肿瘤细胞逃逸Fas系统介导的免疫杀伤的研究
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摘要
对于外周血干细胞移植支持下大剂量化疗治疗的实体瘤患者，治疗后免疫功能重建并清除残留病灶是关键问题。利用基因治疗和免疫治疗手段作为防止肿瘤复发的有效武器有着巨大希望。Fas抗原(APO-1／CD95)与Fas配体(FasL)组成的Fas系统，在将凋亡信号转导入细胞内发挥重要作用。效应细胞FasL结合靶细胞Fas抗原后，在数小时内通过细胞凋亡途径使靶细胞死亡。近年来很多研究发现Fas在多种细胞表面均有表达，而FasL表达只限于少量细胞类型，诸如淋巴细胞、免疫特权器官的细胞和多种恶性肿瘤细胞，同时已证实表达FasL的肿瘤周围激活的Fas阳性淋巴细胞凋亡异常增加。另一方面，肿瘤的发生在很大程度上是因为转化的细胞不能正常凋亡所致。因此，研究如何抑制肿瘤的Fas反击(counterattack)和如何促进肿瘤细胞的凋亡将为肿瘤治疗提供新的途径。通过对FasL功能的深入研究将有助于充分认识FasL基因治疗的潜力，甚至提供一种有效的治疗措施。
根据早期的文献，通过表达Fas配体对Fas介导凋亡途径的抵抗可能会使多种肿瘤对机体免疫系统进行优先攻击或反击。从概念上讲，消除肿瘤的Fas反击(counterattack)是肿瘤免疫治疗的一个令人兴奋的有效目标。我们将把表达Fas的T淋巴细胞和表达FasL的肿瘤细胞的体外相互作用作为本项目的研究对象，利用反义FasL核酸技术拮抗肿瘤细胞的FasL表达或功能，以达到消除肿瘤的Fas反击并延长TIL寿命的目的，这将最终促进免疫功能重建，从而提高抗肿瘤的效果。
天津医科大学硕士研究生学位论文
第一部分
肿瘤细胞逃逸Fas系统介导的免疫杀伤的研究
为研究结肠肿瘤细胞系表达FasL逃逸免疫监视的可能，研究中采用了敏感
的免疫组织化学SABC法观察结肠癌细胞系和JurkatT淋巴细胞系Fas与FasL
的表达，为癌细胞的Fas和FasL的功能提供形态学依据。结果提示SW620结
肠癌细胞FasL呈阳性，阳性物质定位于细胞膜及胞质，胞核为阴性反应;Jurkat
T淋巴细胞FasL呈阳性。JurkatT淋巴细胞呈Fas呈阳性，阳性物质定位于细
胞膜及胞质，胞核为阴性反应;而结肠癌Sw620细胞Fas呈阴性。我们采用
非放射性细胞毒分析，测定sw620结肠癌细胞与Jurkat靶细胞共培养后LDH
释放值检测效应细胞的杀伤效应。同时测定靶细胞自发释放值、靶细胞最大释
放值、效应细胞自发释放值、体积校正值和培养基背景值。应用如下公式计算
杀伤效应:杀伤效应(%)=(实验值一效应细胞自发释放值一靶细胞自发释
放值)/(靶细胞最大释放值一靶细胞自发释放值)x 100。结果提示在结肠
癌细胞系表达功能性的FasL，能杀伤Fas阳性的JurkatT淋巴细胞。
第二部分
人FasL反义真核表达载体的构建和鉴定
为了研究抑制肿瘤的Fas反击(contera沈昵k)的可行性，一种新的重组真核表
达载体peDNA3 .1~hFL一s被构建。将人FasL cDNA亚克隆入人巨细胞病毒
(cMv)启动子控制基因表达质粒peDNA3 .1乃al位点构建真核表达载体
PcDNA3.1-hFL一ASo PcDNA3.1-hFL一S酶切鉴定结果显示分别为1.85kb和
4.55kb片段。基因序列分析确定人FasL cDNA被正确插人载体，证实核昔酸
2
天津医科大学硕士研究生学位论文
序列正确。重组真核表达载体 pCDNA3．lhFL ots的成功构建使进一步研究
FasL的功能成为可能，甚至提供一种有效的治疗措施。
For solid tumor patients undergoing high-dose chemotherapy sustained by peripheral blood stem cell transplantation (PSCH), it is crucial to improve immune reconstitution and get rid of remaining cancer. Gene therapy and irnmuotherapeutic approaches hold great promise as effective weapons against the recurrence of tumor. The Fas/Fas ligand (FasL) system plays an important role in the transduction of apoptotic signal into the cells. Within several hours after the engagement of target cells Fas by effector cells FasL, target cells undergo cell death by way of the Fas-mediated apoptosis. In recent years, numerous studies have demonstrated that Fas is expressed on the surface of cells of various types, whereas FasL expression is restricted to a small number of cell types, such as lymphocytes, cells of the immune-privileged organs and many types of malignant tumor cells. Meanwhile, evidence has pointed to an abnormal increase in apoptosis among activated Fas-positive lymphocytes, mainly in the periphery of the Fas
L-expressing tumors. On the other hand, to a great extent, the occurrence of tumor is due to the fact that the converted cells can not undergo a normal process of apoptosis. Therefore, a research about how to disarm the tumor's Fas counterattack and how to promote tumor cell apoptosis will provide new approaches to the therapy of tumors. A better understanding of FasL function will help to fully realize the potential of the FasL gene therapy and even offer a potential therapeutic intervention.
According to earlier literature, resistance to apoptosis through the Fas receptor pathway coupled with expression of the Fas ligand might enable many cancers to deliver a pre-emptive strike or counterattack against the immune system. Disarming
the Fas counterattack is a conceptually appealing and exciting potential goal for tumor immunotherapy. Our research is about the interaction in vitro between T cells that express Fas and tumor cells that express FasL. The approach of this project is to block the expression or function of FasL on the tumor cell by antisensing FasL, with a view to abrogate the Fas counterattack and prolong TIL's life span, which will ultimately regulate immune recovery and enhance antitumoral efficacy.
