本文選題：食管鱗狀細胞癌 + VEGF-A�。� 參考：《第三軍醫(yī)大學》2016年博士論文

【摘要】：研究背景食管癌(Esophageal cancer,EC)是一種常見的消化系統(tǒng)惡性腫瘤。在我國食管癌發(fā)病率和死亡率均居世界首位,全球范圍內(nèi)因該病造成的死亡患者有一半發(fā)生在我國。其死亡率占據(jù)第二位,僅次于胃癌。食管鱗癌(esophageal squamous cell carcinoma,ESCC)占EC的90%以上,因此本研究主要針對ESCC進行探討。雖然通過早期手術切除及后續(xù)的放療、化療等策略,可以使ESCC患者的生存率有所提高,然而這些治療方式難以達到根治效果,且同時該類患者還伴有不同程度的區(qū)域淋巴結轉移或遠處轉移。腫瘤的發(fā)生、發(fā)展、侵犯、轉移是一個多基因、多分子及多系統(tǒng)共同參與的復雜的病理過程。其中腫瘤新血管的生成(angiogenesis)是重要因素之一。新生血管與腫瘤的生長、侵襲和淋巴結轉移等過程緊密相關。趨化因子受體(chemokine receptor)是G蛋白偶聯(lián)受體超家族的成員,是一類表達在某些特定的細胞膜表面的跨膜受體。近年來的研究表明,趨化因子通過與受體結合,參與新生血管生成,進一步影響腫瘤生成和轉移。不同結構的趨化因子在血管生成中起不同作用,可以作為腫瘤治療的新靶點。最近的研究已證實,趨化因子受體7(chemokine receptor,CCR7)在多種腫瘤細胞中存在過度表達,這提示腫瘤的發(fā)生發(fā)展、浸潤和轉移與CCR7有著密不可分的關系。血管內(nèi)皮生長因子(vascular endothelial growth factor,VEGF),最早亦稱作血管通透因子,其與內(nèi)皮細胞的增殖、遷移和管腔形成密切相關。VEGF家族包括VEGF-A、VEGF-B、VEGF-C及VEGF-D等幾種同類物。酪氨酸激酶在VEGF與其受體VEGFR結合后被激活,繼發(fā)一系列信號通路級聯(lián)反應,隨后激發(fā)腫瘤血管內(nèi)皮發(fā)生增殖、遷移和新生血管管腔形成。核轉錄因子-κB(nuclear transcription factor Kappa B,NF-ΚB),最早是從成熟的B淋巴細胞中分離提取出來的蛋白,為真核細胞轉錄調(diào)節(jié)因子,存在于幾乎所有的動物細胞中。NF-κB具有廣泛的生物學活性,是炎性反應與腫瘤相互聯(lián)系的重要分子蛋白,在炎癥、代謝、人體免疫以及腫瘤的發(fā)生、發(fā)展、侵犯、轉移等方面發(fā)揮著關鍵作用。大量研究表明,NF-κB與腫瘤有密切關系,可以通過調(diào)節(jié)各種不同靶基因,參與多種信號通路,誘導細胞抑制凋亡并促進細胞的增殖、侵襲、轉移,促進腫瘤細胞血管生成、與致癌作用密切相關。目前國內(nèi)外有較多的臨床資料顯示,CCR7的高表達與ESCC的發(fā)生、浸潤及轉移密切相關。但是,對于CCR7在ESCC細胞株中的表達,和ESCC血管形成中的作用,及其與NF-κB、VEGF等分子的相互作用關系目前尚不清楚。目標在本研究中,我們首先檢測CCR7在ESCC細胞株中的表達,進而通過體內(nèi)外實驗,觀察CCR7的異常表達與ESCC新生血管形成的關系,以及CCR7與VEGF、NF-κB及其下游基因的相互作用關系,再進一步探討其中的分子機制。力圖為闡釋食管癌轉移、復發(fā)的機制,以及基于CCR7的靶向治療藥物開發(fā)奠定基礎。第一部分CCR7在ESCC中表達情況方法1、應用生物信息學技術方法回顧性整理并研究了不同報道中ESCC患者腫瘤組織中CCR7表達情況;2、應用逆轉錄-聚合酶鏈反應(RT-PCR)及免疫印跡(WB)方法聯(lián)合檢測CCR7在多種不同種系的ESCC細胞中的表達情況。結果1、針對CCR7基因的表達情況在GEO PROFILE數(shù)據(jù)庫(https://www.ncbi.nlm.nih.gov/gds/)中進行搜索分析,結果發(fā)現(xiàn)ESCC中的CCR7的表達較正常組織中CCR7的水平顯著提高。2、Western Blot結果顯示,所有6株ESCC細胞系(TE-1,TE-2,TE-3,TE-8,TE-12和ECA-109)CCR7蛋白表達均顯著高于正常食管上皮細胞株HEEC。q RT-PCR結果顯示在m RNA水平上,ESCC細胞系中的CCR7表達顯著高于HECC細胞。結果提示,CCR7在食管鱗狀細胞癌基因及蛋白水平均具有高表達的特性。第二部分CCR7在Eca109細胞增殖、遷移和誘導新生血管形成中的作用方法1、建立CCR7穩(wěn)定過表達和沉默的Eca109細胞系。2、應用MMT法檢測過表達和沉默CCR7對Eca109細胞的活性影響。3、采用人臍靜脈內(nèi)皮細胞增殖、遷移實驗和CAM實驗檢測CCR7表達改變對Eca109細胞增殖、遷移及誘導血管生成能力的影響。4、采用Western Blot實驗檢測VEGF-A和VEGF-C在CCR7過表達和沉默的Eca109細胞系中的表達情況。結果1、成功建立CCR7穩(wěn)定過表達和沉默的Eca109細胞系。2、CCR7過表達Eca109細胞增殖、遷移和誘導血管生成能力顯著增強;而CCR7沉默Eca109細胞上述能力顯著減弱。3、CCR7過表達Eca109細胞VEGF-A和VEGF-C蛋白表達顯著增高;而CCR7沉默Eca109細胞上述蛋白表達顯著降低。結果提示,過表達CCR7增強Eca109細胞的細胞存活率,增加內(nèi)皮細胞的遷移,增強血管形成能力,并上調(diào)VEGF-A和VEGF-C蛋白的表達,提高食管鱗癌細胞在體外誘導新血管形成能力。相反,沉默抑制CCR7表達則得到相反的結果。這些結果證實,CCR7能促進食管鱗狀細胞癌的血管生成能力。第三部分CCR7對NF-k B信號通路的調(diào)節(jié)作用方法1、熒光素酶報告分析驗證過表達CCR7和沉默CCR7對NF-k B通路的影響。2、通過Western blot、RT-PCR和ELISA等實驗檢測過表達和沉默CCR7對NF-k B信號通路的影響。結果1、CCR7蛋白的過表達能使NF-k B報告基因的轉錄活性增強。2、通過Wetern blotting實驗結果證實ECA109中過表達CCR7能夠顯著提高磷酸化的IKK和磷酸化的Ik Bα的表達水平,但不能顯著改變IKK和Ik Bα的總蛋白表達水平。3、Western botting實驗和ELISA實驗的結果顯示多種NF-k B靶基因(包括TNF-α、IL-6、IL-8和TGF-β)在過表達CCR7的Eca109細胞中表達上調(diào);而在CCR7沉默的Eca109細胞中表達下調(diào)。