本文關鍵詞： 肝細胞癌 褪黑素 外泌體 巨噬細胞 免疫　出處：《安徽醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：研究背景肝細胞癌(hepatocellular carcinoma,HCC)是消化系統(tǒng)常見的惡性腫瘤之一,死亡率排第五,并且具有高耐藥性和缺乏有效的治療方法。盡管過去的幾十年出現(xiàn)很多先進的治療方法但還是有很多人死于肝癌。外科手術(shù)是非常有效的治療方法,但很多的患者診斷時已失去了手術(shù)機會。全身性化療是晚期肝癌患者的主要治療方法,但不論是單藥還是多藥聯(lián)合治療效果均不理想。傳統(tǒng)的治療方法似乎已經(jīng)達到了平臺期。因此,如何提高HCC治療效果是當今腫瘤研究的熱點。目前臨床腫瘤免疫治療取得一系列突破,明確藥物相關免疫作用及機制,為其更好地應用于臨床提供有力依據(jù),最終達到提高治療效果,延長患者生存期的作用,具有很重要的意義。腫瘤微環(huán)境(tumor microenviroment)通常是由多種不同細胞構(gòu)成,主要包括腫瘤細胞、間質(zhì)細胞、免疫細胞(如樹突狀細胞、巨噬細胞和T淋巴細胞等)組成,其與腫瘤的發(fā)生、發(fā)展和轉(zhuǎn)移有著密切聯(lián)系。免疫細胞在體內(nèi)環(huán)境中起著至關重要的作用,通過免疫監(jiān)視,免疫清除維持機體的正常穩(wěn)定。在腫瘤的發(fā)生、發(fā)展過程中,腫瘤細胞會影響微環(huán)境的功能從而引起免疫抑制狀態(tài)以應對惡劣的生存條件,維持自身生存。巨噬細胞是一種調(diào)節(jié)性很強的細胞,在維持機體內(nèi)環(huán)境穩(wěn)態(tài),維持機體免疫狀態(tài)等方面具有重要作用。多種研究表明,巨噬細胞是腫瘤微環(huán)境中的重要組成部分之一,且腫瘤微環(huán)境中的巨噬細胞在多種因素的作用下常常會發(fā)生表型變化,轉(zhuǎn)變?yōu)槟[瘤相關巨噬細胞(tumor-associated macrophage,TAM),TAM的功能會發(fā)生改變,可以引起微環(huán)境中免疫狀態(tài)的改變,進而發(fā)揮促進腫瘤增殖、侵襲遷移等促腫瘤作用。然而,腫瘤細胞通過何種途徑使原來具有抗腫瘤免疫調(diào)節(jié)作用的巨噬細胞轉(zhuǎn)變?yōu)槊庖咭种频木奘杉毎麖亩龠M腫瘤發(fā)展,能否通過褪黑素影響該途徑從而減輕這種腫瘤導致的免疫抑制機制尚不清楚。Exosome是一類直徑大約為30-100nm的圓形或類圓形囊泡樣結(jié)構(gòu),其包含有大量物質(zhì),在細胞間信號傳遞中發(fā)揮重要作用。因此,深入研究微環(huán)境中的腫瘤細胞影響巨噬細胞,進而改變其免疫功能具有重要的臨床意義。本課題擬探討褪黑素誘導的肝癌細胞外泌體能否影響巨噬細胞的免疫功能及可能的分子機制。目的通過將處于褪黑素誘導與未誘導的肝癌細胞釋放的exosome與巨噬細胞共培養(yǎng),觀察巨噬細胞PD-L1(程序性死亡配體)及炎癥因子的表達狀況,了解褪黑素作用的肝癌細胞對巨噬細胞免疫功能的影響。并通過研究相關的STAT3信號通路,進一步研究褪黑素誘導的肝癌exosome調(diào)控巨噬細胞PD-L1表達的具體機制。方法(1)使用0.1m M的褪黑素與肝癌細胞共培養(yǎng)24小時,分別收集MT處理組(Exo-MT)和未處理組細胞(Exo-con)的上清液,使用Exo Quick-TC試劑盒提取exosome,透射電子顯微鏡(Transmission Electron Microscope,TEM)觀察exosome的形態(tài)、大小,Western-Blot方法檢測exosomes標志蛋白CD63、TSG101以及分子伴侶蛋白Calnexin的表達。(2)使用膜染料PKH67標記exosome,然后將PKH67標記的exosomes與巨噬細胞共同孵育或尾靜脈注射到裸鼠體內(nèi),12h后共聚焦顯微鏡觀察巨噬細胞吞噬exosome的情況。(3)將不同的exosome(Exo-con和Exo-MT)與THP-1巨噬細胞(經(jīng)100ng/ml PMA誘導48小時所得)共培養(yǎng)24小時,分別收集細胞和上清液,使用流式細胞儀檢測PD-L1表達,CBA細胞因子試劑盒檢測炎癥因子表達。(4)分別對裸鼠尾靜脈注射100μl的PBS、Exo-con和Exo-MT,隔日一次,共10次。注射結(jié)束后24h,處死裸鼠收集腹腔巨噬細胞,貼壁2小時后換液棄去未貼壁細胞,剩余細胞培養(yǎng)過夜后收集細胞和上清液,流式細胞儀檢測PD-L1和炎癥因子的表達。(5)分別收集Exo-con和Exo-MT與THP-1巨噬細胞共培養(yǎng)24小時后的細胞,提取總蛋白,Western-Blot方法檢測STAT3與p-STAT3等蛋白水平變化。(6)使用STAT3抑制劑抑制巨噬細胞STAT3表達,Western-Blot方法檢測STAT3通路蛋白變化,流式細胞術(shù)檢測抑制后THP-1巨噬細胞PD-L1表達變化。結(jié)果(1)透射電鏡可見收集到的外泌體呈圓形或類圓形膜性囊泡樣結(jié)構(gòu),直徑為30-100nm,符合外泌體的典型特征。Western-Blot檢測顯示所得的“exosome”表達CD63,但不表達內(nèi)質(zhì)網(wǎng)分子伴侶蛋白Calnexin,進一步證明上清中收集到的是不含細胞成分的外泌體。BCA蛋白定量法對所得外泌體總蛋白進行定量,結(jié)果表明MT作用24h后肝癌細胞分泌的外泌體無明顯增加。(2)激光共聚焦顯微鏡顯示,PKH67標記的外泌體能夠被THP-1巨噬細胞和腹腔巨噬細胞所攝取,流式細胞術(shù)結(jié)果進一步證明腹腔巨噬細胞能夠攝取PKH67標記的外泌體。(3)流式細胞術(shù)和免疫組化結(jié)果均顯示,與對照組和Exo-con組相比,Exo-MT可明顯下調(diào)THP-1巨噬細胞PD-L1。CBA炎癥因子檢測結(jié)果顯示,Exo-con可上調(diào)IL-1β,IL-6,IL-10和TNF-α等炎癥因子的表達,Exo-TM則可以下調(diào)這些細胞因子的表達。類似的,將Exo-con和Exo-MT通過尾靜脈注射到裸鼠體內(nèi),Exo-con注射小鼠的腹腔巨噬細胞PD-L1表達也明顯上升,且細胞上清中的IL-6,IL-10和TNF-α等炎癥因子的表達明顯升高,而Exo-MT組則明顯下降。(4)Western-Blot檢測顯示Exo-MT與THP-1巨噬細胞共培養(yǎng)24小時后能顯著下調(diào)p-STAT3蛋白水平。而Exo-con則明顯上調(diào)了p-STAT3蛋白的表達。提示外泌體可能是通過STAT3信號通路調(diào)節(jié)PD-L1的表達。(5)使用STAT3抑制劑使THP-1巨噬細胞STAT3蛋白明顯下調(diào),流式細胞術(shù)檢測結(jié)果顯示,抑制劑能夠明顯降低Exo-con作用下THP-1巨噬細胞的PD-L1表達水平。進一步證實了STAT3信號通路在外泌體調(diào)控PD-L1表達中的作用。結(jié)論(1)褪黑素不影響肝癌細胞外泌體量的分泌。(2)肝癌細胞分泌以及褪黑素誘導肝癌細胞釋放的外泌體能夠被巨噬細胞吞噬攝取,并通過調(diào)節(jié)PD-L1和炎癥因子表達等方式影響其免疫功能。(3)褪黑素誘導肝癌細胞釋放的外泌體通過下調(diào)STAT3蛋白的表達通路下調(diào)巨噬細胞PD-L1表達。
