重症肌无力患者胸腺切除术远期疗效分析和胸腺及外周血调节性B细胞和T细胞研究

发布时间：2018-06-03 19:21

本文选题：重症肌无力 + 胸腺切除术　；参考：《华中科技大学》2014年博士论文

【摘要】：第一部分重症肌无力患者胸腺切除术远期疗效及影响因素分析目的分析重症肌无力(MG)患者胸腺切除术的远期疗效及影响疗效的相关因素,并比较不同胸腺病理类型患者的临床特点和预后差别。方法回顾性分析1984年1月-2010年12月我院胸腺切除术后规律随访的306例MG患者,以完全稳定缓解(CSR)和广义临床缓解(CSR+PR+MM)为疗效指标,对疗效相关影响因素进行单因素Kaplan-Meier和多因素Cox比例风险回归分析；并对其中174例MG伴胸腺瘤(T-MG)和132例MG不伴胸腺瘤(NT-MG)患者的临床特点和预后进行方差分析或秩检验。结果 306名患者中女性151名,男性155名,平均起病年龄为31.54岁,术前平均症状持续时间为33.43月,胸腺切除术时平均年龄为34.16岁,术前MGFA分级Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴ分别为116(37.91%),117(38.24%),53(17.32%),3(0.98%),17(5.56%),其中274例采用正中劈胸扩大胸腺切除术,32例采用胸腔镜胸腺切除术(VATS)。围手术期死亡率为2.94%(9/306),余297例患者平均随访时间为103月,胸腺切除术总体有效率为81.14%,其中81例达到完全稳定缓解(CSR)(27.27%),172例达到广义临床缓解(CSR+PR+MM)(57.91%),69例症状显著缓解(Improved)(23.23%)。在单因素分析中,胸腺切除术时年龄40岁(P=0.034),术前MGFA分型I型(P=0.001)和MG不伴胸腺瘤(P=0.019)与CSR正相关；同时术前MGFA分型I型(P=0.004),无伴发疾病(P=0.022)和MG不伴胸腺瘤(P=0.019)与广义临床缓解(CSR+PR+MM)正相关；多因素分析中,MGFA分型全身型(OR:2.088,95%CI:1.339-3.265,P=0.001)和有伴发疾病(OR:1.668,95%CI:1.072-2.549,P=0.023)是胸腺切除术后临床缓解的独立危险因素。NT-MG患者完全稳定缓解率和临床缓解率显著高于T-MG患者(CSR:33.33%vs22.42%,P=0.036;临床缓解率：64.39%vs52.72%,P=0.043),而且,与T-MG患者相比,更多的NT-MG患者从胸腺切除术中获益(86.36%vs76.97%,P=0.04)。此外,T-MG患者临床恶化率和死亡率明显高于NT-MG(临床恶化率：15.76%vs6.06%,P=0.009；死亡率：11.49%vs3.03%,P=0.004)。9例围手术期死亡患者均为T-MG,随访阶段9例T-MG(5.45%)死于MG危象,而NT-MG仅2例(1.52%)。结论胸腺切除术是治疗MG患者的有效手段,可获得较高的长期临床缓解率,术前MGFA分型全身型和有伴发疾病是术后临床缓解的独立危险因素。T-MG比NT-MG患者临床症状相对更严重,且长期预后不乐观,因此需要优化对T-MG患者的术前评估和围手术期准备以降低患者围手术期死亡率并提高术后长期生存率。即使患者术后较长时间内达到完全临床缓解,医生仍需严格把握停药指征以防复发。第二部分重症肌无力患者胸腺调节性B细胞和T细胞比例和相关分子表达的研究目的 1.比较CD5+CD1d+CD19+Breg细胞和CD4+CD25+CD127low/Treg细胞在MG不同胸腺病理类型间的比例差异。 2.比较MG胸腺组织中调节性B细胞和T细胞功能相关分子的表达差异。方法 1.采用免疫组化法检测10例MG增生胸腺、14例MG伴胸腺瘤、4例MG萎缩胸腺和15例正常对照(7例儿童和8例成人)胸腺组织CD19、CD5、CD25、 Foxp3分子的分布及表达。 2.采用流式细胞术分析6例MG增生胸腺、12例MG伴胸腺瘤、5例MG萎缩胸腺和13例正常对照(7例儿童和6例成人)胸腺CD5+CD1d+CD19+Breg细胞和CD4+CD25+CD127low/-Treg细胞的比例变化。 3.采用Real time PCR法分析10例MG增生胸腺、16例MG伴胸腺瘤和17例正常对照胸腺组织中调节性B细胞和调节性T细胞功能相关分子转录水平的差异。结果 1.成人萎缩胸腺CD19少量散在分布于胸腺髓质,而儿童正常胸腺可见大量CD19分布于胸腺髓质。MG增生胸腺内CD19分布范围广泛,表达显著增加,主要表达在淋巴滤泡内,尤其在生发中心和髓质胸腺小体周围呈簇状分布;正常对照胸腺组织中CD5分布范围广泛；MG增生胸腺内CD5分布范围也广泛,特征性出现生发中心明区的阳性表达,淋巴滤泡边缘带也可见CD5阳性表达；MG胸腺瘤组织内CD19和CD5的表达与胸腺瘤分型相关,伴有淋巴滤泡形成的B1型胸腺瘤患者,可见淋巴滤泡内CD19表达显著增加,而A型、AB型和B3型胸腺瘤内罕见CD19阳性表达,CD5的表达也显著降低。MG萎缩胸腺CD19和CD5的表达与正常对照类似。 