【摘要】：目的:我國是食管癌(Esophageal Carcinoma,EC)的高發(fā)地區(qū),每年因食管癌死亡者大約15萬人,約占全部惡性腫瘤死亡數(shù)的近四分之一。我國食管癌人口死亡率最低的為云南省(105/10萬),與死亡率最高的河南省相差31倍,可見食管癌的發(fā)病率存在明顯的地域差異。近年來,環(huán)境致癌因素(如真菌毒素、病毒等)在各類腫瘤的發(fā)生發(fā)展中已成為研究熱點之一。人類乳頭瘤病毒(Human papillomavirus HPV)與食管癌發(fā)生的關(guān)系最早是1982年由Syrjanen提出,他的研究發(fā)現(xiàn)40%(24/60)的食管癌發(fā)生的組織學(xué)形態(tài)改變與生殖道濕疣改變極其相似,隨后他與同事用免疫組織化學(xué)法在食管鱗狀上皮細(xì)胞癌(Esophageal squamous Cell Carcinoma,ESCC)中發(fā)現(xiàn)了HPV的結(jié)構(gòu)蛋白。HPV作為重要致癌因素在宮頸癌發(fā)生中的作用已得到證實。隨著研究的深入,有學(xué)者發(fā)現(xiàn)口腔、食管鱗狀細(xì)胞癌的發(fā)生還與HPV感染有關(guān)。2008年,WHO認(rèn)定HPV感染與人口咽部鱗狀細(xì)胞癌發(fā)生存在直接關(guān)系。然而HPV與食管鱗狀細(xì)胞癌的發(fā)生關(guān)系及其作用尚未闡明。Fas屬于腫瘤壞死因子和神經(jīng)生長因子受體家族成員,當(dāng)Fas與它相應(yīng)配體Fas L(CD95L)結(jié)合后,Fas受體三聚體化而活化,激活的受體與FADD結(jié)合,再與caspase-8相互作用使后者活化,形成死亡誘導(dǎo)信號復(fù)合物,再激活一系列的Caspase-1,3,7等,其中Caspase-3的切割底物為PARP,調(diào)控凋亡最終步驟的執(zhí)行。FADD作為Fas通路的關(guān)鍵蛋白,可以觸發(fā)凋亡級聯(lián)反應(yīng)。研究發(fā)現(xiàn)在人結(jié)腸腺癌細(xì)胞系HCT116細(xì)胞中高表達(dá)HPV16 E6,可以通過影響TRAIL蛋白介導(dǎo)的凋亡信號途徑,避免宿主細(xì)胞凋亡。研究還發(fā)現(xiàn)HPV E6蛋白可以誘導(dǎo)FADD和caspase-8的泛素化,從而抑制Fas信號通路的激活來抑制細(xì)胞凋亡的發(fā)生。我們在前期實驗中已經(jīng)了解到HPV感染可以降低食管鱗狀細(xì)胞癌中Fas蛋白的表達(dá),為進(jìn)一步明確HPV在食管鱗狀細(xì)胞癌發(fā)生發(fā)展中的作用,本研究首先明確我省食管鱗狀細(xì)胞癌組織中FADD、caspase-8蛋白的表達(dá)情況,分析HPV感染與FADD、caspase-8蛋白表達(dá)的關(guān)系,其次對正常食管上皮細(xì)胞和高分化食管鱗狀細(xì)胞癌細(xì)胞株轉(zhuǎn)染攜帶HPV16 E6基因片段的質(zhì)粒,在過表達(dá)E6蛋白的情況下觀察Fas/Fas L凋亡通路相關(guān)蛋白(Fas/Fas L、FADD、caspase-8)的變化情況。在整體水平和細(xì)胞水平探討HPV對Fas介導(dǎo)的細(xì)胞凋亡通路相關(guān)蛋白的影響,從細(xì)胞凋亡角度進(jìn)一步闡明HPV與食管癌發(fā)生的關(guān)系及其作用機(jī)制,為食管癌發(fā)生的病毒學(xué)機(jī)制的研究提供新的思路和視角,為食管癌的臨床治療、預(yù)后判斷和預(yù)防提供科學(xué)依據(jù)。方法:1標(biāo)本收集收集河北醫(yī)科大學(xué)第二醫(yī)院2009年1月到2015年12月間符合條件的食管鱗狀細(xì)胞癌(原發(fā)性食管鱗狀細(xì)胞癌,手術(shù)前未經(jīng)放療和化療)的石蠟標(biāo)本,共159例。收集臨床病理資料,包括年齡,性別,淋巴結(jié)轉(zhuǎn)移情況和癌組織的分化程度。2檢測病毒DNA將石蠟組織切成薄片,使用石蠟組織DNA提取盒提取組織DNA。以HPV L1基因MY09/MY11為引物,用PCR方法檢測各實驗組與對照組中HPV L1基因的存在。使用β-actin作為內(nèi)參,使用三蒸水代替引物完成PCR反應(yīng),作為陰性對照,使用確診HPV陽性的宮頸癌石蠟組織提取的DNA作為陽性對照。3檢測食管鱗狀細(xì)胞癌組織的FADD、caspase-8蛋白表達(dá)情況應(yīng)用免疫組化SP法檢測食管鱗狀細(xì)胞癌組織中FADD、caspase-8蛋白表達(dá)情況,FADD、caspase-8蛋白陽性反應(yīng)均定位于細(xì)胞質(zhì)。4 EX-GS3066-M90(HPV16 E6)、EX-NEG-M90(空載)質(zhì)粒提純及濃度檢測。5 HPV16 E6質(zhì)粒轉(zhuǎn)染對體外培養(yǎng)的Het-1A(人正常食管上皮細(xì)胞)和Eca-109(人食管高分化鱗狀細(xì)胞癌株)的Fas介導(dǎo)凋亡通路相關(guān)蛋白(Fas/Fas L、FADD、caspase-8)的影響體外培養(yǎng)Het-1A和Eca-109,轉(zhuǎn)染HPV16 E6質(zhì)粒和空載質(zhì)粒(陰性對照),應(yīng)用半定量RT-PCR、Western blot方法分別檢測兩組Het-1A和Eca-109細(xì)胞FADD、caspase-8的m RNA和蛋白表達(dá)變化情況,分析HPV16 E6蛋白對Fas凋亡通路的可能影響和機(jī)制。為了進(jìn)一步明確HPV16 E6對人食管正常上皮Fas介導(dǎo)的凋亡通路的影響,轉(zhuǎn)染Het-1A后于12h、24h、48h收集細(xì)胞,觀察細(xì)胞內(nèi)Fas/Fas L、FADD和caspase-8蛋白表達(dá)在轉(zhuǎn)染后的變化趨勢。6統(tǒng)計分析所有數(shù)據(jù)采用SPSS19.0軟件,計數(shù)資料采用χ2檢驗及Pearson相關(guān)分析法,P0.05認(rèn)為差異有統(tǒng)計學(xué)意義。計量資料以均數(shù)±標(biāo)準(zhǔn)差((?)±s)表示,單因素方差分析P0.05時認(rèn)為差異有統(tǒng)計學(xué)意義。結(jié)果:1.1食管鱗狀細(xì)胞癌(ESCC)病理形態(tài)學(xué)觀察結(jié)果與臨床病理分析本組ESCC病例共159例,年齡跨度為27-87歲,中位數(shù)為62歲;男性患者124例,女性患者35例;經(jīng)病理形態(tài)學(xué)觀察:淋巴結(jié)轉(zhuǎn)移患者47例;ESCC高分化患者39例,中分化72例,低分化48例。