慢性乙型肝炎患者T淋巴细胞亚群与自然杀伤细胞活性分析

目的 分析慢性乙型肝炎患者外周血T淋巴细胞亚群与自然杀伤细胞(NK)的活性变化.方法 应用流式细胞技术分别检测35名健康入(A组)、31例乙型肝炎病毒携带(B组)、32例慢性乙型肝炎患者(C组)、30例急性乙型肝炎患者(D组),外周血T淋巴细胞CD4+、CD8+以及NK细胞,并分析各组间免疫细胞表达差异性.结果 C、D组CD4+ T淋巴细胞平均值为(32.45±10.45)％、(29.54±11.55)％较A、B组的(38.05±10.15)％、(37.17±11.22)％,明显降低,而CD8+ T淋巴细胞平均值为(36.15±11.56)％、(38.45±10.35)％,较A组的(30.14±11.25)(％),明显升高(P＜0.01); NK细胞则表现为C、D组平均值为(15.23±6.38)％、(12.07±5.95)％,较A、B组的(22.05±7.15)％、(20.32±7.67)％,明显降低(P＜0.05),以D组降低最为明显,A、B组差异无统计学意义.结论 慢性乙型肝炎和急性乙型肝炎患者外周血CD4+T淋巴细胞、NK细胞百分比降低,CD8+ T淋巴细胞升高;通过T淋巴细胞和NK细胞百分比检测可监测慢性乙型肝炎患者的免疫状态,为临床治疗时机的选择提供免疫学依据.     

特发性血小板减少性紫癜患者血清白细胞介素11水平、淋巴细胞亚群及NK细胞变化的临床意义

目的 检测特发性血小板减少性紫癜(ITP)患者血清白细胞介素11(IL-11)水平、淋巴细胞亚群及NK细胞的变化,探讨相关因素在ITP发病中的作用.方法 应用酶联免疫吸附法(ELISA)、流式细胞术分别检测50例ITP(ITP组)和30例健康体检者(对照组)血清IL-11水平、淋巴细胞亚群及NK细胞的变化.结果 ITP组的血小板计数[(30.21±19.40)×109/L]明显低于对照组[(207.21±31.55)×109/L](P＜0.05),ITP组患者血清IL-11水平[(255.72±163.43)ng/L]明显高于对照组[(40.60±5.57)ng/L](P＜0.05),相关分析表明ITP患者血清IL-11水平与血小板计数呈负相关(r=-0.557,P＜0.05);ITP组患者CD3+、CD4+T淋巴细胞百分比及CD4+/CD8+明显低于对照组(P＜0.05),CD8+T淋巴细胞百分比明显高于对照组(P＜0.05);CD3-CD(16+56)+NK细胞百分比明显低于对照组(P＜0.05).结论 IL-11水平、淋巴细胞亚群及NK细胞变化与ITP的发病密切相关,且IL-11水平与血小板计数可能存在负反馈调节作用.

Abstract：

Objective To detect the serum level of interleukin (IL)-1 1, lymphocyte subsets and NK cells in patients with idiopathic thrombocytopenic purpura (ITP), and explore the related factors in the pathogenesis of ITP. Methods The serum level of IL-11, lymphocyte subsets and NK cells were detected by double antibody sandwich enzyme linked immunosorbent assay (ELISA) and flow cytometry in 50 ITP patients (ITP group) and 30 controls (control group). Results The platelet in ITP group [ (30.21 ± 19.40) ×109/L] was lower than that in control group [ (207.21 ± 31.55 ) × 109/L] obviously (P ＜ 0.05 ); the serum level of IL-11 in ITP group [(255.72 ± 163.43) ng/L] was significantly higher than that in control group [ (40.60 ± 5.57 ) ng/L ] (P ＜ 0.05 ). The correlation analysis indicated that the blood serum levels of IL- 11 had negative relationship with the platelet (r = -0.557 ,P ＜ 0.05). The percentage of CD3+ and CD4+ T lymphocyte percentage, CD4+/CD8+ in ITP group were lower and the percentage of CD8+ T lymphocyte was higher than those in control group obviously (P ＜ 0.05 ). The percentage of CD3- CD(16+56) +NK cell in ITP group was lower than that in control group (P ＜ 0.05). Conclusion IL-11, lymphocyte subgroup and NK cell change correlate with ITP morbidity closely, and the IL-11 level and the platelet possibly have the negative feedback control action.     

