發(fā)布時間：2018-06-11 19:29

  本文選題：X射線 + 輻射��； 參考：《遼寧醫(yī)學院》2012年碩士論文

【摘要】：目的 觀察小劑量X射線照射對體外培養(yǎng)不同時間的人外周血樹突狀細胞（dendriticcell, DC）凋亡、成熟能力、抗原遞呈能力和致敏T細胞能力的影響并探討其初步機制，為系統(tǒng)研究小劑量照射對人免疫功能影響提供實驗依據(jù)。 方法 分離人外周血單個核細胞（PBMC），以細胞因子GM-CSF及IL-4誘導分化、腫瘤抗原及TNF-α刺激DC成熟，以LIN抗體純化，HLA-DR和CD11C抗體鑒定。實驗分為三組：A組：DC培養(yǎng)至第2天接受X線照射；B組：DC培養(yǎng)至第6天接受X線照射，A、B組細胞受照劑量分別為0.05Gy、0.1Gy、0.2Gy和0.5Gy；對照組為假照射組。照射后24h及48h分別以Annexin V聯(lián)合PI雙染法檢測DC凋亡率，流式細胞儀檢測細胞膜表面分子標記物CD80、CD83表達量；各組DC培養(yǎng)至第8天時，以DC致敏T細胞，CCK-8(Cell Counting Kit-8)法檢測混合淋巴細胞反應（MLR，mixed lymphocyte reaction)；MTT法檢測A549細胞數(shù)量；ELISA檢測DC細胞培養(yǎng)液中白介素12（IL-12）、淋巴細胞培養(yǎng)液中γ-干擾素（IFN-γ）水平。 結(jié)果 1.按本實驗方法培養(yǎng)的DC成熟度高，純度高，可用于本研究。 2.0.5Gy照射培養(yǎng)不同時間DC，A、B兩組24、48h檢測凋亡率顯著高于對照組(A組24、48h, t值分別2.88，2.54；B組24、48h，t值分別為2.97，3.73,P 0.05)，其它劑量組無顯著差異（P0.05）。 3.0.2Gy、0.5Gy照射后，A組CD80、CD83表達量顯著下降（P0.05），B組CD80、CD83表達量顯著上升（P0.05）；其它劑量組無顯著差異（P0.05）。 4.受到0.2Gy、0.5Gy照射的DC，培養(yǎng)至第8天時其致敏T細胞增殖的數(shù)量，A組顯著低于對照組（t值分別為2.79、3,71，P0.05），B組明顯高于對照組（t值分別為3.60、3.11，P0.05），，其它劑量組無顯著差異（P0.05）；0.2Gy、0.5Gy照射，A組DC分泌IL-12水平明顯下降（t值分別為4.44、6.93，P0.05），B組DC分泌IL-12水平明顯上升（t值分別為3.51、4.12，P0.05），其它劑量組無顯著差異（P0.05）。 5. DC致敏T淋巴細胞對A549的殺傷能力：0.2Gy、0.5Gy照射后，A組明顯低于對照組（t值分別為2.89、2.91, P0.05），B組顯著高于對照組（t值分別為2.91、2.82, P0.05）；0.05Gy和0.1Gy照射后，A組及B組與對照組比較，所測各指標無明顯變化(P0.05)。0.2Gy、0.5Gy照射后，A組DC致敏的T細胞分泌IFN-γ水平下降，而B組DC致敏的T細胞分泌IFN-γ水平明顯上升(P0.05)；0.05Gy和0.1Gy照射后，A組及B組與對照組比較，所測各指標無明顯變化(P0.05)。 結(jié)論 0.5Gy X射線照射后24h及48h，DC的凋亡率顯著增加。培養(yǎng)早期(2d，A組)接受0.2Gy及0.5Gy X射線照射的DC，在培養(yǎng)至第8天時，其成熟能力、抗原遞呈能力、分泌IL-12的能力以及致敏T細胞能力均顯著降低；培養(yǎng)晚期(6d，B組)接受0.2Gy及0.5Gy X射線照射的DC，在培養(yǎng)至第8天時，其成熟能力、抗原遞呈能力、分泌IL-12的能力以及致敏T細胞的能力顯著增強。該發(fā)現(xiàn)對研究小劑量核輻射對人免疫系統(tǒng)功能影響、探索一種體外提高DC功能的新方法提供了實驗依據(jù)。
[Abstract]:Objective to observe the effects of low dose X-ray irradiation on apoptosis, maturation, antigen presentation and sensitized T cells of human peripheral blood dendritic cells (DCs) cultured in vitro for different time and to explore its mechanism. Methods PBMCs were isolated from human peripheral blood mononuclear cells (PBMC) and differentiated by cytokines GM-CSF and IL-4. Tumor antigen and TNF- 偽 stimulated DC maturation. HLA-DR and CD11C antibodies were purified by Lin antibody. The experiment was divided into three groups: group A: DC was cultured to the second day after X ray irradiation, group B: DC was cultured to the 6th day, the irradiation dose of cells in group A was 0. 05 Gy 0. 1 Gy and 0. 5 Gy, respectively, and the control group was sham irradiation group. The apoptotic rate of DC was detected by Annexin V combined with Pi double staining at 24 h and 48 h after irradiation, and the expression of CD80 CD83, a molecular marker on cell membrane, was detected by flow cytometry. The mixed lymphocyte reaction (MLR mixed lymphocyte reactionation) assay was used to detect the number of A549 cells. Elisa was used to detect the levels of IL-12 and IFN- 緯 in DC cell culture medium. The DC cultured by this method has high maturity and purity. It can be used in the present study. The apoptotic rate in the two groups was significantly higher than that in the control group A at 2448 hours, and the t value in group B was 2.97 ~ 3.73 渭 g / h, respectively. There was no significant difference between the other dose groups in the expression of CD80CD83 in group A after irradiation with 0.5 Gy of 0.2Gy or 0.5 Gy, and the expression of CD80CD83 in group A was significantly higher than that in group A after irradiation with 2.0.5 Gy irradiation at different time. The expression of CD80CD83 in group A was significantly higher than that in group A (2.88 鹵2.54). In group B, the expression of CD80 and CD83 increased significantly (P 0.05), but there was no significant difference in other dose groups (P 0.05). The number of T cell proliferation in group A was significantly lower than that in group A (2.79 渭 g / kg), respectively, and the t value of group B was significantly higher than that of group B (3.60 鹵3.11g / ml), respectively. There was no significant difference between other dose groups (P0.05Gy) and 0.2Gy (0.2Gy) in DC cells of group A, and the number of T cell proliferation in group A was significantly lower than that in group B (P < 0.05). The level of IL-12 secreted by DC was significantly increased in group B (4.44 鹵6.93), respectively. The level of IL-12 secreted by DC was 3.51 鹵4.12, P 0.05, respectively, but there was no significant difference in other dose groups. The cytotoxicity of DC sensitized T lymphocytes to A549 was significantly lower in group A than that in group A (2.89 鹵2.91) after 0.5 Gy irradiation, and significantly higher in group B than that in group B (2.91 鹵2.82, P 0.05Gy 0.05Gy and 0.1 Gy, respectively). The levels of IFN- 緯 in DC sensitized T cells in group A decreased, while those in group B increased significantly after irradiation with P0.05Gy, 0.2Gy, 0.5Gy, respectively, and the levels of IFN- 緯 in group A and group B were significantly increased after irradiation with P0.05Gy and 0.1 Gy, and compared with those in group B and group B, respectively, in comparison with those in group B, and that in group B was significantly higher than that in group B, and the level of IFN- 緯 in group A was significantly higher than that in group B. Conclusion the apoptosis rate of DC increased significantly at 24 h and 48 h after 0.5 Gy X-ray irradiation. At the 8th day, the ability of maturation, antigen presentation, IL-12 secretion and sensitized T cells were significantly decreased in group A (group A) exposed to 0.2 Gy and 0.5 Gy X-ray. At the 8th day, the ability of maturation, antigen presentation, IL-12 secretion and sensitized T cells were significantly enhanced in group B (group B) exposed to 0.2Gy and 0.5Gy X-rays at the end of the incubation period, and the ability of antigen presentation, secretion of IL-12 and the ability of sensitized T cells were significantly enhanced at the 8th day of culture. The findings provide an experimental basis for the study of the effects of low dose nuclear radiation on the function of human immune system and the exploration of a new method to improve the function of DC in vitro.
【學位授予單位】：遼寧醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392

