CEA/B7-1、CNA/IL-2核酸疫苗特异性抗结直肠癌免疫的实验研究
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摘要
大肠癌是消化道最常见的恶性肿瘤之一，手术切除是其治疗的主要方法，但手术后常发生复发与转移，是其死亡的主要原因。如果在术中或术后早期进行有效的临床干预，则能显著的提高大肠癌的术后生存时间。但目前临床上预防术后复发与转移的途径主要以化疗为主，但化疗为肿瘤非特异性，敏感性低，全身副作用大，效果欠佳。
肿瘤核酸疫苗是近年崛起的新型生物治疗方法，它可同时诱导机体产生体液免疫和细胞免疫，尤其是细胞免疫，在抗肿瘤免疫中起关键作用，并且应用简便，可直接注射质粒至肌肉，通过肌细胞的MHCⅠ、MHCⅡ传递抗原信息，不必考虑个体间的MHC限制性。与传统性的化疗有本质的区别，是一种生理性的，着眼于调动宿主自身的抗癌能力，通过增强机体固有的抗肿瘤机制达到抑制、杀灭肿瘤细胞及根治肿瘤的目的；而传统的化疗则是借助“外力”杀灭肿瘤细胞，肿瘤细胞消灭不彻底，难以控制肿瘤复发与转移，并给正常组织细胞带来巨大毒副作用。
癌胚抗原(Carcinoembryonic Antigen,CEA)是许多消化道腺癌表达的一种肿瘤相关抗原，大肠癌几乎100％都表达CEA。正常消化道粘膜仅有少量分泌。它的免疫原性较低，临床检测为CEA阳性的病人可以对CEA处于免疫耐受状态。但研究发现，若将CEA与一强抗原共同应用，则可产生较强的抗CEA反应，随着肿瘤抗原筛选技术的发展、完善，对CEA诱发免疫应答的抗原表位也有了清楚的了解，从而引发了将CEA作为肿瘤免疫靶抗原的研究和应用。此外，CEA还是一种粘附因子，可促进肿瘤细胞与正常细胞的结合，在肿瘤细胞的转移中起重要作用，故把CEA作为靶抗原可有效的抑制大肠癌术后的复发与转移。
共刺激分子(Costimulatory factors)是T细胞激活的第二信号。T细
一
胞受体（TCR）与抗原结合产生T细胞活化的第一信号，而T细胞表面的CD28
与抗原提呈细胞（APC）表达的B7分子结合产生第H信号，T细胞只有同时
受到这两种信号的刺激，才能被完全活化。如果只有TCR介导的第一信号，
缺乏B7等共刺激分子提供的第h信号，则可能导致T细胞的凋亡或产生兔
疫无反应性（兔疫耐受）。除B细胞淋巴瘤和部分黑色素瘤外，肿瘤细胞不
表达B7－l（CD80）或B7－2（CD86）分子，因此肿瘤细胞容易导致兔疫耐受。
IL－2是所有细胞因子佐剂中被最广泛研究的细胞因子。IL－2通过分布于T、
B细胞、NK细胞及（淋巴因子激活的）杀伤细胞表面的受体系统发挥作用。
这一分子直接激活单核细胞并对诱导其他巨噬细胞有作用。而且，一些次
级分子如 GM－CSF的释放也由几－2引发，进而激发巨噬细胞的活性。当以
DNA表达质粒形式与抗原在抗原提呈部位共同接种后，IL－2有效地扩增针
对抗原的B细胞及T细胞应答。此外，IL－2是一种重要的T细胞生长因子，
幼稚T细胞转化为TH；或TH。细胞需要它的存在，其佐剂效应以促进细胞兔
疫应答为主。
鉴于此，本实验用RT－PCR的方法自病人手术切除结肠癌组织（CEA阳
性）中取得编码CEA的CDNA，构建其真核表达质粒p1RES－CEA。并以CEA
为肿瘤抗原的代表，构建了 B7－1和 IL－2基因与CEA CDNA的共表达质粒
pIRES－CEA／B7－1和 pIRES－CEA／IL－2。转染 COS7细胞，了解构建质粒的表
达情况。以共表达质粒 pIRES－CEA／B7＊和 pIRES－CEA／IL－2免疫小鼠模型，
研究其抗CEA的免疫应答作用，为临床上预防大肠癌术后复发与转移奠定
实验基础。
结果表明：（）本实验首次成功构建了能在真核细胞中高效表达的内部
核糖体进入位点控制的双重表达重组质粒 PIRES－CEA／B7－1 和
PIRES－CEA／IL－2；（2）共表达重组质粒 PIRES－CEA／B7－l禾 PIRES－CEA／IL－2
在表达CEA、B7八 及CEA、IL－2 量上无明显差异；（3）PIRES－CEA、
国家自然科学基金资助项目4
一
PIRES－CEA／B7l和PIRES－CEA／IL－2能诱导小鼠体内细胞免喇懈蚊，
其中 PIRES－CEA／B7－1和 PIRES－CEA／IL－2较PIRES－CEA免疫 答作用明显
增强。PIRES－CEA／B7－1 和 PIRES－CEA／IL－2 混合接f中应答最强，但与
plm卜优V队－1和p工旺｝nVI卜2靴接种统计学无明显差异：（4）中剂
量、大剂能禾中 PIRES－CEA／B7－1和 PIRES－CEA／IL－2弓I起的细胞 啪兔
觎答强于小剂貉种PIRES－CEA／B7－1和PIRES－CEA／IL－2。跟，中剂量
与大剂量
Colorecta1 cancer is one of the most common ma1ignant carcinoma
of the digestive tract. The main treatment for co1orecta1 cancer is
exci sion, but recurrence or metastasi s often occurs postoperat ive1y,
which common1y resu1ts in death. If we can do something during
operation or in early postoperative period to prevent recurrence or
metastasis, the surviva1 time for patients wi11 be pro1onged
remarkab1e1y. At present, chemica1 treatment is the conunon method used
to achieve this, but it is neither specific nor sensitive.
