【摘要】：目的：以BALB/c小鼠脾臟細(xì)胞在體外與細(xì)粒棘球蚴囊液共培養(yǎng)，簡(jiǎn)單模擬肝囊型包蟲�。–E）患者體內(nèi)環(huán)境，進(jìn)行前期實(shí)驗(yàn)，觀察細(xì)粒棘球蚴（E.g）囊液對(duì)體外培養(yǎng)BABL/c小鼠脾臟細(xì)胞中調(diào)節(jié)性T細(xì)胞（Treg細(xì)胞）相對(duì)特異分子叉頭蛋白3（Foxp3）及轉(zhuǎn)化生長(zhǎng)因子-β1（TGF-β1）下游信號(hào)通路Smad蛋白4（Smad4）表達(dá)的影響。進(jìn)一步試驗(yàn)研究，CE患者和健康志愿者外周血單個(gè)核細(xì)胞（PBMC）中Treg細(xì)胞的比例，以體內(nèi)和體外實(shí)驗(yàn)相比較，觀察抗原B在體外對(duì)健康志愿者PBMC中Treg細(xì)胞的影響，以此來研究抗原B在細(xì)粒棘球蚴所致的免疫逃避中對(duì)Treg細(xì)胞的影響。方法：以BABL/c小鼠作為脾細(xì)胞供者，用研磨法分離脾臟細(xì)胞，隨機(jī)分為試驗(yàn)組（與細(xì)粒棘球蚴囊液共同培養(yǎng)）和對(duì)照組，1×10~5接種于96孔板上，分別在1h，3h，6h，12h提取總RNA，實(shí)時(shí)熒光定量PCR（qRT-PCR）技術(shù)檢測(cè)Foxp3以及Smad4的基因表達(dá)。新疆醫(yī)科大學(xué)第一附屬醫(yī)院25例受試者分為兩組：健康對(duì)照組(healthy control,HC)10例，肝囊性包蟲�。╟ystic echinococcosis, CE）組15例。Ficoll密度梯度離心法分離PBMC，流式細(xì)胞術(shù)（flow cytometry, FCM)檢測(cè)Treg細(xì)胞的表達(dá)；收集5例健康人外周血，體外分離PBMC，分為陰性對(duì)照組，低濃度(終濃度2μg/mL)和高濃度抗原B處理組(終濃度5μg/mL)，分別在96，120和144h收集細(xì)胞，流式細(xì)胞術(shù)檢測(cè)Treg細(xì)胞的表達(dá)。采用配對(duì)t檢驗(yàn)分析組間差異，Pearson相關(guān)性檢驗(yàn)分析CE病人Treg與AgB的相關(guān)性。結(jié)果：qRT-PCR結(jié)果顯示，細(xì)粒棘球蚴囊液處理1h Foxp3表達(dá)量顯著降低（試驗(yàn)組4.577±0.317，對(duì)照組9.274±0.451）；3h，12h Foxp3表達(dá)量顯著升高（試驗(yàn)組8.517±0.978，對(duì)照組3.297±0.408；試驗(yàn)組7.406±0.822，對(duì)照組2.464±0.328）。細(xì)粒棘球蚴囊液處理組1h Smad4表達(dá)量顯著增加（試驗(yàn)組3.862±1.417，對(duì)照組1.689±0.221）；12h Smad4的表達(dá)量顯著降低（試驗(yàn)組1.690±0.248，對(duì)照組3.600±1.081）。并且Foxp3與Smad4二者之間存在負(fù)相關(guān)，差異有統(tǒng)計(jì)學(xué)意義（P＜0.05）。流式細(xì)胞檢測(cè)結(jié)果顯示：與健康對(duì)照組（0.800±0.470）比較，肝囊性包蟲病組Treg細(xì)胞比例（2.540±1.130）顯著升高（P＝0.026，P＜0.05）；與陰性對(duì)照組比較（0.575±0.126，1.067±0.666），體外培養(yǎng)的正常PBMC在抗原B作用120和144h，低濃度組Treg細(xì)胞在PBMC中的比例顯著升高（分別為0.800±0.082，2.200±0.819），差異有統(tǒng)計(jì)學(xué)意義(t＝2.820和t＝2.529，P＜0.01)；高濃度組Treg細(xì)胞在PBMC中的比例顯著降低（分別為0.333±0.115，0.833±0.551），差異有統(tǒng)計(jì)學(xué)意義(t＝2.598和t＝2.836，P＜0.05)。且病人血清中抗原B的表達(dá)量與Treg細(xì)胞呈正相關(guān)(r＝0.739，P＜0.05)。結(jié)論：細(xì)粒棘球蚴囊液上調(diào)小鼠脾臟細(xì)胞中Treg相對(duì)特異分子Foxp3的表達(dá)，下調(diào)TGF-β1的下游信號(hào)通路Smad4的表達(dá)，二者之間存在負(fù)相關(guān)，提示細(xì)粒棘球蚴侵染宿主的過程中可能通過負(fù)調(diào)控TGF-β信號(hào)通路而造成宿主Treg細(xì)胞的表達(dá)升高，進(jìn)而可能參與細(xì)粒棘球蚴感染引起的的免疫逃避。CE病人外周血中Treg細(xì)胞表達(dá)升高，且與AgB呈正相關(guān)。體外實(shí)驗(yàn)進(jìn)一步證實(shí)低濃度細(xì)粒棘球蚴抗原B可能對(duì)正常人外周血單個(gè)核細(xì)胞中Treg細(xì)胞的表達(dá)起到促進(jìn)作用，但隨著抗原B濃度的升高，促進(jìn)作用減弱，，推測(cè)抗原B在細(xì)粒棘球蚴感染所致的免疫逃避中對(duì)Treg細(xì)胞分化的起著重要作用。
[Abstract]:Objective: The spleen cells of BALB/ c mice were co-cultured with the fine-grained echinococcin in vitro, and the in-vivo environment of the patients with hepatic cysticercosis (CE) was simply simulated, and the early-stage experiments were carried out. The expression of Smad-4 (Smad4) in the downstream signal pathway of T cell (Tregs) in the spleen of BABL/ c mice and the expression of Smad-4 (Smad4) were observed in the spleen cells of BABL/ c mice. The ratio of Treg cells in peripheral blood mononuclear cells (PBMC) of CE patients and healthy volunteers was further tested, and the effect of antigen B on Treg cells in PBMC of healthy volunteers was observed in vivo and in vitro. In this way, the effect of the antigen B on Treg cells in the immune escape caused by the fine-grained echinococcus is studied. Methods: BABL/ c mice were used as the donor of the spleen cells, and the spleen cells were isolated by the grinding method. The cells were randomly divided into the test group (co-cultured with the fine-grained echinocular capsule) and the control group, and the total RNA was extracted at 1 h,3 h,6 h and 12 h, respectively, and the total RNA was extracted at 1 h,3 h,6 h and 12 h, respectively. The gene expression of Foxp3 and Smad4 was detected by real-time fluorescence quantitative PCR (qRT-PCR). In the first Affiliated Hospital of Xinjiang Medical University,25 subjects were divided into