胸腺肽α1对严重脓毒症患者的疗效观察回顾性研究与体外实验

发布时间：2018-06-30 18:00

本文选题：胸腺肽α1 + 严重脓毒症　；参考：《南方医科大学》2016年硕士论文

【摘要】：研究背景在美国,每年每10万人中发生脓毒症例数超过200例,发生严重脓毒症的例数也高达50-95例,且脓毒症发病率逐年增高,在过去的20年里,美国住院患者中,脓毒症的发病率每年以8.7%的速度递增。尽管人们一直致力于脓毒症的预防与治疗,严重脓毒症的病死率仍高达20-50%,严重脓毒症已成为重症监护病房中患者的主要死亡原因。起初人们认为强烈的炎症反应是导致脓毒症患者的主要死亡原因,然而,大量的针对早期炎症介质的目标治疗,如抗肿瘤坏死因子a抗体、白介素1受体抗体治疗并没有改善脓毒症患者的临床预后。目前,淋巴细胞凋亡和免疫抑制越来越多的被认为在脓毒症的发病机制中起到关键的作用,有脓毒症动物模型实验和临床试验指出,阻止淋巴细胞凋亡和加强免疫功能可以改善受试者的预后。肿瘤坏死因子a被认为是脓毒症重要的促炎症反应介质之一,但Docke et al指出,肿瘤坏死因子a分泌水平的增加表明单核细胞功能及抗微生物反应能力的恢复,体外脂多糖刺激后肿瘤坏死因子α的分泌量可以作为一个有效的指标来评估脓毒症患者的免疫状态。Hall et al也指出体外刺激全血后引起肿瘤坏死因子α的分泌量可以作为一个有用的生物标志物来监测脓毒症患者的免疫功能。胸腺肽α1是胸腺分泌的一种激素,最早于1972年由Goldstein等首次发现并对其特征进行了描述。胸腺肽a1被认为是一种免疫调节多肽,在临床上已广泛应用于慢性乙型肝炎及丙型肝炎的免疫调理治疗。胸腺肽α1在免疫系统中有很多生物学活性,如激活自然杀伤细胞,刺激T淋巴细胞的增殖、分化和成熟,阻止淋巴细胞凋亡。因此,作为一种免疫调节剂,胸腺肽α1可以用于脓毒症的治疗。有相关临床研究发现,胸腺肽α1治疗严重脓毒症可以取得有益的治疗效果。然而,由于严重脓毒症患者的异质性,胸腺肽α1或许对有些患者有效,对有些患者疗效不明显,这些研究没有指出哪些严重脓毒症患者用胸腺肽α1治疗更有效。本研究主要观察胸腺肽α1对哪些严重脓毒症患者的治疗效果更好,及胸腺肽α1对严重脓毒症患者APACHEⅡ评分、SOFA评分、淋巴细胞计数的影响,同时用脂多糖体外刺激严重脓毒症患者的外周血单个核细胞后分泌的肿瘤坏死因子α的浓度,来评估胸腺肽α1对严重脓毒症患者免疫功能的影响。研究目的1.探讨胸腺肽α1对严重脓毒症患者28天死亡率及生存时间的影响；2.探讨胸腺肽α1对严重脓毒症患者APACHEII评分、SOFA评分、淋巴细胞计数的影响；3.探讨胸腺肽α1对严重脓毒症患者免疫功能的影响。研究方法1.伦理学标准收集2013年1月至2014年12月住入我院重症医学科的严重脓毒症患者为回顾性研究,统计数据时为匿名分析,故免签知情同意书。用脂多糖体外刺激严重脓毒症患者的外周血单个核细胞后分泌的肿瘤坏死因子α的浓度,来评估胸腺肽α1对严重脓毒症患者免疫功能的影响,此体外实验抽取患者血液时均由患者或直系亲属签署知情同意书。本研究符合医学伦理学标准,经医院伦理委员会批准,批准号：2014一ZZYXK-003。2.研究对象收集2013年1月至2014年12月住入我院重症医学科,符合2001年国际脓毒症定义会议制定的严重脓毒症诊断标准的患者。排除标准：年龄小于18岁、妊娠期妇女、患有恶性肿瘤、自身免疫性疾病、血液系统疾病(如再生障碍性贫血、白血病等)、或在过去1个月内使用过激素、免疫抑制剂或其他免疫刺激剂的患者,失访的患者。最终,244例患者纳入本研究。其中男169例,女75例,平均年龄(63.0±16.4)岁。引起严重脓毒症的原发感染灶为：肺部感染183例,腹部感染32例,泌尿系感染11例,其他部位感染18例。体外实验选择2015年1月诊断为严重脓毒症且不符合上述排除标准的患者12例进行抽血。3.数据收集通过浏览患者的临床病历资料,收集患者的性别、年龄、引起严重脓毒症的原发感染灶、APACHEII评分、SOFA评分、淋巴细胞计数和预后等指标(或者电话联系患者家属以获取患者的预后信息)。(以上检测指标均有我院检验科及重症医学科完成)。4.分组(1)244例患者中,根据患者确诊严重脓毒症后是否给予胸腺肽α1治疗分为胸腺肽α1组和非胸腺肽α1组,非胸腺肽α1组根据2008年国际严重脓毒症诊疗指南采用常规治疗方案,胸腺肽α1组在常规治疗基础上联合应用胸腺肽α1皮下注射1.6mg,每12小时1次,连用7天。(2)胸腺肽α1组和非胸腺肽α1组患者根据确诊严重脓毒症后第1天淋巴细胞计数分为三个亚组：≥1.00×109/L亚组,0.50-1.00×109/L亚组和≤0.50×109/L亚组。5.体外实验方案(1)严重脓毒症患者的外周血单个核细胞分离分别抽取12例严重脓毒症患者血液4m1于抗凝真空采血管中,加等体积磷酸缓冲盐溶液稀释,另加3m1人Ficoll分离液至离心管中,然后将稀释后的血液叠加到Ficoll分离液上层。将离心管在水平离心机中离心,2000转/min离心30min,取出离心管,管内液体分为3层,用吸管取出中层含有外周血单个核细胞的Ficoll液,放入另一离心管中,再加等体积磷酸缓冲盐溶液稀释后再离心,1000转/min离心10min,弃去含有血小板的上层液,离心管中沉淀物即为外周血单个核细胞。(2)胸腺肽α1对脂多糖刺激外周血单个核细胞分泌肿瘤坏死因子α的影响将含有10%胎牛血清的RPMI1640培养基平均分成3组：对照组、脂多糖组和脂多糖+胸腺肽a1组,在脂多糖+胸腺肽α1组中加入胸腺肽a1使其浓度达到200μg/ml,对照组和脂多糖组分别加入等体积磷酸缓冲盐溶液,每一组培养基中均加入外周血单个核细胞使其浓度达到106个细胞/ml,然后将3组培养基均置于37℃5% CO2培养箱中培养8小时。结束后,脂多糖组和脂多糖+胸腺肽a1组分别加入含脂多糖的磷酸缓冲盐溶液,使脂多糖终浓度为10μg/ml,对照组加入等体积磷酸缓冲盐溶液,继续培养6小时。结束后,分别取各组细胞培养上清液,用ELISA试剂盒检测肿瘤坏死因子α的浓度。6.统计方法全部数据用SPSS 19.0进行统计分析,对计量资料进行正态性检验,成正态分布者用均数±标准差(x±s)表示,采用两独立样本均数的t检验；呈偏态分布者用中位数与四分位数表示[M(QR)],采用两独立样本的mann-whitney U秩和检验；分类变量用频率表示,采用x2检验；患者的生存时间用Kaplan-Meier分析并使用log-rank进行检验；肿瘤坏死因子α的浓度比较采用单因素方差分析,组间比较采用LSD检验。以上检验均以P0.05为差异具有统计学意义。研究结果1.