MUC1-VNTR核酸疫苗抗胰腺癌的体内外实验研究
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摘要
胰腺癌是各种恶性肿瘤中恶性程度极高的肿瘤之一，其病因至今不完全明确。目前胰腺癌的治疗仍以手术为主，但是手术只能切除肉眼可见的病灶，对转移和微小的病灶无法去除，而这些转移和微小病灶正是造成胰腺癌复发和患者死亡的主要因素。因此寻找对胰腺癌有效治疗手段，是目前国际上的研究热点之一。本文在前期研究的基础上，进一步研究MUC1-VNTR核酸疫苗抗胰腺癌的体内外效应。实验分为两个部分：
第一部分MUC1-VNTR核酸疫苗抗胰腺癌的体内实验研究
第一节小鼠胰腺癌细胞系panc02-MUC1构建
研究目的：
构建表达MUC1的小鼠胰腺癌细胞系panc02-MUC1。
材料和方法：
小鼠胰腺癌细胞系panc02由美国MD Anderson Cancer center赠送，表达全长MUC1的质粒pcDNA3-MUC1由美国匹兹堡大学Dr．Finn赠送。体外以Lipofectamine2000脂质体转染pcDNA3-MUC1质粒到小鼠胰腺癌细胞系panc02中，并以空载质粒pcDNA3转染细胞为对照；转染细胞再经G418压力筛选单克隆细胞株；并于小鼠皮下接种进行成瘤实验。
结果：
构建单克隆细胞株panc02-MUC1经Western blot检测可见MUC1蛋白表达；细胞免疫荧光检测可见细胞膜上有MUC1表达；且动物接种实验证实该细胞可在正常小鼠皮下成瘤，免疫组化检测可见该细胞系在C57BL／6小鼠皮下接种胰腺癌模型中可表达MUC1。
结论：
构建的细胞系为可表达MUC1的单克隆细胞系，且该细胞系可在正常C57BL／6小鼠中成瘤。
第二节MUC1-VNTR核酸疫苗预防小鼠胰腺癌实验研究
研究目的：
以pcDNA3.1-VNTR质粒肌注免疫C57BL／6小鼠，以pcDNA3.1空载质粒及PBS免疫小鼠为对照；研究MUC1-VNTR核酸疫苗免疫小鼠能否预防表达MUC1的小鼠胰腺癌细胞panc02-MUC1的成瘤效应。
材料和方法：
C57BL／6正常小鼠，随机分3组，每组18只：分别为PBS组、Neo组(pcDNA3.1免疫组)、MUC1组(pcDNA3.1-VNTR免疫组)。各组小鼠经右腿胫前肌中部注射100μg／100μl质粒PBS溶液。第1次免疫2周后，以同一质粒再次免疫小鼠，第2次免疫2周后，以同一质粒再次免疫小鼠；第3次免疫结束后5日自内眦静脉采血获得血清并以ELISA法测抗VNTR抗体；取脾细胞悬液体外以VNTR合成肽特异性刺激培养(每组3只)，3天后以LDH法进行CTL杀伤试验；第3次免疫结束后7日，各组小鼠经左侧前肢腋下皮下注射1×10~6／100μl／只panc02-MUC1细胞，7日后接种部位皮下可及肿块，每2-3日测量肿瘤长径及短径，观察MUC1-VNTR核酸疫苗预防小鼠胰腺癌细胞panc02-MUC1的成瘤效应。另6只pcDNA3.1-VNTR质粒免疫小鼠接种panc02-Neo细胞，接种细胞量为1×10~6／100μl／只。肿瘤体积大于1000mm~3时，视该小鼠死亡。
结果：
pcDNA3.1-VNTR质粒免疫的小鼠血清抗VNTR抗体浓度以吸光度表示，该组吸光度显著高于空质粒对照组和PBS对照组(P＜0.05)。pcDNA3.1-VNTR质粒免疫的小鼠脾细胞对表达MUC1的小鼠胰腺癌细胞panc02-MUC1细胞有显著的杀伤效应，明显高于空质粒组及PBS组(P＜0.05)；该杀伤效应可以被MUC1单克隆抗体VU3C6抑制(P＜0.05)；且该脾细胞对不表达MUC1的panc02-Neo细胞无明显杀伤效应(P＜0.05)。pcDNA3．1-VNTR质粒免疫组小鼠对panc02-MUC1细胞生长具有明显抑制作用，第24天时，pcDNA3．1-VNTR质粒免疫组小鼠肿瘤体积显著小于空质粒组及PBS组(P＜0.05)；且该组小鼠生存期明显长于空质粒组及PBS组(P＜0.05)；此外pcDNA3.1-VNTR质粒免疫的小鼠对不表达MUC1的panc02-Neo细胞生长无抑制作用，第28天时该组小鼠皮下肿瘤体积显著大于接种panc02-MUC1细胞组(P＜0.05)；且该组小鼠生存期显著短于接种panc02-MUC1细胞组(P＜0.05)。
结论：
MUC1-VNTR核酸疫苗可以预防表达MUC1的小鼠胰腺癌细胞panc02-MUC1的成瘤效应。
第三节GMCSF联合MUC1-VNTR核酸疫苗治疗小鼠胰腺癌实验研究
研究目的：
以GMCSF联合pcDNA3.1-VNTR质粒治疗荷载panc02-MUC1的C57BL／6小鼠。
材料和方法：
0天C57BL／6正常小鼠经左侧前肢腋下皮下注射panc02-MUC1细胞1×10~6／100μl／只，随机分5组，：G+M(GMCSF+MUC1)(n=10)、M(MUC1)(n=10)、G(GMCSF)(n=10)、Neo(pcDNA3.1)(n=9)及PBS(n=9)组。4天时各组分别经后腿肌肉注射GMCSF50ng／100μl+pcDNA3.1-VNTR质粒100μg／100μl、pcDNA3.1-VNTR质粒100μg／100μl、GMCSF50ng／100μl、peDNA3.1质粒100μg／100μl、PBS100μl；于9天、14天各重复注射一次。肿瘤接种后6-7天开始测量各组小鼠肿瘤长径及短径，小鼠肿瘤体积超过1000mm~3视为小鼠死亡；第3次治疗后11天各组取3只小鼠脾细胞体外行杀伤实验。另12只小鼠分为G+M(GMCSF+MUC1)、M(MUC1)两组，每组6只，0天时经左侧前肢腋下皮下注射1×10~6／100μl／只panc02-Neo细胞，4天时各组分别经后腿肌肉注射GMCSF50ng／100μl+pcDNA3.1-VNTR质粒100μg／100μl、pcDNA3.1-VNTR质粒100μg／100μl；于9天、14天各重复注射一次。肿瘤接种后6-7天开始测量各组小鼠肿瘤长径及短径，小鼠肿瘤体积超过1000mm~3视为小鼠死亡。
结果：
G+M及M组小鼠panc02-MUC1肿瘤细胞生长明显慢于G、Neo及PBS组(P＜0.05)，且G+M组明显慢于M组(P＜0.05)；而GMCSF联合pcDNA3.1-VNTR质粒或单用pcDNA3.1-VNTR质粒治疗panc02-Neo肿瘤，均未能抑制肿瘤生长(P＜0.05)：生存期分析，荷载panc02-MUC1肿瘤细胞小鼠经GMCSF联合pcDNA3.1-VNTR质粒或单用peDNA3.1-VNTR质粒治疗，生存期明显长于G、Neo及PBS组(P＜0.05)，而前两组间生存期无明显差异(P＞0.05)；荷载panc02-Neo肿瘤细胞小鼠GMCSF联合pcDNA3.1-VNTR质粒或单用pcDNA3.1-VNTR质粒治疗，生存期无影响(P＞0.05)。体外杀伤实验结果，GMCSF联合pcDNA3.1-VNTR质粒治疗组小鼠脾细胞对panc02-MUC1细胞有明显杀伤效应，而其他各组均无明显杀伤效应(P＜0.05)；且该脾细胞对panc02-Neo细胞无明显杀伤效应(P＞0.05)。
结论：
GMCSF联合MUC1核酸疫苗或单用MUC1核酸疫苗可以抑制荷载panc02-MUC1肿瘤小鼠肿瘤的生长；且联合GMCSF具有更强的抑制作用，但联合GMCSF对荷瘤小鼠生存期无明显影响。
第二部分MUC1-VNTR核酸疫苗抗胰腺癌的体外实验研究
第一节人外周血树突细胞体外培养及生物学特性研究
研究目的：
从健康人外周血分离外周血单个核细胞(PBMC)，体外经细胞因子诱导培养树突细胞，体外进行生物学特性研究。
材料和方法：
体外以淋巴细胞分离液Fieoll经健康人外周血白细胞悬液分离PBMC，体外以GMCSF、IL4诱导树突细胞，并以TNFα促细胞成熟。FACS法(流式细胞仪)分析PBMC及成熟树突细胞表型；混和淋巴细胞反应(MLR)分析成熟树突细胞刺激同种异体淋巴细胞增殖能力。
结果：
以GMCSF、IL4及TNFα细胞因子联合诱导培养，体外可以从外周血细胞扩增获得成熟树突细胞；成熟树突细胞其细胞表面标志物HLA-DR、CD209、CD86明显升高(P＜0.05)；而淋巴细胞表面标志物CD3／CD8／CD4及单核细胞表面标志物CD14明显降低(P＜0.05)。混和淋巴细胞反应表明成熟树突细胞较未成熟树突细胞有更高的刺激同种异体T淋巴细胞增殖能力(P＜0.05)。
结论：
体外联合GMCSF、IL4及TNFα细胞因子可以从外周血扩增树突细胞，且获得的成熟树突细胞与未成熟树突细胞相比具有更强的抗原递呈功能(P＜0.05)。
第二节转染MUC1-VNTR核酸疫苗树突细胞功能的体外研究
研究目的：
以pcDNA3.1-VNTR质粒体外转染健康人树突细胞，研究其在人树突细胞内的表达情况；以及转染pcDNA3.1-VNTR质粒的树突细胞是否可以刺激自体T细胞增殖；并以Elispot法检测分泌IFN_γ及GranzymeB的CTL细胞。
材料和方法：
体外从健康人外周血PBMC诱导培养树突细胞，于细胞培养第5天，以Lipofectamine2000体外转染pcDNA3.1-VNTR质粒，并以TNFα促细胞成熟。Western blot法检测MUC1-VNTR多肽的表达；pcDNA3.1-VNTR质粒转染的成熟树突细胞与自体T细胞混和培养，刺激T细胞增殖，以~3H测定刺激增殖能力；以Elispot法检测pcDNA3.1-VNTR质粒转染的成熟树突细胞刺激自体T细胞获得分泌IFN_γ及GranzymeB的CTL细胞数。
结果：
pcDNA3.1-VNTR质粒转染的树突细胞可以表达MUC1-VNTR多肽；pcDNA3.1-VNTR质粒转染的成熟树突细胞可以刺激自体T细胞增殖，且在DC：T=1∶10时，刺激自体T细胞增殖能力最强，显著高于空质粒转染组及Lipofecatamine处理组(P＜0.05)；且Elispot法检测pcDNA3.1-VNTR质粒转染的成熟树突细胞激活的分泌IFN_γ及GranzymeB的CTL细胞数量明显高于空质粒转染组及Lipofectamine处理组(P＜0.05)。
结论：
pcDNA3.1-VNTR质粒可以在人体树突细胞中表达MUC1-VNTR多肽；且转染pcDNA3.1-VNTR质粒的成熟树突细胞可以刺激自体初始T细胞增殖；刺激分泌IFN_γ及GranzymeB的CTL细胞增殖。
第三节转染MUC1-VNTR核酸疫苗树突细胞诱导的特异性CTL对表达MUC1胰腺癌细胞的体外杀伤实验
研究目的：
pcDNA3.1-VNTR质粒转染树突细胞，并促成熟后，与自体T细胞体外共培养获得MUC1特异性的CTL，体外研究其对表达MUC1胰腺癌细胞杀伤效应。
材料和方法：
从HLA-A_2+健康人外周血PBMC诱导培养树突细胞，培养至第5日，以Lipofectamine2000转染peDNA3.1-VNTR质粒，并以空质粒转染树突细胞及Lipofectamine2000处理树突细胞为对照；促成熟后树突细胞与自体T细胞共刺激培养获得CTL，体外以LDH法检测该CTL对HLA-A_2+MUC1+胰腺癌细胞Capan-2的杀伤效应。
结果：
转染pcDNA3.1-VNTR质粒的成熟树突细胞诱导的CTL可以特异性地杀伤HLA-A_2+MUC1+胰腺癌细胞Capan-2，该杀伤效应可以被MUC1单克隆抗体VU3C6所抑制；且该CTL对HLA-A_2-MUC1+的胰腺癌细胞Aspc-1无明显的杀伤效应(P＜0.05)，提示该CTL杀伤效应为MUC1特异性的，且为HLA-A_2限制性的。
The anti-pancreatic cancer research of MUC1-VNTR DNA vaccine in vivo and in vitro
Pancreatic cancer is one of the most malignant tumors, and the cause of this disease is still unclear. At present surgical resection is still the major therapeutic method; however the surgery only could remove the visible mass not the invisible metastatic and minimal focus. And these invisible tumor cells are the major recourses of the recurrence of the tumor and induce the death of the patients. To find out ways to cure the pancreatic cancer has become one of the hot focuses of the international research groups. Based on the previous research, we furthered the research on the anti-pancreatic cancer effect of the DNA vaccine MUC1-VNTR in vivo and in vitro. The research is divided into two parts.
Part one
The anti-pancreatic cancer research of MUC1-VNTR DNA vaccine in vivo
Section one The construction of the pancreatic cancer cell line panc02-MUC1
Objective:
To construct the pancreatic cancer cell line panc02-MUC1 that could express the MUC1 protein.
Material and method:
The murine pancreatic cancer cell line panc02 is presented by the American MD Anderson cancer center and the MUC1 plasmid which encoded the whole gene of the MUC1 protein was a gift from Dr. Finn, Pittsburg University. In vitro, we used the Lipofectamine2000 to transfect the plasmid into the pancreatic cancer cell and use the empty plasmid pcDNA3 transfected cell line as control. After transfection, the G418 was used to select the monoclonal pancreatic cancer cell line panc02-MUC1.
Result:
The monoclonal pancreatic cancer cell line panc02-MUC1 we had constructed could express MUC1 by the Western blot and by means of immunofluorescence we found that the MUC1 was expressed on the cell membrane. Then in the animal model, it could form tumors in the normal C57BL/6 mice and immunohistological examination showed that the cell line could express MUC1 in vivo.
Conclusion:
The cell line we constructed is the MUC1 expressing monoclonal and it could form tumor in the normal mice.
Section two The research of preventing murine pancreatic cancer by the MUC1-VNTR DNA vaccine
Objective:
The normal C57BL/6 mice were immunized with the pcDNA3.1-VNTR plasmid i.m. three times, taken the empty plasmid pcDNA3.1 and PBS as control. To study whether the pcDNA3.1-VNTR plasmid immunized mice could prevent from the challenge of the panc02-MUC1 cells.
