严重烧伤后细胞免疫改变及其与高迁移率族蛋白B1的相关性

目的 探讨严重烧伤患者T细胞免疫功能的变化规律及其与高迁移率组蛋白B1(HMGB1)的相关性.方法 采集35例烧伤体表总面积>30%的患者静脉血样,根据是否并发多器官功能障碍综合征(MODS)分组(MODS组13例、非MODS组22例),采集患者伤后第1、3、5、7、14、21、28大的外周静脉血,采用酶联免疫吸附试验检测血浆HMGB1水平;观察外周血T淋巴细胞增殖反应和白细胞介素2(IL-2)的产生能力,并通过流式细胞仪检测CD4+/CD8+T细胞的比值.结果 严重烧伤患者伤后第1天血浆HMGB1水平即明显升高,其中伤后第1、7、21、28天MODS组HMGB1显著高于非MODS组(P<0.05).两组间比较,外周血T淋巴细胞增殖反应和IL-2的产生能力、CIM4+/CD8+T淋巴细胞比值在伤后第1、14、21、28天MODS组显著低于非MODS组(P均<0.05).大面积烧伤患者伤后血浆HMGB1含量与细胞免疫功能指标包括T淋巴细胞增殖活性、IL-2含量、CD4+/CD8+T细胞比值呈显著负相关(P<0.05).结论 大面积烧伤后T淋巴细胞免疫功能紊乱与患者并发MODS密切相关,严重烧伤患者血浆HMGBI水平的持续升高对机体细胞免疫功能异常具有显著影响.     

Tim-3在小鼠心脏移植受者体内不同部位T淋巴细胞上表达的变化

目的 检测激活的T_H1类淋巴细胞标记分子"T淋巴细胞免疫球蛋白域及粘蛋白域蛋白-3(Tim-3)"在受者体内不同部位T淋巴细胞上表达的变化,探讨其与急性排斥反应的关系.方法 建立小鼠同基因和异基因心脏移植模型(简称:同基因组和异基因组);移植术后第3和第6天,分离和制备两组受者外周血、脾脏、引流淋巴结和移植心内淋巴细胞悬液,采用流式细胞仪检测Tim-3阳性细胞在CD4~+ 和CD8~+ T淋细胞中的比值.结果 两组受者术后外周血和脾脏内Tim-3~+/CD4~+以及Tim-3~+/CD8~+ 的比值比较,差异均无统计学意义(P>O.05).与同基因组比较,异基因组引流淋巴结内Tim-3~+/CD4~+ 比值轻度升高(P<0.05);但异基因组术后第6天与第3天比较,差异无统计学意义(P>0.05).与同基因组比较,移植心内Tim-3~+/CD4~+和TiM-3~+/CD8~+比值均显著升高(P<0.01);异基因组术后第6天与第3天比较.移植心TiM-3~+/CD4~+和Tim-3~+/CD8~+比值也显著升高(P<0.01).结论 小鼠异基因心脏移植受者引流淋巴结和移植心内T淋巴细胞上Tim-3的表达升高与急性排斥反应的进展动态相关.     

七氟醚复合右美托咪定对HIV无症状感染者术后免疫系统的影响

目的研究七氟醚复合右美托咪定对人类免疫缺陷病毒(human immunodeficiency,HIV)无症状感染者手术后免疫系统的影响。方法选择在本院接受胆囊切除术、妇科良性肿瘤切除术以及结肠良性肿瘤切除术的HIV无症状感染者60例,男28例,女32例,年龄35～51岁,体重58～62kg,ASAⅠ—Ⅲ级,随机分为3组:七氟醚组(S组)、右美托咪定(D组)和七氟醚复合右美托咪定组(SD组),每组20例。同时选择20例行全麻下胆囊切除术、妇科良性肿瘤切除术以及结肠良性肿瘤切除术的正常患者为对照组(N组)。测定患者手术前和手术后24h外周血血常规(白细胞、中性粒细胞、淋巴细胞、单核细胞)、T淋巴细胞亚群(CD3+、CD4+、CD8+)和HIV病毒载量。结果与手术前比较,手术后24hS组和D组外周血淋巴细胞、CD4+含量、CD4+/CD8+比值明显降低,中性粒细胞、单核细胞、CD8+含量明显升高(P0.05)。与N组比较,术后24hSD组CD4+含量、CD4+/CD8+比值明显降低,CD8+含量明显升高(P 0.05)。与S组比较,SD组术后外周血CD8+含量明显下降,CD4+/CD8+比值明显升高(P0.05)。S组、D组和SD组术前术后病毒载量构成比差异无统计学意义。结论七氟醚复合右美托咪定麻醉可能减轻全麻手术对HIV无症状感染期患者免疫系统的影响。     

