脓毒症大鼠调节性T细胞凋亡对辅助性T细胞漂移的影响及血必净注射液的干预作用

目的 探讨脓毒症大鼠CD4+CD25+调节性T细胞(Treg)凋亡对辅助性T细胞(Th)漂移的影响及血必净注射液的干预作用.方法 将Wistar大鼠随机分为正常组、假手术组、模型组、血必净组,每组8只.行盲肠结扎穿孔术(CLP)制备脓毒症大鼠模型.于第3日采用免疫磁珠法分选各组CD4+CD25+Treg并培养12 h,用流式细胞仪检测Treg凋亡率及叉头翼状螺旋转录因子(Foxp3)和T淋巴细胞毒性相关抗原4(CTLA-4)的表达,用酶联免疫吸附法(ELISA)检测白细胞介素-10(IL-10)的分泌量;再将CD4+CD25+ Treg与CD4+CD25-T细胞1∶1共培养,刀豆素A刺激68 h,ELISA检测Th1、Th2、Th17分泌的γ-干扰素(IFN-γ)、IL-4、IL-17水平.结果 正常组Treg凋亡率[(12.03±0.89)%]与假手术组[(9.48±2.17)%]比较差异无统计学意义;模型组Treg凋亡率[(5.87±0.44)%]明显低于正常组和假手术组;而血必净组的Treg凋亡率[(27.29±2.48)%]显著高于其他各组(P均<0.01).Foxp3、CTLA-4表达和IL-10分泌量随Treg凋亡率增加而减少,相关系数(r)分别为-0.878(P=0.042)、-0.877(P=0.042)、-0.743(P=0.010).与正常组比较,模型组IFN-γ、IL-4水平和IFN-γ/IL-4比值均明显升高[IFN-γ:(254.70±44.88)ng/L比(0.68±0.78)ng/L,IL-4:(8.82±0.61)ng/L比(3.48±0.98)ng/L,IFN-γ/IL-4比值:30.28±4.87比0.23±0.30,P均<0.01];而血必净组IFN-γ[(491.54±84.28)ng/L]和IFN-γ/IL-4比值(45.31±8.01)均显著高于模型组(P<0.01和P<0.05);各组间IL-17水平无明显差异(P均>0.05).结论 脓毒症时Treg凋亡率增加可减轻对效应T细胞的抑制功能,血必净注射液能有效促进脓毒症Treg凋亡,介导Th2向Th1漂移.     

胸腺肽联合血必净注射液对老年脓毒症患者炎症状态及免疫功能的影响

目的探讨胸腺肽联合血必净注射液对老年脓毒症患者炎症状态及免疫功能的影响。方法将70例老年脓毒症患者随机分为对照组和观察组,各35例。在常规治疗基础上,对照组采用胸腺肽治疗,观察组给予胸腺肽联合血必净注射液。治疗前及治疗7 d后,观察2组白细胞计数(WBC)、C-反应蛋白(CRP)、降钙素原(PCT)等炎症指标,Th1、Th2、CD_3~+、CD_4~+、CD_8~+等淋巴T细胞以及急性生理学及慢性健康状况(APACHEⅡ)评分的变化。结果治疗7 d后,2组WBC、CRP、PCT水平明显降低,且观察组同期水平显著低于对照组(P0.05或P0.01)。治疗7 d后,2组CD_3~+、CD_4~+、Th1淋巴细胞比例明显升高,CD_8~+明显降低(P0.05或P0.01),其中观察组升高及降低程度均大于对照组(P0.05或P0.01);Th2细胞比例虽有降低趋势,但组间比较无显著差异(P0.05)。治疗7 d后,2组APACHEⅡ评分较治疗前有所改善,且观察组改善情况优于对照组(P0.01)。结论胸腺肽联合血必净注射液能有效控制老年脓毒症患者的炎症反应,调节免疫状态,从而改善预后。     

血必净注射液治疗脓毒症的临床研究

目的:观察血必净注射液对脓毒症的治疗效果.方法:将60例脓毒症患者随机分为对照组与治疗组.两组均根据药敏结果选用抗生素、早期液体复苏、纠正酸中毒等基础治疗;治疗组在对照组基础上加用血必净注射液.结果:治疗组第5 d、第10 d的APACHE II评分差异明显;治疗组病死率也明显低于对照组;治疗第5d时两组血清TNF-α、CRP、GOT、CK、CK-MB、LDH较入院时均有所降低,且除LDH外,两组其他各项指标均有显著性差异.治疗第10d时两组仅血清TNF-α及CK有显著差异.结论:血必净能降低病死率,有效地降低促炎因子水平,血必净注射液可有效治疗脓毒症.     

