放化疗诱导人直肠癌细胞凋亡及相关基因表达的研究
|
摘要
放化疗作为手术治疗直肠癌的有效辅佐手段，可能与诱导癌细胞凋亡及调控
相关基因的表达有关。为了探讨不同治疗手段诱导人直肠癌细胞凋亡水平的差异
及其相关基因调控机制，本文运用体外培养人HR 8348直肠癌细胞，开展了放疗
和化疗两方面的研究。
一、放疗实验。用不同剂量X-射线（0，2，4，6，8，10Gy）及具有同等放射生
物学效应的相当于X-射线1/3剂量的不同剂量快中子（0，0.67，1.34，2.01，2.68，
3.34Gy）照射细胞，于37℃孵育6h，24h，48h，72h后，进行Hoechest33 342荧光
染色，观察细胞凋亡形态并计数凋亡指数AI（凋亡细胞的百分数）；用电子显微
镜观察凋亡细胞特有的形态学特征；分别对不同剂量（0，2，4，6，8，10Gy）X-射线
及快中子（0，0.67，1.34，2.01，2.68，3.34Gy）照射后24h的细胞用免疫组化方法进行
基因表达（p53及bc1-2）的检测。结果不同剂量X-射线和快中子照射的细胞凋
亡指数，随着照射剂量和受照时间的延长而逐渐增加，在同一时间点，快中子诱导
的细胞凋亡水平明显高于X-射线；不同剂量X-射线照射后的细胞，较阳性对照
组，p53基因表达下调明显，bc1-2基因表达下调不明显；不同剂量快中子照射后
的细胞，p53及bc1-2基因表达下调均不明显。结论 高能X射线和快中子诱导人
直肠癌细胞的凋亡具有明确的时间-剂量相关性，随着受照后时间的延长和剂量
的逐渐增大，凋亡指数渐趋升高，无明显变缓之势；快中子照射人直肠癌细胞，
与X-射线相比较，可引起较大的凋亡反应；此外，照射后p53及bc1-2基因表达
下调，说明此两类基因都参与了细胞凋亡的基因调控。
二、化疗+放疗实验。化疗（C）及放疗合并化疗（R+C）对细胞进行两种不同
的处理，用Hoechest33 342荧光染色法检测凋亡细胞并计数凋亡指数AI（凋亡
细胞的百分数），观察两组凋亡水平的差异；采用琼脂糖凝胶电泳及TUNEL（脱
氧核苷酸末端转移酶介导的缺口末端标记法）实验对凋亡细胞从不同角度进行了
检测；对X-射线、5-FU、CDDP、5-Fu+CDDP、X+5-Fu+CDDP五种不同处理因
素的细胞，用免疫组化方法进行基因表达（p53及bc1-2）的检测。结果 单一使
用5-Fu或CDDP随用药时间的延长及用药剂量的增大，细胞凋亡指数逐渐增加；
C组5-Fu-CDDP联合使用较单一使用5Fu或CDDP，细胞凋亡指数明显增加；R+C
组较C组，细胞凋亡指数明显增加，尤以X-射线+5-Fu-CDDP组凋亡指数最高；
琼脂糖凝胶电泳及TUNEL实验均显示有典型的细胞凋亡；施加五种不同处理因
素的细胞，较阳性对照组，p53及bc1-2均有一定程度的基因表达下调。结论 X-
射线合并5-Fu-CDDP诱导的人直肠癌细胞凋亡反应明显高于单纯化疗或单纯放
疗组；5-Fu-CDDP联合诱导的人直肠癌细胞凋亡反应高于单一使用5-Fu或
CDDP，提示5-Fu-CDDP联合诱导人直肠癌细胞的凋亡有协同或相加作用，5-Fu
和 CDDP联合使用作为放疗增敏剂治疗直肠癌有广阔的应用前景；p53及比毛
基因表达下调，说明在X－射线jFll及CDDP诱导细胞凋亡的过程中，p53及h2
均参与了细胞凋亡的基因调控。
As an effective adjuvant therapy for surgery treatment of rectal cancer, radiotherapy and chemotherapy may be relevant to induced apoptosis of cancer cells and regulation and control of involved genes expression. In order to investigate the difference of apoptosis level induced by a variety of treatments in rectal cancer cells and mechanism of regulation and control of relevant genes, the thesis performs a study in two aspects-radiotherapy and chemotherapy.