急性氯化氢气体中毒患者外周血淋巴细胞亚群的变化

目的 探讨急性氯化氢气体中毒患者外周血淋巴细胞亚群的变化,为临床治疗提供依据.方法 采用美国BD公司生产的FACSCalibur流式细胞仪检测37例急性氯化氢气体中毒患者入院当日或次日淋巴细胞亚群总T细胞(CD3+CD19-)、CD4+T细胞(CD3+CD4+)、CD8+T细胞(CD3+CD8+)、B细胞(CD3-CD19+)和NK细胞(CD3-CD16+CD56+)百分数及CD4+/CD8+比值;并与健康体检的49名健康人进行对照分析,观察淋巴细胞亚群的变化.结果 急性氯化氢气体中毒患者总T细胞百分数及CD4+T细胞百分数明显低于健康组,差异有统计学意义(P＜0.05);CD8+T细胞百分数与健康组的差异无统计学意义(P＞0.05);NK细胞及B淋巴细胞百分数明显高于健康组,差异有统计学意义(P＜0.05);CD4+/CD8+比值明显低于健康组,差异有统计学意义(P＜0.05).结论 急性氯化氢气体中毒患者外周血淋巴细胞亚群水平存在异常,提示急性氧化氢气体中毒患者免疫功能受到影响.     

三氯乙烯药疹样皮炎患者及健康接触工人外周血淋巴细胞亚群分析

目的 研究二氯乙烯(TCE)对接触工人淋巴细胞亚群的影响,寻找TCE药疹样皮炎早期的免疫学效应标志物.方法 选取TCE药疹样皮炎患者28例(病例组),来自发生病例车间的健康ICE作业工人56名(接触组)和未从事过TCE作业的健康工人28名(非接触对照组)作为研究对象.所有研究对象采集静脉血2 ml,EDTA抗凝处理,应用流式细胞技术检测淋巴细胞各亚群的百分比,结合血常规检测结果计算T细胞、CD4+T细胞、CD8+T细胞、B细胞、自然杀伤(NK)细胞绝对数及CD3+ CD4 +/CD3+ CD8+比值,比较各组之间外周血淋巴细胞业群的变化情况.结果 病例组外周血淋巴细胞总数、T细胞、CD4+T细胞、CD8+T细胞绝对数(中位数依次为2810.00、1846.17、831.87、904.05个/μl全血)均明显高于接触组(中位数依次为2101.00、1218.59、643.87、482.81个/μl全血,Z值分别为-3.19、-4.96、-3.22、-4.99,P值均＜0.001)和非接触对照组(中位数依次为1900.00、1223.60、558.60、325.80个/μl全血,Z值分别为- 3.30、-4.46、-3.45、-5.03,P值均＜0.001);病例组CD3+ CI4+/CD3+ CD8+比值和NK细胞绝对数(中位数分别为1.11、255.50个/μl全血)明显低于非接触对照组(中位数分别为1.96、642.60个/μl全血,Z值分别为-3.11、-3.56,P值均＜0.01);接触组CD8+T细胞数(中位数为482.81个/μl全血)明显高于非接触对照组(中立数为325.80个/μl全血,Z=-2.63,P值＜0.01);接触组CD3+ CD4+/CD3+ CD8+比值和NK细胞数(中位数分别为1.27、318.76个/μl全血)均明显低于非接触对照组(中位数分别为1.96、642.60个/μl全血,Z值分别为-2.29、-3.52,P值均＜0.05).结论 TCE接触可导致作业工人外周血淋巴细胞亚群发生变化;总淋巴细胞、T细胞和CD4+T细胞计数升高可作为TCE药疹样皮炎早期的免疫学效应标志物.     

