本文選題：脾臟γδT細(xì)胞 + 抗原　； 參考：《北京協(xié)和醫(yī)學(xué)院》2011年碩士論文

【摘要】：T淋巴細(xì)胞表面表達(dá)兩種抗原受體(TCR)即αβTCR和γδTCR。人們目前研究較多的是αβT細(xì)胞,γδT細(xì)胞所占比例較小,僅占正常人外周血淋巴細(xì)胞總數(shù)的3%～10%,但是在機(jī)體免疫調(diào)節(jié)及免疫監(jiān)視中起著重要的作用。γδT細(xì)胞主要識(shí)別以下類型的抗原：MHC分子；磷酸化抗原；熱休克蛋白；非肽類抗原等。本文首先利用三種不同的抗原(即IPP,HSP70和EP6)刺激小鼠脾臟γδT細(xì)胞,以了解三種抗原在不同濃度和不同方式干預(yù)下小鼠脾臟γδT細(xì)胞的增殖純度。 實(shí)驗(yàn)中主要采用流式細(xì)胞術(shù)檢測(cè)細(xì)胞培養(yǎng)中脾臟γδT細(xì)胞的比例。結(jié)果表明,在IPP,HSP70和EP6持續(xù)刺激下脾臟γδT細(xì)胞在培養(yǎng)第三天時(shí)均出現(xiàn)增殖的高峰,而后增殖有所下降。隨著三種抗原濃度的增加,IPP的濃度在1.25ng/ml時(shí)出現(xiàn)脾臟γδT細(xì)胞的增殖高峰,擴(kuò)殖的百分比為5.45%±0.02%(P0.05)；而后隨著濃度的增加增殖減低。在相同濃度下不同干預(yù)方式中發(fā)現(xiàn),三種抗原一次性單次刺激時(shí)亦均在作用后第三天出現(xiàn)增殖高峰,擴(kuò)增的百分比分別為3.60%±0.27%,4.54%±0.16%,3.20%±0.11%(P0.05)；IPP和EP6組第三天增殖高峰時(shí),持續(xù)性刺激的增殖純度大于一次性單次刺激,擴(kuò)增的百分比分別為(6.38%±0.58%VS 3.60%±0.27%,5.41%±0.11% VS 3.20%±0.11%)(P0.05)。 本實(shí)驗(yàn)證實(shí)了在體外刺激脾臟γδT細(xì)胞時(shí),增殖高峰出現(xiàn)在培養(yǎng)的第三天。不同抗原刺激的γδT細(xì)胞種類不同,因而增殖的純度亦不相同。持續(xù)性的抗原刺激較一次性單次刺激更能促進(jìn)γδT細(xì)胞的增殖。 γδT細(xì)胞的免疫調(diào)節(jié)作用越來越受研究者的重視,已知另一種在免疫反應(yīng)中起重要作用的巨噬細(xì)胞廣泛分布于全身各組織中,是機(jī)體抵抗外界入侵的重要防線。雷帕霉素是一種大環(huán)內(nèi)酯類免疫抑制劑,已知的主要靶點(diǎn)是胞漿內(nèi)的激酶mTOR。通過體外培養(yǎng)LPS誘導(dǎo)的小鼠急性肺損傷模型中脾臟γδT細(xì)胞和肺組織巨噬細(xì)胞,以了解脾臟γδT細(xì)胞和肺組織巨噬細(xì)胞之間是否存在相互作用以及體外雷帕霉素對(duì)這種相互作用是否有影響。 實(shí)驗(yàn)中,建立LPS誘導(dǎo)的小鼠急性肺損傷的模型,采用HE染色固定肺組織并觀察其病理改變；流式細(xì)胞術(shù)檢測(cè)磁珠分選前后肺組織巨噬細(xì)胞和脾臟γδT細(xì)胞的純度,熒光顯微鏡下觀察脾臟γδT細(xì)胞的形態(tài),ELISA法檢測(cè)小鼠脾臟γδT細(xì)胞和肺組織巨噬細(xì)胞培養(yǎng)的上清液中炎癥因子IL-4,IL-12,IFN-γ和TNF-α的分泌水平；Real-time PCR檢測(cè)培養(yǎng)細(xì)胞中IL-4,IL-12,IFN-γ和TNF-α的mRNA水平。 實(shí)驗(yàn)結(jié)果顯示在LPS誘導(dǎo)的急性肺損傷模型中,LPS組支氣管肺泡灌洗液的總細(xì)胞和中性粒細(xì)胞濃度明顯大于PBS組和LPS+RAPA組,總細(xì)胞分別為(85.6±31.2VS 10.8±2.5,48.6±26.0)×104個(gè)/ml,中性粒細(xì)胞的分別為(73.4±28.8 VS 1.5±0.5,39.8±24.8)×104個(gè)/ml(PO.05)。