The first part: radiotherapy test
Subject: To investigate apoptosis induced by x-ray and fast-neutron-ray and relevant gene expression in human rectal carcinoma cell line. Methods: Cells were radiated by various dose of x-ray (OGy, 2Gy, 4Gy, 6Gy, 8Gy, lOGy) and by that of fast-neutron-ray (OGy, 0.67Gy, l.34Gy, 2.OlGy, 2.68Gy, 3.34Gy), which are the same effects of radiobiology as one-third of the equivalent dose of x-ray. After that, cells were incubated for 6h, 24h, 48h, and 72h in the constant temperature of 370C. Fluorescent staining of DNA was used to detect the relation between AL (apoptotic index, percentage of apoptotic cells) and irradiation dose of x-ray and fast-neutron-ray, as well as time after radiation. The special morphological feature of apoptotic cell was observed with electron microscope. In addition, we detect gene expression for cells radiated by various dose of x-ray (0,2,4,6,8,1 OGy) and fast-neutron-ray (0,O.67,l.34,2.Ol,2.68,3.34Gy) Results: AL of cells radiated by various dose of xray and fast-neutron-ray gradually increased with the increase of dose and time in human rectal cancer cell line. Furthermore, at the same point of time, AL of cells induced by fast- neutron-ray was higher than that induced by x-ray. For cells radiated by various dose of x-ray, down-regulated expression of p53 rotein was observed significantly, but bcl-2 protein wasn抰. For cells radiated by various dose of fa~t-~neutron-ray, it wasn抰 observed that the significant down-regulated expression of p53 protein and protein. Conclusion: Our results indicate, the apoptotic response induced by x-ray and fast-neutron-ray in rectal carcinoma cell line is time-dose dependent. The more time and dose increased, the more AL increased, without observing the appearance of plateau. The apoptotic response induced by fast-neutronray was higher than by x-ray in human rectal cancer cell line. Additionally, downregulated expression of p53 protein and bcl-2 protein has shown that p53 tumor suppressor gene and bcl-2 protooncogene are both involved in the control of apoptotic gene expression.
S
The second part: chemotherapy +radiotherapy test
Subject: To investigate aPoptosis induced by the combination of radiotherapy and
chemotheraPy (5-Fu-CDDP) and relevant gene expression in human rectal cancer cell
line. Methods Cells were divided in two groups. One group was treated by
chemotheraPy, and the other was treated by chemotherapy and radiotherapy. By
special fluorescent staining of DNA, we detected aPoptotic cells and numbered AI
(Percentage of aPoptotic cells) for cells treated by the two different means, so that we
have a comparison of apoptotic level fOr cells treated by the two different means.
Apoptosis of cells were evaluated by DNA agrose electrophoresis and TUNEL
(terminal-deoxynucleotidyl transferase mediated d-UTP nick and labeling).
Additionally, Cells were treated by x-ray, 5-Fu, CDDP, 5-Fu-CDDP, x-ray-5-Fu-
CDDP respectively. The expression of p53 protein and bcl-2 protein were detected by
immunohistochemical method fOr the cells treated by five different factors mentioned
above. Results The more time and dose increased in C group by the single use of 5-
Fu or CDDP, the more AI increased; The significam increase of AI has aPpeared by
the combination use of 5-Fu-CDDP compared with the single use of 5-Fu or CDDP;
So has the significan increase of AI by the combination of radiotherapy and
chemotheraPy compared with by the single use of chemotherapy. Especially the group
of the combination of radiotherapy with 5-Fu-CDDP has the h
相关基因的表达有关。为了探讨不同治疗手段诱导人直肠癌细胞凋亡水平的差异
及其相关基因调控机制，本文运用体外培养人HR 8348直肠癌细胞，开展了放疗
和化疗两方面的研究。
一、放疗实验。用不同剂量X-射线（0，2，4，6，8，10Gy）及具有同等放射生
物学效应的相当于X-射线1/3剂量的不同剂量快中子（0，0.67，1.34，2.01，2.68，