杭州地区男性素食者细胞免疫水平与血浆磷脂脂肪酸构成的相关性

目的研究杭州地区男性素食者细胞免疫功能水平及其与血浆磷脂脂肪酸的相关性。方法采用流式细胞术测定89例男性素食者(素食组)和103例健康体检者(对照组)的外周血淋巴细胞亚群,采用气相色谱法测定血浆磷脂脂肪酸的构成。结果与对照组相比,素食组CD3+T淋巴细胞、CD4+T淋巴细胞、CD8+T淋巴细胞、CD16+/56+自然杀伤(NK)细胞百分比及CD4+/CD8+比值均无统计学意义,而素食者CD19+B淋巴细胞低于对照组,差异有非常显著意义(P<0.01)。相关性分析显示,CD3+T淋巴细胞和CD4+T淋巴细胞均与血浆n-3多不饱和脂肪酸(polyunsaturated fatty acids,PUFAs)含量存在负相关(P<0.01),CD19+B淋巴细胞与n-6 PUFAs部分指标存在负相关(P<0.05),NK细胞与n-3 PUFA部分存在正相关(P<0.01),但与总n-3 PUFAs及n-6 PUFAs均无相关性。结论素食者的细胞免疫功能水平无改变,体液免疫功能水平下降,部分原因与血浆磷脂脂肪酸构成变化有关。     

系统性硬化症患者外周血淋巴细胞亚群分布异常的临床研究

目的研究系统性硬化症(SSc)患者与健康人外周血中CD3~+T淋巴细胞、CD4~+T淋巴细胞、CD8~+T淋巴细胞、CD19~+B淋巴细胞和CD3~-CD16~+CD56~+]NK细胞有无差异及临床意义。方法研究浙江省人民医院2013年12月-2018年10月的36例SSc患者和36例健康体检者的流式抗凝血,采用外周血和荧光标记抗体共同孵育的方法,使用BD FACSCantoⅡ流式细胞仪检测CD3~+T淋巴细胞、CD19~+B淋巴细胞、CD3~-CD16~+CD56~+NK细胞、CD4~+T淋巴细胞和CD8~+T淋巴细胞亚群的分布频率。结果 36例SSc患者中CD3~+T淋巴细胞和CD4~+T淋巴细胞频率显著高于对照组(P0.05),CD19~+B淋巴细胞和CD3~-CD16~+CD56~+NK细胞频率显著低于36例对照组,差异均有统计学意义(P 0.05),但CD8~+T淋巴细胞频率在36例SSc患者和36例对照组之间差异无统计学意义(P0.05)。结论 CD3~+T淋巴细胞、CD4~+T淋巴细胞、CD19~+B淋巴细胞和CD3~-CD16~+CD56~+NK细胞的免疫紊乱在SSc患者免疫失调中扮演重要作用。     

儿童噬血细胞性淋巴组织细胞增生症外周血淋巴细胞亚群分析

[目的]研究儿童噬血细胞性淋巴组织细胞增生症(hemophagocytic lymphohistiocytosis,HLH)外周血淋巴细胞活性的改变. [方法]选择2005年1月-2009年12月在本院住院的49例确诊为HLH 且为急性期的患儿为病例组,30例健康儿童为对照组,分别抽取外周血,肝素抗凝,采用流式细胞术方法分别检测两组外周血淋巴细胞CD3+、CD4+、CD8+、CD(16+56)+的表达率. [结果] 病例组CD(16+56)+和CD8+细胞比例显著低于对照组(P＜0.01),CD3+细胞比例高于对照组(P＜0.05),CD4+细胞比例与对照组比较无显著统计学意义(P＞0.05). [结论] NK细胞[CD(16+56)+]和CTL细胞(CD8+)活性的降低对诊断HLH可能具有重要临床意义,T淋巴细胞(CD3+)活性增强对诊断HLH也有一定价值.     

