逆转录病毒载体介导趋化因子MIP-1α和共刺激因子B7-1基因共转染增强抗乳腺癌免疫效应的实验研究
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摘要
机体的抗肿瘤免疫反应主要是由激活的T淋巴细胞介导,T淋巴细胞激活需要两个信号系统,第一信号是由T细胞受体(TCR)与抗原肽-MHC复合物相结合所提供的,第二信号是由辅助分子或共刺激分子通过与T细胞上相应受体结合而传递的,其中以B7分子的共刺激作用最为重要。肿瘤细胞表面往往缺乏或低表达B7分子,是肿瘤细胞逃避宿主免疫系统监视的机制之一。趋化因子可通过趋化免疫活性细胞到达肿瘤部位,还可抑制肿瘤血管生成,产生抗肿瘤效应。本研究通过逆转录病毒载体介导共转染趋化因子MIP-1α基因和共刺激因子B7-1基因,期望通过“招引-活化”的机制,即将免疫细胞聚集到肿瘤局部,并给予激活,达到更强的抗肿瘤效应,为乳腺癌免疫基因治疗提供新的途径。
我们在成功构建的B7-1和MIP-1α基因基础上,应用基因重组技术将MIP-1α和B7-1分别导入带不同选择标记的两种逆转录病毒载体pLXSN和pBabe puro,通过基因转染和克隆筛选等技术,得到了两株分泌不同重组逆转录病毒的包装细胞。通过病毒上清感染、药物筛选等技术得到了MIP-lα和B7-1双基因修饰的大鼠乳腺癌细胞株SHZ-88/mMIP-1α+mB7-1。计数板法绘制细胞生长曲线显示重组逆转录病毒感染对乳腺癌细胞增殖无明显影响。RT-PCR和流式细胞仪分别证实SHZ-88/mMIP-1α+mB7-1有B7-1 mRNA和蛋白水平的表达;RT-PCR、免疫细胞化学分别显示SHZ-88/mMIP-1α+mB7-1有MIP-1αmRNA和蛋白水平的表达。体外趋化实验证实MIP-lα基因修饰的乳腺癌细胞能分泌具有趋化活性的分子;MTT比色法证实B7-1基因修饰大鼠乳腺癌细胞能刺激大鼠脾单个核细胞增殖。动物体内实验表明, MIP-lα和B7-1双基因共修饰乳腺癌细胞的致瘤性消失,能抑制野生型肿瘤细胞的体内生长及延长荷瘤大鼠的生存期,均优于单基因组或对照组(P<0.05)。流式细胞仪及ELISA结果显示MIP-lα和B7-1双基因共修饰乳腺癌细胞接种后,外周血CD4+和CD8+细胞的百分比、CD4+/CD8+比值及IL-2、IFN-γ含量均较单基因组或对照组升高(P<0.05)。HE染色结果显示:SHZ-88/mMIP-1α+mB7-1细胞于肿瘤中心接种后,瘤组织可见大量的淋巴细胞浸润。SHZ-88细胞肿瘤中心注射后只见少量淋巴细胞浸润。肿瘤注射部位对肿瘤体积、CD4+和CD8+细胞的百分比、CD4+/CD8+比值及外周血IL-2、IFN-γ的含量无明显影响(P>0.05)。
本研究得到如下结论:1.通过逆转录病毒载体能介导建立MIP-1α和B7-1基因共表达的大鼠乳腺癌细胞株,具有招引单个核细胞,并将其激活的体外生物学活性。2.共转染MIP-1α和B7-1比MIP-1α或B7-1基因单独转染,能产生更强的抗肿瘤作用,不仅使肿瘤细胞自身不能在动物体内接种成瘤,而且能明显抑制野生型细胞的体内生长及延长荷瘤大鼠的生存期。
Ativated T lymphocytes are a critical component of the immune responseto tumors.They are sufficient to protect against tumors and can eliminate evenestablished cancers in murine tumor models and in humans. At least two sign-als are required to activate naive T lymphocytes. The first signal involves theinteraction of the MHC-peptide with the TCR and CD4 or CD8 receptors onT-lymphocytes. The second signal involves the interaction between costimulatoryfactors such as members of B7 family on the antigen presenting cells (APCs)and CD28 or CTLA-4 on T lymphocytes. Once this second signal is accompli-shed,the first signal will suffice to maintain T-lymphocyte activity. In responseto certain inflammatory cytokines,APCs express costimulatory molecules suchas B7,which is normally expressed on macrophages cells and B lymphocytesand reacts with CD28 to activate T cell. In the absence of the appropriate co-stimulatory signals,engagement of the T cell receptor itself typically leads tounresponsiveness,anergy or apoptosis of the antigen specific T lymphocytes.The human breast carcinoma cells often fail to express B7,thus, the antigenpresented in its absence will be ignored by the immune system. Another impo-rtant aspect of immunity is the recruitment and accumulation of lymphocytesinto taget tissues. Some chemokine family members are chemotactic for selectedpopulations of immunocytes,and may exert indirect effects on antitumor byinhibit angiogenesis. Genetic modification of many types of tumor cells,whichexpress either costimulatory molecules,such as B7-1,or chemokines,such asMIP-lα,can increase the immunogenicity. To augment antitumor effect in vivo, the combination treatment of breast carcinoma cells with costimulatory moleculegenes and chemokine genes mediated by retrovirus was investigated.
