本文選題：腫瘤相關(guān)巨噬細胞 + 自噬�。� 參考：《蘇州大學》2016年博士論文

【摘要】：目的:研究調(diào)節(jié)腫瘤相關(guān)巨噬細胞(Tumor associated macrophages,TAMs)的自噬狀態(tài)對共培養(yǎng)胃癌細胞放射敏感性的影響并探討其可能機制,為胃癌的放射增敏研究提供新的思路;通過觀察自噬調(diào)節(jié)預處理的腫瘤相關(guān)巨噬細胞(TAMs)與胃癌細胞混合接種裸鼠的生長情況及移植瘤的放射敏感性,初步探索通過調(diào)控TAMs自噬狀態(tài)影響腫瘤放射治療效果的可能性。方法:(一)通過選擇激活途徑使用佛波酯(PMA)、人重組白細胞介素-4(interleukin-4,IL-4)激活人單核細胞THP-1分化為Ⅱ型腫瘤相關(guān)巨噬細胞(TAMs)后,采用流式細胞術(shù)鑒定選擇激活途徑激活所得細胞表面CD68、CD204、CD206標志性分子表達情況;分別用不同自噬調(diào)節(jié)劑雷帕霉素(Rapamycin)和巴弗洛霉素A1(Bafilomycin A1)干預其自噬,MDC(Monodansylcardeverine)熒光染色法、Lyso-Tracker Red和Mito-Traker Green免疫熒光標記法檢測線粒體和溶酶體的表達、免疫熒光檢測自噬標記性蛋白MAP1 LC3及透射電鏡觀察自噬調(diào)控劑作用后各組Ⅱ型腫瘤相關(guān)巨噬細胞自噬的發(fā)生情況。(二)采用非接觸共培養(yǎng)方式將自噬上調(diào)組Ⅱ型腫瘤相關(guān)巨噬細胞、空白實驗組、自噬下調(diào)組Ⅱ型腫瘤相關(guān)巨噬細胞與人胃癌MGC803細胞共同培養(yǎng)48h。檢測輻照前后自噬調(diào)節(jié)劑對共培養(yǎng)胃癌MGC803細胞放射反應能力的表達情況,MDC熒光染色法、Lyso-Tracker Red和Mito-Traker Green免疫熒光標記法檢測線粒體和溶酶體的表達、免疫熒光檢測自噬標記性蛋白LC3及透射電鏡觀察自噬調(diào)控劑調(diào)節(jié)各組Ⅱ型腫瘤相關(guān)巨噬細胞自噬并輻照后胃癌MGC803細胞自噬的表達情況。應用Western Blot技術(shù)分別對各組胃癌細胞相關(guān)蛋白caspase 3、7,cathepasin B、D、E、L進行檢測。(三)在體外實驗基礎(chǔ)上,利用雷帕霉素與巴弗洛霉素A1分別對TAMs進行自噬上調(diào)與下調(diào),按照3:1的比例與MGC803胃癌細胞混合接種于BALB/c裸鼠,觀察各種成瘤率、成瘤時間,瘤體體積變化與小鼠體重改變,并對成瘤裸鼠進行局部外照射,比較各組受照后小鼠體重與瘤體體積變化及照后7d瘤體重量,并通過HE染色、免疫組化等方法觀察照后病理改變及相關(guān)蛋白表達變化。結(jié)果:(一)人單核細胞白血病細胞THP-1在通過佛波酯(PMA)、人重組白介素-4(IL-4)依次序貫作用總計72h小時后,檢測所得貼壁細胞表面CD68、CD204、CD206的表達水平,流式細胞檢測顯示經(jīng)上述2種藥物作用后所得的細胞表面CD68、CD204、CD206表達皆高于未經(jīng)處理的THP-1細胞,差異具有統(tǒng)計學意義(P0.05)。針對Ⅱ型腫瘤相關(guān)巨噬細胞自噬水平的LC3免疫熒光及自噬囊泡(Autophagic Vacuoles,AVs)熒光蛋白染色檢測結(jié)果顯示:經(jīng)過一定濃度自噬上調(diào)控劑作用后的Ⅱ型腫瘤相關(guān)巨噬細胞自噬標志性蛋白MAP1 LC3(microtubule-associated protein 1 light chain 3)在撤除干預藥物48h后仍然有顯著表達。結(jié)合MDC染色實驗結(jié)果,Lyso-Tracker Red及Mito-Traker Green免疫熒光雙標記法的檢測結(jié)果,在預處理之后的M2-TAMs自噬狀態(tài)的維持可持續(xù)一段時間,可以作為共培養(yǎng)的研究試劑使用。(二)經(jīng)一定劑量(4gy)照射后,MGC803細胞與不同自噬狀態(tài)M2-TAMs共培養(yǎng),克隆形成實驗顯示自噬上調(diào)組克隆形成數(shù)最多,自噬下調(diào)組克隆數(shù)最少;在放射敏感性方面,M2-TAMs細胞自噬上調(diào)促進了MGC803細胞的克隆形成率,抑制其凋亡,M2-TAMs自噬下調(diào)對MGC803細胞的影響則相反,抑制M2-TAMs自噬有望提高共培養(yǎng)體系中胃癌細胞的放射敏感性。M2-TAMs自噬改變,對腫瘤相關(guān)蛋白的表達也產(chǎn)生顯著影響,自噬上調(diào)組的cathepsin、B、D、E基因表達上調(diào),而caspase-3、7以及cathepsin L表達下調(diào),自噬下調(diào)組為cathepsin、B、D、E表達下調(diào),而caspase-3、7以及cathepsin L表達上調(diào)。(三)在體外實驗基礎(chǔ)上,我們建立胃癌移植瘤裸鼠模型;TAMs自噬上調(diào)組腫瘤體積最大、瘤體最重,自噬上調(diào)組COX-2,PD-L1,TNF-α表達最強,PTEN表達最弱。而自噬下調(diào)組腫瘤最小,瘤體最輕,COX-2,PD-L1,TNF-α表達最少,輻照后自噬下調(diào)組明顯抑制COX-2,PD-L1,TNF-α表達,而促進PTEN表達。結(jié)論:上調(diào)TAMs自噬水平,可降低共培養(yǎng)胃癌細胞的放射敏感性,而抑制TAMs自噬,可增加共培養(yǎng)胃癌細胞的放射敏感性;腫瘤微環(huán)境中TAMs自噬的調(diào)節(jié)狀態(tài),可能是腫瘤放射抵抗的機制之一,抑制TAMs的自噬,有望增加腫瘤的放射敏感性,提高腫瘤放療效果,值得進一步深入研究。
[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.

【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.2

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