Tumor DNA vaccine, a nove1 type of vaccine, e1 icits both humora1
and ce11u1ar irmune responses. Ce11u1ar inununity especial1y has been
proved to p1ay the important ro1es in anti--tumor inununity. Since DNA
vaccines directly inocu1ate and express in myocyte, via MHC I and
MHC 1I of APC or myocyte de1ivering antigen to inunune system, it
is not necessary to consider MHC restriction in different
individuals.
Carcinoembryonic Antigen (CEA) is a re1ated antigen which is
expressed in many adenocarcinomas of the digestive system
adenocarcinomas. A1most a11 co1orecta1 cancers express CEA. In
addition, CEA is a1so an adherence factor that faci1itates the
conjugation of tumor and norma1 ce11s, which plays an important ro1e
in tumor recurrence and metastasis. However, tumor antigen is so weak
in ant igenicity that effective inunune response can not be e1 icited.
An important strategy to overcome this insufficiency is combination
of adjuvant and antigen.
T ce11 activation is dependent upon signa1s de1ivered through
the antigen--specific T ce11 receptors and accessory receptors on the
T ce11. Costimu1atory factors is the second singa1 for T ce11
activation. T--ce11 receptor (TCR) binding with antigen deve1op the
first singa1 for T ce11 to become active. CD28 on the T ce11 surface
binding with B7 which is expressed from APC deve1op second singa1. T
ce11 can on1y be activated when it receives these two singa1s. If there
is only first singa1, without the second singa1, maybe the T ce1l wi11
die or be ionuno1ogica11y unresponsive. Besides B ce11 1yinphoma and
some me1anoma, tumor ce11 do not express B7--1 (CD80) or B7--2 (CD86), so
tumor ce11s can easi1y deve1op immuno1ogica1 to1erance. IL--2 is one
of the cytokines that have been studied wide1y. IL--2 produces a marked
effect through the receptor system with distribution in the T, B, NK
and K ce11s. The mo1ecu1e direct1y activate the monocytes and induce
other histiocytes. When DNA and antigen both inocu1ate, IL--2 uti1 ity
amp1ify the B ce11s and T ce11s respond to the antigen. In
addition, IL--2 is a kind of important T ce11 growth factor. It is needed
for the conversion of irnature T ce11s to TH, or TH, ce11s. Its primary
adjuvant effect is to acce1erate the ce11u1ar inununity response.
In this study, we c1oned the CEA cDNA with RT--PCR and constructed
the pIRES expression vector. Taking Carcinoembryonic antigen (CEA)
as an examp1e of tumor antigens, CEA cDNA was c1oned from patients
suffering from co1orecta1 carcinoma that where CEA positive.
We c1oned the B7--1 and IL--2 cDNA with RT--PCR. The coexpression
plasmid of CEA，B7、land IL－2 were constructed．Further，their
effects as antn－tumor，their aballty to Induce llune response eere
S上tidied．
Theresultsshowedthat：（l）Threeeukaryotlcexpress。onplasmlds，
PIRES－CEA，PIRES－CEA／B7－1，PIRES－CEA／工乙－2，had bPPn Sllccess上＂fly
constructed，transformed Into COS7 cells and expression of two
proteins were demonstrated by ELISA。flow cytometer and elecsy．（2）
Inununlzed mince with coexpressson plasmld and controls，showed that
