本文選題：房水 + 樹突細(xì)胞�。� 參考：《河北醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的:眼處于免疫赦免狀態(tài)和很多因素有關(guān),其中包括解剖因素,如血眼屏障、缺少直接淋巴引流通路等,此外,房水中還存在大量起免疫抑制作用的因子對(duì)免疫細(xì)胞活性有廣泛的調(diào)節(jié)作用。樹突細(xì)胞(dendritic cells, DCs)是體內(nèi)功能最強(qiáng)的抗原遞呈細(xì)胞(antigen-presenting cell, APC),廣泛存在于虹膜、睫狀體和房水流出通道卻沒(méi)有得到應(yīng)有的關(guān)注。本實(shí)驗(yàn)通過(guò)在體外模擬炎癥反應(yīng)環(huán)境,來(lái)觀察房水微環(huán)境對(duì)脂多糖(lipopolysaccharide, LPS)介導(dǎo)的DCs成熟的抑制作用。 方法:取新鮮處死的豬的眼睛,保證球壁完整以防房水被污染和滲漏,用含800U/ml慶大霉素的50%酒精浸泡15min以消毒。于角膜緣處用27G針頭刺入前房,使房水自動(dòng)流入連接的硅化微量離心管,保存于-80°C;應(yīng)用ELISA法測(cè)定房水中PGE_2含量;選用6-8周齡雄性C57BL/6小鼠,培養(yǎng)骨髓源DCs,培養(yǎng)基培養(yǎng)7天,半量換液后,1μg/ml的LPS刺激48h,收集懸浮和半貼壁細(xì)胞即為成熟的DCs。經(jīng)流式細(xì)胞術(shù)鑒定DCs表面特異性分子標(biāo)記物CD11c以觀察細(xì)胞純度。為了研究房水對(duì)DCs成熟狀態(tài)的影響,將未成熟DCs分為三組:對(duì)照組(與1μg/ml LPS共培養(yǎng)48h),實(shí)驗(yàn)1組(與1μg/ml LPS+25%AH共培養(yǎng)48h),實(shí)驗(yàn)2組(與1μg/ml LPS+50%AH共培養(yǎng)48h),并補(bǔ)足rmGM-CSF、培養(yǎng)液,48h后收集細(xì)胞,通過(guò)流式細(xì)胞術(shù)做表型和內(nèi)吞功能的鑒定。尼龍毛柱法分離得到雄性6-8周齡BALB/c小鼠脾臟的T細(xì)胞,應(yīng)用于混合淋巴細(xì)胞反應(yīng)來(lái)觀察DCs刺激T細(xì)胞增殖的能力;根據(jù)文獻(xiàn)結(jié)果于房水中加入10000倍于TGF-β_2濃度的抗TGF-β_2中和抗體(20μg/ml),與DCs作用48h后檢測(cè)拮抗TGF-β_2后房水對(duì)DCs成熟狀態(tài)的影響;第7天于DCs中加入AH6809(EP2受體拮抗劑)至終濃度為100μM,作用30min,以阻斷PGE_2的作用的受體,半量換液加入房水,繼續(xù)培養(yǎng)48h,觀察DCs的成熟狀態(tài)。實(shí)驗(yàn)數(shù)據(jù)應(yīng)用SPSS13.0軟件包進(jìn)行統(tǒng)計(jì)學(xué)分析。 結(jié)果:1 DCs培養(yǎng)7天后,可見(jiàn)較多細(xì)胞呈半懸浮生長(zhǎng),大量細(xì)胞集落形成,集落部分細(xì)胞表面出現(xiàn)長(zhǎng)短不一毛刺樣突起,并可見(jiàn)少量單個(gè)懸浮的典型DCs,經(jīng)流式細(xì)胞術(shù)鑒定DCs表面特異性分子標(biāo)記物CD11c細(xì)胞純度達(dá)到70%。 2房水抑制DCs表型成熟:對(duì)照組呈現(xiàn)出表型成熟狀態(tài),表現(xiàn)為高表達(dá)MHC-II、CD80、CD86;實(shí)驗(yàn)組可見(jiàn)依AH濃度呈劑量依賴性抑制DCs成熟,MHC-II、CD80、CD86相應(yīng)的MFI明顯下降;各組的散點(diǎn)圖表明不同處理組并沒(méi)有影響DCs數(shù)量。 3房水抑制DCs功能成熟:流式細(xì)胞術(shù)檢測(cè)DCs吞噬FITC-dextran的能力發(fā)現(xiàn),房水呈劑量依賴性抑制LPS介導(dǎo)內(nèi)吞功能;尼龍毛柱法分離得到BALB/c小鼠脾臟T細(xì)胞,經(jīng)流式細(xì)胞術(shù)鑒定CD3~+T細(xì)胞純度達(dá)到90%;混合淋巴細(xì)胞反應(yīng)中對(duì)照組DCs經(jīng)LPS刺激后表現(xiàn)為強(qiáng)的刺激T細(xì)胞增殖能力,實(shí)驗(yàn)2組較實(shí)驗(yàn)1組刺激T細(xì)胞能力則減低,并在1:10比例組可觀察到實(shí)驗(yàn)2組的刺激能力是對(duì)照組1/3。 4阻斷細(xì)胞因子拮抗房水的抑制作用:經(jīng)ELISA測(cè)得房水中PGE_2的濃度為2603.919±771.1536pg/ml,遠(yuǎn)遠(yuǎn)小于10~(-6)mol/L,因此選用EP2受體特異性拮抗劑AH6809預(yù)處理DCs1h以阻斷PGE_2細(xì)胞膜作用通路。培養(yǎng)液中加入房水、抗TGF-β_2中和抗體、AH6809和DMSO并沒(méi)有影響CD11c的表達(dá),也即各組DCs具有均一性。房水能夠顯著抑制LPS誘導(dǎo)的DCs MHC-II的高表達(dá),而抗TGF-β_2中和抗體預(yù)處理房水后則完全中和房水的抑制作用。盡管封閉EP2受體對(duì)房水的抑制作用沒(méi)有影響,但是內(nèi)吞功能測(cè)定,同時(shí)拮抗EP2受體和中和TGF-β_2可以協(xié)同提高DCs的內(nèi)吞功能。 結(jié)論:1房水呈劑量依賴性抑制LPS介導(dǎo)的DCs表型和功能的成熟,表現(xiàn)為低表達(dá)MHC-II和共刺激分子CD80、CD86,高的內(nèi)吞功能以及弱的刺激T細(xì)胞增殖的能力。 2房水中TGF-β_2在抑制LPS介導(dǎo)的DCs成熟過(guò)程中起主導(dǎo)作用。 3房水中基礎(chǔ)量PGE_2對(duì)LPS介導(dǎo)的DCs表型成熟沒(méi)有抑制作用,但是可以協(xié)同TGF-β_2提高DCs的內(nèi)吞功能。