一般资料244例患者纳入本研究,其中胸腺肽a1组127例,非胸腺肽α1组117例患者,胸腺肽a1组与非胸腺肽α1组两组患者的性别、年龄比较,差异均无统计学意义(均P0.05)、确诊严重脓毒症后第1天虽然胸腺肽a1组患者APACHEII评分、SOFA评分高于非胸腺肽α1组、淋巴细胞计数低于非胸腺肽a1组,但两组比较,差异也均无统计学意义(均P0.05),根据淋巴细胞计数分层后,两组中各亚组间比较,以上指标差异也均无统计学意义(均P0.05)。2.患者预后比较胸腺肽a1组与非胸腺肽α1组患者28天死亡率分别为42.5%、50.4%,两组间比较,差异无统计学意义(P0.05),两组中淋巴细胞计数≥1.00×10札亚组和0.50-1.00×109/L亚组患者的28天死亡率比较,差异也均无统计学意义(均P0.05),但在淋巴细胞计数≤0.50×109/L亚组,胸腺肽α1组患者28天死亡率显著低于非胸腺肽a1组,差异有统计学意义(P0.05)。3.患者生存时间比较胸腺肽a1组与非胸腺肽a1组患者生存时间比较,差异无统计学意义(log-rank检验,P0.05),两组中淋巴细胞计数≥1.00×109/L亚组和0.50-1.00×109/L亚组患者生存时间比较,差异也均无统计学意义(log-rank检验,均P0.05),但在淋巴细胞计数≤0.50×109几亚组,胸腺肽α1组患者生存时间显著长于非胸腺肽α1组,差异有统计学意义(log-rank检验,P0.05)。4. 严重脓毒症第7天与第1天各指标动态变化值胸腺肽α1组与非胸腺肽α1组比较胸腺肽α1组与非胸腺肽α1组APACHEII评分在诊断严重脓毒症后第7天均显著低于第1天(均P0.05),胸腺肽α1组比非胸腺肽α1组降低更显著(P0.05),胸腺肽a1组SOFA评分在诊断严重脓毒症后第7天显著低于第1天(P0.05),非胸腺肽α1组SOFA评分在诊断严重脓毒症第7天虽低于第1天,但差异无统计学意义(P0.05),胸腺肽α1组与非胸腺肽α1组SOFA评分变化值比较,差异无统计学意义(P0.05),胸腺肽α1组与非胸腺肽α1组淋巴细胞计数在诊断严重脓毒症后第7天均显著高于第1天(均P0.05),但胸腺肽α1组比非胸腺肽α1组变化更显著(P0.05)。5.体外实验结果对照组、脂多糖组和脂多糖+胸腺肽al组三组中肿瘤坏死因子α的浓度比较,差异有统计学意义(P0.05),脂多糖+胸腺肽α1组肿瘤坏死因子α的浓度显著高于对照组与脂多糖组,差异均有统计学意义(均P0.05)。脂多糖组肿瘤坏死因子α的浓度也显著高于对照组,差异有统计学意义(P0.05)。研究结论1.胸腺肽αl可以显著降低淋巴细胞计数≤0.50×109/L的严重脓毒症患者的28天死亡率,延长患者的生存时间；2.胸腺肽α1能够显著降低严重脓毒症患者的APACHEII评分、SOFA评分,提高患者的淋巴细胞计数,改善患者的病情；3.胸腺肽α1能够显著改善严重脓毒症患者的免疫功能。
[Abstract]:In the United States, in the United States, more than 200 cases of sepsis occur in more than 200 cases per 100 thousand people each year. The number of cases of severe sepsis is also up to 50-95, and the incidence of sepsis increases year by year. In the past 20 years, the incidence of sepsis increases at a rate of 8.7% per year in American inpatients, although people have been committed to the prevention and prevention of sepsis. The fatality rate of severe sepsis is still as high as 20-50%, and severe sepsis has become the main cause of death in the intensive care unit. At first it was believed that the strong inflammatory response was the main cause of death in patients with sepsis. However, a large number of targets for early inflammatory mediations, such as anti tumor necrosis factor A antibodies, were found to be the main cause of death. The treatment of the 1 receptor antibody does not improve the clinical prognosis of patients with sepsis. At present, the increasing number of lymphocyte apoptosis and immunosuppression is considered to play a key role in the pathogenesis of sepsis. The experimental and clinical trials of sepsis animal model and clinical trials point out that the prevention of apoptosis and the strengthening of immune function can improve the disease. The tumor necrosis factor A is considered to be one of the important proinflammatory mediators of sepsis, but Docke et al points out that the increase in the secretion of tumor necrosis factor A indicates the recovery of monocyte function and the ability to resist microbial reaction, and the secretion of tumor necrosis factor alpha after lipopolysaccharide stimulation in vitro can be an effective one. The index to evaluate the immune state of patients with