co—dellveryofB7－for IL－2 andCEAcDNAproduces anti一CEA immune
response（SI and IL－4）that are sUperior to those generated by the
nlasmid DNA encoding CEA alone．（3）pIRES－CEA，pIRES－CEA／B7－land
pIRES－CEA／IL－2 can induce mice cellular lllllllunlty and humoral
1mLlfllty In vlvo．The lllllllune resPonse of PIRES－CEA／B7－land
pIRES－CEA／IL
肿瘤核酸疫苗是近年崛起的新型生物治疗方法，它可同时诱导机体产生体液免疫和细胞免疫，尤其是细胞免疫，在抗肿瘤免疫中起关键作用，并且应用简便，可直接注射质粒至肌肉，通过肌细胞的MHCⅠ、MHCⅡ传递抗原信息，不必考虑个体间的MHC限制性。与传统性的化疗有本质的区别，是一种生理性的，着眼于调动宿主自身的抗癌能力，通过增强机体固有的抗肿瘤机制达到抑制、杀灭肿瘤细胞及根治肿瘤的目的；而传统的化疗则是借助“外力”杀灭肿瘤细胞，肿瘤细胞消灭不彻底，难以控制肿瘤复发与转移，并给正常组织细胞带来巨大毒副作用。
癌胚抗原(Carcinoembryonic Antigen,CEA)是许多消化道腺癌表达的一种肿瘤相关抗原，大肠癌几乎100％都表达CEA。正常消化道粘膜仅有少量分泌。它的免疫原性较低，临床检测为CEA阳性的病人可以对CEA处于免疫耐受状态。但研究发现，若将CEA与一强抗原共同应用，则可产生较强的抗CEA反应，随着肿瘤抗原筛选技术的发展、完善，对CEA诱发免疫应答的抗原表位也有了清楚的了解，从而引发了将CEA作为肿瘤免疫靶抗原的研究和应用。此外，CEA还是一种粘附因子，可促进肿瘤细胞与正常细胞的结合，在肿瘤细胞的转移中起重要作用，故把CEA作为靶抗原可有效的抑制大肠癌术后的复发与转移。
共刺激分子(Costimulatory factors)是T细胞激活的第二信号。T细
一
胞受体（TCR）与抗原结合产生T细胞活化的第一信号，而T细胞表面的CD28
与抗原提呈细胞（APC）表达的B7分子结合产生第H信号，T细胞只有同时
受到这两种信号的刺激，才能被完全活化。如果只有TCR介导的第一信号，
缺乏B7等共刺激分子提供的第h信号，则可能导致T细胞的凋亡或产生兔
疫无反应性（兔疫耐受）。除B细胞淋巴瘤和部分黑色素瘤外，肿瘤细胞不
表达B7－l（CD80）或B7－2（CD86）分子，因此肿瘤细胞容易导致兔疫耐受。
IL－2是所有细胞因子佐剂中被最广泛研究的细胞因子。IL－2通过分布于T、
B细胞、NK细胞及（淋巴因子激活的）杀伤细胞表面的受体系统发挥作用。
这一分子直接激活单核细胞并对诱导其他巨噬细胞有作用。而且，一些次
级分子如 GM－CSF的释放也由几－2引发，进而激发巨噬细胞的活性。当以
DNA表达质粒形式与抗原在抗原提呈部位共同接种后，IL－2有效地扩增针
对抗原的B细胞及T细胞应答。此外，IL－2是一种重要的T细胞生长因子，
幼稚T细胞转化为TH；或TH。细胞需要它的存在，其佐剂效应以促进细胞兔
疫应答为主。
鉴于此，本实验用RT－PCR的方法自病人手术切除结肠癌组织（CEA阳
性）中取得编码CEA的CDNA，构建其真核表达质粒p1RES－CEA。并以CEA
为肿瘤抗原的代表，构建了 B7－1和 IL－2基因与CEA CDNA的共表达质粒
pIRES－CEA／B7－1和 pIRES－CEA／IL－2。转染 COS7细胞，了解构建质粒的表
达情况。以共表达质粒 pIRES－CEA／B7＊和 pIRES－CEA／IL－2免疫小鼠模型，
研究其抗CEA的免疫应答作用，为临床上预防大肠癌术后复发与转移奠定
实验基础。
结果表明：（）本实验首次成功构建了能在真核细胞中高效表达的内部
核糖体进入位点控制的双重表达重组质粒 PIRES－CEA／B7－1 和
PIRES－CEA／IL－2；（2）共表达重组质粒 PIRES－CEA／B7－l禾 PIRES－CEA／IL－2
在表达CEA、B7八 及CEA、IL－2 量上无明显差异；（3）PIRES－CEA、
国家自然科学基金资助项目4
一
PIRES－CEA／B7l和PIRES－CEA／IL－2能诱导小鼠体内细胞免喇懈蚊，
其中 PIRES－CEA／B7－1和 PIRES－CEA／IL－2较PIRES－CEA免疫 答作用明显
增强。PIRES－CEA／B7－1 和 PIRES－CEA／IL－2 混合接f中应答最强，但与
plm卜优V队－1和p工旺｝nVI卜2靴接种统计学无明显差异：（4）中剂
量、大剂能禾中 PIRES－CEA／B7－1和 PIRES－CEA／IL－2弓I起的细胞 啪兔
觎答强于小剂貉种PIRES－CEA／B7－1和PIRES－CEA／IL－2。跟，中剂量
与大剂量
Colorecta1 cancer is one of the most common ma1ignant carcinoma
of the digestive tract. The main treatment for co1orecta1 cancer is
exci sion, but recurrence or metastasi s often occurs postoperat ive1y,
which common1y resu1ts in death. If we can do something during
operation or in early postoperative period to prevent recurrence or
metastasis, the surviva1 time for patients wi11 be pro1onged
remarkab1e1y. At present, chemica1 treatment is the conunon method used
to achieve this, but it is neither specific nor sensitive.
Tumor DNA vaccine, a nove1 type of vaccine, e1 icits both humora1
and ce11u1ar irmune responses. Ce11u1ar inununity especial1y has been
proved to p1ay the important ro1es in anti--tumor inununity. Since DNA
vaccines directly inocu1ate and express in myocyte, via MHC I and
MHC 1I of APC or myocyte de1ivering antigen to inunune system, it
is not necessary to consider MHC restriction in different
individuals.
