IL-12锚定修饰exosomes的肾癌疫苗的制备及体外抗肿瘤效应研究
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摘要
第一部分:肾癌源性exosome的提取及鉴定
目的:分离纯化肾癌细胞分泌的exosome,检测exosome免疫相关蛋白的组成,探讨其潜在的临床应用价值。
方法:体外培养人肾癌细胞株RC-2。用超滤和蔗糖/重水密度梯度超速离心法分离纯化exosome,透射电镜观察其形态,Western blot检测其免疫相关蛋白的组成。免疫细胞化学检测肾癌细胞肾癌特异性抗原G250的表达。
结果:用超滤和蔗糖/重水密度梯度超速离心法成功分离出exosome。透射电镜下肾癌源性exosome为类圆碟形,由脂质双分子层包绕的半球体,直径在30～80nm之间。Western blot检测:肾癌源性exosome表达肾癌特异性抗原G250、热休克蛋白70(Heat Shock Protein70,HSP70)和细胞间粘附分子(Intercellular Adhesion Molecule-1,ICAM-1)。免疫细胞化学检测示:人肾癌细胞RC-2高表达肾癌特异性抗原G250。
结论:超滤和蔗糖/重水密度梯度超速离心法可以分离纯化肾癌细胞分泌的exosome,exosome表达免疫相关蛋白HSP70、ICAM-1,其负载肾癌特异性抗原G250,具有免疫原性,可用于制备肾癌疫苗。
第二部分人白细胞介素12基因与GPI信号肽序列融合基因的真核双表达质粒的构建及鉴定
目的:将糖基化磷脂酰肌醇(Glycosylphosphatidylinositol,GPI)信号肽序列与人白细胞介素12基因拼接成融合基因,构建该融合基因的真核双表达质粒。
方法:将人白细胞介素12(IL-12)的P35亚基基因亚克隆至pBudCE4.1质粒,获得重组质粒pBudCE4.1/IL-12A。将人胎盘碱性磷酸酶-1(hPLAP-1)C末端信号肽序列与IL-12的P40亚基基因进行拼接,将融合基因亚克隆至重组质粒pBudCE4.1/IL-12A,获得重组质粒pBudCE4.1/IL-12A/IL-12B-GPI。酶切及测序鉴定质粒。
结果:用KpnⅠ和XhoⅠ双酶切pBudCE4.1/IL-12A质粒,可切下约762bp的目的条带,测序正确。将重组质粒pBudCE4.1/IL-12A/IL-12B -GPI用KpnⅠ、XhoⅠ、BamHⅠ和XbaⅠ酶切,可切下约762bp和1104两个的目的条带,分别是IL-12A链和IL-12B-GPI。测序结果未发生基因突变,与GenBank的编码序列对比,显示碱基序列正确。。
结论:真核双表达质粒pBudCE4.1/IL-12A/IL-12B-GPI的成功构建,为进一步应用IL-12锚定修饰exosome奠定了基础。
第三部分IL-12锚定修饰exosome的肾癌疫苗的制备及鉴定?
