FLIP干扰性小RNA阻断大肠癌细胞免疫逃逸的体外实验研究
摘要
目的：
大肠癌是胃肠道最常见的恶性肿瘤之一，目前各种治疗方法均不能有效地防止其转移和复发，其中最主要的原因是肿瘤组织的凋亡水平下调，从而逃避机体的免疫监视，免遭免疫系统的杀伤和攻击。因此进一步阐释肿瘤的免疫逃逸机制，寻找新的治疗靶点，已成为当今国内外肿瘤治疗的研究热点。本论文聚焦于Fas受体凋亡途径中的抑制蛋白——FLIP，采用RNA干扰技术，比较干扰前后体外培养的大肠癌细胞株凋亡敏感性的变化，阐明在大肠癌中是否存在由FLIP介导的免疫逃逸机制，并为肿瘤的基因治疗提供新的思路。
方法：
体外培养大肠癌细胞株，采用直接免疫荧光流式细胞学技术和半定量RT-PCR法筛选体外实验模型。利用电穿孔，将干扰性小RNA(siRNA)片段转染至HT-29细胞，以相对半定量PCR法计算干扰效率，确定FLIP基因被封阻情况。在激活型抗Fas抗体诱导下，采用Annexin V法和细胞周期分析共同判定转染前后HT-29细胞凋亡敏感性的变化。
结果：
1．综合Fas和FLIP两项指标的测定结果，筛选出HT-29细胞为适宜的体外实验模型(其Fas的表达水平为42.46％，c-FLIP_L mRNA的相对含量为83.36％)。
2．在摸索到的最佳电穿孔条件下(90mOsmol／kg低渗环境，电压500V，时程70μs，外源片段浓度100nM，室温)，将对应目的基因FLIP的siRNA转染入HT-29细胞，FLIP mRNA的相对含量由78.55％下降到27.48％，干扰效率达65.02％。
3．在激活型抗Fas抗体的刺激下，转染后的HT-29细胞凋亡比例由1.76％上升到29.5％，细胞周期分布图上出现“凋亡峰”。
天津医科大学硕士学位论文中文摘要
结论:
1.大肠癌细胞中存在一定水平的FLIP表达，经RNA干扰后，FUPm丑NA表
达水平下调，细胞表面Fas受体表达水平未产生明显变化，细胞对Fas介导
的凋亡敏感性上升，从而证实在大肠癌中存在着由FLIP介导的免疫逃逸机
制。
2.随着FLIP表达水平的下降，在激活型抗Fas抗体的刺激下，大肠癌细胞中
凋亡细胞所占比例增加，提示FLIP作为受体凋亡通路的关键因子，可望成
为肿瘤基因治疗的新靶点。
3.将对应目的基因FLIP的外源siRNA片段转染入体外培养细胞后，高效、特
异地降低了FLIP mRNA的表达，并进而阐明FLIP的生物学功能，提示RNA
干扰作为一种新型基因研究手段拥有较广阔的应用前景。
Objective:
Colorectal cancer is one of the most general intestinal tumors and none of therapies can effectually prevent it from metastasis and recurrence by now. The most possible reason about this phenomenon is that tumor cells reduce their apoptotic sensitivity, evade immune surveillance and survive upon many kinds of signals. Thus, how to exclude the mechanism of tumor evasion and find out new targets of tumor therapy has been the hotspot. Here, we focus on FLIP, one . of the inhibitory proteins of Fas-mediated apoptotic pathway. After RNA interfering, the mRNA expression level of FLIP is decreased and the apoptotic sensitivity is increased in the transfected colorectal cancer cells. We hope our study could exclude whether there is FLIP-associated apoptotic evasion in colorectal cancer cells and put a new way on tumor gene therapy. Methods:
Four colorectal cancer cell lines were cultured in vitro. After direct immunofluoscent FACScan and semi-quantitative RT-PCR analysis, the optimal experimental model was selected. HT-29 was transfected with siRNA by electroporation. The inhibitory of FLIP mRNA was detected by relative semi-quantitative PCR. After that, the transfected cells were treated with agonistic anti-Fas antibody in order to induce apoptosis. We used Annexin V and cell cycle analysis to determine the apoptotic sensitivity of transfected HT-29. Results:
1. Combining the levels of Fas and FLIP, HT-29 was selected as the best experimental model.
2. After transfecting siRNA into HT-29, the expression level of FLIP mRNA was
decreased from 78.55 % to 27.48 % and the interfering effectiveness is 65.02 %. 3. After treated with agonistic anti-Fas antibody, the apoptotic rate of transfected HT-29 was increased from 1.76 % to 29.5 % and we can find an "apoptotic peak" on the cell cycle analysis. Conclusions:
1 . There is FLIP-associated immune evasion mechanism in colorectal cancer cell lines exactly.
2. As an important factor in Fas-mediated apoptotic pathway, FLIP could be one of the effective targets in tumor gene therapy.
3. Since the mRNA level of FLIP is effectually reduced after transfecting siRNA into HT-29 cell line, RNAi will possess expansive applicable prospect in gene silencing area.
大肠癌是胃肠道最常见的恶性肿瘤之一，目前各种治疗方法均不能有效地防止其转移和复发，其中最主要的原因是肿瘤组织的凋亡水平下调，从而逃避机体的免疫监视，免遭免疫系统的杀伤和攻击。因此进一步阐释肿瘤的免疫逃逸机制，寻找新的治疗靶点，已成为当今国内外肿瘤治疗的研究热点。本论文聚焦于Fas受体凋亡途径中的抑制蛋白——FLIP，采用RNA干扰技术，比较干扰前后体外培养的大肠癌细胞株凋亡敏感性的变化，阐明在大肠癌中是否存在由FLIP介导的免疫逃逸机制，并为肿瘤的基因治疗提供新的思路。
方法：
体外培养大肠癌细胞株，采用直接免疫荧光流式细胞学技术和半定量RT-PCR法筛选体外实验模型。利用电穿孔，将干扰性小RNA(siRNA)片段转染至HT-29细胞，以相对半定量PCR法计算干扰效率，确定FLIP基因被封阻情况。在激活型抗Fas抗体诱导下，采用Annexin V法和细胞周期分析共同判定转染前后HT-29细胞凋亡敏感性的变化。
结果：
1．综合Fas和FLIP两项指标的测定结果，筛选出HT-29细胞为适宜的体外实验模型(其Fas的表达水平为42.46％，c-FLIP_L mRNA的相对含量为83.36％)。
2．在摸索到的最佳电穿孔条件下(90mOsmol／kg低渗环境，电压500V，时程70μs，外源片段浓度100nM，室温)，将对应目的基因FLIP的siRNA转染入HT-29细胞，FLIP mRNA的相对含量由78.55％下降到27.48％，干扰效率达65.02％。
3．在激活型抗Fas抗体的刺激下，转染后的HT-29细胞凋亡比例由1.76％上升到29.5％，细胞周期分布图上出现“凋亡峰”。
天津医科大学硕士学位论文中文摘要
结论:
1.大肠癌细胞中存在一定水平的FLIP表达，经RNA干扰后，FUPm丑NA表
达水平下调，细胞表面Fas受体表达水平未产生明显变化，细胞对Fas介导
的凋亡敏感性上升，从而证实在大肠癌中存在着由FLIP介导的免疫逃逸机
制。
2.随着FLIP表达水平的下降，在激活型抗Fas抗体的刺激下，大肠癌细胞中
凋亡细胞所占比例增加，提示FLIP作为受体凋亡通路的关键因子，可望成
为肿瘤基因治疗的新靶点。
3.将对应目的基因FLIP的外源siRNA片段转染入体外培养细胞后，高效、特
异地降低了FLIP mRNA的表达，并进而阐明FLIP的生物学功能，提示RNA
干扰作为一种新型基因研究手段拥有较广阔的应用前景。
Objective:
Colorectal cancer is one of the most general intestinal tumors and none of therapies can effectually prevent it from metastasis and recurrence by now. The most possible reason about this phenomenon is that tumor cells reduce their apoptotic sensitivity, evade immune surveillance and survive upon many kinds of signals. Thus, how to exclude the mechanism of tumor evasion and find out new targets of tumor therapy has been the hotspot. Here, we focus on FLIP, one . of the inhibitory proteins of Fas-mediated apoptotic pathway. After RNA interfering, the mRNA expression level of FLIP is decreased and the apoptotic sensitivity is increased in the transfected colorectal cancer cells. We hope our study could exclude whether there is FLIP-associated apoptotic evasion in colorectal cancer cells and put a new way on tumor gene therapy. Methods:
Four colorectal cancer cell lines were cultured in vitro. After direct immunofluoscent FACScan and semi-quantitative RT-PCR analysis, the optimal experimental model was selected. HT-29 was transfected with siRNA by electroporation. The inhibitory of FLIP mRNA was detected by relative semi-quantitative PCR. After that, the transfected cells were treated with agonistic anti-Fas antibody in order to induce apoptosis. We used Annexin V and cell cycle analysis to determine the apoptotic sensitivity of transfected HT-29. Results:
1. Combining the levels of Fas and FLIP, HT-29 was selected as the best experimental model.
2. After transfecting siRNA into HT-29, the expression level of FLIP mRNA was
decreased from 78.55 % to 27.48 % and the interfering effectiveness is 65.02 %. 3. After treated with agonistic anti-Fas antibody, the apoptotic rate of transfected HT-29 was increased from 1.76 % to 29.5 % and we can find an "apoptotic peak" on the cell cycle analysis. Conclusions:
1 . There is FLIP-associated immune evasion mechanism in colorectal cancer cell lines exactly.
2. As an important factor in Fas-mediated apoptotic pathway, FLIP could be one of the effective targets in tumor gene therapy.
3. Since the mRNA level of FLIP is effectually reduced after transfecting siRNA into HT-29 cell line, RNAi will possess expansive applicable prospect in gene silencing area.
引文
1. Avi Ashkenazi, Vishva M. Dixit. Death receptors: signaling and modulation. Science, 1998, 281: 1305-1308.
2．刘培军。穿孔素研究进展。国外医学免疫学分册，2002，25(2)：69-72。
3. Claude Aussel, Claudette Pelassy, Jean Philippe Breittmayer. CD95 induces an increased phosphatidylserine synthesis that precedes its externalization during programmed cell death. Clinical Cancer Research, 2000, 6: 1529-1535.
4. Peter Juo, Michele Sue-Ann Woo, Calvin J. Kuo, et al. FADD is required for multiple signaling events downstream of the receptor Fas. Cell Growth and Differentiation. 1999, 10: 797-804.
5. Keith P. Wilson, Jo-Anne F. Black, John A. Thomson, et al. Structure and mechanism of interleukin-1β converting enzyme. Nature, 1994, 370: 270-274.
6. Nancy A. Thornberry, Yurt Lazebnik. Caspases: enemies within. Science, 1998, 281: 1312-1316.
7. Fukazawa T, Fujiwara T, Morimoto Y, et al. Differential involvement of the CD95 receptor/ligand system on apoptosis induced by the wild-type p53 gene transfer in human cancer cells. Oncogene, 1999, 18(13): 2189-2199.
8. Martin Irmler, Margot Thome, Michael Hahne, et al. Inhibition of death receptor signals by cellular FLIP. Nature, 1997, 388: 190-195.
9. Kanneboyina Nagaraju, Livia Casciola-Rosen, et al. The inhibition of apoptosis in myositis and in normal muscle cells. J. Immuno., 2000, 164: 5459-5465.