Part I The study on tumor cells escaping from Fas-mediated apoptosis
To investigate the possibility of colon cancer cells escaping from immune surveillance through the expression of FasL on tumor cells, a study was undertaken by using immunohistochemical SABC method. The expression of Fas receptor and Fas ligand in SW620 colon carcinoma cell line and Jurkat T cells was observed so as to supply morphological evidence for the functions of Fas receptor and Fas ligand. It was shown that the Fas ligand of colon carcinoma SW620 cells was positive. The positive substances were distributed in the cell membrane and cytoplasm while the nuclei of the cells were negative, and the Fas ligand of Jurkat T lymphocytes turned out to be positive. Meanwhile, it was also shown that the Fas receptor of Jurkat T cells was positive. The positive substances were distributed in the cell membrane and cytoplasm while the nuclei of the cells were negative, and the Fas receptor of
colon carcinoma SW620 cells was negative. In an effort to examine the cytotoxicity of effective cells, we adopted CytoTox 96 Non-Radioactive Cytotoxicity Assay to measure LDH releasing value after the SW620 cells were co-cultured with the Jurkat T lymphocytes. At the same time, we also measured the values of Effector Cell Spontaneous LDH Release, Target Cell Spontaneous LDH Release, Target Cell Maximum LDH Release, Volume Correction Control and Culture Medium Background. Then the following formula was applied in the calculation of percent
cytotoxicity: Cytotoxicity (%) = (Experimental- Effector Spontaneous - Target Spontaneous) / (Target Maximum - Target Spontaneous) X 100. The result indicates that FasL expression in the colon carcinoma SW620 cells could inversely induce apoptosis of Fas-expressing Jurkat T lymphocytes.
Part
根据早期的文献，通过表达Fas配体对Fas介导凋亡途径的抵抗可能会使多种肿瘤对机体免疫系统进行优先攻击或反击。从概念上讲，消除肿瘤的Fas反击(counterattack)是肿瘤免疫治疗的一个令人兴奋的有效目标。我们将把表达Fas的T淋巴细胞和表达FasL的肿瘤细胞的体外相互作用作为本项目的研究对象，利用反义FasL核酸技术拮抗肿瘤细胞的FasL表达或功能，以达到消除肿瘤的Fas反击并延长TIL寿命的目的，这将最终促进免疫功能重建，从而提高抗肿瘤的效果。
天津医科大学硕士研究生学位论文
第一部分
肿瘤细胞逃逸Fas系统介导的免疫杀伤的研究
为研究结肠肿瘤细胞系表达FasL逃逸免疫监视的可能，研究中采用了敏感
的免疫组织化学SABC法观察结肠癌细胞系和JurkatT淋巴细胞系Fas与FasL
的表达，为癌细胞的Fas和FasL的功能提供形态学依据。结果提示SW620结
肠癌细胞FasL呈阳性，阳性物质定位于细胞膜及胞质，胞核为阴性反应;Jurkat
T淋巴细胞FasL呈阳性。JurkatT淋巴细胞呈Fas呈阳性，阳性物质定位于细
胞膜及胞质，胞核为阴性反应;而结肠癌Sw620细胞Fas呈阴性。我们采用
非放射性细胞毒分析，测定sw620结肠癌细胞与Jurkat靶细胞共培养后LDH
释放值检测效应细胞的杀伤效应。同时测定靶细胞自发释放值、靶细胞最大释
放值、效应细胞自发释放值、体积校正值和培养基背景值。应用如下公式计算
杀伤效应:杀伤效应(%)=(实验值一效应细胞自发释放值一靶细胞自发释
放值)/(靶细胞最大释放值一靶细胞自发释放值)x 100。结果提示在结肠
癌细胞系表达功能性的FasL，能杀伤Fas阳性的JurkatT淋巴细胞。
第二部分
人FasL反义真核表达载体的构建和鉴定
为了研究抑制肿瘤的Fas反击(contera沈昵k)的可行性，一种新的重组真核表
达载体peDNA3 .1~hFL一s被构建。将人FasL cDNA亚克隆入人巨细胞病毒
(cMv)启动子控制基因表达质粒peDNA3 .1乃al位点构建真核表达载体
PcDNA3.1-hFL一ASo PcDNA3.1-hFL一S酶切鉴定结果显示分别为1.85kb和
4.55kb片段。基因序列分析确定人FasL cDNA被正确插人载体，证实核昔酸
2
天津医科大学硕士研究生学位论文
序列正确。重组真核表达载体 pCDNA3．lhFL ots的成功构建使进一步研究
FasL的功能成为可能，甚至提供一种有效的治疗措施。
For solid tumor patients undergoing high-dose chemotherapy sustained by peripheral blood stem cell transplantation (PSCH), it is crucial to improve immune reconstitution and get rid of remaining cancer. Gene therapy and irnmuotherapeutic approaches hold great promise as effective weapons against the recurrence of tumor. The Fas/Fas ligand (FasL) system plays an important role in the transduction of apoptotic signal into the cells. Within several hours after the engagement of target cells Fas by effector cells FasL, target cells undergo cell death by way of the Fas-mediated apoptosis. In recent years, numerous studies have demonstrated that Fas is expressed on the surface of cells of various types, whereas FasL expression is restricted to a small number of cell types, such as lymphocytes, cells of the immune-privileged organs and many types of malignant tumor cells. Meanwhile, evidence has pointed to an abnormal increase in apoptosis among activated Fas-positive lymphocytes, mainly in the periphery of the Fas
L-expressing tumors. On the other hand, to a great extent, the occurrence of tumor is due to the fact that the converted cells can not undergo a normal process of apoptosis. Therefore, a research about how to disarm the tumor's Fas counterattack and how to promote tumor cell apoptosis will provide new approaches to the therapy of tumors. A better understanding of FasL function will help to fully realize the potential of the FasL gene therapy and even offer a potential therapeutic intervention.
