本文選題：姜黃素膠束 + 腫瘤納米疫苗�。� 參考：《華中科技大學(xué)》2016年博士論文

【摘要】：研究目的:腫瘤免疫治療是應(yīng)用免疫學(xué)方法,激發(fā)體內(nèi)免疫系統(tǒng),產(chǎn)生殺傷腫瘤細(xì)胞的特異性因子殺傷腫瘤細(xì)胞、從而抑制腫瘤生長(zhǎng)的治療方式,近年來(lái)備受關(guān)注。但是單獨(dú)使用免疫治療時(shí)反應(yīng)率不是很高,尤其對(duì)于晚期惡性黑色素瘤的治療效果明顯降低。因?yàn)殡S著腫瘤生長(zhǎng),到晚期時(shí),腫瘤微環(huán)境呈免疫抑制狀態(tài),阻止細(xì)胞毒性T淋巴細(xì)胞(CTL)向腫瘤組織浸潤(rùn)。比較可行的方法是將這些能夠刺激免疫反應(yīng)的分子或抗體或核酸等制成納米形式,增強(qiáng)抗原遞呈等免疫作用的同時(shí)與化學(xué)預(yù)防藥物或者化學(xué)治療藥物聯(lián)合使用,降低腫瘤微環(huán)境中的免疫抑制因子水平、加強(qiáng)免疫治療的效果、有效遏制惡性黑色素瘤生長(zhǎng)。而對(duì)于腫瘤抗原明確的模型以及臨床上沒(méi)有找到合適的腫瘤抗原時(shí)的模型采取不同的聯(lián)合給藥方式或許更有臨床實(shí)際應(yīng)用價(jià)值。方法:對(duì)于腫瘤抗原明確的模型,我們采用了磷酸鈣核-殼型納米粒包裹腫瘤多肽分子與佐劑,制成納米疫苗(Trp-Vac);與化學(xué)預(yù)防藥物姜黃素納米膠束(Cur PM)聯(lián)合給予小鼠。我們首先合成了Cur PM,對(duì)其表征,測(cè)試了其在荷瘤小鼠中的組織分布。再聯(lián)合給藥,進(jìn)行腫瘤生長(zhǎng)抑制實(shí)驗(yàn),體內(nèi)細(xì)胞毒性T淋巴細(xì)胞(in vivo CTL)殺傷效應(yīng)實(shí)驗(yàn)和酶聯(lián)免疫斑點(diǎn)檢測(cè)(ELISPOT)等實(shí)驗(yàn),評(píng)估細(xì)胞毒性T細(xì)胞增殖效果。接著用流式細(xì)胞術(shù)測(cè)定腫瘤浸潤(rùn)免疫細(xì)胞數(shù)目變化,實(shí)時(shí)熒光定量PCR測(cè)定腫瘤組織中細(xì)胞因子水平等,最后經(jīng)血液化學(xué)分析評(píng)估其毒性作用。對(duì)于臨床上找不到合適腫瘤抗原的模型,我們可利用化療藥物如順鉑等藥物原位誘導(dǎo)凋亡細(xì)胞產(chǎn)生免疫原性的能力,將順鉑制成納米粒(LPC),靶向到腫瘤組織內(nèi)誘發(fā)腫瘤細(xì)胞凋亡,待其產(chǎn)生有免疫原性的自身腫瘤抗原后,再靜脈注射免疫佐劑的脂質(zhì)體(Cp G-Lipo)加強(qiáng)抗原遞呈。測(cè)量腫瘤體積,觀測(cè)處理后小鼠平均生存時(shí)間,HE染色切片評(píng)估其毒性作用。并進(jìn)行ELISPOT等實(shí)驗(yàn)判定T細(xì)胞增殖效果,并用流式細(xì)胞術(shù)測(cè)定腫瘤浸潤(rùn)免疫細(xì)胞數(shù)目、ELISA測(cè)定腫瘤組織中細(xì)胞因子水平等。結(jié)果與結(jié)論:第一種給藥方式結(jié)果表明,在晚期黑色素瘤荷瘤小鼠中,聯(lián)合給予了納米膠束和納米疫苗的小鼠抗腫瘤作用明顯高于單獨(dú)給予藥物的小組以及對(duì)照組。在免疫器官中,聯(lián)合給藥方式顯著增強(qiáng)體內(nèi)CTL效應(yīng)以及γ干擾素(IFN-γ)這種效應(yīng)細(xì)胞因子的水平。在腫瘤組織內(nèi),聯(lián)合給藥方式一方面顯著降低免疫與腫瘤共同通路-信號(hào)轉(zhuǎn)導(dǎo)及轉(zhuǎn)錄激活因子3-STAT3體內(nèi)表達(dá)水平,另一方面聯(lián)合給藥方式導(dǎo)致了許多免疫抑制細(xì)胞或因子如骨髓源性抑制細(xì)胞(MDSC)、調(diào)節(jié)性T細(xì)胞(Treg)、白細(xì)胞介素6(IL-6)和趨化因子配體2(CCL2)等的顯著性降低;而許多促炎因子如腫瘤壞死因子(TNF-α)和IFN-γ升高,因而減除了微環(huán)境中的免疫抑制狀態(tài),T細(xì)胞浸潤(rùn)增多,利于化學(xué)預(yù)防性藥物及疫苗發(fā)揮作用。第二種給藥方式結(jié)果表明,此聯(lián)合給藥方式產(chǎn)生了很強(qiáng)的誘導(dǎo)腫瘤組織細(xì)胞凋亡以及抑制晚期惡性黑色素瘤生長(zhǎng)的作用。這種很強(qiáng)的協(xié)同抗腫瘤生長(zhǎng)作用很大程度上是因?yàn)榧?xì)胞毒性T細(xì)胞增殖加強(qiáng),以及免疫器官、腫瘤微環(huán)境中的免疫抑制因子減少。因?yàn)檫@些作用,聯(lián)合給藥大大增強(qiáng)了小鼠生存時(shí)間。這種基于納米系統(tǒng)的LPC聯(lián)合Cp G-Lipo減小藥物毒性的同時(shí)延長(zhǎng)了荷瘤鼠生存時(shí)間、減少了腫瘤肺轉(zhuǎn)移、顯示了原位誘導(dǎo)產(chǎn)生腫瘤抗原也是一種有效可行的腫瘤治療方式。創(chuàng)新點(diǎn):(1)本研究中使用的藥物、腫瘤疫苗以及免疫佐劑等都采用了納米遞釋系統(tǒng),并且根據(jù)藥物性質(zhì)的不同設(shè)計(jì)了不同的納米形式,從而靶向到不同器官。(2)這些經(jīng)由納米系統(tǒng)遞釋的藥物對(duì)晚期難以治療的惡性黑色素瘤腫瘤微環(huán)境有很好的靶向聚集與調(diào)節(jié)作用,可以有效解除腫瘤微環(huán)境中免疫抑制狀態(tài),使之有免疫應(yīng)答能力。當(dāng)與納米化的疫苗或者免疫佐劑聯(lián)合使用時(shí),可以促進(jìn)細(xì)胞毒性T淋巴細(xì)胞向腫瘤組織浸潤(rùn),有效遏制晚期惡性黑色素瘤生長(zhǎng)。(3)本研究探索了基于納米遞釋系統(tǒng)的兩種聯(lián)合給藥模式。一種模式是對(duì)于腫瘤抗原明確的模型,采用了靶向樹(shù)突狀細(xì)胞(DC)的納米腫瘤抗原疫苗(TrpVac)與姜黃素兩親性納米膠束(Cur PM)聯(lián)合給藥的模式;另一種是當(dāng)臨床上沒(méi)有找到適合的腫瘤抗原時(shí)適用的聯(lián)合給藥模式,利用核-殼納米遞釋系統(tǒng)將化療藥物順鉑靶向到腫瘤組織中,原位誘導(dǎo)腫瘤自身抗原的產(chǎn)生,再輔以納米免疫佐劑,增強(qiáng)腫瘤抗原遞呈,啟動(dòng)小鼠適應(yīng)性免疫應(yīng)答對(duì)抗腫瘤。
