体外研究贺斯、佳乐施、万汶对成人静脉血辅助性T淋巴细胞分化的影响
摘要
研究背景
机体免疫系统的变化越来越引起人们的关注,其对疾病的预后和转归有重要的影响。组织损伤,严重的应激反应,输血均可抑制机体的免疫系统,延缓患者康复,导致围术期感染、肿瘤复发和转移。有研究表明,输血引起的免疫抑制在其中的作用最为重要。为预防交叉感染,缓解输血引起的免疫抑制,因此血浆代用品成为容量治疗的重要手段。它可以补充血浆容量,维持血流动力学稳定;改善微循环和组织灌注,增加组织氧供;减少并避免血液制品输入并发症。虽然血浆代用品对机体扩容,肝肾功能,凝血方面的作用已有许多研究报告,但其对免疫系统的影响有待进一步研究。贺斯、佳乐施、万汶是常用的胶体液,贺斯和万汶为羟乙基淀粉制剂,佳乐施为明胶制剂。
目的
观察比较贺斯、佳乐施、万汶对成人静脉血辅助性T淋巴细胞(Th细胞)分化的影响。
方法
采集18例健康志愿者外周静脉血20ml,采用随机区组设计,每份血样均参与下列各组研究。根据临床常用剂量设置20%,40%(体积比)两个血液稀释浓度,分为空白对照组(C组)、不同剂量贺斯组[20%(H1组)、40%(H2组)]、不同剂量佳乐施组[20%(G1组)、40%(G2组)]、不同剂量万汶组[20%(V1组)、40%(V2组)]。用两种不同方法评估Th细胞的功能分化情况:(1)胞内细胞因子检测:将全血在体外用相应剂量的药物预先培养24h,然后加入刺激剂(氟波醇乙酯、离子霉素及蛋白质转运抑制剂),刺激4h,采用流式细胞技术测定每份样本中Th1及Th2细胞亚群比例,并计算Th1细胞与Th2细胞的比值(Th1/Th2)。(2)分离外周血单个核细胞(PBMcs),经药物预处理24小时后,加入刺激剂植物血球凝集素继续培养48小时,采用流式蛋白检测技术(CBA)检测培养上清中Th1(IFN-γ、TNF-α、IL-2)及Th2(IL-4、IL-6、IL-10)型细胞因子的浓度。统计分析采用SPSS11.5软件,组间比较采用随机区组设计方差分析,P＜0.05为差异有统计学意义。
结果
胞内细胞因子检测法:与C组比较,Th1亚群比例在H1组、V1组升高(P＜0.05),G2组降低(P＜0.01);Th2亚群比例在H2组、G2组、V2组均降低(P＜0.05);Th1/Th2比值在H2组、G2组、V1组、V2组均升高(P＜0.05)。细胞培养上清细胞因子浓度测定:贺斯和万汶组六种细胞因子除了IL-10(P＜0.01)外均上升;佳乐施组IL-4,IL-6升高(P＜0.01),余不变。
结论
在20%浓度时,贺斯、万汶可促使Th细胞向Th1细胞分化,佳乐施作用不明显;在40%浓度时,贺斯、万汶抑制Th2细胞的分化,佳乐施对Th1和Th2细胞的分化均呈抑制作用。万汶在两种浓度下均可促使Th1/Th2比值升高。
Background
In recent years,more and more attentions have been paid to the function of immune system in patients.The helper T-lymphatcyte(Th) cells are the important active immune cells,which promote the differrentiation of Th1 or Th2.And the ratio of Th1 and Th2 can impact the immune function.Plasma substitutes are widely used to supply intravascular volume.Previous studies have demonstrated the effects of colloids on inflammation response,but little is known about the effects on the differentiation of Th cells.
Objective
To investigate the effects of pretreatment with different concentra- tions of hydroxyethyl starch(HES),Gelofusine and Voluven on the differentiation of human helper T cells in vitro.
Methods
Eighteen healthy volunteers were enrolled in this study.Two clinically relevant degrees of in vitro haemodilution were prepared: 20%and 40%(V/V).In each volunteer,20 ml of blood was taken from peripheral vein and divided into 7 groups:two HES groups[20%(H1)、40%(H2)],two Gelofusine groups[20%(G1)、40%(G2)],two Voluven groups[20%(V1)、40%(V2)],control group(C).Whole blood and mononuclear cells from peripheral blood(PBMCs)were pre-treated with different colloids.To estimate the imbalance of Th1/Th2,the intracellular cytokines(IFN-γ/IL-4)were detected by four-color flow cytometry.The cytokines in the PBMCs culture supernatant were examined by cytometric bead array(CBA).All data represent the mean±SD and are compared by two way analysis of variance(SPSS11.5 software). Differences were considered significant if the P value is less than 0.05.