目的:制备IL-12锚定修饰exosome (exosome-GPI-IL-12,EXO/IL-12)的肾癌疫苗及其蛋白组成和功能鉴定。
方法:将糖基化磷脂酰肌醇信号肽序列与白介素12基因融合构建真核双表达质粒pBudCE4.1/IL-12A/IL-12B-GPI转染肾癌细胞,获得稳定转染细胞株。激光共聚焦显微镜和磷脂酰特异性磷脂酶C(PI-PLC)洗脱的流式细胞仪检测目的蛋白(GPI-IL-12)在肾癌细胞的表达。用超滤和蔗糖/重水密度梯度离心法从稳定转染细胞株培养上清液中制备疫苗EXO/IL-12。透射电镜鉴定其形态,Western blot鉴定EXO/IL-12的标志性分子HSP70、ICAM-1的组成,以及肾癌特异性抗原G250和目的蛋白GPI-IL-12的表达。ELISA测定EXO/IL-12负载IL-12的量。IFN-γ释放实验和CFSE标记淋巴细胞的增殖实验检测膜结合的IL-12的功能。
结果:激光共聚焦显微镜示:稳定转染的肾癌细胞株高表达目的蛋白GPI-IL-12。PI-PLC洗脱的流式细胞仪检测示:IL-12是以锚定蛋白的形式结合在肾癌细胞膜表面。从稳定转染细胞株培养上清液中可以分离纯化出EXO/IL-12疫苗,为类圆碟形、双层膜结构,直径30～80nm,高表达HSP70、ICAM-1、G250和GPI-IL-12。ELISA测定10μg的EXO/IL-12含约80±9.6pg/ml的IL-12。10μg的EXO/IL-12在体外能诱导淋巴细胞分泌IFN-γ的量为174.3±14.6pg/ml ,与exosome的40.7±9.5pg/ml相比,差异显著(P<0.01)。并且,EXO/IL-12在体外能显著诱导淋巴细胞的增殖,其增殖指数为1.45±0.015,与exosome(1.29±0.01)相比,差异显著(P<0.01)。
结论:pBudCE4.1/IL-12A/IL-12B-GPI质粒稳定转染肾癌细胞能制备EXO/IL-12疫苗,该疫苗表达GPI-IL-12,负载肾癌特异性抗原G250,能在体外显著诱导T淋巴细胞的增殖及分泌IFN-γ。结果表明:膜结合的IL-12具有生物学功能,经IL-12锚定修饰的exosome,其免疫原性显著提高。
第四部分IL-12锚定修饰肾癌源性exosome疫苗在体外抗肿瘤效应的研究
目的:探讨EXO/IL-12疫苗在体外诱导淋巴细胞对肾癌细胞的杀伤效应。
方法:从健康自愿者外周血中分离培养淋巴细胞和树突状细胞,扫描电镜鉴定树突状细胞的形态。将树突状细胞负载的EXO/IL-12为刺激因子与淋巴细胞体外共培养48小时,刺激活化的淋巴细胞为效应细胞,以CFSE预标记肾癌细胞为靶细胞,按不同的效-靶比加入,共培养48小时后,用CFSE/PI双标的流式细胞仪分析其对肾癌细胞的杀伤效应,以PBS、EXO、IL-12为对照。选择不同的CFSE预标记靶细胞(人肾癌细胞、人膀胱癌细胞和人结肠癌细胞),用CFSE/PI双标的流式细胞仪检测其杀伤率差异,探讨EXO/IL-12诱导淋巴细胞的杀伤效应是否具有抗原特异性。
结果:结果显示:在效-靶比为50:1时, IL-12诱导活化的淋巴细胞对肾癌细胞的杀伤率为10.34±3.92%,高于用PBS组的0.7±0.26%(P<0.05);EXO组诱导活化的T淋巴细胞对肾癌细胞的杀伤率为24.3±3.8%,明显高于IL-12组和PBS组,差异显著(P<0.01)。EXO/IL-12诱导的淋巴细胞对靶细胞的杀伤作用最强,杀伤率达53.7±3.9%,与各组比较,差异显著(P<0.01)。并且,EXO/IL-12活化的淋巴细胞对肾癌细胞RC-2的杀伤率明显高于对膀胱癌细胞T-24和结肠癌细胞SW480(杀伤率分别为9.8±2.5%和10.8±2.1%),差异有显著性意义(P<0.01)
结论:EXO/IL-12疫苗能在体外诱导淋巴细胞产生较强的抗原特异性细胞毒作用,可望成为治疗肾癌的新疫苗。
PART ONE Separation and identification of exosomes derived from renal cancer cells and primary study of protein composition
Objective:To separate and purify exosomes derived from renal cancer cells and detect their immune associated protein in order to investigate their potential clinical value.
Methods:Exosomes derived from RC-2 cells culture supematants were separated and purified by ultrafiltration and sucrose gradient centrifugation, which were identified by transmission electron microscope. The expressions of HSP70, ICAM-1 and G250 were detected by western blot.
Results:Exosomes were successfully purified by ultrafiltration and sucrose gradient centrifugation, which were 30~80 nm in diameter with typical saucer-shape morphology, and expressed HSP70、ICAM-1 and G250 antigen.
Conclusion:Ultrafiltration and sucrose gradient ultracentrifugation can be used to purify renal cancer cells-secreted exosomes. Exosomes, expressing HSP70, ICAM-1 and G250, might be the novel vaccine for renal cell cancer.