結果顯示,過表達CCR7增強了NF-k B的報告基因的轉錄活性,而抑制CCR7表達則抑制了NF-k B的轉錄活性。CCR7的表達增加,增強了磷酸化IKK和磷酸化Ik Bα的水平,卻沒有顯著改變IKK或Ik Bα總蛋白水平。此外,包括TNF-α,IL-6,IL-8,TGF-β在內(nèi)的NF-k B相關的靶基因水平數(shù),在上調(diào)CCR7的表達和下調(diào)細胞CCR7沉默食管癌細胞分別被上調(diào)和抑制了。表明CCR7對NF-k B信號通路起促進作用。第四部分CCR7下調(diào)對Eca109裸鼠移植瘤生長的影響及機制方法1、建立裸小鼠皮下ESCC移植瘤模型。分組注射1×107個control-si RNA和CCR7-si RNA ECA109細胞后觀察腫瘤的生長。2、Western botting實驗分析了腫瘤中VEGF-A、VEGF-C、TNF-α、IL-6、IL-8、TGF-β和NF-k B通路中磷酸化IKK和磷酸化Ik Bα及IKK或Ik Bα總蛋白的表達水平。結果1、下調(diào)CCR7蛋白的表達,ESCC移植瘤體積顯著減小2、抑制CCR7表達后,VEGF-A、VEGF-C、TNF-α、IL-6、IL-8、TGF-β和NF-κB的蛋白表達水平顯著降低,IKK和Ik Bα的磷酸化水平降低,而IKK和Ik Bα總蛋白水平無顯著改變。結果提示,si RNA沉默CCR7在Eca109裸鼠移植瘤模型的抑制腫瘤療效非常顯著,其可能是通過抑制NF-k B通路來抑制ESCC細胞生長。全文結論1、CCR7在ESCC細胞中表達增高。2、通過構建穩(wěn)定CCR7過表達和沉默的Eca109細胞系,明確了CCR7對Eca109腫瘤新生血管的促進作用。表明CCR7可能對ESCC的發(fā)生發(fā)展和血管生成發(fā)揮重要作用,可能作為ESCC新的治療靶點。3、CCR7蛋白的過表達能使NF-k B報告基因的轉錄活性增強;CCR7能夠顯著提高IKK和Ik Bα的磷酸化水平;且多種NF-k B靶基因(包括TNF-α、IL-6、IL-8和TGF-β)在過表達CCR7的Eca109細胞中表達上調(diào),而在CCR7沉默的Eca109細胞中表達下調(diào)。表明CCR7對NF-k B信號通路起促進作用。4、通過體內(nèi)實驗證實注射CCR7沉默組Eca109細胞裸鼠的腫瘤平均體積比對照組裸鼠明顯減少。且移植瘤中VEGF-A、VEGF-C、TNF-α、IL-6、IL-8和TGF-β的蛋白表達水平顯著降低,IKK和Ik Bα的磷酸化水平降低,而IKK和Ik Bα總蛋白水平無顯著改變。進一步證實CCR7可能通過NF-k B信號通路對ESCC發(fā)揮促進作用。
[Abstract]:Background Esophageal cancer (EC) is a common malignant tumor of the digestive system. The incidence and mortality of esophageal cancer in China are the first in the world, and half of the deaths in the world are in China. The mortality rate is second, only inferior to gastric cancer. Esophageal squamous cell car Cinoma, ESCC) accounts for more than 90% of EC, so this study focuses on ESCC. Although early surgical resection and follow-up radiotherapy, chemotherapy and other strategies can improve the survival rate of patients with ESCC, however, these treatments are difficult to achieve radical effect, and at the same time the patients are accompanied by different degrees of regional lymph node metastasis. Tumor occurrence, development, invasion and metastasis are complex pathological processes involving multiple genes, multiple molecules and multiple systems. The formation of neovascularization (angiogenesis) is one of the important factors. Neovascularization is closely related to tumor growth, invasion and lymph node metastasis. Chemokine receptor (chemokin) E receptor) is a member of the G protein coupling receptor superfamily, a class of transmembrane receptors expressed on certain specific cell membrane surfaces. Recent studies have shown that chemokines are involved in the formation of neovascularization through binding with the receptors and further affect the formation and metastasis of tumors. Chemokines of different structures play a different role in angiogenesis. It can be used as a new target for cancer treatment. Recent studies have shown that chemokine receptor (CCR7) is overexpressed in a variety of tumor cells, which suggests the