MG患者与正常对照间CD25和FoxP3具有相似的组织学分布,但MG增生胸腺和儿童胸腺CD25的表达显著高于正常成人,MG萎缩胸腺CD25表达与正常成人萎缩胸腺无差别,MG胸腺瘤患者CD25的表达显著低于正常对照、MG增生和萎缩胸腺。MG患者胸腺组织FoxP3表达显著低于正常对照,以胸腺瘤患者表达最低。 2.MG患者胸腺组织CD5+CD1d+CD19+Breg匕例显著低于正常对照,MG增生胸腺的CD5+CDld+CD19+Breg细胞比例显著高于MG萎缩胸腺和MG胸腺瘤患者(p0.05)。MG患者胸腺CD4+CD25+CD127-/lowT细胞比例与健康对照间无明显差异, MG增生胸腺、MG萎缩胸腺和MG胸腺瘤间CD4+CD25+CD127/lowT细胞比例无明显差异。 3.MG胸腺增生患者出现多种调节性B细胞功能可能相关分子转录水平相对显著升高,而胸腺瘤患者仅CD5转录水平相对显著低于正常对照,其余均与正常对照间无明显差异；调节性T细胞功能相关分子中,MG患者FoxP3和AIRE转录水平相对显著低于正常对照,但MG胸腺增生患者CTLA-4、GITR和ICAM转录水平相对显著高于正常对照,而MG胸腺瘤患者CTLA-4相对显著低于正常对照。结论 MG患者胸腺Breg细胞比例显著低于正常对照,而且MG胸腺瘤与胸腺增生间Breg相关分子表达差异明显,提示Breg参与MG的发病。MG患者胸腺Treg细胞比例无明显改变,但是FoxP3和AIRE表达显著降低,造成Treg细胞调控功能严重受损。MG患者出现Breg细胞和Treg细胞数量和／或功能改变,可能在MG发病中发挥作用。第三部分胸腺切除术对重症肌无力患者外周血调节性B细胞和T细胞比例的影响目的比较MG患者外周血调节性B细胞(CD5+CD1d+CD19+Breg)和调节性T细胞(CD4+CD25+CD127low/-Treg)比例变化和血清抗体滴度与MG手术治疗的关系,并评估这些指标能否成为MG治疗及预后判断的免疫学指标。方法以2012.10-2013.7武汉市同济医院MG门诊和住院病人为研究对象,采用流式细胞仪分析69例MG患者和10例正常对照外周血中CD5+CD1d+CD19+Breg细胞和CD4+CD25+CD127low/-Treg细胞比例,同时ELISA法检测这些患者血清AChR抗体水平。结果 1.总体MG患者外周血CD5+CD1d+CD19+Breg细胞比例显著低于健康对照(P=0.013),术后患者CD5+CD1d+CD19+Breg细胞比例较术前升高不明显,而且术后加重复发与稳定缓解患者间细胞比例无明显差异。 2.总体MG患者外周血CD4+CD25+T细胞比例与健康对照无明显差异(p= 0.085),但术前患者CD4+CD25+T细胞的比例显著高于健康对照。总体MG患者外周血CD4+CD25+CD127low/-Treg细胞比例与健康对照组间无明显差异,术后患者CD4+CD25+CD127low/-Treg细胞比例有明显升高趋势,但与健康对照间无显著差异；而且术后加重复发与稳定缓解患者间细胞比例无明显差异。MG患者外周血CD5+CD1d+CD19+Breg细胞比例与CD4+CD25+CD127low/-Treg细胞比例间无明显相关性. 3.术后MG患者血清AChR-Ab滴度无明显下降,但术后胸腺瘤患者AChR-Ab滴度显著高于非瘤患者(p0.001),术后加重复发患者AChR-Ab滴度显著高于MG稳定缓解患者(P0.001)。结论 MG患者外周血CD5+CD1d+CD19+Breg细胞比例显著降低,而且胸腺切除术后升高不明显；CD4+CD25+CD127low/-Treg细胞比例与正常对照无明显差异,但术后有明显升高趋势。MG患者术后血清AChR-Ab滴度无明显下降。调节性B细胞参与MG发病,但调节性B细胞不能作为MG预后的可靠指标,而且胸腺切除术可能不通过影响这两类细胞亚群来发挥其治疗效应。
[Abstract]:Part one
Long term outcome and influencing factors of thymectomy in myasthenia gravis patients
objective
To analyze the long-term effect of thymectomy in patients with myasthenia gravis (MG) and the related factors affecting the curative effect, and to compare the clinical characteristics and the difference of prognosis in patients with different thymus pathological types.
Method
