妊娠合并ITP患者血清TPO水平及其鉴别诊断价值研究

发布时间：2018-07-11 10:07

本文选题：免疫性血小板减少症 + 妊娠　；参考：《山东大学》2016年博士论文

【摘要】：免疫性血小板减少症(immune thrombocytopenia, ITP)是临床上最常见的出血性疾病,约占出血性疾病总数的30%。临床表现主要为皮肤粘膜出血,以及胃肠道、泌尿生殖道出血甚至颅内出血,严重者可能危及生命。目前公认本病是一种获得性自身免疫性疾病,血小板自身抗体和／或细胞毒性T淋巴细胞(CTL)所介导的血小板破坏增多和巨核细胞成熟障碍引起的血小板生成减少都在本病的发病机制中具有重要作用。妊娠期合并血小板减少是常见的妊娠期合并症,其发病率约10%,可由多种疾病引起,以妊娠期血小板减少症(gestational thrombocytopenia, GT)最常见；妊娠合并ITP亦是比较常见的原因,约占妊娠合并血小板减少的5%。妊娠期血小板减少症是一种妊娠期的良性疾病,对孕妇和胎儿均无不良影响,临床上一般无需进行治疗；而妊娠合并ITP则可能严重影响母体和胎儿健康,使妊娠及其处理复杂化。母体的IgG型抗血小板抗体可通过胎盘到达胎儿循环,导致胎儿血小板减少及出血,表现为胎儿或新生儿的消化道及颅内出血。因此,尽早鉴别和诊断妊娠合并ITP,并准确评估孕妇和新生儿的出血风险及预后情况,对于疾病的及时治疗和密切监护具有重要意义。血小板生成素(thrombopoietin, TPO)能够与c-Mpl受体特异性结合,进而发挥生物学效应,调节巨核细胞增殖、分化、成熟,并分裂形成有功能的血小板。TPO主要在肝脏内产生,进入血液循环后,与血小板和巨核细胞表面的c-Mpl受体结合而被清除。由于功能正常的肝脏产生TPO的速度基本恒定,因此外周血TPO浓度主要被血小板计数调控,血小板计数越低,TPO水平越高。然而,近年来有多项研究发现,ITP患者血清TPO水平略高于甚至不高于健康对照组；由于ITP患者血小板计数显著降低,有学者认为内源性TPO生成不足为ITP发病机制之一。为了研究妊娠合并ITP患者与妊娠期血小板减少症患者TPO水平是否存在差异,进而讨论TPO水平可否作为鉴别诊断妊娠合并ITP与GT的参考指标,并进一步探讨妊娠合并ITP的发病机制,我们研究了妊娠合并ITP患者、妊娠期血小板减少症患者、正常妊娠女性、非妊娠期育龄女性ITP患者以及正常育龄女性的外周血血清TPO水平,以及妊娠合并ITP患者和非妊娠期育龄女性ITP患者的骨髓细胞学涂片。我们发现,妊娠合并ITP患者的血清TPO水平显著高于GT患者,在35位妊娠合并ITP患者中,29位患者的TPO水平高于500 pg/mL,而全部31位GT患者的血清TPO水平均低于500 pg/mL。因此,我们推测,血清TPO水平可以作为鉴别诊断妊娠合并ITP与GT的参考指标,TPO水平高于500 pg/mL的妊娠女性患者极有可能为妊娠合并ITP患者,需要密切监护和积极治疗。另一方面,我们还发现妊娠合并ITP患者的血清TPO水平显著高于非妊娠期育龄女性ITP患者。近年来多项研究证实,巨核细胞不足所引起的血小板减少症患者TPO水平显著升高,而血小板破坏增多所引起的血小板减少症患者TPO水平较低。因此,我们进一步对比了妊娠合并ITP患者和非妊娠期育龄女性ITP患者的骨髓细胞学涂片,对比结果显示,非妊娠期育龄女性ITP患者骨髓中巨核细胞数目正常或增多,而半数以上的妊娠合并ITP患者骨髓中巨核细胞数目减少。以上结果提示,妊娠合并ITP的发病可能与骨髓中巨核细胞产生血小板减少有关,其发病机制可能与非妊娠期ITP不同。第一部分：妊娠合并ITP患者血清TPO水平及其鉴别诊断价值的研究目的：检测妊娠合并ITP患者、妊娠期血小板减少症患者及非妊娠期育龄女性ITP患者的外周血血清TPO水平,通过比较妊娠合并ITP患者与GT患者及与非妊娠ITP患者TPO水平的差异,分析TPO作为妊娠合并ITP与GT鉴别诊断参考指标的可行性,并进一步探讨妊娠合并ITP的发病机制。方法：1.收集35例妊娠合并ITP患者、31例GT患者、32例正常妊娠女性、32例非妊娠期育龄女性ITP患者及35例正常育龄女性的外周血,检测外周血血小板计数,并分离血清备用。2.酶联免疫吸附试验(ELISA)检测血清中TPO的浓度。3.对23例妊娠合并ITP患者和27例非妊娠期育龄女性ITP患者行髂后上嵴骨髓穿刺,并进行骨髓细胞学涂片,Wright-Giemsa染色,计数巨核细胞。4.改良单克隆抗体俘获血小板抗原技术(MAIPA)检测25例妊娠合并ITP患者及32例非妊娠期育龄女性ITP患者血浆中血小板自体抗体。结果：1.妊娠合并ITP患者血清TPO水平显著高于GT患者。妊娠合并ITP组血清TPO水平显著高于GT组(均数±标准差,1283±646 pg/mL vs.187±64 pg/mL,P0.01)；在35位妊娠合并ITP患者中,29位患者的TPO水平高于500 pg/mL,而全部31位GT患者的血清TPO水平均低于500 pg/mL。这些结果说明,血清TPO水平可以作为鉴别诊断妊娠合并ITP与GT的参考指标,TPO水平高于500 pg/mL的妊娠女性患者更有可能为妊娠合并ITP患者。2.妊娠合并ITP患者血清TPO水平显著高于非妊娠期育龄女性ITP患者。妊娠合并ITP组血清TPO水平显著高于非妊娠期育龄女性ITP组(均数±标准差,1283±646 pg/mL vs.88±41 pg/mL,P0.01).3.大多数妊娠合并ITP患者骨髓巨核细胞数量减少。将骨髓细胞学涂片中每1到3个低倍视野见1个巨核细胞定义为巨核细胞数量正常,每个低倍视野2个巨核细胞为巨核细胞数量增多,每5到10个低倍视野见1个巨核细胞为巨核细胞数量减少。基于上述标准,在23例妊娠合并ITP患者中,14例(60.9%)巨核细胞数量减少,5例(21.7%)正常,4例(17.4%)增多；而在27例非妊娠期育龄女性ITP患者中,10例(37.0%)巨核细胞数量正常,17例(63.0%)增多。以上结果说明,大多数妊娠合并ITP患者骨髓巨核细胞数量减少,而大多数非妊娠期育龄女性ITP患者骨髓巨核细胞数量增多,妊娠与非妊娠ITP发病机制可能存在差异。4.妊娠合并ITP患者与非妊娠期育龄女性ITP患者血小板抗体无显著差异。改良MAIPA法检测血小板抗体,将吸光度大于正常对照组均数+3倍标准差定义为抗血小板抗体阳性。25例妊娠合并ITP患者和32例非妊娠期育龄女性ITP患者中,血小板抗体阳性率分别为44.0%和62.5%,差异不具有显著性(P0.05)。结论：1.妊娠合并ITP患者血清TPO水平显著高于GT患者,TPO水平可作为两种疾病鉴别诊断的参考指标。2.妊娠合并ITP患者血清TPO水平显著高于非妊娠期育龄女性ITP患者。3.大多数妊娠合并ITP患者骨髓巨核细胞数量减少,妊娠与非妊娠期ITP发病机制可能存在差异。第二部分：rhTPO治疗对ITP患者血小板去唾液酸化调控作用的研究目的：通过检测ITP患者和正常对照血小板去唾液酸化水平,分析ITP患者血小板去唾液酸化水平是否存在异常；检测ITP患者在接受重组人血小板生成素(recombinant human thrombopoietin, rhTPO)治疗前后的血小板去唾液酸化水平,并分析其与疗效的关系。从而探讨rhTPO用于ITP治疗时可能存在的作用机制。