Results
Compared with C group,the percentages of Th1 cells increased significantly in H1 and V1 groups(P＜0.05),and decreased in G2 group (P＜0.01);while the percentages of Th2 cells decreased in H2,G2 and V2 groups(P＜0.05).The ratios of Th1/Th2 were increased in H2,G2,V1 and V2 groups(P＜0.05).In two HES groups and two Voluven groups,the levels of Th1 and Th2 cytokines of the supematants increased(P＜0.05), while IL-10 decreased(P＜0.01).In two Gelofusine groups,the levels of IL-4 and IL-6 increased(P＜0.01).
Conclusion
Voluven and Hes encouraged helper cells to differentiate into Th1 cells while Gelofusine inhibited the differentiation of Th1 and Th2 cells. Voluven could increase the Th1/Th2 ratios.
机体免疫系统的变化越来越引起人们的关注,其对疾病的预后和转归有重要的影响。组织损伤,严重的应激反应,输血均可抑制机体的免疫系统,延缓患者康复,导致围术期感染、肿瘤复发和转移。有研究表明,输血引起的免疫抑制在其中的作用最为重要。为预防交叉感染,缓解输血引起的免疫抑制,因此血浆代用品成为容量治疗的重要手段。它可以补充血浆容量,维持血流动力学稳定;改善微循环和组织灌注,增加组织氧供;减少并避免血液制品输入并发症。虽然血浆代用品对机体扩容,肝肾功能,凝血方面的作用已有许多研究报告,但其对免疫系统的影响有待进一步研究。贺斯、佳乐施、万汶是常用的胶体液,贺斯和万汶为羟乙基淀粉制剂,佳乐施为明胶制剂。
目的
观察比较贺斯、佳乐施、万汶对成人静脉血辅助性T淋巴细胞(Th细胞)分化的影响。
方法
采集18例健康志愿者外周静脉血20ml,采用随机区组设计,每份血样均参与下列各组研究。根据临床常用剂量设置20%,40%(体积比)两个血液稀释浓度,分为空白对照组(C组)、不同剂量贺斯组[20%(H1组)、40%(H2组)]、不同剂量佳乐施组[20%(G1组)、40%(G2组)]、不同剂量万汶组[20%(V1组)、40%(V2组)]。用两种不同方法评估Th细胞的功能分化情况:(1)胞内细胞因子检测:将全血在体外用相应剂量的药物预先培养24h,然后加入刺激剂(氟波醇乙酯、离子霉素及蛋白质转运抑制剂),刺激4h,采用流式细胞技术测定每份样本中Th1及Th2细胞亚群比例,并计算Th1细胞与Th2细胞的比值(Th1/Th2)。(2)分离外周血单个核细胞(PBMcs),经药物预处理24小时后,加入刺激剂植物血球凝集素继续培养48小时,采用流式蛋白检测技术(CBA)检测培养上清中Th1(IFN-γ、TNF-α、IL-2)及Th2(IL-4、IL-6、IL-10)型细胞因子的浓度。统计分析采用SPSS11.5软件,组间比较采用随机区组设计方差分析,P＜0.05为差异有统计学意义。
结果
胞内细胞因子检测法:与C组比较,Th1亚群比例在H1组、V1组升高(P＜0.05),G2组降低(P＜0.01);Th2亚群比例在H2组、G2组、V2组均降低(P＜0.05);Th1/Th2比值在H2组、G2组、V1组、V2组均升高(P＜0.05)。细胞培养上清细胞因子浓度测定:贺斯和万汶组六种细胞因子除了IL-10(P＜0.01)外均上升;佳乐施组IL-4,IL-6升高(P＜0.01),余不变。
结论
在20%浓度时,贺斯、万汶可促使Th细胞向Th1细胞分化,佳乐施作用不明显;在40%浓度时,贺斯、万汶抑制Th2细胞的分化,佳乐施对Th1和Th2细胞的分化均呈抑制作用。万汶在两种浓度下均可促使Th1/Th2比值升高。
Background
In recent years,more and more attentions have been paid to the function of immune system in patients.The helper T-lymphatcyte(Th) cells are the important active immune cells,which promote the differrentiation of Th1 or Th2.And the ratio of Th1 and Th2 can impact the immune function.Plasma substitutes are widely used to supply intravascular volume.Previous studies have demonstrated the effects of colloids on inflammation response,but little is known about the effects on the differentiation of Th cells.
Objective
To investigate the effects of pretreatment with different concentra- tions of hydroxyethyl starch(HES),Gelofusine and Voluven on the differentiation of human helper T cells in vitro.
Methods
Eighteen healthy volunteers were enrolled in this study.Two clinically relevant degrees of in vitro haemodilution were prepared: 20%and 40%(V/V).In each volunteer,20 ml of blood was taken from peripheral vein and divided into 7 groups:two HES groups[20%(H1)、40%(H2)],two Gelofusine groups[20%(G1)、40%(G2)],two Voluven groups[20%(V1)、40%(V2)],control group(C).Whole blood and mononuclear cells from peripheral blood(PBMCs)were pre-treated with different colloids.To estimate the imbalance of Th1/Th2,the intracellular cytokines(IFN-γ/IL-4)were detected by four-color flow cytometry.The cytokines in the PBMCs culture supernatant were examined by cytometric bead array(CBA).All data represent the mean±SD and are compared by two way analysis of variance(SPSS11.5 software). Differences were considered significant if the P value is less than 0.05.
