本文選題：重癥肌無力 + 胸腺切除術　； 參考：《華中科技大學》2014年博士論文

【摘要】：第一部分 重癥肌無力患者胸腺切除術遠期療效及影響因素分析 目的 分析重癥肌無力(MG)患者胸腺切除術的遠期療效及影響療效的相關因素,并比較不同胸腺病理類型患者的臨床特點和預后差別。 方法 回顧性分析1984年1月-2010年12月我院胸腺切除術后規(guī)律隨訪的306例MG患者,以完全穩(wěn)定緩解(CSR)和廣義臨床緩解(CSR+PR+MM)為療效指標,對療效相關影響因素進行單因素Kaplan-Meier和多因素Cox比例風險回歸分析；并對其中174例MG伴胸腺瘤(T-MG)和132例MG不伴胸腺瘤(NT-MG)患者的臨床特點和預后進行方差分析或秩檢驗。 結果 306名患者中女性151名,男性155名,平均起病年齡為31.54歲,術前平均癥狀持續(xù)時間為33.43月,胸腺切除術時平均年齡為34.16歲,術前MGFA分級Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴ分別為116(37.91%),117(38.24%),53(17.32%),3(0.98%),17(5.56%),其中274例采用正中劈胸擴大胸腺切除術,32例采用胸腔鏡胸腺切除術(VATS)。圍手術期死亡率為2.94%(9/306),余297例患者平均隨訪時間為103月,胸腺切除術總體有效率為81.14%,其中81例達到完全穩(wěn)定緩解(CSR)(27.27%),172例達到廣義臨床緩解(CSR+PR+MM)(57.91%),69例癥狀顯著緩解(Improved)(23.23%)。在單因素分析中,胸腺切除術時年齡40歲(P=0.034),術前MGFA分型I型(P=0.001)和MG不伴胸腺瘤(P=0.019)與CSR正相關；同時術前MGFA分型I型(P=0.004),無伴發(fā)疾病(P=0.022)和MG不伴胸腺瘤(P=0.019)與廣義臨床緩解(CSR+PR+MM)正相關；多因素分析中,MGFA分型全身型(OR:2.088,95%CI:1.339-3.265,P=0.001)和有伴發(fā)疾病(OR:1.668,95%CI:1.072-2.549,P=0.023)是胸腺切除術后臨床緩解的獨立危險因素。NT-MG患者完全穩(wěn)定緩解率和臨床緩解率顯著高于T-MG患者(CSR:33.33%vs22.42%,P=0.036;臨床緩解率：64.39%vs52.72%,P=0.043),而且,與T-MG患者相比,更多的NT-MG患者從胸腺切除術中獲益(86.36%vs76.97%,P=0.04)。此外,T-MG患者臨床惡化率和死亡率明顯高于NT-MG(臨床惡化率：15.76%vs6.06%,P=0.009；死亡率：11.49%vs3.03%,P=0.004)。9例圍手術期死亡患者均為T-MG,隨訪階段9例T-MG(5.45%)死于MG危象,而NT-MG僅2例(1.52%)。 結論 胸腺切除術是治療MG患者的有效手段,可獲得較高的長期臨床緩解率,術前MGFA分型全身型和有伴發(fā)疾病是術后臨床緩解的獨立危險因素。T-MG比NT-MG患者臨床癥狀相對更嚴重,且長期預后不樂觀,因此需要優(yōu)化對T-MG患者的術前評估和圍手術期準備以降低患者圍手術期死亡率并提高術后長期生存率。即使患者術后較長時間內達到完全臨床緩解,醫(yī)生仍需嚴格把握停藥指征以防復發(fā)。 第二部分 重癥肌無力患者胸腺調節(jié)性B細胞和T細胞比例和相關分子表達的研究 目的 1.比較CD5+CD1d+CD19+Breg細胞和CD4+CD25+CD127low/Treg細胞在MG不 同胸腺病理類型間的比例差異。 2.比較MG胸腺組織中調節(jié)性B細胞和T細胞功能相關分子的表達差異。 方法 1.采用免疫組化法檢測10例MG增生胸腺、14例MG伴胸腺瘤、4例MG萎縮胸腺和15例正常對照(7例兒童和8例成人)胸腺組織CD19、CD5、CD25、 Foxp3分子的分布及表達。 2.采用流式細胞術分析6例MG增生胸腺、12例MG伴胸腺瘤、5例MG萎縮胸腺和13例正常對照(7例兒童和6例成人)胸腺CD5+CD1d+CD19+Breg細胞和CD4+CD25+CD127low/-Treg細胞的比例變化。 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例正常對照胸腺組織中調節(jié)性B細胞和調節(jié)性T細胞功能相關分子轉錄水平的差異。 結果 1.成人萎縮胸腺CD19少量散在分布于胸腺髓質,而兒童正常胸腺可見大量CD19分布于胸腺髓質。