低分化組與高中分化組的淋巴結(jié)轉(zhuǎn)移率無明顯差異(P0.05)。1.2 ESCC組織中HPV的感染情況與臨床病理特征的關(guān)系159例ESCC的HPV感染率為32.1%。HPV陽性組高中分化鱗癌占56.9%,明顯低于HPV陰性組的75.9%;HPV陽性組低分化鱗癌占43.1%,明顯高于HPV陰性組的24.1%(P0.05);HPV感染與年齡,性別,淋巴結(jié)轉(zhuǎn)移之間無明顯差異(P0.05)。2免疫組化結(jié)果2.1 ESCC組織中FADD、caspase-8蛋白表達(dá)情況159例ESCC組織中FADD蛋白表達(dá)陽性81例,陰性78例;caspase-8蛋白表達(dá)陽性93例,陰性66例。癌旁組織中FADD蛋白表達(dá)陽性150例,陰性9例;caspase-8蛋白表達(dá)陽性131例,陰性28例。FADD和caspase-8在ESCC組織中蛋白表達(dá)的陽性率分別為50.9%和58.5%,明顯低于癌旁組織的94.3%和82.4%(P0.05)。2.2 ESCC組織中FADD、caspase-8蛋白表達(dá)與HPV的關(guān)系HPV陽性組FADD蛋白表達(dá)陽性19例,陰性32例,caspase-8蛋白表達(dá)陽性24例,陰性27例;HPV陰性組FADD蛋白表達(dá)陽性62例,陰性46例;caspase-8蛋白表達(dá)陽性69例,陰性39例,FADD和caspase-8在HPV陽性組蛋白表達(dá)的陽性率分別為37.3%和47.1%,明顯低于陰性組的57.4%和63.9%(P0.05)。2.3 ESCC組織中FADD和caspase-8蛋白表達(dá)與臨床病理特征的關(guān)系男性FADD蛋白表達(dá)陽性率為59.7%,明顯高于女性的20.0%(P0.05);FADD蛋白表達(dá)與年齡,淋巴結(jié)轉(zhuǎn)移及腫瘤分化程度之間無明顯差異(P0.05)。男性caspase-8蛋白表達(dá)陽性率63.7%,明顯高于女性的40.0%(P0.05);caspase-8蛋白表達(dá)與年齡,淋巴結(jié)轉(zhuǎn)移及腫瘤分化程度之間無明顯差異(P0.05)。2.4 HPV與FADD和caspase-8蛋白表達(dá)與臨床病理特征的關(guān)系HPV感染陽性的ESCC組織:62歲以上(包括62歲)患者FADD蛋白陽性表達(dá)率24.1%,明顯低于62歲以下患者的54.5%(P0.05);低分化組FADD蛋白陽性表達(dá)率為13.6%,明顯低于高中分化的55.2%;FADD蛋白表達(dá)與性別和淋巴結(jié)轉(zhuǎn)移之間無明顯差異(P0.05)。HPV感染陽性的ESCC組織:低分化組caspase-8蛋白陽性率為27.3%,明顯低于高中分化組的62.1%(P0.05),caspase-8蛋白表達(dá)與年齡,性別和淋巴結(jié)轉(zhuǎn)移之間無明顯差異(P0.05)。3細(xì)胞實驗轉(zhuǎn)染后HPV16 E6、Fas、Fas L、FADD、caspase-8蛋白表達(dá)情況3.1 RT-PCR結(jié)果顯示:Het-1A細(xì)胞轉(zhuǎn)染E6基因后與空載質(zhì)粒組相比,E6基因相對表達(dá)升高(P0.05);FADD、caspase-8基因相對表達(dá)量降低(P0.05);Fas基因相對表達(dá)量升高(P0.05);Fas L無明顯變化(P0.05)。Eca-109細(xì)胞轉(zhuǎn)染E6基因后與空載質(zhì)粒組相比,E6基因相對表達(dá)量升高(P0.05);FADD、caspase-8、Fas基因相對表達(dá)量升高(P0.05);Fas L無明顯變化(P0.05)。Het-1A細(xì)胞轉(zhuǎn)染E6基因后與空載質(zhì)粒組相比,E6基因在12h、24h和48h相對表達(dá)量呈上升趨勢(P0.05);FADD和caspase-8基因在12h、24h和48h相對表達(dá)量呈下降趨勢(P0.05);Fas基因在12h、24h和48h相對表達(dá)量呈上升趨勢(P0.05);Fas L無明顯變化(P0.05)。3.2 Western blot結(jié)果顯示:Het-1A細(xì)胞轉(zhuǎn)染E6基因后與空載質(zhì)粒組相比,E6蛋白相對表達(dá)升高(P0.05);FADD、caspase-8、Fas蛋白相對表達(dá)量降低(P0.05);Fas L無明顯變化(P0.05)。Eca-109細(xì)胞轉(zhuǎn)染E6基因后與空載質(zhì)粒組相比,E6蛋白相對表達(dá)量升高(P0.05);FADD、caspase-8、Fas蛋白相對表達(dá)量降低(P0.05);Fas L無明顯變化(P0.05)。Het-1A細(xì)胞轉(zhuǎn)染E6基因后與空載質(zhì)粒組相比,E6蛋白在12h、24h和48h相對表達(dá)量呈上升趨勢(P0.05);FADD和caspase-8蛋白在12h、24h和48h相對表達(dá)量呈下降趨勢(P0.05);Fas蛋白在12h和24h相對表達(dá)量升高(P0.05),在48h相對表達(dá)量降低(P0.05);Fas L無明顯變化(P0.05)。結(jié)論:1本組食管鱗狀細(xì)胞癌組織HPV的檢出率為32.1%,HPV陽性的癌組織中低分化鱗狀細(xì)胞癌所占比例高于HPV陰性組,提示HPV與腫瘤分化存在相關(guān)關(guān)系。2食管鱗狀細(xì)胞癌中FADD和caspase-8蛋白表達(dá)降低,而且HPV可進(jìn)一步降低兩者的蛋白表達(dá)水平。3 HPV16 E6基因可以降低Het-1A和Eca-109細(xì)胞FADD、caspase-8、和Fas蛋白表達(dá)水平。4組織學(xué)和細(xì)胞學(xué)水平研究結(jié)果提示,HPV可能通過抑制FAS介導(dǎo)的凋亡通路,在食管鱗狀細(xì)胞癌發(fā)生發(fā)展中發(fā)揮一定作用。
