基因工程融合蛋白疫苗的衍生化及肝癌转移动物模型的建立与鉴定
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摘要
肿瘤转移是恶性肿瘤的生物学特征之一,也是肿瘤临床治疗的难点。据报道,60%以上的恶性肿瘤患者于初次诊断时已发现转移[1]。在临床肿瘤患者中,约有80%-90%以上死于侵袭和转移,可以说肿瘤转移是大多数肿瘤患者死亡的主要原因。肝细胞癌由于其组织结构的特点,容易发生早期转移,是肝癌预后不良的原因之一。在肝细胞肝癌患者尸检中发现50%-84.6%有转移[2]。目前肝癌的治疗手段主要有手术治疗、放射治疗、化学治疗、中医药治疗等。随着对肿瘤免疫理论的深入认识和一些新的实验方法的涌现,肿瘤疫苗已成为预防和治疗肿瘤转移、复发的得力手段。黑色素瘤抗原(Melanoma Antigen,MAGE)是最先被发现的人类肿瘤特异性抗原,1991年Boon等首次发现的人类肿瘤免疫排斥抗原-黑色素瘤相关抗原-1(Melanoma associated antigen-1,MAGE-1)是MAGE家族的组成成员。其广泛表达于多种不同组织类型的肿瘤,而在除睾丸和胎盘外的正常组织中均不表达;又因MAGE-1可与人类白细胞抗原(Human Leukocytes Antigen,HLA)分子形成抗原复合物,此抗原复合物可被细胞毒T淋巴细胞(CytotoxicLymphocyte,CTL)的T细胞受体(T cell receptor,TCR)所识别,刺激强烈的CTL反应[3],因此其是肿瘤特异性免疫治疗理想的靶分子。热休克蛋白(Heat Shock Protein, HSP)具有分子伴侣作用,参与肿瘤抗原的加工递呈,在诱导肿瘤免疫反应中发挥着重要作用。本实验室研究已经表明,HSP70与MAGE1融合蛋白能提高MAGE1蛋白疫苗的免疫效果。
为了给MAGE1与HSP70融合蛋白基因工程纳米疫苗在预防肝癌转移方面提供合适的动物模型,本研究主要用人外周血淋巴细胞(Human peripheral blood lymphocyte,huPBL)和肝癌转移细胞系97-高转移(Metastatic hepatocellular carcinoma cell 97-High,MHCC97-H)建立免疫重建荷人高转移肝癌重症联合免疫缺陷(Severe combined immunodeficiency,SCID)小鼠模型,并对其进行鉴定。全文分为以下三个部分:
实验一MHCC97-H细胞中MAGE-1抗原表达的检测
目的:检测MHCC97-H细胞中MAGE-1抗原的表达情况,为MAGE-1/HSP70融合蛋白基因工程纳米疫苗预防肝癌实验提供依据。方法:用免疫组化、免疫荧光、Western Blotting来检测MHCC97-H细胞中MAGE-1的表达情况。结果:检测出MHCC97-H细胞中有MAGE-1抗原的表达,且免疫组化、免疫荧光、Western Blotting的结果是相一致的。结论:MHCC97-H细胞中有MAGE-1抗原的表达,为后面的用肿瘤特异性抗原MAGE-1与热休克蛋白HSP70融合蛋白疫苗(M1H)预防肝癌的转移实验提供了依据。
实验二MAGE1-HSP70融合蛋白的衍生化
目的:为了提高大分子蛋白的载药量,对本实验室已经纯化的肿瘤特异性抗原M1H融合蛋白进行衍生化。方法:运用化学合成方法将肿瘤特异性抗原融合蛋白M1H与棕榈酸交联,使融合蛋白酯化后能更容易进行纳米包裹。结果:成功制备了衍生化M1H融合蛋白。结论:衍生化M1H融合蛋白的成功制备,为下一步用纳米脂质体包裹肿瘤特异性抗原MAGE-1与热休克蛋白HSP70融合蛋白奠定了基础。
实验三免疫重建荷人高转移肝癌SCID鼠模型的建立及其鉴定目的:建立具有人免疫学特性的高转移肝癌SCID鼠模型。方法:SCID小鼠腹腔注射人外周血淋巴细胞,皮下接种人高转移肝癌细胞MHCC97-H,免疫重建人高转移肝癌SCID小鼠模型,并鉴定建立的结果。结果:(1)免疫重建荷人高转移肝癌细胞小鼠的成瘤率为100%,较荷瘤组成瘤潜伏期延长,体积缩小(p<0.05);荷瘤组和人化荷瘤组的肝脏转移率分别为82.5%(5/6)和67%(4/6),人化荷瘤组从皮下转移到肝脏所需时间较荷瘤组延长(p<0.05)。(2)第2、4、6周小鼠血中人IgG含量的测定:同期相比,人化荷瘤组小鼠血中人IgG含量高于人化组(p<0.05)。(3)第6周SCID小鼠外周血中人CD3+T淋巴细胞和人CD20+ B淋巴细胞含量的测定:人化荷瘤组小鼠血中人CD3+T淋巴细胞和人CD20+ B淋巴细胞含量高于人化组(p<0.05)。(4)免疫组化检测人化组和人化荷瘤组小鼠脾脏中存在人CD3+ T淋巴细胞和CD20+ B淋巴细胞。结论:成功建立免疫重建荷人高转移肝癌SCID鼠模型,为肝癌转移的研究及治疗提供了理想的动物模型。