According to earlier literature, resistance to apoptosis through the Fas receptor pathway coupled with expression of the Fas ligand might enable many cancers to deliver a pre-emptive strike or counterattack against the immune system. Disarming
the Fas counterattack is a conceptually appealing and exciting potential goal for tumor immunotherapy. Our research is about the interaction in vitro between T cells that express Fas and tumor cells that express FasL. The approach of this project is to block the expression or function of FasL on the tumor cell by antisensing FasL, with a view to abrogate the Fas counterattack and prolong TIL's life span, which will ultimately regulate immune recovery and enhance antitumoral efficacy.
Part I The study on tumor cells escaping from Fas-mediated apoptosis
To investigate the possibility of colon cancer cells escaping from immune surveillance through the expression of FasL on tumor cells, a study was undertaken by using immunohistochemical SABC method. The expression of Fas receptor and Fas ligand in SW620 colon carcinoma cell line and Jurkat T cells was observed so as to supply morphological evidence for the functions of Fas receptor and Fas ligand. It was shown that the Fas ligand of colon carcinoma SW620 cells was positive. The positive substances were distributed in the cell membrane and cytoplasm while the nuclei of the cells were negative, and the Fas ligand of Jurkat T lymphocytes turned out to be positive. Meanwhile, it was also shown that the Fas receptor of Jurkat T cells was positive. The positive substances were distributed in the cell membrane and cytoplasm while the nuclei of the cells were negative, and the Fas receptor of
colon carcinoma SW620 cells was negative. In an effort to examine the cytotoxicity of effective cells, we adopted CytoTox 96 Non-Radioactive Cytotoxicity Assay to measure LDH releasing value after the SW620 cells were co-cultured with the Jurkat T lymphocytes. At the same time, we also measured the values of Effector Cell Spontaneous LDH Release, Target Cell Spontaneous LDH Release, Target Cell Maximum LDH Release, Volume Correction Control and Culture Medium Background. Then the following formula was applied in the calculation of percent
cytotoxicity: Cytotoxicity (%) = (Experimental- Effector Spontaneous - Target Spontaneous) / (Target Maximum - Target Spontaneous) X 100. The result indicates that FasL expression in the colon carcinoma SW620 cells could inversely induce apoptosis of Fas-expressing Jurkat T lymphocytes.
Part
引文
1.Cheng JH, Zhou T, Liu CD,et al. Protection from Fas-mediated apoptosis by a soluble form of the Fas molecule. Science 1994;263:1759-1762
2.Mariani SM, Matiba B, et al. Regulation of cell surface Apo-1/Fas (CD95) ligand expression by metalloproteases. EUR J Immunol, 1995, 25(8): 2303
3．侯力，吕申。细胞凋亡与肿瘤转移。国外医学—生理、病理科学与临床分册，2000；20(3)：207
4.Green D, Kroemer G.The central executioners of apoptosis or mitochondria? Trends in Cell Biology, 1998;8:267
5．欧阳高亮。细胞凋亡与肿瘤的发生发展和治疗。国外医学—肿瘤学分册，2000；27(5)：266
6.Perter ME, Krammer PH. Mechanisms of CD95-mediated apoptosis. Curr Opin Immumol, 1998; 10(5):545-551
7．郑徽。Caspases与细胞凋亡。癌变·畸变·突变，2000；12(2)：120
8.Dayan CM, Elsegood KA, Maile R. FasL expression on epithelial cells: the Bottazzo-Feldman hypothesis revisited. [J]. Immunol Today, 1997,18(5):203
9.MaA. Fas and the immunologically underprivileged intestine. Gastroenterology, 1997; 113:345-348
10. Joe O'Connell, Michael W. Bennett, Gerald C.O'Sullivan. The Fas counterattack: cancer as a site of immune privilege. IMMUNOLOGY TODAY,1999;20:46-52
11．郝希山主编 简明肿瘤学 北京：人民卫生出版社，第一版，2001：458
12．李世荣。进一步提高我国大肠癌的早期诊断水平。世界华人消化杂志，2001；9：737-738
13．张振书，张亚历。中国大肠癌研究进展。世界华人消化杂志，2001；9：489-494
14．来茂德。中国结直肠癌发生发展相关基因。世界华人消化杂志，2001；9：1227-1232
15．郁宝铭，赵任。大肠癌分子生物学研究的现状。世界华人消化杂志，1999；7：173-175
16．盛剑秋，晨智敏。大肠癌相关基因对大肠癌的筛检。世界华人消化杂志，2001；9：783-785
17. Yuan P, Sun MH, Zhang JS, Zhu XZ, Shi DR. APC and K-ras gene mutation in crypt foci of human colon. World J Gastroenterol, 2001; 7:352-356
18．范如英。大肠癌粪便脱落细胞及其基因的检测。世界华人消化杂志，2001；9：787-789
19．庄小强，赖日权，孙桂华，王小怀，袁世珍。大肠癌P53蛋白与PCNA表达的预后意义。世界华人消化杂志，1999；7：616