Carcinoembryonic Antigen (CEA) is a re1ated antigen which is
expressed in many adenocarcinomas of the digestive system
adenocarcinomas. A1most a11 co1orecta1 cancers express CEA. In
addition, CEA is a1so an adherence factor that faci1itates the
conjugation of tumor and norma1 ce11s, which plays an important ro1e
in tumor recurrence and metastasis. However, tumor antigen is so weak
in ant igenicity that effective inunune response can not be e1 icited.
An important strategy to overcome this insufficiency is combination
of adjuvant and antigen.
T ce11 activation is dependent upon signa1s de1ivered through
the antigen--specific T ce11 receptors and accessory receptors on the
T ce11. Costimu1atory factors is the second singa1 for T ce11
activation. T--ce11 receptor (TCR) binding with antigen deve1op the
first singa1 for T ce11 to become active. CD28 on the T ce11 surface
binding with B7 which is expressed from APC deve1op second singa1. T
ce11 can on1y be activated when it receives these two singa1s. If there
is only first singa1, without the second singa1, maybe the T ce1l wi11
die or be ionuno1ogica11y unresponsive. Besides B ce11 1yinphoma and
some me1anoma, tumor ce11 do not express B7--1 (CD80) or B7--2 (CD86), so
tumor ce11s can easi1y deve1op immuno1ogica1 to1erance. IL--2 is one
of the cytokines that have been studied wide1y. IL--2 produces a marked
effect through the receptor system with distribution in the T, B, NK
and K ce11s. The mo1ecu1e direct1y activate the monocytes and induce
other histiocytes. When DNA and antigen both inocu1ate, IL--2 uti1 ity
amp1ify the B ce11s and T ce11s respond to the antigen. In
addition, IL--2 is a kind of important T ce11 growth factor. It is needed
for the conversion of irnature T ce11s to TH, or TH, ce11s. Its primary
adjuvant effect is to acce1erate the ce11u1ar inununity response.
In this study, we c1oned the CEA cDNA with RT--PCR and constructed
the pIRES expression vector. Taking Carcinoembryonic antigen (CEA)
as an examp1e of tumor antigens, CEA cDNA was c1oned from patients
suffering from co1orecta1 carcinoma that where CEA positive.
We c1oned the B7--1 and IL--2 cDNA with RT--PCR. The coexpression
plasmid of CEA，B7、land IL－2 were constructed．Further，their
effects as antn－tumor，their aballty to Induce llune response eere
S上tidied．
Theresultsshowedthat：（l）Threeeukaryotlcexpress。onplasmlds，
PIRES－CEA，PIRES－CEA／B7－1，PIRES－CEA／工乙－2，had bPPn Sllccess上＂fly
constructed，transformed Into COS7 cells and expression of two
proteins were demonstrated by ELISA。flow cytometer and elecsy．（2）
Inununlzed mince with coexpressson plasmld and controls，showed that
co—dellveryofB7－for IL－2 andCEAcDNAproduces anti一CEA immune
response（SI and IL－4）that are sUperior to those generated by the
nlasmid DNA encoding CEA alone．（3）pIRES－CEA，pIRES－CEA／B7－land
pIRES－CEA／IL－2 can induce mice cellular lllllllunlty and humoral
1mLlfllty In vlvo．The lllllllune resPonse of PIRES－CEA／B7－land
pIRES－CEA／IL
引文
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2. Ford CH, Osbome PA, Rego BG, et al. Bispecific antibody targeting of doxorubicin to carcinoembryonic antigen-expressing colon cancer cell lines in vitro and in vivo. Int J Cancer, 2001, 92(6):851-5.
3. Shapiro M, Scapa E. Elevated carcinoembryonic antigen (CEA) levels in a patient with no malignancy. Hepatogastroenterology, 2000,47(31):163-4.
4．杨镇主编。肿瘤免疫学。湖北科技出版社。1998，P15-185。
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11. Reiter W, Stieber P, Reuter C, et al. Multivariate analysis of the prognostic value of CEA and CA 19-9 serum levels in colorectal cancer. Anticancer Res, 2000 ,20(6D):5195-8.
12. Kem W, Beckert B, Lang N, et al. Hepatic arterial infusion with oxaliplatin, folinic acid, and 5-fluorouracil in patients with hepatic metastases from colorectal cancer: role of carcino-embryonic antigen in assessment of response. Anticancer Res, 2000 ,20(6D):4973-5.
13. Wichmann MW, Lau-Werner U, Muller C, et al. Carcinoembryonic antigen for the detection of recurrent disease following curative resection of colorectal cancer. Anticancer Res, 2000,20(6D):4953-5.
14. White SA, LoBuglio AF, Arani RB et al. Induction of anti-tumor immunity by intrasplenic administration of a carcinoembryonic antigen DNA vaccine. J-Gene-Med, 2000,2(2): 135-40.
15. Foon KA, et al. Immune response to the carcinoembryonic antigen in patients treated with an antiidiotype antibody vaccine. J Clin Invest, 1995, (96):334-342.
16. Foon KA, John WJ, Chakraborty M et al. Clinical and immune responses in resected colon cancer patients treated with anti-idiotype monoclonal antibody vaccine that mimics the carcinoembryonic antigen. J-Clin-Oncol, 1999,17(9): 2889-5.
