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免疫性血小板减少症一线治疗策略和P-gp在激素抵抗中的机制研究

发布时间:2018-09-14 21:14  文章来源:笔耕文化传播
【摘要】:免疫性血小板减少症(immune thrombocytopenia,ITP)是一种获得性的自身免疫异常综合征,约占临床出血性疾病的1/3。经典的ITP发病机制主要是血小板特异性自身抗体介导的血小板破坏增多。近年来研究发现细胞毒性T细胞直接杀伤血小板以及血小板去唾液酸化并在肝内清除也是ITP血小板减少的重要途径。此外,血小板特异性自身抗体和细胞毒性T细胞都能够抑制ITP患者骨髓巨核细胞的成熟和凋亡,导致血小板生成减少。ITP的发病涉及极其复杂的上游自身免疫失调,基本机制是自身免疫的失耐受,糖皮质激素对ITP的自身免疫紊乱状态具有广泛的调节作用。ITP患者存在自身反应性T、B淋巴细胞凋亡障碍,导致异常长生存,体外实验中地塞米松可通过抑制BAFF逆转这种异常。ITP中还存在自身反应性免疫细胞亚群和保护性亚群之间的失平衡,包括Th1/Th2失衡、Th1/Th17/Treg失衡等。研究发现地塞米松能够恢复Th1/Th2平衡以及Th17和Treg细胞的正常数量和功能,还能促进保护性亚群CD8+ Treg以及CD5+ Breg细胞的功能。此外,地塞米松能够调节ITP患者抗原呈递细胞表面功能性分子基团间的失平衡,有助于重建患者自身免疫耐受。糖皮质激素是国内外ITP诊疗指南一致推荐的成人ITP一线治疗方案,包括常规剂量泼尼松治疗和大剂量地塞米松冲击治疗。泼尼松治疗的早期有效率在2/3左右,但长期有效率较低,且由于多数患者进行长期的持续用药,易产生激素相关的不良反应。单臂研究显示大剂量地塞米松治疗新诊断的ITP早期有效率能达到80%以上,起效迅速且不良反应轻微,有望在改善疗效的同时缩短用药周期并降低不良反应。然而此前研究均未直接对比两种治疗方案的疗效和安全性,因此目前仍然需要前瞻性、随机对照研究来提供可靠的循证医学证据,以阐明哪种方案应作为ITP一线治疗的首选。部分患者对糖皮质激素治疗反应不佳或短期内复发,这种现象称为激素抵抗。克服激素抵抗现象,进一步提高糖皮质激素用于ITP一线治疗的疗效,具有重要的临床价值。P-糖蛋白由多药耐药基因MDR-1编码,具有ATP依赖的药物转运泵活性,其过表达或功能上调是导致多种自身免疫性疾病激素抵抗发生的重要机制。研究发现在系统性红斑狼疮和类风湿性关节炎患者外周血单个核细胞(peripheral blood mononuclear cell, PBMC) P-gp表达显著上调,尤其是糖皮质激素疗效不佳的患者,且PBMC对地塞米松的摄取能力显著降低,环孢素、他克莫司等能够抑制P-gp功能,恢复对地塞米松的摄取能力。因此,P-gp在ITP激素抵抗中的作用和机制值得探索。本研究应用前瞻性、多中心、随机对照研究的方法,通过对比大剂量地塞米松和常规剂量泼尼松两种治疗方案的疗效和安全性,为ITP一线治疗策略的优化提供了高等级的循证医学证据。同时检测并分析了患者大剂量地塞米松疗效和PBMC细胞P-gp表达以及细胞对地塞米松摄取能力之间的关系,并尝试应用环孢素调节P-gp功能,揭示了ITP中激素抵抗的机制和潜在的干预途径。第一部分:大剂量地塞米松对比常规剂量泼尼松用于成人原发免疫性血小板减少症一线治疗的前瞻性、多中心、随机对照研究研究目的:对比大剂量地塞米松和常规剂量泼尼松治疗成人原发性ITP的早期疗效,包括有效率和起效时间;对比两种药物的远期疗效,包括随访半年的持续缓解率及评价生存曲线;评价两种药物治疗中患者的不良反应和耐受性,对比其安全性;评价出血症状的转归和对早期疗效的预后价值;评价血小板特异性自身抗体对疗效的预后价值。从而为ITP一线治疗策略的优化提供循证医学证据,并探索糖皮质激素治疗的预后因素。研究方法:1.将未接受过正规治疗的新诊断的ITP患者按照1:1的比例随机分配至大剂量地塞米松组和常规剂量泼尼松组。2.地塞米松组给予地塞米松口服40mg/d,连用4天为1个周期,第1个周期治疗无效的患者于第10d后加用1个周期;泼尼松组给予泼尼松1.Omg/kg·d口服,连用28天,其后有效患者快速减量至最小维持剂量(15mg/d)或停药,无效患者则快速减量至停用。3.主要研究终点是早期有效率和持续有效率。早期有效率的评价依据最新国内外ITP诊疗指南,持续有效率定义为获得早期有效后,外周血小板计数连续六个月维持在30×109/L以上,且没有出血症状或者ITP特异性附加治疗需求。次要研究终点包括出血评分、起效时间、疗效维持时间和不良反应等。4.采用改良MAIPA技术检测患者治疗前外周血小板特异性自身抗体GPIIb/Ⅲa和GPIb/IX。结果:1.地塞米松组早期总有效率和完全反应(complete response, CR)率均显著高于泼尼松组,且起效时间较泼尼松组更为迅速。在地塞米松组,对第1个周期无效的患者加用第2个周期治疗能够显著提高有效率,但是在第2个周期起效的患者获得CR的可能性低于在第1个周期就起效的患者。2.治疗前外周血小板计数低于10×109/L的患者出血症状明显较严重,治疗后地塞米松组出血例数显著低于泼尼松组。治疗前出血评分8分是早期疗效的不良预后因素。3.两组患者随访半年的持续有效率无统计学差异,早期获得CR是进而获得持续有效的阳性预后因素。生存分析显示两组远期结局相当,早期获得CR的患者复发率显著较低。4.血小板特异性自身抗体GPIIb/III日性的患者基线血小板数更低,治疗前出血症状更严重。两种抗体阳性均提示患者获得持续有效的可能性降低。5.地塞米松组不良反应的发生少于泼尼松组。结论:1.1或2个周期的大剂量地塞米松治疗成人新诊断的ITP具有起效迅速,早期有效率高的优点,长期有效率与传统泼尼松治疗相当。2.大剂量地塞米松治疗患者耐受性优于传统泼尼松治疗,患者能够在保证长期有效率的前提下规避长期的激素负荷及相关不良反应风险。3.大剂量地塞米松有望成为ITP一线治疗的首选方案。第二部分:P-gp介导免疫性血小板减少症中激素抵抗的作用和机制研究研究目的:分别检测P-gp在ITP患者PBMC细胞以及外周CD4+T淋巴细胞和CD19+B淋巴细胞表面的表达水平,并根据大剂量地塞米松疗效分层分析;建立超高效液相色谱法(ultra performance liquid chromatography, UPLC)检测细胞培养液中地塞米松浓度的方法学体系,评价PBMC细胞对培养液中地塞米松的摄取能力;研究环孢素对体外培养的PBMC细胞P-gp的抑制作用。从而明确P-gp数量和功能在ITP激素抵抗中的作用和机制,并探索逆转激素抵抗作用的途径。研究方法:1.采集拟进行大剂量地塞米松治疗的新诊断的ITP患者及健康志愿者的外周血,并分离PBMC细胞。2.以PE-CD4、APC-CD19和FITC-P-gp单克隆抗体标记PBMC细胞,流式细胞术检测ITP患者和健康志愿者PBMC细胞、CD4+T淋巴细胞和CD19+B淋巴细胞表面的P-gp表达水平。3.将ITP患者和健康志愿者PBMC细胞分别与地塞米松或地塞米松+环孢素进行体外共培养,收集细胞和培养液上清。4.建立UPLC法检测细胞培养液地塞米松残余浓度的方法学体系,使用系列稀释的标准溶液进行线性检验以及回收率、精密度和稳定性考察。5.流式细胞学检测培养后的PBMC细胞、CD4+T淋巴细胞和CD19+B淋巴细胞表面的P-gp表达水平。UPLC检测细胞培养液中地塞米松残余浓度。结果:1.ITP患者PBMC细胞、CD4+T淋巴细胞和CD19+B淋巴细胞表面的P-gp表达水平均显著高于健康志愿者,大剂量地塞米松治疗无效的患者P-gp表达水平高于有效患者。2. PBMC细胞和地塞米松或环孢素共培养后,表面P-gp表达水平较培养前无明显变化。3.ITP患者PBMC细胞培养后培养液中地塞米松残余浓度明显高于健康对照,大剂量地塞米松治疗无效患者的PBMC细胞培养后地塞米松残余浓度显著高于有效患者。4.环孢素明显降低ITP患者PBMC细胞培养后培养液中地塞米松的残留浓度,培养中加入环孢素后,地塞米松残余浓度下降至健康对照水平。结论:1.大剂量地塞米松治疗无效的患者PBMC细胞表达P-gp增加,对地塞米松的摄取减少,表明P-gp的数量和功能上调是ITP患者激素抵抗的重要机制。2.环孢素能够抑制ITP患者PBMC细胞P-gp功能,增加对地塞米松的摄取,可能成为逆转ITP激素抵抗的干预途径。
