肿瘤相关巨噬细胞的自噬调节对胃癌放射敏感性的影响及机制研究

发布时间：2018-05-15 17:47

本文选题：肿瘤相关巨噬细胞 + 自噬　；参考：《苏州大学》2016年博士论文

【摘要】：目的:研究调节肿瘤相关巨噬细胞(Tumor associated macrophages,TAMs)的自噬状态对共培养胃癌细胞放射敏感性的影响并探讨其可能机制,为胃癌的放射增敏研究提供新的思路;通过观察自噬调节预处理的肿瘤相关巨噬细胞(TAMs)与胃癌细胞混合接种裸鼠的生长情况及移植瘤的放射敏感性,初步探索通过调控TAMs自噬状态影响肿瘤放射治疗效果的可能性。方法:(一)通过选择激活途径使用佛波酯(PMA)、人重组白细胞介素-4(interleukin-4,IL-4)激活人单核细胞THP-1分化为Ⅱ型肿瘤相关巨噬细胞(TAMs)后,采用流式细胞术鉴定选择激活途径激活所得细胞表面CD68、CD204、CD206标志性分子表达情况;分别用不同自噬调节剂雷帕霉素(Rapamycin)和巴弗洛霉素A1(Bafilomycin A1)干预其自噬,MDC(Monodansylcardeverine)荧光染色法、Lyso-Tracker Red和Mito-Traker Green免疫荧光标记法检测线粒体和溶酶体的表达、免疫荧光检测自噬标记性蛋白MAP1 LC3及透射电镜观察自噬调控剂作用后各组Ⅱ型肿瘤相关巨噬细胞自噬的发生情况。(二)采用非接触共培养方式将自噬上调组Ⅱ型肿瘤相关巨噬细胞、空白实验组、自噬下调组Ⅱ型肿瘤相关巨噬细胞与人胃癌MGC803细胞共同培养48h。检测辐照前后自噬调节剂对共培养胃癌MGC803细胞放射反应能力的表达情况,MDC荧光染色法、Lyso-Tracker Red和Mito-Traker Green免疫荧光标记法检测线粒体和溶酶体的表达、免疫荧光检测自噬标记性蛋白LC3及透射电镜观察自噬调控剂调节各组Ⅱ型肿瘤相关巨噬细胞自噬并辐照后胃癌MGC803细胞自噬的表达情况。应用Western Blot技术分别对各组胃癌细胞相关蛋白caspase 3、7,cathepasin B、D、E、L进行检测。(三)在体外实验基础上,利用雷帕霉素与巴弗洛霉素A1分别对TAMs进行自噬上调与下调,按照3:1的比例与MGC803胃癌细胞混合接种于BALB/c裸鼠,观察各种成瘤率、成瘤时间,瘤体体积变化与小鼠体重改变,并对成瘤裸鼠进行局部外照射,比较各组受照后小鼠体重与瘤体体积变化及照后7d瘤体重量,并通过HE染色、免疫组化等方法观察照后病理改变及相关蛋白表达变化。结果:(一)人单核细胞白血病细胞THP-1在通过佛波酯(PMA)、人重组白介素-4(IL-4)依次序贯作用总计72h小时后,检测所得贴壁细胞表面CD68、CD204、CD206的表达水平,流式细胞检测显示经上述2种药物作用后所得的细胞表面CD68、CD204、CD206表达皆高于未经处理的THP-1细胞,差异具有统计学意义(P0.05)。针对Ⅱ型肿瘤相关巨噬细胞自噬水平的LC3免疫荧光及自噬囊泡(Autophagic Vacuoles,AVs)荧光蛋白染色检测结果显示:经过一定浓度自噬上调控剂作用后的Ⅱ型肿瘤相关巨噬细胞自噬标志性蛋白MAP1 LC3(microtubule-associated protein 1 light chain 3)在撤除干预药物48h后仍然有显著表达。结合MDC染色实验结果,Lyso-Tracker Red及Mito-Traker Green免疫荧光双标记法的检测结果,在预处理之后的M2-TAMs自噬状态的维持可持续一段时间,可以作为共培养的研究试剂使用。(二)经一定剂量(4gy)照射后,MGC803细胞与不同自噬状态M2-TAMs共培养,克隆形成实验显示自噬上调组克隆形成数最多,自噬下调组克隆数最少;在放射敏感性方面,M2-TAMs细胞自噬上调促进了MGC803细胞的克隆形成率,抑制其凋亡,M2-TAMs自噬下调对MGC803细胞的影响则相反,抑制M2-TAMs自噬有望提高共培养体系中胃癌细胞的放射敏感性。M2-TAMs自噬改变,对肿瘤相关蛋白的表达也产生显著影响,自噬上调组的cathepsin、B、D、E基因表达上调,而caspase-3、7以及cathepsin L表达下调,自噬下调组为cathepsin、B、D、E表达下调,而caspase-3、7以及cathepsin L表达上调。(三)在体外实验基础上,我们建立胃癌移植瘤裸鼠模型;TAMs自噬上调组肿瘤体积最大、瘤体最重,自噬上调组COX-2,PD-L1,TNF-α表达最强,PTEN表达最弱。而自噬下调组肿瘤最小,瘤体最轻,COX-2,PD-L1,TNF-α表达最少,辐照后自噬下调组明显抑制COX-2,PD-L1,TNF-α表达,而促进PTEN表达。结论:上调TAMs自噬水平,可降低共培养胃癌细胞的放射敏感性,而抑制TAMs自噬,可增加共培养胃癌细胞的放射敏感性;肿瘤微环境中TAMs自噬的调节状态,可能是肿瘤放射抵抗的机制之一,抑制TAMs的自噬,有望增加肿瘤的放射敏感性,提高肿瘤放疗效果,值得进一步深入研究。
[Abstract]:Objective: To study the effect of autophagy regulating the autophagy status of Tumor associated macrophages (TAMs) on the radiosensitivity of co cultured gastric cancer cells and explore its possible mechanism to provide a new idea for the study of radiation sensitization of gastric cancer. By observing the mixed macrophage (TAMs) pretreated with autophagy (TAMs) and gastric cancer cells The growth of nude mice and the radiosensitivity of transplanted tumor were preliminarily explored by regulating the effect of autophagy on TAMs. Methods: (1) using the selective activation pathway to use PMA, recombinant human interleukin -4 (interleukin-4, IL-4) to activate human monocyte THP-1 to differentiate into type II tumor phase After TAMs, flow cytometry was used to identify the expression of CD68, CD204, CD206 markers on the surface of the cell surface activated by selective activation pathway, and the autophagy (Rapamycin) and buffalamycin A1 (Bafilomycin A1) were used to interfere with the autophagy, MDC (Monodansylcardeverine) fluorescence staining, Lyso-Tracker R, respectively. ED and Mito-Traker Green immunofluorescent labeling were used to detect the expression of mitochondria and lysosomes. Immunofluorescence detection of autophagy labeled protein MAP1 LC3 and transmission electron microscopy were used to observe the autophagy of macrophages in type II tumor related macrophages after the action of autophagic regulators. (two) autophagy was up-regulated by autophagy. Guan macrophage, blank test group, autophagy - regulated group II tumor related macrophages and human gastric cancer MGC803 cells co culture 48h. to detect the expression of radioactivity of autophagic modifiers on co cultured MGC803 cells, MDC fluorescence staining, Lyso-Tracker Red and Mito-Traker Green immunofluorescence detection lines The expression of the granules and lysosomes, the immunofluorescence detection of autophagy labeled protein LC3 and the transmission electron microscope to observe autophagy regulating the autophagy of macrophages