本文選題：免疫相關(guān)性全血細(xì)胞減少癥 + 白介素35�。� 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:免疫相關(guān)性全血細(xì)胞減少癥(Immnorelated pancytopenia,IRP)是一種由于某些原因?qū)е聶C(jī)體產(chǎn)生了抗骨髓造血細(xì)胞自身抗體,因此機(jī)體的正常造血功能受到破壞(抑制)所導(dǎo)致的疾病。臨床可主要表現(xiàn)為不同程度的貧血、出血、感染。白介素35(interleukin 35,IL-35)是IL-12家族的新成員。它不僅是一種新型的抗炎因子,也廣泛參與了體內(nèi)免疫應(yīng)答的過程。研究目的為檢測IRP患者IL-35水平的變化及原因,并探究IL-35與Th17細(xì)胞的關(guān)系及其在IRP發(fā)病機(jī)制中的作用。材料、方法:研究對象為IRP患者共43人。其中初治組患者18人;經(jīng)免疫抑制治療(輸血依賴患者經(jīng)大劑量靜脈丙種球蛋白治療)后血象符合IRP療效緩解標(biāo)準(zhǔn)患者(緩解組)25人;正常對照組19人。第一部分IRP患者外周血IL-35水平檢測。ELISA法測定研究對象外周血血漿中IL-35的水平,并與IRP患者外周血血紅蛋白(Hb)、血小板(Plt)、白細(xì)胞計(jì)數(shù)(WBC)、中性粒細(xì)胞百分比(N%)、網(wǎng)織紅細(xì)胞比例(Ret%)及CD5~+CD19~+B細(xì)胞占淋巴細(xì)胞和CD19~+B細(xì)胞的百分比做相關(guān)性分析;流式細(xì)胞術(shù)(FCM)檢測外周血CD4~+CD25~+Foxp3~+調(diào)節(jié)性T(Treg)細(xì)胞(IL-35的主要分泌細(xì)胞)占淋巴細(xì)胞及CD4~+T細(xì)胞的百分比;免疫磁珠分選研究對象CD4~+CD25~+Treg細(xì)胞,實(shí)時(shí)熒光定量PCR(real-time PCR,RT-PCR)檢測IL-35亞單位(Ebi3、p35)m RNA的表達(dá)情況。第二部分探究IL-35與Th17細(xì)胞的關(guān)系。用流式細(xì)胞術(shù)(FCM)檢測IRP患者及正常對照外周血(CD4~+IL17~+)Th17細(xì)胞占CD4~+的比例,并用ELISA法測定研究外周血血漿中IL-17的水平,并與其IL-35水平做相關(guān)性分析。分選除5例IRP初治患者CD4~+T細(xì)胞,與人融合性蛋白IL-35體外混合培養(yǎng)3天,FCM分析其Th17細(xì)胞占CD4~+T細(xì)胞的百分比變化;RT-PCR檢測glycoprotein 130(gp130)、IL-12受體β2鏈(IL-12Rβ2)、維甲酸相關(guān)孤核受體(retinoic acid-related orphan receptor,ROR)γt、IL-17a的m RNA表達(dá)量。結(jié)果:第一部分IRP患者外周血IL-35水平檢測。1.IRP初治組IL-35的水平(20.59±6.05pg/ml)明顯低于緩解組(30.75±10.6pg/ml,P0.01)與正常對照(98.45±57pg/ml,P0.01)。將IRP緩解組患者按照其膜抗體檢查結(jié)果,分為膜抗體陽性組(膜抗體~+組)及膜抗體陰性組(膜抗體-組),并與初治組的IL-35水平進(jìn)行比較發(fā)現(xiàn),初治組外周血血漿IL-35低于膜抗體~+組(29.66±8.41 pg/ml,P=0.013)及膜抗體-組(31.64±8.41 pg/ml,P0.01),IRP患者外周血血漿IL-35水平與臨床指標(biāo)做相關(guān)性分析發(fā)現(xiàn),它與外周血Hb(P0.01,r=0.62)、WBC(P0.01,r=0.429)及Plt(P0.01,r=0.558)水平呈正相關(guān)。CD5~+CD19~+細(xì)胞占淋巴細(xì)胞百分比(P=0.03,r=-0.30)及CD19~+細(xì)胞的百分比(P0.01,r=-0.308)均與血漿IL-35濃度呈負(fù)相關(guān),而其與N%和Ret%之間的關(guān)聯(lián)則并無明顯的統(tǒng)計(jì)學(xué)意義。2.CD4~+T細(xì)胞在淋巴細(xì)胞中所占的比例在初治組、緩解組及正常對照組中分別為(29.80±3.87)%、(34.15±4.48)%、(37.97±6.05)%;初治組及緩解組與對照組相比均有顯著降低(P0.01),而初治組與緩解組相比,也是有統(tǒng)計(jì)學(xué)意義的(P0.01)。CD4~+CD25~+Foxp3~+Treg細(xì)胞在CD4~+T細(xì)胞中所占的百分比,初治組(1.43±0.75%)與緩解組(1.86±0.79)%均比對照組(2.70±0.75)%有顯著降低(P0.01),而初治組與緩解組相比較并無顯著差別(P=0.08)。3.初治組、緩解組及對照組CD4~+CD25~+Treg細(xì)胞內(nèi)IL-35亞單位Ebi3m RNA水平(2-△△CT)分別為(0.16±0.13)、(0.43±0.54)及(1.20±0.80)。初治組及緩解組的m RNA表達(dá)水平與對照組相比均顯著降低(P0.01),且有統(tǒng)計(jì)學(xué)意義,而初治組與緩解組之間相比則并無明顯差異(P=0.124)。另一亞單位p35在三個(gè)組中的m RNA表達(dá)水平(2-△△CT)則分別為(0.23±0.40)、(0.66±0.60)、(1.28±0.87)。其中初治組m RNA表達(dá)水平與正常對照組有顯著差異(P0.01),第二部分探究IL-35與Th17細(xì)胞的關(guān)系1.初治IRP患者Th17(CD4~+IL17~+)細(xì)胞占CD4~+T細(xì)胞的百分比(4.83±2.53)%明顯高于正常對照(1.8035±0.9225)%,(P0.01)。經(jīng)過治療,IRP患者的Th17細(xì)胞百分比有明顯降低(2.214±1.12)%(P0.01),但仍然比正常對照高(P=0.04)。IRP初治組IL-17的水平(273.48±59.74 pg/ml)明顯高于緩解組(206.56±45.09pg/ml,P0.01)及對照組(171.29±27.69 pg/ml,P0.01),而緩解組與對照組相比也有明顯增高(P0.01)。與IL-35濃度的相關(guān)性分析顯示:IRP患者外周血血漿IL-35濃度及IL-17濃度呈明顯負(fù)相關(guān)(r=-0.55,P0.01)。2.