Results
Compared with C group,the percentages of Th1 cells increased significantly in H1 and V1 groups(P＜0.05),and decreased in G2 group (P＜0.01);while the percentages of Th2 cells decreased in H2,G2 and V2 groups(P＜0.05).The ratios of Th1/Th2 were increased in H2,G2,V1 and V2 groups(P＜0.05).In two HES groups and two Voluven groups,the levels of Th1 and Th2 cytokines of the supematants increased(P＜0.05), while IL-10 decreased(P＜0.01).In two Gelofusine groups,the levels of IL-4 and IL-6 increased(P＜0.01).
Conclusion
Voluven and Hes encouraged helper cells to differentiate into Th1 cells while Gelofusine inhibited the differentiation of Th1 and Th2 cells. Voluven could increase the Th1/Th2 ratios.
引文
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3 Heal JM,Chuang Blum berg N,et al.Perioperative blood transfusion and prostate cancer recurrence and survival[J].Am J Surg,1988,156(5):374
4 孙家潭,葛衡江,刘怀琼.围术期输血对机体免疫功能抑制的研究进展[J].《国外医学》麻醉学与复苏分册,2000,21(5):285-288
5 Abbas AK,Murphy KM,Sher A.Functional diversity of helper T lymphocytes.Nature,1996,383:787-793.
6 吴亚娣,孙兵.Th1和Th2细胞的表面受体及其功能调节.上海免疫学杂志,2000,5:318-320.
7 黄咏磊,杭燕南.减少异体输血的新方法和新观点[J].《国外医学》麻醉学与复苏分册,2001,22(4):193-197
8 邢同京,章廉,主编.Th类细胞极化群体的基础与临床.第一版.北京军事医学科学出版社,2002:214-221.
9 Jason J,Lamed J.Single-cell cytokine profiles in normal humans:comparison of flow cytometric reagents and stimilation protocols.J Immunol Methods,1997,207:13-22.
10 Sewell C,Margaret E,North A,et al.Determination of intracellular cytokines by flow-cytometry following whole-blood culture.J Immunol Methods,1997,209:67-74.
11 Sewell C,Margaret E,North A,et al.Determination of intracellular cytokines by flow-cytometry following whole-blood culture.J Immunol Methods,1997,209:67-74.
12 李振,曾耀英,何贤辉.正常成人外周血Th1/Th2细胞亚群体外活化的研究.中国病理生理杂志,2001,17:302-304.
13 Nath N,Giri S,Prasad R,et al.Potential targets of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor for multiple sclerosis therapy.J Immunol,2004,127:1273-1286.
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2 Wag mack Rapien J,Garnett D.et al.Effect of transfusion on immune function in a tranmatized animal model[J].Arch Surg,1986,121(1):50
3 Heal JM,Chuang Blum berg N,et al.Perioperative blood transfusion and prostate cancer recurrence and survival[J].Am J Surg,1988,156(5):374
4 孙家潭,葛衡江,刘怀琼.围术期输血对机体免疫功能抑制的研究进展[J].《国外医学》麻醉学与复苏分册,2000,21(5):285-288
5 Abbas AK,Murphy KM,Sher A.Functional diversity of helper T lymphocytes.Nature,1996,383:787-793.
6 吴亚娣,孙兵.Th1和Th2细胞的表面受体及其功能调节.上海免疫学杂志,2000,5:318-320.
7 黄咏磊,杭燕南.减少异体输血的新方法和新观点[J].《国外医学》麻醉学与复苏分册,2001,22(4):193-197
8 邢同京,章廉,主编.Th类细胞极化群体的基础与临床.第一版.北京军事医学科学出版社,2002:214-221.
9 Jason J,Lamed J.Single-cell cytokine profiles in normal humans:comparison of flow cytometric reagents and stimilation protocols.J Immunol Methods,1997,207:13-22.
10 Sewell C,Margaret E,North A,et al.Determination of intracellular cytokines by flow-cytometry following whole-blood culture.J Immunol Methods,1997,209:67-74.
11 Sewell C,Margaret E,North A,et al.Determination of intracellular cytokines by flow-cytometry following whole-blood culture.J Immunol Methods,1997,209:67-74.
12 李振,曾耀英,何贤辉.正常成人外周血Th1/Th2细胞亚群体外活化的研究.中国病理生理杂志,2001,17:302-304.
13 Nath N,Giri S,Prasad R,et al.Potential targets of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor for multiple sclerosis therapy.J Immunol,2004,127:1273-1286.
14 Beyer R,Harmening U,Rittmeyer O,et al.Use of modified fluid gelatin and hydroxyethyl starch for colloidal volume replacement in major orthopedic surgery.Br J Anaesth,1997;78:44-50
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