免疫相关性全血细胞减少症患者白介素35水平对Th17细胞调节功能的研究

发布时间：2018-05-10 17:37

本文选题：免疫相关性全血细胞减少症 + 白介素35　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的:免疫相关性全血细胞减少症(Immnorelated pancytopenia,IRP)是一种由于某些原因导致机体产生了抗骨髓造血细胞自身抗体,因此机体的正常造血功能受到破坏(抑制)所导致的疾病。临床可主要表现为不同程度的贫血、出血、感染。白介素35(interleukin 35,IL-35)是IL-12家族的新成员。它不仅是一种新型的抗炎因子,也广泛参与了体内免疫应答的过程。研究目的为检测IRP患者IL-35水平的变化及原因,并探究IL-35与Th17细胞的关系及其在IRP发病机制中的作用。材料、方法:研究对象为IRP患者共43人。其中初治组患者18人;经免疫抑制治疗(输血依赖患者经大剂量静脉丙种球蛋白治疗)后血象符合IRP疗效缓解标准患者(缓解组)25人;正常对照组19人。第一部分IRP患者外周血IL-35水平检测。ELISA法测定研究对象外周血血浆中IL-35的水平,并与IRP患者外周血血红蛋白(Hb)、血小板(Plt)、白细胞计数(WBC)、中性粒细胞百分比(N%)、网织红细胞比例(Ret%)及CD5~+CD19~+B细胞占淋巴细胞和CD19~+B细胞的百分比做相关性分析;流式细胞术(FCM)检测外周血CD4~+CD25~+Foxp3~+调节性T(Treg)细胞(IL-35的主要分泌细胞)占淋巴细胞及CD4~+T细胞的百分比;免疫磁珠分选研究对象CD4~+CD25~+Treg细胞,实时荧光定量PCR(real-time PCR,RT-PCR)检测IL-35亚单位(Ebi3、p35)m RNA的表达情况。第二部分探究IL-35与Th17细胞的关系。用流式细胞术(FCM)检测IRP患者及正常对照外周血(CD4~+IL17~+)Th17细胞占CD4~+的比例,并用ELISA法测定研究外周血血浆中IL-17的水平,并与其IL-35水平做相关性分析。分选除5例IRP初治患者CD4~+T细胞,与人融合性蛋白IL-35体外混合培养3天,FCM分析其Th17细胞占CD4~+T细胞的百分比变化;RT-PCR检测glycoprotein 130(gp130)、IL-12受体β2链(IL-12Rβ2)、维甲酸相关孤核受体(retinoic acid-related orphan receptor,ROR)γt、IL-17a的m RNA表达量。结果:第一部分IRP患者外周血IL-35水平检测。1.IRP初治组IL-35的水平(20.59±6.05pg/ml)明显低于缓解组(30.75±10.6pg/ml,P0.01)与正常对照(98.45±57pg/ml,P0.01)。将IRP缓解组患者按照其膜抗体检查结果,分为膜抗体阳性组(膜抗体~+组)及膜抗体阴性组(膜抗体-组),并与初治组的IL-35水平进行比较发现,初治组外周血血浆IL-35低于膜抗体~+组(29.66±8.41 pg/ml,P=0.013)及膜抗体-组(31.64±8.41 pg/ml,P0.01),IRP患者外周血血浆IL-35水平与临床指标做相关性分析发现,它与外周血Hb(P0.01,r=0.62)、WBC(P0.01,r=0.429)及Plt(P0.01,r=0.558)水平呈正相关。CD5~+CD19~+细胞占淋巴细胞百分比(P=0.03,r=-0.30)及CD19~+细胞的百分比(P0.01,r=-0.308)均与血浆IL-35浓度呈负相关,而其与N%和Ret%之间的关联则并无明显的统计学意义。2.CD4~+T细胞在淋巴细胞中所占的比例在初治组、缓解组及正常对照组中分别为(29.80±3.87)%、(34.15±4.48)%、(37.97±6.05)%;初治组及缓解组与对照组相比均有显著降低(P0.01),而初治组与缓解组相比,也是有统计学意义的(P0.01)。