血必净注射液治疗脓毒症的疗效分析

目的：评价血必净注射液对脓毒症的治疗效果。方法：将45例脓毒症患者随机分成2组,对照组（n=22）给予常规治疗;治疗组（n=23）在常规治疗基础上联合应用血必净100ml,静脉滴注1次/d,连用1周。治疗前后对患者进行APCACHEⅡ评分,白细胞计数、中性粒细胞比率及C反应蛋白定量,记录28d死亡率。结果：与对照组比较,治疗组的APCACHEⅡ评分,白细胞计数及C反应蛋白水平明显降低,28d死亡率也明显下降（P〈0.01）。结论：血必净注射液可阻断全身炎症反应综合症（SIRS）的病理反应过程,改善脓毒症患者的预后。     

血必净对老年脓毒症患者多脏器功能的保护作用

目的:探讨血必净对老年脓毒症多脏器功能保护的机制和疗效。方法:将70例老年脓毒症患者随机分为治疗组和对照组。治疗组加用血必净100mL静脉点滴,每天2次,共7d,其余治疗2组相同。于治疗前和治疗后第8天测定血浆肿瘤坏死因子-!(TNF-")和白细胞介素-6(IL-6)等炎性介质及血浆生化指标。结果:2组患者治疗后TNF-α和IL-6均较治疗前下降,治疗组较对照组下降更显著(P均<0.05);2组血浆丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、肌酐(Cr)和尿素氮(BUN)均较治疗前明显降低(P均<0.01),且治疗组前三项指标较对照组下降明显;2组血浆乳酸脱氢酶(LDH)、肌酸激酶(CK)及其同工酶(CK-MB)均较治疗前明显降低(P均<0.01),治疗组较对照组下降更显著(P均<0.05)。结论:血必净对老年脓毒症的肝、肾、心脏功能有较好的保护作用。     

血必净注射液对脓毒症大鼠肾保护作用的研究

目的探讨血必净注射液对脓毒症大鼠的肾保护作用。方法采用盲肠结扎穿孔术（CLP）制备脓毒症模型。健康Wistar大鼠80只按髓机数字表法分为正常对照组、假手术组、模型组和血必净治疗组，后两组又分为术后2、8、24和48h4个时间点亚组，每组8只。检测各组血清肌酐（Cr）、尿素氮（BUN）、Na^＋、K^＋，尿Cr、Na^＋、计算钠排泄分数（FENa），采用放射免疫分析法测定β2-微球蛋白（β2-MG），采用酶联免疫吸附法及蛋白分析试剂盒测定肾组织白细胞介素-6（IL-6）及蛋白含量，通过光镜观察各组大鼠肾组织形态学变化。结果与模型组比较，CLP2h血必净治疗组血清β2-MG水平下降，差异具有显著性（P〈0．01）；CLP8h时血清BUN、肾组织IL-6和24h时肾组织IL-6均有明显下降；CLP48h血清BUN、Cr水平也较模型组下降，差异均具有显著性（P〈0．05或P〈0．01）。血必净可减轻肾组织形态学损伤，阻止损伤范围进一步扩大。结论血必净注射液对脓毒症大鼠具有肾保护效应，其作用环节可能与抑制IL-6的合成与释放有关。     

脓毒症患者辅助性T细胞17和CD4+CD25+调节性T细胞表达及血必净注射液的干预作用

目的 观察脓毒症患者外周血辅助性T细胞17(Th17)及CD4+CD25+调节性T细胞(Treg)的水平,并探讨其意义及血必净注射液的干预作用.方法 ①将64例重症监护病房(ICU)脓毒症患者按疾病严重程度分为脓毒症组(26例)、严重脓毒症组(21例)、脓毒性休克组(17例),同时选取18例健康体检者作为对照组.观察不同严重程度脓毒症患者外周血Th17和CD4+CD25+Treg的表达及其与病情严重程度的关系.②按随机原则将64例患者分为常规治疗组(25例,给予常规集束化治疗)和血必净治疗组(39例,在常规治疗基础上加用血必净注射液50 ml静脉滴注,每日2次),两组均以7 d为1个疗程.入ICU当日和治疗7 d用流式细胞术检测外周血Th17及CD4+CD25+Treg表达,观察血必净注射液的干预作用.结果 ①健康对照组Th17表达率为(0.84±0.29)%,CD4+CD25+Treg表达率为(0.43±0.20)%;脓毒症患者细胞表达均较健康对照组明显升高(均P<0.05),其中Th17表达以严重脓毒症组最高[(3.18±0.84)%],CD4+CD25+Treg 表达以脓毒性休克组最高[(3.28±0.76)%].脓毒症患者CD4+CD25+Treg与急性生理学与慢性健康状况评分系统Ⅰ(APACHE Ⅰ)评分和血乳酸均呈正相关(r1=0.519,r2=0.451,均P=0.01).②与常规治疗组比较,血必净注射液能更有效降低脓毒症患者Th17和CD4+CD25+Treg的异常表达[Th17:(1.72±0.69)%比(2.35±0.81)%,CD4+CD25+Treg:(1.78±1.00)%比(2.30±0.85)%,均P<0.05],纠正免疫平衡紊乱,缩短住ICU时间[(4.7±2.6)d比(7.5±4.3)d,P=0.0023,使脓毒症患者28 d病死率有降低趋势(20.5%比28.0%,P>0.05).结论 脓毒症患者外周血Th17和CD4+CD25+Treg表达增加,且与病情严重程度呈正相关,提示Th17和CD4+CD25+Treg在脓毒症发生发展的免疫机制中可能起着重要作用.血必净注射液能有效降低脓毒症患者Th17和CD4+CD25+Treg的异常表达,有降低脓毒症患者病死率的趋势.