[Abstract]:The research background of hepatocellular carcinoma (hepatocellular, carcinoma, HCC) is one of the most common malignant tumor of the digestive system, the mortality rate is fifth, and has a high resistance and a lack of effective treatment. Although in the past few decades a lot of advanced treatment methods but there are still a lot of people died of liver cancer. Surgical treatment is very effective however, many patients have lost the chance of operation. Systemic chemotherapy is the main method for the treatment of patients with advanced hepatocellular carcinoma, but whether single medicine or treatment effect is not ideal. The traditional treatment method seems to have reached a plateau. Therefore, how to improve the therapeutic effect of HCC is a hot spot in the study of tumor the current clinical. Tumor immunotherapy achieved a series of breakthroughs, and clarify the mechanism of drug related immune function, provide a strong basis for the better clinical application, finally achieve the The high treatment effect, prolong the survival time of patients, and has very important significance. The tumor microenvironment (tumor microenviroment) is usually composed of various cells, including tumor cells, stromal cells, immune cells (such as dendritic cells, macrophages and T lymphocytes, etc.) with the tumors. The development and metastasis are closely linked. Immune cells play a crucial role in the in vivo environment, through immune surveillance and immune clearance to maintain the body's normal and stable. In the occurrence of tumor development, tumor cells will affect the microenvironment function causing immune suppression in response to the harsh living conditions, to maintain their own survival macrophage is a regulator of strong cells in maintaining homeostasis, which plays an important role in maintaining immune state. Many researches showed that the macrophage is swollen One important part of the tumor microenvironment in tumor microenvironment, and macrophages in the role in a variety of factors often occur phenotype into tumor associated macrophages (tumor-associated macrophage, TAM TAM), the function will be changed, can cause the immune status in the micro environment change, and promote the tumor cell proliferation. To promote tumor invasion and migration of tumor cells. However, the mechanism by which to change the original macrophages play a role in the regulation of anti tumor immunity as immunosuppressive macrophages, thereby promoting tumor development, whether through the way to mitigate the effects of melatonin induced inhibition of this tumor immune mechanism is