Material and method:
The normal female 6-7w C57BL/6 mice were randomly divided into 3 groups, 18 mice every group: PBS、Neo(pcDNA3.1)、MUC1(pcDNA3.1-VNTR). We injected a total of 100μg plasmid DNA of pcDNA3.1-VNTR in 100μl of PBS into the anterior tibialis muscle of C57BL/6(every 2 weeks, 3 times). Mice inoculated with either the empty plasmid pcDNA3.1or PBS was used as control. 5 days after the third immunization, the blood of the mice was collected from the inner canthus vein which used for the ELISA assay of anti-VNTR antibodies. 7 days after the third immunization the spleen cells of each group(n=3) were collected and stimulated by the peptide of the MUC1 which used for the LDH cytotoxicity assay and at the same time the remaining mice were inoculated with the pancreatic cancer cell panc02-MUC1 at the interdermal of the left anterior leg armpit(1×10~6/100μl/each mouse). 7 days after the tumor challenge, the length and width of the tumor was calculated with the caliper every 2-3 days, and the volume of the tumor was calculated by the formula Volume= length×width×width/2. When the tumor volume was over 1,000mm~3, the mice was considered as dead. While the other 6 mice immunized with the pcDNA3.1-VNTR plasmid were inoculated with the pancreatic cancer cell line panc02-neo which did not express the MUC1 protein(1×10~6/100μl/each mouse).
Results:
Anti-VNTR specific antibody was found significantly higher in the pcDNA3.1-VNTR immunized mice than the control group (P＜0.05). Cytotoxic assay showed that the intramuscular delivery of the recombinant plasmid into C57BL/6 mice resulted in more efficient induction of CTL lyses specific against VNTR polypeptide than the control group(P＜0.05). And this specific cytotoxity ability could be suppressed by the VNTR-antibody VU3C6 (P＜0.05); meanwhile this cytotoxity was restricted to the MUC1 expressing cell line panc02-MUC1 not the MUCl-negative cell line panc02-Neo (P＜0.05). After the tumor challenging, the tumor growth rate in the pcDNA3.1-VNTR immunized mice was much slower than the control group (P＜0.05) and the survival curve showed that the life time of the pcDNA3.1-VNTR immunized mice was much longer than the control group (P＜0.05). Otherwise the life time of the pcDNA3.1-VNTR immunized mice which were challenged with the panc02-neo cell line was not affected (P＜0.05).
Conclusion:
The recombinant plasmid pcDNA3.1-VNTR could significantly induct VNTR specific CTL response and antibodies response. And the pcDNA3.1-VNTR immunized mice could suppress the growth of the MUC1-positive cell line panc02-MUC 1 not the panc02-Neo.
Section three The research of treating murine pancreatic cancer by the MUC1-VNTR DNA vaccine combined with GMCSF
Objective:
Combining the pcDNA3.1-VNTR plasmid with the murine GMCSF to treat the murine inoculated with MUC1-expressing pancreatic cancer cell line panc02-MUC1.
Material and method:
On the day 0, 48 female C57BL/6 normal mice were inoculated with panc02-MUC1 cell line (1×10~6/100μl/each mouse) in the interderm of the left anterior leg armpit. Then these mice were randomly divided into 5 groups as follows: G+M(GMCSF+MUC1) (n=10)、M(MUC1) (n=10)、G(GMCSF) (n=10)、Neo(pcDNA3.1) (n=9) and the PBS group (n=9). And another 12 mice were inoculated with the MUCl-negative cell line panc02-neo, then were divided into 2 groups as follows: G+M(GMCSF+MUC1)、M(MUC1) (n=6). On day 4, the treatment proposal of each group was as follows respectively: GMCSF50ng/100μl+pcDNA3.1-VNTR100μg/100μl、pcDNA3.1-VNTR 100μg/100μl、GMCSF50ng/100μl、pcDNA3.1 100μg/100μl, PBS100μl; And on the day 9 and 14 repeated it again. As the mice challenged with the cell line panc02-neo, each group was treated with GMCSF50ng/100μl+pcDNA3.1-VNTR100μg/100μl and pcDNA3.1-VNTR 100μg/100μl respectively; and repeated on the day 9 and day 14. After the inoculation of the tumor cells, the length and width of the tumor were measured from the day 6-7 every 2-3 day. The mice were considered dead when the volume of the tumor was over 1000 mm3. Eleven days after the last treatment, 3 mice were taken from each group and the splenic cells were isolated cultured for the VNTR specific CTL cytotoxity assay.
Results:
In the group G+M and M the panc02-MUC1 cell line growth rate was much slower than the group G, Neo and PBS (P＜0.05) and the group G+M was slower than the group M (P＜0.05). However in the mice inoculate with the cell line panc02-neo, neither of them showed treatment effect compared with the mice inoculated with panc02-MUC1 (P＜0.05). About the life time of the mice challenged with panc02-MUC1, the group G+M and M were much longer than the other 3 group (P＜0.05), and these two group had no difference in the survival time(P＞0.05). However after treated with G+M or M, the life time of the mice challenged with panc02-neo was much shorter than the life time of the mice challenged with panc02-MUC1(P＜0.05). The in vitro cytotoxity assay showed that only the splenic cells from the G+M treated group had significant cytotoxity ability(P＜0.05), meanwhile had no cytotoxity ability to the panc02-neo cell line(P＞0.05).
Conclusion:
Both MUC1-VNTR DNA vaccine and MUC1-VNTR DNA vaccine combined with GMCSF could suppress the growth of the panc02-MUC1 cell line not the panc02-neo cell line. And combined with GMCSF the tumor grew much slower than the group M. However the life time did not show much difference in these two groups. As for the cytotoxity assay, only the CTL from the G+M group showed cytotoxity ability to the the panc02-MUC1 cell line.
Part two
The anti-pancreatic cancer research of MUC1-VNTR DNA vaccine in vitro
Section one In vitro induction and culture of the human dendritic cell and the research on the biological characteristics
Objective:
The PBMC was isolated from the normal human peripheral blood and induced to the dendritic cell by the cytokine. And the biological characteristic of the dendritic cell was studied in vitro.
Material and method:
The PBMC was isolated from the normal human leukocyte using Ficoll in vitro. Then after anchorage, the non-anchorage cells were washed away while the anchorage cells were cultured with the cytokine GMCSF and IL4, then induced to maturity by the cytokine TNFα. Using the FACS to analyze the phenotype of the PBMC and the mature dendritic cell. Using the mixed lymphocyte reaction to analyze the ability of the mature dendritic cell to stimulate the homologous lymphocyte proliferation.
Result:
Under the culture combined with the cytokine of GMCSF、IL4 and TNFα, the mature dendritic cell could be induced from the PBMC; and the surface marker of the mature dendritic cell HLA-DR, CD209 and CD86 was much higher than the PBMC(P＜0.05), while the lymphocyte surface marker CD14、CD3/CD4、CD3/CD8 was less than PBMC (P＜0.05). The mixed lymphocyte reaction showed that the mature dendritic cells had much higher ability to stimulate the homologous lymphocyte proliferation than the non-mature dendritic cells (P＜0.05).
Conclusion:
Combined with GMCSF、IL4 and TNFαculture, the dendritic cell could be induced from the PBMC and the mature dendritic cell had much higher antigen-presenting ability than the non-mature dendritic cell.
Section two In vitro research on the function of the MUC1-VNTR DNA transfected dendritic cell
Objective:
To investigate whether the pcDNA3.1-VNTR plasmid transfected dendrtic cell could express the VNTR in vitro and stimulate the proliferation of the autologous T cells. Meanwhile using the Elispot to detect the IFNγand Granzyme B secreting CTL.
Material and method:
The dendritic cells were augmented from the normal human PBMC by the stimulation of the cytokine GMCSF and IL4. On the day 5, the immature dendritic cells were transfected with pcDNA3.1-VNTR plasmid in vitro by Lipofectamine2000, followed with the conculture of the cytokine TNFαto stimulate the maturation of the dendritic cells. 24 hours after transfection the expression of the VNTR was detected by Western blot. On the day 7, the mature dendritic cells were co-cultured with the autologous T cells to investigate wheather they could stimulate the autologous T cell proliferation. And using the Elispot to detect the number of the IFNy and Granzyme B secreting CTL stimulated by the mature pcDNA3.1-VNTR plasmid transfected dendritic cells.
Result:
The pcDNA3.1-VNTR plasmid transfected dendritic cells could express VNTR in vitro; and they could stimulate the autologous T cells to proliferate especially at the ratio of DC:T=1:10 (P＜0.05). The Elispt result showed that the number of the IFNγand Granzyme B secreting CTL in the group pcDNA3.1-VNTR were more than the group pcDNA3.1 and the group Lipofectamine (P＜0.05).
Conclusion:
The pcDNA3.1-VNTR plasmid could be expressed in the human dendritic cells and the pcDNA3.1-VNTR plasmid transfected dendritic cells could stimulate the proliferation of the autologous T cells and induce the IFNT and Granzyme B secreting CTL.
Section three The research on the cytotoxity assay of the MUC1-VNTR specific CTL induced by the MUC1-VNTR DNA vaccine transfected dendritic cell
Objective:
To investigate whether the pcDNA3.1-VNTR plasmid transfected dendritic cells could induce the MUC1-VNTR specific CTL and their ability to kill the MUC1-positive pancreatic cancer cell lines.
Material and method:
The dendritic cells were induced from the healthy HLA-A_2+ human PBMC; then on the day 5, the dendritic cells were transfected with pcDNA3.1-VNTR plasmid by Lipofectamine2000, using the pcDNA3.1 transfected dendritic cells and the Lipofectamine2000 treated dendritic cells as control. After maturation, the dendritic cells were co-cultured with autologous T cells to induce the CTL and using the LDH method to detect the cytotoxity activity to the pancreatic cell line Capan-2(HLA-A2+MUC 1 +).
Result:
The CTL induced by the pcDNA3.1-VNTR plasmid transfected dendritic cells could specifically kill the Capan-2 cell line (HLA-A_2+MUCI+), and the cytotoxity ability could be suppressed by the MUCl-antibody VU3C6. However they could not kill the Aspc- 1 (HLA-A2-MUC 1 +).
Conclusion:
The pcDNA3.1-VNTR plasmid transfected dendritic cells could induce HLA-A_2 restricted VNTR specific CTL.
第一部分MUC1-VNTR核酸疫苗抗胰腺癌的体内实验研究
第一节小鼠胰腺癌细胞系panc02-MUC1构建
研究目的：
构建表达MUC1的小鼠胰腺癌细胞系panc02-MUC1。
材料和方法：
小鼠胰腺癌细胞系panc02由美国MD Anderson Cancer center赠送，表达全长MUC1的质粒pcDNA3-MUC1由美国匹兹堡大学Dr．Finn赠送。体外以Lipofectamine2000脂质体转染pcDNA3-MUC1质粒到小鼠胰腺癌细胞系panc02中，并以空载质粒pcDNA3转染细胞为对照；转染细胞再经G418压力筛选单克隆细胞株；并于小鼠皮下接种进行成瘤实验。
结果：
构建单克隆细胞株panc02-MUC1经Western blot检测可见MUC1蛋白表达；细胞免疫荧光检测可见细胞膜上有MUC1表达；且动物接种实验证实该细胞可在正常小鼠皮下成瘤，免疫组化检测可见该细胞系在C57BL／6小鼠皮下接种胰腺癌模型中可表达MUC1。
结论：
构建的细胞系为可表达MUC1的单克隆细胞系，且该细胞系可在正常C57BL／6小鼠中成瘤。
第二节MUC1-VNTR核酸疫苗预防小鼠胰腺癌实验研究
研究目的：
以pcDNA3.1-VNTR质粒肌注免疫C57BL／6小鼠，以pcDNA3.1空载质粒及PBS免疫小鼠为对照；研究MUC1-VNTR核酸疫苗免疫小鼠能否预防表达MUC1的小鼠胰腺癌细胞panc02-MUC1的成瘤效应。
材料和方法：
C57BL／6正常小鼠，随机分3组，每组18只：分别为PBS组、Neo组(pcDNA3.1免疫组)、MUC1组(pcDNA3.1-VNTR免疫组)。各组小鼠经右腿胫前肌中部注射100μg／100μl质粒PBS溶液。第1次免疫2周后，以同一质粒再次免疫小鼠，第2次免疫2周后，以同一质粒再次免疫小鼠；第3次免疫结束后5日自内眦静脉采血获得血清并以ELISA法测抗VNTR抗体；取脾细胞悬液体外以VNTR合成肽特异性刺激培养(每组3只)，3天后以LDH法进行CTL杀伤试验；第3次免疫结束后7日，各组小鼠经左侧前肢腋下皮下注射1×10~6／100μl／只panc02-MUC1细胞，7日后接种部位皮下可及肿块，每2-3日测量肿瘤长径及短径，观察MUC1-VNTR核酸疫苗预防小鼠胰腺癌细胞panc02-MUC1的成瘤效应。另6只pcDNA3.1-VNTR质粒免疫小鼠接种panc02-Neo细胞，接种细胞量为1×10~6／100μl／只。肿瘤体积大于1000mm~3时，视该小鼠死亡。
结果：
pcDNA3.1-VNTR质粒免疫的小鼠血清抗VNTR抗体浓度以吸光度表示，该组吸光度显著高于空质粒对照组和PBS对照组(P＜0.05)。pcDNA3.1-VNTR质粒免疫的小鼠脾细胞对表达MUC1的小鼠胰腺癌细胞panc02-MUC1细胞有显著的杀伤效应，明显高于空质粒组及PBS组(P＜0.05)；该杀伤效应可以被MUC1单克隆抗体VU3C6抑制(P＜0.05)；且该脾细胞对不表达MUC1的panc02-Neo细胞无明显杀伤效应(P＜0.05)。pcDNA3．1-VNTR质粒免疫组小鼠对panc02-MUC1细胞生长具有明显抑制作用，第24天时，pcDNA3．1-VNTR质粒免疫组小鼠肿瘤体积显著小于空质粒组及PBS组(P＜0.05)；且该组小鼠生存期明显长于空质粒组及PBS组(P＜0.05)；此外pcDNA3.1-VNTR质粒免疫的小鼠对不表达MUC1的panc02-Neo细胞生长无抑制作用，第28天时该组小鼠皮下肿瘤体积显著大于接种panc02-MUC1细胞组(P＜0.05)；且该组小鼠生存期显著短于接种panc02-MUC1细胞组(P＜0.05)。
