The correlation between the RG1 and the clinicopathological features and the prognosis of patients, and to analyze whether LRG1 is an independent prognostic factor that affects the survival of the patients. Analysis of the relationship between LRGI and MVD-CD34, and to determine the effect of LRG1 on angiogenesis, the effect of.2. LRG1 on the angiogenesis of colorectal cancer in vitro: the use of lentivirus construction gene overexpressed genes to knock down cell lines. The effect of LRG1 on the invasion and migration of each stable cancer cell line was detected by Transwell invasion test and scratch test, and the effects of LRG1 on the proliferation, migration and angiogenic ability of human umbilical vein endothelial cells (HUVEC) were determined by MTT experiment, Transwell migration and tubule formation, and the effect of.3. LRG1 on the angiogenesis of colorectal cancer was influenced by.3. LRG1. In vivo study: using overexpressed and controlled cell lines to establish a subcutaneous tumor model in nude mice. Measure the volume changes of the tumor, take thirtieth days to take out the weight of the tumor, and make tissue sections for HE staining, CD34 and VEGFA, to determine the effect of LRG1 on the angiogenesis of.4. LRG1 to promote the angiogenesis of colorectal cancer: detection of LRG1 Overexpression and knockdown cell line VEGFA expression and secretion of VEGFA levels; detection of PI3K/AKT and MEK/ERK pathway related molecular protein levels; detection of the distribution of HIF-1 alpha cytoplasm and nucleus; determine whether LRG1 mediates the role of the HIF-1a shift into the nucleus to play a transcription factor; detection of VEGFA expression in cells under the action of signaling pathway inhibitors Whether or not the level changes, and whether the HUVECs tube ability decreased, further confirmed that LRG1 through AKT, ERK double pathway activation of VEGFA induced angiogenesis. Experimental results: 1. a total of 312 cases of stage III colorectal cancer were included in this subject. The immunohistochemical staining results showed that the high expression of LRG1 in the carcinoma tissues of 60.9% cases, only 11.9% patients in the normal para cancerous tissue The expression of LRG1 was higher than that of normal tissue adjacent to the cancer tissue (X2=35.412, P 0.001). Single factor analysis showed that LRG1 was poorly differentiated from tumor, and T4 staging was associated with vascular infiltration (P=0.035,0.028,0.007). The multivariate analysis showed that LRG1 was independent of the survival of OS and disease free survival in 312 patients with stage III colorectal cancer. Prognostic factors (HR=1.517, P=0.020; HR=1.754, P=0.013). And LRG1 level with MVD positive correlation (t=-8.603, P0.001).2. selection LRG1 expressed the lowest DLD1 and the highest expression of HT29 cell lines. In vitro, LRG1 transcriptional level decreased by 35% ~ 96%. in vitro experiments showed that LRG1 promoted invasion and migration of cancer cells. Further functional experiments were carried out by co cultured HUVECs cells. The overexpression of LRG1 gene promoted the proliferation of vascular endothelial cells, migration and tubular function (P0.01) tumor growth curve of.3. nude mice. The results showed that LRG1 promoted the growth of transplanted tumor in nude mice (P0.01). The average weight of the overexpressed group was greater than that of the control group (P0.05). The tumor body was made into tissue section and stained with HE. The results showed that the transplanted tumor infiltrated the muscle layer in the overexpressed group. LRG1 promoted the growth of the tumor and the infiltration of.CD34-MVD and VEGFA fluorescence staining demonstrated that the overexpression group promoted the formation of blood vessels. The expression and secretion of VEGFA in the.4.DLD1 cells expressed in the.4.DLD1 cells and the control group were all higher than those in the control group; the VEGFA expression and secretion level decreased by.Western Blot results in the control group compared with the control group. The results showed that LRG1 promoted PI3K/AKT/HIF-1 alpha and Ras/MEK1/ERK1/2 pathway activation. And LRG1 induced HIF-1 alpha from cytoplasm to cytoplasm. After the inhibition of AKT and ERK pathway, the expression of VEGFA in DLD1-LRG1 group decreased, and the formation ability of LRG1 induced endothelial cells was inhibited. Conclusion LRG1 was highly expressed in colorectal cancer tissues, and the expression level was significantly higher than that of normal tissues adjacent to the carcinoma. And the T score of colorectal cancer was compared with that of colorectal cancer. .LRG1 is an independent prognostic factor affecting OS and DFS in colorectal cancer. The positive correlation between.LRG1 and MVD is a significant positive correlation between.LRG1 and MVD, which not only promotes the invasion and migration of tumor cells, but also promotes the proliferation, migration and angiogenic ability of vascular endothelial cells. In vivo tumorigenesis experiments show that LRG1 promotes transplanted tumor. Infiltration to the myometrium also increases the expression of CD34-MVD and VEGFA. In tumor cells, LRG1 can increase the angiogenesis mediated by VEGFA secretion through the PI3K/AKT/HIF1 - and MEK/ERK double pathways.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R735.34

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