重组质粒Her2-Hsp70的构建及抗肿瘤作用研究

发布时间：2018-04-29 11:19

本文选题：热休克蛋白 + DNA疫苗　；参考：《河南大学》2012年硕士论文

【摘要】：背景乳腺癌是女性常见的恶性肿瘤，发病率正在逐年上升，常规治疗对晚期患者的痊愈难以奏效，免疫治疗以激发或增强机体的免疫功能为手段，选择性地针对Her2阳性肿瘤细胞，特异性地发挥杀伤作用，进而达到控制和杀伤肿瘤细胞的目的。许多资料显示，此法与常规治疗联合可显著提高肿瘤的综合治疗效果。原癌基因Her2是生长因子受体家族成员之一，在25％～30％的侵润性的乳腺癌中过表达，Her2的表达与肿瘤的转移和预后不良有关，而在正常组织中低表达，因而成为良好的肿瘤免疫生物治疗的靶分子。大量资料表明热休克蛋白70可以作为一种强有力的分子佐剂,可作为肿瘤抗原肽的分子载体,在肿瘤抗原呈递和激活CD8+T细胞方面发挥重要作用。HSP70与MHC分子相互作用是抗原识别和呈递的重要环节。由于HSP70分子与MHC分子有极相似的多肽结合沟,HSP70能够促进MHC分子对肿瘤特异性多肽抗原的识别和呈递;且HSP70会像MHC一样能够作为肿瘤特异性多肽抗原载体与MHC分子共同参与抗原的识别和呈递。本研究中将分子佐剂HSP70与人Her2基因融合，构建重组质粒pcDNA3.1/Her2-HSP70,观察其体内诱导小鼠免疫应答的能力和抗小鼠乳腺癌免疫治疗的作用。通过本实验希望对乳腺癌的治疗找到有效的基因疫苗，为乳腺癌的治疗提供实验和理论依据。目的构建重组质粒pcDNA3.1/Her2-HSP70,观察其诱导免疫应答的能力和抗肿瘤免疫治疗的作用。方法 RT-PCR扩增得到人乳腺癌细胞Her2和Hsp70DNA,构建pcDNA3.1/Her2、pcDNA3.1/HSP70、pcDNA3.1/Her2-HSP70重组质粒,将pcDNA3.1、pcDNA3.1/Her2分别转染小鼠乳腺癌4T-1细胞株，获得稳定表达细胞株，应用western-blot技术检测。将乳腺癌细胞株4T-1/Her2接种于小鼠皮下，建立肿瘤动物模型，再肌肉注射基因疫苗pcDNA3.1/Her2-HSP70,同时联合佐剂BCG、GM-CSF注射，间隔7d，共3次,以pcDNA3.1/Her2、pcDNA3.1/hsp70、pcDNA3.1为对照,观察小鼠肿瘤的生长；三周后处死小鼠，剥瘤称重，肿瘤组织HE染色，小鼠脾细胞用FCM检测T细胞表面标志即膜分子CD3、CD4、CD8的变化及小鼠血清抗Her2抗体的变化。ELISA法检测小鼠血清IL-4、IFN-γ水平。结果构建了重组质粒，经酶切和DNA测序分析,结果表明该基因的序列与设计的序列完全相同。获得了稳定表达4T-1/Her2的细胞株，根据氨基酸序列本实验扩增的Her2分子量约为69kDa的蛋白；western-blot检测到转染4T-1/Her2中Her2蛋白的表达，而4T-1及转染pcDNA3.1的4T-1中没有检测到Her2蛋白的表达。动物实验表明，用重组质粒pcDNA3.1/Her2-HSP70联合佐剂BCG、GM-CSF免疫的BALB/c小鼠，增强了T细胞的免疫应答，上调分子IFN-γ的表达，肿瘤的生长受到抑制。Her2-Hsp70基因疫苗及佐剂应用具有较强的抗肿瘤效应；病理切片结果显示融合质粒组切片中坏死组织较多，瘤细胞体积变小；流式细胞仪检测结果显示：Her2-hsp70+佐剂免疫小鼠后，CD8细胞比例较高，CD4/CD8比值较小，说明Hsp70及佐剂在肿瘤抗原呈递和激活CD8+T细胞方面发挥重要作用。结论异种抗原Her2有效地打破了抗肿瘤免疫耐受，HSP70则加强了抗原递呈作用，GM-CSF和BCG有效刺激了细胞因子IFN-γ的产生，重组质粒pcDNA3.1/Her2-HSP70基因疫苗具有较强的抗小鼠乳腺癌作用。
[Abstract]:background
Breast cancer is a common malignant tumor in women. The incidence of the disease is increasing year by year. Routine treatment is difficult for the recovery of advanced patients. Immunotherapy is used to stimulate or enhance the immune function of the body as a means of selectively targeting Her2 positive tumor cells to kill and kill tumor cells, and then to control and kill tumor cells. Many data show that the combination of this method and conventional therapy can significantly improve the comprehensive therapeutic effect of the tumor. The proto oncogene Her2 is one of the members of the growth factor receptor family, overexpressed in 25% ~ 30% of the invasive breast cancer. The expression of Her2 is associated with the metastasis and poor prognosis of the tumor, and is low in normal tissue, thus becoming a good one. A good target for tumor immunotherapy.
A large number of data suggest that heat shock protein 70 can be used as a powerful molecular adjuvant, as a molecular vector for tumor antigen peptides, play an important role in tumor antigen presentation and activation of CD8+T cells. The interaction between.HSP70 and MHC molecules is an important ring for antigen recognition and presentation. Because HSP70 molecules are very similar to MHC molecules HSP70 can promote the recognition and presentation of tumor specific polypeptide antigens by MHC molecules, and HSP70, like MHC, can participate in the identification and presentation of antigen as a tumor specific polypeptide antigen and MHC molecule. This study combines the molecular adjuvant HSP70 with human Her2 gene to construct the recombinant plasmid pcDNA3.1/Her2-HSP7. 0, observe the ability of inducing immune response in mice in vivo and the effect of anti mouse breast cancer immunotherapy. Through this experiment, we hope to find effective gene vaccine for the treatment of breast cancer, and provide experimental and theoretical basis for the treatment of breast cancer.