3.34Gy）照射细胞，于37℃孵育6h，24h，48h，72h后，进行Hoechest33 342荧光
染色，观察细胞凋亡形态并计数凋亡指数AI（凋亡细胞的百分数）；用电子显微
镜观察凋亡细胞特有的形态学特征；分别对不同剂量（0，2，4，6，8，10Gy）X-射线
及快中子（0，0.67，1.34，2.01，2.68，3.34Gy）照射后24h的细胞用免疫组化方法进行
基因表达（p53及bc1-2）的检测。结果不同剂量X-射线和快中子照射的细胞凋
亡指数，随着照射剂量和受照时间的延长而逐渐增加，在同一时间点，快中子诱导
的细胞凋亡水平明显高于X-射线；不同剂量X-射线照射后的细胞，较阳性对照
组，p53基因表达下调明显，bc1-2基因表达下调不明显；不同剂量快中子照射后
的细胞，p53及bc1-2基因表达下调均不明显。结论 高能X射线和快中子诱导人
直肠癌细胞的凋亡具有明确的时间-剂量相关性，随着受照后时间的延长和剂量
的逐渐增大，凋亡指数渐趋升高，无明显变缓之势；快中子照射人直肠癌细胞，
与X-射线相比较，可引起较大的凋亡反应；此外，照射后p53及bc1-2基因表达
下调，说明此两类基因都参与了细胞凋亡的基因调控。
二、化疗+放疗实验。化疗（C）及放疗合并化疗（R+C）对细胞进行两种不同
的处理，用Hoechest33 342荧光染色法检测凋亡细胞并计数凋亡指数AI（凋亡
细胞的百分数），观察两组凋亡水平的差异；采用琼脂糖凝胶电泳及TUNEL（脱
氧核苷酸末端转移酶介导的缺口末端标记法）实验对凋亡细胞从不同角度进行了
检测；对X-射线、5-FU、CDDP、5-Fu+CDDP、X+5-Fu+CDDP五种不同处理因
素的细胞，用免疫组化方法进行基因表达（p53及bc1-2）的检测。结果 单一使
用5-Fu或CDDP随用药时间的延长及用药剂量的增大，细胞凋亡指数逐渐增加；
C组5-Fu-CDDP联合使用较单一使用5Fu或CDDP，细胞凋亡指数明显增加；R+C
组较C组，细胞凋亡指数明显增加，尤以X-射线+5-Fu-CDDP组凋亡指数最高；
琼脂糖凝胶电泳及TUNEL实验均显示有典型的细胞凋亡；施加五种不同处理因
素的细胞，较阳性对照组，p53及bc1-2均有一定程度的基因表达下调。结论 X-
射线合并5-Fu-CDDP诱导的人直肠癌细胞凋亡反应明显高于单纯化疗或单纯放
疗组；5-Fu-CDDP联合诱导的人直肠癌细胞凋亡反应高于单一使用5-Fu或
CDDP，提示5-Fu-CDDP联合诱导人直肠癌细胞的凋亡有协同或相加作用，5-Fu
和 CDDP联合使用作为放疗增敏剂治疗直肠癌有广阔的应用前景；p53及比毛
基因表达下调，说明在X－射线jFll及CDDP诱导细胞凋亡的过程中，p53及h2
均参与了细胞凋亡的基因调控。
As an effective adjuvant therapy for surgery treatment of rectal cancer, radiotherapy and chemotherapy may be relevant to induced apoptosis of cancer cells and regulation and control of involved genes expression. In order to investigate the difference of apoptosis level induced by a variety of treatments in rectal cancer cells and mechanism of regulation and control of relevant genes, the thesis performs a study in two aspects-radiotherapy and chemotherapy.
The first part: radiotherapy test
Subject: To investigate apoptosis induced by x-ray and fast-neutron-ray and relevant gene expression in human rectal carcinoma cell line. Methods: Cells were radiated by various dose of x-ray (OGy, 2Gy, 4Gy, 6Gy, 8Gy, lOGy) and by that of fast-neutron-ray (OGy, 0.67Gy, l.34Gy, 2.OlGy, 2.68Gy, 3.34Gy), which are the same effects of radiobiology as one-third of the equivalent dose of x-ray. After that, cells were incubated for 6h, 24h, 48h, and 72h in the constant temperature of 370C. Fluorescent staining of DNA was used to detect the relation between AL (apoptotic index, percentage of apoptotic cells) and irradiation dose of x-ray and fast-neutron-ray, as well as time after radiation. The special morphological feature of apoptotic cell was observed with electron microscope. In addition, we detect gene expression for cells radiated by various dose of x-ray (0,2,4,6,8,1 OGy) and fast-neutron-ray (0,O.67,l.34,2.Ol,2.68,3.34Gy) Results: AL of cells radiated by various dose of xray and fast-neutron-ray gradually increased with the increase of dose and time in human rectal cancer cell line. Furthermore, at the same point of time, AL of cells induced by fast- neutron-ray was higher than that induced by x-ray. For cells radiated by various dose of x-ray, down-regulated expression of p53 rotein was observed significantly, but bcl-2 protein wasn抰. For cells radiated by various dose of fa~t-~neutron-ray, it wasn抰 observed that the significant down-regulated expression of p53 protein and protein. Conclusion: Our results indicate, the apoptotic response induced by x-ray and fast-neutron-ray in rectal carcinoma cell line is time-dose dependent. The more time and dose increased, the more AL increased, without observing the appearance of plateau. The apoptotic response induced by fast-neutronray was higher than by x-ray in human rectal cancer cell line. Additionally, downregulated expression of p53 protein and bcl-2 protein has shown that p53 tumor suppressor gene and bcl-2 protooncogene are both involved in the control of apoptotic gene expression.
