MICA基因修饰的口腔鳞癌细胞疫苗诱导抗肿瘤免疫应答的实验研究
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摘要
目的
研究MHC-Ⅰ类链相关基因A修饰的口腔鳞癌细胞疫苗对免疫重建荷瘤SCID鼠皮下移植瘤生长的抑制作用及诱导机体抗肿瘤免疫应答的有效性,并探讨其可能的作用机制。
方法
密度梯度离心法分离人外周静脉血淋巴细胞(hu-PBL),腹腔注射至已用环磷酰胺抑制骨髓造血功能的SCID鼠,建立人免疫功能重建Hu-PBL/SCID动物模型,人IgG ELISA检测试剂盒检测鼠外周血清中人IgG含量以评价重建效果。
60Co放射源致死剂量照射转染pEGFP-N1-MICA真核表达质粒并稳定过表达MICA的Tb-pEGFP-N1-MICA细胞,并以转染空白质粒的Tb-pEGFP-N1细胞及未转染的Tca8113-Tb细胞作为对照,制备灭活肿瘤细胞疫苗,分别腹腔注射接种免疫功能重建Hu-PBL/SCID鼠,共3次。2周后皮下接种Tca8113-Tb细胞,观察成瘤情况,测量肿瘤体积及重量,计算抑瘤率。
取SCID鼠外周血及脾脏,流式细胞术检测外周血单个核细胞(PBMC)和脾细胞表面NKG2D的表达,LDH释放法检测PBMC和脾细胞对肿瘤细胞的杀伤活性。
采用SPSS16.0软件包两独立样本t检测或方差分析对数据进行统计学处理,P<0.05为差异具有统计学意义。
结果
1.SCID鼠腹腔注射hu-PBL免疫重建1周后,其外周血清中即能检测到人IgG,且在5周内随时间的延长而升高。
2.接种灭活肿瘤细胞疫苗的Hu-PBL/SCID鼠皮下注射肿瘤细胞后均能顺利成瘤,成瘤率100%。但Tb-pEGFP-N1-MICA疫苗组与Tca8113-Tb和Tb-pEGFP-N1疫苗组比较,移植瘤体积与重量均明显较少(P<0.05),抑瘤率为63.26%。
3.流式细胞术及LDH释放法检测结果显示Tb-pEGFP-N1-MICA疫苗组Hu-PBL/SCID鼠PBMC和脾细胞表面NKG2D的表达及杀伤活性均显著高于Tca8113-Tb和Tb-pEGFP-N1疫苗组,差异有统计学意义(P<0.05)。
结论
1.SCID鼠腹腔注射细胞密度为2.5×108/mL的Hu-PBL 0.2mL后,其外周血清中可持续检测到人IgG,并随时间延长而增高,表明成功建立了免疫功能重建的hu-PBL/SCID动物模型。
2.MICA基因修饰的口腔鳞癌细胞疫苗能诱导机体更为强烈的抗肿瘤免疫应答能力,对移植瘤生长具有显著的抑制作用。其机制可能是上调了免疫效应细胞NKG2D的表达,从而促进对肿瘤靶细胞的杀伤。
Objective
To investigate the inhibition effect of tumor growing and the potency of MHC class I chain-related gene A (MICA) modified oral squamous cell carcinoma cells vaccine to bearing tumor SCID mice.
Methods
Hu-PBL was separated by density gradient centrifugation method, and then was injected into abdominal cavity of SCID mice whose hematopoietic function of bone marrow was inhibited by Cyclophosphamide to build the hu-PBL/SCID model. Then the level of human IgG in the sera of hu-PBL/SCID mice was detected with ELISA Kit to judge the effect of immunologic reconstruction.
