本文選題：樹突狀細(xì)胞 + 細(xì)胞因子　； 參考：《瀘州醫(yī)學(xué)院》2010年碩士論文

【摘要】： 目的：ω-3多不飽和脂肪酸(ω-3 polyunsaturated fatty acids,ω-3 PUFAs)是營養(yǎng)免疫中小分子物質(zhì)的重要組成部分,其主要成分包括二十碳五烯酸(EPA)和二十二碳六烯酸(DHA)。樹突狀細(xì)胞(Dendritic cells, DCs)具有激活免疫排斥和誘導(dǎo)免疫耐受的雙重潛力,兩者此消彼長。經(jīng)DCs誘導(dǎo)耐受是延長移植物存活的有效方法。本研究通過人外周血單個核細(xì)胞來源的樹突狀細(xì)胞的體外培養(yǎng),對DCs進(jìn)行形態(tài)學(xué)觀察、細(xì)胞因子及免疫表型的檢測,探討ω-3多不飽和脂肪酸對樹突狀細(xì)胞成熟狀態(tài)及免疫學(xué)功能的影響。方法：取新鮮健康成人外周血,經(jīng)淋巴細(xì)胞分離液梯度分離,取中間白膜層細(xì)胞,通過貼壁處理,獲得單個核細(xì)胞,加入含10%胎牛血清的RPMI 1640完全培養(yǎng)基、人重組集落刺激因子(rhGM-CSF)、人重組白細(xì)胞介素-4(rhIL-4),使其終濃度為：rhGM-CSF 500U/ml, rhIL-4250U/ml,隔日半量換液,誘導(dǎo)5天,獲得未成熟樹突狀細(xì)胞(immature DC, imDC),然后以ω-3多不飽和脂肪酸二十碳五烯酸(EPA),二十二碳六烯酸(DHA)或飽和脂肪酸硬脂酸(SA)處理細(xì)胞24h,并將其分為5組：未成熟DCs組、成熟DCs組、EPA處理組、DHA處理組和SA處理組。內(nèi)毒素脂多糖(LPS)誘導(dǎo)細(xì)胞成熟后,細(xì)胞爬片Wright-Giemsa染色觀察各組樹突狀細(xì)胞形態(tài)；采用半定量反轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)檢測樹突狀細(xì)胞共刺激分子CD80 mRNA、CD86 mRNA及表面抗原提呈分子人類白細(xì)胞抗原-DR(HLA-DR) mRNA的表達(dá)；采用酶聯(lián)免疫吸附試驗(ELISA)法檢測各組細(xì)胞上清中白細(xì)胞介素-12(IL-12)及腫瘤壞死因子-α(TNF-α)的含量。結(jié)果：未成熟DCs組CD80 mRNA.CD86 mRNA及HLA-DR mRNA的相對表達(dá)量為0.369±0.023、0.573±0.033及0.467±0.020,成熟DCs組這3種分子高表達(dá),其相對表達(dá)量為0.767±0.017、1.160±0.047及0.801±0.016,成熟DCs組與未成熟DCs組之間相比,有顯著性差異(P0.01)；EPA處理組CD80 mRNA.CD86mRNA及HLA-DRmRNA的相對表達(dá)量分別為0.547±0.034、0.849±0.043及0.657±0.023,DHA處理組CD80mRNA.CD86mRNA及HLA-DRmRNA的相對表達(dá)量分別為0.465±0.032.0.875±0.042及0.619±0.019,EPA和DHA處理組與成熟DCs組相比有統(tǒng)計學(xué)意義(P0.01),而SA處理組CD80mRNA.CD86mRNA及HLA-DRmRNA的相對表達(dá)量分別為0.757±0.020、1.132±0.064及0.792±0.018,與成熟組相比差異無統(tǒng)計學(xué)意義(P0.05)。細(xì)胞上清IL-12的含量,5個組分別為190.73±16.26、768.05±62.73.358.71±52.79.412.63±62.64.709.48±47.81 pg／ml,細(xì)胞上清TNF-α的含量,5個組分別為637.61±38.40、2405.73±125.48、1605.64±138.39.1807.38±140-34.2353.34±194.74 pg／ml,EPA及DHA處理組與成熟DCs組相比差異有統(tǒng)計學(xué)意義(P0.01),而SA組與成熟DCs組相比差異無統(tǒng)計學(xué)意義(P0.05)。結(jié)論：ω-3多不飽和脂肪酸可以在體外下調(diào)樹突狀細(xì)胞共刺激分子CD80.CD86及表面抗原提呈分子HLA-DR的表達(dá),降低其細(xì)胞因子白細(xì)胞介素-12及腫瘤壞死因子-α的釋放,減弱樹突狀細(xì)胞的抗原提呈能力,且對樹突狀細(xì)胞的體外成熟有抑制作用,從而增強(qiáng)免疫耐受性,提示ω-3多不飽和脂肪酸可能減輕器官移植中的排斥反應(yīng)。
[Abstract]:Objective: Omega -3 polyunsaturated fatty acids (omega -3 polyunsaturated fatty acids, Omega -3 PUFAs) are important components of medium and small molecular substances in nutritional immunization. Their main components include twenty carbon five enoic acid (EPA) and twenty-two carbon six enoic acid (DHA). Dendritic cells (Dendritic cells, DCs) have double immunological rejection and induction of immune tolerance. DCs induced tolerance is an effective method for prolonging the survival of the grafts. This study was conducted through the culture of dendritic cells derived from human peripheral blood mononuclear cells, morphological observation, cytokine and immunophenotype detection of DCs, and the study of Omega -3 polyunsaturated fatty acids on the mature state and immunity of dendritic cells. Methods: the influence of the function of the epidemic. Methods: the fresh healthy adult peripheral blood was extracted by the gradient separation of lymphocyte separation liquid, and the interleukin cells were obtained. The single nucleus cells were obtained by adherent treatment, and the RPMI 1640 complete medium containing 10% fetal bovine serum was added. The