缺氧诱导因子-1α与EphA2在食管鳞状细胞癌中的表达及其相关性研究

目的 检测缺氧诱导因子-1α (HIF-1α) 蛋白与EphA2蛋白在食管鳞状细胞癌中的表达定位并探讨二者表达的相关性.方法 采用细胞免疫荧光法检测HIF-1α与EphA2在食管鳞状细胞癌细胞株Eca109中的表达与定位;采用免疫组化法检测HIF-1α与EphA2在41例食管鳞状细胞癌和25例正常食管组织中的表达;常氧和缺氧条件下,Western 印迹法检测食管鳞状细胞癌细胞株Eca109和TE13经RNA干扰(RNAi)技术特异性沉默HIF-1α后EphA2表达的变化.结果 ①细胞免疫荧光结果显示HIF-1α和EphA2均在Eca109细胞胞质表达.②免疫组化结果显示HIF-1α在食管鳞状细胞癌和正常食管组织中的阳性表达率分别为70.73%(29/41)和0%(0/25),两者比较差异有统计学意义(P<0.05).EphA2在食管鳞状细胞癌和正常食管组织中的阳性表达率分别为78.04%(32/41)和28%(7/25),两者比较差异有统计学意义(P<0.05).相关性检验提示HIF-1α和EphA2在食管鳞状细胞癌中表达呈正相关性(r=0.5654,χ~2=13.11,P<0.05).③ Western印迹结果显示在食管鳞状细胞癌细胞株Eca109和TE13中,EphA2表达随HIF-1α的沉默而受抑制.结论 HIF-1α与EphA2在食管鳞状细胞癌组织中呈高表达,且二者表达呈正相关;EphA2表达受HIF-1α的调控,可能为HIF-1α的靶基因.     

Role of stress-activated MAP kinase P38 in cisplatin- and DTT-induced apoptosis of the esophageal carcinoma cell line Eca109

AIM: To study the role of P38 kinase in esophageal cancer cell apoptosis induced by genotoxin, cisplatin and the unfolded protein response (UPR) inducer, dithiothreitol (DTT). METHODS: Esophageal carcinoma cell line Eca109 was cultured in RPMI 1640 medium to 70% confluency and treated with either cisplatin, DTT, or cisplatin plus DTT in the presence or absence of P38 inhibitor, SB203580. The untreated cells served as the control. The esophageal carcinoma cell apoptosis was detected by agarose gel DNA ladder analysis and quantified by flow cytometry. The P38 phosphorylation was detected by immunohis-tochemistry using antibodies specific to phosphorylated P38 protein. RESULTS: (1) Both cisplatin and DTT induced apoptosis in the esophageal cancer cell line Eca109 as shown by DNA ladder formation; (2) As detected by antibodies specific for the phosphorylated P38 protein (p-P38), both cisplatin and DTT treatments activated the stress-activated enzyme, MAP kinase P38. The number of positive cells was about 50% for the treatment groups, comparing to that of 10% for untreated group. DTT treatment, but not cisplatin treatment, induces nuclear localization of p-P38; (3) As measured by flow cytometry, inhibition of P38 activity by SB203580 blocks DTT- and cisplatin-induced apoptosis. The rates for DTT, cisplatin, and DTT plus cisplatin-induced apoptosis were 16.8%, 17.1%, and 21.4%, respectively. Addition of the SB compound during the incubation reduced the apoptotic rate to about 7.6% for all the treatment groups, suggesting that P38 activation is essential for cisplatin- and DTT-induced apoptosis in Eca109 cells. CONCLUSION: (1) Both DTT and cisplatin were able to induce apoptosis in esophageal cancer cell line Eca109; (2) P38 MAP kinase is essential for DTT- and cisplatin- induced apoptosis in Eca109 cells; (3) P38 activation may be the common signaling component relaying the multiple upstream signaling events to the downstream cell death program.     