A retrospective analysis was made of 306 MG patients following the regular follow-up of thymectomy in our hospital in December -2010 January 1984. With complete stability remission (CSR) and generalized clinical remission (CSR+PR+MM) as the therapeutic index, a single factor Kaplan-Meier and multiple factor Cox proportional risk regression analysis were carried out on the effect related factors, and 174 cases of MG accompanied by thymoma (T-M) were analyzed. G and 132 patients with MG without thymoma (NT-MG) were analyzed by ANOVA or rank test.
Result
There were 151 women and 155 men in 306 patients. The average onset age was 31.54 years, the average duration of the preoperative symptoms was 33.43 months, the average age of the thymectomy was 34.16 years, and the preoperative MGFA grade I, II, III, IV, and V were 116 (37.91%), 117 (38.24%), 53 (17.32%), 3 (0.98%). 32 cases were treated with thoracoscopic thymectomy (VATS). The peri operative mortality was 2.94% (9/306), the average follow-up time of the remaining 297 patients was 103 months, the total effective rate of thymectomy was 81.14%, of which 81 cases reached complete stability remission (CSR) (27.27%), 172 cases reached generalized clinical remission (57.91%), 69 cases significantly alleviated (Improve D) (23.23%). In single factor analysis, the age of thymectomy is 40 years (P=0.034), MGFA type I (P=0.001) and MG without thymoma (P=0.019) are positively related to CSR; at the same time, MGFA type I type (P=0.004), no associated disease (P=0.022), no thoracic adenoma and generalized clinical remission; multifactor analysis MGFA genotyping (OR:2.088,95%CI:1.339-3.265, P=0.001) and associated disease (OR:1.668,95%CI:1.072-2.549, P=0.023) were independent risk factors for clinical remission after thymectomy. The total stability remission rate and clinical remission rate of.NT-MG patients were significantly higher than those of T-MG patients (CSR:33.33%vs22.42%, P=0.036; clinical remission rates: 64.39%vs52.72%, P. =0.043), and more NT-MG patients benefited from thymectomy compared with T-MG patients (86.36%vs76.97%, P=0.04). In addition, the clinical deterioration rate and mortality rate of T-MG patients were significantly higher than that of NT-MG (clinical deterioration rate: 15.76%vs6.06%, P=0.009; mortality: 11.49%vs3.03%, P=0.004) all deaths in the perioperative period were 9. Case T-MG (5.45%) died of MG crisis while NT-MG had only 2 cases (1.52%).