recombinant human colony stimulating factor (rhGM-CSF) and human recombinant interleukin -4 (rhIL-4) were reorganized. The concentration is: rhGM-CSF 500U/ml, rhIL-4250U/ml, half a day for fluid replacement, induction for 5 days, to obtain immature dendritic cells (immature DC, imDC), and then use Omega -3 polyunsaturated fatty acids twenty carbon five enoic acid (EPA), twenty-two carbon six enoic acid (DHA) or saturated fatty acid hard fatty acid (SA) to treat cell 24h, and divide it into 5 groups: immature DCs group, mature Group DCs, EPA treatment group, DHA treatment group and SA treatment group. After endotoxin lipopolysaccharide (LPS) induced cell maturation, cell crawling Wright-Giemsa staining was used to observe the morphology of dendritic cells. Semi quantitative reverse transcriptional polymerase chain reaction (RT-PCR) was used to detect CD80 mRNA in dendritic cells, CD86 mRNA and surface antigen presented molecules. The expression of leucocyte antigen -DR (HLA-DR) mRNA and the content of interleukin -12 (IL-12) and tumor necrosis factor - alpha (TNF- alpha) in the supernatant of each group were detected by enzyme linked immunosorbent assay (ELISA). Results: the relative expression of CD80 mRNA.CD86 mRNA and HLA-DR mRNA.CD86 was 0.369 + 0.033 and 0.467 + 0.020 in the immature DCs group. The high expression of these 3 molecules in mature DCs group was 0.767 + 0.017,1.160 + 0.047 and 0.801 + 0.016. Compared with immature DCs group, the mature DCs group had significant difference (P0.01), and the relative expression of CD80 mRNA.CD86mRNA and HLA-DRmRNA in EPA treatment group was 0.547 + 0.034,0.849 + 0.043 and 0.657 + 0.023, DHA treatment group CD80mRNA The relative expressions of.CD86mRNA and HLA-DRmRNA were 0.465 + 0.032.0.875 + 0.042 and 0.619 + 0.019 respectively. Compared with the mature DCs group, the EPA and DHA treatment groups were statistically significant (P0.01), while the relative expressions of CD80mRNA.CD86mRNA and HLA-DRmRNA were 0.757 + 0.064 and 0.792 + 0.018 respectively in SA treatment group, and there were no differences compared with those of the mature group. Study significance (P0.05). The content of IL-12 in cell supernatant was 190.73 + 16.26768.05 + 16.26768.05 + 62.73.358.71 + 52.79.412.63 + 62.64.709.48 + 47.81 pg / ml, and the content of TNF- alpha in cell supernatant was 637.61 + 38.402405.73 + 125.481605.64 + 138.39.1807.38 + 194.74 194.74 The difference between group Cs and group SA was statistically significant (P0.01), but there was no significant difference between group SA and mature DCs group (P0.05). Conclusion: Omega -3 polyunsaturated fatty acids can reduce the expression of CD80.CD86 and surface antigen presentation of molecular HLA-DR in dendritic cells in vitro, and reduce the cytokines of interleukin -12 and the cause of cancer death. The release of sub - alpha reduces the antigen presenting ability of dendritic cells and inhibits the maturation of dendritic cells in vitro, thus enhancing immune tolerance, suggesting that Omega -3 polyunsaturated fatty acids may mitigate the rejection in organ transplantation.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392.4

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本文編號：1941116