17. Yip D, Strickl and AH, Karapetis CS, et al. Immunomodulation therapy in colorectal carcinoma. Cancer Treat Rev, 2000 ,26(3):169-90.
18. Maxwell-Armstrong CA, Durrant LG, Scholefield JH. Colorectal cancer vaccines. Br J Surg, 1998,85(2):149-54.
19. Hollingshead MG, Tonkonogy SL, De Buysscher EV.A method for the accurate determination of the specificity of monoclonat antibodies reactive with porcine immunoglobulins. Vet Immunol Immunopathol, 1985,10(2-3):167-75.
20. Pervin S, Chakraborty M, Bhattacharya-Chatterjee M, et al. Induction of antitumor immunity by an anti-idiotype antibody mimicking carcinoembryonic antigen. Cancer Res, 1997,57(4):728-34.
21. Kaufman H, Kantor J, Irvine K, et al. Antitumor activity and immune responses induced by a recombinant carcinoembryonic antigen-vaccinia virus vaccine. J Natl Cancer Inst, 1992, 84(14):1084-91.
22. Butler D, Maurice J,O'Brien C. Vaccines:a roller-coaster of hopes. Nature, 1997,86:537-538.
23. Yamana H, Itoh K. Specific immunotherapy with cancer vaccines. Gan-To-Kagaku-Ryoho, 2000,27(10): 1477-88.
24．孙树汉。核酸疫苗及其在疾病免疫防治中的应用。第二军医大学学报，2000，21(6)：519-521。
25. Slavik JM, Hutchcroft JE, Bierer BE. CD28/CTLA-4 and CD80/CD86 families: signaling and function. Immunol-Res, 1999, 19(1): 1-24.
26．何球藻主编。细胞与分子免疫学。上海科学技术文献出版社。1997。
27. Hamilton JM, et al. Phase Ⅰ study of recombinant vaccinia virus(rv) that expresses human carcinoembryonic antigen in adult patients with adenocarcinomas. Proc Am Assoc Clin Oncol Aunu Meet, 1994,961.
28. Conry RM, et al. Immune response to the carcinoembryonic antigen polynucleotide vaccine. Cancer Res, 1994, (54):1164-1171.
29. Conry RM, et al. Polynucleotide-mediated immunization therapy of cancer. Semin Oncol, 1996, (23):135-139.
30. Conry RM, et al. Phase a trial of a polynucleotide antitumor immunization to human carcinoembryonic antigen in patients with metastatic colorectal cancer. Hun Gene Ther 1996, (7):755-759.
31. Conry RM, et al. Selected strategies to augment polynucleotide immunization. Gene Ther, 1996, (3):67-74.
32．叶传忠，陈仕平，裴雪涛等。内部核糖体进入位点控制hytk基因和绿色荧光蛋白的表达。基础医学与临床，2000，20(4)：31-36。
33. Misteli T, Spector DL. Application of the green fluorescent protein in cell biology and biotechnology. Nature Biotechnology, 1997,15:961-964.
34. Grabstein KH, Eisenman J, Shanebeck K. Cloning of a T cell growth factor that interacts with the chain of the interleukin-2 receptor. Science, 1994,264(5161)965.
35. Degiannis D, Kalteziotis V, Thalassinos A. Effect of cyclosporine and sirolimus on interleukin-15-driven proliferation of OKT3-preactivated human lymphocytes. Transplant Proc, 1998,30(3):948.
36. Kantor J, et al. Immunogenicity and safety of a recombinant vaccinia expression the carcinoembryonic antigen gene in a non human primate. Cancer Res, 1992, (52):6217-6222.
37. Conry RM, Khazaeli MB, Saleh MN, et al. Phase Ⅰ trial of a recombinant vaccinia virus encoding carcinoembryonic antigen in metastatic adenoearcinoma: comparison of intradermal versus subcutaneous administration. Clin Cancer Res, 1999,5(9):2330-7.
38. Marshall JL, et al. Phase Ⅰ study in cancer patients of a replication-defective avipox recombinant vaccine that expresses human carcinoembryonic antigen. J Clin Oncol, 1999, 17(1):332-7.
39. Conry RM, Allen KO, Lee S, et al. Human autoantibodies to carcinoembryonic antigen (CEA) induced by a vaccinia-CEA vaccine. Clin Cancer Res, 2000,6(1):34-41.
40．谢建云等。疫苗佐剂的分类及作用方式。“国外医学”预防、诊断、治疗用生物制品分册，1997，(20)：253-258。
41. Chen LP, McGowan P, Ashe S, et al. Tumor immunogenicity determines the effect of B7 costimulation on T cell-mediated tumor immunity. J Exp Med, 1994,179:523-532.
42. Tao G, Hu J, Zou H. Therapeutic antitumor response to cervical cancer in mice immunized with U14 vaccines transfected with costimulatory B7 gene. Chin Med J (Engl), 2001,114(6):623-7.
43．曹世龙主编。肿瘤学新理论与新技术。上海科技教育出版社。1997，P112-133。
44. Tighe H. Gene vaccination:plasmid DNA more than just a blueprint. Immunol Today, 1998, (19):89-97.
45. Heicappell R, et al. Prevention of metastatic spread by postoperative immunotherapy with virally modified autologous tumor lls. Int J Cancer, 1986, (37):569-577.