Abstract：

Objective To study the level and significance of T helper 17(Th17)and CD4+CD25+regulatory T cells(Treg)in peripheral blood of patients with sepsis and to evaluate the effects of Xuebijing of Anhui Provincial Hospital were divided into three groups:sepsis group(n=26),severe sepsis group (n=21),and septic shock group(n=17).Eighteen healthy individuals served as controls.The comparison in the expression of Th17 and CD4+CD25+Treg within groups and the correlation between their levels and group(n=25,received routine bundle treatment)and Xuebijing treatment group(n=39,received bundle treatment+Xuebijing treatment).Patients in Xuebijing treated group were given 50 ml Xuebijing injection two times per day in addition to routine bundle treatment.Seven days constituted one course of treatment.The expressions of Th17 and CD4+CD25+Treg of 64 patients on the 1 day and 7 days after treatment were detected by flow cytometry.The effects of Xuebijing injection on the patients were evaluated.Results in control group,and they were lower than that of patients with sepsis(P<0.05).The expression rate of Th17 was higher in severe sepsis group [(3.18±0.84)%]than that of other two groups(P<0.05).Moreover,The expression rate of CD4+CD25+Treg was highest [(3.28±0.76)%]in septic shock group (P<0.05),and it was positive correlated with acute physiology and chronic health evaluation Ⅰ(APACHE routine group,our study indicated that Xuebijing injection could reduce the abnormal expression of Th17[(1.72±0.69)%vs.(2.35±0.81)%,P<0.05] and CD4+CD25+Treg[(1.78±1.00)% vs.(2.30±0.85)%,P<0.05] and decrease length of stay in ICU[(4.7±2.6)days vs.(7.5±4.3)days,P=0.002].It also lowered 28-day mortality of patients with sepsis,but the difference between two groups was not significant(20.5%vs.28.0%,P>0.05).Conclusion The expression of Th17 and CD4+CD25+Treg was increased in sepsis patients and was positively correlated with severity of sepsis,suggesting that they may play an important role in pathogenesis of sepsis.Xuebijing injection could decrease the abnormal expression of Th17 and CD4+CD25+Treg and tend to decrease the fatality rate of sepsis.     

血必净注射液联合胸腺肽α1治疗慢性肝衰竭的疗效观察

目的观察血必净注射液联合胸腺肽α1对慢性肝衰竭患者的疗效。方法将62例慢性肝衰竭的患者随机分成对照组20例、治疗I组21例和治疗Ⅱ组21例。3组均给予保肝、降酶、退黄治疗及抗感染等内科综合治疗。治疗I组加用血必净50 ml/d,疗程2周;治疗Ⅱ组加用血必净50 ml/d,联合胸腺肽α1 1.6 mg,隔天一次,疗程均2周。观察3组患者治疗前后肝功能、中性粒细胞比率、凝血参数、血小板、内毒素水平的变化。结果治疗两周后,治疗Ⅱ组内毒素水平下降和肝功能指标改善与对照组比较差异有显著性(P<0.05与P<0.01),且治疗Ⅱ组疗效优于治疗I组,但治疗Ⅰ、Ⅱ组有效率与对照组比较差异无显著意义(P>0.05)。结论血必净注射液联合胸腺肽a1治疗慢性肝衰竭能明显降低内毒素血症,改善患者的肝功能。     

不同剂量血必净对内毒素诱导调节性T细胞凋亡的影响

目的 探讨血必净注射液对内毒素诱导CD4+CD25+调节性T细胞(Treg)凋亡影响的剂量-效应关系.方法 体外培养免疫磁珠法分选大鼠脾脏CD4+CD25+Treg,分为对照组、抗CD3/CD28组、抗CD3/CD28+脂多糖(LPS)组、抗CD3/CD28+血必净组(1、5、25 mg/ml)和抗CD3/CD28+LPS+血必净组(1、5、25 mg/ml).以刺激第3日为观察时间点,采用流式细胞术分析Treg凋亡率、叉头翼状螺旋转录因子(Foxp3)和T淋巴细胞毒性相关抗原4(CTLA-4)表达水平,酶联免疫吸附法(ELISA)检测白细胞介素-10(IL-10)分泌情况.结果 抗CD3/CD28+血必净各组CD4+CD25+Treg凋亡率高于对照组和抗CD3/CD28组,抗CD3/CD28+LPS+血必净各组凋亡率较对照组和抗CD3/CD28+LPS组明显升高(P<0.05或P<0.01);且凋亡率随血必净刺激剂量增加而升高,其中5 mg/ml血必净组凋亡率接近50%.Foxp3和CTLA-4的表达及IL-10分泌随凋亡率增加而逐渐降低,不同剂量血必净组间比较差异有统计学意义(P<0.05或P<0.01).结论 血必净注射液能显著促进内毒素介导Treg的凋亡,并呈一定程度的剂量-效应关系.     