结论：
MUC1-VNTR核酸疫苗可以预防表达MUC1的小鼠胰腺癌细胞panc02-MUC1的成瘤效应。
第三节GMCSF联合MUC1-VNTR核酸疫苗治疗小鼠胰腺癌实验研究
研究目的：
以GMCSF联合pcDNA3.1-VNTR质粒治疗荷载panc02-MUC1的C57BL／6小鼠。
材料和方法：
0天C57BL／6正常小鼠经左侧前肢腋下皮下注射panc02-MUC1细胞1×10~6／100μl／只，随机分5组，：G+M(GMCSF+MUC1)(n=10)、M(MUC1)(n=10)、G(GMCSF)(n=10)、Neo(pcDNA3.1)(n=9)及PBS(n=9)组。4天时各组分别经后腿肌肉注射GMCSF50ng／100μl+pcDNA3.1-VNTR质粒100μg／100μl、pcDNA3.1-VNTR质粒100μg／100μl、GMCSF50ng／100μl、peDNA3.1质粒100μg／100μl、PBS100μl；于9天、14天各重复注射一次。肿瘤接种后6-7天开始测量各组小鼠肿瘤长径及短径，小鼠肿瘤体积超过1000mm~3视为小鼠死亡；第3次治疗后11天各组取3只小鼠脾细胞体外行杀伤实验。另12只小鼠分为G+M(GMCSF+MUC1)、M(MUC1)两组，每组6只，0天时经左侧前肢腋下皮下注射1×10~6／100μl／只panc02-Neo细胞，4天时各组分别经后腿肌肉注射GMCSF50ng／100μl+pcDNA3.1-VNTR质粒100μg／100μl、pcDNA3.1-VNTR质粒100μg／100μl；于9天、14天各重复注射一次。肿瘤接种后6-7天开始测量各组小鼠肿瘤长径及短径，小鼠肿瘤体积超过1000mm~3视为小鼠死亡。
结果：
G+M及M组小鼠panc02-MUC1肿瘤细胞生长明显慢于G、Neo及PBS组(P＜0.05)，且G+M组明显慢于M组(P＜0.05)；而GMCSF联合pcDNA3.1-VNTR质粒或单用pcDNA3.1-VNTR质粒治疗panc02-Neo肿瘤，均未能抑制肿瘤生长(P＜0.05)：生存期分析，荷载panc02-MUC1肿瘤细胞小鼠经GMCSF联合pcDNA3.1-VNTR质粒或单用peDNA3.1-VNTR质粒治疗，生存期明显长于G、Neo及PBS组(P＜0.05)，而前两组间生存期无明显差异(P＞0.05)；荷载panc02-Neo肿瘤细胞小鼠GMCSF联合pcDNA3.1-VNTR质粒或单用pcDNA3.1-VNTR质粒治疗，生存期无影响(P＞0.05)。体外杀伤实验结果，GMCSF联合pcDNA3.1-VNTR质粒治疗组小鼠脾细胞对panc02-MUC1细胞有明显杀伤效应，而其他各组均无明显杀伤效应(P＜0.05)；且该脾细胞对panc02-Neo细胞无明显杀伤效应(P＞0.05)。
结论：
GMCSF联合MUC1核酸疫苗或单用MUC1核酸疫苗可以抑制荷载panc02-MUC1肿瘤小鼠肿瘤的生长；且联合GMCSF具有更强的抑制作用，但联合GMCSF对荷瘤小鼠生存期无明显影响。
第二部分MUC1-VNTR核酸疫苗抗胰腺癌的体外实验研究
第一节人外周血树突细胞体外培养及生物学特性研究
研究目的：
从健康人外周血分离外周血单个核细胞(PBMC)，体外经细胞因子诱导培养树突细胞，体外进行生物学特性研究。
材料和方法：
体外以淋巴细胞分离液Fieoll经健康人外周血白细胞悬液分离PBMC，体外以GMCSF、IL4诱导树突细胞，并以TNFα促细胞成熟。FACS法(流式细胞仪)分析PBMC及成熟树突细胞表型；混和淋巴细胞反应(MLR)分析成熟树突细胞刺激同种异体淋巴细胞增殖能力。
结果：
以GMCSF、IL4及TNFα细胞因子联合诱导培养，体外可以从外周血细胞扩增获得成熟树突细胞；成熟树突细胞其细胞表面标志物HLA-DR、CD209、CD86明显升高(P＜0.05)；而淋巴细胞表面标志物CD3／CD8／CD4及单核细胞表面标志物CD14明显降低(P＜0.05)。混和淋巴细胞反应表明成熟树突细胞较未成熟树突细胞有更高的刺激同种异体T淋巴细胞增殖能力(P＜0.05)。
结论：
体外联合GMCSF、IL4及TNFα细胞因子可以从外周血扩增树突细胞，且获得的成熟树突细胞与未成熟树突细胞相比具有更强的抗原递呈功能(P＜0.05)。
第二节转染MUC1-VNTR核酸疫苗树突细胞功能的体外研究
研究目的：
以pcDNA3.1-VNTR质粒体外转染健康人树突细胞，研究其在人树突细胞内的表达情况；以及转染pcDNA3.1-VNTR质粒的树突细胞是否可以刺激自体T细胞增殖；并以Elispot法检测分泌IFN_γ及GranzymeB的CTL细胞。
材料和方法：
体外从健康人外周血PBMC诱导培养树突细胞，于细胞培养第5天，以Lipofectamine2000体外转染pcDNA3.1-VNTR质粒，并以TNFα促细胞成熟。Western blot法检测MUC1-VNTR多肽的表达；pcDNA3.1-VNTR质粒转染的成熟树突细胞与自体T细胞混和培养，刺激T细胞增殖，以~3H测定刺激增殖能力；以Elispot法检测pcDNA3.1-VNTR质粒转染的成熟树突细胞刺激自体T细胞获得分泌IFN_γ及GranzymeB的CTL细胞数。
结果：
pcDNA3.1-VNTR质粒转染的树突细胞可以表达MUC1-VNTR多肽；pcDNA3.1-VNTR质粒转染的成熟树突细胞可以刺激自体T细胞增殖，且在DC：T=1∶10时，刺激自体T细胞增殖能力最强，显著高于空质粒转染组及Lipofecatamine处理组(P＜0.05)；且Elispot法检测pcDNA3.1-VNTR质粒转染的成熟树突细胞激活的分泌IFN_γ及GranzymeB的CTL细胞数量明显高于空质粒转染组及Lipofectamine处理组(P＜0.05)。
结论：
pcDNA3.1-VNTR质粒可以在人体树突细胞中表达MUC1-VNTR多肽；且转染pcDNA3.1-VNTR质粒的成熟树突细胞可以刺激自体初始T细胞增殖；刺激分泌IFN_γ及GranzymeB的CTL细胞增殖。
第三节转染MUC1-VNTR核酸疫苗树突细胞诱导的特异性CTL对表达MUC1胰腺癌细胞的体外杀伤实验
研究目的：
pcDNA3.1-VNTR质粒转染树突细胞，并促成熟后，与自体T细胞体外共培养获得MUC1特异性的CTL，体外研究其对表达MUC1胰腺癌细胞杀伤效应。
材料和方法：
从HLA-A_2+健康人外周血PBMC诱导培养树突细胞，培养至第5日，以Lipofectamine2000转染peDNA3.1-VNTR质粒，并以空质粒转染树突细胞及Lipofectamine2000处理树突细胞为对照；促成熟后树突细胞与自体T细胞共刺激培养获得CTL，体外以LDH法检测该CTL对HLA-A_2+MUC1+胰腺癌细胞Capan-2的杀伤效应。
结果：
转染pcDNA3.1-VNTR质粒的成熟树突细胞诱导的CTL可以特异性地杀伤HLA-A_2+MUC1+胰腺癌细胞Capan-2，该杀伤效应可以被MUC1单克隆抗体VU3C6所抑制；且该CTL对HLA-A_2-MUC1+的胰腺癌细胞Aspc-1无明显的杀伤效应(P＜0.05)，提示该CTL杀伤效应为MUC1特异性的，且为HLA-A_2限制性的。
The anti-pancreatic cancer research of MUC1-VNTR DNA vaccine in vivo and in vitro
Pancreatic cancer is one of the most malignant tumors, and the cause of this disease is still unclear. At present surgical resection is still the major therapeutic method; however the surgery only could remove the visible mass not the invisible metastatic and minimal focus. And these invisible tumor cells are the major recourses of the recurrence of the tumor and induce the death of the patients. To find out ways to cure the pancreatic cancer has become one of the hot focuses of the international research groups. Based on the previous research, we furthered the research on the anti-pancreatic cancer effect of the DNA vaccine MUC1-VNTR in vivo and in vitro. The research is divided into two parts.
Part one
The anti-pancreatic cancer research of MUC1-VNTR DNA vaccine in vivo
Section one The construction of the pancreatic cancer cell line panc02-MUC1
Objective:
To construct the pancreatic cancer cell line panc02-MUC1 that could express the MUC1 protein.
Material and method:
The murine pancreatic cancer cell line panc02 is presented by the American MD Anderson cancer center and the MUC1 plasmid which encoded the whole gene of the MUC1 protein was a gift from Dr. Finn, Pittsburg University. In vitro, we used the Lipofectamine2000 to transfect the plasmid into the pancreatic cancer cell and use the empty plasmid pcDNA3 transfected cell line as control. After transfection, the G418 was used to select the monoclonal pancreatic cancer cell line panc02-MUC1.
Result:
The monoclonal pancreatic cancer cell line panc02-MUC1 we had constructed could express MUC1 by the Western blot and by means of immunofluorescence we found that the MUC1 was expressed on the cell membrane. Then in the animal model, it could form tumors in the normal C57BL/6 mice and immunohistological examination showed that the cell line could express MUC1 in vivo.
Conclusion:
The cell line we constructed is the MUC1 expressing monoclonal and it could form tumor in the normal mice.
Section two The research of preventing murine pancreatic cancer by the MUC1-VNTR DNA vaccine
Objective:
The normal C57BL/6 mice were immunized with the pcDNA3.1-VNTR plasmid i.m. three times, taken the empty plasmid pcDNA3.1 and PBS as control. To study whether the pcDNA3.1-VNTR plasmid immunized mice could prevent from the challenge of the panc02-MUC1 cells.
Material and method:
The normal female 6-7w C57BL/6 mice were randomly divided into 3 groups, 18 mice every group: PBS、Neo(pcDNA3.1)、MUC1(pcDNA3.1-VNTR). We injected a total of 100μg plasmid DNA of pcDNA3.1-VNTR in 100μl of PBS into the anterior tibialis muscle of C57BL/6(every 2 weeks, 3 times). Mice inoculated with either the empty plasmid pcDNA3.1or PBS was used as control. 5 days after the third immunization, the blood of the mice was collected from the inner canthus vein which used for the ELISA assay of anti-VNTR antibodies. 7 days after the third immunization the spleen cells of each group(n=3) were collected and stimulated by the peptide of the MUC1 which used for the LDH cytotoxicity assay and at the same time the remaining mice were inoculated with the pancreatic cancer cell panc02-MUC1 at the interdermal of the left anterior leg armpit(1×10~6/100μl/each mouse). 7 days after the tumor challenge, the length and width of the tumor was calculated with the caliper every 2-3 days, and the volume of the tumor was calculated by the formula Volume= length×width×width/2. When the tumor volume was over 1,000mm~3, the mice was considered as dead. While the other 6 mice immunized with the pcDNA3.1-VNTR plasmid were inoculated with the pancreatic cancer cell line panc02-neo which did not express the MUC1 protein(1×10~6/100μl/each mouse).
Results:
Anti-VNTR specific antibody was found significantly higher in the pcDNA3.1-VNTR immunized mice than the control group (P＜0.05). Cytotoxic assay showed that the intramuscular delivery of the recombinant plasmid into C57BL/6 mice resulted in more efficient induction of CTL lyses specific against VNTR polypeptide than the control group(P＜0.05). And this specific cytotoxity ability could be suppressed by the VNTR-antibody VU3C6 (P＜0.05); meanwhile this cytotoxity was restricted to the MUC1 expressing cell line panc02-MUC1 not the MUCl-negative cell line panc02-Neo (P＜0.05). After the tumor challenging, the tumor growth rate in the pcDNA3.1-VNTR immunized mice was much slower than the control group (P＜0.05) and the survival curve showed that the life time of the pcDNA3.1-VNTR immunized mice was much longer than the control group (P＜0.05). Otherwise the life time of the pcDNA3.1-VNTR immunized mice which were challenged with the panc02-neo cell line was not affected (P＜0.05).
Conclusion:
The recombinant plasmid pcDNA3.1-VNTR could significantly induct VNTR specific CTL response and antibodies response. And the pcDNA3.1-VNTR immunized mice could suppress the growth of the MUC1-positive cell line panc02-MUC 1 not the panc02-Neo.
Section three The research of treating murine pancreatic cancer by the MUC1-VNTR DNA vaccine combined with GMCSF
Objective:
Combining the pcDNA3.1-VNTR plasmid with the murine GMCSF to treat the murine inoculated with MUC1-expressing pancreatic cancer cell line panc02-MUC1.
Material and method:
On the day 0, 48 female C57BL/6 normal mice were inoculated with panc02-MUC1 cell line (1×10~6/100μl/each mouse) in the interderm of the left anterior leg armpit. Then these mice were randomly divided into 5 groups as follows: G+M(GMCSF+MUC1) (n=10)、M(MUC1) (n=10)、G(GMCSF) (n=10)、Neo(pcDNA3.1) (n=9) and the PBS group (n=9). And another 12 mice were inoculated with the MUCl-negative cell line panc02-neo, then were divided into 2 groups as follows: G+M(GMCSF+MUC1)、M(MUC1) (n=6). On day 4, the treatment proposal of each group was as follows respectively: GMCSF50ng/100μl+pcDNA3.1-VNTR100μg/100μl、pcDNA3.1-VNTR 100μg/100μl、GMCSF50ng/100μl、pcDNA3.1 100μg/100μl, PBS100μl; And on the day 9 and 14 repeated it again. As the mice challenged with the cell line panc02-neo, each group was treated with GMCSF50ng/100μl+pcDNA3.1-VNTR100μg/100μl and pcDNA3.1-VNTR 100μg/100μl respectively; and repeated on the day 9 and day 14. After the inoculation of the tumor cells, the length and width of the tumor were measured from the day 6-7 every 2-3 day. The mice were considered dead when the volume of the tumor was over 1000 mm3. Eleven days after the last treatment, 3 mice were taken from each group and the splenic cells were isolated cultured for the VNTR specific CTL cytotoxity assay.
Results:
In the group G+M and M the panc02-MUC1 cell line growth rate was much slower than the group G, Neo and PBS (P＜0.05) and the group G+M was slower than the group M (P＜0.05). However in the mice inoculate with the cell line panc02-neo, neither of them showed treatment effect compared with the mice inoculated with panc02-MUC1 (P＜0.05). About the life time of the mice challenged with panc02-MUC1, the group G+M and M were much longer than the other 3 group (P＜0.05), and these two group had no difference in the survival time(P＞0.05). However after treated with G+M or M, the life time of the mice challenged with panc02-neo was much shorter than the life time of the mice challenged with panc02-MUC1(P＜0.05). The in vitro cytotoxity assay showed that only the splenic cells from the G+M treated group had significant cytotoxity ability(P＜0.05), meanwhile had no cytotoxity ability to the panc02-neo cell line(P＞0.05).
Conclusion:
Both MUC1-VNTR DNA vaccine and MUC1-VNTR DNA vaccine combined with GMCSF could suppress the growth of the panc02-MUC1 cell line not the panc02-neo cell line. And combined with GMCSF the tumor grew much slower than the group M. However the life time did not show much difference in these two groups. As for the cytotoxity assay, only the CTL from the G+M group showed cytotoxity ability to the the panc02-MUC1 cell line.
Part two
The anti-pancreatic cancer research of MUC1-VNTR DNA vaccine in vitro
Section one In vitro induction and culture of the human dendritic cell and the research on the biological characteristics
Objective:
The PBMC was isolated from the normal human peripheral blood and induced to the dendritic cell by the cytokine. And the biological characteristic of the dendritic cell was studied in vitro.