46．卢大儒主编。医学分子遗传学。复旦大学出版社。1997，P165-243。
47. Xiang R, Silletti S, Lode HN, et al. Protective immunity against human carcinoembryonic antigen (CEA) induced by an oral DNA vaccine in CEA-transgenic mice. Clin Cancer Res, 2001,7(3 Suppl):856s-864s.
48. Song K, Chang Y, Prudhomme GJ. IL-12 plasmid-enhanced DNA vaccination against carcinoembryonic antigen(CEA) studied in immune-gene knockout mice. Gene Ther, 2000,7(18):1527-35.
49. Nishimura N, Nishioka Y, Shinohara T, et al. Enhanced Efficiency by Centrifugal Manipulation of Adenovirus-Mediated Interleukin 12 Gene Transduction into Human Monocyte-Derived Dendritic Cells. Hum Gene Ther, 2001,12(4):333-346.
50. Buchanan RM, Briles DE, Arulanandam BP, et al. IL-12-mediated increases in protection elicited by pneumococcal and meningococcal conjugate vaccines. Vaccine, 2001, 28(19):2020-2028.
51. Stevenson MM, Su Z, Sam H, et al. Modulation of host responses to blood-stage malaria by interleukin-12: from therapyto adjuvant activity. Microbes Infect, 2001,3(1):49-59.
52. Hall MA, McGlinn E, Coakley G, et al. Genetic polymorphism of IL-12 p40 gene in immune-mediated disease. Genes Immun, 2000,1(3):219-24.
53. Dai-Z; Lakkis-FG. The role of cytokines, CTLA-4 and costimulation in transplant tolerance and rejection. Curr-Opin-Immunol. 1999, 11(5): 504-8
54. Burger JA, Mendoza RB, Kipps TJ. Plasmids encoding granulocyte-macrophage colony-stimulating factor and CD154 enhance the immune response to genetic vaccines. Vaccine, 2001,19(15-16):2181-2189.
55. Wei L, Dai JX, Sun SH. Construction and expression of coexpression plasmid of carcinoembryonic antigen and Newcastle disease virus HN gene. Sheng Wu Gong Cheng Xue Bao. 2000,16(5):641-4.
56. Zhong H, Cao C, Li P, et al. Induction of protective immune responses in rhesus monkey by immunization with recombinant plasmids of polyvalent epitopes of Plasmodium falciparum using cholera toxin B as adjuvant. Yi Chuan Xue Bao, 2000,27(11):966-71.
57. Krieg AM, Yi AK, Matson S, et al. CpG motifs in bacterial DNA trigger direct B-cell activation .Nature, 1995,374:546-549.
58. Krieg AM, et al. Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motif. Proc Natl Acad Sci USA, 1998, (95):12631-12636.
59. Shoda LK, Kegerreis KA, Suarez CE, et al. Immunostimulatory CpG-modified plasmid DNA enhances IL-12, TNF-alpha, and NO production by bovine macrophages. J Leukoc Biol, 2001,70(1):103-12.
60. Moss RB, Diveley J, Jensen FC, et al. HIV-Specific CD4(+) and CD8(+) immune responses are generated with a gp120-depleted, whole-killed HIV-1 immunogen with CpG immunostimulatory sequences of DNA. J Interferon Cytokine Res, 2000,20(12):1131-7.
61. von Hunolstein C, Mariotti S, Teloni R, et al. The adjuvant effect of synthetic oligodeoxynucleotide containing CpG motif converts the anti-Haemophilus influenzae type b glycoconjugates into efficient anti-polysaccharide and anti-carrier polyvalent vaccines. Vaccine, 2001,19(23-24):3058-66.
2. Ford CH, Osbome PA, Rego BG, et al. Bispecific antibody targeting of doxorubicin to carcinoembryonic antigen-expressing colon cancer cell lines in vitro and in vivo. Int J Cancer, 2001, 92(6):851-5.
3. Shapiro M, Scapa E. Elevated carcinoembryonic antigen (CEA) levels in a patient with no malignancy. Hepatogastroenterology, 2000,47(31):163-4.
4．杨镇主编。肿瘤免疫学。湖北科技出版社。1998，P15-185。
5. Kantor J, et al. Antitumor activity and immune response induced by a recombinant carcinoembryonic antigen-vaccinia vaccine. J Natl Cancer Inst, 1992, (84):1084-1092.
6. Krop-Watorek A, Lisowska E. Carcinoembryonic antigen as an adhesion molecule. Arch Immunol Ther Exp (Warsz), 1998,46(3):129-36.
7. Maxwell P. Carcinoembryonic antigen: cell adhesion molecule and useful diagnostic marker. Bt J Biomed Sci, 1999,56(3):209-14.
8. Riesen W, Kessler AC, Ehrhardt V. Individual variations of carcinoembryonic antigen (CEA) in serum of healthy subjects. Clin Chem, 1987 ,33(11):2123.
9. Flynn AA, Boxer GM, Begent RH, et al. Relationship between tumour morphology, antigen and antibody distribution measured by fusion of digital phosphor and photographic images. Cancer Immunol Immunother, 2001 ,50(2):77-81.