PART TWO Construction of mammalian co-expression plasmid of chemric IL-12-GPI cDNA
Objective:To construct a mammalian co-expression plasmid of pBudCE4.1/IL-12A/IL-12B-GPI.
Methods:A mammalian co-expression plasmid of GPI-anchor- IL-12 (human) were constructed by subcloning IL-12A (P35) gene and a fusion gene containing GPI-anchor signal sequence of hPLAP-1 and IL-12B (P40) in pBudCE4.1. .
Results:Plasmid pBudCE4.1/IL-12A was digested with KpnI and XhoI, and resulted in band of IL-12A-chain(762 bp). The correct sequence was identified by DNA sequencing. The recombinant plasmids pBudCE4.1/IL-12A/IL-12B-GPI were digested with KpnI, XhoI, BamHI andXbaI, and resulted in two bands, 1104 and 762 bp, named IL-12A-chain and IL-12B-GPI, respectively. The correct sequence was identified by DNA sequencing, showing that co-expression plasmid of pBud-CE4.1/IL-12A/IL-12B-GPI was successfully constructed.
Conclusion The recombinant plasmid may provide foundation for further preparation of tumor vaccine.
PART THREE The preparation and identification of renal-cancer vaccine of exosomes
Objective:To prepare IL-12-anchored exosomes(EXO/IL-12) derived from renal cancer cells and identify their structure and function.
Methods:RC-2 transfectants expressing GPI-IL-12 (RC-2-GPI-IL-12) were established by transfecting pBudCE4.1/IL-12A /IL-12B-GPI plasmid. Confocal laser scanning microscopy and flow cytometry analyzed the expression of fusion protein. TEM and Western blot identified the morphology and characteristic molecules of exosomes separated by ultrafiltration and sucrose gradient centrifugation. The function of anchored IL-12 exosomes was determined by IFN-γrelease assay and CFSE-based cell proliferation.
Results:Confocal laser scanning microscopy and flow cytometry analysis of the RC-2-GPI-IL-12 transfectants showed expression of IL-12 on the cell surface via a GPI-moiety. EXO/IL-12 were purified by ultrafiltration and sucrose gradient centrifugation, which were 30~80 nm in diameter with typical saucer-shape morphology, and expressing HSP70, ICAM-1, G250 and GPI-IL-12. 80±9.6pg/ml of IL-12 was detected in 10μg EXO/IL-12 and it significantly induced cell proliferation and the release of IFN-γ.
Conclusion:The vaccine of EXO/IL-12 can be obtained from the culture supernatant of IL-12-anchored RC-2 cells, exprssing IL-12 and tumor associated antigen G250, could efficiently increase induciont of cell proliferation and the release of IFN-γof T lymphocytes in vitro.
PART FOUR EXO/IL-12 increases induction of specific antitumor response in vitro:
Objective:To evaluate antitumor effect of EXO/IL-12 in vitro.
Methods:Lymphocytes and dendritic cells were isolated from human peripheral blood mononuclear for cytotoxicity assay. The morphology of DC was identified by SEM. Exosome-loaded DCs promoted activation of autologous T cells used as effector cells. Autologous DCs and T cells were co-inoculated with EXO/IL-12 (10μg) for 48h, PBS, IL-12, EXO was used as control. Target cells were RC-2, T-24 (bladder cancer cell line) or SW480 (colon cancer cell line). Cytotoxicity assays based on PI costaining of CFSE-labeled target cells were performed. All cells were harvested and resuspended in PBS and analyzed by flow cytometry. Killing rate was assessed by the percentage of PI- costaining cells out of gated CFSE-labeled cells.
Results:The results showed that initiation by EXO and EXO/IL-12 efficiently killed tumor cells as compared with PBS and IL-12 group (P<0.01); however, initiation by EXO/IL-12 exhibited the highest killing rate (P<0.01). To further explore whether EXO/IL-12 could induce antigen-specific cytotoxic effect, we used T-24, SW480 and RC-2 as target cells and evaluated the difference in killing rate. These results suggested that exosomes derived from renal cancer cell could activate T cells in antigen-specific manner through DCs, resulting in more efficient killing of renal cancer cells as compared with T-24 and sw480 cells (P<0.01).