方法：1.入组22例一线治疗失败或复发的慢性ITP患者,治疗前收取患者外周血。同时入组24例健康志愿者作为正常对照,收取外周血,离心获取血小板。2.对入组的慢性ITP患者给予重组人血小板生成素(rhTPO)治疗(300U/kg/d),治疗起效后或14天仍不起效时停止rhTPO治疗,再次收取外周血,离心获取血小板。3.用PE-Cy5-CD41a单克隆抗体标记血小板,并同时使用FITC-RCA-1单克隆抗体标记血小板表面去唾液酸化后所暴露的β-Gal残基,流式细胞术检测RCA-I的平均荧光强度,并以此反映血小板表面去唾液酸化水平。结果：1.接受rhTPO治疗的慢性ITP患者,40.9%在14天内达到完全反应(complete response, CR)标准并停药,31.8%在14天后达到反应(response, R)标准,27.3%在治疗14天后仍无反应(no response, NR)。rhTPO治疗过程中,ITP患者均未出现严重不良反应。2.ITP患者血小板去唾液酸化水平显著高于正常对照组(P0.01)。3.接受rhTPO治疗后,ITP患者血小板去唾液酸化水平降低,治疗前后存在显著性差异(P0.01)。结论：1.血小板去唾液酸化水平升高可能与ITP的发病机制有关。2. rhTPO用于ITP患者的治疗安全且有效,并可显著降低ITP患者的血小板去唾液酸化水平,这可能是rhTPO治疗ITP的作用机制之一。
[Abstract]:Immune thrombocytopenia (ITP) is the most common haemorrhagic disease in clinical. The clinical manifestations of 30%., which account for the total number of hemorrhagic diseases, are mainly caused by bleeding of the skin and mucous membrane, as well as gastrointestinal tract, urogenital tract hemorrhage and even intracranial hemorrhage. Immune diseases, platelet autoantibodies and / or cytotoxic T lymphocyte (CTL) mediated platelet destruction and thrombocytopenia caused by megakaryocyte maturation disorders are all important in the pathogenesis of this disease. Pregnancy associated thrombocytopenia is a common complication of pregnancy, with an incidence of about 10%, Gestational thrombocytopenia (GT) is the most common cause of pregnancy induced thrombocytopenia (GT). Pregnancy combined with ITP is also a common cause of thrombocytopenia in pregnancy with thrombocytopenia. Thrombocytopenia in pregnancy is a benign disease of pregnancy. There is no adverse effect on pregnant women and fetus, and there is no need in clinical practice. The pregnancy combined with ITP may seriously affect the maternal and fetal health and complicate the pregnancy and its treatment. The maternal IgG antiplatelet antibody can reach the fetal circulation through the placenta, resulting in fetal thrombocytopenia and bleeding, as well as the digestive tract and intracranial hemorrhage of the fetus or newborn. Therefore, the early identification and diagnosis of pregnancy induced pregnancy Pregnancy with ITP, and accurately assessing the risk of bleeding and prognosis of pregnant and newborn babies, is of great significance for timely treatment and close monitoring of the disease. Thrombopoietin (TPO) can specifically combine with the c-Mpl receptor, and then play a biological effect, regulate megakaryocyte proliferation, differentiation, maturation, and split to form. The function of platelet.TPO is produced mainly in the liver. After entering the blood circulation, it is cleared with the c-Mpl receptor on the surface of the blood platelets and megakaryocytes. The rate of TPO in the normal liver is basically constant, so the concentration of TPO in the peripheral blood is mainly regulated by the platelet count, the lower the platelet count, the higher the TPO level. However, in recent years, the higher the TPO level. A number of studies have found that the level of serum TPO in ITP patients is slightly higher or not higher than that in the healthy control group. Due