干扰素γ诱导蛋白10基因与肿瘤抗原修饰树突状细胞抗前列腺癌实验研究
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摘要
干扰素γ诱导蛋白10基因与肿瘤抗原修饰树突状细胞抗前列腺癌实验研究
目的：
1．构建干扰素γ诱导蛋白10(IFN-γ inducible protein-10, IP-10)真核表达质粒(IP-10／pcDNA3.1)并大量制备。
2．建立从小鼠骨髓前体细胞中分离、培养和扩增树突状细胞(dendritic cell, DC)的方法；研究DC发育、分化和成熟过程中形态、结构以及免疫生物学特性的改变；探讨影响DC增殖、分化的有关因素。
3．将RM-1前列腺癌细胞的裂解产物(Lysate)和IP-10基因共同修饰DC，构建前列腺癌DC瘤苗(IP-10／Lysate-DC)；体内、外实验检测该瘤苗抗前列腺癌免疫治疗作用及免疫保护作用。
方法：
提取小鼠脾脏总RNA，逆转录多聚酶链反应(RT-PCR)扩增小鼠IP-10基因并加上信号肽基因序列后，克隆至pcDNA3.1(+)质粒，构建IP-10／pcDNA3.1表达载体并转化、抽提和纯化。分离骨髓前体细胞，在细胞因子GM-CSF和IL-4联合作用下，经手法筛选，大量制备骨髓DC(bone marrow derived DC, BM-DC)。采用形态学(光镜、电镜)、免疫细胞化学(FCM)以及免疫生物学(MLR)等方法研究DC的生物免疫学特性。将RM-1前列腺癌细胞的裂解产物负载DC后，通过脂质体转染法将IP-10基因导入DC，制备IP-10／Lysate-DC前列腺癌瘤苗；RT-PCR和Western blot检测转染DC IP-10的表达，测定转染DC上清对T细胞的趋化指数，检测DC瘤苗刺激T细胞的增殖以及诱导特异性CTL的杀伤作用。建立小鼠RM-1前列腺癌模型，观察该瘤苗对荷瘤小鼠的免疫治疗作用；预先应用瘤苗接种小鼠后，以RM-1细胞攻击小鼠，观察瘤苗诱导的免疫保护作用；ELISA方法检测接种DC瘤苗小鼠CTL的IL-2和IFN-γ表达水平。
结果：
1．所构建的IP-10／pcDNA3.1表达质粒，经酶切电泳和DNA正、反向测序鉴定，证实重组质粒构建正确，插入片断包含IP-10基因序列和单核细胞趋化蛋白1信号肽基因序列。
2．在细胞因子GM-CSF或GM-CSF加IL-4诱导下，体外培养7天后，可从每
中文摘要
只小鼠的骨髓前体细胞中获得5一10xl护个Dc。形态学观察可见，培养早
期的DC突起少而短，胞内有许多囊泡，细胞器少;细胞表面低表达B7分子
和MHC分子，刺激T细胞增殖能力极弱，但吞噬能力强;培养7天时，细
胞呈现典型的DC形态，表面伸出许多树枝样突起，胞内囊泡结构减少，细
胞器成分增加，吞噬功能减弱，但B7分子和MHC分子表达明显上调，能强
烈刺激T细胞增殖。GM一CSF加IL一4组的DC得率及细胞功能均明显高于
GM一CSF组。
3.将RNI一1前列腺癌细胞的裂解产物负载培养5天后的DC，通过DOI人P脂质
体转染法可以成功的将IP一10基因导入Dc，构建IP一10/Lysate一DC前列腺癌
瘤苗;经RpPCR和M尾Stem blot检测证实转染DC的IP一10 mRNA和蛋白表
达明显增强，其细胞上清对T淋巴细胞有显著趋化作用。
4.混合淋巴细胞反应显示，IP一10/Lysate一DC瘤苗刺激同基因型小鼠脾脏T细胞
增殖能力明显提高。四氮吟蓝(MTT)还原法检测显示，用负载RM一1细胞
裂解产物的DC(Lysate一DC)免疫小鼠，其脾脏T细胞经RM一1细胞诱导后，
对RM一1肿瘤细胞具有特异性杀伤作用，但杀伤作用明显低于IP一10/切sate一DC
免疫组小鼠，差异有显著性意义(p<0.01)。
5.RM一1前列腺癌荷瘤小鼠模型构建成功，成瘤率为100%。体内实验显示，
Lysate一DC治疗组小鼠肿瘤生长减慢，生存期延长;应用IP一10/切sate一Dc的
治疗作用较切sate一DC进一步增强，接种RM一1肿瘤细胞75天后，
IP一10/Lysate一Dc治疗组仍有50%的小鼠存活，差异有显著性意义(p<0 .01)。
6，组织学检查发现，经IP一10/Lysate一DC瘤苗治疗的小鼠，肿瘤组织局部坏死明
显，在瘤体周围和瘤体内有大量炎细胞浸润，切sate一DC治疗组也可见较明显
的炎细胞浸润，但少于IP一10/Ly sate一Dc组。
7.免疫保护实验发现，经IP一10/Lysate一DC免疫的小鼠接种R人1一1肿瘤细胞后，
肿瘤生长缓慢，小鼠生存期延长，在RM一1细胞攻击后75天，经
lP一10/切sate一DC免疫的小鼠仍有67%无肿瘤生长;Lysate一DC也具有免疫保
护作用，但明显弱于IP一10/Lysate一DC，免疫小鼠只有17%无肿瘤生长，差异
有显著性意义(p<0 .05)。
8.ELISA检测显示，经IP一10/场sate一DC免疫的小鼠，其CTL的IL一2和IFN一Y
表达水平明显升高，与Lysate一DC免疫小鼠相比，差异有显著性意义(p<0.05)。
结论:
1.所构建的IP一1帅cDNA3.1表达质粒正确。
2.小鼠骨髓单个核细胞在细胞因子GM一CSF和IL一4诱导下，通过手法筛选，能
中文摘要
扩增获得大量功能性DC。GM一CSF是DC生长的必须因子，GM一CSF和IL一4
