【作者】 赵菲；

【导师】 李世拥；

【作者基本信息】 第三军医大学 ， 外科学， 2007， 博士

【摘要】 目的:结直肠癌是常见的恶性肿瘤,其预后较差,生存率低,其中结直肠癌的浸润转移是术后复发和导致患者死亡的重要原因。结直肠癌转移是一个多因素、多阶段、多步骤的复杂生物学过程,癌细胞对肿瘤内部缺氧微环境的耐受与适应是发生转移的基础。转录因子缺氧诱导因子-1α（hypoxia inducible factor-1 alpha,HIF-1α）作为感受不同氧环境下氧分压的中枢调控子,除调节细胞内氧稳态外,尚能够调节相关基因的转录活性,积极参与肿瘤的侵袭、转移等病理生理过程。因其作用机制复杂,目前仍缺乏深入了解。为此,本课题在既往对结直肠癌侵袭转移研究的基础上,探讨肿瘤发生发展过程中HIF-1α与FasL的表达,以期进一步了解结直肠癌的侵袭转移机制,为临床防治提供新的靶点与理论依据。方法:1.采用分子克隆方法,将我室已构建的FasL-pcDNA3.1（+）质粒与pcDNA3.1（-）质粒进行重组,得到新的FasL-pcDNA3.1（-）质粒并进行鉴定。通过脂质体转染法将空质粒、FasL-pcDNA3.1（+）与FasL-pcDNA3.1（-）质粒分别转染人直肠癌HR-8348细胞,构建侵袭力不同的结直肠癌细胞HR-8348_L、HR-8348_F和HR-8348_As并加以鉴定;以未转染细胞HR-8348_B为空白对照,观察细胞形态,检测各组细胞的侵袭能力与增殖速度。2.采用化学缺氧法构建四组直肠癌HR-8348细胞缺氧模型,免疫细胞化学方法检测各组细胞缺氧12h后HIF-1α表达水平,计算阳性表达百分率;Western blot方法定量检测缺氧0h、6h、12h及24h各组细胞内HIF-1α的表达;并在缺氧12h时相下,应用流式细胞仪测定各组细胞周期的分布,MTT法测定各组细胞增殖能力的改变,TUNEL法测定各组细胞凋亡状况。3.采用免疫组织化学方法,对120例结直肠癌组织及癌旁组织、30例结肠腺瘤组织及28例结直肠癌肝转移组织中HIF-1α与FasL的表达情况进行检测,并分析HIF-1α表达与结肠癌临床病理特征和FasL水平之间的关系。结果1.新构建质粒FasL-pcDNA3.1（-）符合要求,FasL酶切片断及PCR片断大小正确,测序正确率为99.2%;各质粒转染成功,RT-PCR方法可分别自HR-8348_F细胞和HR-8348_As细胞中得到FasL和As-FasL片断;倒置显微镜下观察各组细胞,见HR-8348_F细胞异型性更明显;单层细胞体外侵袭实验见HR-8348_F细胞穿透Transwell滤膜的细胞数目为12.930±2.434,显著多于HR-8348_B、HR-8348_L和HR-8348_As细胞（分别为8.133±1.959,7.670±2.093和7.870±1.685）（P＜0.05）;HR-8348_B、HR-8348_L、HR-8348_F和HR-8348_As细胞的群体倍增时间分别为28.3h、41.0h、24h和35.4h。2.缺氧12h后,HR-8348_F细胞HIF-1α蛋白阳性率为76.0%,显著高于HR-8348_B、HR-8348_L和HR-8348_As细胞（分别为48.5%,43.0%和39.5%）（P＜0.05）,而后三组细胞间无显著性差异（P＞0.05）。Western blot检测示HIF-1α蛋白于120kD处显色;比较不同缺氧时相各组细胞内HIF-1α的表达水平,缺氧0h与6h,各组样品中HIF-1α表达微量;缺氧12h,HR-8348_F细胞内HIF-1α水平较0h和6h时明显增高（P＜0.05）,而HR-8348_B、HR-8348_L及HR-8348_As细胞内HIF-1α表达与6h时无明显变化（P＞0.05）;缺氧24h,HR-8348_F细胞内HIF-1α表达仍处于较高水平,但与缺氧12h表达量比较差别不显著（P＞0.05）,HR-8348_B、HR-8348_L及HR-8348_As细胞内HIF-1α表达水平则较缺氧12h略有下降,但不具备统计学意义（P＞0.05）;组间比较,缺氧0h、6h时各组细胞HIF-1α蛋白水平无显著性差异（P＞0.05）,缺氧12h、24h HR-8348_F细胞HIF-1α水平显著高于HR-8348_B、HR-8348_L及HR-8348_As细胞（P＜0.01）。测定缺氧状态下的细胞生物学特性,见HR-8348_F细胞的增殖指数（60.43±3.61）显著高于HR-8348_B、HR-8348_L和HR-8348_As细胞（40.01±3.22,41.30±3.96和35.87±4.28）（P＜0.05）,增殖抑制率和凋亡指数（17.30±2.08和13.10±1.06）显著低于HR-8348_B、HR-8348_L、HR-8348_As细胞（33.70±4.56和21.60±1.33,34.20±4.12和20.10±1.17,38.00±4.79和23.90±1.25）（P＜0.05）。3.HIF-1α阳性率在结直肠癌旁粘膜中为5.8%（7/120）,在结肠腺瘤组织中为20.0%（6/30）,在结直肠癌原发灶和转移灶中分别为71.7%（86/120）和89.3%（25/28）;癌原发灶和肝转移灶中HIF-1α的表达水平显著高于腺瘤和癌旁组织（P＜0.01）,腺瘤组织中HIF-1α水平亦高于癌旁粘膜组织（P＜0.05）,而癌原发灶与肝转移灶中HIF-1α水平无明显差异（P＞0.05）。FasL的阳性表达率在癌旁粘膜、结肠腺瘤、癌原发灶和肝转移灶中分别为15.0%（18/120）、23.3%（7/30）、69.2%（83/120）和100.0%（28/28）;癌原发灶和转移灶中FasL的表达水平明显高于腺瘤和癌旁组织（P＜0.01）,而腺瘤和癌旁组织中、癌原发灶与肝转移灶中FasL水平均无显著性差异（P＞0.05）。结直肠癌原发灶中HIF-1α的表达水平与患者性别、年龄、病理组织学类型及分化程度无关（P＞0.05）,而与区域淋巴结转移状况及Dukes分期密切相关（P＜0.05）。发生肝转移患者的癌原发灶中HIF-1α和FasL阳性表达率分别为92.9%（26/28）和85.7%（24/28）,与无肝转移组（分别为65.2%,60/92和64.1%,59/92）相比均差异显著（P＜0.01）;HIF-1α与FasL的表达强度有等级相关性（r=0.924,P＜0.01）。结论:1.应用分子克隆技术将正义FasL片断转染入结直肠癌细胞内可构建具有高侵袭力的细胞,结直肠癌细胞内FasL的表达强度与其侵袭能力呈正相关;2.结直肠癌细胞内FasL表达增强可促进缺氧状态下细胞HIF-1α表达增高,促进细胞对微环境缺氧的适应,导致细胞的增殖加速,凋亡减少,侵袭能力进一步增强;3.结直肠癌组织中HIF-1α的表达水平与FasL水平呈正相关,并与肿瘤细胞的生物学行为关系密切,HIF-1α表达水平可作为预测结直肠癌侵袭转移情况的参考指标之更多还原