development of tumor, infiltration and metastasis are inseparable from CCR7. Vascular endothelial growth factor (vascular endothelial growth factor, VEGF), the most Early also known as vascular permeability factor, which is closely related to the proliferation, migration and lumen formation of endothelial cells,.VEGF families include VEGF-A, VEGF-B, VEGF-C and VEGF-D. Tyrosine kinase is activated by VEGF and its receptor VEGFR, secondary to a series of signal pathway cascade reactions, followed by stimulating the proliferation of tumor vascular endothelial cells. Migration and neovascular lumen formation. Nuclear transcription factor - kappa B (nuclear transcription factor Kappa B, NF- B), the earliest proteins isolated from mature B lymphocytes, are eukaryotic cell transcriptional regulators, which exist in almost all animal cells with extensive biological activity in.NF- kappa B, and are inflammatory reactions and tumors. Important molecular proteins linked to each other play a key role in inflammation, metabolism, human immunity and the occurrence, development, invasion and metastasis of cancer. A large number of studies have shown that NF- kappa B is closely related to tumors. It can regulate various target genes and participate in multiple signaling pathways, inducing cells to inhibit apoptosis and promote cell proliferation. Invasion, metastasis, metastasis and promoting angiogenesis of tumor cells is closely related to carcinogenesis. At present, there are many clinical data at home and abroad that the high expression of CCR7 is closely related to the occurrence of ESCC, infiltration and metastasis. However, the expression of CCR7 in the ESCC cell line, the role of ESCC in the formation of blood tube, and the interaction with NF- kappa B, VEGF and other molecules In this study, we first detected the expression of CCR7 in the ESCC cell line, and then we observed the relationship between the abnormal expression of CCR7 and the angiogenesis of ESCC, as well as the interaction between CCR7 and VEGF, NF- kappa B and its downstream genes, and further explored the molecular mechanism of the molecular mechanism. To illustrate the mechanism of esophageal cancer metastasis, recurrence, and the development of target therapy based on CCR7. The first part is the expression of CCR7 in ESCC 1. The expression of CCR7 in the tumor tissues of ESCC patients in different reports is reviewed and studied by bioinformatics technique; and 2, the use of reverse transcription polymerase chain reaction (RT PCR) (RT-PCR) and immunoblotting (WB) method combined to detect the expression of CCR7 in ESCC cells of various different lines. Results 1, the expression of CCR7 gene was searched and analyzed in GEO PROFILE database (https://www.ncbi.nlm.nih.gov/gds/). The results showed that the expression of CCR7 in ESCC was significantly higher than that