Angiopoietin-1 targeted RNA interference suppresses angiogenesis and tumor growth of esophageal cancer

AIM： To determine the inhibitory effect of the adenovirusbased angiopoietin-1 （Ang-1） targeted small interfering RNA expression system （Ad/Ang-1si） on the expression of the Ang-1 gene, cell growth and apoptosis in human esophageal cancer cell line Eca109. METHODS： siRNA-expressing adenovirus targeting Ang-1 gene was constructed using the Ad Easy System. Cultured Eca109 cells were transfected with Ad/Ang-1si （Eca109/Ang-1si）, and Ad/si was used to infect Eca109 cells as control （Eca109/si）. Ang-1 gene expression and concentration was determined with RT-PCR and ELISA, respectively. Human umbilical vein endothelial cell （HUVEC） migration and proliferation were analyzed. After s.c. injection into athymic nu/nu mice, the tumor growth, vessel density and apoptosis of each group was also determined. RESULTS： HUVEC migration induced by conditioned medium from Ang-1si-transfected Eca109 cells was significantly less than that induced by conditioned medium from Eca109 cells and control adenovirustransfected Eca109 cells. Furthermore, after s.c. injection into athymic nu/nu mice, the tumor growth and cell apoptosis of Ad/Ang-1si -expressing Eca109 cells was significantly lower than that of parental or control adenovirus-transfected cells. Vessel density assessed by CD31 immunohistochemical analysis and Ang-1 expression by RT-PCR were also decreased. CONCLUSION： The targeting Ang-1 may provide a therapeutic option for esophageal cancer.     

Effect of S1P5 on proliferation and migration of human esophageal cancer cells

AIM:To investigate the sphingosine 1phosphate (S1P) receptor expression profile in human esophageal cancer cells and the effects of S1P5 on proliferation and migration of human esophageal cancer cells. METHODS: S1P receptor expression profile in human esophageal squamous cell carcinoma cell line Eca109 was detected by semiquantitative reverse trans cription polymerase chain reaction. Eca109 cells were stably transfected with S1P5EGFP or controlEGFP constructs. The relation between the responses of cell prol...     

Expression of Egr-1 gene and its correlation with the oncogene proteins in non-irradiated and irradiated esophageal squamous cell carcinoma

We study the expression of early growth response gene-1 (Egr-1 gene) in non-irradiated and irradiated human esophageal cancer tissues, and its relationship with the expression of C-fos, C-jun onco-proteins as well as Egr-1 target gene proteins P53, Rb and Bax expression. In situ hybridization (ISH) and immunohistochemistry (IHC) were used respectively to detect Egr-1 mRNA, Egr-1, C-fos, C-jun, P53, Rb and Bax proteins in 80 surgically resected non-irradiated and irradiated tumor specimens of esophageal squamous cell carcinoma. Egr-1 gene mRNA and Bax protein were located in the cytoplasm, whereas Egr-1, C-fos, C-jun, P53, Rb proteins were located in the nuclei. Egr-1 was expressed in nine out of 40 cases (22.5%) of non-irradiated and 23 of 40 cases (57.5%) of irradiated tumor specimens. No correlation was found between Egr-1 gene expression and C-fos, C-jun onco-proteins expression, neither was any correlation disclosed between Egr-1 gene expression with its target gene protein expression. Patients who underwent radiotherapy with Egr-1 overexpressed in their cancer tissue had better prognosis. Radiotherapy up-regulates Egr-1 expression in esophageal carcinoma. Egr-1 overexpression may be a potential radiation response gene marker and may play an important role in prognosis of esophageal squamous cell carcinoma.     

RNA干扰Akt对食管癌细胞株Eca109生物学特性的影响

目的:探讨RNA干扰沉默Akt对人食管鳞癌细胞体外增殖、迁移及血管生成拟态(VM)形成的影响.方法:应用倒置荧光显微镜观察Akt的干扰质粒转染食管癌细胞Eca109后绿色荧光蛋白的表达;采用Western blot方法检测Akt蛋白的表达;四甲基偶氮唑蓝(MTT)法检测转染前后细胞增殖能力的变化;Transwell方法...     

