本文關(guān)鍵詞： 胰腺癌 腫瘤微環(huán)境 納米藥物 抗PD-L1抗體 放化療　出處：《第二軍醫(yī)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：胰腺癌細(xì)胞由于缺少特異性的腫瘤標(biāo)志物,前期癥狀又隱匿,因此治療窗口小;另外胰腺癌解剖位置特殊,造成手術(shù)難度大,而且術(shù)后易復(fù)發(fā),并不延長生存期。因此,對于絕大多數(shù)的中晚期胰腺癌,放化療以及綜合療法成為優(yōu)選方案。然而,臨床胰腺癌的特殊的腫瘤微環(huán)境明顯降低了這些治療的效果。胰腺癌的腫瘤微環(huán)境包括富含透明質(zhì)酸的稠密基質(zhì)屏障、血管生長分布異質(zhì)性等嚴(yán)重阻礙了藥物的瘤內(nèi)富集,降低了免疫細(xì)胞(如T和NK細(xì)胞)的浸潤導(dǎo)致腫瘤細(xì)胞免疫耐受;而基質(zhì)內(nèi)的胰腺星狀細(xì)胞及還原環(huán)境又促進胰腺癌細(xì)胞耐輻射損傷;同時,腫瘤組織內(nèi)PD-L1的表達及其他負(fù)向調(diào)控因子也抑制了效應(yīng)T細(xì)胞的殺傷作用。因此,通過打破基質(zhì)屏障促進藥物滲透,抑制胰腺星狀細(xì)胞,調(diào)節(jié)免疫環(huán)境來調(diào)控胰腺癌腫瘤微環(huán)境來提高放化療治療效果顯得尤為重要。該設(shè)想的關(guān)鍵是如何針對上述不同靶點設(shè)計體系,協(xié)同治療。納米藥物由于獨特的增強滲透阻滯(EPR)效應(yīng)以及環(huán)境響應(yīng)性等特點為上述設(shè)想提供了完美的解決方案。本研究中,我們針對胰腺癌微環(huán)境中的基質(zhì)成分透明質(zhì)酸、谷胱甘肽、胰腺星狀細(xì)胞、效應(yīng)T細(xì)胞等因素,巧妙利用二硫鍵將支化-聚乙烯亞胺(b-PEI)偶聯(lián),并負(fù)載星狀細(xì)胞抑制劑LDE225,同時在載體表面修飾抗PD-L1抗體(Avelumab)及透明質(zhì)酸酶,設(shè)計靶向這些因素的可瘤內(nèi)崩解的納米免疫體系。動靜態(tài)激光光散射儀(Dynamic/Static laser light scattering,DLLS/SLLS),HPLC(高效液相色譜),透射電子顯微鏡(TEM)及Zeta電位等檢測結(jié)果都提示了載藥納米免疫體系的成功合成,并且得到了載體類似凝膠的拓?fù)浣Y(jié)構(gòu),載體體系的分子量,抗體和酶的負(fù)載總數(shù)量為130個左右;同時,ELISA及酶功能試驗都表明載體上修飾的抗體及透明質(zhì)酸酶都保持了良好的功能,而借助免疫熒光我們發(fā)現(xiàn)納米免疫體系在模擬細(xì)胞外基質(zhì)環(huán)境中有更好的滲透能力。因此課題所制備的納米免疫體系具有良好的結(jié)構(gòu)和生物功能。我們驗證了胰腺癌腫瘤微環(huán)境的基質(zhì)屏障影響了小分子藥物的滲透,同時胰腺星狀細(xì)胞對胰腺癌細(xì)胞的放射損傷有一定保護作用。胰腺癌PANC-1細(xì)胞及SW1990細(xì)胞PD-L1表達不明顯,胰腺癌BxPC-3細(xì)胞和耐輻射的PANC-1細(xì)胞的PD-L1卻高表達PD-L1。細(xì)胞水平的評價發(fā)現(xiàn),納米免疫體系的生物相容性良好,流式及激光共聚焦的結(jié)果驗證了載藥納米免疫體系與細(xì)胞表面PD-L1良好結(jié)合。載藥納米免疫體系能對胰腺星狀細(xì)胞或者胰腺癌細(xì)胞產(chǎn)生殺傷作用,同時聯(lián)合放療后能明顯提高殺傷效果。3D共培養(yǎng)克隆形成實驗表明在同一照射劑量下,載藥納米免疫體系能突破表面的基質(zhì)屏障滲透進入共培養(yǎng)環(huán)境,釋放藥物抑制細(xì)胞,從而增強了放療敏感性。我們用人外周血的單個核細(xì)胞評價了納米免疫體系誘導(dǎo)的ADCC(Antibody-dependent cellular cytotoxicity,ADCC)作用,在利用IFN-γ使胰腺癌細(xì)胞PD-L1上調(diào)后,納米免疫體系產(chǎn)生了更強的ADCC作用,這可能由于胰腺癌細(xì)胞表面結(jié)合的納米免疫體系偶聯(lián)著大量抗體,引起多個NK細(xì)胞的識別而釋放更多的穿孔素和顆粒酶,產(chǎn)生了更強的免疫殺傷效果。而當(dāng)用滲透過基質(zhì)屏障的納米免疫體系和抗體來進行ADCC實驗時,我們發(fā)現(xiàn)納米免疫體系的ADCC作用要更強,這也從側(cè)面反映了納米免疫體系的良好滲透能力。我們進一步通過皮下荷瘤小鼠(Bal B/c裸鼠)系統(tǒng)研究了納米免疫體系聯(lián)合放療的體內(nèi)抑瘤效果,結(jié)果表明載藥納米免疫體系具有較為突出的抑瘤效果,而與放療聯(lián)用后,抑瘤效果也增強了,說明了載藥納米免疫體系聯(lián)合放療有一定協(xié)同增效的效果。本課題的通過精細(xì)構(gòu)建的納米免疫體系消融腫瘤組織中豐富的透明質(zhì)酸,抑制活化的胰腺星狀細(xì)胞,調(diào)節(jié)腫瘤內(nèi)的免疫環(huán)境,有效實現(xiàn)協(xié)同增強放化療效果。本課題的順利開展為設(shè)計新型納米免疫抗體,提升臨床胰腺癌的綜合治療效果提供了新的思路和方案,對臨床治療胰腺癌及機制研究具有重要的指導(dǎo)意義。
[Abstract]:Pancreatic cancer cells due to the lack of specific tumor markers, early symptoms and hidden, so the treatment of small window; in addition to pancreatic cancer special anatomical position, resulting in difficult surgery, and postoperative recurrence, does not prolong the survival period. Therefore, for the vast majority of advanced pancreatic cancer, chemotherapy and combined therapy as the preferred scheme. However, clinical pancreatic cancer specific