【參考文獻】

相關期刊論文 前6條

1 吳明媛;朱一蓓;周菊英;戴俊;黃勇;夏瑜;強亦忠;張學光;;X射線照射對人樹突狀細胞免疫功能的影響[J];輻射防護;2006年01期

2 黎力;濾泡樹突狀細胞:一些新的概念和進展[J];上海免疫學雜志;2002年03期

3 朱一蓓,王月丹,席泓,顧宗江,古濤,謝煒,強亦忠,張學光;輻射對樹突狀細胞的影響及其機理的研究[J];中華放射醫(yī)學與防護雜志;2002年05期

4 劉曉冬,馬淑梅,劉樹錚;電離輻射對小鼠腹腔巨噬細胞IL-12和NF-κB活性的影響[J];中華放射醫(yī)學與防護雜志;2003年01期

5 王獻理，蘇士杰，尹洪淑，馮樂平，于開君;低劑量輻射對小鼠種植腫瘤及其放療的影響[J];中華放射醫(yī)學與防護雜志;1996年03期

6 于洪升,宋愛琴,盧彥達,邱文生,沈方臻;Effects of low-dose radiation on tumor growth, erythrocyte immune function and SOD activity in tumor-bearing mice[J];Chinese Medical Journal;2004年07期

本文編號：2006400