Material and method:
The PBMC was isolated from the normal human leukocyte using Ficoll in vitro. Then after anchorage, the non-anchorage cells were washed away while the anchorage cells were cultured with the cytokine GMCSF and IL4, then induced to maturity by the cytokine TNFα. Using the FACS to analyze the phenotype of the PBMC and the mature dendritic cell. Using the mixed lymphocyte reaction to analyze the ability of the mature dendritic cell to stimulate the homologous lymphocyte proliferation.
Result:
Under the culture combined with the cytokine of GMCSF、IL4 and TNFα, the mature dendritic cell could be induced from the PBMC; and the surface marker of the mature dendritic cell HLA-DR, CD209 and CD86 was much higher than the PBMC(P＜0.05), while the lymphocyte surface marker CD14、CD3/CD4、CD3/CD8 was less than PBMC (P＜0.05). The mixed lymphocyte reaction showed that the mature dendritic cells had much higher ability to stimulate the homologous lymphocyte proliferation than the non-mature dendritic cells (P＜0.05).
Conclusion:
Combined with GMCSF、IL4 and TNFαculture, the dendritic cell could be induced from the PBMC and the mature dendritic cell had much higher antigen-presenting ability than the non-mature dendritic cell.
Section two In vitro research on the function of the MUC1-VNTR DNA transfected dendritic cell
Objective:
To investigate whether the pcDNA3.1-VNTR plasmid transfected dendrtic cell could express the VNTR in vitro and stimulate the proliferation of the autologous T cells. Meanwhile using the Elispot to detect the IFNγand Granzyme B secreting CTL.
Material and method:
The dendritic cells were augmented from the normal human PBMC by the stimulation of the cytokine GMCSF and IL4. On the day 5, the immature dendritic cells were transfected with pcDNA3.1-VNTR plasmid in vitro by Lipofectamine2000, followed with the conculture of the cytokine TNFαto stimulate the maturation of the dendritic cells. 24 hours after transfection the expression of the VNTR was detected by Western blot. On the day 7, the mature dendritic cells were co-cultured with the autologous T cells to investigate wheather they could stimulate the autologous T cell proliferation. And using the Elispot to detect the number of the IFNy and Granzyme B secreting CTL stimulated by the mature pcDNA3.1-VNTR plasmid transfected dendritic cells.
Result:
The pcDNA3.1-VNTR plasmid transfected dendritic cells could express VNTR in vitro; and they could stimulate the autologous T cells to proliferate especially at the ratio of DC:T=1:10 (P＜0.05). The Elispt result showed that the number of the IFNγand Granzyme B secreting CTL in the group pcDNA3.1-VNTR were more than the group pcDNA3.1 and the group Lipofectamine (P＜0.05).
Conclusion:
The pcDNA3.1-VNTR plasmid could be expressed in the human dendritic cells and the pcDNA3.1-VNTR plasmid transfected dendritic cells could stimulate the proliferation of the autologous T cells and induce the IFNT and Granzyme B secreting CTL.
Section three The research on the cytotoxity assay of the MUC1-VNTR specific CTL induced by the MUC1-VNTR DNA vaccine transfected dendritic cell
Objective:
To investigate whether the pcDNA3.1-VNTR plasmid transfected dendritic cells could induce the MUC1-VNTR specific CTL and their ability to kill the MUC1-positive pancreatic cancer cell lines.
Material and method:
The dendritic cells were induced from the healthy HLA-A_2+ human PBMC; then on the day 5, the dendritic cells were transfected with pcDNA3.1-VNTR plasmid by Lipofectamine2000, using the pcDNA3.1 transfected dendritic cells and the Lipofectamine2000 treated dendritic cells as control. After maturation, the dendritic cells were co-cultured with autologous T cells to induce the CTL and using the LDH method to detect the cytotoxity activity to the pancreatic cell line Capan-2(HLA-A2+MUC 1 +).
Result:
The CTL induced by the pcDNA3.1-VNTR plasmid transfected dendritic cells could specifically kill the Capan-2 cell line (HLA-A_2+MUCI+), and the cytotoxity ability could be suppressed by the MUCl-antibody VU3C6. However they could not kill the Aspc- 1 (HLA-A2-MUC 1 +).
Conclusion:
The pcDNA3.1-VNTR plasmid transfected dendritic cells could induce HLA-A_2 restricted VNTR specific CTL.
引文
1. Evans, D. B., Abbruzzese, J. L. & Willett, C. G. in Principles and Practice of Oncology 6th edn (ed. DeVita, V. T.)1126-1161 (J. B. Lippincott, Philadelphia, 2001).
2. Li, D. et al. Pancreatic cancer. Lancet. 2004; 363: 1049-1057.
3. Spratlin, J. et al. The absence of human equilibrative nucleoside transporter 1 is associated with reducedsurvival in patients with gemcitabine-treated pancreas adenocarcinoma. Clin. Cancer Res. 2004; 10: 6956-6961.
4. Laheru, D., Biedrzycki, B. & Jaffee, E. M. Immunologic approaches to the management of pancreatic cancer. Cancer J. 2001; 7: 324-337.
5. Pardoll, D. & Allison, J. Cancer Immunotherapy: breaking the barriers to harvest the crop. Nature Med. 2004; 10: 887-892.
6. Mendelsohn, J. & Baselga, J. The EGF receptor family as targets for cancer therapy. Oncogene. 2000; 19: 6550-6565.
7. Schwarte-Waldhoff, I., Volpert, O. V. & Bouck, N. P. SMAD4/DPC4-mediated tumor suppression through suppression ofangiogenesis. Proc. Natl Acad. Sci. USA. 2000; 97: 9624-9629.
8. Xiong, H. Q. et al. Cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor, in combination with gemcitabine for advanced pancreatic Cancer: a multicenter phase Ⅱ Trial. J. Clin. Oncol. 2004; 22: 2610-2616.
9. Kindler, H. L, et al. Bevacizumab plus gemcitabine in patients with advanced pancreatic cancer: Updated results of a multi-center phase Ⅱ trial. J Clin Oncol. 2004; 22(Suppl): 4009.
10. Bos, J. L. ras oncogenes in human cancer: a review. Cancer Res. 1989; 49: 4682-4689.
11. Finn, O.J. et al. MUC-1 epithelial tumor mucin-based immunity and vaccines. Immunol. Rev.1995; 145:61-89.
12. Hammarstrom, S. The carcinoembryonic antigen (CEA) family:structures, suggested functions and expression in normal and malignant tissues. Semin. Cancer Biol. 1999; 9:67-81.
13. Achtar, M. et al. Mutant ras vaccine in advanced cancers.Proc. Am. Soc. Clin. Oncol.2003; 22:A677.
14. Morse, M. et al. Phase I study of immunization with dendritic cells modified with recombinant fowlpox encoding carcinoembryonic antigen (CEA) and the triad of costimulatory molecules CD54, CD58 and CD80 (rF-CEA(6D)-TRICOM) in patients with advanced malignancies. Proc. Am. Soc. Clin. Oncol. 2004; 23: A2508.
15. Finn, O. J. et aL A phase Ib study of a MUC1 pulsed autologous dendritic cell vaccine as adjuvant therapy in patients with resected pancreatic or biliary tumors. Proc. Am. Soc. Clin. Oncol.2004; 23:A2578.
16. Toshiaki Hayashi, Tohru Takahashi, Satoshi Motoya, et al. MUC1 Mucin Core Protein Binds to the Domain 1 of ICAM-l.Digestion.2001; 63:S87-92.
17. Makiko Yamamoto,Ajit Bharti, Yongqing Li,et al. Interaction of the DF3/MUC1 Breast Carcinoma-associated Antigen and β-Catenin in Cell Adhesion. J Biol Chem. 1997;272(19):12492-12494.
18. Li Y, Bharti A, Chen D, Gong J,et al. Interaction of glycogen synthase kinase 3beta with the DF3/MUC1 carcinoma-associated antigen and beta-catenin. Mol Cell Biol. 1998; 18( 12):7216-7224.
19. Yuko Hiraga, Shinji Tanaka, Ken Haruma, et al. Immunoreactive MUC1 Expression at the Deepest Invasive Portion Correlates with Prognosis of Colorectal Cancer. Oncology. 1998; 55:307-319.
20. Luttges J, Feyerabend B, Buchelt T, et al. The mucin profile of noninvasive and invasive mucinous cystic neoplasms of the pancreas. Am J Surg Pathol. 2002; 26(4):466-471.
21. Kashiwagi H, Kijima H, Dowaki S,et al. DF3 expression in human gallbladder carcinoma: significance for lymphatic invasion. Int J Oncol. 2000; 16(3):455-459.
22. Rahn JJ, Dabbagh L, Pasdar M, et al. The importance of MUC1 cellular localization in patients with breast carcinoma: an immunohistologic study of 71 patients and review of the literature. Cancer. 2001; 91(11):1973-1982.
23. Kohlgraf KG, Gawron AJ, Higashi M, et al. Contribution of the MUC1 tandem repeat and cytoplasmic tail to invasive and metastatic properties of a pancreatic cancer cell line. Cancer Res. 2003; 63(16):5011-5020.
24. Van de Wiel-van Kemenade E, Ligtenberg MJL, et al.Episialin (MUC1) inhibits cytotoxic lymphocyte-target cell interaction. J Immunol 1993; 151:767-776.
25. Zhang K, Sikut R, Hansson GC. A MUC1 mucin secreted from a colon carcinoma cell line inhibits target cell lysis by natural killer cells. Cell Immunol. 1997; 176(2):158-165.
26. Claude D. Gimmi, Briggs W. Morrison, Brigitte A. Breast cancer-associated antigen, DF3/MUC1, induces apoptosis of activated human T cells.Nature Medicine. 1996; 2:1367-1370.
27. Adachi, Hinoda, Nishimori, et al. Increased sensitivity of gastric cancer cells to Natural killer and lymphokine-Activated killer cells by antisense suppression of N-Actetylgalactosaminytransferase. J Immunol. 1997; 159(6):2645-2651.
28. Krause, Turner.Are selectins involved in metastasis? Clin Exp Metastasis. 1999; 17(3):183-192.
29. Butts C, Murray N, Maksymiuk A, Randomized phase IIB trial of BLP25 liposome vaccine in stage IIIB and IV non-small-cell lung cancer. J Clin Oncol. 2005;23(27):6674-6681.
30. Henderson RA, Konitsky WM, Barratt-Boyes SM, et al.Retroviral expression of MUC-1 human tumor antigen with intact repeat structure and capacity to elicit immunity in vivo. J Immunother. 1998; 21(4):247-256.
31. Pecher G, Haring A, Kaiser L, et al. Mucin gene (MUC1) transfected dendritic cells as vaccine: Results of a phase I/II clinical trial. Cancer Immunol Immunother, 2002; 51:669-673.
32. Wierecky J, Muller MR, Wirths S, Immunologic and clinical responses after vaccinations with peptide-pulsed dendritic cells in metastatic renal cancer patients. Cancer Res. 2006; 66(11):5910-5918.
33. Graham RA, Burchell JM, Beverley P. Intramuscular immunisation with MUC1 cDNA can protect C57 mice challenged with MUC1-expressing syngeneic mouse tumour cells. Int J Cancer. 1996; 65(5):664-670.
34. Snyder LA, Goletz TJ, Gunn GR A MUC1/IL-18 DNA vaccine induces anti-tumor immunity and increased survival in MUC1 transgenic mice. Vaccine. 2006;24(16):3340-3352.
35．吴文川，靳大勇，秦新裕等．建立胰腺癌黏液蛋白核芯肽-连续重复序列核酸疫苗的实验研究．中华普通外科杂志．2006；21(5)：357-359．
36. Donnelly JJ, Ulmer JB, Liu MA. Protective efficacy of intramuscular immunization with naked DNA. Ann NY Acad Sci. 1995; 772: 40-46.
1. Hollingsworth, Strawhecker, Caffrey, et al. Expression of MUC 1, MUC2, MUC3, and MUC4 mucin mRNA in human pancreatic and intestinal tumor cell lines. Int J Cancer, 1994, 57: 198-203.
2. Finn, O. J. et al. MUC-1 epithelial tumor mucin-based immunity and vaccines. Immunol. Rev. 1995; 145: 61-89.
3. Ciernik IF, Berzofsky JA, Carbone DP. Induction of cytotoxic T lymphocytes and antitumor immunity with DNA vaccines expressing single T cell epitopes. J Immunol. 1996; 156: 2369-2375.
4．张立新，李春海．MUCl粘蛋白的免疫生物学作用及其在肿瘤生物学治疗中的作用．中国肿瘤生物治疗杂志．2000；7：165-170．
5．彭英芳，钟辉，蔡民华．MUC1蛋白的结构功能及作用．生物技术通讯．2001；12：130-131．
6. Pietersz GA, Li W, Osinski C, Apostolopoulos V, et al. Definition of MHC-restricted CTL epitopes from non-variable number of tandem repeat sequence of MUC1. Vaccine. 2000; 18: 2059-2071.
7. Kontani K, Taguchi O, Ozaki Y, et al. Dendritic cell vaccine immunotherapy of cancer targeting MUC1 mucin. Int J Mol Med. 2003; 12: 493-502.
8. Fu X, Guadagni F, Hoffman RM. A metastatic nude-mouse model of human pancreatic cancer constructed orthotopically with histologically intact patient specimens. Proc Natl Acad Sci U SA 1992; 89: 5645-5649.
9. An Z, Wang X, Kubota T, et al. A clinical nude mouse metastatic model for highly malignant human pancreatic cancer. Anticancer Res. 1996; 16: 627-631.
10．刘秋珍，脱朝伟，张艳华．三株人体胰腺癌裸小鼠原位移植模型的建立与应用中国医学影像技术．1997；1 3(6)：498-500．
11．李敏杰，陈永珍，何杨．人胰腺癌裸小鼠模型的建立及其生物学特性的研究．苏州大学学报(医学版)．2003；23(6)：649-651．
12. Corbett TH, Roberts BJ, Leopold WR, et al. Induction and chemotherapeutic response of two transplantable ductal adenocarcinomas of the pancreas in C57BL/6 mice. Cancer Res. 1984; 44: 717-726.
13. Keith R. Jerome, Dawen Bu, Olivera J. Finn. Expression of Tumor-associated Epitopes on Epstein-Barr Virus-immortalized B-Cells and Burkitt's Lymphomas Transfected with Epithelial Mucin Complementary DNA. Cancer Res. 1992; 52: 5985-5990.
14. Devesa SS, Blot WJ, Stone BJ, et al. Recent Cancer trends in the United States. J Natl Cancer Inst. 1995; 87: 175-82.
15. Michand DS. Epideminology of pancreatic cancer. Minerva Chir. 2004; 59: 99-111.
16. Grcenlee RT, Munay T, Bolden S, et al. Cancer statistics 2000. CA Cancer J Chin. 2000; 50: 7-33.
17. Lowenfels AB, Maisouneuve P. Epidemiology and prevention of pancreatic cancer. Jpu J Chin oncol. 2004; 34: 238-249.
18. Wang L, Yang GH, Lu XH. Pancreatic cancer mortality inChina 1991-2000. World J of Gastroenterol. 2003; 9: 1819-1823.
19. Li, D. et al. Pancreatic cancer. Lancet. 2004; 363, 1049-1057.
20．吴文川，靳大勇，秦新裕等．建立胰腺癌黏液蛋白核芯肽-连续重复序列核酸疫苗的实验研究．中华普通外科杂志．2006；21(5)：357-359．
21. Morikane K, Muto T, Tempero R, et al. Organ-specific pancreatic tumor growth properties and tumor immunity. Cancer Immunol Immunother. 1997; 47: 287-296.