10. Margot Thome, Pascal Schneider, Kay Hofmann, et al. Viral FLICE-inhibitory proteins(FLIPs) prevent apoptosis induced by death receptors. Nature, 1997, 386: 517-521.
11. Lars E. French, Jurg Tschopp. The inhibitory of death receptor signaling,
FLICE-inhibitory protein defines a new class of tumor progression factors. J. Exp. Med., 1999, 190: 1025-1031.
12. Shimin Hu, Claudius Vincenz, Jian Ni, et al. Ⅰ-FLICE, a novel inhibitor of tumor necrosis factor receptor-1 and CD-95-induced apoptosis. J. Bio. Chem., 1997, 272(28): 17255-17257.
13. Sabine K.irchoff, Wolfgang W. Muller, Andreas Krueger, et al. TCR-mediated up-regulation of c-FLIPs correlateds with resistance toward CD95-mediated apoptosis by blocking death-inducing signaling complex activity. J. Immuno., 2000, 165: 6293-6300.
14. Elke S., Bergmarm-Leitner, Scott I. Abrams. Differential role of Fas/Fas ligand interactions in cytolysis of primary and metastatic colon carcinoma cell lines by human antigen-specific CD8+ CTL. J. Immuno., 2000, 164: 4941-4954.
15. Robert E. Hewitt, Andrew McMarlin, David Kleiner, et al. Validation of a model of colon cancer progression. J. Pathol., 2000, 192: 446-454.
16. Sayda M. Elbashir, Jens Harborth, Winfried Lendeckel, et al. Duplexes of 21-nucleotide RNAs mediated RNA interference in cultured mammalian cells. Nature, 2001, 411: 494-498.
17. Y Aoki, DP Cioca, H Oidaira, et al. RNA interference may be more potent than antisense RNA in human cancer cell lines. Clinical and Experimental Pharmacology and Physiology, 2003, 30: 96-102.
18. Gewirtz, Alan M. MD. Oligonucleotide therapeutics: a step forward. J. Clin. Onco., 2000, 18: 286-293.
19. Noelle Sevilir Williams, Richard B. Gaynor, Shane Scoggin, et al. Identification and validation of genes involved in the pathogenesis of colorectal cancer using cDNA microarrays and RNA interference. Clinical Cancer Research, 2003, 9: 931-946.
20. Jenn-Yah Yu, Stacy L. DeRuiter, David L. Turner. RNA interference by
expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Sci. USA., 2002, 99(9): 6047-6052.
21. Patrick J. Paddison, Amy A. Caudy, Gregory J. Hannon. Stable suppression of gene expression by RNAi in mammalian cells. Proc. Natl. Acad. Sci. USA., 2002, 99(3): 1443-1448.
22. Andrew Thorburn. Death receptor-induced cell killing. Cellular Siganling, 2004, 16: 139-144.
23. Peter Moiler, Karin Koretz, Frank Leithauser, et al. Expression of APO-1, a member of the NGF/TNF receptor superfamily, in normal and neoplastic colon epithelium. Int. J. Cancer, 1994, 57: 371-377.
24．夏成青，刑长虹，唐小奈。结肠癌组织中Fas表达的定量分析。临床与实验病理学杂志，2002，18(3)：307-309。
25. Byung-Kyu Ryu, Min-Goo Lee, Sung-Gil Chi, et al. Increased expression of cFLIPL in colonic adenocarcinoma. J Pathol, 2001, 194: 15-19.
26. Que FG, Phan VA, Phan VH, et al. Cholangiocarcinomas express Fas ligand and disable the Fas receptor. Hepatology, 1993, 30(6): 1398-1404.
27. Nagata, S. Apoptosis by death factor. Cell, 1997, 88: 355-365.
28. Klas C., Debatin K. M., Jonker R. R. et al. Activation interferes with the APO-1 pathway in mature human T cells. Int. Immunol., 1993, 5: 625-630.