to the significant decrease in the platelet count in ITP patients, some scholars believe that endogenous TPO deficiency is one of the pathogenesis of ITP. In order to study the difference in the TPO level between the patients with pregnancy combined with ITP and the pregnancy stage of small plate reduction. Whether TPO level can be used as a reference index for differential diagnosis of pregnancy combined with ITP and GT, and to further explore the pathogenesis of pregnancy combined with ITP, we studied TPO water in peripheral blood serum of pregnant women with ITP, gestational thrombocytopenia, normal pregnancy, non pregnant women of childbearing age ITP and normal women of childbearing age. We found that the serum TPO level of patients with pregnancy combined with ITP and non pregnant women of childbearing age ITP was significantly higher than that of GT patients. In the 35 pregnancy combined with ITP, the level of TPO in 29 patients was higher than that of 500 pg/mL, while the serum TPO levels of all 31 patients were lower than those of 500 pg/mL.. Therefore, we speculate that serum TPO level can be used as a reference for differential diagnosis of pregnancy combined with ITP and GT. Women with TPO levels higher than 500 pg/mL are highly likely to be pregnant with ITP patients, requiring intensive care and active treatment. On the other hand, we also found that serum TPO levels in patients with pregnancy combined with ITP are significantly higher than those in non pregnancy. ITP patients of childbearing age. In recent years, a number of studies have confirmed that the level of TPO in patients with thrombocytopenia caused by megakaryocyte deficiency is significantly higher and the level of TPO in patients with thrombocytopenia caused by increased platelet destruction is lower. Therefore, we further compare the bone marrow of patients with ITP and non pregnant women of childbearing age ITP. Cytological smears showed that the number of megakaryocytes in the bone marrow of non pregnant women of childbearing age was normal or increased, and the number of megakaryocytes in bone marrow of more than half of the pregnant women with ITP decreased. The results suggested that the incidence of ITP in pregnancy may be related to the thrombocytopenia of megakaryocytes in the bone marrow, and the pathogenesis of the disease may be associated with the pathogenesis of ITP. It may be different from non pregnancy ITP. Part 1: the study of serum TPO level and differential diagnostic value of ITP patients with pregnancy: detection of TPO levels in peripheral blood serum of patients with pregnancy combined with ITP, gestational thrombocytopenia and non pregnant women of childbearing age ITP, by comparing pregnancy with ITP patients and GT patients and The difference in the level of TPO in the non pregnant ITP patients was analyzed, and the feasibility of TPO as a reference index for the differential diagnosis of pregnancy combined with ITP and GT was analyzed, and the pathogenesis of ITP in pregnancy was further explored. Methods: 1. a total of 35 pregnant women with ITP, 