未分化骨髓间充质干细胞的免疫学特性研究

目的 研究骨髓间充质干细胞表面MHC-Ⅰ、MHC-Ⅱ类抗原与共刺激分子CD80（B7—1）和CD86（B7—2）的表达及探讨MSC的抗原性。方法 采用流式细胞技术检测MSC表面MHC-Ⅰ、MHC-Ⅱ类抗原与共刺激分子CDS0（B7—1）和CD86（B7—2）的表达，测定淋巴细胞MSC混合反应的强度。结果 MSC表达高水平的MHCⅠ类抗原，不表达MHCⅡ类抗原，低表达CD86（B7—2），不表达CD80（B7—1），IFN-γ刺激48h后，MHCⅠ类抗原表达增加，MHCⅡ类抗原表达，CD80（B7—1）、CD86（B7—2）也均有表达。未分化的MSC经γ-干扰素（IFN-γ）刺激之后，不会引起很强的免疫排斥反应，表现为弱免疫原性。结论 未分化的MSC抗原性较弱，具有同种异体移植的可能。     

围手术期静脉营养治疗对胃恶性肿瘤患者细胞免疫功能的影响

目的探讨围手术期静脉营养治疗对胃癌患者细胞免疫功能的影响. 方法 57例随机分为两组对照组不给予营养治疗,实验组手术前后给予静脉营养治疗各1周.观察比较手术前后外周血淋巴细胞总数,T细胞亚群(CD3+,CD4+,CD8+)及CD4+/ CD8+比值的变化. 结果对照组术后外周血淋巴细胞总数增加(P<0.05),CD4+/ CD8+比值升高(P<0.01).实验组术后外周血淋巴细胞总数、T细胞亚群及CD4+/ CD8+比值均明显改善(P<0.01).实验组的细胞免疫功能明显优于对照组(P<0.01). 结论围手术期使用静脉营养治疗可明显改善胃癌患者的细胞免疫功能.     

白介素-12在多器官功能不全小鼠免疫失调中的作用

目的:探讨多器官功能不全(MODS)小鼠在伤后不同时相点白介素-12(IL-12)水平的变化趋势及其与创伤后免疫失衡的关系.方法:选择6～8周龄雄性C57BL/6小鼠96只,随机分为正常组,伤后3～24h组、3～7d组和12d组.腹腔注射酵母多糖复制MODS动物模型,用间接酶标法行脾脏IL-12p40的免疫组织化学标记,采用ELISA法分别检测血清与脾脏组织匀浆上清中IL-12p40和IL-12p70的水平,流式细胞术检测外周血T细胞亚群.结果:在MODS病程早期和晚期的"两次打击"中,小鼠脾脏组织与血清中IL-12p40含量显著增多,IL-12p70相对减少,同时伴有CD4/CD8比值下降.结论:MODS小鼠发生免疫抑制可能与免疫器官中抗原提呈细胞高表达IL-12p40有一定相关性.     