食管鳞癌中微小核糖核酸let-7的表达及病理生物学意义

目的 探讨微小RNA(miRNA)1et-7对食管鳞癌细胞增殖的影响及食管鳞癌组织中let-7表达水平与临床病理特征间的关系.方法 利用RNA干扰(RNAi)和细胞转染技术将食管鳞癌细胞Eca109分别转染入let-7、let-7抑制剂及随机序列.以正常培养的Eca109细胞为阴性对照组.噻唑蓝(MTT)比色法检测各组Eca109细胞的增殖情况.实时荧光定量聚合酶链反应(qRTPCR)检测各组细胞、45例食管鳞癌组织及其癌旁组织中let-7的表达水平,并分析其与食管鳞癌临床病理特征间的关系.结果 转染后72 h,转染let-7组吸光度(A)值较阴性对照组明显降低(P=0.005),转染let-7抑制剂组A值较阴性对照组明显升高(P=0.029).与阴性对照组let-7表达量比较,转染let-7组表达增加33%(1.33比1.00,P=0.039),转染let-7抑制剂组表达降低50%(0.50比1.00,P=0.014).食管鳞癌组织和癌旁组织中let-7相对表达量的比值为0.66±0.47,差异有统计学意义(P=0.001).汉族患者食管鳞癌组织中let-7相对表达量(0.48±0.43)低于哈萨克族(0.88±0.51,P=0.019).低分化食管鳞癌组织中let-7相对表达量(0.42±0.30)低于高分化食管鳞癌组织(0.84±0.38,P=0.015).有淋巴结转移者食管鳞癌组织中let-7相对表达量(0.50±0.35)低于无淋巴结转移者(0.80±0.52,P=0.032).结论 let-7对食管鳞癌的发生、发展起抑制作用,其表达水平与组织分化程度、淋巴结转移及民族相关.

Abstract：

Objective To estimate the effect of microRNA (miRNA) let-7 expression on human esophageal squamous cell carcinoma(ESCC) and the relationship between let-7 level and clinicopathological parameters. Methods ESCC cell line (Eca109) was transfected with let-7 or its inhibitor by RNAi and cell transfection techniques. Normal cultured Eca109 cell was served as negative control. The proliferation of Eca109 cell was detected by MTT. The expression of let-7 in Eca109 cells and 45 paired ESCC tissues and corresponding para-cancerous tissues were measured using real-time quantitative polymerase chain reaction (qRT-PCR). The relationship between let-7 level and clinicopathological parameters in patients with ESCC was analyzed. Results The A value of let-7 in Eca109 cells transfected with let-7 was lower than negative control (P=0.005), while it was higher in Eca109 cells transfected inhibitor than that in negative control 72 hours after transfection. In comparison with negative control, the expression of let-7 in Eca109 cells transfected with let-7 was increased 33% (1.33 vs 1.00,P=0. 039) and it was decreased 50% in Eca109 cells transfected with inhibitor (0.50 vs 1.00,P=0. 014). The ratio of let-7 expression in ESCC tissue and para-cancerous tissue was 0.66 ± 0.47 with significant differece (P= 0.001). Moreover, The level of let-7 expression in Han patients with ESCC was lower than Kazakh patients with ESCC (0.48±0.43 vs 0. 88±0.51,P=0. 019). The level of let-7 expression in poorly differentiated ESCC tissue was lower than well differentiated ESCC tissue (0.42±0.30 vs 0.84±0.38,P=0. 015). The level of let-7 expression in patients with lymph node metastasis was lower than those without lymph node metastasis (0.50±0.35vs 0. 80±0.52,P=0. 032) . Conclusion It is demonstrated that let-7 can inhibit the carcinogenesis and development of ESCC. The level of let-7 expression is associated with cell differentiation,lymph node metastasis and nationalities.     

Relationship between Egr—1 gene expression and apoptosis in esophageal carcinoma and precancerous lesions

AIM: To study the expression of early growth response gene-1 (Egr-1 gene) and Bcl-X/(L) protein and its relationship with the cell apoptosis in human esophageal carcinoma (EC) and precancerous lesions. METHODS: In situ hybridization(ISH), immunohistochemistry (IHC) and TUNEL method were used respectively to detect Egr-1mRNA, Egr-1 protein, apoptosis related-protein Bcl-X/(L) and cell apoptosis in situ from 66 cases of esophageal squamous cell carcinoma and their upper cut edge and paracancerous mucosa. RESULTS: Egr-1 gene in situ hybridization, Bcl-X/(L) immunohistochemistry positive products were located in the cytoplasm, while Egr-1 immunohistochemistry and TUNEL positive signal were located in the nuclei. The apoptosis index(AI) and the frequency of apoptosis occurrence were increased gradually from precancerous lesion to cancer (P<0.01) and the expression of Egr-1mRNA and Egr-1 protein in dysplasia was the highest among all specimens (P<0.01). The AI of Egr-1 positive cancer tissues was much higher than that of Egr-1 negative cancer tissues (P<0.01), while the AI of Bcl-X/(L) positive cancer tissues was much lower than that of Bcl-X/(L) negative cancer tissues (P<0.01). The AI and Egr-1 expression were not correlated with invasiveness and lymphatic metastasis in EC. CONCLUSION: Cell apoptosis was present through esophageal carcinogenesis. The expression of Egr-1 mRNA and Egr-1 protein were high in precancerous lesion of esophagus. The AI was increased significantly in Egr-1 positive squamous cell carcinoma. Egr-1 might promote apoptotic effect. Egr-1 expression and cell apoptosis may have an important biological significance in esophageal carcinogenesis.     