联合应用，可增加DC的得率，增强DC的免疫功能。
3.通过脂质体转染法将IP一10/P cDNA3.1导入DC，获得有效的IP一10表达，转
染DC的上清对T淋巴细胞有显著趋化作用。
4，RM一1前列腺癌细胞裂解产物和IP一10基因共同修饰DC所构建的前列腺癌
Dc瘤苗(IP一10/切sate一DC)，能够明显刺激T细胞的增殖，所诱导的特异性
cTL分泌IL一2和IFN一Y能力增强，对RM一1细胞具有显著杀伤作用。
5.将IP一10/Lysate一Dc瘤苗回?
Objective:
To construct recombinant plasmid IP-10/pcDNA3.1 by inserted IFN-y inducible protein-10 (IP-10) DNA fragments into pcDNA3.1(+) vector. To establish the methods of isolation, purification and propagation of mouse bone marrow-derived dendritic cell (DC), investigate the changes of morphological structure and bio-immunological characteristics of DC during its development, differentiation and maturation, study the related factors involved in proliferation and polarization of DC. To construct the DC vaccine pulsed with whole tumor cell lysate and modified with IP-10 gene. To detect and analyze the specific anti-tumor activity, the immunotherapeutic function and immune protection of DC vaccine both in vitro and in vivo,
Methods:
The cDNA fragment encoding the murine IP-10 was obtained from mRNA of murine T cells by using Reversal transcript-polymerase chain reaction (RT-PCR) and contained a signal peptide derived from the human monocyte chemoattractant protein 1, then it was inserted into the eukaryotic expression vector pcDNA3.1(+). The mouse bone marrow progenitors were isolated and cultured with cytokines of GM-CSF plus IL-4, and a large amount of BM-DC were harvested after special selection and culture for 7 days. The bio-immunological features and functions of DC in different stages were studied by using methods of light- and electro-microscopy, HRP phagocytosis assay, mixed leucocyte reaction (MLR), flow cytometry (FCM), and so on. Mouse bone marrow DC were pulsed with cell lysate from RM-1 prostate cancer cell line and then transfected with the recombinant plasmid IP-10/pcDNA3.1 by DOTAP liposome. The expression of IP-10 was assayed by Western blot and chemotaxis assay. The ability of stimulating syngeneic T cells and the eff
ect of inducing specific kill activity of DC vaccine were detected. The immunotherapeutic effect of DC vaccine on mice with prostate cancer was assessed. The immunological protection induced by DC vaccine was studied by the challenge of tumor cell injection followed the inoculation.