IFN-γ上调MHC-Ⅱ分子表达对KC免疫原性的影响

目的观察在炎症因子IFN-γ的作用下,角质形成细胞（Keratinocyte,KC）免疫原性的变化情况。方法 KC体外传代培养,按不同的INF-γ浓度分组后作用于传代的KC。将高表达MHC-Ⅱ分子的KC与异体淋巴细胞混合培养后,观察淋巴细胞的增殖情况;同时,将KC植入异体小鼠皮下,观察免疫排斥迹象。结果在6 000 U/mL IFN-γ的作用下,MHC-Ⅱ+的KC超过90%。高表达MHC-Ⅱ分子的传代KC无明显刺激异体淋巴细胞增殖的作用,植入异体小鼠皮下后也无明显淋巴细胞浸润现象。结论传代后的KC在IFN-γ作用下高表达MHC-Ⅱ分子,表达MHC-Ⅱ分子的KC无明显免疫原性。     

不同免疫状态肝移植受者外周血T淋巴细胞亚群及共刺激分子的表达及意义

目的探讨不同免疫状态肝移植受者外周血T淋巴细胞亚群(CD4+、CD8+)、共刺激分子(CD80、CD86)的表达,及其判断肝移植受者不同免疫状态的价值。方法 52例肝移植受者,分为免疫耐受组28例、急性排斥反应组(排斥组)13例、药物不良反应组(药物反应组)11例。采用流式细胞术测定三组患者外周血CD4+、CD8+及CD80、CD86的表达水平,并计算CD4+/CD8+。结果与免疫耐受组和药物反应组比较,排斥组患者外周血CD8+水平较低,而CD4+、CD4+/CD8+、CD80、CD86均较高(P0.05～0.01)。与免疫耐受组比较,药物反应组患者外周血CD8+、CD80、CD86水平明显升高(P0.05～0.01),而CD4+、CD4+/CD8+差异无统计学意义(均为P0.05)。结论免疫T淋巴细胞亚群(CD4+、CD8+及CD4+/CD8+)、CD80、CD86可作为判断肝移植受者免疫状态的指标。     

MHC-Ⅱ类分子表达的调控与其在器官移植中的应用

主要组织相容性复合体-Ⅱ类分子(major histocompatibility compelx classⅡmolecules,MHC-Ⅱmolecules)向T淋巴细胞提呈抗原肽,在免疫系统特异性识别和免疫应答中起着至关重要的作用.MHC-Ⅱ类分子的表达受到精细的调控,其调控主要发生在转录水平,一系列反式作用因子(trans-acting factor)参与了MHC-Ⅱ类基因转录活性的调节.同时,MHC-Ⅱ类分子在同种异体器官移植中起着重要作用,是参与移植排斥反应的强反应抗原.MHC-Ⅱ类分子配型与移植物的排斥反应以及移植物的存活时间有着密切的关系.     

MHC-Ⅱ类分子表达的调控与其在器官移植中的应用

主要组织相容性复合体-Ⅱ类分子(major histocompatibility compelx classⅡmolecules,MHC-Ⅱmolecules)向T淋巴细胞提呈抗原肽,在免疫系统特异性识别和免疫应答中起着至关重要的作用.MHC-Ⅱ类分子的表达受到精细的调控,其调控主要发生在转录水平,一系列反式作用因子(trans-acting factor)参与了MHC-Ⅱ类基因转录活性的调节.同时,MHC-Ⅱ类分子在同种异体器官移植中起着重要作用,是参与移植排斥反应的强反应抗原.MHC-Ⅱ类分子配型与移植物的排斥反应以及移植物的存活时间有着密切的关系.     

Relationship between HMGB1 content and MHC-Ⅱ expression in circulating monocytes and spleen of mice challenged with zymosan