混合培養(yǎng)3天后,a組(IRP初治患者CD4~+T淋巴細(xì)胞空白組)Th17細(xì)胞占CD4~+T細(xì)胞百分比(6.94±1.88)%與b組(CD4~+T淋巴細(xì)胞與anti-CD3抗體,anti-CD28抗體,TGF-β,IL-6混合培養(yǎng))相比(10.95±3.72),明顯降低(P=0.044),b組與c組(CD4~+T淋巴細(xì)胞與anti-CD3抗體,anti-CD28抗體,TGF-β,IL-6,IL-35混合培養(yǎng))(7.06±2.75)%相比,Th17細(xì)胞百分比有所增高(P=0.048)。3.混合培養(yǎng)三組(a組、b組、c組)檢測gp130的m RNA表達(dá)水平分別為(1.57±1.45)(6.17±5.91)、(13.13±10.47),c組與a相比有明顯增高(P=0.023),另一受體亞單位IL-12Rβ2的m RNA表達(dá)量分別為(2.50±2.87)、(3.15±4.53)、(8.03±6.51),盡管c組在三組中表達(dá)量最高,但每組之間相比均無統(tǒng)計(jì)學(xué)意義。RORγt的m RNA表達(dá)量在三組間分別為(1.11±0.50)、(6.44±5.93)、(1.57±0.87),b組m RNA表達(dá)水平最高(與a組相比P=0.032,與c組相比P=0.046)。IL-17a的m RNA表達(dá)量在b組中最高(10.95±3.72),與a組(6.94±1.88,P=0.018)及c組(7.06±2.75,P=0.023)相比均有統(tǒng)計(jì)學(xué)意義。結(jié)論:1.IRP患者外周血血漿IL-35水平較正常對照組明顯降低;緩解組外周血血漿IL-35水平較初治組明顯增高。CD4~+CD25~+Foxp3~+細(xì)胞比例的降低及該細(xì)胞中IL-35m RNA表達(dá)水平的降低可能是IL-35水平降低的原因之一。2.IRP患者Th17細(xì)胞及血漿IL-17水平較正常對照組明顯增高。體外混合培養(yǎng)實(shí)驗(yàn)證明IL-35可抑制IRP患者CD4~+T淋巴細(xì)胞分化為Th17細(xì)胞,這種抑制是通過IL-35對其受體亞單位gp130的激活進(jìn)而抑制轉(zhuǎn)錄調(diào)節(jié)因子RORγt所實(shí)現(xiàn)的。
[Abstract]:Objective: Immnorelated pancytopenia (IRP) is a kind of anti hematopoietic cell autoantibody that causes the body to produce anti hematopoietic cell autoantibodies for some reason. Therefore, the normal hematopoiesis of the body is caused by the destruction (inhibition) of the disease. The main clinical manifestation is different degree of anemia, bleeding, infection. Interleukin 35. (interleukin 35, IL-35) is a new member of the IL-12 family. It is not only a new type of anti-inflammatory factor, but also widely participates in the process of immune response in the body. The purpose of this study is to detect the changes and causes of IL-35 level in IRP patients, and to explore the relationship between IL-35 and Th17 cells and its role in the pathogenesis of IRP. Materials and methods: the object of study is IR. There were 43 patients with P, of which 18 were in the primary treatment group; the hemogram after the immunosuppressive therapy (blood transfusion dependent patients treated with high dose intravenous gamma globulin) conformed to 25 of the IRP remission standard patients (remission group); the normal control group was 19. The peripheral blood level of peripheral blood in the first part of IRP patients was measured by.ELISA method in the peripheral blood plasma IL-3 5 of the levels were associated with peripheral blood hemoglobin (Hb), platelet (Plt), leukocyte count (WBC), neutrophils percentage (N%), reticulocyte proportion (Ret%) and the percentage of CD5~+CD19~+B cells in lymphocytes and CD19~+B cells; flow cytometry (FCM) was used to detect CD4~+CD25~+Foxp3~+ regulatory T (Treg) in peripheral blood. Cell (IL-35's main secretory cells) accounted for the percentage of lymphocytes and CD4~+T cells; immunomagnetic beads were selected to study CD4~+CD25~+Treg cells, and real-time quantitative PCR (RT-PCR) was used to detect the expression of IL-35 subunit (Ebi3, p35) m RNA. The second part explored the relationship between IL-35 and m RNA cells. Patients and normal control peripheral blood (CD4~+IL17~+) Th17 cells accounted for the proportion of CD4~+, and ELISA method was used to