EphA2在缺氧条件下的表达及其对食管癌细胞体外三维培养的影响

目的:探讨在正常氧分压和缺氧条件下,食管癌细胞中上皮细胞激酶A2(EphA2)表达变化及其对体外三维培养的影响.方法:正常氧分压及缺氧条件下培养食管癌Ecal09及TE13细胞,RT-PCR及Western blot 分别监测细胞中EphA2表达的变化:EphA2 miRNA干扰质粒转染Ecal09和TE13细胞后,采...     

磁性纳米控释紫杉醇对食管癌Eca109细胞株生长的影响

目的:观察不同浓度磁性纳米控释紫杉醇对食管癌细胞Eca109的影响及与不同化疗药物的对比.方法:取对数生长期的食管癌Eca109细胞作细胞增殖抑制实验,实验组分别给予不同浓度的磁性纳米控释紫杉醇和5-氟尿嘧啶、泰素,同时设立二甲基亚砜(DMSO)和RPMI1640液对照,测定24,48,72h三个时间段的吸光度值,计算抑制率.经不同浓度的磁性纳米控释紫杉醇作用72h后,用电镜观察细胞超微结构,同时用流式细胞仪测定细胞周期和细胞凋亡.结果:MTT实验显示磁性纳米控释紫杉醇可抑制食管癌细胞增殖,与5-氟尿嘧啶,泰素相比,具有缓释性(P<0.01);电镜可发现药物作用组细胞核固缩、解聚以及凋亡小体;流式细胞仪检测显示G1峰前有明显的凋亡峰;细胞周期分析提示磁性纳米控释紫杉醇可将Eca109细胞阻滞于G2-M期,且与浓度相关.结论:磁性纳米控释紫杉醇对人食管癌细胞Eca109的生长有明显的抑制作用,使细胞分裂阻滞于G2-M期,并诱导细胞凋亡,且具有缓释效果.     

p27kip1基因转移对食管癌细胞生存素表达和端粒酶活性的影响

背景：p27kip1是新近发现的一种抑癌基因，早期研究表明p27kip1基因转移能显著抑制食管癌细胞和人食管癌裸鼠移植瘤的生长，表明该基因疗法可能是食管癌治疗的新途径，但其抑癌机制尚未完全阐明。目的：研究p27kip1基因转移对食管癌细胞生存素（survivin）表达和端粒酶活性的影响。从而阐明p27kip1的抑癌机制，为p27kip1基因治疗食管癌提供理论依据。方法：将携带p27kip1基因的重组腺病毒（Ad—p27kip1）和LacZ重组腺病毒（Ad—LacZ）分别转染食管癌细胞系Eca9706，观察细胞形态变化。以免疫细胞化学染色和蛋白质印迹法检测p27kip1和生存素的表达．以端粒重复序列扩增程序（TRAP）聚合酶链反应（PCR）-酶联免疫吸附测定（ELISA）检测端粒酶活性。结果：经Ad—p27kip1转染后，Eta9706细胞变圆，呈葡萄串样聚集以致脱落。细胞p27kip1表达明显增强，生存素表达降低，端粒酶活性显著受抑制。结论：p27kip1基因抑制食管癌细胞生长的作用机制可能与下调生存素表达和抑制端粒酶活性有关。     

HIF-1α induces VE-cadherin expression and modulates vasculogenic mimicry in esophageal carcinoma cells