1. Wolff JA, Malone RW, Williams P, et al. Direct gene transfer into mouse muscle in vivo. Science. 1990; 247: 1465-1468.
2. Ulmer, Donnelly, Parker, et al. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science. 1993; 259: 1745-49.
3. Sanjay gurunathan, Dennis M. klinman, Robert Seder. DNA Vaccines: Immunology, application, and optimization. Annual review of immunology. 2000; 18: 927-974.
4．吴文川，靳大勇，秦新裕等．建立胰腺癌黏液蛋白核芯肽-连续重复序列核酸疫苗的实验研究．中华普通外科杂志．2006；21(5)：357-359．
5．吴文川，靳大勇，秦新裕等．胰腺癌MUCl-VNTR核酸疫苗的构建和体外转染．中华实验外科杂志．2005；22(9)：1081-1083．
6. Gong JL, Chen, Kashiwaba M, et al.Induction of antitumor activity by immunization with fusions of dendritic and carcinoma cells. Nature Medicine.1997; 3: 558-561.
7. Richard M. Tempero, Michelle L et al. CD4+ Lymphocytes Provide MUCl-Specific Tumor Immunity In Vivo That Is Undetectable In Vitro and Is Absent in MUCl Transgenic Mice. J Immunol. 1998; 161: 5500-5506.
8. Mukherjee P, Ginardi AR, Madsen CS, et al. Mice with spontaneous pancreatic cancer naturally develops MUC-l-specific CTLs that eradicate tumors when adoptively transferred. J Immunol. 2000; 165: 3451-3460.
9. Tempero RM, Rowse GJ, Gendler SJ, Hollingsworth MA. Passively transferred anti-MUC1 antibodies cause neither autoimmune disorders nor immunity against transplanted tumors in MUC1 transgenic mice. Int J Cancer. 1999; 80: 595-599.
10. Snyder LA, Goletz TJ, Gunn GR, et al. A MUC1/IL-18 DNA vaccine induces anti-tumor immunity and increased survival in MUC1 transgenic mice. Vaccine. 2006; 24(16): 3340-3352.
11. Heiko Johnen a Hagen Kulbe a Gabriele Pecher. Long-term tumor growth suppression in mice immunized, with naked DNA of the human tumor antigen mucin (MUC1). Cancer immunology and immunotherapy. 50(77): 356-360.
12. Donnelly JJ, Ulmer JB, Liu MA. Protective efficacy of intramuscular immunization with naked DNA. Ann NY Acad Sci. 1995; 772: 40-46.
13. Donnelly, Friedman, Ulmer, et al. Further protection against drift of influenza virus in a gerret model by DNA vaccination. Vaccine. 1997; 15: 865-868.
14. Rodriguez F, An LL, Harkins S, et al. DNA immunization with minigenes: Low frequency of memory Cytotoxic T Lymphocytes and inefficient antiviral protection are rectified by ubiquitination. J Virol. 1998; 72: 5174-5181.
15．台桂香，张吉凤，朱迅．重组人MUC1-MBP融合蛋白的抗肿瘤作用．中国肿瘤生物治疗杂志．2003；10：202-205．
16. Romani N, Gruner S, Brang D, et al. Proliferating dendritic cell progenitors in human blood. J Exp Med. 1994; 180: 83-93.
17. Berlyn KA, Ponniah S, Stass SA, et al. Developing dendritic cell polynucleotide vaccination for prostate cancer immunotherapy. J Biotechnol. 1999; 73: 155-179.
18. Schnurr M, Galambos P, Scholz C, et al. Tumor cell lysate-pulsed human dendritic cells induce a T-cell response against pancreatic carcinoma cells: an in vitro model for the assessment of tumor vaccines. Cancer Res. 2001; 61: 6445-6450.
19. Henderson RA, Nimgaonkar MT, Watkins SC, et al. Human dendritic cells genetically engineered to express high levels of the human epithelial tumor antigen mucin (MUC-1). Cancer Res. 1996; 56: 3763-3770.
20. Kontani K, Taguchi O, Ozaki Y, et al. Dendritic cell vaccine immunotherapy of cancer targeting MUC1 mucin. Int J Mol Med. 2003; 12: 493-502.
21. Banchereau J, Bazan F, Blanchard D,Briere F, Galizzi LP, van Kooten C, Liu YJ, Rousset F, Saeland S. The CD40 antigen and its ligand. Annu. Rev.Immunol. 1994;12:881-922
22. Ridge JP, Di Rosa F,Matzinger RA conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell.Nature. 1998;393:474-478.
23. Bennett SR, Carbone FR, Karamalis F, et al. Help for cytotoxic-T cell responses is mediated by CD40 signalling. Nature. 1998; 393(6684):478-480.
24. Schoenberger, Toes, Voort EL, et al. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions.Nature.1998; 393(4):480-483.
25. Seder RA, Paul WE. Acquisition of lymphokine- producing phenotype by CD; T Cells. Annu Rev Immunol. 1994; 12:635-673.
26. O'Garra A. Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity. 1998; 8: 275-283.
1. Geissler M, Tokushige K, Chante CC, et al. Cellular and humoral immune response to hepatitis B virus structural proteins in mice after DNA-based immunization. Gastroenterology. 1997; 112: 1307-1320.
2. Goebels N, Michaelis D, Wekerie, et al. Human myoblasts as antigen-presenting cells. J Immunol. 1992; 149: 661-667.
3. Sanjay gurunathan, Dennis, klinman, Robert Seder. DNA Vaccines: Immunology, application, and optimization. Annual review of immunology. 2000; 18: 927-974.
4. Corr M, Lee DJ, Carson DA, et al. Gene vaccination with plasmid DNA: Mechanism of CTL priming. J Exp Med. 1996, 184: 1555-1560.
5. Doe B, Selby M, Barnett S, et al. Induction of cytotoxic T lymphocytes by intramuscular immunization with plasmid DNA is facilitated by bone marrow-derived cells. Proc Natl Acad Sci USA. 1996; 93: 8578-8583.
6. Xiang, Z., and Ertl, H. C. Manipulation of the immune response to a plasmid-encoded viral antigen by coinoculation with plasmids expressing cytokines. Immunity. 1995; 2: 129-135.
7. Kim. In vivo engineering of a cellular immune response by coadministration of IL-12 expression vector with a DNA immunogen.Jouranl of Immunology. 1997; 158: 816-826.
8. Snyder LA, Goletz TJ, Gunn GR, et al. A MUC1/IL-18 DNA vaccine induces anti-tumor immunity and increased survival in MUC1 transgenic mice. Vaccine. 2006; 24(16): 3340-3352.
9. Shawn M, Sumida, Paul F. et al Recruitment and expansion of dendritic cells in vivo potentiate the immunogenicity of plasmid DNA vaccinesarticle: J. Clin. Invest. 2004; 114: 1334-1342.
10．吴文川，靳大勇，秦新裕等．胰腺癌MUCl-VNTR核酸疫苗的构建和体外转染．中华实验外科杂志．2005；22(9)：1081-1083．
11. Wenxin Sun, Haili Qian, Xueyan Zhang, et al. Induction of protective and therapeutic antitumor immunity using a novel tumor-associated antigen-specific DNA vaccine. Immunology and cell biology. 2006; 84: 440-447.
12. Mwangi W, Brown WC, Splitter GA, et al. Enhancement of antigen acquisition by dendritic cells and MHC class Ⅱ-restricted epitope presentation to CD4~+ T cells using VP22 DNA vaccine vectors that promote intercellular spreading following initial transfection. J Leukoc Biol, 2005; 4: 876-881.
13. Ohta K, Yamaguchi Y, Miyahara E, et al. Novel system for generating cytotoxic effector lymphocytes using carcinoembryonic antigen(CEA) peptide and culture dendritic cells. Anticancer research. 2002; 22: 2597-2606.
14. Rafiq K, Bergtold A, Clynes R. Immune complex-mediated antigen presentation induces tumor immunity. J Clin Invest. 2002; 110(1): 71-79.
15. Kusakabe K, Xin KQ, Katoh H, et al. The timing of GM-CSF expression plasmid administration influences the Th1/Th2 response induced by HIV-l-specific DNA vaccine[J]. J Immunol. 2000; 164(6): 3102-3111.
16. Barouch DH, Santra S, Tenner-Racz K, et al. Potent CD4+ T cell responses elicited by a bicistronic HIV-1 DNA vaccine expressing gp120 and GM-CSF. J Immunol. 2002; 168: 562-568.
17．袁时芳，李开宗，王岭等．MUCl基因疫苗诱导小鼠特异性CTL和体液免疫应答．细胞与分子免疫学杂志．2004；20(6)：737-740．
18．袁时芳，王岭，李开宗．MUCl基因免疫抑制H22肝癌生长的实验研究．世界华人消化杂志．2003；11(9)：1322-1325．
19. Heiko Johnen, Hagen Kulbe, Gabriele Pecher. Long-term tumor growth suppression in mice immunized with naked DNA of the human tumor antigen mucin (MUC1) Cancer Immunology, Immunotherapy. 2004; 50(7): 356-360.
20. Vitaly Vasilevko, Anahit Ghochikyan, Nadya Sadzikava, et al. Immunization with a vaccine that combines the expression of MUC1 and B7 co-stimulatory molecules prolongs the survival of mice and delays the appearance of mouse mammary tumors. Clinical and Experimental Metastasis. 2003; 20 (6): 489-498.
21. Fong CL, Mok CL, Hui KM. Intramuscular immunization with plasmid coexpressing tumour antigen and Flt-3L results in potent tumour regression. Gene Ther. 2006; 13(3): 245-256.
22. Mika Kamata, Kaori Denda-Nagai, Nobuyoshi Kubota, et al. Vaccination of mice with MUC1 cDNA suppresses the development of lung metastases. Clinical and Experimental Metastasis. 2003; 19(8): 689-696.
1. Sanjay gurunathan, Dennis, klinman, Robert Seder. DNA Vaccines: Immunology, application, and optimization. Annual review of immunology. 2000; 18: 927-974.
2. Corr M, Lee DJ, Carson DA, et al. Gene vaccination with plasmid DNA: Mechanism of CTL priming. J Exp Med. 1996; 184: 1555-1560.
3. Doe B, Selby M, Barnett S, et al. Induction of cytotoxic T lymphocytes by intramuscular immunization with plasmid DNA is facilitated by bone marrow-derived cells. Proc Natl Acad Sci USA. 1996; 93: 8578-8583.
4. Figdor CG, de Vries IJM, Lesterhuis WJ, Melief CJM. Dendritic cell. Immunotherapy: mapping the way. Nat Med. 2004; 10: 475-480.
5. Suss G, Shortman K. A subclass of dendritic cells kills CD4+ T cells via Fas/Fas-ligand-induced apoptosis. J Exp Med. 1996; 183: 1789-1796.
6. Banchereau J, F Briere, C Caux, et al. Immunobiology of dendritic cells. Annu Rev Immuno. 2002; 18: 767-811.
7. Bernhard H, Disis ML, Heinfeld S, et al. Generation of immunostimulatory dendritic cells from human CD34 +hematopoietic progenitor cells of the bone marrow and peripheral blood[J]. Cancer Res. 1995; 55: 1099-1104.
8. Mackensen A, Herbst B, Chen JL, et al. Phase I study in melanoma patients of a vaccine with peptide pulsed dendritic cells generated in vitro from CD34+ hematopoietic progenitor cells. Int J Cancer. 2000; 86: 385-392.
9. Heiser A, Coleman D, Dannull J, et al. Autologous dendritic cells transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors. J Clin Invest. 2002; 109 (3): 409-417.
10. Romani N, Gruner S, Brang D, et al. Proliferating dendritic cell progenitors in human blood. J Exp Med. 1994; 180: 83-93.
11. Sallusto F, Lanzavecchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colonystimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med. 1994; 179(4): 1109-1118
12. Peters JH, Xu H, Ruppert J, et al. Signals required for differentiating of dendritic cells from human monocytes in vitro. Adv Exp Med Biol. 1993; 329: 275-280.
13．谢芳艺，王胜军，彭光勇等．人外周血单核细胞体外诱导树突细胞及鉴定．南京医科大学报．2001；21(2)：111-113．
14．史敦云，张琼丽，李明．人外周血单核细胞体外诱导成熟和激活的树突细胞．山西医药杂志．2002；31(5)：384-386．
1. Heiko Johnen, Hagen Kulbe, Gabriele Pecher. Long-term tumor growth suppression in mice immunized with naked DNA of the human tumor antigen mucin (MUC1) Cancer Immunology, Immunotherapy.2004; 50(7): 356-360.
2. Brossart P, Wirths S, Stuhler G, et al. Induction of cytotoxic T-lymphocyte responses in vivo after vaccinations with peptide-pulsed dendritic cells. Blood. 2000; 96: 3102-3108.
3. Nestle FO, Alijagic S, Gilliet M, et al. Vaccination of melanoma patients with peptide or tumor lysate pulsed dendritic cells. Nat Med. 1998; 4(3): 328-332.
4. De Bruijn M. L. et al. Immunization with human papillomavirus type 16 (HPV16) oncoprotein-loaded dendritic c ells as well as protein in adjuvant induces MHC class I-restricted protection ofHPV16-induced tumor cells. Cancer research. 1998; 58: 724-731.
5. Homma S, Toda G, Gong J, Kufe D, Ohno T. Preventive antitumor activity against hepatocellular carcinoma (HCC) induced by immunization with fusions of dendritic cells and HCC cells in mice. J Gastroenterol. 2001; 36: 764-771.
6. Kikuchi T, Akasaki B, Irie M, et al. Results of a phase clinical trial of vaccination of glioma patients with fusion of dendritic and glioma cells. Cancer Immunol Immunother, 2001; 50 (7): 337-344.
7. Henderson RA, Nimgaonkar MT, Watkins SC, et al. Hu man dendritic cells genetically engineered to express high levels of the human epithelial tumor antigen mucin (MUC-1). Cancer Res. 1996; 56(16): 3763-3770.
8. Szabolcs, Gallardo, Ciocon, et al. Retrovirally transduced human dendritic cells expressed a nomal phenotype and potent T cell stimulatory capacity. Blood. 1997; 90: 2160-2167.
9. Alijagic S, Moiler P, Artuc M, et al. Dendritic cells generated from peripheral blood transfected with human tyrosinase induces specific T cell activation. Eur J Immunol, 1995; 25: 3100-3107.
10. Pecher G, Haring A, Kaiser L, et al. Mucin gene (MUC1) transfected dendritic cells as vaccine: Results of a phase Ⅰ /Ⅱ clinical trial. Cancer Immunol Immunother. 2002; 51 (11-12): 669-673.