S
The second part: chemotherapy +radiotherapy test
Subject: To investigate aPoptosis induced by the combination of radiotherapy and
chemotheraPy (5-Fu-CDDP) and relevant gene expression in human rectal cancer cell
line. Methods Cells were divided in two groups. One group was treated by
chemotheraPy, and the other was treated by chemotherapy and radiotherapy. By
special fluorescent staining of DNA, we detected aPoptotic cells and numbered AI
(Percentage of aPoptotic cells) for cells treated by the two different means, so that we
have a comparison of apoptotic level fOr cells treated by the two different means.
Apoptosis of cells were evaluated by DNA agrose electrophoresis and TUNEL
(terminal-deoxynucleotidyl transferase mediated d-UTP nick and labeling).
Additionally, Cells were treated by x-ray, 5-Fu, CDDP, 5-Fu-CDDP, x-ray-5-Fu-
CDDP respectively. The expression of p53 protein and bcl-2 protein were detected by
immunohistochemical method fOr the cells treated by five different factors mentioned
above. Results The more time and dose increased in C group by the single use of 5-
Fu or CDDP, the more AI increased; The significam increase of AI has aPpeared by
the combination use of 5-Fu-CDDP compared with the single use of 5-Fu or CDDP;
So has the significan increase of AI by the combination of radiotherapy and
chemotheraPy compared with by the single use of chemotherapy. Especially the group