重型甲型H1N1流感患者外周血免疫细胞变化及其临床意义

目的 通过动态观察重型甲型H1N1流感(重症及危重症)患者外周血淋巴细胞的数量变化,探讨患者的免疫病理机制及淋巴细胞亚群检测在甲型H1N1流感诊断中的临床意义.方法 采用流式细胞技术检测2009年10-12月在我院住院治疗的41例重症、危重症甲型H1N1流感患者和同期41例轻症患者的外周血T、B和NK细胞数量.结果 在发病初期,重症及危重症患者外周血总T细胞、CD8+T细胞、NK细胞较轻症甲型H1N1流感患者显著降低(P＜0.01),CD4+T细胞较轻症患者降低(P＜0.05);危重症患者总T细胞、CD4+T细胞、CD8+T细胞、NK细胞低于重症患者(P＜0.01).恢复期重症及危重症甲型H1N1流感患者总T细胞、CD8+T细胞低于轻症患者(P＜0.05),NK细胞低于轻症患者(P＜0.01),B细胞高于轻症患者(P＜0.05).结论 重症及危重症甲型H1N1流感患者存在明显的细胞免疫损伤.     

尖锐湿疣患者外周血T细胞及自然杀伤细胞水平与其复发的关系

目的 探讨尖锐湿疣患者外周血T细胞亚群及NK细胞水平及其与尖锐湿疣临床复发的关系.方法 用免疫荧光法检测杭州市第一人民医院就诊的94例尖锐湿疣病例和50例正常对照组外周血中T细胞亚群及NK细胞水平,C02激光清除病例疣体后每2周复查1次,随访持续12周,并统计复发率和复发次数,并比较复发和未复发病例、不同型别HPV患者外周血T细胞亚群水平.结果 病例总体复发率为65.96％ (62/94),平均复发次数为2.23次.尖锐湿疣病例外周血CD4+T细胞占(32.41±8.14)％,低于对照组(t=4.52,P＜0.01);CD8+T细胞占(31.56±7.95)％,高于对照组(t=5.44,P＜0.01);NK细胞占(19.86±6.29)％,低于对照组(t=2.12,P＜0.05).与尖锐湿疣未复发病例相比,复发病例外周血CD8+T细胞较高,占(33.64±7.71)％;而CD4/CD8比值较低,为(0.97±0.31),差异均有统计学意义(t=3.79、4.34,P均＜0.01).与HPV16/18单一型别感染和HPV6/11/16/18型合并感染病例相比,HPV6/11单一型别感染病例外周血CD8+T细胞水平较低、CD47CD8+比值较高(q=3.37～3.45,P均＜0.05).结论 尖锐湿疣患者存在细胞免疫缺陷,而高危型HPV感染病例有更高的免疫缺陷风险.CD8+T细胞水平和CD4+/CD8+比值有助于判断尖锐湿疣预后.     

河南地区健康儿童淋巴细胞亚群参考区间的建立

目的建立适合河南地区年龄、性别相关的健康儿童淋巴细胞亚群参考区间。方法采用多阶段分层整群随机抽样的方法,抽取河南省5个地市1 027名<7岁健康儿童,分婴儿组(4~12月龄,n=45)、幼儿组(1~3岁,n=190)和学龄前组(3~7岁,n=792)进行研究。用流式细胞术双平台法检测淋巴细胞亚群百分比和绝对计数。结果学龄前组不同性别健康儿童之间,淋巴细胞免疫亚群CD3^+、CD3^+CD4^+、CD3^+CD8^+、CD4^+/CD8^+和CD3^+CD16^+CD56^+百分比差异均有统计学意义(P值均<0.01),绝对计数CD3^+CD4^+、CD3^+CD8^+、CD3^+CD16^+CD56^+差异均有统计学意义(P值均<0.01);不同年龄组健康儿童之间淋巴细胞亚群百分比和绝对计数差异均有统计学意义(P值均<0.01)。与国内已发表的淋巴细胞亚群百分比相关文献比较,相同年龄段淋巴细胞亚群相对计数参考区间明显不同。结论年龄、性别影响健康儿童淋巴细胞亚群的分布,本研究可为建立河南省健康学龄前儿童年龄和性别相关的淋巴细胞亚群参考区间提供依据。     

肝实质细胞和肝非实质细胞及非肝细胞的共培养

生物人工肝有望成为肝衰竭患者有效的体外肝支持治疗手段,而建立生物人工肝的前提是要获得足够数量高活性和良好功能的肝细胞.大量研究发现,肝细胞共培养能明显增强肝细胞功能,保持肝细胞的表型.此文就近年来肝细胞和非实质细胞、非肝细胞共培养的效果、方法和可能的机制作了综述.     