The use of retroviral vectors for gene therapy has drawn much attentionand currently is the choice for the transferral of therapeutic genes in a varietyof approved protocols both in the U.S.A and in Europe. We have generatedtwo retroviral constructs that contained one destination gene and one resistancemarker respectively: B7-1 gene or MIP-1αgene,G418 resistance or puromycinresistance. We transfected SHZ-88 cell line with MIP-lαor B7-1 gene respecti-vely. Moreover,we also transfected the tumor cells with both MIP-lαand B7-1genes. By one or two drug screening test,some positive clones,which expressMIP-1αand (or) B7-1,has been obtained in 14 days. None of the transfectants,including the multi-gene tranfectant,was demonstrated that altered grow rateand cell appearance in vitro. The expression of MIP-1αand B7-1 mRNA ingene-transfected SHZ-88 cell was showed by RT-PCR. The expression of B7-1on gene-transfected SHZ-88 cell was showed by flow cytometry; While the ex-pression of MIP-lαon gene-transfected SHZ-88 cell was confirmed by immu-nohistochemistry. When mB7-1 transfected cells were used as stimulators ofallogeneic spleen lymphocytes in mixed lymphocyte-tumor culture(MLTC),MTTassay indicated that T-lymphocyte proliferations were significantly increased.Chemotaxis assay demonstated that mMIP-lαmodified cells can secreted subst-antial levels of chemokine activity for activated rat spleen lymphocytes. Thetumor volumes indicated the injection of MIP-lα-transfected or B7-1-transfectedSHZ-88 cells subcutaneously(s.c.) could inhibit the growth of SHZ-88 cellsand prolonged the animal tumor-bearing life span in vivo compared with thecontrol group (P<0.05). The inhibitory effect was enhanced when the MIP-lα+B7-1 co-transfected SHZ-88 cells were used (P<0.05). Flow cytometry showedthat the level of CD4+、CD8+ and CD4+ / CD8+ ratio in the MIP-lα+ B7-1 gr-oup was significantly higher than that of the single gene-transfected or control group(P<0.05);EILSA showed that the content of serum IL-2 and IFN-γinMIP-lα+B7-1 group was significantly higher than that in the single gene-trans- fected or control group (P<0.05). HE stain showed more lymphocyte infiltrationwere found in the inoculationsites of SHZ-88 / MIP-1α+B7-1 cells than in that ofMIP-lαor B7-1-transfected SHZ-88 cells. These results suggest that the comb-ination of B7-1 gene and MIP-1αgene can augment antitumor effect in vivo.The antitumor effects are not significant difference after gene-transfected cells wereinjected intumor center or opposite side axillary fossa.(P>0.05).
The main conclusions of the in vitro and in vivo experimental studies areas follows:
1. A new rat breast cancer cel1 strain,SHZ-88 / mMIP-1α+mB7-1, whichcould co-express MIP-1α+B7-1 gene stably,was established successfully,pos-sessing biologic activity in vitro;2. The injection of MIP-lα+B7-1 gene transfe-cted SHZ-88 cells could induce stronger antitumor effect than that of B7-1 or MIP-1αgene transfected SHZ-88 cells in vivo.