Objective: To observe the regularity of change in high mobility group protein box 1 (HMGB1) content in serum and spleen of mice with multiple organ dysfunction syndrome (MODS), to analyze the correlation between HMGB1 content and major histocompatibility complex (MHC)-Ⅱ-I-Ab expression on monocytes in blood and spleen, and to explore the effect of HMGB1 on immune function of circulating monocytes and splenocytes. Methods: One hundred 8-week-old male 57BL/6 mice were randomly divided into normal group and experimental group subdivided into 8 subgroups: 3, 8, 12 hours, 1, 2, 3, 5-7 days and 10-12 days post zymosan injection (PZI). MODS model was replicated by injecting zymosan into the peritoneal cavity. At each time point, blood and spleen were collected to detect HMGB1 content and the rate of I-Ab positive monocytes. Results: In normal and PZI 3-hour, 8-hour mice, serum HMGB1 was not detected, but it significantly increased at PZI 12 hours. In spleen of normal mice, there was low level of HMGB1 expression. In zymosan-treated mice, HMGB1 started to rise in spleen at PZI 3 hours. Subsequently, HMGB1 content in both serum and spleen significantly increased, and it reached the peak level in 1-2 days, decreased in 5 days, and then increased in 10-12 days. The number of I-Ab positive monocytes in circulating blood and spleen decreased at 1-2 days (t=9.589, 4.432, P<0.01) and 10-12 days following the challenge, forming a two trough like decrease, just corresponding with two-peak increase of HMGB1. However, at 3 hours after zyrnosan challenge, I-Ab expression on circulating monocytes was downregulated (t=5.977, P<0.01), while that in spleen upregulated (t=4.814, P<0.01). Conclusion: In mice with MODS, up-regulated HMGB1 expression can regulate I-Ab expression on monocytes to depress their ability of presenting antigen, which results in immune disturbance contributing development of MODS.     

重组hIFN-α-2b-BCG对小鼠原位膀胱肿瘤免疫效应的研究

目的 探讨重组hIFN-α-2b-BCG对原位膀胱肿瘤小鼠体内抗肿瘤免疫反应及其作用机制.方法 构建C57BL/6原位膀胱肿瘤小鼠模型,采用流式细胞仪对小鼠膀胱灌注治疗后的淋巴细胞亚群进行分析,并测定小鼠血清中mTNF-α和mIL-12的水平,了解小鼠全身免疫状况.肿瘤组织冰冻切片进行T细胞亚群的免疫组织化学分析,免疫组化检测肿瘤Fas表达,了解膀胱局部免疫反应. 结果 重组BCG灌注治疗后小鼠外周血CD4+ T细胞比例明显增高,CD8+ T细胞比例无明显变化,CD4+ /CD8+比例为2.63,与野生BCG组(2.10)比较差异有统计学意义(P＜0.05).荷瘤小鼠外周血中Th1型细胞因子mTNF-α和mIL-12灌注治疗后比PBS对照组大幅提高,重组BCG组mTNF-α为806 pg/ml,mIL-12为860 pg/ml,与野生BCG组及野生BCG联合IFN组比较差异无统计学意义(P＞0.05).重组BCG组瘤组织内CD3、CD4和CD5检测强阳性,显著高于PBS对照组(P＜0.05),重组BCG组和野生BCG加IFN组瘤组织CD4+、重组BCG组CD8+检测值显著高于野生BCG组(P＜0.05).BCG灌注后荷瘤小鼠膀胱肿瘤表达Fas明显高于PBS对照组(P＜0.05). 结论 重组hIFN-α-2b-BCG具有在小鼠体内调节系统及局部免疫能力的作用,纠正荷瘤小鼠淋巴细胞亚群的比例失调,增强局部淋巴细胞浸润,具有增强Th1型细胞因子产生作用,上调荷瘤小鼠Fas的表达,诱导对膀胱肿瘤细胞的免疫攻击.