conclusion
Thymectomy is an effective method for the treatment of MG patients with a higher long-term clinical remission rate. Pre operation MGFA typing and associated disease are independent risk factors for postoperative clinical remission..T-MG is more severe than NT-MG, and the long-term prognosis is not optimistic. Therefore, it is necessary to optimize the preoperative assessment and circumference of T-MG patients. The operation period is prepared to reduce the peri operative mortality and increase the long-term survival rate. Even if the patient has complete clinical remission within a long time after the operation, the doctor still needs to strictly grasp the indication of drug withdrawal to prevent recurrence.
The second part
Proportion of thymic regulatory B cells and T cells and expression of related molecules in myasthenia gravis patients
objective
1. comparison of CD5+CD1d+CD19+Breg cells and CD4+CD25+CD127low/Treg cells in MG
The difference in the proportion of the pathological types of the thymus.
2. to compare the expression differences of regulatory B cells and T cell function related molecules in MG thymus.
Method
1. the distribution and expression of CD19, CD5, CD25, Foxp3 molecules were detected in 10 cases of MG hyperplasia thymus, 14 cases of MG with thymoma, 4 cases of MG atrophic thymus and 15 cases of normal control (7 children and 8 adults).
2. the proportion of thymus CD5+CD1d+CD19+Breg cells and CD4+CD25+CD127low/-Treg cells in 6 cases of MG hyperplasia thymus, 12 cases of MG with thymoma, 5 cases of MG atrophic thymus and 13 normal controls (7 children and 6 adults) were analyzed by flow cytometry.
3. the transcriptional levels of regulatory B cells and regulatory T cell function related molecular transcription levels in 10 cases of MG hyperplasia thymus, 16 cases of MG with thymoma and 17 normal control thymus were analyzed by Real time PCR.
Result
1. a small amount of CD19 in the adult thymus is scattered in the thymus medulla, while a large number of CD19 in the normal thymus of children are distributed in the thymus medullary.MG hyperplasia thymus, the CD19 distribution is widely distributed, the expression is significantly increased, mainly in the lymphoid follicles, especially in the germinal center and the medullary thymus small body, and in the normal thymus gland tissue, C The distribution of D5 is extensive, and the distribution of CD5 in the MG hyperplasia thymus is also extensive, the positive expression of the bright area of the germinal center and the positive expression of CD5 in the marginal zone of the lymphoid follicle are also found. The expression of CD19 and CD5 in the MG thymoma tissue is related to the thymoma typing, and the CD19 in the B1 type thymoma with lymphoid follicle formation can be seen in the lymphoid follicle CD19 The expression was significantly increased, while the expression of CD19 in type A, AB and B3 thymoma was rare, and the expression of CD5 also significantly decreased the expression of CD19 and CD5 in the.MG atrophic thymus similar to that of the normal control.
The histological distribution of CD25 and FoxP3 between MG and normal controls was similar, but the expression of MG hyperplasia thymus and thymus CD25 was significantly higher than that of normal adults. The CD25 expression in MG atrophied thymus was not different from that of normal adult atrophic thymus. The expression of CD25 in MG thymoma patients was significantly lower than that of normal controls. MG hyperplasia and atrophic thymus gland.MG patients' thymus tissues were significantly lower than normal controls. The expression of FoxP3 was significantly lower than that of normal controls, and was lowest in patients with thymoma.
The CD5+CD1d+CD19+Breg dagger of thymus tissue in 2.MG patients was significantly lower than that of normal controls. The proportion of CD5+CDld+CD19+Breg cells in MG hyperplasia thymus was significantly higher than that of MG atrophic thymus and MG thymoma patients (P0.05).MG patients with no significant difference in the proportion of thymus CD4+CD25+CD127-/lowT cells from healthy controls, MG hyperplasia thymus, MG atrophic thymus and MG thymoma. There was no significant difference in the proportion of CD4+CD25+CD127/lowT cells.