Results:
1. By partial nucleotide sequencing, the cDNA was consistent with the reported mIP-10 cDNA in the GeneBank, and then was inserted into the eukaryotic expression vector pcDNA3.1(+) to construct recombinant plasmid IP-10/pcDNA3.1.
2. About 5~10X106 DC were harvested from bone marrow progenitors per mouse followed culturing the cells with GM-CSF plus IL-4 for 7 days. In early stage, DC possessed short processes and fewer organelles, but a large numbers of vacuoles in the cytoplasm. They expressed very low level of B7-1, B7-2, MHC class-I and class-II molecules and failed to stimulate allogeneic T cell proliferation, but could pino-phagocytose vigorously HRP granules. After 7 days culture, they exhibited morphologically the distinct dendritic shape with long processes, increased organelle and decreased fraction of vacuoles. Simultaneously, they expressed higher level of surface MHC and B7 molecules and stronger activity of stimulating T cell proliferation.
3. The IP-10 plasmid vector was successfully transfected into DC and the DC tranfected with IP-10 gene were capable of synthesizing and secreting IP-10 chemokine, which could increase the preferential chemotaxis of DC to T cells. The DC vaccine modified with tumor cell lysate and IP-10 gene (IP-10/Lysate-DC) was a more potent stimulator in MLR. The mice immunized with DC pulsed with RM-1 cell lysate (Lysate-DC) exhibited a specific CTL response, but the highest CTL activity against RM-1 cells was induced by immunization with IP-10/Lysate-DC (p<0.01).
4. In the mice model with pre-established subcutaneous RM-1 prostate cancer, vaccination with Lysate-DC could inhibit tumor growth, but the immunization of IP-10/Lysate-DC inhibited the tumor growth most significantly when compared with Lysate-DC (p<0.01). The survival time of the mice treated with IP-10/Lysate-DC was also greatly extended (p<0.01).
5. Histological examination showed that the most obvious tumor nec
目的：
1．构建干扰素γ诱导蛋白10(IFN-γ inducible protein-10, IP-10)真核表达质粒(IP-10／pcDNA3.1)并大量制备。
2．建立从小鼠骨髓前体细胞中分离、培养和扩增树突状细胞(dendritic cell, DC)的方法；研究DC发育、分化和成熟过程中形态、结构以及免疫生物学特性的改变；探讨影响DC增殖、分化的有关因素。