CD4~+CD25~+Foxp3~+Treg细胞在CD4~+T细胞中所占的百分比,初治组(1.43±0.75%)与缓解组(1.86±0.79)%均比对照组(2.70±0.75)%有显著降低(P0.01),而初治组与缓解组相比较并无显著差别(P=0.08)。3.初治组、缓解组及对照组CD4~+CD25~+Treg细胞内IL-35亚单位Ebi3m RNA水平(2-△△CT)分别为(0.16±0.13)、(0.43±0.54)及(1.20±0.80)。初治组及缓解组的m RNA表达水平与对照组相比均显著降低(P0.01),且有统计学意义,而初治组与缓解组之间相比则并无明显差异(P=0.124)。另一亚单位p35在三个组中的m RNA表达水平(2-△△CT)则分别为(0.23±0.40)、(0.66±0.60)、(1.28±0.87)。其中初治组m RNA表达水平与正常对照组有显著差异(P0.01),第二部分探究IL-35与Th17细胞的关系1.初治IRP患者Th17(CD4~+IL17~+)细胞占CD4~+T细胞的百分比(4.83±2.53)%明显高于正常对照(1.8035±0.9225)%,(P0.01)。经过治疗,IRP患者的Th17细胞百分比有明显降低(2.214±1.12)%(P0.01),但仍然比正常对照高(P=0.04)。IRP初治组IL-17的水平(273.48±59.74 pg/ml)明显高于缓解组(206.56±45.09pg/ml,P0.01)及对照组(171.29±27.69 pg/ml,P0.01),而缓解组与对照组相比也有明显增高(P0.01)。与IL-35浓度的相关性分析显示:IRP患者外周血血浆IL-35浓度及IL-17浓度呈明显负相关(r=-0.55,P0.01)。2.混合培养3天后,a组(IRP初治患者CD4~+T淋巴细胞空白组)Th17细胞占CD4~+T细胞百分比(6.94±1.88)%与b组(CD4~+T淋巴细胞与anti-CD3抗体,anti-CD28抗体,TGF-β,IL-6混合培养)相比(10.95±3.72),明显降低(P=0.044),b组与c组(CD4~+T淋巴细胞与anti-CD3抗体,anti-CD28抗体,TGF-β,IL-6,IL-35混合培养)(7.06±2.75)%相比,Th17细胞百分比有所增高(P=0.048)。3.混合培养三组(a组、b组、c组)检测gp130的m RNA表达水平分别为(1.57±1.45)(6.17±5.91)、(13.13±10.47),c组与a相比有明显增高(P=0.023),另一受体亚单位IL-12Rβ2的m RNA表达量分别为(2.50±2.87)、(3.15±4.53)、(8.03±6.51),尽管c组在三组中表达量最高,但每组之间相比均无统计学意义。RORγt的m RNA表达量在三组间分别为(1.11±0.50)、(6.44±5.93)、(1.57±0.87),b组m RNA表达水平最高(与a组相比P=0.032,与c组相比P=0.046)。IL-17a的m RNA表达量在b组中最高(10.95±3.72),与a组(6.94±1.88,P=0.018)及c组(7.06±2.75,P=0.023)相比均有统计学意义。结论:1.IRP患者外周血血浆IL-35水平较正常对照组明显降低;缓解组外周血血浆IL-35水平较初治组明显增高。CD4~+CD25~+Foxp3~+细胞比例的降低及该细胞中IL-35m RNA表达水平的降低可能是IL-35水平降低的原因之一。2.IRP患者Th17细胞及血浆IL-17水平较正常对照组明显增高。体外混合培养实验证明IL-35可抑制IRP患者CD4~+T淋巴细胞分化为Th17细胞,这种抑制是通过IL-35对其受体亚单位gp130的激活进而抑制转录调节因子RORγt所实现的。
[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.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R55

【参考文献】