two groups: the health control group (HC) in 10 cases, and the cystic echinococcosis (CE) group in 15 cases. The expression of Treg cells was detected by Ficoll density gradient centrifugation, and the expression of Treg cells was detected by flow cytometry (FCM). The peripheral blood of 5 healthy subjects was collected, and the PBMCs were isolated in vitro, divided into a negative control group, a low concentration (final concentration of 2. mu.g/ mL) and a high concentration antigen B treatment group (final concentration of 5. mu.g/ mL). Cells were collected at 96,120 and 144h, respectively, and the expression of Treg cells was detected by flow cytometry. The correlation between Treg and AgB in the CE patients was analyzed by Pearson correlation test using the paired t-test analysis group differences. Results: The results of qRT-PCR showed that the expression of Foxp3 was significantly reduced by qRT-PCR (4.577-0.317 in the test group and 9.274-0.451 in the control group). The expression of Foxp3 in the 3-h,12-h Foxp3 was significantly higher (8.517-0.978 in the test group, 3.297-0.408 in the control group, 7.406-0.822 in the test group and 2.464-0.328 in the control group). The expression of Smad4 was significantly increased in the treatment group (3.862-1.417 in the test group and 1.689-0.221 in the control group), and the expression of the 12-h Smad4 was significantly decreased (1.690-0.248 in the test group and 3.600-1.081 in the control group). There was a negative correlation between Foxp3 and Smad4 (P <0.05). The results of flow cytometry showed that, compared with the healthy control group (0.800-0.470), the proportion of Treg cells (2.540-1.130) in the hepatic-cystic hydatid disease group was significantly higher (P = 0.026, P <0.05); compared with the negative control group (0.575-0.126, 1.067-0.666), the normal PBMCs were cultured in vitro for 120 and 144h. The proportion of Treg cells in the low-concentration group was significantly higher in PBMCs (0.800, 0.082, 2.200-0.819, respectively), and the difference was statistically significant (t = 2.820 and t = 2.529, P <0.01), and the proportion of Treg cells in the high-concentration group was significantly lower in PBMCs (0.333-0.115, 0.833-0.551, respectively). The difference was statistically significant (t = 2.598 and t = 2.836, P <0.05). And the expression of the antigen B in the serum of the patient was positively correlated with the Treg cell (r = 0.739, P <0.05). Conclusion: The expression of Treg relative to the specific molecular Foxp3 in the spleen cells of the mouse is up-regulated by the fine-grained echinocular capsule, and there is a negative correlation between the expression of the downstream signal pathway Smad4 of the TGF-CD1. It is suggested that the expression of the host Treg cells may be increased by the negative regulation of the TGF-1 signaling pathway in the process of infecting the host, and the immune escape caused by the infection of the fine-grained echinococcus may be involved. The expression of Treg cells in peripheral blood of CE patients was elevated and was positively correlated with AgB. in vitro, it is further confirmed that the low-concentration fine-grained echinocandin antigen B can promote the expression of Treg cells in the peripheral blood mononuclear cells of a normal person, but with the increase of the concentration of the antigen B, the promoting effect is weakened, It is assumed that the antigen B plays an important role in the differentiation of Treg cells in the immune escape caused by the infection of the fine-grained echinococcus.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 唐潔;李柏青;阮潔;張林杰;;新生兒臍帶血CD4~+CD25~(high)調(diào)節(jié)性T細(xì)胞Foxp3的表達(dá)[J];細(xì)胞與分子免疫學(xué)雜志;2008年04期