3．将RM-1前列腺癌细胞的裂解产物(Lysate)和IP-10基因共同修饰DC，构建前列腺癌DC瘤苗(IP-10／Lysate-DC)；体内、外实验检测该瘤苗抗前列腺癌免疫治疗作用及免疫保护作用。
方法：
提取小鼠脾脏总RNA，逆转录多聚酶链反应(RT-PCR)扩增小鼠IP-10基因并加上信号肽基因序列后，克隆至pcDNA3.1(+)质粒，构建IP-10／pcDNA3.1表达载体并转化、抽提和纯化。分离骨髓前体细胞，在细胞因子GM-CSF和IL-4联合作用下，经手法筛选，大量制备骨髓DC(bone marrow derived DC, BM-DC)。采用形态学(光镜、电镜)、免疫细胞化学(FCM)以及免疫生物学(MLR)等方法研究DC的生物免疫学特性。将RM-1前列腺癌细胞的裂解产物负载DC后，通过脂质体转染法将IP-10基因导入DC，制备IP-10／Lysate-DC前列腺癌瘤苗；RT-PCR和Western blot检测转染DC IP-10的表达，测定转染DC上清对T细胞的趋化指数，检测DC瘤苗刺激T细胞的增殖以及诱导特异性CTL的杀伤作用。建立小鼠RM-1前列腺癌模型，观察该瘤苗对荷瘤小鼠的免疫治疗作用；预先应用瘤苗接种小鼠后，以RM-1细胞攻击小鼠，观察瘤苗诱导的免疫保护作用；ELISA方法检测接种DC瘤苗小鼠CTL的IL-2和IFN-γ表达水平。
结果：
1．所构建的IP-10／pcDNA3.1表达质粒，经酶切电泳和DNA正、反向测序鉴定，证实重组质粒构建正确，插入片断包含IP-10基因序列和单核细胞趋化蛋白1信号肽基因序列。
2．在细胞因子GM-CSF或GM-CSF加IL-4诱导下，体外培养7天后，可从每
中文摘要
只小鼠的骨髓前体细胞中获得5一10xl护个Dc。形态学观察可见，培养早
期的DC突起少而短，胞内有许多囊泡，细胞器少;细胞表面低表达B7分子
和MHC分子，刺激T细胞增殖能力极弱，但吞噬能力强;培养7天时，细
胞呈现典型的DC形态，表面伸出许多树枝样突起，胞内囊泡结构减少，细
胞器成分增加，吞噬功能减弱，但B7分子和MHC分子表达明显上调，能强
烈刺激T细胞增殖。GM一CSF加IL一4组的DC得率及细胞功能均明显高于
GM一CSF组。
3.将RNI一1前列腺癌细胞的裂解产物负载培养5天后的DC，通过DOI人P脂质
体转染法可以成功的将IP一10基因导入Dc，构建IP一10/Lysate一DC前列腺癌
瘤苗;经RpPCR和M尾Stem blot检测证实转染DC的IP一10 mRNA和蛋白表
达明显增强，其细胞上清对T淋巴细胞有显著趋化作用。
4.混合淋巴细胞反应显示，IP一10/Lysate一DC瘤苗刺激同基因型小鼠脾脏T细胞
增殖能力明显提高。四氮吟蓝(MTT)还原法检测显示，用负载RM一1细胞
裂解产物的DC(Lysate一DC)免疫小鼠，其脾脏T细胞经RM一1细胞诱导后，
对RM一1肿瘤细胞具有特异性杀伤作用，但杀伤作用明显低于IP一10/切sate一DC
免疫组小鼠，差异有显著性意义(p<0.01)。
5.RM一1前列腺癌荷瘤小鼠模型构建成功，成瘤率为100%。体内实验显示，
Lysate一DC治疗组小鼠肿瘤生长减慢，生存期延长;应用IP一10/切sate一Dc的
治疗作用较切sate一DC进一步增强，接种RM一1肿瘤细胞75天后，
IP一10/Lysate一Dc治疗组仍有50%的小鼠存活，差异有显著性意义(p<0 .01)。
6，组织学检查发现，经IP一10/Lysate一DC瘤苗治疗的小鼠，肿瘤组织局部坏死明
显，在瘤体周围和瘤体内有大量炎细胞浸润，切sate一DC治疗组也可见较明显
的炎细胞浸润，但少于IP一10/Ly sate一Dc组。
7.免疫保护实验发现，经IP一10/Lysate一DC免疫的小鼠接种R人1一1肿瘤细胞后，
肿瘤生长缓慢，小鼠生存期延长，在RM一1细胞攻击后75天，经
lP一10/切sate一DC免疫的小鼠仍有67%无肿瘤生长;Lysate一DC也具有免疫保
护作用，但明显弱于IP一10/Lysate一DC，免疫小鼠只有17%无肿瘤生长，差异
有显著性意义(p<0 .05)。
8.ELISA检测显示，经IP一10/场sate一DC免疫的小鼠，其CTL的IL一2和IFN一Y
表达水平明显升高，与Lysate一DC免疫小鼠相比，差异有显著性意义(p<0.05)。
结论:
1.所构建的IP一1帅cDNA3.1表达质粒正确。
2.小鼠骨髓单个核细胞在细胞因子GM一CSF和IL一4诱导下，通过手法筛选，能
中文摘要
扩增获得大量功能性DC。GM一CSF是DC生长的必须因子，GM一CSF和IL一4
联合应用，可增加DC的得率，增强DC的免疫功能。
3.通过脂质体转染法将IP一10/P cDNA3.1导入DC，获得有效的IP一10表达，转
染DC的上清对T淋巴细胞有显著趋化作用。
4，RM一1前列腺癌细胞裂解产物和IP一10基因共同修饰DC所构建的前列腺癌
Dc瘤苗(IP一10/切sate一DC)，能够明显刺激T细胞的增殖，所诱导的特异性
cTL分泌IL一2和IFN一Y能力增强，对RM一1细胞具有显著杀伤作用。
5.将IP一10/Lysate一Dc瘤苗回?