血必净联合血液净化治疗严重脓毒症临床研究

目的:探讨血必净联合血液净化治疗严重脓毒症的临床疗效。方法:将84例严重脓毒症患者分为A组(常规治疗组)26例、B组(常规治疗+血必净组)20例、C组(常规治疗+血液净化组)22例、D组(常规治疗+血必净+血液净化组)16例,观察各组治疗前及治疗后24,48,72 h血小板计数、凝血酶原时间、活化部分凝血酶时间、纤维蛋白原、血肌酐、尿素氮、尿量以及氧合指数、APACHEⅡ评分,比较各组多器官功能障碍综合征的发生率。结果:治疗后4组血小板计数、凝血酶原时间、活化部分凝血酶时间、纤维蛋白原、尿量、血肌酐、尿素氮、氧合指数较治疗前均有改善(P<0.05),但B,C,D组改善明显,D组疗效更显著(P<0.01);治疗后APACHEⅡ评分及多器官功能障碍综合征发生率明显下降,D组下降更明显(P<0.01)。结论:血必净联合血液净化治疗严重脓毒症可预防多器官功能障碍综合征的发生,降低病死率。     

Xuebijing in the treatment of patients with sepsis

Background

There are more than 18 million patients diagnosed with sepsis every year. In China, Xuebijing (XBJ) injection is a traditional medicine that is widely used in the treatment of sepsis. However, the efficacy of XBJ in treatment of randomized controlled trials (RCTs) remains unclear. This meta-analysis was to evaluate the clinical efficacy of XBJ based on randomized case-control studies.

脓毒症大鼠肺基质金属蛋白酶-2/9表达与血必净干预

目的 观察创伤弧菌脓毒症大鼠肺基质金属蛋白酶(MMP)-2/9和组织型金属蛋白酶抑制剂(TIMP)-1/2的动态表达及血必净干预.方法 温州医学院生命科学院实验室,清洁级SD大鼠110只,随机(随机数字法)分为正常对照组(A组,n=10)、创伤弧菌脓毒症组(B组,n=50,采用大鼠左下肢皮下注射创伤弧菌悬液制作创伤弧菌脓毒症大鼠模型)及血必净干预组(C组,n=50,感染后0.5 h腹腔注射血必净4 mL/kg).B、C组大鼠于染菌后1,6,12,24,48 h麻醉后活杀,留取右肺标本.观察大鼠行为学变化,采用考马斯亮蓝法测肺通透性,采用逆转录-聚合酶链式反应(RT-PCR)法测肺MMP-2/9,TIMP-1/2 mRNA的表达,免疫组化法和双抗体夹心酶联免疫吸附法(ELISA)测肺MMP-2/9和TIMP-1/2的表达.数据采用单因素方差分析,并用LSD-t法进行组间两两比较,以P＜0.05为差异有统计学意义.结果 B组和C组肺通透性显著高于A组,C组显著低于B组.B组和C组MMP-2/9,TIMP-1/2mRNA显著升高,B组分别于6 h(0.344±0.108),6 h(1.230±0.377),12 h(0.523±0.098),12 h(0.280±0.070)达高峰(P＜0.05),C组分别于12 h(0.256±0.074),6 h(0.968±0.225),12 h(0.746±0.316),12 h(0.356±0.035)达高峰(P＜0.05),C组MMP-2/9mRNA升高趋势显著低于B组(P＜0.05),TIMP-1/2mRNA显著高于B组(P＜0.05).B组和C组MMP-2/9,TIMP-1/2蛋白也升高,B组分别于12 h(0.692±0.191),12 h(0.061±0.017),24 h(1384.42±91),24 h(41.04±3.60)达高峰(P＜0.05);C组分别于24 h(0.217±0.065),12 h(0.045±0.013),24 h(1617.22±103),24 h(47.66±3.58)达高峰(P＜0.05);C组MMP-2/9蛋白升高趋势低于B组(P＜0.05),TIMP-1/2蛋白早期与B组差别不大,后期显著高于B组(P＜0.05).结论 MMP/TIMP比例失衡是创伤弧菌脓毒症大鼠肺损伤机制之一,血必净可促进MMP/TIMP比例恢复平衡,对创伤弧菌脓毒症大鼠肺损伤具有保护作用.

