【作者】 张曙光；

【导师】 张林；

【作者基本信息】 中国医科大学 ， 外科学， 2008， 博士

【摘要】 目的食管癌是人类常见的恶性肿瘤之一,发病率较高,在地区间差别较大。它的特征是早期诊断比较困难且预后差,我国食管癌的发病率目前居世界第一位,发病人数占发病总数的60%,且男性的发病率是女性的2倍。全世界每年新发食管癌病例约30万。我国食管癌发病率为13/10万,但是食管癌的确切发病机制尚不明确,且对于食管癌的治疗主要是手术治疗为主联合放疗和化疗的综合治疗,但是现有方法已经达到其治疗的极限,只有深入研究其发病机制,并寻找新的治疗方式才能提高食管癌的治愈率。现有的研究发现,肿瘤的发生与其凋亡率降低有关,最重要的凋亡通路就是Caspase通路。X染色体连锁的凋亡抑制基因（X-linkedinhibitor of apoptosis protein,XIAP）是凋亡抑制基因家族中重要的成员之一,其编码的蛋白XIAP通过选择性的抑制Caspase-3,-7和-9,并参与其它途径来抑制细胞的凋亡,XIAP是该基因家族中唯一能够同时抑制起始和效应阶段的IAP。本研究拟在探索XIAP与食管癌之间的关系,从三个方面探讨XIAP与食管癌的发生发展以及对食管癌化疗敏感性的影响。第一,通过免疫组化、RT-PCR和Western Blot检测XIAP在食管鳞癌和正常食管组织中的表达,并检测其在多株食管鳞癌细胞系中的表达情况;第二,通过化学合成法合成的XIAP特异的siRNA,观察能否通过RNAi方法降低XIAP在食管癌细胞中的表达;第三,若能通过RNAi方法降低食管癌细胞中XIAP的表达,探讨XIAP低表达对食管癌细胞凋亡的影响,更重要的是观察其对多种化疗药物敏感性影响。材料与方法1、临床标本及组织芯片制作:食管鳞癌及癌旁正常食管配对组织取自安徽医科大学胸外科,由2位以上资深病理学家诊断为食管鳞癌,所有病人术前均未经放化疗。新鲜组织采用手术分离,并在术后立即置于液氮中冻存,选取110对食管癌配对组织常规石蜡包埋。组织芯片在Beecher组织芯片仪制作完成,在蜡块上设计14×7共98点组织阵列,打孔并将供体蜡块标记部位取出的组织柱推入预先设计的相应孔内;对蜡块进行连续切片,裱于防脱片载玻片上。2、食管鳞癌细胞培养:本研究中选用的人食管鳞癌细胞系,其中TE10、KYSE30、KYSE70、KYSE150、KYSE410、KYSE450、KYSE510细胞系由日本Kyoto University的Shimada Y博士惠赠,EC9706由王明荣教授惠赠。食管癌细胞被培养在RPMI1640培养基中,这个培养基含有10%胎牛血清,不含有抗生素,生长环境是5%CO2、37℃的细胞培养箱中培养。3、siRNA合成及细胞转染:XIAP-siRNA由Invitrogen公司设计合成,并在Hela细胞中验证其对XIAP的抑制效率。我们采用脂质体法,应用Lipofectamine2000转染试剂进行转染,siRNA转染浓度为25nm。4、RT-PCR半定量法检测XIAP在组织标本和细胞中的表达:食管癌组织、癌旁正常食管组织和食管癌细胞均按Trizol法提取总RNA,然后进行RT-PCR,最终的结果应用Gel-pro Analyzer软件进行半定量分析。5、Western Blot半定量法检测XIAP在组织标本和细胞中的表达:食管癌组织、癌旁正常食管组织和食管癌细胞按照常规方法提取胞浆总蛋白,进行蛋白定量后,进行Western Blot,最终的结果应用Gel-pro Analyzer软件进行半定量分析。6、免疫组织化学染色及结果分析:采用微波法修复抗原,应用ABC免疫组化试剂盒按照ABC法进行免疫组化染色,免疫组化染色结果采用二级记分法进行判定,结果判定由两位病理科医生分别独立完成,取二者平均值作为最终结果。7、流式细胞术检测细胞凋亡:同时收集悬浮和贴壁生长的细胞,PI法染色,应用FACSCalibur flow cytometer流式细胞仪检测,CellQuest软件进行结果分析,sub-G1-G0区细胞被认定为凋亡细胞。8、化疗药物处理及MTT法检测细胞活性:按照实验设计对接种于96孔板的细胞进行siRNA转染,并加入相应浓度的化疗药物,在设定的时间点应用MTT法检测细胞活性。9、应用SPSS11.0软件进行统计分析,XIAP在食管癌和正常组织表达的差异应用非配对T检验进行,XIAP的表达与临床病理因素之间的关系用卡方检验和Sperman’s相关分析进行分析。IC₅₀的计算采用Probit分析法计算,不同组之间的均数比较采用Student-Neuman-Keuls多样本均数检验进行分析。P＜0.05认为有统计学意义。结果1、首先我们通过免疫组织化学、RT-PCR和Western Blot的方法检测XIAPmRNA和XIAP蛋白在食管鳞癌组织、癌旁正常食管组织和食管鳞癌细胞系中的表达,不同的方法检测的结果一致显示其在食管癌组织中的表达显著高于其在正常食管组织中的表达（统计分析均显示P＜0.01）,同样其在大多数食管癌细胞系中高表达,但是其在食管癌组织中的表达与食管癌患者的临床病理因素如:患者的年龄、性别、食管癌的分化程度和TNM分期无统计学相关。2、我们选择了两株XIAP高表达细胞系KYSE150、EC9706和两株XIAP低表达细胞系TE10、KYSE510,进行XIAP-siRNA转染,选取转染后24小时、48小时和72小时三个时间点,通过RT-PCR和Western Blot检测发现,XIAP-siRNA在mRNA和蛋白水平均能够有效地降低XIAP的表达,对XIAP的抑制率可以达到90%以上。3、应用XIAP-siRNA进行RNA干扰后食管癌细胞XIAP低表达,我们检测了其对细胞凋亡的影响,结果显示在XIAP高表达细胞系,XIAP的表达降低能够显著增加细胞的凋亡,而在XIAP低表达细胞系中XIAP的低表达对细胞的凋亡率没有影响（P＜0.01）,这两方面结果提示食管癌细胞凋亡的增加确实是由XIAP的表达降低引起的。4、在研究XAIP低表达后对食管癌细胞化疗敏感性的影响中,结果发现,在XIAP高表达细胞系KYSE150、EC9706中,降低XIAP的表达能够显著增加所有细胞对所有化疗药物紫杉醇（PTX）、顺铂（DDP）、依托泊苷（VP-16）和氟尿嘧啶（5-Fu）的敏感性;而对于XIAP低表达系,在TE10仅能增加对四种中的三种化疗药物紫杉醇（PTX）、顺铂（DDP）和依托泊苷（VP-16）的敏感性,在KYSE150中仅能增强对紫杉醇（PTX）敏感性,且这两株细胞中,XIAP低表达对化疗药物敏感性的增加远不如在XIAP高表达细胞系中显著。结论1、X染色体连锁的凋亡抑制基因XIAP在食管鳞癌组织中明显高于其在正常食管组织中的表达,但是与患者的临床病理因素（年龄、性别、食管癌的分化程度和TNM分期）无关,基于此,可为食管鳞癌的病理诊断提供了一个新的免疫组织化学指标,可能会对食管癌的诊断提供一定的辅助作用。2、XIAP特异的siRNA能够显著降低食管癌细胞中XIAP的表达,在降低XIAP后,能够显著增加XIAP高表达食管癌细胞的细胞凋亡,进一步验证了XIAP主要通过Csapase凋亡通路在食管癌生长中的抑制凋亡和促进生长的作用。3、通过RNAi降低XIAP表达后,能够显著增强XIAP高表达细胞系对紫杉醇（PTX）、顺铂（DDP）、依托泊苷（VP-16）和氟尿嘧啶（5-Fu）的敏感性,结合在降低XIAP后,能够显著增加XIAP高表达食管癌细胞的细胞凋亡,本研究为增加食管癌化疗敏感性提供了基础研究方面的依据,有可能为食管癌的临床治疗带来新的突破。更多还原

