【作者】 戎叶飞；

【导师】 秦新裕； 靳大勇； 秦莹； 吴文川；

【作者基本信息】 复旦大学 ， 外科学， 2007， 博士

【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1×10⁶／100μl／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1×10⁶／100μl／只。肿瘤体积大于1000mm³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1×10⁶／100μl／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1000mm³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1×10⁶／100μl／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1000mm³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-A₂+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-A₂+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-A₂+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-A₂-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-A₂限制性的。更多还原

【Abstract】 The anti-pancreatic cancer research of MUC1-VNTR DNA vaccine in vivo and in vitroPancreatic 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 oneThe anti-pancreatic cancer research of MUC1-VNTR DNA vaccine in vivoSection one The construction of the pancreatic cancer cell line panc02-MUC1Objective: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 vaccineObjective: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（1×10⁶/100μl/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 1,000mm³, 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（1×10⁶/100μl/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 GMCSFObjective: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 （1×10⁶/100μl/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 twoThe anti-pancreatic cancer research of MUC1-VNTR DNA vaccine in vitroSection one In vitro induction and culture of the human dendritic cell and the research on the biological characteristicsObjective: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 cellObjective: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 cellObjective: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-A₂+ 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-A₂+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-A₂ restricted VNTR specific CTL.更多还原