20．许沈华，冯建国，李德川，牟潮舟，楼荣灿。大肠癌患者外周血CD~(44)水平与临床病理的相关性研究。世界华人消化杂志，2000；8：432-435
21．秦环龙，林擎天。p53，nm23一H1和CEA表达与大肠癌术后肝脏转移和局部复发的关系。世界华人消化杂志，2000；8：589-590
22．谷化平，倪灿荣，詹熔洲。大肠癌CD15，CD44v6和nm23H1的mRNA表达与转移及预后的相关性。世界华人消化杂志，2000；8：887-891
23．乔庆，吴金生，张静，马庆久，赖大年。凋亡相关基因bcl-2，bax在人类大肠腺癌中的表达意义。世界华人消化杂志，1999；7：936-938
24．周志宏，沈志样。大肠癌肿瘤坏死因子的研究进展。世界华人消化杂志，2000；8：700-701
25．沈云芳，庄惠，沈健伟，陈士筷。细胞凋亡与肿瘤。世界华人消化杂志，1999；7：267-268
26．顾小红，李奇芬，王宇明。丁型肝炎患者肝组织中Fas／FasL和肝细胞凋亡表达。世界华人消化杂志，2000；8：35-38
27．李瑛，李家邦，陈军，陈松。Hp~+消化性溃疡患者胃粘膜上皮细胞凋亡／增生与Fas／FasL表达的关系。世界华人消化杂志，2001；9：1404-1408
28．李琳，陈乃玲，陈佩兰、慢性肝病患者肝组织Fas抗原的表达及意义。世界华人消化杂志，1999；7：84-85
29．梁云，陆斌，崔贞福，李晓冬，郭亚军，刘彦君。肝细胞癌中Fas／FasL表达的意义。世界华人消化杂志，2001；9：1364-1368
30．陆星海，王江滨，张俊格。凋亡相关因子FasL在大肠癌组织中的表达规律研究。世界华人消化杂志，2000；8：1184-1186
31. M.MUSCHEN,C.MOERS, U.WARSKULAT, J.EVEN. CD95 ligand expression as a mechanism of Immune escape in breast cancer, Immunology 2000;99:69-77
32．丁尔迅，张伟，王强等。细胞因子上调大肠癌细胞Fas配体表达。癌症，2000；19(11)：966-968
33．周京旭。Fas和FasL与成熟T淋巴细胞的免疫反应调节。国外医学—免疫学分册，2000；24(4)：246
34．李莉，李先兴，张玲珍等。淋巴细胞Fas和配体FasL表达变化及其抗肿瘤活性的关系。解放军医学杂志，1999；24(1)：28
35．肖雪媛，杨贵贞，杨祟敬。淋巴细胞表面APO-1受体的表达与其凋亡敏感性的相关性研究。中国实验临床免疫学杂志，1998；10(3)：163
36. Hahne M, Rimoldi M, Schroter M et al. Melanoma cell expression of Fas(Apo-1/CD95) ligand: implications for tumor immune escape. Science, 1996,Nov 22;274:1363
37. O'Connell J, O'Sullivan GC, Collins JK et al. The Fas counterattack:Fas-mediated T cell killing by colon cancer cells expressing Fas ligand. J Exp Med, 1996,Sep 1; 184(3): 1075
38． Strand S, Hofmann WJ, Hug H et al. Lymphocyte apoptosis induced by CD95 (APO-1/Fas) ligand-expressing tumor cells-a mechanism of immune evasion? Nat Med, 1996,Dec;2(12) :1361
39． Lee SH, Jang JJ, Lee JY et al. Immunohistochemical analysis of Fas ligand expression in sarcomas. Sarcomas express high level of FasL in vivo. APMIS,1998,Nov;106(11) :1035
40． Ng CS, Lo ST, Chan JK. Peripheral T and putative natural killer cell lymphomas commonly coexpress CD95 and CD95 ligand. Hum Pathol,1999,Jan;30(1) :48-53
41． Hatake K, Tomizuka H, Dceda M et al. Apoptosis-gene expression in hematopoietic system: normal and pathological conditions (Review). Int J Mol Med,1998,Jan;1(1) : 121-129
42． Niehans GA, Brunner T, Frizelle SP, et al. Human lung carcinomas express Fas ligand. Cancer Res,1997,Mar 15;57(6) : 1007-1012
43． Saas P, Walker PR, Hahne M, et al. Fas ligand expression by astrocytoma in vivo: maintaining immune privilege in the brain? J Clin Invest,1997,Mar15;99(6) ;1173-1178
44． Uzzo RG, Rayman P, et al. Mechanisms of apoptosis in T cells from patients with renal cell carcinoma. Clin Cancer Res, 1999, 5(5) : 1219
45． Mller P, Koretz K, Leith user F, et al. Expression of APO-1(CD95) , a member of the NGF/TNF receptor superfamily, in normal and neoplastic colon epithelium.Int J Cancer 1994;57:371-377
46． Bennett MW, O' Connell J, Houston A, Kelly J, O' Sullivan GC, Collins JK . Fas ligand upregulation is an early event in colonic carcinogenesis. J Clin Pathol 2001;54:598-640
47． Strater J, Wellisch I, Riedl S, et al. CD(Apo-1/Fas)-mediated apoptosis in colon epithelial cells: a possible role in ulcerative colitis. Gastroenterology 1997;113:160-167
48． Morimoto Y, Hizuta A, Ding EX, et al. Functional expression of Fas and Fas
ligand on human intestinal intraepithelial lymphocytes. Clin Exp Immunol 1999;116:84-89
49． Reyher UV, Starter J, Kittstein W, et al. Colon carcinoma cells as different mechanisms to escape CD95-mediated apoptosis. Cancer Res, 1998;58:526
50． Ungefroten H, Voss M, Jansen M, et al. Human Pancrestic adenocarcinomas express Fas and FasL yet are resistant to Fas-mediated apoptosis. Cancer Res,1998;58:1741
51． Butler LM, Hewwtt PJ, Butler WJ,et al. Down-regulation of Fas expression in colon cancer is not a result of alletic lossor gene rearrangement. Br J Cancer, 1998;77:1454
52． Meterissian SH, Kontogiannea M, Po J, et al. Apoptosis induced in human colorectal carcinoma by anti-Fas antibody. Ann Surg Oncol 1997;4:169-175
53． Kushlinskii NE, Britvin TA, Abbasova SG,et al. Soluble fas antigen in the serum of patients with colon cancer. Bull Exp Biol Med 2001;131:361-363