【Abstract】 ObjectiveMalignant cells commonly have defects in cell death control and apoptosis, and inhibition of apoptosis can lead to tumorigenesis and resistance to therapy. Most chemotherapeutic drugs counteract cancer by inducing cell apoptosis. Apoptosis resistance enables cancer cells to survive, although exposed to many proapoptotic factors, such as cytotoxic drugs, anoxemia, and radialization Cancer cells escape apoptosis by a number of mechanism among which overexpression of antiapoptotic genes has been shown to play a critical role, such as overexpression of some members of the IAP gene family or the Bcl-2 family. The inhibitor of apoptosis proteins （IAPs） have been identified as acting downstream of Bcl-2 by inhibiting caspases. To date, eight members of IAPs have been identified in humans, and x-linked inhibitor of apoptosis protein （XIAP） is the most potent one. XIAP can inhibit caspases-3, -7 and -9, and is the only member of IAP family able to directly inhibit both the initiation and execution phase of the caspase cascade crucial to mediate the controlled demise of malignant cells. Esophageal squamous cell carcinoma, the major histologic form of esophageal cancer, is one of the most frequent fatal malignancies in the world, especially in the northern part of China. Treatment of ESCC has primarily relied on classical modalities including surgery, radiotherapy and chemotherapy or a combination of these methods, but the outcome has not improved significantly. Most of the treatment failures are due to relapse after surgery or metastatic disease resistant to systemic therapy. Apoptosis resistance in ESCC accounts for its poor response to chemotherapy and enhanced metastasis. Therefore, it is necessary to search for new treatment strategies. Whether XIAP could be a therapeutic target in ESCC was still unknown.In this study, the expression of XIAP in ESCC cases with reference to normal mucosa, as well as the expression of XIAP in ESCC cell lines, was evaluated. Then we investigated whether the downregulation of XIAP expression in ESCC cell lines by siRNA could enhance cell apoptosis and the chemotherapeutic effects of Paclitaxel, Cisplatin, Fluorouracil and Etoposide, the chemotherapeutic agents used currently in the treatment of patients with ESCC.Materials and Methods1. Patients and Tissue Specimens: Specimens of cancer tissues and matched adjacent normal mucosa were taken from 110 consecutive patients. All patients in this study had undergone curative tumor resection in the Department of Thoracic Surgery, the First Affiliated Hospital of Anhui Medical University between Oct 2004 and Sept 2005. None of the patients had received radiotherapy or chemotherapy before surgery. Tumor tissues were dissected from the resected specimens and the normal tissue blocks were taken from the distal resection margin. The specimens for RT-PCR and Western blot were snap-frozen in liquid nitrogen, and the specimens for immunohistochemistry were fixed in 4% polyformaldehyde and embedded in paraffin.2. Cell Lines and Cell Culture: Human ESCC cell lines TE10, KYSE30, KYSE70, KYSE150, KYSE410, KYSE450, KYSE510 and EC9706 were grown in RPMI1640 medium with 10% fetal bovine serum. The human esophageal squamous cancer cell line KYSE series was a generous gift from Dr. Shimada Y and EC9706 was kindly provided by Prof. Mingrong Wang.3. Tissue Microarray Construction, Immunohistochemistry and Evaluation of the IHC Staining: Tissue microarrays （TMA） containing 110 ESCC cases were constructed with a Beecher Instruments for Tissue Array. After the construction of the array