11. Pecher G, Spahn G, Schirrmann T, et al. Mucin gene ( MUC1 ) transfer into human dendritic cells by cationic liposomes and recombinant adenovirus. Anticancer Res, 2001; 21 (4A): 2591-2596.
12. Banchereau J, F Briere, C Caux, et al. Immunobiology of dendritic cells. Annu Rev Immuno. 2002; 18: 767-811.
13. Figdor CG, de Vries IJM, Lesterhuis WJ, Melief CJM. Dendritic cell. Immunotherapy: mapping the way. Nat Med. 2004; 10: 475-480.
14. Hollingsworth, Strawhecker, Caffrey, et al. Expression of MUC1, MUC2, MUC3, and MUC4 mucin mRNA in human pancreatic and intestinal tumor cell lines. Int J Cancer. 1994; 57: 198-203.
15．张立新，李春海．MUC1粘蛋白的免疫生物学作用及其在肿瘤生物学治疗中的作用．中国肿瘤生物治疗杂志，2000，7：165-170．
16．袁时芳，李开宗，王岭等．MUCl基因疫苗诱导小鼠特异性CTL和体液免疫应答．细胞与分子免疫学杂志．2004；20(6)：737-740．
17．袁时芳，王岭，李开宗．MUCl基因免疫抑制H22肝癌生长的实验研究．世界华人消化杂志．2003；11(9)：1322-1325．
18. Snyder LA, Goletz TJ, Gunn GR, et al. A MUC1/IL-18 DNA vaccine induces anti-tumor immunity and increased survival in MUC1 transgenic mice. Vaccine. 2006; 24(16): 3340-3352.
19. Waldman. Immunotherapy: past, present and future. Nat. Med. 2003; 9: 269-277. 20. Pardoll DM. Cancer vaccines. Nat. Med. 1998; 4: 525-523
1. Waldman. Immunotherapy: past, present and future. Nat. Med. 2003. 9:269-277.
2. Pardoll DM. Cancer vaccines. Nat. Med. 1998; 4: 525-523.
3. Lee PP, Yee C, Savage PA, et al.Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients. Nat Med. 1999; 5:677-685.
4. Angela M, Tim G, Carsten Z, et al. Generation of activated and antigen-specific T cells with cytotoxic activity after co-culture with dendritic cells. Cancer immunol immunother. 2002; 51:25-32.
5. Heiko Johnen a Hagen Kulbe a Gabriele Pecher.Long-term tumor growth suppression in mice immunized with naked DNA of the human tumor antigen mucin (MUC 1). Cancer immunology and immunotherapy. 2001; 50:356-360
6. Koido S, Kashiwaba M, Chen D, Gendler S, Kufe D. Induction of antitumor immunity by vaccination of dendritic cells transfected with MUC1 RNA. J Immunol. 2000; 165:5713-5719.
7. Pecher G, Haring A, Kaiser L, et al. Mucin gene (MUC1) transfected dendritic cells as vaccine: Results of a phase I/II clinical trial. Cancer Immunol Immunother.2002; 51: 669-673.
1. SJ Gendler, CA Lancaster, J Taylor-Papadimitriou, et al. Molecular cloning and expression of human tumor-associated polymorphic epithelial mUcin. J. Biol. Chem. 1990; 265(25), 15286-15293.
2. Seregni E, Botti C, Massaron S, Lombardo C, et al. Structure, function and gene expression of epithelial mucins. Tumori. 1997; 83(3): 625-632.
3. Joyce TP, Joy MB, Timothy Plunkett, et al. MUC1 and the immunobiology of cancer. Journal of mammary gland biology and neoplasia. 2002; 7(2): 209-218.
4. Toshiaki Hayashi, Tohru Takahashi, Satoshi Motoya, et al. MUC1 Mucin Core Protein Binds to the Domain 1 of ICAM-1. Digestion. 2001; 63: S87-92.
5. Makiko Yamamoto, Ajit Bharti, Yongqing Li, et al. Interaction of the DF3/MUC1 Breast Carcinoma-associated Antigen and β-Catenin in Cell Adhesion. J Biol Chem. 1997; 272(19): 12492-12494.
6. Li Y, Bharti A, Chen D, Gong J, et al. Interaction of glycogen synthase kinase 3beta with the DF3/MUC1 carcinoma-associated antigen and beta-catenin. Mol Cell Biol. 1998; 18(12): 7216-7224.
7. Ren J, Li Y, Kufe D. Protein kinase C delta regulates function of the DF3/MUC1 carcinoma antigen in beta-catenin signaling. J Biol Chem. 2002; 277(20):17616-17622.
8. Joyce A. Schroeder, Melissa C,et al. Transgenic MUC1 interacts with EGFR and correlates with map kinase activation in the mouse mammary gland. J Biol Chem. 2001; 276(16): 13057-13064.
9. Yuko Hiraga, Shinji Tanaka, Ken Haruma, et al. Immunoreactive MUC1 Expression at the Deepest Invasive Portion Correlates with Prognosis of Colorectal Cancer. Oncology.1998; 55:307-319.
10. Luttges J, Feyerabend B, Buchelt T, et al. The mucin profile of noninvasive and invasive mucinous cystic neoplasms of the pancreas. Am J Surg Pathol. 2002; 26(4):466-471.
11. Kashiwagi H, Kijima H, Dowaki S,et al. DF3 expression in human gallbladder carcinoma: significance for lymphatic invasion. Int J Oncol. 2000; 16(3):455-459.
12. Rahn JJ, Dabbagh L, Pasdar M, et al. The importance of MUC1 cellular localization in patients with breast carcinoma: an immunohistologic study of 71 patients and review of the literature. Cancer. 2001; 91(11): 1973-82.
13. Bramwell ME, Wiseman G, Shotton DM. Electron-microscopic studies of the CA antigen, epitectin. J Cell Sci. 1986; 86:249-261.
14. MJ Ligtenberg, F Buijs, HL Vos, et al.Suppression of cellular aggregation by high levels of episialin. Cancer Res. 1992; 52: 2318-2324.
15. Wesseling, SW van der Valk, HL Vos, et al. Episialin (MUC1) overexpression inhibits integrin-mediated cell adhesion to extracellular matrix components. The Journal of Cell Biology.1995; 129:255-265.
16. Kohlgraf KG, Gawron AJ, Higashi M, et al. Contribution of the MUC1 tandem repeat and cytoplasmic tail to invasive and metastatic properties of a pancreatic cancer cell line. Cancer Res. 2003; 63(16):5011-20.
17. Van de Wiel-van Kemenade E, Ligtenberg MJL, et al.Episialin (MUC1) inhibits cytotoxic lymphocyte-target cell interaction. J Immunol 1993; 151:767-776.
18. Zhang K, Sikut R, Hansson GC. A MUC1 mucin secreted from a colon carcinoma cell line inhibits target cell lysis by natural killer cells. Cell Immunol. 1997; 176(2):158-165.
19. Claude D. Gimmi, Briggs W. Morrison, Brigitte A. Breast cancer-associated antigen, DF3/MUC1, induces apoptosis of activated human T cells.Nature Medicine. 1996; 2: 1367-1370.
20. Hinoda Y, Takahashi T, Hayashi T, Enhancement of reactivity of anti-MUC1 core protein antibody and killing activity of anti-MUCl cytotoxic T cells by deglycosylation of target tissues or cells.J Gastroenterol. 1998; 33(2):164-171.
21. Adachi, Hinoda Y, Nishimori I, et al. Increased sensitivity of gastric cancer cells to Natural killer and lymphokine-Activated killer cells by antisense suppression of N-Actetylgalactosaminytransferase. J Immunol. 1997; 159(6):2645-2651.
22. Krause T, Turner G A.Are selectins involved in metastasis? Clin Exp Metastasis. 1999; 17 (3): 183-192.
23. Rye, Michael A, McGuckin. MUCl: Antibodies and Immunoassays. Tumor Biology .2001; 22:269-272.
24. Apollina Goel, Sam Augustine, Janina Baranowska-Kortylewicz,et al.Single-Dose versus Fractionated Radioimmunotherapy of Human Colon Carcinoma Xenografts Using ~(131)I-labeled Multivalent CC49 Single-chain Fvs. Clinical Cancer Research. 2001; 7:175-184.
25. Avichezer D, Taylor-Papadimitriou J, Arnon R. A short synthetic peptide (DTRPAP) induces anti-mucin (MUC-1) antibody, which is reactive with human ovarian and breast cancer cells. Cancer Biochem Biophys. 1998;16(1-2):113-128
26. Butts C, Murray N, Maksymiuk A, Randomized phase IIB trial of BLP25 liposome vaccine in stage IIIB and IV non-small-cell lung cancer. J Clin Oncol. 2005;23(27):6674-6681.
27. Henderson RA, Konitsky WM, Barratt-Boyes SM, et al.Retroviral expression of MUC-1 human tumor antigen with intact repeat structure and capacity to elicit immunity in vivo. J Immunother. 1998; 21(4):247-256.
28. Pecher G, Haring A, Kaiser L, et al. Mucin gene (MUCl) transfected dendritic cells as vaccine: Results of a phase I/II clinical trial. Cancer Immunol Immunother, 2002; 51:669-673.
29. Wierecky J, Muller MR, Wirths S, Immunologic and clinical responses after vaccinations with peptide-pulsed dendritic cells in metastatic renal cancer patients. Cancer Res. 2006; 66(11):5910-5918.
30. Graham RA, Burchell JM. Beverley P. Intramuscular immunisation with MUCl cDNA can protect C57 mice challenged with MUC1-expressing syngeneic mouse tumour cells. Int J Cancer. 1996; 65(5):664-670.
31. Snyder LA, Goletz TJ, Gunn GR A MUCI/IL-18 DNA vaccine induces anti-tumor immunity and increased survival in MUC1 transgenic mice. Vaccine. 2006; 24(16): 3340-3352.
32．吴文川，靳大勇，秦新裕等．胰腺癌MUCl-VNTR核酸疫苗的构建和体外转染．中华实验外科杂志．2005；22(9)：1081．
2. Li, D. et al. Pancreatic cancer. Lancet. 2004; 363: 1049-1057.
3. Spratlin, J. et al. The absence of human equilibrative nucleoside transporter 1 is associated with reducedsurvival in patients with gemcitabine-treated pancreas adenocarcinoma. Clin. Cancer Res. 2004; 10: 6956-6961.
4. Laheru, D., Biedrzycki, B. & Jaffee, E. M. Immunologic approaches to the management of pancreatic cancer. Cancer J. 2001; 7: 324-337.
5. Pardoll, D. & Allison, J. Cancer Immunotherapy: breaking the barriers to harvest the crop. Nature Med. 2004; 10: 887-892.
6. Mendelsohn, J. & Baselga, J. The EGF receptor family as targets for cancer therapy. Oncogene. 2000; 19: 6550-6565.
7. Schwarte-Waldhoff, I., Volpert, O. V. & Bouck, N. P. SMAD4/DPC4-mediated tumor suppression through suppression ofangiogenesis. Proc. Natl Acad. Sci. USA. 2000; 97: 9624-9629.
8. Xiong, H. Q. et al. Cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor, in combination with gemcitabine for advanced pancreatic Cancer: a multicenter phase Ⅱ Trial. J. Clin. Oncol. 2004; 22: 2610-2616.
9. Kindler, H. L, et al. Bevacizumab plus gemcitabine in patients with advanced pancreatic cancer: Updated results of a multi-center phase Ⅱ trial. J Clin Oncol. 2004; 22(Suppl): 4009.
10. Bos, J. L. ras oncogenes in human cancer: a review. Cancer Res. 1989; 49: 4682-4689.
11. Finn, O.J. et al. MUC-1 epithelial tumor mucin-based immunity and vaccines. Immunol. Rev.1995; 145:61-89.
12. Hammarstrom, S. The carcinoembryonic antigen (CEA) family:structures, suggested functions and expression in normal and malignant tissues. Semin. Cancer Biol. 1999; 9:67-81.
13. Achtar, M. et al. Mutant ras vaccine in advanced cancers.Proc. Am. Soc. Clin. Oncol.2003; 22:A677.
14. Morse, M. et al. Phase I study of immunization with dendritic cells modified with recombinant fowlpox encoding carcinoembryonic antigen (CEA) and the triad of costimulatory molecules CD54, CD58 and CD80 (rF-CEA(6D)-TRICOM) in patients with advanced malignancies. Proc. Am. Soc. Clin. Oncol. 2004; 23: A2508.
15. Finn, O. J. et aL A phase Ib study of a MUC1 pulsed autologous dendritic cell vaccine as adjuvant therapy in patients with resected pancreatic or biliary tumors. Proc. Am. Soc. Clin. Oncol.2004; 23:A2578.
16. Toshiaki Hayashi, Tohru Takahashi, Satoshi Motoya, et al. MUC1 Mucin Core Protein Binds to the Domain 1 of ICAM-l.Digestion.2001; 63:S87-92.
17. Makiko Yamamoto,Ajit Bharti, Yongqing Li,et al. Interaction of the DF3/MUC1 Breast Carcinoma-associated Antigen and β-Catenin in Cell Adhesion. J Biol Chem. 1997;272(19):12492-12494.
18. Li Y, Bharti A, Chen D, Gong J,et al. Interaction of glycogen synthase kinase 3beta with the DF3/MUC1 carcinoma-associated antigen and beta-catenin. Mol Cell Biol. 1998; 18( 12):7216-7224.
19. Yuko Hiraga, Shinji Tanaka, Ken Haruma, et al. Immunoreactive MUC1 Expression at the Deepest Invasive Portion Correlates with Prognosis of Colorectal Cancer. Oncology. 1998; 55:307-319.
20. Luttges J, Feyerabend B, Buchelt T, et al. The mucin profile of noninvasive and invasive mucinous cystic neoplasms of the pancreas. Am J Surg Pathol. 2002; 26(4):466-471.
21. Kashiwagi H, Kijima H, Dowaki S,et al. DF3 expression in human gallbladder carcinoma: significance for lymphatic invasion. Int J Oncol. 2000; 16(3):455-459.
22. Rahn JJ, Dabbagh L, Pasdar M, et al. The importance of MUC1 cellular localization in patients with breast carcinoma: an immunohistologic study of 71 patients and review of the literature. Cancer. 2001; 91(11):1973-1982.
23. Kohlgraf KG, Gawron AJ, Higashi M, et al. Contribution of the MUC1 tandem repeat and cytoplasmic tail to invasive and metastatic properties of a pancreatic cancer cell line. Cancer Res. 2003; 63(16):5011-5020.
24. Van de Wiel-van Kemenade E, Ligtenberg MJL, et al.Episialin (MUC1) inhibits cytotoxic lymphocyte-target cell interaction. J Immunol 1993; 151:767-776.
25. Zhang K, Sikut R, Hansson GC. A MUC1 mucin secreted from a colon carcinoma cell line inhibits target cell lysis by natural killer cells. Cell Immunol. 1997; 176(2):158-165.
26. Claude D. Gimmi, Briggs W. Morrison, Brigitte A. Breast cancer-associated antigen, DF3/MUC1, induces apoptosis of activated human T cells.Nature Medicine. 1996; 2:1367-1370.
27. Adachi, Hinoda, Nishimori, et al. Increased sensitivity of gastric cancer cells to Natural killer and lymphokine-Activated killer cells by antisense suppression of N-Actetylgalactosaminytransferase. J Immunol. 1997; 159(6):2645-2651.