of the combination of radiotherapy with 5-Fu-CDDP has the h
引文
1．陈君石等译，食物、营养和癌症预防，第一版，上海，上海医科大学出版社，1999：
2．李正生等，肿瘤，1998，18：114-117
3. BaBa S et al, Int Surg, 1994, 79:123-6
4. Tannapfel A et al. Strahlenther Onkol, 1998, 174:6295-9
5. Desai G.R et al, Dis Colon Rectum, 1996,39:101090-6
6. Asano M et al, Gan To Kagaku Ryoho, 1997, 24:155-60
7. Ishikawa K et al. Jpn J Clin Oncol, 1998,28:6, 374-7
8. Okabo S et al. Gan To Kagaku Ryoho, 1997, 24:7, 849-54
9. Kerr J.F.R et al, J Pathol, 1972, 107:41-44.
10. White E et al, Genes Dev, 1996, 10:1
11. Wyllie AH et al, Cancer and Metastasis Review, 1992, 11: 95-103
12. Bicknell, G. R et al. Biochem Biophys Res Commun 1995, 207:40-7.
13. Yoshihide Tsujimoto, Oncogene, 1989, 4:1331-1336
14. Lin,Y et al, Genes Der 1999,13:2514-26
15. Amsterdam,A et al. Steroids 1998,63(5-6):314-8.
16. Lee,S.J et al, J Biol Chem 1998,273:10618-23.
17 Force T et al, J Biol Chem, 1991, 266:4295-4302
18. Renard DC et al, Biochem J, 1992, 284:507-512
19. Trump BJ et al, FASEB J, 1995, 9:219-228
20．李春海等，肿瘤分子生物学进展，北京，军事医学科学出版社，1996
21．叶棋浓等，蛋白酶与细胞凋亡，国外医学分子生物学分册，1996，18：256-260
22. Tranganos E et al, Int J Oncol, 1993, 2:47-59
23. Brunner T et al, Nature, 1995, 373,441-443
24. Hockenbery DM et al, Nature, 1990, 348:334-336
25. Delia D et al, Blood, 1992, 79:1291-1298
26. Nunez G et al, J Immunol, 1990, 144:3602-3610
27. Tsujimoto Y, Cell, 1986, 44:817-829
28. Vaux DL et al, Nature, 1985,338, 440-442
29. Korsmeyer S J, Immunol Today, 1992, 13:285-288
30. Hockenbery DM et al, Nature, 1990, 348:334-336
31. Hockenbery DM et al, Proc Natl Acad Sci USA, 1991, 88:6981-6985
32. Yang E et al, Cell, 1995, 80:285-291
33. Kastan MB et al, Cancer Metas Rev, 1995,14; 3-15
34. Yonish-Rouach E et al, Nature, 1991,352:345-437
35. Dehw A et al, J Biol Chem, 1992, 267:10709-10715
36. Williams GT et al, Cell, 1993, 74:777-779
37. Bissonnette BP et al, Nature, 1992, 359:552-554
38. Thornberry NA et al, Nature, 1992, 356:768-774
39. Ray CAM et al, Cell, 1992-69:597-604
40. Kumar S et al, Genes Dev, 1994, 8:1613-1626
41. Walker,P.R et al,Biochem Cell Biol 1997,75:287-99.
42. Merino,J.J et al, Invest Clin 1998,39:323-58.
43. Dickinson,J.L et al, Cell Death Differ 1998,5:163-71
44. Reuther,J.Y et al, J Biol Chem 1999, 274:20664-70
45. Lowe SW et al, Science, 1994, 266:807-810
46. Vral A et al, Int J Radiat Biol 1998, 73:289-95
47. Akagi Y et al, Int J Radiat Biol, 1993, 64:47-56
48. Ling CC et al, Radiother Oncol, 1994, 32:129-136
49. Radford IR et al, Int J Radiat Biol, 1994, 65:229-239
50. Fukunaga Johnson N, Carcinogenesis, 1995, 16:1761-1767
51. Olive PL, Radiat Res, 1996, 146:595-602
52. Schwartz JL, Mutagenesis, 1995, 10:227-233
53. Ling CC et al, Cancer Res, 1995, 55:202-5212
54. Harms RM, Mutat Res, 1996, 366:171-179
55. Aldridge DR, Br J Cancer, 1995, 71:571-577
56. Levine EL, Radiother Oncol, 1995, 37:1-9
57. Kaspers GJ et al, Leuk Lymphoma, 1994, 13:187-201
58. Guchelaar HJ et al, Pharm World Sci, 1997, 19:119-125
59. Kerbel RS et al, Invasion Metastasis, 1994, 14:50-60
60. Dalton WS et al, Semin Oncol, 1999, 26:23-27
61. Bergman PJ et al, Vet Clin North Am Small Anim Pract, 1997, 27:47-57
62. Coukos G et al, Obstet Gynecol, 1998, 91:783-792
63. Weller M, Cell Tissue Res, 1998, 292:435-445
64. Lara PN Jr et al, Cancer Invest, 1999, 17:137-144
65. Deiry WS et al, Curr Opin Oncol, 1997, 9:79-87
66．J.萨姆布鲁克等主编，分子克隆实验指南，第二版，北京科学出版社，1992：309-313
67．刘鼎新等，细胞生物学研究方法与技术，第二版，北京医科大学中国协和医科大学联合出版社，1997：70-73
68．姜泊等，分子生物学常用实验方法，北京，人民军医出版社，1996：174-175