block, multiple 4μm sections were cut with a microtome using an adhesive-coated tape sectioning system. H&E-stained sections were used for histological verification of tumor and normal tissues on the arrayed samples.Then immunohistochemistry （IHC） was performed using a diaminobenzidine-base detection method. Omitting the primary antibody from the immunohistochemical procedure and replacing it with antibody diluent acted as negative controls.The percentage of XIAP positive cells was determined semiquantitatively by assessing the entire sample. Each sample was assigned to one of the following categories: 0 （0-4%）, 1 （5-24%）, 2 （25-49%）, 3 （50-74%）, or 4 （75-100%）. The intensity of immunostaining was determined as 0 （negative）, 1+ （weak）, 2+ （moderate）, or 3+ （strong）. A final immunoreactive score between 0 and 12 was calculated by multiplying the percentage of positive cells with the staining intensity score. In this study, scores of 8-12 were defined as XIAP "high expression", and scores of 0-7 were defined as "negative or reduced expression". All slides were evaluated for immunostaining independently by two observers with no prior knowledge of patients’ clinical data.4. siRNA Transfection and The Effect Confirmation: siRNA targeting XIAP （Hs_BIRC4₅ HP Validated siRNA 1027400, Cat. SI00299446） was designed and synthesized by Qiagen company. The control （nonsilencing） siRNA was designed by Qiagen company and synthesized by GeneChem company. Transfection was performed in 50%-60% confluent cells using Lipofectamine2000 Reagent according to the manufacture’s protocol. The downregulation of XIAP by siRNA was confirmed by RT-PCR and Western blot at the indicated time points.5. RT-PCR: Total RNA was extracted from frozen tissues or cells using TRIzol reagent according to the instructions of the manufacturer. Five micrograms of total RNAs of each sample were reverse transcribed to the first strand of cDNA primed with random hexamers using Superscript? First-Strand Synthesis System for RT-PCR kit. The products were electrophoresed by 1.2% agarose-gel and semi-quantified by Gel-pro Analyzer image analysis software. 6. Western Blot Analysis: For Western blot analysis, tissues or cells were lysed with the buffer. The protein concentrations were determined using the BCA Protein Assay kit. Thirty micrograms of protein were separated on 10% SDS-PAGE gels and transferred to PVDF membrane. After blocking, the membrane was incubated with anti-XIAP antibody （1:1000） at 4℃overnight. After washing, the membrane was incubated with secondary antibody at a dilution 1:3000 at room temperature for 1 hour. Proteins were detected with the ECL kit and anti-β-actin antibody was used as loading control. Densitometry was performed by Gel-pro Analyzer software.7. Flow Cytometric Assay: Cells were seeded in 6-well plates. Forty-eight hours after transfection, both floating and attached cells （use trypsin） were collected. Cells were incubated with 5μg/ml propidium iodide and 50μg/ml RNase-A in PBS for 30 min in 37℃. Flow activated cell sorter analysis was carried out using a FACSCalibur flow cytometer with CellQuest software. A total of 10,000 cells were measured per sample. The sub-G₁-G₀ cell fraction was considered as representative of apoptotic cells.8.Treatments with Chemotherapeutic Agents and Measurement of Cell Viability: Growth inhibition of ESCC cells were determined by the colorimetric