Abstract：

Objective To detect the expression of MMP-2/9 and TIMP-1/2 in the lung of Vibrio vulnificus sepsis rats and observe the intervention of Xuebijing injection. Method One hundred and ten SD rats of clean grade were randomly(random number) divided into normal control group (group A, n = 10),Vibrio vulnificus sepsis group (group B, n = 50. Sepsis was reproduced in rats with subcutaneous injection in left lower limb with Vibrio vulnificus) and Xuebijin intervention group ( group C, n = 50. Rats were intraperitoneal(ip) with the dose of Xuebijing 4mL/kg at the time of 30 min later after infection). The rats in group B and C were sacrificed at 1 h, 6 h, 12 h, 24 h, 48 h after infection, the expression of MMP-2/9 and TIMP-1/2 were examined by PCR, Immunohistochemistry or ELISA methods, the lung permeability were measured by Coomassie Brilliant Blue method. Experimental data used single factor analysis of variance, and between groups by LSD method for pairwise comparison,P ＜0.05 statistically significant difference. Results The lung permeability increased both in group B and C compared with group A,and in group B were relatively higher. The lung MMP-2/9, TIMP-1/2mRNA expression in groups B and C compared with in group A was markedly higher, and reached the peak at 6 h(0. 344 ± 0. 108 ),6 h ( 1. 230 ± 0.377 ), 12 h (0.523 ±0.098),12 h(0.280±0.070) (P＜0.05) in group B while at 12 h(0.256 ±0.074),6 h(0.968±0.225) ,12 h(0.746 ±0. 316) ,12 h(0.356 ±0.035) (P ＜0. 05) in group C; the MMP-2/9mRNA expression in group C decreased(P＜0. 05) compared with the group B while the TIMP-1/2mRNA expression increased(P＜0. 05). The lung MMP-2/9, TIMP-1/2 protein expression in groups B and C compared with the group A(0.345±0.109) also increased, and the peak was at 12 h (0. 692 ± 0. 191 ), 12 h (0. 061 ±0.017) ,24 h(1384.42 ±91) ,24 h(41.04 ±3.60)in group B while at 24 h(0. 217 ±0.065) ,12 h(0. 045± 0. 013 ) ,24 h ( 1617.22 ± 103 ) ,24 h (47.66 ± 3.58 )in group C, the MMP-2/9 protein expression in group C was lower than in group B(P＜0.05), the TIMP-1/2 protein expression in group C was similar to in group B early while marked increased(P＜0.05)later. Conclusions MMP/TIMP imbalance was one of the mechanisms of the lung injury in the rats with Vibrio vulnificus sepsis, Xuebijing could restore the balance of MMP/TIMP ratio.     

血必净注射液对脂多糖刺激大鼠调节性T细胞凋亡及其介导效应T细胞免疫功能的影响

目的 探讨中药血必净注射液对体外脂多糖(LPS)刺激CD4+CD25+调节性T细胞(Treg)凋亡及其介导效应T细胞(Teff)增殖反应、分泌白细胞介素-2(IL-2)功能的影响.方法 40只雄性清洁级Wistar大鼠,实验前禁食12 h.免疫磁珠法分选大鼠脾脏CD4+CD25+Treg,分为对照组(n=8)、抗CD3/CD28组(n=8)、抗CXB/CD28+LPS组(/1,=8)、抗CD3/CD28+血必净组(n=8)和抗CD3/CD28+LPS+血必净组(n=8).培养3 d后流式细胞术检测CD4+CD25+Treg凋亡率、叉头翼状螺旋转录因子(Foxp3)和T淋巴细胞毒性相关抗原4(CTLA-4)表达,酶联免疫吸附试验检测IL-10分泌.同时,CD4+CD25+Treg与CD4+CD25-T细胞1:1培养,刀豆素A刺激68 h后分析Treg对Teff增殖的抑制效应,并检测上清中IL-2/可溶性IL-2受体水平.结果 抗CD3/CD28诱导Treg凋亡率为(33.70±3.06%),显著高于对照组(12.84±0.84)%;抗CD3/CD28+LPS+血必净组(45.13±2.70)%凋亡率明显高于抗CD3/CD28+LPS组[(29.41±1.63)%,P＜0.01].同时,CD4+CLY25+Treg roxp3和CTLA-4表达及IL-10分泌随凋亡增加而降低.抗CD3/CD28+血必净组(31.26%)Teff增殖反应平均抑制率显著低于对照组(54.48%,P＜0.05),抗CD3/CD28+LPS+血必净组抑制率与抗CD3/CD28+LPS组比较亦明显下降(P＜0.01).此外,抗CD3/CD28+血必净组和抗CD3/CD28+LPS+血必净组IL-2分泌水平均显著高于抗CD3/CD28+LPS组(P＜0.01).结论 LPS刺激可明显上调大鼠Treg对Teff的抑制功能,血必净注射液能通过促进Treg凋亡而有效改善其对Teff的抑制效应.     