[Abstract]:Objective: the immune pardon is related to many factors, including anatomical factors, such as the blood barrier, the lack of direct lymphatic drainage and so on. In addition, there are a large number of immunosuppressive factors that have extensive regulation on immune cell activity in aqueous humor. Dendritic cells (DCs) is the strongest resistant body in the body. Antigen-presenting cell (APC), which is widely present in the iris, ciliary body and aqueous efflux channel, has not received due attention. This experiment was conducted to observe the inhibitory effect of aqueous microenvironment on the maturation of lipopolysaccharide (LPS) mediated by lipopolysaccharide (LPS) by simulating the inflammatory response environment in vitro.
Methods: take the eyes of fresh and dead pigs, ensure that the wall of the ball is intact to prevent the water from being polluted and leaked, and soaked in 50% alcohol containing gentamicin 800U/ml to disinfect it with 15min. At the edge of the cornea, the anterior chamber is inserted into the anterior chamber with 27G needle, so that the aqueous humor automatically flows into the connected silicified micro centrifuge tube and is stored in -80 degree C; PGE_2 content in aqueous humor is determined by ELISA; selection of PGE_2 in aqueous humor is used. With 6-8 weeks male C57BL/6 mice, bone marrow source DCs was cultured, culture medium was cultured for 7 days. After half volume, 1 u g/ml LPS stimulated 48h, and suspended and half adherent cells were collected to identify the specific molecular marker of DCs surface to observe the purity of DCs surface specific molecular marker, which was mature DCs. flow cytometry. In order to study the effect of aqueous humor on the mature state of DCs, it would not be possible. The mature DCs is divided into three groups: the control group (co culture 48h with 1 mu LPS), the experiment 1 groups (1 mu g/ml LPS+25%AH co culture 48h), the experiment 2 groups (1 mu g/ml LPS+50%AH co culture 48h), and fill the rmGM-CSF, culture liquid, collect the cells after 48h, and identify the phenotypic and endocytosis by flow cytometry. The nylon wool column method was used to separate the male 6-8 weeks old age The T cells of mouse spleen were used in mixed lymphocyte reaction to observe the ability of DCs to stimulate the proliferation of T cells. According to the results, the anti TGF- beta _2 neutralization antibody (20 u g/ml) was added to the aqueous humor of 10000 times the concentration of TGF- beta _2 in aqueous humor, and the effect of the antagonism of the aqueous humor on the mature state after TGF- beta _2 was detected after DCs action 48h. EP2 receptor antagonist) to the final concentration of 100 mu M, the action of 30min, to block the effect of PGE_2 receptor, half of the liquid into the aqueous humor, continue to cultivate 48h, observe the mature state of DCs. The experimental data were statistically analyzed by the SPSS13.0 package.