29. Ulrike von Reyher, Join Strater, Walter Kittstein, et al. Colon carcinoma cells use different mechanism to escape CD95-mediated apoptosis. Cancer Research, 1998, 58: 526-534.
30. Roberto R. Bullani, Bertrand Huard, Isabelle Viard-Leveugle, et al. Selective expression of FLIP in malignant melanocytic skin lesions. J Invest Dermatol, 2001, 117: 360-364.
31. Kataoka T, Schroter M, Hahne M, et al. FLIP prevents apoptosis induced by death receptors but not by perforin/granzyme B, chemtheraputic drugs, and
gamma irradiation. J Immuno, 1998, 161 (8): 3936-3942.
32．彭志海，刑同海，裘国强等。结肠腺癌细胞株Fas／FmsL的表达与凋亡的关系。中国癌症杂志，2001，11(1)：25-28。
33. Nita ME, Nagawa H, Tominaga O, et al. 5-Fluorouracil induces apoptosis in human colon cancer cell lines with modulation of Bcl-2 family proteins. Br J Cancer, 1998, 78(7): 986-992.
34. Joyce A. Wilson, Sumedha Jayasena, Anastaisa Khvorova, et al. RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. PNAS, 2003, 100(5): 2783-2788.
35. Jenny Dunne, Bettina Drescher, Heidemarie Riehle, et al. Theapparent uptake of fluorescently labeled siRNAs by electroporated cells depends on the fluorochrome. Oligonucleotides, 2003, 13: 375-380.
36. Udit N. Verma, Rama M. Surabhi, Aurelia Schmaltieg, et al. Small interfering RNAs directed against β-catenin inhibit the in vitro and in vivo growth of colon cancer cells. Clinical Cancer Research, 2003, 9: 1291-1300.
37. Daniela Siegrnund, Philipp Hadwiger, Klaus Pfizenmaier, et al. Selective inhibition of FLICE-like inhibitory protein (FLIP) expression with small interfering RNA oligonucleotides (siRNAs) is sufficient to sensitize tumor cells for TRAIL-induced apoptosis. Molecular Medicine, 2002, 8(11): 725-732.
38. Kanneboyina Nagaraju, Livia Casciola-Rosen, Antony Rosen, et al. The inhibition of apoptosis in myositis and in normal muscle cells. J Immuno, 2000, 164: 5459-5465.
39. Hirokazu Kinoshita, Hideshi Yoshkawa, Kazuhiko Shiikr, et al. Cisplatin (CDDP). sensitizes human osteosarcoma cell to Fas/CD95-mediated apoptosis by down-regulating FLIP-L expression. Int. J. Cancer, 2000, 88: 986-991.
40. Simone Fulda, Eric Meyer, Klaus-Michael Debatin. Metabolic inhibitors sensitize for CD95 (APO-1/Fas)-induced apoptosis by down-regulating
Fas-associated death domain-like interleukin 1-converting enzyme inhibitory protein expression. Cancer Research, 2000, 60: 3947-3956.
41. Harris Perlman, Lisa J. Pagliari, Constantinos Georganas, Toshiaki Mano, et al. FLICE-inhibitory protein expression during macrophage differentiation confers resistance to Fas-mediated apoptosis. J. Exp. Med., 1999, 190: 1679-1688.
42. Daniel E. Johnson, Brian R. Gastman, Eva Wiechowski, et al. Inhibitor of apoptosis protein hILP undergoes caspase-mediated cleavage during T lymphocyte apoptosis. Cancer Research, 2000, 60: 1818-1823.
43. Andreas Krueger, Ingo Schmitz, Sven Baumann, et al. Cellular FLICE-inhibitory protein splice variants inhibit different steps of caspase-8 activation at the CD95 death-inducing signaling complex. J. Bio. Chem., 2001, 276(23): 20633-20640.
44. Andreas Krueger, Sven Baumann, Peter H. Krammer, et al. FLICE-inhibitory proteins: regulators of death receptor-mediated apoptosis. Mol. Cell. Bio., 2001, 21(24): 8247-8254.