sepsis.Hall et al also points out that the secretion of TNF - alpha induced by in vitro stimulation of whole blood can be used as a useful biomarker to monitor the immune function of patients with sepsis. Thymosin alpha 1 is a hormone secreted in the thymus, first found in 1972 by Goldstein and so on. Its characteristics are described. Thymosin A1 is considered as an immunomodulatory polypeptide that has been widely used in the immunomodulatory treatment of chronic hepatitis B and hepatitis C. Thymosin alpha 1 has many biological activities in the immune system, such as activating natural killer cells, stimulating the proliferation, differentiation and maturation of T lymphocytes, and preventing lymphatic lymph nodes. Apoptosis. Therefore, as an immunomodulator, thymosin alpha 1 can be used in the treatment of sepsis. Related clinical studies have found that thymosin alpha 1 in the treatment of severe sepsis can achieve beneficial effects. However, due to the heterogeneity of patients with severe sepsis, thymosin alpha 1 may be effective in some patients, and some patients are not effective. Obviously, these studies did not point out which patients with severe sepsis were more effective with thymosin alpha 1. This study was mainly to observe the effect of thymosin alpha 1 on patients with severe sepsis, and the effect of thymosin alpha 1 on APACHE II score, SOFA score, lymphocytic count, and in vitro stimulation of lipopolysaccharide. The concentration of tumor necrosis factor alpha secreted by peripheral blood mononuclear cells in patients with severe sepsis to assess the effect of thymosin alpha 1 on the immune function of patients with severe sepsis. Purpose 1. to investigate the effect of thymosin alpha 1 on 28 day mortality and survival time in patients with severe sepsis; 2. to explore A of thymosin alpha 1 for patients with severe sepsis. The effect of PACHEII score, SOFA score and lymphocyte count; 3. to explore the effect of thymosin alpha 1 on the immune function of patients with severe sepsis. Methods 1. ethics standards were collected from January 2013 to December 2014 for severe sepsis in the Department of intensive medicine of our hospital for a retrospective study. Agreement. The concentration of TNF - alpha secreted by the peripheral blood mononuclear cells of patients with severe sepsis in vitro by using lipopolysaccharide to evaluate the effect of thymosin alpha 1 on the immune function of patients with severe sepsis. Ethical standards, approved by the hospital ethics committee, approval number: 2014 one ZZYXK-003.2. research subjects were collected from January 2013 to December 2014 in the Department of severe medicine in our hospital, which conformed to patients with severe sepsis diagnosed by the 2001 International Sepsis definition conference. Exclusion criteria: age less than 18 years, pregnant women, and malignant Tumors, autoimmune diseases, hematological