related to the type II tumor and the expression of autophagy in MGC803 cells after irradiation. The Western Blot technique was applied to the gastric cancer cell related proteins caspase 3,7, cathepasin B, D, E, L. (three) on the basis of in vitro experiment, the autophagy was up-regulated and down regulated by rapamycin and buffalomycin A1, and the MGC803 gastric cancer cells were inoculated in BALB/c nude mice according to the proportion of 3:1, and the tumorigenesis rate, the tumorigenesis time, the volume change of the tumor body and the weight change of the mice were observed, and the tumor nude mice were localized. The changes of body weight and tumor volume and 7d tumor weight after illumination were compared and the changes of pathological changes and related protein expression were observed by HE staining and immunohistochemistry. Results: (1) the sequence of THP-1 in human monocytic leukemia cells was 72 in sequence through PF (PMA) and recombinant human interleukin -4 (IL-4). After H hours, the expression level of CD68, CD204 and CD206 on the surface of the adherent cells was detected. Flow cytometry showed that the expression of CD68, CD204 and CD206 on the surface of cells after these 2 drugs was higher than that of untreated THP-1 cells, and the difference was statistically significant (P0.05). LC3 immunization against the autophagy level of macrophages in type II tumor related macrophages. The results of fluorescence and autophagic vesicles (Autophagic Vacuoles, AVs) fluorescent protein staining showed that the autophagic related macrophage autophagic marker protein MAP1 LC3 (microtubule-associated protein 1 light chain 3) after a certain concentration of autophagic regulators (microtubule-associated protein 1 light chain 3) was still significantly expressed after the withdrawal of the intervention drug 48h. The results of the color test, the results of Lyso-Tracker Red and Mito-Traker Green immunofluorescence double labeling method, can be used as co culture research reagent for the sustained period of M2-TAMs autophagy after pretreatment. (two) after a certain dose (4Gy) irradiation, MGC803 cells are co cultured with different autophagic states M2-TAMs and cloned. The formation of autophagy up-regulated group has the highest clone formation number, and the number of autophagy down-regulation group is the least. In the radiosensitivity, the autophagy up regulation of M2-TAMs cells promotes the clone formation rate of MGC803 cells and inhibits its apoptosis. The effect of autophagy down regulation of M2-TAMs on MGC803 cells is opposite. The inhibition of M2-TAMs autophagy is expected to improve the stomach of the co culture system. The radiosensitivity of the cancer cells was altered by autophagy.M2-TAMs, and the expression of tumor related proteins was also significantly affected. The expression of cathepsin, B, D, E gene was up regulated in the up regulation group, and the expression of caspase-3,7 and cathepsin L was down regulated. The down regulation group of autophagy was cathepsin, B, D, down regulation. (three) in body On the basis of external experiment, we established the nude mouse model of gastric cancer transplant tumor. The TAMs up regulation group had the largest tumor volume, the heaviest tumor, the COX-2, PD-L1, TNF- alpha expression in the autophagy up group was the strongest, the PTEN expression was the weakest. The least tumor in the autophagy group, the least tumor body, the least expression of COX-2, PD-L1, TNF- a, and the decrease of COX-2, PD-L1, TNF in the autophagy reduction group after irradiation. - alpha expression and promote PTEN expression. Conclusion: up regulation of TAMs autophagy can reduce the radiosensitivity of co cultured gastric cancer cells, and inhibit TAMs autophagy, and increase the radiosensitivity of co cultured gastric cancer cells. The regulation state of TAMs autophagy in tumor microenvironment may be one of the mechanisms of tumor ejection resistance, inhibiting autophagy of TAMs is expected to increase. The radiosensitivity of tumors will enhance the effectiveness of radiotherapy.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

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