The Tb-pEGFP-N1-MICA cells transfected the pEGFP-N1-MICA plasmid and overexpressed MICA were irradiated with 60Co radioactive source in fatal dose to prepare the inactivated tumor cell vaccine, the cells transfected blank plasmid and no transfection were irradiated as control. Then the vaccine was injected into abdominal cavity of SCID mice to reconstruct the immunologic function. Two weeks later, the tumor bearing hu-PBL/SCID model was establish by subcutaneous injection of the Tca8113-Tb cells, then the growth of tumor was observed, the volume and weight of tumor was measured to research the inhibition of tumor with tumor cell vaccine. And the rate of tumour inhibition was calculated.
The expression of NKG2D and the cytotoxicity in vitro to Tb cells of peripheral blood mononuclear cells (PBMCs) and spleen cells were measured by flow cytometry and lactate dehydrogenase (LDH) release assay.
The data was analyzed with SPSS 16.0 software package,it is significant when P<0.05.
Results
1. Human IgG can be detected in mice's peripheral sera one week after the the hu-PBL/SCID model was built,and it kept rising in 5 weeks.
2. The tumor mass could be touched in all of the Hu-PBL/SCID mice after inoculating the inactivated vaccine,the tumor formation rate is 100%. Compared with the Tca8113-Tb and the Tb-pEGFP-N1 group,the volume and weight of transplantable tumor in Tb-pEGFP-N1-MICA group decreased obviously (P<0.05).The rate of tumour inhibition is 63.26%.
3. The results of flow cytometry and LDH release assay suggested that the expression of NKG2D and the cytotoxicity in Tb-pEGFP-N1-MICA group was higer than that of the Tb and Tb-pEGFP-N1 group, the differences was statically significant (P<0.05)
Conclusion
1. After injecting 0.2mL Hu-PBL the density of which is 2.5×108/mL, into abdominal cavity of SCID mice,human IgG can be detected in its peripheral sera and it kept rising, suggested that we successed to establish the hu-PBL/SCID model.
2. The MICA gene modified oral squamous cell carcinoma vaccine can enhance the ability of antitumor immune response. The mechanism may be up-regulated NKG2D and promoted the proliferation,then improved its cytotoxicity effect in tumor target cells.
研究MHC-Ⅰ类链相关基因A修饰的口腔鳞癌细胞疫苗对免疫重建荷瘤SCID鼠皮下移植瘤生长的抑制作用及诱导机体抗肿瘤免疫应答的有效性,并探讨其可能的作用机制。
方法
密度梯度离心法分离人外周静脉血淋巴细胞(hu-PBL),腹腔注射至已用环磷酰胺抑制骨髓造血功能的SCID鼠,建立人免疫功能重建Hu-PBL/SCID动物模型,人IgG ELISA检测试剂盒检测鼠外周血清中人IgG含量以评价重建效果。
60Co放射源致死剂量照射转染pEGFP-N1-MICA真核表达质粒并稳定过表达MICA的Tb-pEGFP-N1-MICA细胞,并以转染空白质粒的Tb-pEGFP-N1细胞及未转染的Tca8113-Tb细胞作为对照,制备灭活肿瘤细胞疫苗,分别腹腔注射接种免疫功能重建Hu-PBL/SCID鼠,共3次。2周后皮下接种Tca8113-Tb细胞,观察成瘤情况,测量肿瘤体积及重量,计算抑瘤率。
取SCID鼠外周血及脾脏,流式细胞术检测外周血单个核细胞(PBMC)和脾细胞表面NKG2D的表达,LDH释放法检测PBMC和脾细胞对肿瘤细胞的杀伤活性。
采用SPSS16.0软件包两独立样本t检测或方差分析对数据进行统计学处理,P<0.05为差异具有统计学意义。
结果
1.SCID鼠腹腔注射hu-PBL免疫重建1周后,其外周血清中即能检测到人IgG,且在5周内随时间的延长而升高。
2.接种灭活肿瘤细胞疫苗的Hu-PBL/SCID鼠皮下注射肿瘤细胞后均能顺利成瘤,成瘤率100%。但Tb-pEGFP-N1-MICA疫苗组与Tca8113-Tb和Tb-pEGFP-N1疫苗组比较,移植瘤体积与重量均明显较少(P<0.05),抑瘤率为63.26%。
3.流式细胞术及LDH释放法检测结果显示Tb-pEGFP-N1-MICA疫苗组Hu-PBL/SCID鼠PBMC和脾细胞表面NKG2D的表达及杀伤活性均显著高于Tca8113-Tb和Tb-pEGFP-N1疫苗组,差异有统计学意义(P<0.05)。