69. Yusuf AH, Blood, 1997,89:1845-1853
2．李正生等，肿瘤，1998，18：114-117
3. BaBa S et al, Int Surg, 1994, 79:123-6
4. Tannapfel A et al. Strahlenther Onkol, 1998, 174:6295-9
5. Desai G.R et al, Dis Colon Rectum, 1996,39:101090-6
6. Asano M et al, Gan To Kagaku Ryoho, 1997, 24:155-60
7. Ishikawa K et al. Jpn J Clin Oncol, 1998,28:6, 374-7
8. Okabo S et al. Gan To Kagaku Ryoho, 1997, 24:7, 849-54
9. Kerr J.F.R et al, J Pathol, 1972, 107:41-44.
10. White E et al, Genes Dev, 1996, 10:1
11. Wyllie AH et al, Cancer and Metastasis Review, 1992, 11: 95-103
12. Bicknell, G. R et al. Biochem Biophys Res Commun 1995, 207:40-7.
13. Yoshihide Tsujimoto, Oncogene, 1989, 4:1331-1336
14. Lin,Y et al, Genes Der 1999,13:2514-26
15. Amsterdam,A et al. Steroids 1998,63(5-6):314-8.
16. Lee,S.J et al, J Biol Chem 1998,273:10618-23.
17 Force T et al, J Biol Chem, 1991, 266:4295-4302
18. Renard DC et al, Biochem J, 1992, 284:507-512
19. Trump BJ et al, FASEB J, 1995, 9:219-228
20．李春海等，肿瘤分子生物学进展，北京，军事医学科学出版社，1996
21．叶棋浓等，蛋白酶与细胞凋亡，国外医学分子生物学分册，1996，18：256-260
22. Tranganos E et al, Int J Oncol, 1993, 2:47-59
23. Brunner T et al, Nature, 1995, 373,441-443
24. Hockenbery DM et al, Nature, 1990, 348:334-336
25. Delia D et al, Blood, 1992, 79:1291-1298
26. Nunez G et al, J Immunol, 1990, 144:3602-3610
27. Tsujimoto Y, Cell, 1986, 44:817-829
28. Vaux DL et al, Nature, 1985,338, 440-442
29. Korsmeyer S J, Immunol Today, 1992, 13:285-288
30. Hockenbery DM et al, Nature, 1990, 348:334-336
31. Hockenbery DM et al, Proc Natl Acad Sci USA, 1991, 88:6981-6985
32. Yang E et al, Cell, 1995, 80:285-291
33. Kastan MB et al, Cancer Metas Rev, 1995,14; 3-15
34. Yonish-Rouach E et al, Nature, 1991,352:345-437
35. Dehw A et al, J Biol Chem, 1992, 267:10709-10715
36. Williams GT et al, Cell, 1993, 74:777-779
37. Bissonnette BP et al, Nature, 1992, 359:552-554
38. Thornberry NA et al, Nature, 1992, 356:768-774
39. Ray CAM et al, Cell, 1992-69:597-604
40. Kumar S et al, Genes Dev, 1994, 8:1613-1626
41. Walker,P.R et al,Biochem Cell Biol 1997,75:287-99.
42. Merino,J.J et al, Invest Clin 1998,39:323-58.
43. Dickinson,J.L et al, Cell Death Differ 1998,5:163-71
44. Reuther,J.Y et al, J Biol Chem 1999, 274:20664-70
45. Lowe SW et al, Science, 1994, 266:807-810
46. Vral A et al, Int J Radiat Biol 1998, 73:289-95
47. Akagi Y et al, Int J Radiat Biol, 1993, 64:47-56
48. Ling CC et al, Radiother Oncol, 1994, 32:129-136
49. Radford IR et al, Int J Radiat Biol, 1994, 65:229-239
50. Fukunaga Johnson N, Carcinogenesis, 1995, 16:1761-1767
51. Olive PL, Radiat Res, 1996, 146:595-602
52. Schwartz JL, Mutagenesis, 1995, 10:227-233
53. Ling CC et al, Cancer Res, 1995, 55:202-5212
54. Harms RM, Mutat Res, 1996, 366:171-179
55. Aldridge DR, Br J Cancer, 1995, 71:571-577
56. Levine EL, Radiother Oncol, 1995, 37:1-9
57. Kaspers GJ et al, Leuk Lymphoma, 1994, 13:187-201
58. Guchelaar HJ et al, Pharm World Sci, 1997, 19:119-125
59. Kerbel RS et al, Invasion Metastasis, 1994, 14:50-60
60. Dalton WS et al, Semin Oncol, 1999, 26:23-27
61. Bergman PJ et al, Vet Clin North Am Small Anim Pract, 1997, 27:47-57
62. Coukos G et al, Obstet Gynecol, 1998, 91:783-792
63. Weller M, Cell Tissue Res, 1998, 292:435-445
64. Lara PN Jr et al, Cancer Invest, 1999, 17:137-144
65. Deiry WS et al, Curr Opin Oncol, 1997, 9:79-87
66．J.萨姆布鲁克等主编，分子克隆实验指南，第二版，北京科学出版社，1992：309-313
67．刘鼎新等，细胞生物学研究方法与技术，第二版，北京医科大学中国协和医科大学联合出版社，1997：70-73
68．姜泊等，分子生物学常用实验方法，北京，人民军医出版社，1996：174-175
69. Yusuf AH, Blood, 1997,89:1845-1853