not clear.Exosome is a diameter of about a round or oval sac 30-100nm bubble like structure, which comprises a large number of substances in the cell signal transduction play an important role. Therefore, deep The influence of microenvironment of tumor cells in macrophages, and then change the immune function has important clinical significance. This paper intends to explore the liver cancer cell exosome induced by melatonin can influence the immune function of macrophages and its possible molecular mechanism. The purpose of this is exosome and the release of melatonin induced and non induced macrophage cell co culture, observation of macrophage PD-L1 (programmed death ligand) and expression of inflammatory factors, to understand the influence of melatonin on liver cancer cells and macrophage immune function. Through the STAT3 signaling pathway related research, further study of liver exosome PD-L1 expression of macrophage specific regulation mechanism of melatonin induced by melatonin. Methods (1) with liver cancer cells the use of 0.1M M were cultured for 24 hours, were collected in MT treatment group (Exo-MT) and untreated cells (Exo-con). By using Exo Quick-TC exosome extraction kit, transmission electron microscopy (Transmission Electron, Microscope, TEM) to observe the morphology of exosome, size, Western-Blot method to detect exosomes protein markers CD63, TSG101 and the expression of molecular chaperone protein Calnexin. (2) the use of membrane dye PKH67 labeled exosome, then the PKH67 labeled exosomes were co cultured with macrophages sterile or intravenous injection into nude mice after 12h confocal microscopy to observe the macrophage phagocytosis of exosome. (3) exosome will be different (Exo-con and Exo-MT) and THP-1 (by 100ng/ml macrophages induced by PMA for 48 hours from 24 hours) were cultured, the cells were collected and the supernatant, using flow cytometry to detect the expression of PD-L1 the expression of inflammatory cytokines, CBA cell factor detection kit. (4) respectively for the injection of 100 L PBS, Exo-con and Exo-MT, once every other day, a total of 10 times 24h. After the injection, mice were sacrificed to collect peritoneal macrophages adherent after 2 hours for liquid discarding non adherent cells, the remaining cells were cultured overnight and the supernatant were collected after the expression of PD-L1 and inflammatory factors, flow cytometry. (5) were collected from Exo-con and Exo-MT and THP-1 macrophages after 24 hours cell co culture, total protein extraction, change of Western-Blot method for detection of STAT3 and p-STAT3 protein level. (6) the use of STAT3 inhibitors to inhibit macrophage STAT3 expression, Western-Blot method for detection of STAT3 pathway protein changes were detected by flow cytometry after inhibition of THP-1 macrophage PD-L1 expression. Results (1) transmission electron microscopy showed that exosomes were collected round or oval membranous