AIM: To investigate whether hypoxia inducible factor (HIF)-1α modulates vasculogenic mimicry (VM) by upregulating VE-cadherin expression in esophageal squamous cell carcinoma (ESCC).METHODS: Esophageal squamous cancer cell lines Eca109 and TE13 were transfected with plasmids harboring small interfering RNAs targeting HIF-1α or VE-cadherin. The proliferation and invasion of esophageal carcinoma cells were detected by MTT and Transwell migration assays. The formation of tubular networks of cells was analyzed by 3D culture in vitro. BALB/c nude mice were used to observe xenograft tumor formation. The relationship between the expression of HIF-1α and VE-cadherin, ephrinA2 (EphA2) and laminin5γ2 (LN5γ2) was measured by Western blot and real-time polymerase chain reaction.RESULTS: Knockdown of HIF-1α inhibited cell proliferation (32.3% ± 6.1% for Eca109 cells and 38.6% ± 6.8% for TE13 cells, P < 0.05). Both Eca109 and TE13 cells formed typical tubular networks. The number of tubular networks markedly decreased when HIF-1α or VE-cadherin was knocked down. Expression of VE-cadherin, EphA2 and LN5γ2 was dramatically inhibited, but the expression of matrix metalloproteinase 2 had no obvious change in HIF-1α-silenced cells. Knockdown of VE-cadherin significantly decreased expression of both EphA2 and LN5γ2 (P < 0.05), while HIF-1α expression was unchanged. The time for xenograft tumor formation was 6 ± 1.2 d for Eca109 cells and Eca109 cells transfected with HIF-1α Neo control short hairpin RNA (shRNA) vector, and 8.4 ± 2.1 d for Eca109 cells transfected with an shRNA against HIF-1α. Knockdown of HIF-1α inhibited vasculogenic mimicry (VM) and tumorigenicity in vivo.CONCLUSION: HIF-1α may modulate VM in ESCC by regulating VE-cadherin expression, which affects VM formation through EphA2 and LN5γ2.     

负载食管癌抗原的DC诱导特异性CTL对食管癌细胞体外杀伤作用观察

目的观察负载食管癌抗原的树突状细胞（DC）活化的特异性细胞毒性T淋巴细胞（CTLs）对食管癌细胞的体外杀伤作用。方法冻融法获取食管癌细胞抗原,联合应用粒细胞—巨噬细胞集落刺激因子（rhGM-CSF）、白细胞介素4（IL-4）和肿瘤坏死因子α（TNF-α）诱导培养外周血DC并负载肿瘤抗原,激活自体T淋巴细胞,制备特异性CTLs。将其加入食管癌细胞中培养48 h,用MTT法检测食管癌细胞裂解率,ELISA法检测γ干扰素（γ-IFN）水平。结果负载食管癌抗原的DC激活的CTLs表现出对食管癌Eca109细胞的特异性杀伤作用,γ-IFN水平为（1 625±37.55）pg/ml;而对A549细胞仅有微弱的杀伤作用,γ-IFN水平为（169.04±13.81）pg/ml。未负载食管癌抗原DC刺激的CTLs对食管癌细胞几无杀伤作用。结论负载食管癌抗原的DC激活的CTLs在体外对食管癌细胞能产生高效而特异性的杀伤作用。     

Bile salts inhibit growth and induce apoptosis of human esophageal cancer cell line

AIM: To explore the effect of six bile salts, including glycoc holate (GC), glycochenodeoxycholate (GCDC), glycodeoxy cholate (GDC), taurocholate (TC), taurochenodeoxycholate (TCDC), taurodeoxycholate (TDC), and two bile acids including cholic acid (CA) and deoxycholic acid (DCA) on esophageal cancer Ecal09 cell line. METHODS: Eca109 cells were exposed to six bile salts, two bile acids and the mixed bile salts at different concentrations for 24-72 h. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to detect the cell proliferation. Apoptotic morphology was observed by phase-contrast video microscopy and deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Sub-G1 DNA fragmentations and early apoptosis cells were assayed by flow cytometry (FCM) with propidium iodide (PI) staining and annexin V-FITC conjugated with PI staining. Apoptosis DNA ladders on agarose were observed. Activation of caspase-3 was assayed by FCM with FlTC-conjugated monoclonal rabbit anti-active caspase-3 antibody and expressions of Bcl-2 and Bax proteins were examined immunocytochemically in 500 μmol/L-TC-induced apoptosis cells. RESULTS: Five bile salts except for GC, and two bile acids and the mixed bile salts could initiate growth inhibition of Ecal09 cells in a dose- and time-dependent manner. TUNEL, FCM, and DNA ladder assays all demonstrated apoptosis induced by bile salts and bile acids at 500 μmol/L, except for GC. Early apoptosis cell percentages in Eca109 cells treated with GCDC, GDC, TC, TCDC, TDC, CA at 500 μmol/L for 12 h, DCA at 500 μmol/L for 6 h, and mixed bile salts at 1 000 μmol/L for 12 h were 7.5%, 8.7%, 14.8%, 8.9%, 7.8%, 9.3%, 22.6% and 12.5%, respectively, all were significantly higher than that in control (1.9%). About 22% of the cell population treated with TC at 500 μmol/L for 24 h had detectable active caspase-3, and were higher than that in the control (1%). Immunocytochemical assay suggested that TC down-regulated Bcl-2 protein level and up-regulated Bax protein level. CONCLUSION: GCDC, GDC, TC, TCDC, TDC, CA and DCA, except for GC, can inhibit growth and induce apoptosis of esophageal cancer Ecal09 cells. Activation of caspase-3, decreased Bcl-2 protein and increased Bax protein are involved in TC-induced apoptosis of Ecal09 cells.     