54． Joe O'Connell, Michael W.Bennett, O'ullivan GC, et al. Fas ligand expression in primary colon adenocarcinomas : evidence that the Fas counterattack is a prevalent mechanism of immune evasion in human colon cancer. J Pathol 1998; 186:240-246
55． Shimoyama M, Kanda T, liu L, et al. Expression of Fas ligand is an early event in colorectal carcinogenesis. J Surg Oncol 2001;76:63-68
56． O' Connell J, O' Sullivan GC, Collins JE, et al. The Fas counterattack : Fas-mediated T cell killing by colon cancer cells expressing Fas ligand. J Exp Med, 1996,184(3) : 1075
57． Mann B, Gratchev GC, Collins JK, et al. FasL is more frequently expressed in liver metastases of colorectal cancer than in matched primary carcinomas. Br J Cancer 1999;79:1262-1269
58． Yoong KF, Afford SC, Randhawa S, et al. Fas/Fas ligand interaction in human colorectal hepatic metastases. Am J Pathol 1999; 154:693-703
59． Song E, Chen J, Ouyang N, et al. Soluble Fas ligand released by colon
adenocarcinoma cells induces host lymphocyte apoptosis: an ative mode of immune evasion in colon cancer. Br J Cancer 2001 ;85:1047-1054
60． Pitti RM, Marsters SA, Lawrence DA, et al. Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer. Nature 1998;396:699-703
61． Von Reyher U, Strrer J, Kittstein W, et al. Colon carcinoma cells use different mechanisms to escape CD95-mediated apoptosis. Cancer Res 1998;58:526-534
62． Sawa E, Takahashi M, Kamishohara M, et al. Structural modification of Fas C-terminal tripeptide and its effects on the inhibitory activity of Fas/FAP-1 binding. J Med Chem 1999;42:3289-3299
63． Ryu BK, Lee MG, Chi SG, et al. Increased expression of cFLIPL in colonic adenocarcinoma. J Pathol 2001;194:15-19
64． Xu X, Fu XY, Plate J, et al. IFN-r induced cell growth inhibition by Fas-mediated apoptosis : requirement of STAT1 protein for up-regulation of Fas and FasL expression. Cancer Res 1998;58:2832-2837
65． Joe O'Connell, Michael W.Bennett, Nally K, et al. Interferon-gamma sensitizes colonic epithelial cell lines to physiological and therapeutic inducers of colonocyte apoptosis. J Cell Physiol 2000;185:331-338
66． Koshiji M, Adachi Y, Sogo S, et al. Apoptosis of colorectal adenocarcinoma (COLO201) by tumor necrosis factor-alpha(TNF-alpha)and/or interferon-gamma(IFN-gamma),resulting from down-modulation of Bcl-2 expression, din Exp Immunol 1998;111:211-218
67． Song E, Chen J, Ouyang N, et al. Kupffer cells of cirrhotic rat lives sensitize colon cancer cells to Fas-mediated apoptosis. Br J Cancer 2001;84:1265-1271
68． Houghton JA, Harwood FG, Gibson AA, et al. The fas signaling pathway is functional in colon carcinoma cells and induces apoptosis. Clin Cancer Res 1997;3(12ptl):2205-2209
69． Fukazawa T, Fujiwara T, Morimoto Y, et al. Differential involvement of the CD95(Fas/APO-1) receptor/ligand system on apoptosis induced by the wild-type
p53 gene transfer in human cancer cells. Oncogene 1999;18:2189-2199
70． Mllauer L, Gruber P, Sebinger D, et al. Mutations in apoptosis genes : a pathogenetic factor for human disease . Mutat Res 2001 ;488:211-231
71． Micheau O, Solary E, Hammann A, et al. Fas ligand-indepcndent, FADD-mediated activation of the Fas death pathway by anticancer drugs. J Immunol 1995;154:1239-1245
72． Linsley PS, Ledbetter JA. The role of the CD28 receptor during T cell responses to antigen. J Exp Med,1999;190(7) :891
73． Le A, Villunger A, Kos M, et al. Modulation of Apo-1/Fas(CD95) -induced programmed cell death in myeloma cells by interferon-alpha. Eur J Immunol 1996;26(12) :3119
74． 郑世营，张志德，李德春等。腺病毒介导FasL基因转移及对胃癌细胞生长和凋亡的影响。中国肿瘤临床，2001；28(12) ：905
75． El Deiry WS, Tokino T, Velculescu VE, et al. WAF1, a potential mediator of p53 tumor suppression. Cell, 1993;75(4) :817
76． Spillare EA, Okamoto A, Hagiwara K et al. Suppression of growth in vitro and tumorigenicity in vivo of human carcinoma cell lines by transfected p16INK. Mol Carcinog, 1996;16(1) :53
77． Eguchi Y. Antisense RNA. Annu.Rev.Biochem. 1991,60:631-52
78． Denhardt DT. Mechanism of action of antisense RNA. Ann.N.Y. Acad. Sci. 1992;660:70-76
79． Zamecnik P, Stephenson M. Proc Natl Acad Sci USA, 1978;75:280-284