objective
The recombinant plasmid pcDNA3.1/Her2-HSP70 was constructed to observe its ability to induce immune response and the effect of anti-tumor immunotherapy.
Method
Her2 and Hsp70DNA of human breast cancer cells were amplified by RT-PCR, and pcDNA3.1/Her2, pcDNA3.1/HSP70, pcDNA3.1/Her2-HSP70 recombinant plasmids were constructed. PcDNA3.1 and pcDNA3.1/Her2 were transfected into the mice breast cancer 4T-1 cell lines respectively. The stable expression cell lines were obtained and the Western-blot technique was used to detect the mammary cancer cell strain in mice subcutaneous. The tumor animal model, and then intramuscular injection of gene vaccine pcDNA3.1/Her2-HSP70, combined with adjuvant BCG, GM-CSF injection, interval 7d, 3 times, with pcDNA3.1/Her2, pcDNA3.1/hsp70, pcDNA3.1 as the control to observe the growth of mice tumor; three weeks later, the mice were killed, the tumor was weighed, the tumor tissue was stained HE, and the mouse splenocytes were detected by FCM to detect the surface of T cell surface. The changes of membrane molecules CD3, CD4 and CD8 and the changes of serum anti Her2 antibodies in mice were detected by.ELISA. The levels of IL-4 and IFN- gamma were detected by.ELISA.
The recombinant plasmid was constructed and analyzed by enzyme digestion and DNA sequencing. The results showed that the sequence of the gene was exactly the same as that of the designed sequence. The cell line that expressed 4T-1/Her2 steadily was obtained. The Her2 molecular weight of Her2 was about 69kDa according to the amino acid sequence experiment; Western-blot was used to detect the expression of Her2 protein in the transfected 4T-1/Her2, and 4T-1 and The expression of Her2 protein was not detected in 4T-1 transfected with pcDNA3.1. Animal experiments showed that the recombinant plasmid pcDNA3.1/Her2-HSP70 combined with BCG, GM-CSF immune BALB/c mice enhanced the immune response of T cells and increased the expression of IFN- gamma. The growth of the tumor was strongly resistant to the inhibition of the.Her2-Hsp70 gene vaccine and the adjuvant application. The results of pathological section showed that the necrosis tissue of the fusion plasmid group was more and the tumor cell volume became smaller, and the flow cytometry results showed that after Her2-hsp70+ adjuvant immunized mice, the proportion of CD8 cells was higher and the ratio of CD4/CD8 was small, indicating that Hsp70 and adjuvant play important roles in the presentation of tumor antigen and the activation of CD8+T cells. Use. Conclusion
The xenogeneic antigen Her2 effectively breaks the anti-tumor immune tolerance, while HSP70 strengthens the antigen presenting effect, GM-CSF and BCG effectively stimulate the production of cytokine IFN- gamma, and the recombinant plasmid pcDNA3.1/Her2-HSP70 gene vaccine has a strong anti mouse breast cancer effect.

【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

【参考文献】