of CCR7 in the normal tissue. The results of.2, Western Blot showed that all 6 ESCC cell lines (TE-1, TE-2, TE-3, TE-8, TE-12 and ECA-109) were significantly higher than the normal esophageal epithelial cell lines, and the expression in the cell line was significantly higher than that of the cells. Second CCR7 in the proliferation, migration and induction of Eca109 cells in the proliferation, migration and induction of neovascularization of Eca109 cells, the establishment of a CCR7 stable overexpressed and silent Eca109 cell line.2, the use of MMT to detect and silence the activity of CCR7 to Eca109 cells, and to use human umbilical vein endothelial cells to proliferate and migrate. The effects of CCR7 expression on the proliferation, migration and angiogenesis of Eca109 cells were detected by the test and CAM test..4 was used to detect the expression of VEGF-A and VEGF-C in the CCR7 overexpressed and silent Eca109 cell lines by Western Blot. Results 1, the CCR7 stable overexpression and silent Eca109 cell lines were successfully established. Cell proliferation, migration and induction of angiogenesis significantly increased, while the above ability of CCR7 silenced Eca109 cells significantly weakened.3, CCR7 overexpressed Eca109 cells, VEGF-A and VEGF-C protein expression increased significantly, while CCR7 silenced Eca109 cells expressed a significant decrease in the expression of the above protein. The results suggested that overexpression of CCR7 enhanced the cell survival rate of Eca109 cells, and increased the cell survival rate of Eca109 cells. The migration of endothelial cells enhanced angiogenesis, increased the expression of VEGF-A and VEGF-C proteins and enhanced the ability to induce neovascularization in esophageal squamous cell carcinoma cells in vitro. On the contrary, silence inhibition of CCR7 expression was the opposite. These results confirmed that CCR7 could promote the angiogenesis of squamous cell carcinoma of the esophagus. Third part CCR7 pairs The regulation of NF-k B signaling pathway 1, luciferase report analysis verified the effect of overexpression of CCR7 and silent CCR7 on the NF-k B pathway. The effect of expression and silence CCR7 on the signaling pathway was detected by Western blot, RT-PCR and ELISA. Results 1, the overexpression of the protein could increase the transcriptional activity of the reporter gene. The strong.2, through the Wetern blotting experiment, confirmed that the overexpression of CCR7 in ECA109 could significantly increase the expression level of IKK and phosphorylated Ik B alpha, but could not significantly change the total protein expression level of IKK and Ik B alpha. The expression of beta expression was up regulated in the Eca109 cells overexpressing CCR7, and down regulated in the CCR7 silent Eca109 cells. The results showed that overexpression of CCR7 enhanced