80． Dachs GU, Doupherty GJ, Stratford IJ, et al. Targeting gene therapy to cancer: a review. Oncol Res 1997;9:313-25
81． Steiner MS, Anthony CT, Holt JT. Antisense c-myc retroviral vector suppresses established human prostate cancer. Human Gene Therapy. 1998;9:747-755
82． Liu XD, Turbyville T, Fritz A,et al. Inhibition of Insulin-like growth factor I receptor expression in neuroblastoma cells induces the regression of
tumors in mice. Cancer Res. 1998; 58:5432-5438
83.Fakhrai H, Dorigo O, Shawler DL, et al. Eradication of established intracranial rat gliomas by transforming growth factor β antisense gene therapy. Proc.Natl. Acad. Sci. USA,1996;93:2909-2914
84．吴乃虎主编 基因工程原理 北京：科学出版社，第二版，1999：137-152，310-318
85．J.萨母布鲁克，EF.弗里奇，T.曼尼阿蒂斯主编 分子克隆实验指南 北京：科学出版社，第二版，1995：40-55
86.Helene C. Control of oncogene expression by antisense nucleic acids. Eur J Cancer 1994;30AA: 1721-1726
87.Gibson I. Antisense approaches to the gene therapy of cancer. Cancer and Metastasis. Rev 1996; 15:287-299
88.Chrostine VB & Catherine V. Do natural antisense transcripts make sense in eukaryotes? Gene 1998,211:1-9
89.Roth JA. Modification of mutant K-ras gene expression in non-small cell lung cancer(NSXLC). Human Gene Therapy 1996;7:785
90．曹蘶。反义技术及其在医药领域中应用的最新进展。军事医学科学院院刊1994；18(2)：149-152
91．卢圣栋主编 现代分子生物学实验技术 北京：中国协和医科大学出版社，第二版，1999：256-90
2.Mariani SM, Matiba B, et al. Regulation of cell surface Apo-1/Fas (CD95) ligand expression by metalloproteases. EUR J Immunol, 1995, 25(8): 2303
3．侯力，吕申。细胞凋亡与肿瘤转移。国外医学—生理、病理科学与临床分册，2000；20(3)：207
4.Green D, Kroemer G.The central executioners of apoptosis or mitochondria? Trends in Cell Biology, 1998;8:267
5．欧阳高亮。细胞凋亡与肿瘤的发生发展和治疗。国外医学—肿瘤学分册，2000；27(5)：266
6.Perter ME, Krammer PH. Mechanisms of CD95-mediated apoptosis. Curr Opin Immumol, 1998; 10(5):545-551
7．郑徽。Caspases与细胞凋亡。癌变·畸变·突变，2000；12(2)：120
8.Dayan CM, Elsegood KA, Maile R. FasL expression on epithelial cells: the Bottazzo-Feldman hypothesis revisited. [J]. Immunol Today, 1997,18(5):203
9.MaA. Fas and the immunologically underprivileged intestine. Gastroenterology, 1997; 113:345-348
10. Joe O'Connell, Michael W. Bennett, Gerald C.O'Sullivan. The Fas counterattack: cancer as a site of immune privilege. IMMUNOLOGY TODAY,1999;20:46-52
11．郝希山主编 简明肿瘤学 北京：人民卫生出版社，第一版，2001：458
12．李世荣。进一步提高我国大肠癌的早期诊断水平。世界华人消化杂志，2001；9：737-738
13．张振书，张亚历。中国大肠癌研究进展。世界华人消化杂志，2001；9：489-494
14．来茂德。中国结直肠癌发生发展相关基因。世界华人消化杂志，2001；9：1227-1232
15．郁宝铭，赵任。大肠癌分子生物学研究的现状。世界华人消化杂志，1999；7：173-175
16．盛剑秋，晨智敏。大肠癌相关基因对大肠癌的筛检。世界华人消化杂志，2001；9：783-785
17. Yuan P, Sun MH, Zhang JS, Zhu XZ, Shi DR. APC and K-ras gene mutation in crypt foci of human colon. World J Gastroenterol, 2001; 7:352-356
18．范如英。大肠癌粪便脱落细胞及其基因的检测。世界华人消化杂志，2001；9：787-789
19．庄小强，赖日权，孙桂华，王小怀，袁世珍。大肠癌P53蛋白与PCNA表达的预后意义。世界华人消化杂志，1999；7：616
20．许沈华，冯建国，李德川，牟潮舟，楼荣灿。大肠癌患者外周血CD~(44)水平与临床病理的相关性研究。世界华人消化杂志，2000；8：432-435
21．秦环龙，林擎天。p53，nm23一H1和CEA表达与大肠癌术后肝脏转移和局部复发的关系。世界华人消化杂志，2000；8：589-590
22．谷化平，倪灿荣，詹熔洲。大肠癌CD15，CD44v6和nm23H1的mRNA表达与转移及预后的相关性。世界华人消化杂志，2000；8：887-891
23．乔庆，吴金生，张静，马庆久，赖大年。凋亡相关基因bcl-2，bax在人类大肠腺癌中的表达意义。世界华人消化杂志，1999；7：936-938
24．周志宏，沈志样。大肠癌肿瘤坏死因子的研究进展。世界华人消化杂志，2000；8：700-701
25．沈云芳，庄惠，沈健伟，陈士筷。细胞凋亡与肿瘤。世界华人消化杂志，1999；7：267-268
26．顾小红，李奇芬，王宇明。丁型肝炎患者肝组织中Fas／FasL和肝细胞凋亡表达。世界华人消化杂志，2000；8：35-38
27．李瑛，李家邦，陈军，陈松。Hp~+消化性溃疡患者胃粘膜上皮细胞凋亡／增生与Fas／FasL表达的关系。世界华人消化杂志，2001；9：1404-1408
28．李琳，陈乃玲，陈佩兰、慢性肝病患者肝组织Fas抗原的表达及意义。世界华人消化杂志，1999；7：84-85
29．梁云，陆斌，崔贞福，李晓冬，郭亚军，刘彦君。肝细胞癌中Fas／FasL表达的意义。世界华人消化杂志，2001；9：1364-1368
30．陆星海，王江滨，张俊格。凋亡相关因子FasL在大肠癌组织中的表达规律研究。世界华人消化杂志，2000；8：1184-1186
31. M.MUSCHEN,C.MOERS, U.WARSKULAT, J.EVEN. CD95 ligand expression as a mechanism of Immune escape in breast cancer, Immunology 2000;99:69-77
32．丁尔迅，张伟，王强等。细胞因子上调大肠癌细胞Fas配体表达。癌症，2000；19(11)：966-968
33．周京旭。Fas和FasL与成熟T淋巴细胞的免疫反应调节。国外医学—免疫学分册，2000；24(4)：246
34．李莉，李先兴，张玲珍等。淋巴细胞Fas和配体FasL表达变化及其抗肿瘤活性的关系。解放军医学杂志，1999；24(1)：28
35．肖雪媛，杨贵贞，杨祟敬。淋巴细胞表面APO-1受体的表达与其凋亡敏感性的相关性研究。中国实验临床免疫学杂志，1998；10(3)：163
36. Hahne M, Rimoldi M, Schroter M et al. Melanoma cell expression of Fas(Apo-1/CD95) ligand: implications for tumor immune escape. Science, 1996,Nov 22;274:1363