Conclusion:The vaccine of EXO/IL-12 could more effectively increase induction of antigen-specific antitumor immune response in vitro.
目的:分离纯化肾癌细胞分泌的exosome,检测exosome免疫相关蛋白的组成,探讨其潜在的临床应用价值。
方法:体外培养人肾癌细胞株RC-2。用超滤和蔗糖/重水密度梯度超速离心法分离纯化exosome,透射电镜观察其形态,Western blot检测其免疫相关蛋白的组成。免疫细胞化学检测肾癌细胞肾癌特异性抗原G250的表达。
结果:用超滤和蔗糖/重水密度梯度超速离心法成功分离出exosome。透射电镜下肾癌源性exosome为类圆碟形,由脂质双分子层包绕的半球体,直径在30～80nm之间。Western blot检测:肾癌源性exosome表达肾癌特异性抗原G250、热休克蛋白70(Heat Shock Protein70,HSP70)和细胞间粘附分子(Intercellular Adhesion Molecule-1,ICAM-1)。免疫细胞化学检测示:人肾癌细胞RC-2高表达肾癌特异性抗原G250。
结论:超滤和蔗糖/重水密度梯度超速离心法可以分离纯化肾癌细胞分泌的exosome,exosome表达免疫相关蛋白HSP70、ICAM-1,其负载肾癌特异性抗原G250,具有免疫原性,可用于制备肾癌疫苗。
第二部分人白细胞介素12基因与GPI信号肽序列融合基因的真核双表达质粒的构建及鉴定
目的:将糖基化磷脂酰肌醇(Glycosylphosphatidylinositol,GPI)信号肽序列与人白细胞介素12基因拼接成融合基因,构建该融合基因的真核双表达质粒。
方法:将人白细胞介素12(IL-12)的P35亚基基因亚克隆至pBudCE4.1质粒,获得重组质粒pBudCE4.1/IL-12A。将人胎盘碱性磷酸酶-1(hPLAP-1)C末端信号肽序列与IL-12的P40亚基基因进行拼接,将融合基因亚克隆至重组质粒pBudCE4.1/IL-12A,获得重组质粒pBudCE4.1/IL-12A/IL-12B-GPI。酶切及测序鉴定质粒。
结果:用KpnⅠ和XhoⅠ双酶切pBudCE4.1/IL-12A质粒,可切下约762bp的目的条带,测序正确。将重组质粒pBudCE4.1/IL-12A/IL-12B -GPI用KpnⅠ、XhoⅠ、BamHⅠ和XbaⅠ酶切,可切下约762bp和1104两个的目的条带,分别是IL-12A链和IL-12B-GPI。测序结果未发生基因突变,与GenBank的编码序列对比,显示碱基序列正确。。
结论:真核双表达质粒pBudCE4.1/IL-12A/IL-12B-GPI的成功构建,为进一步应用IL-12锚定修饰exosome奠定了基础。
第三部分IL-12锚定修饰exosome的肾癌疫苗的制备及鉴定?
目的:制备IL-12锚定修饰exosome (exosome-GPI-IL-12,EXO/IL-12)的肾癌疫苗及其蛋白组成和功能鉴定。
方法:将糖基化磷脂酰肌醇信号肽序列与白介素12基因融合构建真核双表达质粒pBudCE4.1/IL-12A/IL-12B-GPI转染肾癌细胞,获得稳定转染细胞株。激光共聚焦显微镜和磷脂酰特异性磷脂酶C(PI-PLC)洗脱的流式细胞仪检测目的蛋白(GPI-IL-12)在肾癌细胞的表达。用超滤和蔗糖/重水密度梯度离心法从稳定转染细胞株培养上清液中制备疫苗EXO/IL-12。透射电镜鉴定其形态,Western blot鉴定EXO/IL-12的标志性分子HSP70、ICAM-1的组成,以及肾癌特异性抗原G250和目的蛋白GPI-IL-12的表达。ELISA测定EXO/IL-12负载IL-12的量。IFN-γ释放实验和CFSE标记淋巴细胞的增殖实验检测膜结合的IL-12的功能。
结果:激光共聚焦显微镜示:稳定转染的肾癌细胞株高表达目的蛋白GPI-IL-12。PI-PLC洗脱的流式细胞仪检测示:IL-12是以锚定蛋白的形式结合在肾癌细胞膜表面。从稳定转染细胞株培养上清液中可以分离纯化出EXO/IL-12疫苗,为类圆碟形、双层膜结构,直径30～80nm,高表达HSP70、ICAM-1、G250和GPI-IL-12。ELISA测定10μg的EXO/IL-12含约80±9.6pg/ml的IL-12。10μg的EXO/IL-12在体外能诱导淋巴细胞分泌IFN-γ的量为174.3±14.6pg/ml ,与exosome的40.7±9.5pg/ml相比,差异显著(P<0.01)。并且,EXO/IL-12在体外能显著诱导淋巴细胞的增殖,其增殖指数为1.45±0.015,与exosome(1.29±0.01)相比,差异显著(P<0.01)。