综上所述,本实验为本实验室研究出的“恶性肿瘤基因工程纳米疫苗”NL(M1H)对表达MAGE1抗原的肝癌细胞的转移的预防奠定了基础。本研究为研制使用安全、高效的肿瘤疫苗展示了良好的前景,为本疫苗的进一步研究提供了条件。
The metastasis is one of the biological characteristics of malignant tumor, and it is also difficult to be cured. It is reported that, 60% of malignant tumor has metastasized before the initial diagnosis. In the clinic, about 80%-90% die from invasion and metastasis. That is to say, metastasis may be the main reason for the death of most malignant tumor patients. Because of the characteristic of the liver tumor, it is apt to metastasize. So it is one of the reasons for the unfavorable prognosis. Atuopsy showed that there is 50%-84.6% metastasis existed in the hepatic cellular cancer’s body. The main therapies for the liver cancer include operation, radiotherapy, chemotherapy and Chinese traditional medicine etal.With further knownledgement in tumor immunity theory and emerge of new experiment technology,tumor vaccines have become one of the most attractive methods to prevent the metastasis of liver cancer. The Melanoma Antigen (MAGE) is the earliest discovered human specific Antigen. The Melanoma associated antigen-1 (MAGE-1) was the first reported immunologic tumor rejection antigen discovered by boon in 1991, which is one of important numbers in the MAGE family.It is expressed in most types of malignant tumor except testicle and placenta. Furthermore, MAGE-1 can cohere to human leukocytes antigen (HLA) molecules to form antigen compounds and this antigen compounds can be recognized by the tumor cell receptor (TCR) of cytotoxic T lymphocytes (CTL). The recognition will lead to specific CTL to kill tumor cells. So MAGE-1 has been used as an ideal target molecule for tumor immunotherapy. As molecular chaperone, heat shock protein (HSP)take part in processing and presentation of tumor antigen and plays an important role in arousing antitumor immunity .Our researches have demonstrated that fusion protein of MAGE-1 and HSP70 can enhance tumor vaccines efficiency.