【Abstract】 Background:Colorectal carcinoma is one of the common malignancies with poor prognosis and low survival rate,and the infiltration and metastasis are the key agents leading to death of patients.The metastasis of colorectal carcinoma is a comprehensive biological procedure with multiple factors,multiple stages and multiple steps.The tolerance and accommodation of cells to intratumoral hypoxia is the basement of tumor metastasis. Hypoxia inducible factor-1 alpha（HIF-1α）,which is the center regulator of oxygen pressure, can adjust related genetic transcription,participate in tumor invasion and metastasis as well as adjust the intracellular oxygen steady state.The regulation mechanism of HIF-1αin invasion and metastasis of tumor is so complex that it hasn’t been understood clearly.Objective:To study the expression of HIF-1αand FasL,to expolre the mechanisms of invasion and metastasis in colorectal carcinoma,and to provid a new target and theory basement for tumor therapy.Methods:1.With the method of molecular cloning,the FasL-pcDNA3.1（+） and pcDNA3.1（-） plasmid were reconstructed to get the new FasL-pcDNA3.1（-） plasmid which had been verificated.With the method of lipid infection,the empty plasmid,FasL-pcDNA3.1（+） and FasL-pcDNA3.1（-） were transfected into human rectal carcinoma cells HR-8348,then HR-8348 cells with different invasive ability,HR-8348_L,HR-8348_F and HR-8348_As were constructed and verificated,and the non-transfected cell HR-8348_B was blank.The cells shape were observed,the invasion ability and proliferation speed were detected in all groups.2.Hypoxia models for HR-8348_B,HR-8348_L,HR-8348_F and HR-8348_As were constructed with chemical modelling.Then the expression of HIF-1αin all groups was quantitated at 0h,6h,12h and 24h after hypoxia with Western blot.At 12h after hypoxia, the HIF-1αlevels were detected with immunocytochemistry and the percents of positive cell were calculated;besides,cell distributions of different cell life cycle were detected with flow cytometry;cell reproductive activities were detected with the method of MTT;and cell apoptosis status was assessed with TUNEL.3.The expression of HIF-1αand FasL were detected by immunohistochemistry in the specimens from 120 colon cancer patients and 30 colon adenoma patients,and the association of HIF-1αexpression level and clinical pathological characteristics,including FasL expression level of colorectal cancer were analyzed.Results1.The new constructed plasmid FasL-pcDNA3.1（-） was consistent with the requirement,which size of enzyme cutting parts was right,and the exactitude ratio of sequencing was 99.2%.The part of FasL and As-FasL could be got from HR-8348_F cell and HR-8348_As cell with RT-PCR,respectively,certificating the transfections of all plasmids were successful.With inverted microscope,the heteromorphism of HR-8348_F cell was more obviously.The number of HR-8348_F cells（12.930±2.434） which penetrated Transwell was