我们在成功构建的B7-1和MIP-1α基因基础上,应用基因重组技术将MIP-1α和B7-1分别导入带不同选择标记的两种逆转录病毒载体pLXSN和pBabe puro,通过基因转染和克隆筛选等技术,得到了两株分泌不同重组逆转录病毒的包装细胞。通过病毒上清感染、药物筛选等技术得到了MIP-lα和B7-1双基因修饰的大鼠乳腺癌细胞株SHZ-88/mMIP-1α+mB7-1。计数板法绘制细胞生长曲线显示重组逆转录病毒感染对乳腺癌细胞增殖无明显影响。RT-PCR和流式细胞仪分别证实SHZ-88/mMIP-1α+mB7-1有B7-1 mRNA和蛋白水平的表达;RT-PCR、免疫细胞化学分别显示SHZ-88/mMIP-1α+mB7-1有MIP-1αmRNA和蛋白水平的表达。体外趋化实验证实MIP-lα基因修饰的乳腺癌细胞能分泌具有趋化活性的分子;MTT比色法证实B7-1基因修饰大鼠乳腺癌细胞能刺激大鼠脾单个核细胞增殖。动物体内实验表明, MIP-lα和B7-1双基因共修饰乳腺癌细胞的致瘤性消失,能抑制野生型肿瘤细胞的体内生长及延长荷瘤大鼠的生存期,均优于单基因组或对照组(P<0.05)。流式细胞仪及ELISA结果显示MIP-lα和B7-1双基因共修饰乳腺癌细胞接种后,外周血CD4+和CD8+细胞的百分比、CD4+/CD8+比值及IL-2、IFN-γ含量均较单基因组或对照组升高(P<0.05)。HE染色结果显示:SHZ-88/mMIP-1α+mB7-1细胞于肿瘤中心接种后,瘤组织可见大量的淋巴细胞浸润。SHZ-88细胞肿瘤中心注射后只见少量淋巴细胞浸润。肿瘤注射部位对肿瘤体积、CD4+和CD8+细胞的百分比、CD4+/CD8+比值及外周血IL-2、IFN-γ的含量无明显影响(P>0.05)。
本研究得到如下结论:1.通过逆转录病毒载体能介导建立MIP-1α和B7-1基因共表达的大鼠乳腺癌细胞株,具有招引单个核细胞,并将其激活的体外生物学活性。2.共转染MIP-1α和B7-1比MIP-1α或B7-1基因单独转染,能产生更强的抗肿瘤作用,不仅使肿瘤细胞自身不能在动物体内接种成瘤,而且能明显抑制野生型细胞的体内生长及延长荷瘤大鼠的生存期。
Ativated T lymphocytes are a critical component of the immune responseto tumors.They are sufficient to protect against tumors and can eliminate evenestablished cancers in murine tumor models and in humans. At least two sign-als are required to activate naive T lymphocytes. The first signal involves theinteraction of the MHC-peptide with the TCR and CD4 or CD8 receptors onT-lymphocytes. The second signal involves the interaction between costimulatoryfactors such as members of B7 family on the antigen presenting cells (APCs)and CD28 or CTLA-4 on T lymphocytes. Once this second signal is accompli-shed,the first signal will suffice to maintain T-lymphocyte activity. In responseto certain inflammatory cytokines,APCs express costimulatory molecules suchas B7,which is normally expressed on macrophages cells and B lymphocytesand reacts with CD28 to activate T cell. In the absence of the appropriate co-stimulatory signals,engagement of the T cell receptor itself typically leads tounresponsiveness,anergy or apoptosis of the antigen specific T lymphocytes.The human breast carcinoma cells often fail to express B7,thus, the antigenpresented in its absence will be ignored by the immune system. Another impo-rtant aspect of immunity is the recruitment and accumulation of lymphocytesinto taget tissues. Some chemokine family members are chemotactic for selectedpopulations of immunocytes,and may exert indirect effects on antitumor byinhibit angiogenesis. Genetic modification of many types of tumor cells,whichexpress either costimulatory molecules,such as B7-1,or chemokines,such asMIP-lα,can increase the immunogenicity. To augment antitumor effect in vivo, the combination treatment of breast carcinoma cells with costimulatory moleculegenes and chemokine genes mediated by retrovirus was investigated.