Abstract：

Objective To study local and systemic immune response in an animal model treated with recombinant hIFN-α-2b-BCG instillation. Methods The MB49 orthotopic bladder cancer model in C57BL/6 mice was established and treated separately with rBCG, wild BCG, wild BCG combined with IFN-α-2b and PBS as the control. The changes of lymphocyte subgroups in peripheral blood were analyzed with FCM, and mTNF-α and mIL-12 in peripheral blood of mice were detected with ELISA.Immunohistochemistry was carried out to detect the local immune reaction, T cell subsets and FAS, in bladder cancer after being treated with rBCG or wBCG. Results The content of CD4+ T lymphocyte was up-regulated in the rBCG group. The CD4+/CD8+ ratio of 2. 63 was up-regulated than pretreatment, significantly different than that of wBCG group(P＜0.05). ELISA assay showed that BCG significantly up-regulated the level of mTNF-α and mIL-12 in serum of orthotopie murine bladder cancer mice. The mTNF-α 806 pg/ml, mIL-12 860 pg/ml in rBCG group, was not significantly higher than those in wBCG group and combination group. The immunocompetent cell numbers with CD3, CD4,CD8 phenotype increased significantly in the tumor tissue of BCG treated group than the control(P＜0.05). The results of CD4+ in rBCG group and the combination group, and CD8+ in rBCG group were significantly higher than that of the wBCG(P＜0.05). The expression of Fas in tumor tissues treated with intravesical BCG was increased(P＜0. 05). Conclusions The recombinant IFN-α-2b-BCG can retrieve the disproportion of systemic lymphocyte subgroups, and increases Th1-type factors and local Fas expression in orthotopic murine bladder cancer. The recombinant IFN-α-2b-BCG is effective in regulating local and systemic immune reaction in orthotopic murine bladder cancer model.     

高容量血液滤过对多脏器功能障碍综合征犬CD14+单核细胞白细胞-DR抗原表达的影响

目的 观察高容量血液滤过(HVHF)对多脏器功能障碍综合征(MODS)犬CD14+单核细胞犬白细胞DR抗原(DLA-DR)表达的影响.方法 通过2次打击建立犬MODS模型,随机分HVHF组和MODS组,HVHF组建模后给予HVHF治疗24h,MODS组不给HVHF治疗.监测CD14+单核细胞DLA-DR表达及主要器官功能指标变化.结果 与实验前(6.52±1.47)比较,MODS组CD14+单核细胞DLA-DR明显降低(P＜0.01).在T5、T7、T8、T9时间点HVHF组CD14+单核细胞DLA-DR水平显著高于MODS组(P＜0.05).HVHF组主要器官功能明显改善.结论 HVHF能改善器官功能,提高CD14+单核细胞DLA-DR的表达,有助于重建机体内稳态.     

Toll样受体4在高迁移率族蛋白B1影响小鼠调节性T细胞免疫功能中的作用

目的 探讨腹腔注射高迁移率族蛋白B1(HMGB1)后不同基因型小鼠天然调节性T细胞(CD4~+CD25~+Treg)免疫功能的改变及其受体作用机制.方法 分别给C3H/HeN和C3H/HeJ[分别为Toll样受体4(TLR4)野生型(TLR4~(+/+))和天然突变型(TLR4~(-/-)]小鼠腹腔注射不同剂量HMGB1(0、10、20μg/只),饲养48 h后采用免疫磁珠法分离小鼠脾脏CD4~+CD25~+Treg.体外培养12 h后采用流式细胞仪检测CD4~+CD25~+Treg表达细胞毒性T淋巴细胞相关抗原-4(CTLA-4)表达强度,并应用酶联免疫吸附试验(ELISA)检测CD4~+CD25~+Treg生成白细胞介素(IL)-10量.结果 与对照组比较,20 μg HMGB1攻击后C3H/HeN小鼠CD4~+CD25~+Treg表达CTLA-4水平显著下降(78.70±11.42与60.76±7.64,P<0.01),同时细胞牛成IL-10量也明显降低[(96.89±6.25)ng/L与(47.11±4.25)ng/L,P<0.01];但不同剂量HMGB1攻击可引起C3H/HeJ小鼠CD4~+CD25~+Treg表达CTLA-4明显上调和生成IL-10量不同程度地增加(P<0.01).结论 HMGB1攻击可显著影响CD4~+CD25~+Treg介导的免疫状态,TLR4在HMGB1诱导CD4~+CD25~+Treg免疫活性过程中发挥了重要负向调控作用.     