Germ cells and pluripotent stem cells in the mouse

For many years, attempts to achieve long-term culture of mouse primordial germ cells (PGCs) proved unsuccessful, even when feeder layers were used and individual growth factors were added to the medium. However, when three growth factors were added simultaneously to the medium, some of the cells continued to proliferate indefinitely. Similar to embryonic stem cell lines, these embryonic germ (EG) cell lines were capable of giving rise to embryoid bodies in vitro, and colonizing all cell lineages in chimeras, including the germline. Initially, EG cells were made from PGCs before migration, 8.5 days post coitum (dpc), and after entry into the genital ridge, 11.5 and 12.5 dpc. New EG cell lines from 9.5 dpc (migrating) and 11.5 dpc PGCs, carrying either a LacZ or GFP transgene, are described here. The developmental potential of the new EG cell lines in vitro, in vivo in chimeras, and in tissue aggregates in organ culture was studied. The EG cells were compared with PGCs at the stage from which the EG cells were derived. The two cell types show several similarities, but also some differences in gene expression and cell behaviour, which require further exploration.     

Generation and functional assessment of antigen-specific T cells stimulated by fusions of dendritic cells and allogeneic breast cancer cells

We have reported that fusions of patient-derived dendritic cells (DC) and autologous breast cancer cells induce T-cell responses against autologous tumors. However, the preparation of fusion cells requires patient-derived tumor cells, and these are not always available in the clinical setting. In the present study, we explore an alternative approach to constructing DC-breast cancer fusion vaccine by using breast cancer-cell lines. DC generated from HLA-A*0201-positive donor were fused to HLA-A*0201+ allogeneic MCF7 breast cancer cells. These fusion cells co-expressed tumor-associated antigens and DC-derived costimulatory and MHC molecules. Both CD4 and CD8 T cells were activated by the fusion cells as demonstrated by the production of IFN-gamma. The fusion cells induced strong antigen-specific cytotoxic T lymphocytes (CTL) activity against their parent cells. The lysis of targets was restricted by HLA-A*0201, since killing was blocked by the anti-HLA-A2 mAb. Similar CTL activity against HLA-A*0201-positive targets was induced when T cells were cocultured with fusions of DC and HLA-A*0201-negative allogeneic BT20 breast cancer cells. In addition, administration of T cells stimulated by DC-breast cancer fusion cells regressed 7-day-old tumors and rendered mice free of disease up to 90 days. These results suggest that tumor-cell lines can be used as a fusion partner in the construction of DC-tumor fusion vaccine. Such fusion cells hold promise since they can be used as a vaccine for active immunotherapy or as stimulators to activate and expand T cells for adoptive immunotherapy.     

奈达铂对宫颈癌Hela及Siha细胞的作用

目的:比较奈达铂(NDP)体外对人宫颈腺癌细胞Hela细胞及鳞癌细胞Siha的抑制作用并探讨其作用机制。方法:以2.5～40μg/ml NDP分别作用于Hela及Siha细胞24 h,48 h,72 h,MTT法检测抑制率,计算半数抑制浓度(IC50);利用流式细胞术(FCM)观察细胞凋亡率及周期变化;半定量PCR法分析基因半胱氨酸天门冬氨酸蛋白酶3(Caspase 3,Caspase 9)及基质金属蛋白酶2(MMP-2)的表达变化。结果:NDP对宫颈癌Hela细胞及Siha细胞均有抑制作用,呈时间-剂量依赖性,NDP对Hela细胞的抑制率大于Siha细胞;FCM显示,随时间、浓度增加,Hela及Siha细胞凋亡率增加,相同浓度和作用时间下,Hela细胞的凋亡率大于Siha细胞(P<0.05),S期细胞的比例增加,G0/G1期比例减少,G2/M期比例变化不明显;与对照组相比,Caspase 3,Caspase 9表达增加,MMP-2表达减少。结论:NDP可抑制宫颈腺癌细胞Hela及鳞癌细胞Si-ha的增殖,NDP体外对Hela细胞的抑制作用更强。NDP的作用机制与诱导细胞凋亡、细胞周期阻滞、上调Caspase3和Caspase9的表达有关;下调MMP-2的表达可能有利于降低宫颈癌细胞的侵袭力。     