Results: after 7 days of 1 DCs culture, more cells were found to be semi suspended, a large number of cell colonies were formed, and the surface of the colonies appeared on the surface of the colony. A small number of single suspended typical DCs were seen, and the purity of the DCs surface specific molecular marker CD11c cells was identified as 70%. by flow cytometry.
2 aqueous humor inhibited DCs phenotypic maturation: the control group showed a phenotypic maturity, showing a high expression of MHC-II, CD80, CD86; the experimental group showed a dose dependent inhibition of DCs maturity with AH concentration, MHC-II, CD80, and CD86 corresponding MFI significantly decreased; the scatter plot of each group showed that the number of different groups did not affect DCs.
3 aqueous humor inhibited DCs function maturation: the ability to detect DCs phagocytosis by flow cytometry found that the aqueous humor showed a dose dependent inhibitory LPS mediated endocytosis, the BALB/c mouse spleen T cells were isolated by nylon hairy column method, and the purity of CD3~+T cells was 90% by flow cytometry, and the control group of the control group was stimulated by LPS stimulation in the control group of mixed lymphocyte reaction. The ability to stimulate the proliferation of T cells was strongly stimulated, and the ability of stimulating T cells in the 2 groups was lower than that in the experimental 1 groups, and the stimulation ability of the 2 groups was observed at 1:10 in the control group as the control group of 1/3..
4 blocking the inhibitory effect of cytokine on aqueous humor: the concentration of PGE_2 in aqueous humor was measured by ELISA, which was 2603.919 + 771.1536pg/ml, far less than 10~ (-6) mol/L, so EP2 receptor specific antagonist AH6809 pretreated DCs1h to block the pathway of PGE_2 cell membrane. The expression of CD11c was also affected by the homogeneity of DCs in each group. Aqueous humor could significantly inhibit the high expression of DCs MHC-II induced by LPS, while the anti TGF- beta _2 neutralization antibody pretreated aqueous humor was completely neutralized with aqueous humor. Although the inhibitory effect of the enclosed EP2 receptor on aqueous humor was not affected, the endocytosis was measured and the EP2 receptor was antagonized. Neutralization of TGF- beta _2 can enhance the endocytosis function of DCs.
Conclusion: 1 aqueous humor shows a dose dependent inhibition of LPS mediated DCs phenotype and function maturity, which is characterized by low expression of MHC-II and co stimulatory molecules CD80, CD86, high endocytosis and weak stimulation of T cell proliferation.
2 TGF- _2 in aqueous humor plays a leading role in inhibiting LPS mediated DCs maturation.
3 PGE_2 in aqueous humor has no inhibitory effect on LPS mediated DCs phenotype maturation, but it can synergism with TGF- beta _2 to enhance endocytosis of DCs.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 ;[J];;年期

2 ;[J];;年期

3 ;[J];;年期

4 ;[J];;年期

5 ;[J];;年期

6 ;[J];;年期

7 ;[J];;年期

8 ;[J];;年期

9 ;[J];;年期

10 ;[J];;年期

相關(guān)會(huì)議論文 前10條

1 王薇;劉U，

本文編號(hào)：2085731