31 GT patients, 32 normal pregnant women, 32 non pregnant women of childbearing age and 35 normal childbearing were collected. The peripheral blood of the aged women, the peripheral blood platelet count, and the separation of serum.2. enzyme linked immunosorbent assay (ELISA) to detect the concentration of TPO in serum.3. were performed on 23 cases of pregnant women with ITP and 27 non pregnant women of childbearing age in the posterior superior iliac crest bone marrow puncture, and the bone marrow cytology smear, Wright-Giemsa staining, and megakaryocyte were counted by Wright-Giemsa staining. Cell.4. modified monoclonal antibody captive platelet antigen technique (MAIPA) was used to detect the blood platelet autoantibodies in 25 cases of pregnancy combined with ITP and 32 non pregnant women of childbearing age ITP. Results: the serum TPO level of the 1. pregnant ITP patients was significantly higher than that of the GT patients. The serum TPO level in the group ITP of pregnancy was significantly higher than that of the GT group. The standard deviation, 1283 + 646 pg/mL vs.187 + 64 pg/mL, P0.01). In 29 patients with ITP patients with 35 pregnancy, the level of TPO was higher than that of 500 pg/mL, and the serum TPO levels of all 31 GT patients were lower than 500 pg/mL.. The female patients with pregnant women were more likely to be significantly higher in serum TPO levels in patients with.2. pregnancy combined with ITP than in non pregnant women of childbearing age ITP. The serum TPO level of group ITP pregnant with pregnancy was significantly higher than that of non pregnant women of childbearing age ITP group (1283 + 646 pg/mL vs.88 + 41 pg/mL, P0.01) The number of megakaryocyte in bone marrow of TP patients decreased. 1 megakaryocytes were defined as megakaryocyte in 1 megakaryocytes in bone marrow smears and 1 megakaryocytes were defined as megakaryocyte, 2 megakaryocytes per low field of vision were megakaryocytes, and 1 megakaryocytes were reduced by 1 megakaryocytes for every 5 to 10 low fields. Based on the above criteria, 23 Among ITP patients with ITP, the number of megakaryocytes decreased, 5 cases (21.7%) were normal and 4 (17.4%) increased, while 10 (37%) megakaryocytes were normal and 17 (63%) increased in 27 non pregnant women of childbearing age ITP. The results showed that the number of megakaryocytes in most patients with ITP was reduced, and most of the megakaryocytes were reduced in most patients with ITP. The number of megakaryocyte in bone marrow of ITP patients in non pregnant women of childbearing age increased, and the pathogenesis of pregnancy and non pregnancy ITP may exist differences between.4. pregnancy and ITP patients and non pregnant women of childbearing age ITP. The improved MAIPA method was used to detect platelet antibody, and the absorbance was greater than that of normal control group +3 times standard difference. The positive rate of platelet antibody was 44% and 62.5% in.25 patients with ITP and 32 non pregnant women of childbearing age. The difference was not significant (P0.05). Conclusion: the level of TPO in