结论:pBudCE4.1/IL-12A/IL-12B-GPI质粒稳定转染肾癌细胞能制备EXO/IL-12疫苗,该疫苗表达GPI-IL-12,负载肾癌特异性抗原G250,能在体外显著诱导T淋巴细胞的增殖及分泌IFN-γ。结果表明:膜结合的IL-12具有生物学功能,经IL-12锚定修饰的exosome,其免疫原性显著提高。
第四部分IL-12锚定修饰肾癌源性exosome疫苗在体外抗肿瘤效应的研究
目的:探讨EXO/IL-12疫苗在体外诱导淋巴细胞对肾癌细胞的杀伤效应。
方法:从健康自愿者外周血中分离培养淋巴细胞和树突状细胞,扫描电镜鉴定树突状细胞的形态。将树突状细胞负载的EXO/IL-12为刺激因子与淋巴细胞体外共培养48小时,刺激活化的淋巴细胞为效应细胞,以CFSE预标记肾癌细胞为靶细胞,按不同的效-靶比加入,共培养48小时后,用CFSE/PI双标的流式细胞仪分析其对肾癌细胞的杀伤效应,以PBS、EXO、IL-12为对照。选择不同的CFSE预标记靶细胞(人肾癌细胞、人膀胱癌细胞和人结肠癌细胞),用CFSE/PI双标的流式细胞仪检测其杀伤率差异,探讨EXO/IL-12诱导淋巴细胞的杀伤效应是否具有抗原特异性。
结果:结果显示:在效-靶比为50:1时, IL-12诱导活化的淋巴细胞对肾癌细胞的杀伤率为10.34±3.92%,高于用PBS组的0.7±0.26%(P<0.05);EXO组诱导活化的T淋巴细胞对肾癌细胞的杀伤率为24.3±3.8%,明显高于IL-12组和PBS组,差异显著(P<0.01)。EXO/IL-12诱导的淋巴细胞对靶细胞的杀伤作用最强,杀伤率达53.7±3.9%,与各组比较,差异显著(P<0.01)。并且,EXO/IL-12活化的淋巴细胞对肾癌细胞RC-2的杀伤率明显高于对膀胱癌细胞T-24和结肠癌细胞SW480(杀伤率分别为9.8±2.5%和10.8±2.1%),差异有显著性意义(P<0.01)
结论:EXO/IL-12疫苗能在体外诱导淋巴细胞产生较强的抗原特异性细胞毒作用,可望成为治疗肾癌的新疫苗。
PART ONE Separation and identification of exosomes derived from renal cancer cells and primary study of protein composition
Objective:To separate and purify exosomes derived from renal cancer cells and detect their immune associated protein in order to investigate their potential clinical value.
Methods:Exosomes derived from RC-2 cells culture supematants were separated and purified by ultrafiltration and sucrose gradient centrifugation, which were identified by transmission electron microscope. The expressions of HSP70, ICAM-1 and G250 were detected by western blot.
Results:Exosomes were successfully purified by ultrafiltration and sucrose gradient centrifugation, which were 30~80 nm in diameter with typical saucer-shape morphology, and expressed HSP70、ICAM-1 and G250 antigen.
Conclusion:Ultrafiltration and sucrose gradient ultracentrifugation can be used to purify renal cancer cells-secreted exosomes. Exosomes, expressing HSP70, ICAM-1 and G250, might be the novel vaccine for renal cell cancer.