In patients with 3.MG thymus hyperplasia, a number of regulatory B cell function related molecular transcriptional levels were raised relatively significantly, while only CD5 transcriptional levels in thymoma patients were significantly lower than those of normal controls, and the rest were not significantly different from those of normal controls; of the regulatory T cell function related molecules, the FoxP3 and AIRE transcriptional levels of MG patients were relatively significant. The CTLA-4, GITR and ICAM transcriptional levels in patients with MG thymus hyperplasia were significantly higher than those of normal controls, while CTLA-4 in MG thymoma patients was significantly lower than that of normal controls.
conclusion
The proportion of Breg cells in thymus of MG patients was significantly lower than that of normal controls, and the expression of Breg related molecules between MG thymoma and thymic hyperplasia was distinct, suggesting that the proportion of Treg cells in thymus gland of.MG patients with Breg participation in MG was not significantly changed, but the expression of FoxP3 and AIRE decreased significantly, resulting in serious impairment of Treg cell regulation function. Changes in cell number and / or function of Treg cells may play a role in the pathogenesis of MG.
The third part
Effect of thymectomy on the ratio of regulatory B cells and T cells in peripheral blood of patients with myasthenia gravis
objective
The relationship between the changes in the proportion of B cells (CD5+CD1d+CD19+Breg) and regulatory T cells (CD4+CD25+CD127low/-Treg) in the peripheral blood of MG patients and the serum antibody titer and MG surgical treatment, and to assess whether these indicators could be an immunological index for the treatment of MG and the prognosis of the patients.
Method
The proportion of CD5+CD1d+CD19+Breg and CD4+CD25+CD127low/-Treg cells in 69 cases of MG and 10 normal controls was analyzed by flow cytometry, and the level of serum AChR antibody in these patients was detected by ELISA method by using flow cytometry in the 2012.10-2013.7 Wuhan Tongji Hospital. The flow cytometry was used to analyze the proportion of CD5+CD1d+CD19+Breg and CD4+CD25+CD127low/-Treg cells in the peripheral blood of the patients.
Result
1. the proportion of CD5+CD1d+CD19+Breg cells in peripheral blood of patients with total MG was significantly lower than that of healthy controls (P=0.013). The proportion of CD5+CD1d+CD19+Breg cells in patients after operation was not significantly higher than that before operation, and there was no significant difference in the proportion of cells between recurrent and stable remission patients after operation.
2. the proportion of CD4+CD25+T cells in peripheral blood of patients with MG was not significantly different from that of healthy controls (p=
0.085), but the proportion of CD4+CD25+T cells in patients before operation was significantly higher than that in healthy controls. There was no significant difference in the proportion of peripheral blood CD4+CD25+CD127low/-Treg cells from the control group in the overall MG patients, and the proportion of CD4+CD25+CD127low/-Treg cells in the postoperative patients was significantly higher, but there was no significant difference between the control group and the healthy control, and the recurrence was aggravated after the operation. There was no significant difference in the proportion of cells with stable remission patients. There was no significant correlation between the proportion of CD5+CD1d+CD19+Breg cells in peripheral blood and the proportion of CD4+CD25+CD127low/-Treg cells in.MG patients.
There was no significant decrease in serum AChR-Ab titer in patients with MG after 3., but the AChR-Ab titer of patients with thymoma was significantly higher than that of non tumor patients (p0.001), and the AChR-Ab titer in the patients with repeated operation was significantly higher than that of the patients with MG stable remission (P0.001).
conclusion
The proportion of CD5+CD1d+CD19+Breg cells in peripheral blood of MG patients decreased significantly, and the increase of CD4+CD25+CD127low/-Treg cells was not obvious after thymectomy, but there was no significant difference in the proportion of CD4+CD25+CD127low/-Treg cells with normal controls, but there was no significant decrease in serum AChR-Ab titer in.MG patients after operation. The modulating B cells were involved in the pathogenesis of MG, but the regulatory B cells were involved. It can not be used as a reliable index for prognosis of MG, and thymectomy may not play its therapeutic effect by affecting these two cell subsets.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R746.1

【共引文献】