RNA干扰HIF-1α对食管鳞癌血管生成拟态的影响

背景:研究表明大量高度恶性肿瘤组织中存在血管生成拟态(VM),其分子机制已形成相关假说,但确切机制和关键信号通路尚未明确。目的:探讨缺氧诱导因子(HIF)-1α在食管鳞癌VM形成中的作用。方法:设计合成3种HIF-1α-siRNA质粒,测序后瞬时转染293T细胞,蛋白质印迹法鉴定质粒的干扰效果。将筛选出的pGCsi-HIF3质粒稳定转染食管鳞癌细胞株Eca109和TE13,以蛋白质印迹法检测干扰效果,三维培养法观察VM形成情况,蛋白质印迹法检测VE-cadherin、EphA2、LN5γ2和MMP2蛋白表达。结果:成功构建了3个靶位的质粒,其中pGCsiHIF3的干扰效果最好。pGCsi-HIF3稳定转染食管鳞癌细胞株Eca109和TE13后,与对照组相比,HIF-1α表达明显降低,细胞体外管道形成能力被明显抑制(P0.05)。常氧下转染组细胞中VE-cadherin、EphA2、LN5γ2均显著下调(P0.05),MMP2表达无明显差异,且缺氧条件下上述指标蛋白表达无明显增加。结论:Eca109和TE13细胞能形成管状结构,HIF-1α可能通过调节VE-cadherin、EphA2、LN5γ2等的表达而调节食管鳞癌VM的形成。     

p16-INK4alpha重表达对人食管癌细胞系EC109生长的抑制作用

目的探讨用腺病毒介导野生型的p16基因在食管癌细胞系表达,研究重表达野生型p16基因对食管癌细胞系EC109生长的抑制作用,为寻找食管癌致癌机制的研究及其基因治疗提供理论基础.方法用基因重组技术,将pcDNA3-p16中的野生型p16基因用Kpn I/BamH I进行双酶切,克隆入腺病毒表达载体pAdCMV中,将重组质粒pad-CMV-p16与腺病毒质粒JM17用Lipofectamime 2000共转染293细胞,产生重组腺病毒.用重组腺病毒感染人食管癌细胞系EC109,在感染后的不同时段,用免疫荧光、打点杂交方法检测p16基因在细胞中的表达用MTT法及流式细胞仪观察p16基因的表达对食管癌细胞株生长的影响.结果经酶切鉴定野生型p16基因克隆入腺病毒表达载体中,与JM17共转染293细胞后,可产生具有感染活性的重组腺病毒,用重组腺病毒感染食管癌细胞株EC109细胞后,可抑制食管癌细胞的生长,同对照组相比其最大抑制率可达52.7%.Multipcycle分析软件分析对细胞周期影响表明,处于G0～G1期的细胞为41%～63%感染后的细胞经Dot blot和Westernblot杂交证实,有外源的p16mRNA及蛋白的表达.结论重组腺病毒可将野生型p16基因导入人食管癌细胞系EC109中,野生型p16基因在p16基因功能缺失的细胞中重表达能抑制癌细胞恶性生长.p16基因功能缺失是食管癌致癌因素之一