In order to offer the animal model for the protective effects against liver cancer metastasis by Nanoliposome-encapsulated Tumor Specific Antigen Protein Vaccine of fusion protein of MAGE-1 and HSP70, this research used Human peripheral blood lymphocyte(huPBL)and high Metastatic hepatocellular carcinoma cell 97 (MHCC97) established immune reconstruction human metastatic hepatocelluar carcinoma model and indeficated it. The research was divided into three parts as following:
Experiment 1: The MAGE-1 protein expression in the MHCC97-H cells Objective: To detect MAGE-1 antigen expression in the MHCC97-H cells,lay a basis for the experiment of protective effects against liver cancer metastasis. Methods: Using the methods of Immunohistochemistry, immunofluorescence, and western blotting to detect the MAGE-1 antigen expression in the MHCC97-H cells. Results: The MAGE-1 antigen expresses in the MHCC97-H cells. The result of the Immunohistochemistry, immunofluorescence, and western blotting are coherent. Conclusion: The MAGE-1 antigen’s expression in the MHCC97-H cells offers the basis for the experiment of MAGE-1 and HSP70 fusion protein (M1H) protective effects against liver cancer metastasis. Experiment 2: The derivatization of MAGE1-HSP70 fusion protein Objiective: To make the macromolecule protein carries more dosage.Methods: The recombinant fusion protein was cross linked with Palmitic acid by chemical methods. The purified recombinant fusion protein was esterified and then was wrapped up with nano-liposome.Results: The derivatization of MAGE1-HSP70 fusion protein was prepared successfully.Conclusion: The derivatization of MAGE1-HSP70 fusion protein lays a basis for the experiment of protective effects against liver cancer metastasis.
Experiment 3: Establishment and identification of HU-PBL-SCID mice model of human high metastatic hepatocelluar carcinoma
Objective: To explore the effective methods of establishing a human metastatic hepatocelluar carcinoma model in human peripheral blood lymphocyte (hu-PBL) engrafted to severe combined immunodeficient (SCID) mice. Methods: the immunological features of mice were evaluated after intra-peritoneal injection of hu-PBL and subcutaneous implantation of human high metastatic hepatocelluar carcinoma cells (MHCC97-Hs).Results: (1) subcutaneous tumors developed in all the mice given hu-PBL and MHCC97-Hs. The latency period was significantly prolonged, and the tumor size was marked depressed, as compared with mice given human MHCC97-Hs alone (p<0.05). The tumor’s live metastatic rate in the mice given human MHCC97-Hs alone was 82.5 %(5/6) .While in the mice given hu-PBL and MHCC97-Hs was 67%(4/6). The tumor’s metastatic time needed in the mice given hu-PBL and MHCC97-Hs was longer than the mice given human MHCC97-Hs alone(p<0.05). (2) In the 2nd, 4th and 6th week, the amount of the human IgG in the mice given hu-PBL and MHCC97-Hs was lager than the mice given MHCC97-Hs alone(p<0.05). (3)In the 6th week, the number of the human CD3+ T lymphocytes and CD20+ B lymphocytes in the mice given hu-PBL and MHCC97- Hs was lager than the mice given MHCC97-Hs alone(p<0.05). (4)Immunohistochemical staining revealed presence of remarkable human CD3+ T lymphocytes and CD20+ B lymphocytes in SCID mice’s spleen. Conclusion: A human high metastatic hepatocelluar carcinoma model has been established hu-PBL engrafted to SCID mice, which is an ideal animal model for preclinical research and treatment for metastatic of hepatocelluar carcinoma.
In this study, the Nanoliposome-encapsulated Tumor Specific Antigen Protein Vaccine NL (M1H) could protect hepatocelluar carcinoma metastasis which express MAGE-1.It may develop a new route to design a more efficient and safe tumor vaccine. And this study will lay a basis for the further development of the vaccine.