37. O'Connell J, O'Sullivan GC, Collins JK et al. The Fas counterattack:Fas-mediated T cell killing by colon cancer cells expressing Fas ligand. J Exp Med, 1996,Sep 1; 184(3): 1075
38． Strand S, Hofmann WJ, Hug H et al. Lymphocyte apoptosis induced by CD95 (APO-1/Fas) ligand-expressing tumor cells-a mechanism of immune evasion? Nat Med, 1996,Dec;2(12) :1361
39． Lee SH, Jang JJ, Lee JY et al. Immunohistochemical analysis of Fas ligand expression in sarcomas. Sarcomas express high level of FasL in vivo. APMIS,1998,Nov;106(11) :1035
40． Ng CS, Lo ST, Chan JK. Peripheral T and putative natural killer cell lymphomas commonly coexpress CD95 and CD95 ligand. Hum Pathol,1999,Jan;30(1) :48-53
41． Hatake K, Tomizuka H, Dceda M et al. Apoptosis-gene expression in hematopoietic system: normal and pathological conditions (Review). Int J Mol Med,1998,Jan;1(1) : 121-129
42． Niehans GA, Brunner T, Frizelle SP, et al. Human lung carcinomas express Fas ligand. Cancer Res,1997,Mar 15;57(6) : 1007-1012
43． Saas P, Walker PR, Hahne M, et al. Fas ligand expression by astrocytoma in vivo: maintaining immune privilege in the brain? J Clin Invest,1997,Mar15;99(6) ;1173-1178
44． Uzzo RG, Rayman P, et al. Mechanisms of apoptosis in T cells from patients with renal cell carcinoma. Clin Cancer Res, 1999, 5(5) : 1219
45． Mller P, Koretz K, Leith user F, et al. Expression of APO-1(CD95) , a member of the NGF/TNF receptor superfamily, in normal and neoplastic colon epithelium.Int J Cancer 1994;57:371-377
46． Bennett MW, O' Connell J, Houston A, Kelly J, O' Sullivan GC, Collins JK . Fas ligand upregulation is an early event in colonic carcinogenesis. J Clin Pathol 2001;54:598-640
47． Strater J, Wellisch I, Riedl S, et al. CD(Apo-1/Fas)-mediated apoptosis in colon epithelial cells: a possible role in ulcerative colitis. Gastroenterology 1997;113:160-167
48． Morimoto Y, Hizuta A, Ding EX, et al. Functional expression of Fas and Fas
ligand on human intestinal intraepithelial lymphocytes. Clin Exp Immunol 1999;116:84-89
49． Reyher UV, Starter J, Kittstein W, et al. Colon carcinoma cells as different mechanisms to escape CD95-mediated apoptosis. Cancer Res, 1998;58:526
50． Ungefroten H, Voss M, Jansen M, et al. Human Pancrestic adenocarcinomas express Fas and FasL yet are resistant to Fas-mediated apoptosis. Cancer Res,1998;58:1741
51． Butler LM, Hewwtt PJ, Butler WJ,et al. Down-regulation of Fas expression in colon cancer is not a result of alletic lossor gene rearrangement. Br J Cancer, 1998;77:1454
52． Meterissian SH, Kontogiannea M, Po J, et al. Apoptosis induced in human colorectal carcinoma by anti-Fas antibody. Ann Surg Oncol 1997;4:169-175
53． Kushlinskii NE, Britvin TA, Abbasova SG,et al. Soluble fas antigen in the serum of patients with colon cancer. Bull Exp Biol Med 2001;131:361-363
54． Joe O'Connell, Michael W.Bennett, O'ullivan GC, et al. Fas ligand expression in primary colon adenocarcinomas : evidence that the Fas counterattack is a prevalent mechanism of immune evasion in human colon cancer. J Pathol 1998; 186:240-246
55． Shimoyama M, Kanda T, liu L, et al. Expression of Fas ligand is an early event in colorectal carcinogenesis. J Surg Oncol 2001;76:63-68
56． O' Connell J, O' Sullivan GC, Collins JE, et al. The Fas counterattack : Fas-mediated T cell killing by colon cancer cells expressing Fas ligand. J Exp Med, 1996,184(3) : 1075
57． Mann B, Gratchev GC, Collins JK, et al. FasL is more frequently expressed in liver metastases of colorectal cancer than in matched primary carcinomas. Br J Cancer 1999;79:1262-1269
58． Yoong KF, Afford SC, Randhawa S, et al. Fas/Fas ligand interaction in human colorectal hepatic metastases. Am J Pathol 1999; 154:693-703
59． Song E, Chen J, Ouyang N, et al. Soluble Fas ligand released by colon
adenocarcinoma cells induces host lymphocyte apoptosis: an ative mode of immune evasion in colon cancer. Br J Cancer 2001 ;85:1047-1054
60． Pitti RM, Marsters SA, Lawrence DA, et al. Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer. Nature 1998;396:699-703
61． Von Reyher U, Strrer J, Kittstein W, et al. Colon carcinoma cells use different mechanisms to escape CD95-mediated apoptosis. Cancer Res 1998;58:526-534