determine the level of IL-17 in peripheral blood plasma and the correlation analysis with the level of IL-35. In addition, 5 cases of IRP first treated patients CD4~+T cells, mixed with human fusion protein IL-35 in vitro culture for 3 days, FCM analysis of Th17 cells accounted for CD4~+T cells. Percentage changes; RT-PCR glycoprotein 130 (gp130), IL-12 receptor beta 2 chain (IL-12R beta 2), retinoic acid related nucleus receptor (retinoic acid-related orphan receptor, ROR) gamma t. Group (30.75 + 10.6pg/ml, P0.01) and normal control (98.45 + 57pg/ml, P0.01). The patients in the IRP remission group were divided into membrane antibody positive group (membrane antibody + group) and membrane antibody negative group (membrane antibody group), and compared with the IL-35 level in the primary treatment group, the plasma IL-35 in the primary treatment group was lower than that of the membrane antibody + group (29.66 + (29.66 +). 8.41 pg/ml, P=0.013) and membrane antibody group (31.64 + 8.41 pg/ml, P0.01). The correlation analysis between the level of peripheral blood plasma IL-35 and clinical indexes in IRP patients showed that it was positively correlated with the level of Hb (P0.01, r=0.62), WBC (P0.01, r=0.429) and the levels of peripheral blood. The percentage (P0.01, r=-0.308) was negatively correlated with the concentration of plasma IL-35, but the association with N% and Ret% had no significant statistical significance. The proportion of.2.CD4~+T cells in the lymphocyte was in the primary treatment group, the remission group and the normal control group were (29.80 + 3.87)%, (34.15 + 4.48)%, (37.97 + 6.05)%, and the primary and remission group. Compared with the control group, it was significantly lower (P0.01), but compared with the remission group, the percentage of.CD4~+CD25~+Foxp3~+Treg cells in the CD4~+T cells was significantly higher than that in the remission group. The initial treatment group (1.43 + 0.75%) and the remission group (1.86 + 0.79)% were significantly lower than the control group (2.70 + 0.75)% (2.70 + 0.75)% (P0.01), while the primary and remission groups were significantly lower than those in the control group (P0.01). There was no significant difference (P=0.08) in the early treatment group of.3.. The Ebi3m RNA level of IL-35 subunit in CD4~+CD25~+Treg cells in the remission group and the control group (2- Delta Delta CT) was (0.16 + 0.13), (0.43 + 0.54) and (1.20 + 0.80). The m RNA expression level of the primary and remission group was significantly lower than that of the control group (P0.01), and the initial treatment group was statistically significant. There was no significant difference compared with the remission group (P=0.124). The m RNA expression level of the other subunit p35 (2- Delta Delta CT) was (2- Delta Delta CT), respectively (0.23 + 0.40), (0.66 + 0.60), (1.28 + 0.87). The expression level of M RNA in the primary treatment group was significantly different from that of the normal control group (P0.01). The second part explored the relationship between