结论
1.SCID鼠腹腔注射细胞密度为2.5×108/mL的Hu-PBL 0.2mL后,其外周血清中可持续检测到人IgG,并随时间延长而增高,表明成功建立了免疫功能重建的hu-PBL/SCID动物模型。
2.MICA基因修饰的口腔鳞癌细胞疫苗能诱导机体更为强烈的抗肿瘤免疫应答能力,对移植瘤生长具有显著的抑制作用。其机制可能是上调了免疫效应细胞NKG2D的表达,从而促进对肿瘤靶细胞的杀伤。
Objective
To investigate the inhibition effect of tumor growing and the potency of MHC class I chain-related gene A (MICA) modified oral squamous cell carcinoma cells vaccine to bearing tumor SCID mice.
Methods
Hu-PBL was separated by density gradient centrifugation method, and then was injected into abdominal cavity of SCID mice whose hematopoietic function of bone marrow was inhibited by Cyclophosphamide to build the hu-PBL/SCID model. Then the level of human IgG in the sera of hu-PBL/SCID mice was detected with ELISA Kit to judge the effect of immunologic reconstruction.
The Tb-pEGFP-N1-MICA cells transfected the pEGFP-N1-MICA plasmid and overexpressed MICA were irradiated with 60Co radioactive source in fatal dose to prepare the inactivated tumor cell vaccine, the cells transfected blank plasmid and no transfection were irradiated as control. Then the vaccine was injected into abdominal cavity of SCID mice to reconstruct the immunologic function. Two weeks later, the tumor bearing hu-PBL/SCID model was establish by subcutaneous injection of the Tca8113-Tb cells, then the growth of tumor was observed, the volume and weight of tumor was measured to research the inhibition of tumor with tumor cell vaccine. And the rate of tumour inhibition was calculated.
The expression of NKG2D and the cytotoxicity in vitro to Tb cells of peripheral blood mononuclear cells (PBMCs) and spleen cells were measured by flow cytometry and lactate dehydrogenase (LDH) release assay.
The data was analyzed with SPSS 16.0 software package,it is significant when P<0.05.
Results
1. Human IgG can be detected in mice's peripheral sera one week after the the hu-PBL/SCID model was built,and it kept rising in 5 weeks.
2. The tumor mass could be touched in all of the Hu-PBL/SCID mice after inoculating the inactivated vaccine,the tumor formation rate is 100%. Compared with the Tca8113-Tb and the Tb-pEGFP-N1 group,the volume and weight of transplantable tumor in Tb-pEGFP-N1-MICA group decreased obviously (P<0.05).The rate of tumour inhibition is 63.26%.
3. The results of flow cytometry and LDH release assay suggested that the expression of NKG2D and the cytotoxicity in Tb-pEGFP-N1-MICA group was higer than that of the Tb and Tb-pEGFP-N1 group, the differences was statically significant (P<0.05)
Conclusion
1. After injecting 0.2mL Hu-PBL the density of which is 2.5×108/mL, into abdominal cavity of SCID mice,human IgG can be detected in its peripheral sera and it kept rising, suggested that we successed to establish the hu-PBL/SCID model.
2. The MICA gene modified oral squamous cell carcinoma vaccine can enhance the ability of antitumor immune response. The mechanism may be up-regulated NKG2D and promoted the proliferation,then improved its cytotoxicity effect in tumor target cells.
引文
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