10. Holubec L, Jr Topolcan O, Pikner R, et al. The significance of CEA, CA19-9 and CA72-4 in the detection of colorectal carcinoma recurrence. Anticancer Res, 2000,20(6D):5237-44.
11. Reiter W, Stieber P, Reuter C, et al. Multivariate analysis of the prognostic value of CEA and CA 19-9 serum levels in colorectal cancer. Anticancer Res, 2000 ,20(6D):5195-8.
12. Kem W, Beckert B, Lang N, et al. Hepatic arterial infusion with oxaliplatin, folinic acid, and 5-fluorouracil in patients with hepatic metastases from colorectal cancer: role of carcino-embryonic antigen in assessment of response. Anticancer Res, 2000 ,20(6D):4973-5.
13. Wichmann MW, Lau-Werner U, Muller C, et al. Carcinoembryonic antigen for the detection of recurrent disease following curative resection of colorectal cancer. Anticancer Res, 2000,20(6D):4953-5.
14. White SA, LoBuglio AF, Arani RB et al. Induction of anti-tumor immunity by intrasplenic administration of a carcinoembryonic antigen DNA vaccine. J-Gene-Med, 2000,2(2): 135-40.
15. Foon KA, et al. Immune response to the carcinoembryonic antigen in patients treated with an antiidiotype antibody vaccine. J Clin Invest, 1995, (96):334-342.
16. Foon KA, John WJ, Chakraborty M et al. Clinical and immune responses in resected colon cancer patients treated with anti-idiotype monoclonal antibody vaccine that mimics the carcinoembryonic antigen. J-Clin-Oncol, 1999,17(9): 2889-5.
17. Yip D, Strickl and AH, Karapetis CS, et al. Immunomodulation therapy in colorectal carcinoma. Cancer Treat Rev, 2000 ,26(3):169-90.
18. Maxwell-Armstrong CA, Durrant LG, Scholefield JH. Colorectal cancer vaccines. Br J Surg, 1998,85(2):149-54.
19. Hollingshead MG, Tonkonogy SL, De Buysscher EV.A method for the accurate determination of the specificity of monoclonat antibodies reactive with porcine immunoglobulins. Vet Immunol Immunopathol, 1985,10(2-3):167-75.
20. Pervin S, Chakraborty M, Bhattacharya-Chatterjee M, et al. Induction of antitumor immunity by an anti-idiotype antibody mimicking carcinoembryonic antigen. Cancer Res, 1997,57(4):728-34.
21. Kaufman H, Kantor J, Irvine K, et al. Antitumor activity and immune responses induced by a recombinant carcinoembryonic antigen-vaccinia virus vaccine. J Natl Cancer Inst, 1992, 84(14):1084-91.
22. Butler D, Maurice J,O'Brien C. Vaccines:a roller-coaster of hopes. Nature, 1997,86:537-538.
23. Yamana H, Itoh K. Specific immunotherapy with cancer vaccines. Gan-To-Kagaku-Ryoho, 2000,27(10): 1477-88.
24．孙树汉。核酸疫苗及其在疾病免疫防治中的应用。第二军医大学学报，2000，21(6)：519-521。
25. Slavik JM, Hutchcroft JE, Bierer BE. CD28/CTLA-4 and CD80/CD86 families: signaling and function. Immunol-Res, 1999, 19(1): 1-24.
26．何球藻主编。细胞与分子免疫学。上海科学技术文献出版社。1997。
27. Hamilton JM, et al. Phase Ⅰ study of recombinant vaccinia virus(rv) that expresses human carcinoembryonic antigen in adult patients with adenocarcinomas. Proc Am Assoc Clin Oncol Aunu Meet, 1994,961.
28. Conry RM, et al. Immune response to the carcinoembryonic antigen polynucleotide vaccine. Cancer Res, 1994, (54):1164-1171.
29. Conry RM, et al. Polynucleotide-mediated immunization therapy of cancer. Semin Oncol, 1996, (23):135-139.
30. Conry RM, et al. Phase a trial of a polynucleotide antitumor immunization to human carcinoembryonic antigen in patients with metastatic colorectal cancer. Hun Gene Ther 1996, (7):755-759.
31. Conry RM, et al. Selected strategies to augment polynucleotide immunization. Gene Ther, 1996, (3):67-74.
32．叶传忠，陈仕平，裴雪涛等。内部核糖体进入位点控制hytk基因和绿色荧光蛋白的表达。基础医学与临床，2000，20(4)：31-36。
33. Misteli T, Spector DL. Application of the green fluorescent protein in cell biology and biotechnology. Nature Biotechnology, 1997,15:961-964.
34. Grabstein KH, Eisenman J, Shanebeck K. Cloning of a T cell growth factor that interacts with the chain of the interleukin-2 receptor. Science, 1994,264(5161)965.
35. Degiannis D, Kalteziotis V, Thalassinos A. Effect of cyclosporine and sirolimus on interleukin-15-driven proliferation of OKT3-preactivated human lymphocytes. Transplant Proc, 1998,30(3):948.
36. Kantor J, et al. Immunogenicity and safety of a recombinant vaccinia expression the carcinoembryonic antigen gene in a non human primate. Cancer Res, 1992, (52):6217-6222.