MG增生胸腺內CD19分布范圍廣泛,表達顯著增加,主要表達在淋巴濾泡內,尤其在生發(fā)中心和髓質胸腺小體周圍呈簇狀分布;正常對照胸腺組織中CD5分布范圍廣泛；MG增生胸腺內CD5分布范圍也廣泛,特征性出現(xiàn)生發(fā)中心明區(qū)的陽性表達,淋巴濾泡邊緣帶也可見CD5陽性表達；MG胸腺瘤組織內CD19和CD5的表達與胸腺瘤分型相關,伴有淋巴濾泡形成的B1型胸腺瘤患者,可見淋巴濾泡內CD19表達顯著增加,而A型、AB型和B3型胸腺瘤內罕見CD19陽性表達,CD5的表達也顯著降低。MG萎縮胸腺CD19和CD5的表達與正常對照類似。 MG患者與正常對照間CD25和FoxP3具有相似的組織學分布,但MG增生胸腺和兒童胸腺CD25的表達顯著高于正常成人,MG萎縮胸腺CD25表達與正常成人萎縮胸腺無差別,MG胸腺瘤患者CD25的表達顯著低于正常對照、MG增生和萎縮胸腺。MG患者胸腺組織FoxP3表達顯著低于正常對照,以胸腺瘤患者表達最低。 2.MG患者胸腺組織CD5+CD1d+CD19+Breg匕例顯著低于正常對照,MG增生胸腺的CD5+CDld+CD19+Breg細胞比例顯著高于MG萎縮胸腺和MG胸腺瘤患者(p0.05)。MG患者胸腺CD4+CD25+CD127-/lowT細胞比例與健康對照間無明顯差異, MG增生胸腺、MG萎縮胸腺和MG胸腺瘤間CD4+CD25+CD127/lowT細胞比例無明顯差異。 3.MG胸腺增生患者出現(xiàn)多種調節(jié)性B細胞功能可能相關分子轉錄水平相對顯著升高,而胸腺瘤患者僅CD5轉錄水平相對顯著低于正常對照,其余均與正常對照間無明顯差異；調節(jié)性T細胞功能相關分子中,MG患者FoxP3和AIRE轉錄水平相對顯著低于正常對照,但MG胸腺增生患者CTLA-4、GITR和ICAM轉錄水平相對顯著高于正常對照,而MG胸腺瘤患者CTLA-4相對顯著低于正常對照。 結論 MG患者胸腺Breg細胞比例顯著低于正常對照,而且MG胸腺瘤與胸腺增生間Breg相關分子表達差異明顯,提示Breg參與MG的發(fā)病。MG患者胸腺Treg細胞比例無明顯改變,但是FoxP3和AIRE表達顯著降低,造成Treg細胞調控功能嚴重受損。MG患者出現(xiàn)Breg細胞和Treg細胞數(shù)量和／或功能改變,可能在MG發(fā)病中發(fā)揮作用。 第三部分 胸腺切除術對重癥肌無力患者外周血調節(jié)性B細胞和T細胞比例的影響 目的 比較MG患者外周血調節(jié)性B細胞(CD5+CD1d+CD19+Breg)和調節(jié)性T細胞(CD4+CD25+CD127low/-Treg)比例變化和血清抗體滴度與MG手術治療的關系,并評估這些指標能否成為MG治療及預后判斷的免疫學指標。 方法 以2012.10-2013.7武漢市同濟醫(yī)院MG門診和住院病人為研究對象,采用流式細胞儀分析69例MG患者和10例正常對照外周血中CD5+CD1d+CD19+Breg細胞和CD4+CD25+CD127low/-Treg細胞比例,同時ELISA法檢測這些患者血清AChR抗體水平。 結果 1.總體MG患者外周血CD5+CD1d+CD19+Breg細胞比例顯著低于健康對照(P=0.013),術后患者CD5+CD1d+CD19+Breg細胞比例較術前升高不明顯,而且術后加重復發(fā)與穩(wěn)定緩解患者間細胞比例無明顯差異。 2.總體MG患者外周血CD4+CD25+T細胞比例與健康對照無明顯差異(p= 0.085),但術前患者CD4+CD25+T細胞的比例顯著高于健康對照�？傮wMG患者外周血CD4+CD25+CD127low/-Treg細胞比例與健康對照組間無明顯差異,術后患者CD4+CD25+CD127low/-Treg細胞比例有明顯升高趨勢,但與健康對照間無顯著差異；而且術后加重復發(fā)與穩(wěn)定緩解患者間細胞比例無明顯差異。MG患者外周血CD5+CD1d+CD19+Breg細胞比例與CD4+CD25+CD127low/-Treg細胞比例間無明顯相關性. 3.術后MG患者血清AChR-Ab滴度無明顯下降,但術后胸腺瘤患者AChR-Ab滴度顯著高于非瘤患者(p0.001),術后加重復發(fā)患者AChR-Ab滴度顯著高于MG穩(wěn)定緩解患者(P0.001)。 結論 MG患者外周血CD5+CD1d+CD19+Breg細胞比例顯著降低,而且胸腺切除術后升高不明顯；CD4+CD25+CD127low/-Treg細胞比例與正常對照無明顯差異,但術后有明顯升高趨勢。MG患者術后血清AChR-Ab滴度無明顯下降。調節(jié)性B細胞參與MG發(fā)病,但調節(jié)性B細胞不能作為MG預后的可靠指標,而且胸腺切除術可能不通過影響這兩類細胞亞群來發(fā)揮其治療效應。
[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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R746.1

【共引文獻】

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