[Abstract]:Objective: China is a high incidence area of Esophageal Carcinoma (EC), about 150 thousand people died of esophageal cancer every year, accounting for nearly 1/4 of the deaths of all malignant tumors. The lowest mortality rate of esophageal cancer in China is in Yunnan province (105/10 million), which is 31 times the difference between the highest death rate in Henan Province, and the incidence of esophageal cancer is found to exist. In recent years, environmental carcinogens (such as mycotoxins, viruses, etc.) have become one of the hotspots in the development of various tumors. The relationship between human papillomavirus (Human papillomavirus HPV) and the occurrence of esophageal cancer was first proposed by Syrjanen in 1982. His study found a group of 40% (24/60) of the cancer of the esophagus. The morphological changes were very similar to the changes in genital warts, and then he and his colleagues found the structural protein.HPV of HPV in Esophageal squamous Cell Carcinoma, ESCC as an important carcinogenic factor in the carcinogenesis of the cervix. Researchers found that the occurrence of oral and esophageal squamous cell carcinoma is also associated with HPV infection for.2008 years. WHO affirms that HPV infection has a direct relationship with the occurrence of squamous cell carcinoma of the pharynx. However, the relationship between HPV and the occurrence of squamous cell carcinoma of the esophagus and its role do not clarify that.Fas is a member of the tumor's bad cause of death and a member of the nerve growth factor receptor family, when Fas After binding with its corresponding ligand Fas L (CD95L), the Fas receptor is trimeric and activated, the activated receptor is combined with FADD, and then the latter activates the latter to form a death induced signal complex, and then activates a series of Caspase-1,3,7, in which the Caspase-3's cutting substrate is PARP, and the execution of.FADD as a Fa.FADD is used as Fa. The key protein in the s pathway can trigger the apoptosis cascade reaction. The study found that the high expression of HPV16 E6 in human colon adenocarcinoma cell line HCT116 cells can prevent the apoptosis of host cells by affecting the apoptosis signaling pathway mediated by TRAIL protein. The study also found that HPV E6 protein can induce the ubiquitination of FADD and caspase-8, thus inhibiting the Fas signal. The activation of the pathway inhibits the occurrence of apoptosis. In our previous experiments, we have learned that HPV infection can reduce the expression of Fas protein in the squamous cell carcinoma of the esophagus, and to further clarify the role of HPV in the development of squamous cell carcinoma of the esophagus. This study first identified the FADD, caspase-8 protein in the tissue of the squamous cell carcinoma of our province. The relationship between HPV infection and the expression of FADD and caspase-8 protein was analyzed. Secondly, the plasmids carrying HPV16 E6 gene fragment were transfected into normal esophageal epithelial cells and highly differentiated squamous cell carcinoma cell lines, and the changes of the Fas/Fas L apoptotic pathway Guan Danbai (Fas/Fas L, FADD, Caspase-8) were observed under the overexpression of E6 protein. To explore the effect of HPV on