Objective:
To construct recombinant plasmid IP-10/pcDNA3.1 by inserted IFN-y inducible protein-10 (IP-10) DNA fragments into pcDNA3.1(+) vector. To establish the methods of isolation, purification and propagation of mouse bone marrow-derived dendritic cell (DC), investigate the changes of morphological structure and bio-immunological characteristics of DC during its development, differentiation and maturation, study the related factors involved in proliferation and polarization of DC. To construct the DC vaccine pulsed with whole tumor cell lysate and modified with IP-10 gene. To detect and analyze the specific anti-tumor activity, the immunotherapeutic function and immune protection of DC vaccine both in vitro and in vivo,
Methods:
The cDNA fragment encoding the murine IP-10 was obtained from mRNA of murine T cells by using Reversal transcript-polymerase chain reaction (RT-PCR) and contained a signal peptide derived from the human monocyte chemoattractant protein 1, then it was inserted into the eukaryotic expression vector pcDNA3.1(+). The mouse bone marrow progenitors were isolated and cultured with cytokines of GM-CSF plus IL-4, and a large amount of BM-DC were harvested after special selection and culture for 7 days. The bio-immunological features and functions of DC in different stages were studied by using methods of light- and electro-microscopy, HRP phagocytosis assay, mixed leucocyte reaction (MLR), flow cytometry (FCM), and so on. Mouse bone marrow DC were pulsed with cell lysate from RM-1 prostate cancer cell line and then transfected with the recombinant plasmid IP-10/pcDNA3.1 by DOTAP liposome. The expression of IP-10 was assayed by Western blot and chemotaxis assay. The ability of stimulating syngeneic T cells and the eff
ect of inducing specific kill activity of DC vaccine were detected. The immunotherapeutic effect of DC vaccine on mice with prostate cancer was assessed. The immunological protection induced by DC vaccine was studied by the challenge of tumor cell injection followed the inoculation.
Results:
1. By partial nucleotide sequencing, the cDNA was consistent with the reported mIP-10 cDNA in the GeneBank, and then was inserted into the eukaryotic expression vector pcDNA3.1(+) to construct recombinant plasmid IP-10/pcDNA3.1.
2. About 5~10X106 DC were harvested from bone marrow progenitors per mouse followed culturing the cells with GM-CSF plus IL-4 for 7 days. In early stage, DC possessed short processes and fewer organelles, but a large numbers of vacuoles in the cytoplasm. They expressed very low level of B7-1, B7-2, MHC class-I and class-II molecules and failed to stimulate allogeneic T cell proliferation, but could pino-phagocytose vigorously HRP granules. After 7 days culture, they exhibited morphologically the distinct dendritic shape with long processes, increased organelle and decreased fraction of vacuoles. Simultaneously, they expressed higher level of surface MHC and B7 molecules and stronger activity of stimulating T cell proliferation.
3. The IP-10 plasmid vector was successfully transfected into DC and the DC tranfected with IP-10 gene were capable of synthesizing and secreting IP-10 chemokine, which could increase the preferential chemotaxis of DC to T cells. The DC vaccine modified with tumor cell lysate and IP-10 gene (IP-10/Lysate-DC) was a more potent stimulator in MLR. The mice immunized with DC pulsed with RM-1 cell lysate (Lysate-DC) exhibited a specific CTL response, but the highest CTL activity against RM-1 cells was induced by immunization with IP-10/Lysate-DC (p<0.01).
4. In the mice model with pre-established subcutaneous RM-1 prostate cancer, vaccination with Lysate-DC could inhibit tumor growth, but the immunization of IP-10/Lysate-DC inhibited the tumor growth most significantly when compared with Lysate-DC (p<0.01). The survival time of the mice treated with IP-10/Lysate-DC was also greatly extended (p<0.01).
5. Histological examination showed that the most obvious tumor nec
引文
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