the transcriptional activity of the NF-k B reporter gene, while inhibition of CCR7 expression inhibited the increase of the expression of the NF-k B transcriptional activity.CCR7, and increased the level of phosphorylated IKK and phosphorylated Eca109. There was a significant change in the total protein level of IKK or Ik B alpha. In addition, the number of NF-k B related target genes, including TNF- alpha, IL-6, IL-8 and TGF- beta, was up-regulated and inhibited in the up regulation of CCR7 and down regulated cell CCR7 silent esophageal cancer cells. The effect of the growth of the tumor and the mechanism method 1, the subcutaneous ESCC tumor model of nude mice was established. The growth.2 of the tumor was observed after group injection of 1 x 107 control-si RNA and CCR7-si RNA ECA109 cells. Western botting experiment was used to analyze VEGF-A, VEGF-C, TNF- alpha and phosphorylation of phosphorylation and phosphorylation in the tumor. Or the expression level of Ik B alpha total protein. Results 1, down regulation of the expression of CCR7 protein, the volume of ESCC transplanted tumor significantly decreased by 2. After the inhibition of CCR7 expression, the protein expression level of VEGF-A, VEGF-C, TNF- a, IL-6, IL-8, TGF- beta and NF- kappa decreased significantly, but there was no significant change in the level of the total protein level. The inhibitory effect of RNA silencing CCR7 on the tumor model of Eca109 nude mice is very significant. It may be to inhibit the growth of ESCC cells by inhibiting the NF-k B pathway. Conclusion 1, CCR7 is expressed in.2 in ESCC cells. By constructing a Eca109 cell line that stable CCR7 over expression and silence, the promotion of CCR7 to neovascularization of tumor cells is clear. It shows that CCR7 may play an important role in the development and angiogenesis of ESCC, may be a new therapeutic target for ESCC,.3, and the overexpression of CCR7 protein can enhance the transcriptional activity of the NF-k B reporter gene, and CCR7 can significantly increase the phosphorylation level of IKK and Ik B alpha. The expression of overexpressed CCR7 Eca109 cells was up-regulated and down regulated in CCR7 silent Eca109 cells. It showed that CCR7 promoted.4 in NF-k B signaling pathway. Through in vivo experiments, the average volume of tumor in nude mice with Eca109 cells injected with CCR7 silencing group was significantly lower than that of the control group. The protein expression level of TGF- beta and IKK decreased significantly, and the phosphorylation level of IKK and Ik B alpha was decreased, while the total protein level of IKK and Ik B alpha was not significantly changed. It was further confirmed that CCR7 may play a role in ESCC through the NF-k B signaling pathway.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.1

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