褪黑素诱导肝癌细胞释放的外泌体对巨噬细胞免疫功能的影响及机制研究

发布时间：2018-01-28 23:50

本文关键词： 肝细胞癌褪黑素外泌体巨噬细胞免疫　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景肝细胞癌(hepatocellular carcinoma,HCC)是消化系统常见的恶性肿瘤之一,死亡率排第五,并且具有高耐药性和缺乏有效的治疗方法。尽管过去的几十年出现很多先进的治疗方法但还是有很多人死于肝癌。外科手术是非常有效的治疗方法,但很多的患者诊断时已失去了手术机会。全身性化疗是晚期肝癌患者的主要治疗方法,但不论是单药还是多药联合治疗效果均不理想。传统的治疗方法似乎已经达到了平台期。因此,如何提高HCC治疗效果是当今肿瘤研究的热点。目前临床肿瘤免疫治疗取得一系列突破,明确药物相关免疫作用及机制,为其更好地应用于临床提供有力依据,最终达到提高治疗效果,延长患者生存期的作用,具有很重要的意义。肿瘤微环境(tumor microenviroment)通常是由多种不同细胞构成,主要包括肿瘤细胞、间质细胞、免疫细胞(如树突状细胞、巨噬细胞和T淋巴细胞等)组成,其与肿瘤的发生、发展和转移有着密切联系。免疫细胞在体内环境中起着至关重要的作用,通过免疫监视,免疫清除维持机体的正常稳定。在肿瘤的发生、发展过程中,肿瘤细胞会影响微环境的功能从而引起免疫抑制状态以应对恶劣的生存条件,维持自身生存。巨噬细胞是一种调节性很强的细胞,在维持机体内环境稳态,维持机体免疫状态等方面具有重要作用。多种研究表明,巨噬细胞是肿瘤微环境中的重要组成部分之一,且肿瘤微环境中的巨噬细胞在多种因素的作用下常常会发生表型变化,转变为肿瘤相关巨噬细胞(tumor-associated macrophage,TAM),TAM的功能会发生改变,可以引起微环境中免疫状态的改变,进而发挥促进肿瘤增殖、侵袭迁移等促肿瘤作用。然而,肿瘤细胞通过何种途径使原来具有抗肿瘤免疫调节作用的巨噬细胞转变为免疫抑制的巨噬细胞从而促进肿瘤发展,能否通过褪黑素影响该途径从而减轻这种肿瘤导致的免疫抑制机制尚不清楚。Exosome是一类直径大约为30-100nm的圆形或类圆形囊泡样结构,其包含有大量物质,在细胞间信号传递中发挥重要作用。因此,深入研究微环境中的肿瘤细胞影响巨噬细胞,进而改变其免疫功能具有重要的临床意义。本课题拟探讨褪黑素诱导的肝癌细胞外泌体能否影响巨噬细胞的免疫功能及可能的分子机制。目的通过将处于褪黑素诱导与未诱导的肝癌细胞释放的exosome与巨噬细胞共培养,观察巨噬细胞PD-L1(程序性死亡配体)及炎症因子的表达状况,了解褪黑素作用的肝癌细胞对巨噬细胞免疫功能的影响。并通过研究相关的STAT3信号通路,进一步研究褪黑素诱导的肝癌exosome调控巨噬细胞PD-L1表达的具体机制。方法(1)使用0.1m M的褪黑素与肝癌细胞共培养24小时,分别收集MT处理组(Exo-MT)和未处理组细胞(Exo-con)的上清液,使用Exo Quick-TC试剂盒提取exosome,透射电子显微镜(Transmission Electron Microscope,TEM)观察exosome的形态、大小,Western-Blot方法检测exosomes标志蛋白CD63、TSG101以及分子伴侣蛋白Calnexin的表达。(2)使用膜染料PKH67标记exosome,然后将PKH67标记的exosomes与巨噬细胞共同孵育或尾静脉注射到裸鼠体内,12h后共聚焦显微镜观察巨噬细胞吞噬exosome的情况。(3)将不同的exosome(Exo-con和Exo-MT)与THP-1巨噬细胞(经100ng/ml PMA诱导48小时所得)共培养24小时,分别收集细胞和上清液,使用流式细胞仪检测PD-L1表达,CBA细胞因子试剂盒检测炎症因子表达。(4)分别对裸鼠尾静脉注射100μl的PBS、Exo-con和Exo-MT,隔日一次,共10次。注射结束后24h,处死裸鼠收集腹腔巨噬细胞,贴壁2小时后换液弃去未贴壁细胞,剩余细胞培养过夜后收集细胞和上清液,流式细胞仪检测PD-L1和炎症因子的表达。(5)分别收集Exo-con和Exo-MT与THP-1巨噬细胞共培养24小时后的细胞,提取总蛋白,Western-Blot方法检测STAT3与p-STAT3等蛋白水平变化。(6)使用STAT3抑制剂抑制巨噬细胞STAT3表达,Western-Blot方法检测STAT3通路蛋白变化,流式细胞术检测抑制后THP-1巨噬细胞PD-L1表达变化。结果(1)透射电镜可见收集到的外泌体呈圆形或类圆形膜性囊泡样结构,直径为30-100nm,符合外泌体的典型特征。Western-Blot检测显示所得的“exosome”表达CD63,但不表达内质网分子伴侣蛋白Calnexin,进一步证明上清中收集到的是不含细胞成分的外泌体。BCA蛋白定量法对所得外泌体总蛋白进行定量,结果表明MT作用24h后肝癌细胞分泌的外泌体无明显增加。(2)激光共聚焦显微镜显示,PKH67标记的外泌体能够被THP-1巨噬细胞和腹腔巨噬细胞所摄取,流式细胞术结果进一步证明腹腔巨噬细胞能够摄取PKH67标记的外泌体。(3)流式细胞术和免疫组化结果均显示,与对照组和Exo-con组相比,Exo-MT可明显下调THP-1巨噬细胞PD-L1。CBA炎症因子检测结果显示,Exo-con可上调IL-1β,IL-6,IL-10和TNF-α等炎症因子的表达,Exo-TM则可以下调这些细胞因子的表达。类似的,将Exo-con和Exo-MT通过尾静脉注射到裸鼠体内,Exo-con注射小鼠的腹腔巨噬细胞PD-L1表达也明显上升,且细胞上清中的IL-6,IL-10和TNF-α等炎症因子的表达明显升高,而Exo-MT组则明显下降。(4)Western-Blot检测显示Exo-MT与THP-1巨噬细胞共培养24小时后能显著下调p-STAT3蛋白水平。而Exo-con则明显上调了p-STAT3蛋白的表达。提示外泌体可能是通过STAT3信号通路调节PD-L1的表达。(5)使用STAT3抑制剂使THP-1巨噬细胞STAT3蛋白明显下调,流式细胞术检测结果显示,抑制剂能够明显降低Exo-con作用下THP-1巨噬细胞的PD-L1表达水平。进一步证实了STAT3信号通路在外泌体调控PD-L1表达中的作用。结论(1)褪黑素不影响肝癌细胞外泌体量的分泌。(2)肝癌细胞分泌以及褪黑素诱导肝癌细胞释放的外泌体能够被巨噬细胞吞噬摄取,并通过调节PD-L1和炎症因子表达等方式影响其免疫功能。(3)褪黑素诱导肝癌细胞释放的外泌体通过下调STAT3蛋白的表达通路下调巨噬细胞PD-L1表达。
[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.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

【参考文献】