tumor microenvironment significantly reduces the effectiveness of treatment of pancreatic cancer. The tumor microenvironment including hyaluronic acid rich dense matrix barrier, blood vessel growth heterogeneity has seriously hindered the intratumoral drug accumulation, decreased immune cells (such as T and NK cells). The infiltration of tumor cells induced immune tolerance; and pancreatic stellate cells within the stroma and reducing environment and promote pancreatic cancer cells resistant to radiation damage; at the same time, the expression of PD-L1 in tumor tissues and other negative regulatory factors Also inhibits the cytotoxic function of T cells. Therefore, by breaking the matrix barrier to promote drug permeation, inhibition of pancreatic stellate cells, regulating the immune environment to regulate pancreatic tumor microenvironment is particularly important to improve the therapeutic effect of chemotherapy is put. The key idea is how to according to the different target system design, collaborative nano treatment. The drug due to the enhanced permeability block unique (EPR) effect and environmental response characteristics provides a perfect solution for the above assumption. In this study, we aimed at the matrix components of pancreatic cancer microenvironment in hyaluronic acid, glutathione, pancreatic stellate cells, T cells and other effect factors, use of two disulfide bonds will branched polyethylenimine (b-PEI) coupling, and load stellate cell inhibitor LDE225, and modification of anti PD-L1 antibody on the surface of the carrier (Avelumab) and hyaluronidase, designed to target these factors The immune system can be disintegrated in nano tumor. The static and dynamic laser light scattering (Dynamic/Static laser light scattering, DLLS/SLLS), HPLC (HPLC), transmission electron microscopy (TEM) and Zeta potential test results that the successful synthesis of the drug loaded nano immune system, and obtained the similar topology vector the gel carrier system, molecular weight, the total quantity of antibody and enzyme load was about 130; at the same time, ELISA and enzyme function test showed that the antibody carrier modification and hyaluronidase have maintained a good function, and by immunofluorescence. We found that the immune system has better ability to penetrate nano in simulated cell the matrix in the environment. Therefore the topic nano immune system prepared has good structure and biological function. We verified the matrix barrier of pancreatic cancer tumor microenvironment affects the permeability of small molecule drugs At the same time, the radiation damage of pancreatic stellate cells on pancreatic cancer cells had a protective effect on pancreatic carcinoma cell lines PANC-1 and SW1990. The expression of PD-L1 was not obvious, pancreatic carcinoma BxPC-3 cells and PANC-1 cells of the radiation