本文選題：熱休克蛋白 + DNA疫苗��； 參考：《河南大學》2012年碩士論文

【摘要】：背景 乳腺癌是女性常見的惡性腫瘤，發(fā)病率正在逐年上升，常規(guī)治療對晚期患者的痊愈難以奏效，免疫治療以激發(fā)或增強機體的免疫功能為手段，選擇性地針對Her2陽性腫瘤細胞，特異性地發(fā)揮殺傷作用，進而達到控制和殺傷腫瘤細胞的目的。許多資料顯示，此法與常規(guī)治療聯(lián)合可顯著提高腫瘤的綜合治療效果。原癌基因Her2是生長因子受體家族成員之一，在25％～30％的侵潤性的乳腺癌中過表達，Her2的表達與腫瘤的轉(zhuǎn)移和預(yù)后不良有關(guān)，而在正常組織中低表達，因而成為良好的腫瘤免疫生物治療的靶分子。 大量資料表明熱休克蛋白70可以作為一種強有力的分子佐劑,可作為腫瘤抗原肽的分子載體,在腫瘤抗原呈遞和激活CD8+T細胞方面發(fā)揮重要作用。HSP70與MHC分子相互作用是抗原識別和呈遞的重要環(huán)節(jié)。由于HSP70分子與MHC分子有極相似的多肽結(jié)合溝,HSP70能夠促進MHC分子對腫瘤特異性多肽抗原的識別和呈遞;且HSP70會像MHC一樣能夠作為腫瘤特異性多肽抗原載體與MHC分子共同參與抗原的識別和呈遞。本研究中將分子佐劑HSP70與人Her2基因融合，構(gòu)建重組質(zhì)粒pcDNA3.1/Her2-HSP70,觀察其體內(nèi)誘導小鼠免疫應(yīng)答的能力和抗小鼠乳腺癌免疫治療的作用。通過本實驗希望對乳腺癌的治療找到有效的基因疫苗，為乳腺癌的治療提供實驗和理論依據(jù)。 目的 構(gòu)建重組質(zhì)粒pcDNA3.1/Her2-HSP70,觀察其誘導免疫應(yīng)答的能力和抗腫瘤免疫治療的作用。 方法 RT-PCR擴增得到人乳腺癌細胞Her2和Hsp70DNA,構(gòu)建pcDNA3.1/Her2、pcDNA3.1/HSP70、pcDNA3.1/Her2-HSP70重組質(zhì)粒,將pcDNA3.1、pcDNA3.1/Her2分別轉(zhuǎn)染小鼠乳腺癌4T-1細胞株，獲得穩(wěn)定表達細胞株，應(yīng)用western-blot技術(shù)檢測。將乳腺癌細胞株4T-1/Her2接種于小鼠皮下，建立腫瘤動物模型，再肌肉注射基因疫苗pcDNA3.1/Her2-HSP70,同時聯(lián)合佐劑BCG、GM-CSF注射，間隔7d，共3次,以pcDNA3.1/Her2、pcDNA3.1/hsp70、pcDNA3.1為對照,觀察小鼠腫瘤的生長；三周后處死小鼠，剝瘤稱重，腫瘤組織HE染色，小鼠脾細胞用FCM檢測T細胞表面標志即膜分子CD3、CD4、CD8的變化及小鼠血清抗Her2抗體的變化。ELISA法檢測小鼠血清IL-4、IFN-γ水平。結(jié)果 構(gòu)建了重組質(zhì)粒，經(jīng)酶切和DNA測序分析,結(jié)果表明該基因的序列與設(shè)計的序列完全相同。獲得了穩(wěn)定表達4T-1/Her2的細胞株，根據(jù)氨基酸序列本實驗擴增的Her2分子量約為69kDa的蛋白；western-blot檢測到轉(zhuǎn)染4T-1/Her2中Her2蛋白的表達，而4T-1及轉(zhuǎn)染pcDNA3.1的4T-1中沒有檢測到Her2蛋白的表達。動物實驗表明，用重組質(zhì)粒pcDNA3.1/Her2-HSP70聯(lián)合佐劑BCG、GM-CSF免疫的BALB/c小鼠，增強了T細胞的免疫應(yīng)答，上調(diào)分子IFN-γ的表達，腫瘤的生長受到抑制。Her2-Hsp70基因疫苗及佐劑應(yīng)用具有較強的抗腫瘤效應(yīng)；病理切片結(jié)果顯示融合質(zhì)粒組切片中壞死組織較多，瘤細胞體積變小；流式細胞儀檢測結(jié)果顯示：Her2-hsp70+佐劑免疫小鼠后，CD8細胞比例較高，CD4/CD8比值較小，說明Hsp70及佐劑在腫瘤抗原呈遞和激活CD8+T細胞方面發(fā)揮重要作用。結(jié)論 異種抗原Her2有效地打破了抗腫瘤免疫耐受，HSP70則加強了抗原遞呈作用，GM-CSF和BCG有效刺激了細胞因子IFN-γ的產(chǎn)生，重組質(zhì)粒pcDNA3.1/Her2-HSP70基因疫苗具有較強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.

【學位授予單位】：河南大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392

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