Fas mediated apoptosis pathway related proteins at the overall level and cell level, further elucidate the relationship between HPV and the pathogenesis of esophageal cancer from the angle of cell apoptosis, and provide new ideas and perspectives for the study of the virological mechanism of the occurrence of esophageal cancer, for the clinical treatment of esophageal cancer, the prognosis and the prediction of the prognosis. Methods: 1 specimens were collected and collected from second hospitals of Hebei Medical University from January 2009 to December 2015. The paraffin specimens of esophageal squamous cell carcinoma (primary squamous cell carcinoma of the esophagus, without radiotherapy and chemotherapy) were collected from January 2009 to December 2015. A total of 159 cases were collected, including age, sex and lymph node metastasis. The differentiation degree of the cancer tissue.2 detected the paraffin tissue into thin slices, the paraffin tissue DNA extraction box was used to extract the tissue DNA. and the HPV L1 gene MY09/MY11 was used as primer. The HPV L1 gene in the experimental group and the control group was detected by PCR method. The beta -actin was used as the internal parameter to make the PCR reaction with three water instead of primers. In sex control, the FADD of esophageal squamous cell carcinoma tissue was detected by the DNA extracted from the paraffin tissue of cervix cancer with positive HPV. The expression of caspase-8 protein was detected by immunohistochemical SP method and the expression of FADD, Caspase-8 protein in the tissues of squamous cell carcinoma of the esophagus was detected by immunohistochemistry, and the positive reaction of caspase-8 protein was located in the cells. Qualitative.4 EX-GS3066-M90 (HPV16 E6), EX-NEG-M90 (unloaded) plasmid purification and concentration detection of.5 HPV16 E6 plasmids transfection on Het-1A (human normal esophageal epithelial cells) and Eca-109 (human esophageal highly differentiated squamous cell carcinoma strain) HPV16 E6 plasmids and negative plasmids (negative controls) were transfected into two groups of Het-1A and Eca-109 cells FADD, Caspase-8 m RNA and protein expression changes were detected by semi quantitative RT-PCR and Western blot methods. Effect of mediated apoptosis pathway, after transfection of Het-1A, cells were collected at 12h, 24h, 48h, and the expression of Fas/Fas L, FADD and caspase-8 protein in the cells was observed after transfection. All data were analyzed by SPSS19.0 software. The count data were analyzed by chi 2 test and Pearson correlation analysis. P0.05 believed the difference was statistically significant. Measurement was statistically significant. The results showed that the difference was statistically significant when the single factor variance analysis ((?) + s) showed that the difference was statistically significant when the single factor variance analysis was P0.05. Results: 1.1 the pathological morphological