45. Carsten Scaffidi, Ingo Schmitz, Peter H. Krammer, et al. The role of c-FLIP in modulation of CD95-induced apoptosis. J. Bio. Chem., 1999, 274(3): 1541-1548.
2．刘培军。穿孔素研究进展。国外医学免疫学分册，2002，25(2)：69-72。
3. Claude Aussel, Claudette Pelassy, Jean Philippe Breittmayer. CD95 induces an increased phosphatidylserine synthesis that precedes its externalization during programmed cell death. Clinical Cancer Research, 2000, 6: 1529-1535.
4. Peter Juo, Michele Sue-Ann Woo, Calvin J. Kuo, et al. FADD is required for multiple signaling events downstream of the receptor Fas. Cell Growth and Differentiation. 1999, 10: 797-804.
5. Keith P. Wilson, Jo-Anne F. Black, John A. Thomson, et al. Structure and mechanism of interleukin-1β converting enzyme. Nature, 1994, 370: 270-274.
6. Nancy A. Thornberry, Yurt Lazebnik. Caspases: enemies within. Science, 1998, 281: 1312-1316.
7. Fukazawa T, Fujiwara T, Morimoto Y, et al. Differential involvement of the CD95 receptor/ligand system on apoptosis induced by the wild-type p53 gene transfer in human cancer cells. Oncogene, 1999, 18(13): 2189-2199.
8. Martin Irmler, Margot Thome, Michael Hahne, et al. Inhibition of death receptor signals by cellular FLIP. Nature, 1997, 388: 190-195.
9. Kanneboyina Nagaraju, Livia Casciola-Rosen, et al. The inhibition of apoptosis in myositis and in normal muscle cells. J. Immuno., 2000, 164: 5459-5465.
10. Margot Thome, Pascal Schneider, Kay Hofmann, et al. Viral FLICE-inhibitory proteins(FLIPs) prevent apoptosis induced by death receptors. Nature, 1997, 386: 517-521.
11. Lars E. French, Jurg Tschopp. The inhibitory of death receptor signaling,
FLICE-inhibitory protein defines a new class of tumor progression factors. J. Exp. Med., 1999, 190: 1025-1031.
12. Shimin Hu, Claudius Vincenz, Jian Ni, et al. Ⅰ-FLICE, a novel inhibitor of tumor necrosis factor receptor-1 and CD-95-induced apoptosis. J. Bio. Chem., 1997, 272(28): 17255-17257.
13. Sabine K.irchoff, Wolfgang W. Muller, Andreas Krueger, et al. TCR-mediated up-regulation of c-FLIPs correlateds with resistance toward CD95-mediated apoptosis by blocking death-inducing signaling complex activity. J. Immuno., 2000, 165: 6293-6300.
14. Elke S., Bergmarm-Leitner, Scott I. Abrams. Differential role of Fas/Fas ligand interactions in cytolysis of primary and metastatic colon carcinoma cell lines by human antigen-specific CD8+ CTL. J. Immuno., 2000, 164: 4941-4954.
15. Robert E. Hewitt, Andrew McMarlin, David Kleiner, et al. Validation of a model of colon cancer progression. J. Pathol., 2000, 192: 446-454.
16. Sayda M. Elbashir, Jens Harborth, Winfried Lendeckel, et al. Duplexes of 21-nucleotide RNAs mediated RNA interference in cultured mammalian cells. Nature, 2001, 411: 494-498.
17. Y Aoki, DP Cioca, H Oidaira, et al. RNA interference may be more potent than antisense RNA in human cancer cell lines. Clinical and Experimental Pharmacology and Physiology, 2003, 30: 96-102.
18. Gewirtz, Alan M. MD. Oligonucleotide therapeutics: a step forward. J. Clin. Onco., 2000, 18: 286-293.
19. Noelle Sevilir Williams, Richard B. Gaynor, Shane Scoggin, et al. Identification and validation of genes involved in the pathogenesis of colorectal cancer using cDNA microarrays and RNA interference. Clinical Cancer Research, 2003, 9: 931-946.