diseases (such as aplastic anemia, leukemia, etc.), or patients who have used hormones, immunosuppressive agents or other immunostimulants within the past 1 months, and in 244 patients, including 169 men and 75 women, with an average age of (63 + 16.4) years of age. The primary infection was: 183 cases of pulmonary infection, 32 cases of abdominal infection, 11 cases of urinary tract infection, and 18 cases of other infections. In vitro, 12 cases of severe sepsis diagnosed as severe sepsis in January 2015 and 12 patients who did not meet the above exclusion criteria were collected by scanning the patient's clinical records to collect the sex and age of the patients. Primary infection foci of severe sepsis, APACHEII score, SOFA score, lymphocyte count and prognosis (or telephone contact with the patient's family to obtain the patient's prognosis information). (all of the above tests were performed in the Department of laboratory and the Department of critical medicine) in the.4. group (1) in 244 patients, after the patient was diagnosed with severe sepsis. The treatment of thymosin alpha 1 was divided into groups of thymosin alpha 1 and non thymosin alpha 1 group. Non thymosin alpha 1 group was treated with conventional therapy according to the 2008 International severe sepsis guidelines. The thymosin alpha 1 group was combined with thymosin alpha 1 subcutaneous injection of 1.6mg, 1 times per 12 small, and 7 days. (2) thymosin alpha 1 and non thymus. The patients in the 1 group of peptide alpha 1 were divided into three subgroups on the first day after diagnosis of severe sepsis: 1 x 109/L subgroup, 0.50-1.00 x 109/L subgroup and < 0.50 x 109/L subgroup.5. extracorporeal experimental scheme (1) separation of peripheral blood mononuclear cells from severe sepsis patients, 12 cases of severe sepsis patients' blood 4m1 in anticoagulant vacuum In the blood vessels, an equal volume of phosphate buffer solution was diluted and 3M1 human Ficoll was added to the centrifuge tube, and then the diluted blood was superimposed on the upper layer of the Ficoll separation solution. Centrifuge tube was centrifuged in a horizontal centrifuge and 2000 /min centrifuged 30min, the centrifuge tube was removed, the liquid body in the tube was divided into 3 layers, and the middle layer of peripheral blood was taken out with a sucker to contain peripheral blood single. The Ficoll solution of the nuclear cells was placed in another centrifuge tube and then diluted with an equal volume of phosphoric acid buffer solution and then centrifuged. 