28. Krause, Turner.Are selectins involved in metastasis? Clin Exp Metastasis. 1999; 17(3):183-192.
29. Butts C, Murray N, Maksymiuk A, Randomized phase IIB trial of BLP25 liposome vaccine in stage IIIB and IV non-small-cell lung cancer. J Clin Oncol. 2005;23(27):6674-6681.
30. Henderson RA, Konitsky WM, Barratt-Boyes SM, et al.Retroviral expression of MUC-1 human tumor antigen with intact repeat structure and capacity to elicit immunity in vivo. J Immunother. 1998; 21(4):247-256.
31. Pecher G, Haring A, Kaiser L, et al. Mucin gene (MUC1) transfected dendritic cells as vaccine: Results of a phase I/II clinical trial. Cancer Immunol Immunother, 2002; 51:669-673.
32. Wierecky J, Muller MR, Wirths S, Immunologic and clinical responses after vaccinations with peptide-pulsed dendritic cells in metastatic renal cancer patients. Cancer Res. 2006; 66(11):5910-5918.
33. Graham RA, Burchell JM, Beverley P. Intramuscular immunisation with MUC1 cDNA can protect C57 mice challenged with MUC1-expressing syngeneic mouse tumour cells. Int J Cancer. 1996; 65(5):664-670.
34. Snyder LA, Goletz TJ, Gunn GR A MUC1/IL-18 DNA vaccine induces anti-tumor immunity and increased survival in MUC1 transgenic mice. Vaccine. 2006;24(16):3340-3352.
35．吴文川，靳大勇，秦新裕等．建立胰腺癌黏液蛋白核芯肽-连续重复序列核酸疫苗的实验研究．中华普通外科杂志．2006；21(5)：357-359．
36. Donnelly JJ, Ulmer JB, Liu MA. Protective efficacy of intramuscular immunization with naked DNA. Ann NY Acad Sci. 1995; 772: 40-46.
1. Hollingsworth, Strawhecker, Caffrey, et al. Expression of MUC 1, MUC2, MUC3, and MUC4 mucin mRNA in human pancreatic and intestinal tumor cell lines. Int J Cancer, 1994, 57: 198-203.
2. Finn, O. J. et al. MUC-1 epithelial tumor mucin-based immunity and vaccines. Immunol. Rev. 1995; 145: 61-89.
3. Ciernik IF, Berzofsky JA, Carbone DP. Induction of cytotoxic T lymphocytes and antitumor immunity with DNA vaccines expressing single T cell epitopes. J Immunol. 1996; 156: 2369-2375.
4．张立新，李春海．MUCl粘蛋白的免疫生物学作用及其在肿瘤生物学治疗中的作用．中国肿瘤生物治疗杂志．2000；7：165-170．
5．彭英芳，钟辉，蔡民华．MUC1蛋白的结构功能及作用．生物技术通讯．2001；12：130-131．
6. Pietersz GA, Li W, Osinski C, Apostolopoulos V, et al. Definition of MHC-restricted CTL epitopes from non-variable number of tandem repeat sequence of MUC1. Vaccine. 2000; 18: 2059-2071.
7. Kontani K, Taguchi O, Ozaki Y, et al. Dendritic cell vaccine immunotherapy of cancer targeting MUC1 mucin. Int J Mol Med. 2003; 12: 493-502.
8. Fu X, Guadagni F, Hoffman RM. A metastatic nude-mouse model of human pancreatic cancer constructed orthotopically with histologically intact patient specimens. Proc Natl Acad Sci U SA 1992; 89: 5645-5649.
9. An Z, Wang X, Kubota T, et al. A clinical nude mouse metastatic model for highly malignant human pancreatic cancer. Anticancer Res. 1996; 16: 627-631.
10．刘秋珍，脱朝伟，张艳华．三株人体胰腺癌裸小鼠原位移植模型的建立与应用中国医学影像技术．1997；1 3(6)：498-500．
11．李敏杰，陈永珍，何杨．人胰腺癌裸小鼠模型的建立及其生物学特性的研究．苏州大学学报(医学版)．2003；23(6)：649-651．
12. Corbett TH, Roberts BJ, Leopold WR, et al. Induction and chemotherapeutic response of two transplantable ductal adenocarcinomas of the pancreas in C57BL/6 mice. Cancer Res. 1984; 44: 717-726.
13. Keith R. Jerome, Dawen Bu, Olivera J. Finn. Expression of Tumor-associated Epitopes on Epstein-Barr Virus-immortalized B-Cells and Burkitt's Lymphomas Transfected with Epithelial Mucin Complementary DNA. Cancer Res. 1992; 52: 5985-5990.
14. Devesa SS, Blot WJ, Stone BJ, et al. Recent Cancer trends in the United States. J Natl Cancer Inst. 1995; 87: 175-82.
15. Michand DS. Epideminology of pancreatic cancer. Minerva Chir. 2004; 59: 99-111.
16. Grcenlee RT, Munay T, Bolden S, et al. Cancer statistics 2000. CA Cancer J Chin. 2000; 50: 7-33.
17. Lowenfels AB, Maisouneuve P. Epidemiology and prevention of pancreatic cancer. Jpu J Chin oncol. 2004; 34: 238-249.
18. Wang L, Yang GH, Lu XH. Pancreatic cancer mortality inChina 1991-2000. World J of Gastroenterol. 2003; 9: 1819-1823.
19. Li, D. et al. Pancreatic cancer. Lancet. 2004; 363, 1049-1057.
20．吴文川，靳大勇，秦新裕等．建立胰腺癌黏液蛋白核芯肽-连续重复序列核酸疫苗的实验研究．中华普通外科杂志．2006；21(5)：357-359．
21. Morikane K, Muto T, Tempero R, et al. Organ-specific pancreatic tumor growth properties and tumor immunity. Cancer Immunol Immunother. 1997; 47: 287-296.
1. Wolff JA, Malone RW, Williams P, et al. Direct gene transfer into mouse muscle in vivo. Science. 1990; 247: 1465-1468.
2. Ulmer, Donnelly, Parker, et al. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science. 1993; 259: 1745-49.
3. Sanjay gurunathan, Dennis M. klinman, Robert Seder. DNA Vaccines: Immunology, application, and optimization. Annual review of immunology. 2000; 18: 927-974.
4．吴文川，靳大勇，秦新裕等．建立胰腺癌黏液蛋白核芯肽-连续重复序列核酸疫苗的实验研究．中华普通外科杂志．2006；21(5)：357-359．
5．吴文川，靳大勇，秦新裕等．胰腺癌MUCl-VNTR核酸疫苗的构建和体外转染．中华实验外科杂志．2005；22(9)：1081-1083．
6. Gong JL, Chen, Kashiwaba M, et al.Induction of antitumor activity by immunization with fusions of dendritic and carcinoma cells. Nature Medicine.1997; 3: 558-561.
7. Richard M. Tempero, Michelle L et al. CD4+ Lymphocytes Provide MUCl-Specific Tumor Immunity In Vivo That Is Undetectable In Vitro and Is Absent in MUCl Transgenic Mice. J Immunol. 1998; 161: 5500-5506.
8. Mukherjee P, Ginardi AR, Madsen CS, et al. Mice with spontaneous pancreatic cancer naturally develops MUC-l-specific CTLs that eradicate tumors when adoptively transferred. J Immunol. 2000; 165: 3451-3460.
9. Tempero RM, Rowse GJ, Gendler SJ, Hollingsworth MA. Passively transferred anti-MUC1 antibodies cause neither autoimmune disorders nor immunity against transplanted tumors in MUC1 transgenic mice. Int J Cancer. 1999; 80: 595-599.
10. Snyder LA, Goletz TJ, Gunn GR, et al. A MUC1/IL-18 DNA vaccine induces anti-tumor immunity and increased survival in MUC1 transgenic mice. Vaccine. 2006; 24(16): 3340-3352.
11. Heiko Johnen a Hagen Kulbe a Gabriele Pecher. Long-term tumor growth suppression in mice immunized, with naked DNA of the human tumor antigen mucin (MUC1). Cancer immunology and immunotherapy. 50(77): 356-360.
12. Donnelly JJ, Ulmer JB, Liu MA. Protective efficacy of intramuscular immunization with naked DNA. Ann NY Acad Sci. 1995; 772: 40-46.
13. Donnelly, Friedman, Ulmer, et al. Further protection against drift of influenza virus in a gerret model by DNA vaccination. Vaccine. 1997; 15: 865-868.
14. Rodriguez F, An LL, Harkins S, et al. DNA immunization with minigenes: Low frequency of memory Cytotoxic T Lymphocytes and inefficient antiviral protection are rectified by ubiquitination. J Virol. 1998; 72: 5174-5181.
15．台桂香，张吉凤，朱迅．重组人MUC1-MBP融合蛋白的抗肿瘤作用．中国肿瘤生物治疗杂志．2003；10：202-205．
16. Romani N, Gruner S, Brang D, et al. Proliferating dendritic cell progenitors in human blood. J Exp Med. 1994; 180: 83-93.
17. Berlyn KA, Ponniah S, Stass SA, et al. Developing dendritic cell polynucleotide vaccination for prostate cancer immunotherapy. J Biotechnol. 1999; 73: 155-179.
18. Schnurr M, Galambos P, Scholz C, et al. Tumor cell lysate-pulsed human dendritic cells induce a T-cell response against pancreatic carcinoma cells: an in vitro model for the assessment of tumor vaccines. Cancer Res. 2001; 61: 6445-6450.
19. Henderson RA, Nimgaonkar MT, Watkins SC, et al. Human dendritic cells genetically engineered to express high levels of the human epithelial tumor antigen mucin (MUC-1). Cancer Res. 1996; 56: 3763-3770.
20. Kontani K, Taguchi O, Ozaki Y, et al. Dendritic cell vaccine immunotherapy of cancer targeting MUC1 mucin. Int J Mol Med. 2003; 12: 493-502.
21. Banchereau J, Bazan F, Blanchard D,Briere F, Galizzi LP, van Kooten C, Liu YJ, Rousset F, Saeland S. The CD40 antigen and its ligand. Annu. Rev.Immunol. 1994;12:881-922
22. Ridge JP, Di Rosa F,Matzinger RA conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell.Nature. 1998;393:474-478.
23. Bennett SR, Carbone FR, Karamalis F, et al. Help for cytotoxic-T cell responses is mediated by CD40 signalling. Nature. 1998; 393(6684):478-480.
24. Schoenberger, Toes, Voort EL, et al. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions.Nature.1998; 393(4):480-483.
25. Seder RA, Paul WE. Acquisition of lymphokine- producing phenotype by CD; T Cells. Annu Rev Immunol. 1994; 12:635-673.
26. O'Garra A. Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity. 1998; 8: 275-283.
1. Geissler M, Tokushige K, Chante CC, et al. Cellular and humoral immune response to hepatitis B virus structural proteins in mice after DNA-based immunization. Gastroenterology. 1997; 112: 1307-1320.
2. Goebels N, Michaelis D, Wekerie, et al. Human myoblasts as antigen-presenting cells. J Immunol. 1992; 149: 661-667.
3. Sanjay gurunathan, Dennis, klinman, Robert Seder. DNA Vaccines: Immunology, application, and optimization. Annual review of immunology. 2000; 18: 927-974.
4. Corr M, Lee DJ, Carson DA, et al. Gene vaccination with plasmid DNA: Mechanism of CTL priming. J Exp Med. 1996, 184: 1555-1560.
5. Doe B, Selby M, Barnett S, et al. Induction of cytotoxic T lymphocytes by intramuscular immunization with plasmid DNA is facilitated by bone marrow-derived cells. Proc Natl Acad Sci USA. 1996; 93: 8578-8583.
6. Xiang, Z., and Ertl, H. C. Manipulation of the immune response to a plasmid-encoded viral antigen by coinoculation with plasmids expressing cytokines. Immunity. 1995; 2: 129-135.
7. Kim. In vivo engineering of a cellular immune response by coadministration of IL-12 expression vector with a DNA immunogen.Jouranl of Immunology. 1997; 158: 816-826.
8. Snyder LA, Goletz TJ, Gunn GR, et al. A MUC1/IL-18 DNA vaccine induces anti-tumor immunity and increased survival in MUC1 transgenic mice. Vaccine. 2006; 24(16): 3340-3352.
9. Shawn M, Sumida, Paul F. et al Recruitment and expansion of dendritic cells in vivo potentiate the immunogenicity of plasmid DNA vaccinesarticle: J. Clin. Invest. 2004; 114: 1334-1342.
10．吴文川，靳大勇，秦新裕等．胰腺癌MUCl-VNTR核酸疫苗的构建和体外转染．中华实验外科杂志．2005；22(9)：1081-1083．
11. Wenxin Sun, Haili Qian, Xueyan Zhang, et al. Induction of protective and therapeutic antitumor immunity using a novel tumor-associated antigen-specific DNA vaccine. Immunology and cell biology. 2006; 84: 440-447.
12. Mwangi W, Brown WC, Splitter GA, et al. Enhancement of antigen acquisition by dendritic cells and MHC class Ⅱ-restricted epitope presentation to CD4~+ T cells using VP22 DNA vaccine vectors that promote intercellular spreading following initial transfection. J Leukoc Biol, 2005; 4: 876-881.
13. Ohta K, Yamaguchi Y, Miyahara E, et al. Novel system for generating cytotoxic effector lymphocytes using carcinoembryonic antigen(CEA) peptide and culture dendritic cells. Anticancer research. 2002; 22: 2597-2606.
14. Rafiq K, Bergtold A, Clynes R. Immune complex-mediated antigen presentation induces tumor immunity. J Clin Invest. 2002; 110(1): 71-79.
15. Kusakabe K, Xin KQ, Katoh H, et al. The timing of GM-CSF expression plasmid administration influences the Th1/Th2 response induced by HIV-l-specific DNA vaccine[J]. J Immunol. 2000; 164(6): 3102-3111.
16. Barouch DH, Santra S, Tenner-Racz K, et al. Potent CD4+ T cell responses elicited by a bicistronic HIV-1 DNA vaccine expressing gp120 and GM-CSF. J Immunol. 2002; 168: 562-568.
17．袁时芳，李开宗，王岭等．MUCl基因疫苗诱导小鼠特异性CTL和体液免疫应答．细胞与分子免疫学杂志．2004；20(6)：737-740．
18．袁时芳，王岭，李开宗．MUCl基因免疫抑制H22肝癌生长的实验研究．世界华人消化杂志．2003；11(9)：1322-1325．
19. Heiko Johnen, Hagen Kulbe, Gabriele Pecher. Long-term tumor growth suppression in mice immunized with naked DNA of the human tumor antigen mucin (MUC1) Cancer Immunology, Immunotherapy. 2004; 50(7): 356-360.
20. Vitaly Vasilevko, Anahit Ghochikyan, Nadya Sadzikava, et al. Immunization with a vaccine that combines the expression of MUC1 and B7 co-stimulatory molecules prolongs the survival of mice and delays the appearance of mouse mammary tumors. Clinical and Experimental Metastasis. 2003; 20 (6): 489-498.