Specific cytotoxic activity of T lymphocyte clones derived from a patient with gliosarcoma

Eleven lymphocyte clones were established from the peripheral blood lymphocytes of a patient with gliosarcoma by means of autologous tumor stimulation and the limiting-dilution technique with recombinant interleukin-2. Ten of the 11 clones were cytotoxic against the autologous tumor cell line GI-1. Seven of the 10 clones were also cytotoxic against allogeneic brain-tumor lines and HeLa cells, one clone was cytotoxic against several target cells, and two clones were specifically cytotoxic against GI-1 and allogeneic brain-tumor cells. One of the 11 clones was not cytotoxic against any target cells tested. Lymphokine-activated killer cells induced by recombinant interleukin-2 alone exhibited cytotoxic activity against all target tumor cells tested. Surface phenotypic analysis revealed that all lymphocyte clones expressed CD3 antigen, some expressed CD4 antigen, and others expressed CD8 antigen. These clones seemed to be antigen-specific cytotoxic T lymphocyte clones. Analysis with these antigen-specific cytotoxic T lymphocyte clones may be useful in the elucidation of tumor-specific or tumor-associated antigens on autologous tumor cells.     

烧伤延迟复苏对CD14+单核细胞HLA-DR表达率和外周血淋巴细胞凋亡率的影响

目的:探讨烧伤延迟复苏对CD14 单核细胞人类白细胞组织相容性抗原-DR(HLA-DR)表达率和外周血淋巴细胞凋亡率的影响及其机制。方法:50例烧伤面积大于30%的烧伤患者,按伤后是否及时有效复苏分为延迟复苏组和非延迟复苏组,于伤后1、3、7、14、28d取外周血。流式细胞术检测CD14 单核细胞HLA-DR表达率和外周血淋巴细胞凋亡率。20例健康体检者外周血检测上述指标作为对照。结果:烧伤患者外周血CD14 单核细胞HLA-DR表达率明显低于正常对照组(P均<0.01),延迟复苏患者明显低于非延迟复苏患者(P<0.05或P<0.01)。烧伤患者外周血淋巴细胞凋亡率明显高于正常对照组(P<0.05),延迟复苏患者明显高于非延迟复苏患者,伤后7和28d差异有显著性(P<0.05)。结论:烧伤患者免疫功能低下,动态检测患者的免疫功能状态对患者预后具有指导意义。     

Correlation between radiculalgia and counts of T lymphocyte subsets in the peripheral blood of patients with lumbar disc herniation

Objective: To determine the correlation between the degree of radiculalgia and counts of T lymphocyte subsets in the peripheral blood of patients with lumbar disc herniation. Methods: Forty‐nine patients with lumbar disc herniation (group A) were divided into three subgroups according to Visual Analogue Scale (VAS) pain scores (group A₁: n= 12, VAS 0–4.0; A₂: n= 24, VAS 4.1–7.0; A₃: n= 13, VAS 7.1–10.0. Twenty health blood donors who volunteered to be involved in the study comprised the control group (group B). Peripheral blood counts of various T lymphocyte subsets were measured in each group. Results: (i) The counts of CD4+ T and CD4+/CD8+ lymphocytes were higher in group A than in group B, and the difference between the two groups was statistically significant (P < 0.05). There were also statistically significant differences between group A and group B in the counts of CD3+ and CD8+ T lymphocytes (P < 0.05); (ii) There was no correlation between the VAS scores and the counts of CD3+ T lymphocytes (r= 0.194, P > 0.05). A strong significant correlation was observed between the VAS scores and counts of CD4+ T lymphocytes (r= 0.542, P < 0.05), CD4+/CD8+ (r= 0.468, P < 0.05), which increased with increasing VAS scores in the three subgroups of group A (P < 0.05). However there was a significant negative linear correlation between CD8+ T lymphocyte counts and pain scores (r=?0.462, P < 0.05). Conclusion: Our results suggest that changes in T lymphocyte subsets in peripheral blood take place after prolapse of lumbar intervertebral discs. The current results may provide support for involvement of immunologic mechanisms in low back pain secondary to herniation of the lumbar disc. T lymphocytes may play an important role in the development of symptoms in patients with lumbar intervertebral disc herniation.