1000 turns /min to centrifuge 10min and discard the superfluid containing platelets. The precipitates in the centrifuge tube were peripheral blood mononuclear cells. (2) thymosin alpha 1 secreted TNF - alpha on lipopolysaccharide stimulated peripheral blood mononuclear cells The RPMI1640 medium containing 10% fetal bovine serum was divided into 3 groups: the control group, the lipopolysaccharide group and the lipopolysaccharide + thymosin A1 group, the concentration of thymosin A1 in the lipopolysaccharide + thymosin alpha 1 group was 200 g/ml, the control group and the lipopolysaccharide group were added to the constant volume phosphoric acid salt solution respectively, and each group was added to the periphery. The concentration of blood mononuclear cells reached 106 cells /ml, and then the 3 groups were incubated in the incubator of 37 centigrade 5% CO2 for 8 hours. After the end, the lipopolysaccharide group and the lipopolysaccharide + thymosin A1 group were added to the phosphate buffer solution containing lipopolysaccharide, and the final concentration of lipopolysaccharide was 10 g/ ml, and the control group was added to the equal volume phosphate buffer solution. After 6 hours, the cell culture supernatant was taken and the concentration of TNF - A was detected by ELISA kit. All data were analyzed by SPSS 19, and the normal distribution of the data was tested. The normal distribution was expressed with the average number of.6. s (x + s), and the t test of two independent samples was used. [M (QR) was expressed in the median and four digits, and the Mann-Whitney U rank sum test of the two independent samples was used. The classification variable was expressed by the frequency, and the x2 test was used. The patient's survival time was analyzed by Kaplan-Meier and tested with log-rank; the concentration of the tumor necrosis factor alpha was compared with the single factor variance analysis. LSD test was used between groups. The above tests were statistically significant with P0.05. 1. general data of the study were included in the study, including 127 cases of thymosin A1 group, 117 non thymosin alpha 1 group, and two groups of thymosin A1 and non thymosin alpha 1 group, the difference was not statistically significant (P0. 05), first days after severe sepsis, although the APACHEII score of the thymosin A1 group was higher than the non thymosin alpha 1 group, the lymphocyte count was lower than the non thymosin A1 group, but the difference was also not statistically significant (all P0.05). After the lymphocyte count sublayer, the difference of the above indexes between the two groups was also no more. Statistically significant (P0.05).2. patients' prognosis compared with thymosin A1 group and non thymosin alpha 1 group, the 28 day mortality rate was 42.5%, 50.4%, the difference between the two groups was not statistically significant (P0.05), the two group of lymphocyte count > 1 * 10 Zart group and 0.50-1.00 x 109/L subgroup of the 28 day mortality compared, the difference was also not statistically significant Meaning (all P0.05), but in the lymphocyte count less than 0.50 x 109/L subgroup, the mortality of 28 days in the group of thymosin alpha 1 was significantly lower than the non thymosin A1 group, the