人子宫内膜细胞原代培养与间质细胞MTS比较

目的探究原代人子宫在位内膜细胞分离及培养的简易方法,比较分离培养单一间质细胞和混合培养间质细胞之间的差异。方法采用单纯胰蛋白酶,分段消化人子宫内膜组织,经过两次筛网过滤、离心纯化技术,分离、纯化人子宫内膜间质细胞(ESC)和腺上皮细胞(EEC),采用免疫荧光方法对细胞进行鉴定,采用Cell Titer96~ Aqueous One Solution Reagent(MTS)方法监测细胞之间增长速度。结果间质细胞多呈梭形或多角形,纯度可达97%以上。腺上皮细胞多似蝌蚪形或多角形,纯度可达93%以上。混合培养细胞比单一培养细胞增殖能力强。结论采用单纯胰蛋白酶分段消化法和二次筛网过滤法可成功分离子宫内膜腺上皮细胞和间质细胞,具有获得细胞成活率高、生长稳定的优点,混合培养间质细胞,保留细胞间整体性,可为进一步研究子宫内膜细胞提供一定的实验基础。     

Differentiation of embryonic and adult stem cells into insulin producing cells

Replacement of insulin producing cells represents an almost ideal treatment for patients with diabetes mellitus type 1. Transplantation of pancreatic islets of Langerhans is successful in experienced centers. The wider application of this therapy, however, is limited by the lack of donor organs. Insulin producing cells generated from stem cells represent an attractive alternative. Stem cells with the potential to differentiate into insulin producing cells include embryonic stem cells (ESC) as well as adult stem cells from various tissues including the pancreas, liver, bone marrow and adipose tissue. The use of human ESC is hampered by ethical concerns but research with human ESC may help us to decipher important steps in the differentiation process in vitro since almost all information available on pancreas development are based on animal studies. The present review summarizes the current knowledge on the development of insulin producing cells from embryonic and adult stem cells with special emphasis on pancreatic, hepatic and human mesenchymal stem cells.     

右旋氨基酸氧化酶在胃癌和正常胃黏膜细胞中的表达

目的:研究右旋氨基酸氧化酶(DAO)在胃癌和正常胃黏膜细胞中的表达. 方法:以裸鼠肝、肾组织为对照,采用定量PCR法,检测7株胃癌细胞和胃黏膜细胞株GES-1中DAO的表达.用RT-PCR法检测46份新鲜胃癌组织和正常胃黏膜组织标本中DAO的表达. 结果:DAO在裸鼠肝和肾组织中有表达,在肾组织中表达量较高;7株胃癌细胞和正常胃黏膜细胞株GES-1中均无DAO表达;除1份胃癌组织标本中DAO有表达外,其余胃癌组织和正常胃黏膜组织中均无DAO表达. 结论:胃癌细胞和正常胃黏膜细胞中均无DAO表达,右旋甲硫氨酸替代左旋甲硫氨酸用于胃癌的营养治疗,可避免甲硫氨酸饥饿对肝、肾等重要器官的不利影响.     

Activity of natural killer cells and granulocytes in peripheral blood and separated cells]

The natural killing of K 562 cells by whole blood from normal subjects was comparable with that shown by separated mononuclear cells. In order to establish the conditions for a reliable natural killer assay by using very small numbers of effector cells in whole blood, the isotope uptake of target cells was increased by a modified labelling method, which permitted the use of fewer target cells in the assay. The natural cytotoxicity of whole blood was augmented by interferon to the same extent as observed with separated mononuclear cells. The chemiluminescence of granulocytes in whole blood comparable with that of separated granulocytes. Taken together, these methods are considerably less tedious than the conventional methods, technique is also economical, and the results may reflect in vivo cytolytic processes much better.