脓毒症大鼠肠系膜淋巴结与机体免疫功能相关性研究

目的 探讨脓毒症大鼠肠系膜淋巴结(MLN)与机体促炎/抗炎免疫状态变化及其相关性.方法 雄性Wistar大鼠96只,按随机数字表法分为假手术组和模型组.采用盲肠结扎穿孔术(CLP)制备大鼠脓毒症模型,于术后0、6、12、24、48和72 h各取8只大鼠腹主动脉血和MLN组织,采用流式细胞仪测定全血与MLN中辅助性T细胞1/2(Th1/Th2)比值及调节性T细胞(Treg细胞)占CD4+T淋巴细胞的比例.结果 模型组腹主动脉血中Th1/Th2比值于术后6 h显著升高,12 h达高峰,随后持续降低;与假手术组比较,模型组术后6、12、24 h Th1/Th2比值显著升高(0.82±0.15比0.60±0.22,1.23±0.44比0.76±0.31,0.85±0.25比0.66±0.32),72 h显著降低(0.41±0.16比0.59±0.13,P＜0.05或P＜0.01).模型组MLN中Th1/Th2比值于术后6 h达峰值,然后显著下降;与假手术组比较,模型组术后6 h Th1/Th2比值显著升高(1.01±0.16比0.52±0.13),12、24、48、72 h显著下降(0.34±0.11比0.53±0.09,0.23±0.08比0.51±0.09,0.17±0.07比0.47±0.15,0.16±0.06比0.53±0.11,P＜0.05或P＜0.01).模型组MLN中Treg细胞比例于术后12、24、48、72 h显著高于假手术组(9.62±0.69比7.65±0.67,9.84±0.74比8.08±1.06,10.95±2.09比7.83±1.15,10.81±1.34比8.35±1.12,P＜0.05或P＜0.01).MLN中Th1/Th2比值与Treg细胞比例呈线性负相关(r=-0.882,P＜0.05).结论 严重腹腔感染导致肠源性内毒素通过MLN系统移位,MLN免疫功能障碍加速内毒素人血,进而影响全身的免疫功能,产生Th1向Th2的漂移.MLN免疫功能降低可能与内毒素介导MLN中Treg细胞比例升高有关.

Abstract：

Objective To investigate the changes in T helper cells (Th1 and Th2) of mesenteric lymph node (MLN) and blood Th1 and Th2 cells in septic rats. Methods Ninety-six male Wistar rats were randomly divided into two groups according to random number table method: sham operation group and model group. Intra-abdominal infection with sepsis was induced by cecal ligation and puncture (CLP). Eight rats in each group were sacrificed after collection of blood samples and MLN samples at 0, 6, 12, 24, 48 and 72 hours after CLP. The ratio of Th1/Th2 and the percentage of regulatory T cell (Treg) in CD4+ T cells in blood and MLN were respectively determined by flow cytometry. Results In model group, the ratio of Th1/Th2 in abdominal aorta blood increased significantly at 6 hours and reached summit at 12 hours, then it decreased persistently, and when compared with sham group, the ratio of Th1/Th2 was significantly higher at 6, 12, 24 hours (0.82±0.15 vs. 0. 60±0. 22, 1. 23±0. 44 vs. 0. 76±0. 31, 0. 85±0. 25 vs. 0. 66±0. 32)and lower at 72 hours (0. 41±0.16 vs. 0. 59±0. 13, P＜0. 05 or P＜0. 01). The ratio of Th1/Th2 in MLN of model group reached summit at 6 hours, then decreased significantly, and when compared with sham group, the ratio of Th1/Th2 was significantly higher at 6 hours (1. 01±0.16 vs. 0. 52±0.13) and lower at 12, 24, 48 and 72 hours (0. 34±0.11 vs. 0. 53±0. 09, 0. 23±0. 08 vs. 0. 51±0. 09, 0.17±0. 07 vs. 0. 47±0.15, 0.16±0. 06 vs. 0. 53±0.11, P＜0. 05 or P＜0. 01). Compared with sham group, the percentage of Treg in MLN of model group was increased at 12, 24, 48 and 72 hours after CLP (9. 62±0. 69 vs. 7. 65±0.67, 9. 84±0. 74 vs. 8.08±1. 06, 10. 95±2. 09 vs. 7. 83±1.15, 10. 81±1. 34 vs. 8. 35±1.12, P＜0. 05 or P＜0. 01). There was a negative correlation between the ratio of Th1/Th2 and the percentage of Treg in MLN (r=-0. 882, P＜0. 05). Conclusion Cellular immune function of MLN is suppressed during severe intra-abdominal infection, which induces translocation of gut-derived endotoxin to mesenteric lymphatics,resulting in corporal immuno-suppression, with manifestation of Th1/Th2 cell shift. Immuno-suppression of MLN is related to a higher percentage of Treg due to the effect of endotoxin.     

血必净注射液联合生大黄粉治疗中老年脓毒症患者的临床效果

目的 探讨血必净注射液联合生大黄粉治疗中老年脓毒症患者的临床效果.方法 回顾性选取2020年6月至2021年8月江苏大学附属医院重症医学科收治的92例脓毒症患者作为研究对象,依据住院期间的治疗方案将患者分为对照组(n=46)和观察组(n=46).对照组采用传统西医治疗,包括抗感染、液体复苏、器官功能支持等常规治疗,观察...     