the serum of ITP patients with 1. pregnancy was significantly higher than that of the GT patients, and TPO level could be used as a reference for the differential diagnosis of two diseases. Serum TPO level in.2. pregnancy combined with ITP patients was significantly higher than that in non pregnant women of childbearing age ITP patients with.3. and ITP patients, the number of megakaryocytes decreased, and there may be differences in ITP pathogenesis between pregnancy and non pregnancy. The second part: the study of rhTPO therapy on the regulation of blood acidification in ITP patients By detecting the level of acidification of the ITP patients and the normal control platelets, the abnormality of the blood platelet acidification level in ITP patients was analyzed, and the level of blood platelet acidification before and after the treatment of recombinant human thrombopoietin (rhTPO) with recombinant human thrombopoietin (rhTPO) was detected in patients with ITP, and the effect was analyzed and the effect was analyzed. To explore the possible mechanism of action of rhTPO for ITP treatment. Methods: 1. patients with 22 patients who had failed or relapsed in group 1. were treated to receive peripheral blood before treatment. At the same time, 24 healthy volunteers were enrolled as normal controls, receiving peripheral blood, and centrifugation to obtain platelet.2. for chronic ITP patients who were enrolled in the group. Group human thrombopoietin (rhTPO) therapy (300U/kg/d), the treatment of rhTPO after the onset of effect or 14 days was still not effective, the peripheral blood was collected again, the platelet.3. was centrifuged to mark platelets with PE-Cy5-CD41a monoclonal antibody, and the FITC-RCA-1 monoclonal antibody was used to mark the exposure of the beta -Gal residue after the acidification of the platelet surface. Base, flow cytometry was used to detect the average fluorescence intensity of RCA-I and to reflect the level of acidification on the surface of the platelets. Results: 1. of chronic ITP patients treated with rhTPO, 40.9% had complete response (complete response, CR) standards and stopped drugs within 14 days, 31.8% were 14 days after the reaction (response, R), 27.3% in 14 days after treatment. During the treatment of no response (NR).RhTPO, there was no severe adverse reaction in ITP patients with.2.ITP, the level of blood platelet acidification was significantly higher than that of the normal control group (P0.01).3. accepted rhTPO treatment, the level of platelet acidification decreased in ITP patients, and there was a significant difference before and after treatment (P0.01). Conclusion: 1. platelets are gone. The increase of salivary acidification level may be related to the pathogenesis of ITP..2. rhTPO is safe and effective for the treatment of ITP patients, and can significantly reduce the level of platelet sialic acidification in ITP patients, which may be one of the mechanisms of rhTPO for the treatment of ITP.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R714.25

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