MTT cell viability/proliferation assay. In brief, cells were seeded in 96-well plates with 4000 cells per well. Twenty-four hours after transfection, the chemotherapeutics were added in varying concentrations to each well. Cells were incubated for 72 hours, then the media were replaced with 100μl MTT, dissolved in RPMI1640 at the final concentration of 0.5mg/ml. The plates were incubated for an additional 4 hours, then the medium was aspirated off leaving the dark blue formazan product in the bottom of the wells. The absorbency was detected at 570nm on Bio-Rad model 550-microplate Reader after 200μl of DMSO were added to each well to dissolve the formazan crystals. The percentage of dead （or growth inhibited） cells was normalized to untreated controls. All of the experiments were carried out at least three times in triplicate.9. Statistical Analysis: Statistical analysis was done using the SPSS statistical software. The difference between XIAP expression in tumor tissues and normal tissues was performed by unpaired Student’s t test. The correlation between XIAP expression and clinicopathologic characteristics was analyzed using Chi-square test and Spearman’s correlation analysis. 50% inhibiting concentration （IC50） was calculated using Probit analysis. Differences in mean values between control treatment and combination treatments were analyzed using Student-Neuman-Keuls analysis. P<0.05 was considered statistically significant.Results1. Overexpression of XIAP in Human ESCC Tissues at mRNA and Protein Level: RT-PCR analysis of XIAP mRNA expression in patient-matched normal and tumor tissues showed that XIAP mRNA was up-regulated in ESCC tissues compared with their normal counterparts （P<0.01）. Western blot analysis showed that XIAP protein was overexpressed in ESCC tissues （P<0.01）. And this corresponds to the result of immunohistochemistry detection of TMA samples.2. XIAP Expression with Clinical Features: To further characterize the expression of XIAP with clinical features, we performed tissue microarray analysis of 110 paired ESCC tissue specimens, among them, 102 samples could be assessed due to the loss of paraffin sections from slides. XIAP was localized to cytoplasm and rather diffuse. XIAP protein showed significantly high expression in ESCC compared with normal mucosa （P=0.000）. However, the increased expression of XIAP in ESCC tissues did not show obvious correlation with any of the clinicopathologic characteristics, including patients’ age, gender, and tumor histo-pathological features.3. Expression Pattern of XIAP in ESCC Cell Lines: Multiple ESCC cell lines were examined for XIAP protein expression by Western blot analysis. The highest level of XIAP expression was detected in KYSE30 KYSE150, KYSE410, KYSE450 and EC9706 cell, whereas low XIAP expression was observed in TE10, KYSE70 and KYSE510 cells. We chose both XIAP high expression cell lines KYSE150, EC9706 and XIAP low expression cell lines TE10, KYSE510 in subsequent experiments.4. Expression of XIAP was Blocked Efficiently by RNAi: The expression of XIAP was examined by RT-PCR and Western blot at 24, 48, and 72hours after siRNA transfection. We found that XIAP specific siRNA can efficiently block XIAP expression both at mRNA and protein level. And the efficiency can reach more than 95% at protein level in all the cell lines.5. Effect of RNAi on Apoptosis: The apoptosis rate was11.63%±0.058% in