2 袁勁;吳軻;向芙莉;曾寧;周鴻敏;陳忠華;;轉(zhuǎn)化生長(zhǎng)因子β_1對(duì)同種反應(yīng)性T細(xì)胞增殖能力及CD25表達(dá)的影響[J];醫(yī)學(xué)研究生學(xué)報(bào);2007年06期

3 周伯平;陳心春;李美忠;鄧群益;樂曉華;吳馳;余衛(wèi)業(yè);張維;王火生;付向東;;CD4~+CD25~+FoxP3~+調(diào)節(jié)性T細(xì)胞與結(jié)核病的關(guān)系[J];中華傳染病雜志;2007年05期

4 汪明;;人畜共患寄生蟲病危害與公共衛(wèi)生意義(上)[J];動(dòng)物保健;2006年08期

5 福軍亮;徐東平;趙平;陳黎明;張暉;周春保;姚金霞;榮義輝;王福生;;急慢性乙型肝炎患者外周血調(diào)節(jié)性T細(xì)胞鑒定與臨床意義分析[J];中華醫(yī)學(xué)雜志;2006年22期

6 趙建斌;龔匡隆;張津萍;鐘銘英;尚淑賢;鄭波;王千秋;;梅毒患者外周血CD4+CD25~(bright)調(diào)節(jié)性T細(xì)胞的檢測(cè)[J];中華皮膚科雜志;2006年05期

7 牟達(dá);何芳;;TGF-β與MAPK細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)通路的交互調(diào)節(jié)及其在心血管疾病中的作用[J];國(guó)際病理科學(xué)與臨床雜志;2006年02期

8 李琦;王雅琴;譚天偉;王子鎬;;mdlA基因在畢赤酵母中的高效表達(dá)及表達(dá)產(chǎn)物性質(zhì)研究[J];微生物學(xué)通報(bào);2006年01期

9 張亞樓,盧曉梅,張琰,張金輝,阿不都熱依木阿吉,劉輝,馬旭東,溫浩;細(xì)粒棘球蚴囊液對(duì)外周血淋巴細(xì)胞的影響[J];中國(guó)寄生蟲病防治雜志;2005年02期

10 許隆祺;我國(guó)西部地區(qū)重大寄生蟲病的危害及對(duì)防治工作的反思[J];中國(guó)寄生蟲病防治雜志;2002年01期

相關(guān)博士學(xué)位論文 前1條

1 陳新華;抗原B誘導(dǎo)泡球蚴肝移植后免疫耐受的機(jī)制研究[D];浙江大學(xué);2006年

相關(guān)碩士學(xué)位論文 前1條

1 吐爾洪江·吐遜;Th17細(xì)胞/調(diào)節(jié)性T細(xì)胞偏倚在肝包蟲病所致免疫逃避中的意義[D];新疆醫(yī)科大學(xué);2010年

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