發(fā)布時間：2019-05-15 11:49

【摘要】：目的：以BALB/c小鼠脾臟細胞在體外與細粒棘球蚴囊液共培養(yǎng)，簡單模擬肝囊型包蟲�。–E）患者體內(nèi)環(huán)境，進行前期實驗，觀察細粒棘球蚴（E.g）囊液對體外培養(yǎng)BABL/c小鼠脾臟細胞中調(diào)節(jié)性T細胞（Treg細胞）相對特異分子叉頭蛋白3（Foxp3）及轉(zhuǎn)化生長因子-β1（TGF-β1）下游信號通路Smad蛋白4（Smad4）表達的影響。進一步試驗研究，CE患者和健康志愿者外周血單個核細胞（PBMC）中Treg細胞的比例，以體內(nèi)和體外實驗相比較，觀察抗原B在體外對健康志愿者PBMC中Treg細胞的影響，以此來研究抗原B在細粒棘球蚴所致的免疫逃避中對Treg細胞的影響。方法：以BABL/c小鼠作為脾細胞供者，用研磨法分離脾臟細胞，隨機分為試驗組（與細粒棘球蚴囊液共同培養(yǎng)）和對照組，1×10~5接種于96孔板上，分別在1h，3h，6h，12h提取總RNA，實時熒光定量PCR（qRT-PCR）技術(shù)檢測Foxp3以及Smad4的基因表達。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，流式細胞術(shù)（flow cytometry, FCM)檢測Treg細胞的表達；收集5例健康人外周血，體外分離PBMC，分為陰性對照組，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收集細胞，流式細胞術(shù)檢測Treg細胞的表達。采用配對t檢驗分析組間差異，Pearson相關(guān)性檢驗分析CE病人Treg與AgB的相關(guān)性。結(jié)果：qRT-PCR結(jié)果顯示，細粒棘球蚴囊液處理1h Foxp3表達量顯著降低（試驗組4.577±0.317，對照組9.274±0.451）；3h，12h Foxp3表達量顯著升高（試驗組8.517±0.978，對照組3.297±0.408；試驗組7.406±0.822，對照組2.464±0.328）。細粒棘球蚴囊液處理組1h Smad4表達量顯著增加（試驗組3.862±1.417，對照組1.689±0.221）；12h Smad4的表達量顯著降低（試驗組1.690±0.248，對照組3.600±1.081）。并且Foxp3與Smad4二者之間存在負相關(guān)，差異有統(tǒng)計學(xué)意義（P＜0.05）。流式細胞檢測結(jié)果顯示：與健康對照組（0.800±0.470）比較，肝囊性包蟲病組Treg細胞比例（2.540±1.130）顯著升高（P＝0.026，P＜0.05）；與陰性對照組比較（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)計學(xué)意義(t＝2.820和t＝2.529，P＜0.01)；高濃度組Treg細胞在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的表達量與Treg細胞呈正相關(guān)(r＝0.739，P＜0.05)。結(jié)論：細粒棘球蚴囊液上調(diào)小鼠脾臟細胞中Treg相對特異分子Foxp3的表達，下調(diào)TGF-β1的下游信號通路Smad4的表達，二者之間存在負相關(guān)，提示細粒棘球蚴侵染宿主的過程中可能通過負調(diào)控TGF-β信號通路而造成宿主Treg細胞的表達升高，進而可能參與細粒棘球蚴感染引起的的免疫逃避。CE病人外周血中Treg細胞表達升高，且與AgB呈正相關(guān)。體外實驗進一步證實低濃度細粒棘球蚴抗原B可能對正常人外周血單個核細胞中Treg細胞的表達起到促進作用，但隨著抗原B濃度的升高，促進作用減弱，，推測抗原B在細粒棘球蚴感染所致的免疫逃避中對Treg細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)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392

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