The use of retroviral vectors for gene therapy has drawn much attentionand currently is the choice for the transferral of therapeutic genes in a varietyof approved protocols both in the U.S.A and in Europe. We have generatedtwo retroviral constructs that contained one destination gene and one resistancemarker respectively: B7-1 gene or MIP-1αgene,G418 resistance or puromycinresistance. We transfected SHZ-88 cell line with MIP-lαor B7-1 gene respecti-vely. Moreover,we also transfected the tumor cells with both MIP-lαand B7-1genes. By one or two drug screening test,some positive clones,which expressMIP-1αand (or) B7-1,has been obtained in 14 days. None of the transfectants,including the multi-gene tranfectant,was demonstrated that altered grow rateand cell appearance in vitro. The expression of MIP-1αand B7-1 mRNA ingene-transfected SHZ-88 cell was showed by RT-PCR. The expression of B7-1on gene-transfected SHZ-88 cell was showed by flow cytometry; While the ex-pression of MIP-lαon gene-transfected SHZ-88 cell was confirmed by immu-nohistochemistry. When mB7-1 transfected cells were used as stimulators ofallogeneic spleen lymphocytes in mixed lymphocyte-tumor culture(MLTC),MTTassay indicated that T-lymphocyte proliferations were significantly increased.Chemotaxis assay demonstated that mMIP-lαmodified cells can secreted subst-antial levels of chemokine activity for activated rat spleen lymphocytes. Thetumor volumes indicated the injection of MIP-lα-transfected or B7-1-transfectedSHZ-88 cells subcutaneously(s.c.) could inhibit the growth of SHZ-88 cellsand prolonged the animal tumor-bearing life span in vivo compared with thecontrol group (P<0.05). The inhibitory effect was enhanced when the MIP-lα+B7-1 co-transfected SHZ-88 cells were used (P<0.05). Flow cytometry showedthat the level of CD4+、CD8+ and CD4+ / CD8+ ratio in the MIP-lα+ B7-1 gr-oup was significantly higher than that of the single gene-transfected or control group(P<0.05);EILSA showed that the content of serum IL-2 and IFN-γinMIP-lα+B7-1 group was significantly higher than that in the single gene-trans- fected or control group (P<0.05). HE stain showed more lymphocyte infiltrationwere found in the inoculationsites of SHZ-88 / MIP-1α+B7-1 cells than in that ofMIP-lαor B7-1-transfected SHZ-88 cells. These results suggest that the comb-ination of B7-1 gene and MIP-1αgene can augment antitumor effect in vivo.The antitumor effects are not significant difference after gene-transfected cells wereinjected intumor center or opposite side axillary fossa.(P>0.05).
The main conclusions of the in vitro and in vivo experimental studies areas follows:
1. A new rat breast cancer cel1 strain,SHZ-88 / mMIP-1α+mB7-1, whichcould co-express MIP-1α+B7-1 gene stably,was established successfully,pos-sessing biologic activity in vitro;2. The injection of MIP-lα+B7-1 gene transfe-cted SHZ-88 cells could induce stronger antitumor effect than that of B7-1 or MIP-1αgene transfected SHZ-88 cells in vivo.
引文
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[1] 胡锦跃,朱建高,李官成,等.趋化因子 MCP-3 基因和 B7 基因共转染诱导抗大肠癌主动免疫[J].中国肿瘤生物治疗杂志,2002, 9(2):77-81.
[2] Sica GL,Choi IH,Zhu G,et al.B7-H4,a molecule of the B7 family,negativelyregulates T cell immunity[J].Immunity,2003,18(6):849-861.
[3] He YF,Zhang GM,Wang YH,et al. Blocking programmed death-1 ligand PD-155interactions by local gene therapy results in enhancement of antitumor effe-ct of secondary lymphoid tissue chemokine[J].Immunol,2004,173(8):4919-4928.
[4] Tahiro Shin,Kiyoshi Yoshimura,Takako Shin,et al.In vivo costimulatory roleof B7-DC in tuning T helper cell 1 and cytotoxic T lymphocyte responses [J].JEM,2005,201(10):1531-1541.
[5] Kaufman HL,Deraffele G,Mitcham J,et al.Targeting the local tumor microe-nvironment with vaccinia virus expressing B7.1 for the treatment of melano-ma[J].J Clin Invest,2005,115(7):1903-1912.