为了给MAGE1与HSP70融合蛋白基因工程纳米疫苗在预防肝癌转移方面提供合适的动物模型,本研究主要用人外周血淋巴细胞(Human peripheral blood lymphocyte,huPBL)和肝癌转移细胞系97-高转移(Metastatic hepatocellular carcinoma cell 97-High,MHCC97-H)建立免疫重建荷人高转移肝癌重症联合免疫缺陷(Severe combined immunodeficiency,SCID)小鼠模型,并对其进行鉴定。全文分为以下三个部分:
实验一MHCC97-H细胞中MAGE-1抗原表达的检测
目的:检测MHCC97-H细胞中MAGE-1抗原的表达情况,为MAGE-1/HSP70融合蛋白基因工程纳米疫苗预防肝癌实验提供依据。方法:用免疫组化、免疫荧光、Western Blotting来检测MHCC97-H细胞中MAGE-1的表达情况。结果:检测出MHCC97-H细胞中有MAGE-1抗原的表达,且免疫组化、免疫荧光、Western Blotting的结果是相一致的。结论:MHCC97-H细胞中有MAGE-1抗原的表达,为后面的用肿瘤特异性抗原MAGE-1与热休克蛋白HSP70融合蛋白疫苗(M1H)预防肝癌的转移实验提供了依据。
实验二MAGE1-HSP70融合蛋白的衍生化
目的:为了提高大分子蛋白的载药量,对本实验室已经纯化的肿瘤特异性抗原M1H融合蛋白进行衍生化。方法:运用化学合成方法将肿瘤特异性抗原融合蛋白M1H与棕榈酸交联,使融合蛋白酯化后能更容易进行纳米包裹。结果:成功制备了衍生化M1H融合蛋白。结论:衍生化M1H融合蛋白的成功制备,为下一步用纳米脂质体包裹肿瘤特异性抗原MAGE-1与热休克蛋白HSP70融合蛋白奠定了基础。
实验三免疫重建荷人高转移肝癌SCID鼠模型的建立及其鉴定目的:建立具有人免疫学特性的高转移肝癌SCID鼠模型。方法:SCID小鼠腹腔注射人外周血淋巴细胞,皮下接种人高转移肝癌细胞MHCC97-H,免疫重建人高转移肝癌SCID小鼠模型,并鉴定建立的结果。结果:(1)免疫重建荷人高转移肝癌细胞小鼠的成瘤率为100%,较荷瘤组成瘤潜伏期延长,体积缩小(p<0.05);荷瘤组和人化荷瘤组的肝脏转移率分别为82.5%(5/6)和67%(4/6),人化荷瘤组从皮下转移到肝脏所需时间较荷瘤组延长(p<0.05)。(2)第2、4、6周小鼠血中人IgG含量的测定:同期相比,人化荷瘤组小鼠血中人IgG含量高于人化组(p<0.05)。(3)第6周SCID小鼠外周血中人CD3+T淋巴细胞和人CD20+ B淋巴细胞含量的测定:人化荷瘤组小鼠血中人CD3+T淋巴细胞和人CD20+ B淋巴细胞含量高于人化组(p<0.05)。(4)免疫组化检测人化组和人化荷瘤组小鼠脾脏中存在人CD3+ T淋巴细胞和CD20+ B淋巴细胞。结论:成功建立免疫重建荷人高转移肝癌SCID鼠模型,为肝癌转移的研究及治疗提供了理想的动物模型。
综上所述,本实验为本实验室研究出的“恶性肿瘤基因工程纳米疫苗”NL(M1H)对表达MAGE1抗原的肝癌细胞的转移的预防奠定了基础。本研究为研制使用安全、高效的肿瘤疫苗展示了良好的前景,为本疫苗的进一步研究提供了条件。
The metastasis is one of the biological characteristics of malignant tumor, and it is also difficult to be cured. It is reported that, 60% of malignant tumor has metastasized before the initial diagnosis. In the clinic, about 80%-90% die from invasion and metastasis. That is to say, metastasis may be the main reason for the death of most malignant tumor patients. Because of the characteristic of the liver tumor, it is apt to metastasize. So it is one of the reasons for the unfavorable prognosis. Atuopsy showed that there is 50%-84.6% metastasis existed in the hepatic cellular cancer’s body. The main therapies for the liver cancer include operation, radiotherapy, chemotherapy and Chinese traditional medicine etal.With further knownledgement in tumor immunity theory and emerge of new experiment technology,tumor vaccines have become one of the most attractive methods to prevent the metastasis of liver cancer. The Melanoma Antigen (MAGE) is the earliest discovered human specific Antigen. The Melanoma associated antigen-1 (MAGE-1) was the first reported immunologic tumor rejection antigen discovered by boon in 1991, which is one of important numbers in the MAGE family.It is expressed in most types of malignant tumor except testicle and placenta. Furthermore, MAGE-1 can cohere to human leukocytes antigen (HLA) molecules to form antigen compounds and this antigen compounds can be recognized by the tumor cell receptor (TCR) of cytotoxic T lymphocytes (CTL). The recognition will lead to specific CTL to kill tumor cells. So MAGE-1 has been used as an ideal target molecule for tumor immunotherapy. As molecular chaperone, heat shock protein (HSP)take part in processing and presentation of tumor antigen and plays an important role in arousing antitumor immunity .Our researches have demonstrated that fusion protein of MAGE-1 and HSP70 can enhance tumor vaccines efficiency.