vesicle like structures, diameter of 30-100nm, with typical characteristics of.Western-Blot detection of exosomes showed the "exosome" expression of CD63, but not the expression of endoplasmic reticulum points Chaperone protein Calnexin, further evidence collected in the supernatant of the total protein secreted exosomes.BCA protein quantitative method without cells quantitatively, results show that the exosome secretion of human hepatoma cells MT after 24h were not significantly increased. (2) laser confocal microscopy showed that excretion PKH67 marker can be THP-1 macrophages and macrophage uptake, flow cytometry results prove that peritoneal macrophages can exosome uptake of PKH67 labeled (3). Flow cytometry and immunohistochemical results showed that, compared with the control group and Exo-con group, Exo-MT could significantly decrease THP-1 macrophage inflammatory cytokine PD-L1.CBA test results show that Exo-con can upregulate the expression of IL-10 beta IL-1, IL-6, and TNF- alpha and other inflammatory factors, the expression of Exo-TM can be down regulated these cytokines. Similarly, Exo-con and Exo-MT through the tail vein Injection into nude mice, Exo-con mice injected with peritoneal macrophage PD-L1 expression also increased significantly, and the cells in the supernatant of IL-6, IL-10 and TNF- alpha expression of inflammatory cytokines increased significantly, while Exo-MT group decreased significantly. (4) Exo-MT and THP-1 Western-Blot showed that macrophage cells were cultured after 24 hours could significantly reduce p-STAT3 protein level. Exo-con up-regulated the expression of p-STAT3 protein. Suggesting that exosomes may regulate the expression of PD-L1 through STAT3 signaling pathway. (5) the use of STAT3 inhibitors to THP-1 macrophage STAT3 protein decreased obviously, the results of flow cytometry showed that inhibitors can significantly reduce the expression level of THP-1 macrophage PD-L1 under Exo-con further confirmed the role of STAT3 signaling pathway in the expression regulation of PD-L1 in exosomes. Conclusion (1) does not affect the secretion of melatonin excretion amount of hepatoma cells (2) liver. Cancer cell secretion and excretion of melatonin induced liver cancer cells release can be macrophage uptake, and by regulating the expression of inflammatory factors and PD-L1 etc. affect the immune function. (3) exosomes induce melatonin release in hepatocellular carcinoma cells was down regulated the expression of PD-L1 pathway by down regulating the expression of STAT3 protein.

【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.7

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相關期刊論文 前1條

1 Antonio Facciorusso;Gaetano Serviddio;Nicola Muscatiello;;Local ablative treatments for hepatocellular carcinoma:An updated review[J];World Journal of Gastrointestinal Pharmacology and Therapeutics;2016年04期

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本文編號：1471901