significantly more than that of HR-8348_B（8.133±1.959）,HR-8348_L（7.670±2.093） and HR-8348_As cells（7.870±1.685）,respectively（P＜0.05） in cell monolayer intrafiltration experiment in vitro.The population doubling time of HR-8348_B,HR-8348_L,HR-8348_F and HR-8348_As was 28.3h,41.0h,24h and 35.4h,respectively.2.At 12h after hypoxia,the positive rate of HIF-1αexpression was higher in HR-8348_F cells（76.0%） than in HR-8348_B（48.5%）,HR-8348_L（43.0%） and HR-8348_As cells（39.5%）,respectively（P＜0.05）,and there was no significant differences among those latter 3 groups（P＞0.05）.HIF-1αprotein was coloration at 120kD by Western blot,and the expression level of HIF-1αwas not markedly different in all groups at 0h and 6h after hypoxia（P＞0.05）,but was significantly higher in HR-8348_F cell than in HR-8348_B, HR-8348_L and HR-8348_As cell（P＜0.01） at 12h and 24h after hypoxia.Under the environment of hypoxia,the proliferation index was significantly higher in HR-8348_F （60.43±3.61） than in HR-8348_B（40.01±3.22）,HR-8348_L（41.30±3.96） and HR-8348_As cell （35.87±4.28）,respectively（P＜0.05）,however,both inhibition rate of proliferation and apoptotic index were significantly lower in HR-8348_F（17.30±2.08 and 13.10±1.06） thanin HR-8348_B（33.70±4.56 and 21.60±1.33）,HR-8348_L（34.20±4.12 and 20.10±1.17）, HR-8348_As（38.00±4.79 and 23.90±1.25）,respectively（P＜0.05）.3.The positive rate of HIF-1αexpression was 71.7%（86/120） in colorectal cancer and 89.3%（25/28） in hepatic metastasis,which were obviously higher than that in colon adenoma（20.0%,6/30） and normal tissue（5.8%,7/120）（P＜0.01）.Similarly,the positive rate of FasL expression in colorectal cancer（69.2%,83/120） and hepatic metastasis（100.0%, 28/28） were obviously higher than that in colon adenoma（23.3%,7/30） and normal tissue （15.0%,18/120）.There was no relation between HIF-1αexpression and the sex,age, pathohistology type and differentiation degree of colon cancer patients（P＞0.05）,but close relation with lymph nodes state and Dukes staging（P＜0.05）.Furthermore,the positive rate of HIF-1αand FasL expression in primary focus with hepatic metastasis（92.9%,26/28 and 85.7%,24/28） were markedly higher than those without hepatic metastasis（65.2%,60/92 and 64.1%,59/92）（P＜0.01）.Expression of HIF-1αand FasL were positive correlation in metastasis of colorectal carcinoma（r=0.924,P＜0.01）.Conclusions:1.The expression intensity of intracellular FasL is positive correlation with the invasive ability in colorectal carcinoma.2.The expression enhancement of intracellular FasL in colorectal carcinoma could increase the expression of HIF-1α,which could promote tumor cells to adapt hypoxia,and lead to accelerated proliferation and reduced apoptosis of cells.3.The expression level of HIF-1αin colorectal cancer tissue is positive correlated to the level of FasL,which is highly correlated to the cytobiologic characteristics of colorectal carcinoma,and may be a valuable reference to estimate tumor invasion and metastasis.更多还原