resistant evaluation of PD-L1 is high expression level of PD-L1. cells, the immune system of nano good biocompatibility, flow cytometry and the results of confocal laser to verify the drug loaded immune system and cell surface PD-L1. Good combination of drug loaded immune system could produce cytotoxic effect on pancreatic stellate cells or pancreatic cancer cells, at the same time combined with radiotherapy can significantly improve the killing effect of.3D cloning experiment showed that in the same dose of co culture, drug loading nano immune system can break through the barrier on the surface of substrate penetration into the co culture environment, the release of drugs that inhibit cell, thereby enhancing the sensitivity of radiotherapy. We use mononuclear human peripheral blood To evaluate the immune system cells induced by nano ADCC (Antibody-dependent cellular cytotoxicity, ADCC), in the use of IFN- to gamma PD-L1 in pancreatic cancer cells increased after the immune system produce nano ADCC, this may be due to the nano immune system combined with the surface of pancreatic cancer cells is coupled to a large number of antibodies, by identifying a plurality of NK cells the release of perforin and granzyme more, have stronger immune effect. When using permeable matrix barrier nano immune system and antibody to ADCC experiments, we found that the effect of nano ADCC immune system is stronger, it also reflected from the good penetration ability of nano immune system we further by subcutaneous tumor bearing mice (Bal B/c mice) were studied by nano system combined with radiotherapy in vivo antitumor effect, the results showed that the drug loaded nano immune system The inhibitory effect is more prominent, and in combination with radiotherapy, anti-tumor effect was also enhanced, the drug loaded nano immune system combined with radiotherapy have synergistic effect. The immune system constructed by Nano fine ablation of hyaluronic acid rich in tumor tissue, inhibit the activation of pancreatic stellate cells in the regulation of tumor, immune environment, effectively realize the synergistic efficacy of radiotherapy and chemotherapy. The smooth development of this subject is the design of new nano antibody, provides new ideas and methods to enhance the clinical effect of comprehensive treatment of pancreatic cancer, has important guiding significance to study the mechanism and clinical treatment of pancreatic cancer.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.9

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相關(guān)期刊論文 前2條

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2 Agnieszka Anna Rucki;Lei Zheng;;Pancreatic cancer stroma:understanding biology leads to new therapeutic strategies[J];World Journal of Gastroenterology;2014年09期

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本文編號：1506927