62． Sawa E, Takahashi M, Kamishohara M, et al. Structural modification of Fas C-terminal tripeptide and its effects on the inhibitory activity of Fas/FAP-1 binding. J Med Chem 1999;42:3289-3299
63． Ryu BK, Lee MG, Chi SG, et al. Increased expression of cFLIPL in colonic adenocarcinoma. J Pathol 2001;194:15-19
64． Xu X, Fu XY, Plate J, et al. IFN-r induced cell growth inhibition by Fas-mediated apoptosis : requirement of STAT1 protein for up-regulation of Fas and FasL expression. Cancer Res 1998;58:2832-2837
65． Joe O'Connell, Michael W.Bennett, Nally K, et al. Interferon-gamma sensitizes colonic epithelial cell lines to physiological and therapeutic inducers of colonocyte apoptosis. J Cell Physiol 2000;185:331-338
66． Koshiji M, Adachi Y, Sogo S, et al. Apoptosis of colorectal adenocarcinoma (COLO201) by tumor necrosis factor-alpha(TNF-alpha)and/or interferon-gamma(IFN-gamma),resulting from down-modulation of Bcl-2 expression, din Exp Immunol 1998;111:211-218
67． Song E, Chen J, Ouyang N, et al. Kupffer cells of cirrhotic rat lives sensitize colon cancer cells to Fas-mediated apoptosis. Br J Cancer 2001;84:1265-1271
68． Houghton JA, Harwood FG, Gibson AA, et al. The fas signaling pathway is functional in colon carcinoma cells and induces apoptosis. Clin Cancer Res 1997;3(12ptl):2205-2209
69． Fukazawa T, Fujiwara T, Morimoto Y, et al. Differential involvement of the CD95(Fas/APO-1) receptor/ligand system on apoptosis induced by the wild-type
p53 gene transfer in human cancer cells. Oncogene 1999;18:2189-2199
70． Mllauer L, Gruber P, Sebinger D, et al. Mutations in apoptosis genes : a pathogenetic factor for human disease . Mutat Res 2001 ;488:211-231
71． Micheau O, Solary E, Hammann A, et al. Fas ligand-indepcndent, FADD-mediated activation of the Fas death pathway by anticancer drugs. J Immunol 1995;154:1239-1245
72． Linsley PS, Ledbetter JA. The role of the CD28 receptor during T cell responses to antigen. J Exp Med,1999;190(7) :891
73． Le A, Villunger A, Kos M, et al. Modulation of Apo-1/Fas(CD95) -induced programmed cell death in myeloma cells by interferon-alpha. Eur J Immunol 1996;26(12) :3119
74． 郑世营，张志德，李德春等。腺病毒介导FasL基因转移及对胃癌细胞生长和凋亡的影响。中国肿瘤临床，2001；28(12) ：905
75． El Deiry WS, Tokino T, Velculescu VE, et al. WAF1, a potential mediator of p53 tumor suppression. Cell, 1993;75(4) :817
76． Spillare EA, Okamoto A, Hagiwara K et al. Suppression of growth in vitro and tumorigenicity in vivo of human carcinoma cell lines by transfected p16INK. Mol Carcinog, 1996;16(1) :53
77． Eguchi Y. Antisense RNA. Annu.Rev.Biochem. 1991,60:631-52
78． Denhardt DT. Mechanism of action of antisense RNA. Ann.N.Y. Acad. Sci. 1992;660:70-76
79． Zamecnik P, Stephenson M. Proc Natl Acad Sci USA, 1978;75:280-284
80． Dachs GU, Doupherty GJ, Stratford IJ, et al. Targeting gene therapy to cancer: a review. Oncol Res 1997;9:313-25
81． Steiner MS, Anthony CT, Holt JT. Antisense c-myc retroviral vector suppresses established human prostate cancer. Human Gene Therapy. 1998;9:747-755
82． Liu XD, Turbyville T, Fritz A,et al. Inhibition of Insulin-like growth factor I receptor expression in neuroblastoma cells induces the regression of
tumors in mice. Cancer Res. 1998; 58:5432-5438
83.Fakhrai H, Dorigo O, Shawler DL, et al. Eradication of established intracranial rat gliomas by transforming growth factor β antisense gene therapy. Proc.Natl. Acad. Sci. USA,1996;93:2909-2914
84．吴乃虎主编 基因工程原理 北京：科学出版社，第二版，1999：137-152，310-318
85．J.萨母布鲁克，EF.弗里奇，T.曼尼阿蒂斯主编 分子克隆实验指南 北京：科学出版社，第二版，1995：40-55
86.Helene C. Control of oncogene expression by antisense nucleic acids. Eur J Cancer 1994;30AA: 1721-1726
87.Gibson I. Antisense approaches to the gene therapy of cancer. Cancer and Metastasis. Rev 1996; 15:287-299
88.Chrostine VB & Catherine V. Do natural antisense transcripts make sense in eukaryotes? Gene 1998,211:1-9
89.Roth JA. Modification of mutant K-ras gene expression in non-small cell lung cancer(NSXLC). Human Gene Therapy 1996;7:785
90．曹蘶。反义技术及其在医药领域中应用的最新进展。军事医学科学院院刊1994；18(2)：149-152
91．卢圣栋主编 现代分子生物学实验技术 北京：中国协和医科大学出版社，第二版，1999：256-90