In order to offer the animal model for the protective effects against liver cancer metastasis by Nanoliposome-encapsulated Tumor Specific Antigen Protein Vaccine of fusion protein of MAGE-1 and HSP70, this research used Human peripheral blood lymphocyte(huPBL)and high Metastatic hepatocellular carcinoma cell 97 (MHCC97) established immune reconstruction human metastatic hepatocelluar carcinoma model and indeficated it. The research was divided into three parts as following:
Experiment 1: The MAGE-1 protein expression in the MHCC97-H cells Objective: To detect MAGE-1 antigen expression in the MHCC97-H cells,lay a basis for the experiment of protective effects against liver cancer metastasis. Methods: Using the methods of Immunohistochemistry, immunofluorescence, and western blotting to detect the MAGE-1 antigen expression in the MHCC97-H cells. Results: The MAGE-1 antigen expresses in the MHCC97-H cells. The result of the Immunohistochemistry, immunofluorescence, and western blotting are coherent. Conclusion: The MAGE-1 antigen’s expression in the MHCC97-H cells offers the basis for the experiment of MAGE-1 and HSP70 fusion protein (M1H) protective effects against liver cancer metastasis. Experiment 2: The derivatization of MAGE1-HSP70 fusion protein Objiective: To make the macromolecule protein carries more dosage.Methods: The recombinant fusion protein was cross linked with Palmitic acid by chemical methods. The purified recombinant fusion protein was esterified and then was wrapped up with nano-liposome.Results: The derivatization of MAGE1-HSP70 fusion protein was prepared successfully.Conclusion: The derivatization of MAGE1-HSP70 fusion protein lays a basis for the experiment of protective effects against liver cancer metastasis.
Experiment 3: Establishment and identification of HU-PBL-SCID mice model of human high metastatic hepatocelluar carcinoma
Objective: To explore the effective methods of establishing a human metastatic hepatocelluar carcinoma model in human peripheral blood lymphocyte (hu-PBL) engrafted to severe combined immunodeficient (SCID) mice. Methods: the immunological features of mice were evaluated after intra-peritoneal injection of hu-PBL and subcutaneous implantation of human high metastatic hepatocelluar carcinoma cells (MHCC97-Hs).Results: (1) subcutaneous tumors developed in all the mice given hu-PBL and MHCC97-Hs. The latency period was significantly prolonged, and the tumor size was marked depressed, as compared with mice given human MHCC97-Hs alone (p<0.05). The tumor’s live metastatic rate in the mice given human MHCC97-Hs alone was 82.5 %(5/6) .While in the mice given hu-PBL and MHCC97-Hs was 67%(4/6). The tumor’s metastatic time needed in the mice given hu-PBL and MHCC97-Hs was longer than the mice given human MHCC97-Hs alone(p<0.05). (2) In the 2nd, 4th and 6th week, the amount of the human IgG in the mice given hu-PBL and MHCC97-Hs was lager than the mice given MHCC97-Hs alone(p<0.05). (3)In the 6th week, the number of the human CD3+ T lymphocytes and CD20+ B lymphocytes in the mice given hu-PBL and MHCC97- Hs was lager than the mice given MHCC97-Hs alone(p<0.05). (4)Immunohistochemical staining revealed presence of remarkable human CD3+ T lymphocytes and CD20+ B lymphocytes in SCID mice’s spleen. Conclusion: A human high metastatic hepatocelluar carcinoma model has been established hu-PBL engrafted to SCID mice, which is an ideal animal model for preclinical research and treatment for metastatic of hepatocelluar carcinoma.
In this study, the Nanoliposome-encapsulated Tumor Specific Antigen Protein Vaccine NL (M1H) could protect hepatocelluar carcinoma metastasis which express MAGE-1.It may develop a new route to design a more efficient and safe tumor vaccine. And this study will lay a basis for the further development of the vaccine.
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