observation of esophageal squamous cell carcinoma (ESCC) and clinicopathological analysis were 159 cases in this group of ESCC cases, the age span was 27-87 years and the median was 62 years, 124 cases in male and 35 in female patients. Morphological observation: 47 cases of lymph node metastasis, 39 cases of ESCC high differentiation, 72 cases of middle differentiation, 48 cases of low differentiation. There was no significant difference in lymph node metastasis rate between the low differentiation group and the high school differentiation group (P0.05) the relationship between the HPV infection and the clinicopathological features in the.1.2 ESCC tissue 159 cases of HPV infection rate of ESCC were the grade of differentiated high school in the 32.1%.HPV positive group. Cancer accounted for 56.9%, obviously lower than 75.9% in HPV negative group, and 43.1% in HPV positive group, significantly higher than 24.1% in HPV negative group (P0.05). There was no significant difference between HPV infection and age, sex, lymph node metastasis (P0.05).2 immunization results 2.1 ESCC tissues FADD, Caspase-8 protein expression in 159 cases ESCC tissue of FADD protein expression Yang There were 81 cases, 78 cases negative, 93 cases of caspase-8 protein expression positive, 66 cases negative, 150 cases of FADD protein expression in the paracancerous tissues, 9 cases negative, 131 cases of caspase-8 protein expression, 50.9% and 58.5% in ESCC tissues of negative.FADD and caspase-8 negative, obviously lower than 94.3% and 82.4% (P0.05).2.2 E (P0.05).2.2 E adjacent to the paracancerous tissue. The relationship between the expression of FADD, Caspase-8 protein and HPV in SCC tissue was 19 cases of FADD positive expression in HPV positive group, 32 cases negative, 24 cases of caspase-8 protein expression, 27 cases negative, 62 cases of FADD protein expression in HPV negative group and 46 cases negative, 69 cases of caspase-8 protein expression positive, 39 cases of negative sex, FADD and caspase-8 in the positive group protein expression positive. The rates were 37.3% and 47.1% respectively, which were significantly lower than those of 57.4% and 63.9% (P0.05).2.3 ESCC of the negative group. The expression of FADD and caspase-8 protein was associated with the clinicopathological features, the positive rate of the male FADD protein was 59.7%, significantly higher than that of the female (P0.05), and there was no significant difference between the expression of FADD protein and age, lymph node metastasis and the degree of tumor differentiation. Difference (P0.05). The positive rate of male caspase-8 protein expression was 63.7%, significantly higher than that of female 40% (P0.05); there was no significant difference between the expression of caspase-8 protein and age, lymph node metastasis and tumor differentiation (P0.05) the relationship between.2.4 HPV and FADD and caspase-8 protein expression and clinical pathophysiology: ESCC tissue