difference was statistically significant (P0.05) the survival time of the.3. patients compared with the thymosin A1 group and the non thymosin A1 group, the difference was not statistically significant (log-rank test, P0.05), two groups. The survival time of the lymphocyte count > 1 x 109/L subgroup and the 0.50-1.00 x 109/L subgroup was not statistically significant (log-rank test, P0.05), but in the lymphocyte count < 0.50 x 109 subgroups, the survival time of the thymosin alpha 1 group was significantly longer than that of the non thymic peptide alpha 1 group, the difference was statistically significant (log-rank test, P0.05). .4., seventh days and first days of severe sepsis, the dynamic change values of thymosin alpha 1 and non thymosin alpha 1 were compared with the APACHEII score of thymosin alpha 1 and non thymosin alpha 1 in the diagnosis of severe sepsis significantly lower than first days (all P0.05), thymosin a 1 group was more significantly lower than the non thymic peptide a 1 group (P0.05), thymosin A1 group SOFA The score was significantly lower than first days after the diagnosis of severe sepsis (P0.05). The SOFA score of the non thymosin alpha 1 group was lower than first days in the diagnosis of severe sepsis, but the difference was not statistically significant (P0.05). The difference between the thymosin alpha 1 group and the non thymosin alpha 1 group was not statistically significant (P0.05), the thymosin a 1 group and the non thymus gland were not significant (P0.05). The lymphocyte count in the 1 group of peptide alpha 1 was significantly higher than first days after the diagnosis of severe sepsis (all P0.05), but the change of thymosin alpha 1 was more significant than that of non thymosin alpha 1 group (P0.05) in the control group of.5. in vitro, and the difference was statistically significant between the lipopolysaccharide group and the group three of the lipopolysaccharide + thymosin al Group (P0.0 5) the concentration of TNF - alpha in the lipopolysaccharide + thymosin alpha 1 group was significantly higher than that in the control group and the lipopolysaccharide group. The difference was statistically significant (P0.05). The concentration of TNF - alpha in lipopolysaccharide group was also significantly higher than that in the control group (P0.05). The study concluded that 1. thymosin alpha l could significantly reduce the lymphocyte count less than 0.50 The 28 day mortality of patients with severe sepsis by X 109/L, prolonging the patient's survival time, and 2. thymosin alpha 1 can significantly reduce the APACHEII score of severe sepsis, SOFA score, increase the patient's lymphocyte count, improve the patient's condition, and 3. thymosin alpha 1 can significantly improve the immune function of patients with severe sepsis.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R459.7

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