脾臟γδT細(xì)胞與肺組織巨噬細(xì)胞按1:1混合培養(yǎng)時(shí),LPS組上清液IFN-γ高于PBS組,RAPA組和LPS+RAPA組(174.17±42.16 VS 16.80±8.80,12.19±4.14,7.16±3.03)pg/ml,(P0.05)。IL-4高于PBS組和LPS+RAPA組(73.82±61.71 VS 19.19±10.01,12.35±1.86)pg/ml,(P0.05)。而TNF-α只高于RAPA組和LPS+RAPA組(34.24±8.08 VS 12.20±4.56,10.56±2.99)pg/ml,(P0.05)�；旌吓囵B(yǎng)時(shí)LPS組IFN-γmRNA的表達(dá)明顯高于PBS組(10.67±4.62 VS 1),(P0.05)。 本研究中體外將脾臟γδT細(xì)胞和肺組織巨噬細(xì)胞分離純化后進(jìn)行培養(yǎng),使得細(xì)胞生長的內(nèi)環(huán)境受外界干擾較少,較真實(shí)的反映細(xì)胞的生物學(xué)活性。本實(shí)驗(yàn)結(jié)果證實(shí)了體內(nèi)實(shí)驗(yàn)中RAPA能抑制LPS誘導(dǎo)小鼠急性肺損傷的支氣管肺泡灌洗液中總細(xì)胞和中性粒細(xì)胞的增殖,體外實(shí)驗(yàn)中顯示了脾臟γδT細(xì)胞和肺組織巨噬細(xì)胞單獨(dú)培養(yǎng)時(shí)未見明顯的變化,但混合培養(yǎng)時(shí)細(xì)胞因子分泌產(chǎn)生很大變化,說明脾臟γδT細(xì)胞和肺組織巨噬細(xì)胞可能存在細(xì)胞間的相互作用從而影響了各自的生物學(xué)活性。在混合培養(yǎng)時(shí),LPS能刺激脾臟γδT細(xì)胞和肺組織巨噬細(xì)胞分泌較多的IFN-γ和IL-4,并能增加混合培養(yǎng)細(xì)胞中IFN-γmRNA的表達(dá)。RAPA能抑制混合培養(yǎng)分泌的IFN-γ,IL-4和TNF-α,說明RAPA抑制了脾臟γδT細(xì)胞和肺組織巨噬細(xì)胞的相互作用,該抑制作用對(duì)基因轉(zhuǎn)錄水平?jīng)]有任何影響。其具體作用機(jī)制還需要我們?cè)诮窈蟮难芯恐刑接懸詫ふ已装Y疾病治療的新靶點(diǎn)。
[Abstract]:On the surface of T lymphocyte, two kinds of antigen receptor (TCR), that is, alpha beta TCR and gamma delta TCR., are widely studied. The proportion of gamma delta T cells is small, which accounts for only 3% to 10% of the total number of lymphocytes in normal human peripheral blood, but plays an important role in immune regulation and immune surveillance. The antigen: MHC molecule, phosphorylation antigen, heat shock protein, non peptide antigen and so on. First of all, three different antigens (IPP, HSP70 and EP6) were used to stimulate the spleen Delta T cells of the spleen in order to understand the proliferation purity of the three kinds of antigen in the spleen of the spleen of the mice under the intervention of different concentrations and different ways.