IL-35 and Th17 cells 1. at the beginning of IR. The percentage of Th17 (CD4~+IL17~+) cells (4.83 + 2.53)% of CD4~+T cells in P patients was significantly higher than that of normal controls (1.8035 + 0.9225)%, (P0.01). The percentage of Th17 cells in IRP patients decreased significantly (2.214 + 1.12)% (P0.01), but still higher than normal control (P=0.04).IRP group IL-17 (273.48 + 59.74 pg/ml) was significantly higher than that of the slow control group (273.48 + 59.74 pg/ml). The solution group (206.56 + 45.09pg/ml, P0.01) and the control group (171.29 + 27.69 pg/ml, P0.01), while the remission group was also significantly higher than the control group (P0.01). The correlation analysis with the IL-35 concentration showed that the concentration of IL-35 and the concentration of IL-17 in the peripheral blood plasma of IRP patients were negatively correlated (r=-0.55, P0.01) for 3 days. Lymphocyte blank group) Th17 cells accounted for the percentage of CD4~+T cells (6.94 + 1.88)% (10.95 + 3.72) compared with group B (CD4~+T lymphocyte and anti-CD3 antibody, anti-CD28 antibody, TGF- beta, IL-6 mixed culture), significantly decreased (P=0.044), B and C group (7.06 + 2.7) (7.06 + 2.7) 5)%, the percentage of Th17 cells increased (P=0.048).3. mixed culture three groups (a group, B group, C group) and gp130 m RNA expression level was (1.57 + 1.45) (6.17 + 5.91), (13.13 + 10.47), C group was significantly higher than a (P=0.023) and (2.50 + 2.87), (3.15 + 4.53), respectively (3.15 + 4.53), (3.15 + 4.53), (3.15 + 4.53), (3.15 + 4.53). Although the expression of group C in the three groups was the highest, there was no statistically significant m RNA expression of.ROR gamma t in each group (1.11 + 0.50), (6.44 + 5.93), (1.57 + 0.87), and the highest expression level of M RNA in group B (10.95 + 3.72) in group B (10.95 + 3.72), compared with C group (10.95 + 3.72). .94 + 1.88, P=0.018) and C group (7.06 + 2.75, P=0.023) were statistically significant compared. Conclusion: the level of peripheral blood plasma IL-35 in 1.IRP patients was significantly lower than that in the normal control group; the level of IL-35 in the peripheral blood plasma of the remission group was significantly higher than that in the primary treatment group and the decrease of the proportion of.CD4~+CD25~+Foxp3~+ cells and the decrease of IL-35m RNA expression in this cell may be reduced. One of the reasons for the decrease of IL-35 level is that the level of Th17 cells and plasma IL-17 in.2.IRP patients is significantly higher than that in the normal control group. In vitro mixed culture experiments show that IL-35 can inhibit the differentiation of CD4~+T lymphocytes into Th17 cells in IRP patients. This inhibition is through the activation of IL-35 on its receptor subunit gp130 and the inhibition of the transcriptional regulator ROR gamma t. Realized.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R55

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 付蓉;劉惠;王s，

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