[Abstract]:Immune thrombocytopenia (ITP) is an acquired autoimmune disorder syndrome, accounting for about one third of clinical hemorrhagic diseases. The classical pathogenesis of ITP is increased platelet destruction mediated by platelet-specific autoantibodies. In addition, platelet-specific autoantibodies and cytotoxic T cells can inhibit the maturation and apoptosis of bone marrow megakaryocytes in ITP patients, leading to thrombocytopenia. The mechanism is autoimmune intolerance. Glucocorticoids have a wide range of regulatory effects on ITP autoimmune disorders. ITP patients have autoreactive T and B lymphocyte apoptosis disorders, leading to abnormal long-term survival. Dexamethasone can reverse this abnormality by inhibiting BAFF in vitro. There are also autoreactive immune cells in ITP. Studies have shown that dexamethasone can restore the balance of Th1/Th2 and the normal number and function of Th17 and Treg cells. It also promotes the function of CD8+Treg and CD5+Breg cells. In addition, dexamethasone can regulate antigen presentation in ITP patients. Glucocorticoid is the first-line treatment for adult ITP recommended by the ITP guidelines at home and abroad, including conventional dose prednisone therapy and high dose dexamethasone pulse therapy. Single-arm studies have shown that high-dose dexamethasone is effective in the treatment of newly diagnosed ITP at an early stage of more than 80%, with rapid onset and mild adverse reactions. It is expected to shorten the medication cycle and reduce adverse reactions while improving the efficacy. However, previous studies have not directly compared the efficacy and safety of the two regimens, so prospective, randomized controlled studies are still needed to provide reliable evidence-based medical evidence to clarify which regimen should be the first choice for ITP treatment. Some patients have poor response to glucocorticoid therapy or relapse in the short term. Phenomenon is called hormone resistance. Overcoming hormone resistance and further improving the efficacy of glucocorticoids in the first-line treatment of ITP have important clinical value. The study found that the expression of P-gp in peripheral blood mononuclear cells (PBMC) was significantly up-regulated in SLE and RA patients, especially in patients with poor glucocorticoid therapy, and the uptake of dexamethasone by PBMC was significantly decreased, cyclosporine, tacrolimus and so on. It can inhibit the function of P-gp and restore the ability of dexamethasone uptake. Therefore, the role and mechanism of P-gp in ITP hormone resistance is worth exploring. This study applied a prospective, multicenter, randomized controlled study to compare the efficacy and safety of high-dose dexamethasone and routine-dose prednisone in the first-line treatment of ITP. The relationship between the efficacy of high-dose dexamethasone and the expression of P-gp in P BMC cells and the ability of cells to uptake dexamethasone was examined and analyzed. Cyclosporin was used to regulate the function of