脓毒症大鼠肠系膜淋巴结与机体免疫功能相关性研究

Objective To investigate the changes in T helper cells (Th1 and Th2) of mesenteric lymph node (MLN) and blood Th1 and Th2 cells in septic rats. Methods Ninety-six male Wistar rats were randomly divided into two groups according to random number table method: sham operation group and model group. Intra-abdominal infection with sepsis was induced by cecal ligation and puncture (CLP). Eight rats in each group were sacrificed after collection of blood samples and MLN samples at 0, 6, 12, 24, 48 and 72 hours after CLP. The ratio of Th1/Th2 and the percentage of regulatory T cell (Treg) in CD4+ T cells in blood and MLN were respectively determined by flow cytometry. Results In model group, the ratio of Th1/Th2 in abdominal aorta blood increased significantly at 6 hours and reached summit at 12 hours, then it decreased persistently, and when compared with sham group, the ratio of Th1/Th2 was significantly higher at 6, 12, 24 hours (0.82±0.15 vs. 0. 60±0. 22, 1. 23±0. 44 vs. 0. 76±0. 31, 0. 85±0. 25 vs. 0. 66±0. 32)and lower at 72 hours (0. 41±0.16 vs. 0. 59±0. 13, P＜0. 05 or P＜0. 01). The ratio of Th1/Th2 in MLN of model group reached summit at 6 hours, then decreased significantly, and when compared with sham group, the ratio of Th1/Th2 was significantly higher at 6 hours (1. 01±0.16 vs. 0. 52±0.13) and lower at 12, 24, 48 and 72 hours (0. 34±0.11 vs. 0. 53±0. 09, 0. 23±0. 08 vs. 0. 51±0. 09, 0.17±0. 07 vs. 0. 47±0.15, 0.16±0. 06 vs. 0. 53±0.11, P＜0. 05 or P＜0. 01). Compared with sham group, the percentage of Treg in MLN of model group was increased at 12, 24, 48 and 72 hours after CLP (9. 62±0. 69 vs. 7. 65±0.67, 9. 84±0. 74 vs. 8.08±1. 06, 10. 95±2. 09 vs. 7. 83±1.15, 10. 81±1. 34 vs. 8. 35±1.12, P＜0. 05 or P＜0. 01). There was a negative correlation between the ratio of Th1/Th2 and the percentage of Treg in MLN (r=-0. 882, P＜0. 05). Conclusion Cellular immune function of MLN is suppressed during severe intra-abdominal infection, which induces translocation of gut-derived endotoxin to mesenteric lymphatics,resulting in corporal immuno-suppression, with manifestation of Th1/Th2 cell shift. Immuno-suppression of MLN is related to a higher percentage of Treg due to the effect of endotoxin.     

Complement C3 depletion links to the expansion of regulatory T cells and compromises T-cell immunity in human abdominal sepsis: A prospective pilot study

Purpose

Complement depletion commonly occurred in septic patients, but this problem was often underestimated during the treatment process. This study was designed to determine the association between complement depletion and T-cell immunosuppression.

Methods

From November 2011 to March 2012, patients with severe abdominal sepsis were prospectively enrolled in a single center. The baseline levels of complement C3 were used to stratify subjects into 2 groups. Plasma levels of complement components, percentage of regulatory T cells (Tregs), and T-cell immunity indexes were monitored at times after admission. The relationship between C3 depletion and T-cell response was investigated, with clinical outcomes explored meanwhile.

Results

A total of 60 patients aged 43.9 ± 11.3 years were included within the period. C3 depletion, occurring in 65% of enrolled subjects, was strongly correlated with Treg expansion (P = .001) and decreased CD4⁺/CD8⁺ ratio (P = .008). This depletion was also related to prolonged hospital stay (P = .001), delayed time to operation (P < .001), increased postoperative complications (P = .036), and hospital expenditure (P < .001).

Conclusions

Complement C3 depletion was found to be linked to the expansion of Tregs during abdominal sepsis. Such depletion and associated immunosuppression should be paid close attention in the critical care.     