21. Fong CL, Mok CL, Hui KM. Intramuscular immunization with plasmid coexpressing tumour antigen and Flt-3L results in potent tumour regression. Gene Ther. 2006; 13(3): 245-256.
22. Mika Kamata, Kaori Denda-Nagai, Nobuyoshi Kubota, et al. Vaccination of mice with MUC1 cDNA suppresses the development of lung metastases. Clinical and Experimental Metastasis. 2003; 19(8): 689-696.
1. Sanjay gurunathan, Dennis, klinman, Robert Seder. DNA Vaccines: Immunology, application, and optimization. Annual review of immunology. 2000; 18: 927-974.
2. Corr M, Lee DJ, Carson DA, et al. Gene vaccination with plasmid DNA: Mechanism of CTL priming. J Exp Med. 1996; 184: 1555-1560.
3. Doe B, Selby M, Barnett S, et al. Induction of cytotoxic T lymphocytes by intramuscular immunization with plasmid DNA is facilitated by bone marrow-derived cells. Proc Natl Acad Sci USA. 1996; 93: 8578-8583.
4. Figdor CG, de Vries IJM, Lesterhuis WJ, Melief CJM. Dendritic cell. Immunotherapy: mapping the way. Nat Med. 2004; 10: 475-480.
5. Suss G, Shortman K. A subclass of dendritic cells kills CD4+ T cells via Fas/Fas-ligand-induced apoptosis. J Exp Med. 1996; 183: 1789-1796.
6. Banchereau J, F Briere, C Caux, et al. Immunobiology of dendritic cells. Annu Rev Immuno. 2002; 18: 767-811.
7. Bernhard H, Disis ML, Heinfeld S, et al. Generation of immunostimulatory dendritic cells from human CD34 +hematopoietic progenitor cells of the bone marrow and peripheral blood[J]. Cancer Res. 1995; 55: 1099-1104.
8. Mackensen A, Herbst B, Chen JL, et al. Phase I study in melanoma patients of a vaccine with peptide pulsed dendritic cells generated in vitro from CD34+ hematopoietic progenitor cells. Int J Cancer. 2000; 86: 385-392.
9. Heiser A, Coleman D, Dannull J, et al. Autologous dendritic cells transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors. J Clin Invest. 2002; 109 (3): 409-417.
10. Romani N, Gruner S, Brang D, et al. Proliferating dendritic cell progenitors in human blood. J Exp Med. 1994; 180: 83-93.
11. Sallusto F, Lanzavecchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colonystimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med. 1994; 179(4): 1109-1118
12. Peters JH, Xu H, Ruppert J, et al. Signals required for differentiating of dendritic cells from human monocytes in vitro. Adv Exp Med Biol. 1993; 329: 275-280.
13．谢芳艺，王胜军，彭光勇等．人外周血单核细胞体外诱导树突细胞及鉴定．南京医科大学报．2001；21(2)：111-113．
14．史敦云，张琼丽，李明．人外周血单核细胞体外诱导成熟和激活的树突细胞．山西医药杂志．2002；31(5)：384-386．
1. Heiko Johnen, Hagen Kulbe, Gabriele Pecher. Long-term tumor growth suppression in mice immunized with naked DNA of the human tumor antigen mucin (MUC1) Cancer Immunology, Immunotherapy.2004; 50(7): 356-360.
2. Brossart P, Wirths S, Stuhler G, et al. Induction of cytotoxic T-lymphocyte responses in vivo after vaccinations with peptide-pulsed dendritic cells. Blood. 2000; 96: 3102-3108.
3. Nestle FO, Alijagic S, Gilliet M, et al. Vaccination of melanoma patients with peptide or tumor lysate pulsed dendritic cells. Nat Med. 1998; 4(3): 328-332.
4. De Bruijn M. L. et al. Immunization with human papillomavirus type 16 (HPV16) oncoprotein-loaded dendritic c ells as well as protein in adjuvant induces MHC class I-restricted protection ofHPV16-induced tumor cells. Cancer research. 1998; 58: 724-731.
5. Homma S, Toda G, Gong J, Kufe D, Ohno T. Preventive antitumor activity against hepatocellular carcinoma (HCC) induced by immunization with fusions of dendritic cells and HCC cells in mice. J Gastroenterol. 2001; 36: 764-771.
6. Kikuchi T, Akasaki B, Irie M, et al. Results of a phase clinical trial of vaccination of glioma patients with fusion of dendritic and glioma cells. Cancer Immunol Immunother, 2001; 50 (7): 337-344.
7. Henderson RA, Nimgaonkar MT, Watkins SC, et al. Hu man dendritic cells genetically engineered to express high levels of the human epithelial tumor antigen mucin (MUC-1). Cancer Res. 1996; 56(16): 3763-3770.
8. Szabolcs, Gallardo, Ciocon, et al. Retrovirally transduced human dendritic cells expressed a nomal phenotype and potent T cell stimulatory capacity. Blood. 1997; 90: 2160-2167.
9. Alijagic S, Moiler P, Artuc M, et al. Dendritic cells generated from peripheral blood transfected with human tyrosinase induces specific T cell activation. Eur J Immunol, 1995; 25: 3100-3107.
10. Pecher G, Haring A, Kaiser L, et al. Mucin gene (MUC1) transfected dendritic cells as vaccine: Results of a phase Ⅰ /Ⅱ clinical trial. Cancer Immunol Immunother. 2002; 51 (11-12): 669-673.
11. Pecher G, Spahn G, Schirrmann T, et al. Mucin gene ( MUC1 ) transfer into human dendritic cells by cationic liposomes and recombinant adenovirus. Anticancer Res, 2001; 21 (4A): 2591-2596.
12. Banchereau J, F Briere, C Caux, et al. Immunobiology of dendritic cells. Annu Rev Immuno. 2002; 18: 767-811.
13. Figdor CG, de Vries IJM, Lesterhuis WJ, Melief CJM. Dendritic cell. Immunotherapy: mapping the way. Nat Med. 2004; 10: 475-480.
14. Hollingsworth, Strawhecker, Caffrey, et al. Expression of MUC1, MUC2, MUC3, and MUC4 mucin mRNA in human pancreatic and intestinal tumor cell lines. Int J Cancer. 1994; 57: 198-203.
15．张立新，李春海．MUC1粘蛋白的免疫生物学作用及其在肿瘤生物学治疗中的作用．中国肿瘤生物治疗杂志，2000，7：165-170．
16．袁时芳，李开宗，王岭等．MUCl基因疫苗诱导小鼠特异性CTL和体液免疫应答．细胞与分子免疫学杂志．2004；20(6)：737-740．
17．袁时芳，王岭，李开宗．MUCl基因免疫抑制H22肝癌生长的实验研究．世界华人消化杂志．2003；11(9)：1322-1325．
18. Snyder LA, Goletz TJ, Gunn GR, et al. A MUC1/IL-18 DNA vaccine induces anti-tumor immunity and increased survival in MUC1 transgenic mice. Vaccine. 2006; 24(16): 3340-3352.
19. Waldman. Immunotherapy: past, present and future. Nat. Med. 2003; 9: 269-277. 20. Pardoll DM. Cancer vaccines. Nat. Med. 1998; 4: 525-523
1. Waldman. Immunotherapy: past, present and future. Nat. Med. 2003. 9:269-277.
2. Pardoll DM. Cancer vaccines. Nat. Med. 1998; 4: 525-523.
3. Lee PP, Yee C, Savage PA, et al.Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients. Nat Med. 1999; 5:677-685.
4. Angela M, Tim G, Carsten Z, et al. Generation of activated and antigen-specific T cells with cytotoxic activity after co-culture with dendritic cells. Cancer immunol immunother. 2002; 51:25-32.
5. Heiko Johnen a Hagen Kulbe a Gabriele Pecher.Long-term tumor growth suppression in mice immunized with naked DNA of the human tumor antigen mucin (MUC 1). Cancer immunology and immunotherapy. 2001; 50:356-360
6. Koido S, Kashiwaba M, Chen D, Gendler S, Kufe D. Induction of antitumor immunity by vaccination of dendritic cells transfected with MUC1 RNA. J Immunol. 2000; 165:5713-5719.
7. Pecher G, Haring A, Kaiser L, et al. Mucin gene (MUC1) transfected dendritic cells as vaccine: Results of a phase I/II clinical trial. Cancer Immunol Immunother.2002; 51: 669-673.
1. SJ Gendler, CA Lancaster, J Taylor-Papadimitriou, et al. Molecular cloning and expression of human tumor-associated polymorphic epithelial mUcin. J. Biol. Chem. 1990; 265(25), 15286-15293.
2. Seregni E, Botti C, Massaron S, Lombardo C, et al. Structure, function and gene expression of epithelial mucins. Tumori. 1997; 83(3): 625-632.
3. Joyce TP, Joy MB, Timothy Plunkett, et al. MUC1 and the immunobiology of cancer. Journal of mammary gland biology and neoplasia. 2002; 7(2): 209-218.
4. Toshiaki Hayashi, Tohru Takahashi, Satoshi Motoya, et al. MUC1 Mucin Core Protein Binds to the Domain 1 of ICAM-1. Digestion. 2001; 63: S87-92.
5. Makiko Yamamoto, Ajit Bharti, Yongqing Li, et al. Interaction of the DF3/MUC1 Breast Carcinoma-associated Antigen and β-Catenin in Cell Adhesion. J Biol Chem. 1997; 272(19): 12492-12494.
6. Li Y, Bharti A, Chen D, Gong J, et al. Interaction of glycogen synthase kinase 3beta with the DF3/MUC1 carcinoma-associated antigen and beta-catenin. Mol Cell Biol. 1998; 18(12): 7216-7224.
7. Ren J, Li Y, Kufe D. Protein kinase C delta regulates function of the DF3/MUC1 carcinoma antigen in beta-catenin signaling. J Biol Chem. 2002; 277(20):17616-17622.
8. Joyce A. Schroeder, Melissa C,et al. Transgenic MUC1 interacts with EGFR and correlates with map kinase activation in the mouse mammary gland. J Biol Chem. 2001; 276(16): 13057-13064.
9. Yuko Hiraga, Shinji Tanaka, Ken Haruma, et al. Immunoreactive MUC1 Expression at the Deepest Invasive Portion Correlates with Prognosis of Colorectal Cancer. Oncology.1998; 55:307-319.
10. Luttges J, Feyerabend B, Buchelt T, et al. The mucin profile of noninvasive and invasive mucinous cystic neoplasms of the pancreas. Am J Surg Pathol. 2002; 26(4):466-471.
11. Kashiwagi H, Kijima H, Dowaki S,et al. DF3 expression in human gallbladder carcinoma: significance for lymphatic invasion. Int J Oncol. 2000; 16(3):455-459.
12. Rahn JJ, Dabbagh L, Pasdar M, et al. The importance of MUC1 cellular localization in patients with breast carcinoma: an immunohistologic study of 71 patients and review of the literature. Cancer. 2001; 91(11): 1973-82.
13. Bramwell ME, Wiseman G, Shotton DM. Electron-microscopic studies of the CA antigen, epitectin. J Cell Sci. 1986; 86:249-261.
14. MJ Ligtenberg, F Buijs, HL Vos, et al.Suppression of cellular aggregation by high levels of episialin. Cancer Res. 1992; 52: 2318-2324.
15. Wesseling, SW van der Valk, HL Vos, et al. Episialin (MUC1) overexpression inhibits integrin-mediated cell adhesion to extracellular matrix components. The Journal of Cell Biology.1995; 129:255-265.
16. Kohlgraf KG, Gawron AJ, Higashi M, et al. Contribution of the MUC1 tandem repeat and cytoplasmic tail to invasive and metastatic properties of a pancreatic cancer cell line. Cancer Res. 2003; 63(16):5011-20.
17. Van de Wiel-van Kemenade E, Ligtenberg MJL, et al.Episialin (MUC1) inhibits cytotoxic lymphocyte-target cell interaction. J Immunol 1993; 151:767-776.
18. Zhang K, Sikut R, Hansson GC. A MUC1 mucin secreted from a colon carcinoma cell line inhibits target cell lysis by natural killer cells. Cell Immunol. 1997; 176(2):158-165.
19. Claude D. Gimmi, Briggs W. Morrison, Brigitte A. Breast cancer-associated antigen, DF3/MUC1, induces apoptosis of activated human T cells.Nature Medicine. 1996; 2: 1367-1370.
20. Hinoda Y, Takahashi T, Hayashi T, Enhancement of reactivity of anti-MUC1 core protein antibody and killing activity of anti-MUCl cytotoxic T cells by deglycosylation of target tissues or cells.J Gastroenterol. 1998; 33(2):164-171.
21. Adachi, Hinoda Y, Nishimori I, et al. Increased sensitivity of gastric cancer cells to Natural killer and lymphokine-Activated killer cells by antisense suppression of N-Actetylgalactosaminytransferase. J Immunol. 1997; 159(6):2645-2651.
22. Krause T, Turner G A.Are selectins involved in metastasis? Clin Exp Metastasis. 1999; 17 (3): 183-192.
23. Rye, Michael A, McGuckin. MUCl: Antibodies and Immunoassays. Tumor Biology .2001; 22:269-272.
24. Apollina Goel, Sam Augustine, Janina Baranowska-Kortylewicz,et al.Single-Dose versus Fractionated Radioimmunotherapy of Human Colon Carcinoma Xenografts Using ~(131)I-labeled Multivalent CC49 Single-chain Fvs. Clinical Cancer Research. 2001; 7:175-184.
25. Avichezer D, Taylor-Papadimitriou J, Arnon R. A short synthetic peptide (DTRPAP) induces anti-mucin (MUC-1) antibody, which is reactive with human ovarian and breast cancer cells. Cancer Biochem Biophys. 1998;16(1-2):113-128
26. Butts C, Murray N, Maksymiuk A, Randomized phase IIB trial of BLP25 liposome vaccine in stage IIIB and IV non-small-cell lung cancer. J Clin Oncol. 2005;23(27):6674-6681.
27. Henderson RA, Konitsky WM, Barratt-Boyes SM, et al.Retroviral expression of MUC-1 human tumor antigen with intact repeat structure and capacity to elicit immunity in vivo. J Immunother. 1998; 21(4):247-256.
28. Pecher G, Haring A, Kaiser L, et al. Mucin gene (MUCl) transfected dendritic cells as vaccine: Results of a phase I/II clinical trial. Cancer Immunol Immunother, 2002; 51:669-673.
29. Wierecky J, Muller MR, Wirths S, Immunologic and clinical responses after vaccinations with peptide-pulsed dendritic cells in metastatic renal cancer patients. Cancer Res. 2006; 66(11):5910-5918.
30. Graham RA, Burchell JM. Beverley P. Intramuscular immunisation with MUCl cDNA can protect C57 mice challenged with MUC1-expressing syngeneic mouse tumour cells. Int J Cancer. 1996; 65(5):664-670.
31. Snyder LA, Goletz TJ, Gunn GR A MUCI/IL-18 DNA vaccine induces anti-tumor immunity and increased survival in MUC1 transgenic mice. Vaccine. 2006; 24(16): 3340-3352.
32．吴文川，靳大勇，秦新裕等．胰腺癌MUCl-VNTR核酸疫苗的构建和体外转染．中华实验外科杂志．2005；22(9)：1081．