[Abstract]:Objective: tumor immunotherapy is the application of immunology, to stimulate the immune system in the body, to kill tumor cell specific factors to kill tumor cells, and to inhibit the growth of tumor, and has attracted much attention in recent years. However, the response rate of immunotherapy alone is not very high, especially for advanced malignant melanoma. The effect of the treatment is obviously reduced, because with the growth of the tumor, the tumor microenvironment is immunosuppressive and the cytotoxic T lymphocyte (CTL) is prevented from infiltrating to the tumor tissue at the late stage. The more feasible method is to make the molecules or antibodies or nucleic acid, which can stimulate the immune response, to make nanoscale form and enhance the immunization of the antigen presenting. At the same time, combined with chemical prophylaxis or chemical treatment drugs, reducing the level of immunosuppressive factors in the tumor microenvironment, strengthening the effect of immunotherapy, effectively inhibiting the growth of malignant melanoma, and taking different combinations of the tumor antigen model and the clinical model that did not find the appropriate tumor antigen. The combination of drug delivery methods may be more practical and practical. Method: for the clear model of tumor antigen, we used the calcium phosphate core shell nanoparticles to wrap the tumor polypeptide molecules and adjuvant to make the nano vaccine (Trp-Vac), and the chemical prophylactic drug curcumin nano micelle (Cur PM) was combined to give mice. We first synthesized Cur PM, and The tissue distribution in the tumor bearing mice was tested, and then combined with the drug, the tumor growth inhibition test, the cytotoxic T lymphocyte (in vivo CTL) killing effect and the enzyme linked immunosorbent assay (ELISPOT) were used to evaluate the proliferation of cytotoxic T cells. Then the tumor infiltration immunity was measured by flow cytometry. Changes in the number of cells and the determination of cytokine levels in tumor tissues by real time fluorescence quantitative PCR, and finally by hematological analysis to evaluate their toxic effects. For the clinical model, we can use chemotherapeutic drugs such as cisplatin and other drugs in situ to induce apoptotic cells to produce immunogenicity. Rice grain (LPC), target the tumor cells to induce apoptosis in the tumor tissue. After producing the immunogenicity of the tumor antigen, the antigen presentation is strengthened by the liposome (Cp G-Lipo) of the immuno adjuvant. The tumor volume is measured. The average survival time of the mice after the observation is observed and the HE staining section is used to evaluate the toxic effect of the tumor. And ELISPOT and so on are carried out. The effect of T cell proliferation was determined and the number of tumor infiltrating immune cells was measured by flow cytometry and the level of cytokine in tumor tissue was measured by ELISA. Results and conclusion: the first method of administration showed that in the advanced melanoma tumor bearing mice, the anti tumor effect of the combination of nano micelle and nano vaccine was significantly higher in mice with advanced melanoma. In the immune organs, the combined administration of drugs significantly enhanced the CTL effect and the level of interferon gamma (IFN- gamma) effect cytokine levels in the immune organs. In tumor tissue, combined administration of drugs significantly reduced the co pathway of immunization and tumor, signal transduction and transcription activator 3-STAT3 body. The level of internal expression, on the other hand, leads to a significant reduction in many immunosuppressive cells or factors such as myeloid suppressor cells (MDSC), regulatory T cells (Treg), interleukin 6 (IL-6) and chemokine ligand 2 (CCL2), while many proinflammatory factors such as tumor necrosis factor (TNF- alpha) and IFN- gamma are reduced. The immunosuppressive state in the microenvironment, the increase of T cell infiltration, is beneficial to the chemical prophylactic drugs and vaccines. The results of second drug delivery methods show that the combined administration of the drugs has a strong effect on inducing apoptosis of tumor tissue cells and inhibiting the growth of advanced malignant melanoma. This is a very strong synergistic antitumor growth. To a large extent, it is due to the proliferation of cytotoxic T cells, as well as immune organs, and the reduction of immunosuppressive factors in the microenvironment of the tumor. The combination of these effects greatly enhances the survival time of mice. This nano system based LPC combined with Cp G-Lipo reduces the drug toxicity and prolongs the survival time of the tumor bearing mice and reduces the survival time of the mice. Tumor lung metastasis, showing that in situ induction of tumor antigen is also an effective and feasible method of cancer treatment. Innovation: (1) the drugs used in this study, tumor vaccine and immune adjuvant have adopted the nano delivery system, and different nano forms are designed according to the different drug properties, so as to target different devices. (2) these drugs, which are delivered by the nano system, have good target aggregation and regulation on the malignant melanoma microenvironment of late treatment, which can effectively relieve the immunosuppressive state in the tumor microenvironment and make it immune response. When combined with the nanoscale vaccine or immune adjuvant, it can promote the refinement. Cytotoxic T lymphocytes infiltrate into tumor tissue and effectively inhibit the growth of advanced malignant melanoma. (3) this study explored the two modes of joint delivery based on the nano delivery system. A model is a clear model for tumor antigen, and the nano tumor antigen vaccine (TrpVac) and curcumin are two affinity with targeted dendritic cells (DC). The combination of nano micelle (Cur PM) combined with drug delivery; the other is a joint delivery mode when the suitable tumor antigen is not found in the clinic. The nuclear shell nano delivery system is used to target the chemotherapy drug cisplatin into the tumor tissue and in situ induce the production of the tumor autoantigen, and the nano immuno adjuvant is used to enhance the tumor antigen presentation. The adaptive immune response in mice was initiated against the tumor.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R739.5

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