20. Jenn-Yah Yu, Stacy L. DeRuiter, David L. Turner. RNA interference by
expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Sci. USA., 2002, 99(9): 6047-6052.
21. Patrick J. Paddison, Amy A. Caudy, Gregory J. Hannon. Stable suppression of gene expression by RNAi in mammalian cells. Proc. Natl. Acad. Sci. USA., 2002, 99(3): 1443-1448.
22. Andrew Thorburn. Death receptor-induced cell killing. Cellular Siganling, 2004, 16: 139-144.
23. Peter Moiler, Karin Koretz, Frank Leithauser, et al. Expression of APO-1, a member of the NGF/TNF receptor superfamily, in normal and neoplastic colon epithelium. Int. J. Cancer, 1994, 57: 371-377.
24．夏成青，刑长虹，唐小奈。结肠癌组织中Fas表达的定量分析。临床与实验病理学杂志，2002，18(3)：307-309。
25. Byung-Kyu Ryu, Min-Goo Lee, Sung-Gil Chi, et al. Increased expression of cFLIPL in colonic adenocarcinoma. J Pathol, 2001, 194: 15-19.
26. Que FG, Phan VA, Phan VH, et al. Cholangiocarcinomas express Fas ligand and disable the Fas receptor. Hepatology, 1993, 30(6): 1398-1404.
27. Nagata, S. Apoptosis by death factor. Cell, 1997, 88: 355-365.
28. Klas C., Debatin K. M., Jonker R. R. et al. Activation interferes with the APO-1 pathway in mature human T cells. Int. Immunol., 1993, 5: 625-630.
29. Ulrike von Reyher, Join Strater, Walter Kittstein, et al. Colon carcinoma cells use different mechanism to escape CD95-mediated apoptosis. Cancer Research, 1998, 58: 526-534.
30. Roberto R. Bullani, Bertrand Huard, Isabelle Viard-Leveugle, et al. Selective expression of FLIP in malignant melanocytic skin lesions. J Invest Dermatol, 2001, 117: 360-364.
31. Kataoka T, Schroter M, Hahne M, et al. FLIP prevents apoptosis induced by death receptors but not by perforin/granzyme B, chemtheraputic drugs, and
gamma irradiation. J Immuno, 1998, 161 (8): 3936-3942.
32．彭志海，刑同海，裘国强等。结肠腺癌细胞株Fas／FmsL的表达与凋亡的关系。中国癌症杂志，2001，11(1)：25-28。
33. Nita ME, Nagawa H, Tominaga O, et al. 5-Fluorouracil induces apoptosis in human colon cancer cell lines with modulation of Bcl-2 family proteins. Br J Cancer, 1998, 78(7): 986-992.
34. Joyce A. Wilson, Sumedha Jayasena, Anastaisa Khvorova, et al. RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. PNAS, 2003, 100(5): 2783-2788.
35. Jenny Dunne, Bettina Drescher, Heidemarie Riehle, et al. Theapparent uptake of fluorescently labeled siRNAs by electroporated cells depends on the fluorochrome. Oligonucleotides, 2003, 13: 375-380.
36. Udit N. Verma, Rama M. Surabhi, Aurelia Schmaltieg, et al. Small interfering RNAs directed against β-catenin inhibit the in vitro and in vivo growth of colon cancer cells. Clinical Cancer Research, 2003, 9: 1291-1300.
37. Daniela Siegrnund, Philipp Hadwiger, Klaus Pfizenmaier, et al. Selective inhibition of FLICE-like inhibitory protein (FLIP) expression with small interfering RNA oligonucleotides (siRNAs) is sufficient to sensitize tumor cells for TRAIL-induced apoptosis. Molecular Medicine, 2002, 8(11): 725-732.
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