of HPV infection positive: over 62 years of age (packet The positive expression rate of FADD protein was 24.1%, which was significantly lower than 54.5% (P0.05) in patients under 62 years of age, and the positive rate of FADD protein in low differentiation group was 13.6%, significantly lower than that in high school differentiation, and there was no significant difference between FADD protein expression and sex and lymph node metastasis (P0.05).HPV positive ESCC tissue: low differentiation group caspase-8 protein The positive rate was 27.3%, obviously lower than that of the high school group (62.1% (P0.05)). There was no significant difference between the expression of caspase-8 protein and age, sex and lymph node metastasis (P0.05) HPV16 E6 after transfection of.3 cells, Fas, Fas L, FADD, Caspase-8 protein expression 3.1 RT-PCR nodes showed that the transfected cells were compared with the empty plasmid group after transfection. The relative expression of gene increased (P0.05), the relative expression of FADD, Caspase-8 gene decreased (P0.05), the relative expression of Fas gene increased (P0.05), and Fas L had no obvious change (P0.05).Eca-109 cells transfected to the E6 gene, and the relative expression of the E6 gene was higher than that of the empty plasmid group. The relative expression of E6 gene in 12h, 24h and 48h showed an upward trend (P0.05) in 12h, 24h and 48h after transfection of E6 gene without significant change (P0.05), and the relative expression of FADD and caspase-8 genes decreased. P0.05).3.2 Western blot results showed that the relative expression of E6 protein increased (P0.05) and the relative expression of FADD, Caspase-8, Fas protein decreased (P0.05) in Het-1A cells transfected with E6 gene, and the relative expression of FADD, Caspase-8, Fas protein decreased (P0.05). The relative expression of DD, Caspase-8 and Fas decreased (P0.05), and there was no obvious change in Fas L (P0.05).Het-1A cells transfected to the E6 gene and E6 protein in 12h. The expression amount increased (P0.05), the relative expression of 48h decreased (P0.05) and Fas L had no obvious change (P0.05). Conclusion: 1 the detection rate of HPV in esophageal squamous cell carcinoma was 32.1%, and the proportion of low differentiated squamous cell carcinoma in HPV positive carcinoma tissues was higher than that in the negative group. It was suggested that HPV was associated with tumor differentiation in.2 esophageal squamous cell carcinoma. The expression of ADD and caspase-8 protein decreased, and HPV could further reduce the protein expression level of both.3 HPV16 E6 gene, which could reduce FADD, Caspase-8, and Fas protein expression level.4 histology and cytology, suggesting that the apoptosis pathway may be inhibited in the squamous cell carcinoma of the esophagus. Play a role in the development of life.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R735.1

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