脓毒症大鼠肺基质金属蛋白酶-2/9表达与血必净干预

Objective To detect the expression of MMP-2/9 and TIMP-1/2 in the lung of Vibrio vulnificus sepsis rats and observe the intervention of Xuebijing injection. Method One hundred and ten SD rats of clean grade were randomly(random number) divided into normal control group (group A, n = 10),Vibrio vulnificus sepsis group (group B, n = 50. Sepsis was reproduced in rats with subcutaneous injection in left lower limb with Vibrio vulnificus) and Xuebijin intervention group ( group C, n = 50. Rats were intraperitoneal(ip) with the dose of Xuebijing 4mL/kg at the time of 30 min later after infection). The rats in group B and C were sacrificed at 1 h, 6 h, 12 h, 24 h, 48 h after infection, the expression of MMP-2/9 and TIMP-1/2 were examined by PCR, Immunohistochemistry or ELISA methods, the lung permeability were measured by Coomassie Brilliant Blue method. Experimental data used single factor analysis of variance, and between groups by LSD method for pairwise comparison,P ＜0.05 statistically significant difference. Results The lung permeability increased both in group B and C compared with group A,and in group B were relatively higher. The lung MMP-2/9, TIMP-1/2mRNA expression in groups B and C compared with in group A was markedly higher, and reached the peak at 6 h(0. 344 ± 0. 108 ),6 h ( 1. 230 ± 0.377 ), 12 h (0.523 ±0.098),12 h(0.280±0.070) (P＜0.05) in group B while at 12 h(0.256 ±0.074),6 h(0.968±0.225) ,12 h(0.746 ±0. 316) ,12 h(0.356 ±0.035) (P ＜0. 05) in group C; the MMP-2/9mRNA expression in group C decreased(P＜0. 05) compared with the group B while the TIMP-1/2mRNA expression increased(P＜0. 05). The lung MMP-2/9, TIMP-1/2 protein expression in groups B and C compared with the group A(0.345±0.109) also increased, and the peak was at 12 h (0. 692 ± 0. 191 ), 12 h (0. 061 ±0.017) ,24 h(1384.42 ±91) ,24 h(41.04 ±3.60)in group B while at 24 h(0. 217 ±0.065) ,12 h(0. 045± 0. 013 ) ,24 h ( 1617.22 ± 103 ) ,24 h (47.66 ± 3.58 )in group C, the MMP-2/9 protein expression in group C was lower than in group B(P＜0.05), the TIMP-1/2 protein expression in group C was similar to in group B early while marked increased(P＜0.05)later. Conclusions MMP/TIMP imbalance was one of the mechanisms of the lung injury in the rats with Vibrio vulnificus sepsis, Xuebijing could restore the balance of MMP/TIMP ratio.     

免疫调理治疗对脓毒症患者血CD4+CD25+T细胞的影响

目的 了解脓毒症患者外周血调节性T细胞(CD4+CD25+Tregs)比例水平及其与机体细胞免疫之间的关系,并探讨免疫调理治疗对其水平的影响.方法 选择滕州市中心人民医院住院治疗的脓毒症患者40例,随机分成治疗组和对照组,治疗组加用乌司他丁和胸腺肽α1作抗炎和免疫调节治疗,分别于治疗前和治疗后第3,第8天抽外周静脉血,检测T淋巴细胞亚群、CD4+CD25+Tregs,TNF-α,IL-6,IL-10水平,同时APACHEⅡ评分.结果 治疗前两组患者CD4+CD25+Tregs细胞水平明显升高,总淋巴细胞数、T细胞比例下降,其中以CD4+T细胞下降更明显,CD4+/CD8+明显下降,IL-6,TNF-αt水平升高;治疗后,两组患者CD4+C125+Tregs降低,总淋巴细胞数、CD4+/CD8+比值升高,APACHEⅡ评分和IL-6,TNF-αt水平均下降,治疗组改善更明显.结论 外周血CD4+CD25+Tregs细胞比例水平,可以作为评价机体免疫能力及预后新指标;联合应用胸腺肽α1和乌司他丁治疗脓毒症,可提高患者免疫力,显著降低APACHEⅡ评分、IL-6,TNF-α水平,改善患者病情.

Abstract：

Objective To investigate the percentage of CD4 + C125 +Tregs in peripheral blood of patients with sepsis and its effect on cell immunity so as to unravel the effect of immunomodulatory therapy on it. Method Fourty patients with sepsis in ICU were randomly (random number) divided into experimental group and control group . The patients of experimental group were treated with Ulinastatin and immunoregulation agent (Thymosin αl) as well. The blood specimens were collected just before treatment, 3 days and 8 days after treatment. The percentages of CD4 + CD25 + Tregs and lymphocyte subsets were detected by using FCM (flow cytometry), and TNF-α, IL-6 and IL-10 assayed by using ELISA, and APACHE Ⅱ scores were calculated. Results Before treatment, the percentage of CD4 + CD25 + Tregs increased, and the number of lymphocytes and the percentage of T lymphocytes decreased, especially the CD4 + T lymphocytes and CD4+/CD8+ decreased more markedly, and the levels of IL-6 and TNF-α increased. After treatment,the percentage of CD4+ CD25 + Tregs was decreased, the number of lymphocytes and CD4 +/CD8 + increased, and the levels of APACHE Ⅱ score, IL-6 and TNF-α decreased especially in the experimental group decreased more significantly (P ＜ 0. 05). Conclusions The percentage of CD4 + CD25+ Tregs in peripheral blood can reflect the immune status of patients with sepsis and become a novel indicator to estimate the progress of sepsis, and the immunity and prognosis of patients. Treating the patients with Thymosin αl and Ulinastatin can raise their immunity, decrease the levels of IL-6, TNF-α and APACHE Ⅱ score and improve their prognosis.