PART TWO Construction of mammalian co-expression plasmid of chemric IL-12-GPI cDNA
Objective:To construct a mammalian co-expression plasmid of pBudCE4.1/IL-12A/IL-12B-GPI.
Methods:A mammalian co-expression plasmid of GPI-anchor- IL-12 (human) were constructed by subcloning IL-12A (P35) gene and a fusion gene containing GPI-anchor signal sequence of hPLAP-1 and IL-12B (P40) in pBudCE4.1. .
Results:Plasmid pBudCE4.1/IL-12A was digested with KpnI and XhoI, and resulted in band of IL-12A-chain(762 bp). The correct sequence was identified by DNA sequencing. The recombinant plasmids pBudCE4.1/IL-12A/IL-12B-GPI were digested with KpnI, XhoI, BamHI andXbaI, and resulted in two bands, 1104 and 762 bp, named IL-12A-chain and IL-12B-GPI, respectively. The correct sequence was identified by DNA sequencing, showing that co-expression plasmid of pBud-CE4.1/IL-12A/IL-12B-GPI was successfully constructed.
Conclusion The recombinant plasmid may provide foundation for further preparation of tumor vaccine.
PART THREE The preparation and identification of renal-cancer vaccine of exosomes
Objective:To prepare IL-12-anchored exosomes(EXO/IL-12) derived from renal cancer cells and identify their structure and function.
Methods:RC-2 transfectants expressing GPI-IL-12 (RC-2-GPI-IL-12) were established by transfecting pBudCE4.1/IL-12A /IL-12B-GPI plasmid. Confocal laser scanning microscopy and flow cytometry analyzed the expression of fusion protein. TEM and Western blot identified the morphology and characteristic molecules of exosomes separated by ultrafiltration and sucrose gradient centrifugation. The function of anchored IL-12 exosomes was determined by IFN-γrelease assay and CFSE-based cell proliferation.
Results:Confocal laser scanning microscopy and flow cytometry analysis of the RC-2-GPI-IL-12 transfectants showed expression of IL-12 on the cell surface via a GPI-moiety. EXO/IL-12 were purified by ultrafiltration and sucrose gradient centrifugation, which were 30~80 nm in diameter with typical saucer-shape morphology, and expressing HSP70, ICAM-1, G250 and GPI-IL-12. 80±9.6pg/ml of IL-12 was detected in 10μg EXO/IL-12 and it significantly induced cell proliferation and the release of IFN-γ.
Conclusion:The vaccine of EXO/IL-12 can be obtained from the culture supernatant of IL-12-anchored RC-2 cells, exprssing IL-12 and tumor associated antigen G250, could efficiently increase induciont of cell proliferation and the release of IFN-γof T lymphocytes in vitro.
PART FOUR EXO/IL-12 increases induction of specific antitumor response in vitro:
Objective:To evaluate antitumor effect of EXO/IL-12 in vitro.
Methods:Lymphocytes and dendritic cells were isolated from human peripheral blood mononuclear for cytotoxicity assay. The morphology of DC was identified by SEM. Exosome-loaded DCs promoted activation of autologous T cells used as effector cells. Autologous DCs and T cells were co-inoculated with EXO/IL-12 (10μg) for 48h, PBS, IL-12, EXO was used as control. Target cells were RC-2, T-24 (bladder cancer cell line) or SW480 (colon cancer cell line). Cytotoxicity assays based on PI costaining of CFSE-labeled target cells were performed. All cells were harvested and resuspended in PBS and analyzed by flow cytometry. Killing rate was assessed by the percentage of PI- costaining cells out of gated CFSE-labeled cells.
Results:The results showed that initiation by EXO and EXO/IL-12 efficiently killed tumor cells as compared with PBS and IL-12 group (P<0.01); however, initiation by EXO/IL-12 exhibited the highest killing rate (P<0.01). To further explore whether EXO/IL-12 could induce antigen-specific cytotoxic effect, we used T-24, SW480 and RC-2 as target cells and evaluated the difference in killing rate. These results suggested that exosomes derived from renal cancer cell could activate T cells in antigen-specific manner through DCs, resulting in more efficient killing of renal cancer cells as compared with T-24 and sw480 cells (P<0.01).
Conclusion:The vaccine of EXO/IL-12 could more effectively increase induction of antigen-specific antitumor immune response in vitro.
引文
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