P-gp, revealing the mechanism of hormone resistance in ITP and potential intervention pathways. Part I: Prospective, multicenter, randomized controlled trial of high-dose dexamethasone versus conventional-dose prednisone in the first-line treatment of adult idiopathic immune thrombocytopenia Objective: To compare the early efficacy of high-dose dexamethasone with conventional-dose prednisone in the treatment of adult idiopathic ITP, including efficacy and onset time. To compare the long-term efficacy of the two drugs, including six-month follow-up continuous remission rate and survival curve; to evaluate the adverse reactions and tolerability of the two drugs, and to compare their safety; to evaluate the prognostic value of hemorrhagic symptoms and early curative effect; and to evaluate the prognostic value of platelet-specific autoantibodies for therapeutic effect Methods: 1. Patients with newly diagnosed ITP who had not received regular treatment were randomly assigned to high dose dexamethasone group and routine dose prednisone group at a ratio of 1:1. Dexamethasone group was given dexamethasone. Patients in the prednisone group were given prednisone orally at a dose of 1.Omg/kg/d for 28 days, and then the effective patients were quickly reduced to the minimum maintenance dose (15mg/d) or discontinued, while those in the ineffective group were rapidly reduced to discontinuation. Effectiveness and persistent efficacy. According to the latest ITP guidelines at home and abroad, persistent efficiency was defined as a peripheral platelet count that remained above 30 *109/L for six months without bleeding symptoms or ITP-specific additional treatment requirements. Secondary endpoints included bleeding scores and effectiveness. Results: 1. The total effective rate and complete response (CR) rate in dexamethasone group were significantly higher than those in prednisone group, and the effective time was faster than that in prednisone group. In the dexamethasone group, patients who had no response to the first cycle were significantly more likely to receive CR in the second cycle than in the first cycle. 2. Patients with peripheral platelet counts below 10 *109/L had significantly worse bleeding symptoms before treatment and after treatment. The number of bleeding cases in the dexamethasone group was significantly lower than that in the prednisone group. Before treatment, 8 points of bleeding score was a poor prognostic factor for early curative effect. 3. There was no significant difference in the sustained effective rate between the two groups after six months of follow-up. Early CR was a positive prognostic factor for achieving sustained and effective prognosis. Patients with platelet-specific autoantibodies GPIIb/III had lower baseline platelet counts and worse bleeding symptoms before treatment. Positive antibodies suggested a lower likelihood of sustained efficacy. 5. Adverse reactions in dexamethasone group were less than those in prednisone group. Conclusion: 1.1 or 2 cycles High-dose dexamethasone for newly diagnosed adult ITP has the advantages of rapid onset and high early response rate. The long-term response rate is comparable to that of traditional prednisone therapy. 