37. Conry RM, Khazaeli MB, Saleh MN, et al. Phase Ⅰ trial of a recombinant vaccinia virus encoding carcinoembryonic antigen in metastatic adenoearcinoma: comparison of intradermal versus subcutaneous administration. Clin Cancer Res, 1999,5(9):2330-7.
38. Marshall JL, et al. Phase Ⅰ study in cancer patients of a replication-defective avipox recombinant vaccine that expresses human carcinoembryonic antigen. J Clin Oncol, 1999, 17(1):332-7.
39. Conry RM, Allen KO, Lee S, et al. Human autoantibodies to carcinoembryonic antigen (CEA) induced by a vaccinia-CEA vaccine. Clin Cancer Res, 2000,6(1):34-41.
40．谢建云等。疫苗佐剂的分类及作用方式。“国外医学”预防、诊断、治疗用生物制品分册，1997，(20)：253-258。
41. Chen LP, McGowan P, Ashe S, et al. Tumor immunogenicity determines the effect of B7 costimulation on T cell-mediated tumor immunity. J Exp Med, 1994,179:523-532.
42. Tao G, Hu J, Zou H. Therapeutic antitumor response to cervical cancer in mice immunized with U14 vaccines transfected with costimulatory B7 gene. Chin Med J (Engl), 2001,114(6):623-7.
43．曹世龙主编。肿瘤学新理论与新技术。上海科技教育出版社。1997，P112-133。
44. Tighe H. Gene vaccination:plasmid DNA more than just a blueprint. Immunol Today, 1998, (19):89-97.
45. Heicappell R, et al. Prevention of metastatic spread by postoperative immunotherapy with virally modified autologous tumor lls. Int J Cancer, 1986, (37):569-577.
46．卢大儒主编。医学分子遗传学。复旦大学出版社。1997，P165-243。
47. Xiang R, Silletti S, Lode HN, et al. Protective immunity against human carcinoembryonic antigen (CEA) induced by an oral DNA vaccine in CEA-transgenic mice. Clin Cancer Res, 2001,7(3 Suppl):856s-864s.
48. Song K, Chang Y, Prudhomme GJ. IL-12 plasmid-enhanced DNA vaccination against carcinoembryonic antigen(CEA) studied in immune-gene knockout mice. Gene Ther, 2000,7(18):1527-35.
49. Nishimura N, Nishioka Y, Shinohara T, et al. Enhanced Efficiency by Centrifugal Manipulation of Adenovirus-Mediated Interleukin 12 Gene Transduction into Human Monocyte-Derived Dendritic Cells. Hum Gene Ther, 2001,12(4):333-346.
50. Buchanan RM, Briles DE, Arulanandam BP, et al. IL-12-mediated increases in protection elicited by pneumococcal and meningococcal conjugate vaccines. Vaccine, 2001, 28(19):2020-2028.
51. Stevenson MM, Su Z, Sam H, et al. Modulation of host responses to blood-stage malaria by interleukin-12: from therapyto adjuvant activity. Microbes Infect, 2001,3(1):49-59.
52. Hall MA, McGlinn E, Coakley G, et al. Genetic polymorphism of IL-12 p40 gene in immune-mediated disease. Genes Immun, 2000,1(3):219-24.
53. Dai-Z; Lakkis-FG. The role of cytokines, CTLA-4 and costimulation in transplant tolerance and rejection. Curr-Opin-Immunol. 1999, 11(5): 504-8
54. Burger JA, Mendoza RB, Kipps TJ. Plasmids encoding granulocyte-macrophage colony-stimulating factor and CD154 enhance the immune response to genetic vaccines. Vaccine, 2001,19(15-16):2181-2189.
55. Wei L, Dai JX, Sun SH. Construction and expression of coexpression plasmid of carcinoembryonic antigen and Newcastle disease virus HN gene. Sheng Wu Gong Cheng Xue Bao. 2000,16(5):641-4.
56. Zhong H, Cao C, Li P, et al. Induction of protective immune responses in rhesus monkey by immunization with recombinant plasmids of polyvalent epitopes of Plasmodium falciparum using cholera toxin B as adjuvant. Yi Chuan Xue Bao, 2000,27(11):966-71.
57. Krieg AM, Yi AK, Matson S, et al. CpG motifs in bacterial DNA trigger direct B-cell activation .Nature, 1995,374:546-549.
58. Krieg AM, et al. Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motif. Proc Natl Acad Sci USA, 1998, (95):12631-12636.
59. Shoda LK, Kegerreis KA, Suarez CE, et al. Immunostimulatory CpG-modified plasmid DNA enhances IL-12, TNF-alpha, and NO production by bovine macrophages. J Leukoc Biol, 2001,70(1):103-12.
60. Moss RB, Diveley J, Jensen FC, et al. HIV-Specific CD4(+) and CD8(+) immune responses are generated with a gp120-depleted, whole-killed HIV-1 immunogen with CpG immunostimulatory sequences of DNA. J Interferon Cytokine Res, 2000,20(12):1131-7.
61. von Hunolstein C, Mariotti S, Teloni R, et al. The adjuvant effect of synthetic oligodeoxynucleotide containing CpG motif converts the anti-Haemophilus influenzae type b glycoconjugates into efficient anti-polysaccharide and anti-carrier polyvalent vaccines. Vaccine, 2001,19(23-24):3058-66.