[6] Kurt RA,Baher A,Wisner KP,et a1.Chemokine receptor desensitization in tu-mor-bearing mice[J].Cell lmmunol,2001,207(2):81-88.
[7] Curiel TJ,Coukos G,Zou L,et al.Specific recruitment of regulatory T cells inovarian carcinoma fosters immune privilege and predicts reduced survival[J].Nat Med,2004,l0(9):942-949.
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[1] 胡锦跃,朱建高,李官成,等.趋化因子 MCP-3 基因和 B7 基因共转染诱导抗大肠癌主动免疫[J].中国肿瘤生物治疗杂志,2002, 9(2):77-81.
[2] Sica GL,Choi IH,Zhu G,et al.B7-H4,a molecule of the B7 family,negativelyregulates T cell immunity[J].Immunity,2003,18(6):849-861.
[3] He YF,Zhang GM,Wang YH,et al. Blocking programmed death-1 ligand PD-155interactions by local gene therapy results in enhancement of antitumor effe-ct of secondary lymphoid tissue chemokine[J].Immunol,2004,173(8):4919-4928.
[4] Tahiro Shin,Kiyoshi Yoshimura,Takako Shin,et al.In vivo costimulatory roleof B7-DC in tuning T helper cell 1 and cytotoxic T lymphocyte responses [J].JEM,2005,201(10):1531-1541.
[5] Kaufman HL,Deraffele G,Mitcham J,et al.Targeting the local tumor microe-nvironment with vaccinia virus expressing B7.1 for the treatment of melano-ma[J].J Clin Invest,2005,115(7):1903-1912.
[6] Kurt RA,Baher A,Wisner KP,et a1.Chemokine receptor desensitization in tu-mor-bearing mice[J].Cell lmmunol,2001,207(2):81-88.
[7] Curiel TJ,Coukos G,Zou L,et al.Specific recruitment of regulatory T cells inovarian carcinoma fosters immune privilege and predicts reduced survival[J].Nat Med,2004,l0(9):942-949.
[8] 吴宜林,陶光实,陆琳,等.趋化因子 MCP-3 协同 B7 诱导小鼠抗宫颈癌的主动免疫效果[J].中国肿瘤生物治疗杂志,2001,8(2):98-101.
[9] Delman KA,Zager JS,Bennett JJ,et al.Efficacy of multiagent herpes simplexvirus amplicon-mediated immunotherapy as adjuvant treatment for experimen-tal hepatic cancer[J].Ann Surg,2002,236(3):337-343..
[10] He YF,Zhang GM,Wang XH,et al.Blocking Programmed Death-1 Ligand-PD-1Interactions by local gene therapy results in enhancement of antitumor eff-ect of secondary lymphoid tissue chemokine[J].J Immunol,2004,173(8):4919-4928.
[11] Strieter RM,Belperio JA,Phillips RJ,et al.CXC chemokines in angiogenesis of cancer[J].Semin Cancer Biol,2004,14(3):195-200.
[12] Ben-Baruch A.The multifaceted roles of chemokines in malignancy[J].CancerMetastasis Rev,2006,25(3):357-371.
[13] Goldberg-Bittman L,Sagi-Assif O,Meshel T,et al.Cellular characteristics of neuroblastoma cells:Regulation by the ELR-CXC chemokine CXCL10 andexpression of a CXCR3-like receptor[J].Cytokine,2005,29(3)105-117.
[14] Conti I, Rollins BJ.CCL2 (monocyte chemoattractant protein-1) and cancer[J].Semin Cancer Biol,2004,14(3):149-154.
[15] Suyama T,Furuya M,Nishiyama M,et al.Up-regulation of the interferon gam-ma (IFN-gamma)-inducible chemokines IFN-inducible T-cell alpha chemoatt-ractant and monokine induced by IFN-gamma and of their receptor CXCreceptor 3 in human renal cell carcinoma[J].Cancer,2005,103(2):258-267.
[16] Adler EP,Lemken CA,Katchen NS,et al.A dual role for tumor-derived che-mokine RANTES (CCL5)[J].Immunology Lett,2003,90(2-3),187-194.
[17] Okada N,Gao JQ,Sasaki A,et al.Anti-tumor activity of chemokine is affectedby both kinds of tumors and the activation state of the host’s immunesystem:implications for chemokine-based cancer immunotherapy[J]. BiochemBiophys Res Commun,2004,317(1):68-76.