2. High-dose dexamethasone is more tolerable than traditional prednisone therapy. Patients can avoid long-term hormone load and related factors on the premise of long-term efficacy. Risk of adverse reactions. 3. High-dose dexamethasone is expected to be the preferred first-line treatment for ITP. Part II: Role and mechanism of hormone resistance in immune thrombocytopenia mediated by P-gp. Objective: To detect the expression of P-gp on PBMC cells, peripheral CD4 + T cells and CD19 + B lymphocytes in patients with ITP, respectively. The methodological system of ultra performance liquid chromatography (UPLC) was established to determine the concentration of dexamethasone in cell culture medium, and the uptake of dexamethasone by PBMC cells in culture medium was evaluated. Methods: 1. PBMC cells were isolated from peripheral blood of newly diagnosed ITP patients and healthy volunteers to be treated with high-dose dexamethasone. 2. PBMC cells were isolated by PE-CD4, APC-CD19 and FITC-P-gp. PBMC cells were labeled with monoclonal antibodies, and the expression of P-gp on the surface of PBMC cells, CD4+T lymphocytes and CD19+B lymphocytes in ITP patients and healthy volunteers were detected by flow cytometry. A methodological system for the determination of residual concentration of dexamethasone in cell culture medium by UPLC was established. Linear test, recovery, precision and stability were performed using a series of diluted standard solutions. 5. The expression of P-gp on the surface of cultured PBMC cells, CD4+T lymphocytes and CD19+B lymphocytes was detected by flow cytometry. Results: 1. The expression of P-gp on PBMC, CD4 + T lymphocyte and CD19 + B lymphocyte in ITP patients was significantly higher than that in healthy volunteers. The expression of P-gp in patients with high dose of dexamethasone treatment ineffective was higher than that in effective patients. 2. After co-culture of PBMC cells with dexamethasone or cyclosporine, the expression of P-gp on the surface of PBMC cells was significantly higher than that in healthy volunteers. The residual concentration of dexamethasone in PBMC culture medium of patients with ITP was significantly higher than that of healthy controls. The residual concentration of dexamethasone in PBMC cells of patients with high dose of dexamethasone treatment ineffective was significantly higher than that of effective patients. 4. Cyclosporin significantly reduced the residual concentration of PBMC cells of patients with ITP after culture. The residual concentration of dexamethasone in nutrient solution decreased to the level of healthy control after adding cyclosporine in culture. Conclusion: 1. The expression of P-gp in PBMC cells increased and the uptake of dexamethasone decreased in patients with high dose of dexamethasone ineffective treatment, indicating that the up-regulation of P-gp quantity and function is an important mechanism of hormone resistance in ITP patients. Cyclosporin can inhibit the P-gp function of PBMC cells in ITP patients and increase the uptake of dexamethasone, which may be an intervention way to reverse ITP hormone resistance.
【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】:R558.2

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2 梁燕;特发性血小板减少性紫癜糖皮质激素受体亚型表达及其与激素抵抗的关系[D];大连医科大学;2012年

3 李香;原发性肾病综合征激素疗效差异的相关因素分析[D];中南大学;2012年



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