KYSE150 and 11.30%±0.100% in EC9706 respectively, which were significantly higher than mock and control siRNA transfection （P<0.01, Fig.5）. However, no obvious apoptosis was observed in either TE10 or KYSE150 cells whose XIAP expression was low.6. XIAP Downregulation Sensitizes ESCC Cells to Chemotherapeutics: In KYSE150 and EC9706 cells, our result demonstrated that the cells exposed to XIAP siRNA in the presence of Paclitaxel, Cisplatin, Fluorouracil, or Etoposide showed a significant decrease in IC₅₀ compared with control siRNA, mock transfection or no-treatment. As shown in Fig.6B, XIAP siRNA treatment significantly enhanced the growth inhibitory effect of Paclitaxel, Cisplatin, Fluorouracil, and Etoposide in both cell lines. The control siRNA had either no effect or only a minimal effect.But for TE10 cell, the IC₅₀ of Paclitaxel, Cisplatin, or Fluorouracil, three of the four drugs, could be decreased sightly by XIAP siRNA; and for KYSE150 cell, only the IC₅₀ of Paclitaxel could be decreased by XIAP siRNA compared with control siRNA, mock transfection or no-treatment group. Similarly, the growth inhibitory effect of Paclitaxel, Cisplatin and Fluorouracil could be enhanced slightly by XIAP siRNA in TE10 cell; and XIAP siRNA could only enhance the growth inhibitory effect of Paclitaxel in KYSE510 cell. Moreover, the degree of either the decrease of IC₅₀ or the enhancement of growth inhibitory of the drugs in TE10 or KYSE510 was much smaller than in KYSE150 or EC9706 respectively （P<0.05）Conclusion1. In this study, we detected XIAP expression using RT-PCR, Western blot and immunohistochemistry on ESCC tissues and the adjacent normal tissues. XIAP was highly expressed in ESCC tumors compared with normal tissues. Different methods showed similar results. However, there was no significant correlation between XIAP expression and clinicopathologic characteristics. Because of its significantly higher expression in ESCC compared with normal mucosa, it may be a feasible marker for the diagnosis of ESCC.2. XIAP downregulation could increase apoptosis in XIAP high expression ESCC cell lines, KYSE150 and EC9706, but could not increase apoptosis in XIAP low expression ESCC cell lines, TE10 and KYSE150. From this result we could draw a conclusion that the apoptosis was XIAP dependent. And this confirmed the role that XIAP and caspase apoptosis pathway played in pathogenesis of ESCC.3. Compared with XIAP low expression cell lines, TE10 and KYSE510, XIAP siRNA mediated decrease in XIAP expression resulted in enhanced tumor cell killing by Paclitaxel, Cisplatin, Fluorouracil, and Etoposide in greater degree in KYSE510 and EC9706. And XIAP siRNA could enhance the growth inhibitory effect of these drugs more significantly in XIAP high expression cell lines than the low expression cell lines. These data indicated that combination treatments were more effective at inducing cytotoxic effect in tumor cells, compared with treatment of either siRNA or anticancer drug alone. Furthermore, together with the result of the experiment in apoptosis detection, it is clear that the ESCC cell killing was XIAP dependent and downregulation of XIAP did sensitize ESCC cells to chemotherapeutics. These results suggest that XIAP siRNA combined with Paclitaxel, Cisplatin, Fluorouracil, and Etoposide may be a feasible strategy to enhance the effects of chemotherapy in patients with ESCC.更多还原