In the experiment, the proportion of splenic Delta T cells in cell culture was detected by flow cytometry. The results showed that the proliferation peak of splenic Delta T cells in IPP, HSP70 and EP6 stimulated third days, and then the proliferation decreased. With the increase of the concentration of three antigens, the concentration of IPP in 1.25ng/ml appeared in the spleen of the spleen Delta T fine. The proliferating peak of the cell was 5.45% + 0.02% (P0.05), and then the proliferation decreased with the increase of concentration. At the same concentration, the proliferation peak was found at third days after the action of three kinds of antigen, 3.60% + 0.27%, 4.54% + 0.16%, 3.20% + 0.11% (P0. 05); when the proliferation peak at third days in IPP and EP6 groups, the purity of the sustained stimulation was greater than that of one time single stimulus, and the percentage of the amplification was (6.38% + 0.58%VS 3.60% + 0.27%, 5.41% + 0.11% VS 3.20% + 0.11%) (P0.05).
The experiment confirmed that the proliferation peak appeared at third days in vitro stimulation of the spleen Delta T cells. The species of gamma delta T cells stimulated by different antigens were different, and the purity of the proliferation was different.
The immunomodulatory effect of gamma delta T cells is becoming more and more important. Another known macrophage, which plays an important role in the immune response, is widely distributed throughout the body and is an important defense against the invasion of the outside world. Rapamycin is a macrolide immunosuppressant. The main target is the kinase mT in the cytoplasm. OR. through in vitro culture of LPS induced mouse acute lung injury model of splenic Delta T cells and lung tissue macrophages, in order to understand whether there is a interaction between the splenic Delta T cells and the macrophages of the lung tissue and whether there is any effect of rapamycin on this interaction in vitro.
In the experiment, the model of LPS induced acute lung injury in mice was established. The lung tissue was fixed by HE staining and its pathological changes were observed. The purity of the macrophages and splenic Delta T cells in the lung tissue were detected by flow cytometry before and after the separation of magnetic beads. The morphology of the splenic Delta T cells was observed under the fluorescence microscope. The spleen Delta T cells and lungs of the mice were detected by ELISA method. The secretion level of inflammatory factors IL-4, IL-12, IFN- gamma and TNF- alpha in supernatant of macrophage culture was organized, and Real-time PCR was used to detect mRNA levels of IL-4, IL-12, IFN- gamma and TNF- alpha in cultured cells.
The results showed that in the model of LPS induced acute lung injury, the concentration of total cells and neutrophils in the bronchoalveolar lavage fluid in group LPS was significantly greater than that of group PBS and LPS+RAPA, and the total cells were (85.6 + 31.2VS 10.8 + 2.5,48.6 + 26) x 104 /ml respectively, and the neutrophils were divided into (73.4 + 28.8 VS 1.5, 0.5,39.8 + 24.8) x 104 /ml (PO.) 05) when the splenic Delta T cells and macrophages were mixed with 1:1 in the lung tissue, the supernatant of the LPS group was higher than the PBS group, the RAPA group and the LPS+RAPA group (174.17 + 42.16 VS 16.80 + 8.80,12.19 + 4.14,7.16 + 3.03) pg/ml, which were higher than those of 73.82 + 61.71 (73.82 + 61.71 and 19.19 + 1.86). Group and LPS+RAPA group (34.24 + 8.08 VS 12.20 + 4.56,10.56 + 2.99) pg/ml, (P0.05). The expression of IFN- y mRNA in group LPS was significantly higher than that in group PBS (10.67 + 4.62 VS 1) at mixed culture, (P0.05).
In this study, the splenic Delta T cells and lung tissue macrophages were isolated and purified in this study. The internal environment of the cell growth was less disturbed and the biological activity of the cells was truly reflected. The results of the experiment confirmed that in the experiment, RAPA could inhibit the bronchoalveolar lavage fluid induced by LPS in the acute lung injury of rats. The proliferation of total and neutrophils showed that there was no obvious change in the splenic Delta T cells and lung tissue macrophages in vitro, but the secretion of cytokines produced a great change in mixed culture, indicating that there might be intercellular interactions between the splenic Delta T cells and the macrophages in the lung tissue. In mixed culture, LPS stimulates the secretion of more IFN- gamma and IL-4 by macrophages in the spleen Delta T cells and lung tissue, and can increase the expression of IFN- gamma mRNA in mixed culture cells, which can inhibit the IFN- gamma, IL-4 and TNF- alpha in mixed culture, indicating that RAPA inhibits the interaction between the spleen and the macrophages of the lung tissue. The inhibitory effect has no effect on the transcriptional level of the gene. Its specific mechanism also requires us to explore new targets for the treatment of inflammatory diseases in future studies.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1

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