G—CSF测定在新生儿疾病诊断中的应用

应用ＥＬＩＳＡ法对本科住院的１５０例新生儿进行了粒细胞集落刺激因子的检测。结果为６０例细菌感染性疾病的新生儿Ｇ－ＣＳＦ阳性检出率为９９％，２５例非细菌感染的新生儿Ｇ－ＣＳＦ阳性率为８％，２０例正常新生儿Ｇ－ＣＳＦ检测全部为阴性，表明Ｇ－ＣＳＦ检测可用于新生儿感染性疾病的鉴别诊断，并对临床应用抗生素有较大的指导意义。     

人树突状细胞体外直接抑瘤作用研究

目的：诱导获得人外周血树突状细胞(DCs)，研究其体外直接抑瘤作用及机制。方法：自正常人外周血分离获得单核细胞，体外rhGM-CSF和rhIL-4联合诱导培养，观察细胞形态并检测其相关表型；利用MTT法检测所诱导DCs及其培养上清对不同肿瘤细胞系的体外直接抑瘤效应。结果：诱导5—7天后的悬浮细胞具有典型的DCs形态，流式分析显示HLA-DR表达率为64．02％，CD14表达率为2．34％；抑瘤实验显示：DCs对HT29、Hela及HepG2．2．15三种肿瘤细胞系的生长具有明显抑制，其抑制率分别为20．16％，25．44％，75．41％，而对Lovo和HepG2两种肿瘤细胞系，则无明显的抑制作用。DCs培养上清均未见明显的抑瘤效应。结论：人类DCs可对某些肿瘤细胞的生长产生直接抑制作用，但对不同的肿瘤细胞其作用不同。此作用可能由DCs与肿瘤细胞的直接接触而触发，而与DCs分泌的细胞因子关系不大。     

人IL—2／GM—CSF融合基因的克隆及序列测定

通过ＰＣＲ技术，对人ＩＬ２和ＧＭＣＳＦ基因分别改构，克隆了通过Ｐｒｏ（ＣＣＣ）连接臂相连的ＩＬ２／ＧＭＣＳＦ融合基因。经酶切鉴定和序列测定证实后，将上述融合基因克隆至原核表达载体ｐＬＹ４，为表达具有ＩＬ２，ＧＭＣＳＦ双功能活性的新型融合蛋白，以及发现细胞因子的新生物学功能等研究提供了有利条件     

云芝多糖增强巨噬细胞M—CSF的表达与分泌

为揭示云芝多糖作用与巨噬细胞Ｍ－ＣＳＦ表达与分泌的关系,采用活性测定,斑点杂交等方法,将云芝多糖对小鼠腹腔巨噬细胞Ｍ－ＣＳＦ表达及分泌的影响进行了探讨。结果显示,腹腔注射云芝多糖可以提高小鼠腹腔巨噬细胞培养上清中的Ｍ－ＣＳＦ活性,并使巨噬细胞Ｍ－ＣＳＦ ｍＲＮＡ的含量增加;应用ｍＲＮＡ合成抑制剂及蛋白合成抑制剂的研究发现,ａｃｔｉｎｏｍｙｃｉｎ Ｄ及ｃｙｃｌｏｈｅｘｉｍｉｄｅｆ均能阻断云芝多糖对小     

M-CSF和IL-10对人单核细胞表面分子表达的影响

本文采用流式细胞术检测人外周血单核细胞表面CD14、CD2 3、CD6 4、CD11b、CD18、CD2 9表达 ,研究巨噬细胞集落刺激因子 (M CSF )和白细胞介素 (IL ) 10对单核细胞炎症效应和免疫效应功能的影响。结果显示 ,(1)M CSF诱导单核细胞表面CD14及CD2 3分子的表达 (P <0 0 5 )。IL 10抑制CD2 3的表达 ,促进CD6 4的表达 ,并协同M CSF诱导单核细胞CD14的表达 ,拮抗M CSF对CD2 3的诱导作用。M CSF能协同IL 10对单核细胞CD6 4的诱导作用 ;(2 )M CSF能诱导单核细胞表面CD11b及CD18的表达 (P <0 0 5 )。结论 :M CSF通过促进单核细胞表面CD11b、CD18、CD14、CD2 3的表达及协同促进CD6 4的表达而促进单核细胞粘附、炎性渗出、IgG及IgE依赖的细胞杀伤和吞噬功能 ,促进单核 巨噬细胞在炎症反应、体液免疫应答效应阶段发挥效应细胞功能。IL 10通过促进CD6 4的表达 ,增强体液免疫应答效应阶段单核 巨噬细胞的免疫效应功能 ,但通过抑制CD2 3的表达 ,下调IgE介导的体液免疫效应。     

垂直感染登革病毒对白纹伊蚊滞育卵孵化及F1代成蚊产卵量的影响

本文用低氧水刺激感染登革Ⅱ型病毒的白纹伊蚊滞育卵孵化,按时间顺序共收集孵化的7批幼虫,仅在最先孵化的两批幼虫中检测到病毒,而在此之后孵化的幼虫都没有检测到病毒,推测感染病毒的滞育卵在低氧水的刺激作用下比没有感染病毒的滞育卵更易解除滞育,使得先孵化的蚊虫检测的阳性率较高。感染滞育卵孵化的幼虫最低感染率为1∶131.25(0.76%),明显低于非滞育卵最低感染率1∶82.5(1.21%),即垂直感染登革病毒影响卵的孵化和低龄幼虫的成活。单管饲养法获得经感染滞育卵孵化的F1代单只雌蚊产卵量,被感染的白纹伊蚊平均单只雌蚊产卵量与未感染雌蚊没有显著性差异(P>0.05),即垂直感染病毒对F1代雌蚊产卵量无显著影响。     

M—CSF和IFN—γ基因转染的巨噬细胞的体外生物学特性

利用缺陷型腺病毒的介导将与巨噬细胞功能密切相关的细胞因子Ｍ－ＣＳＦ和ＩＮＦ－γ基因联全转入小鼠腹腔巨噬细胞，对其体外生物学特性进行了观察。     

几种细胞因子对登革病毒感染U937细胞的影响研究

本文作者研究了几种细胞因子（ｒＩＬ－１，ｒＩＬ－２，ｒＩＬ－６，ｒＴＮＦ－α，ｎＩＦＮ－α和ｎＩＦＮ－γ）对Ｄ２Ｖ感染Ｕ９３７细胞的影响，结果显示：除ｎＩＦＮ－γ对ＤＶ感染Ｕ９３７细胞的感染无影响外，其余几种细胞因子都可使Ｄ２Ｖ感染Ｕ９３７细胞的感染率明显降低，为研究细胞因子在ＤＶ感染中的作用作了初步探索。     

IL-6与登革病毒感染相互关系的实验研究

目的：研究ＩＬ－６对登革Ⅱ型病毒（Ｄ２Ｖ）复制的影响以及细胞感染Ｄ２Ｖ后ＩＬ－６产生能力的变化。方法：以人单核细胞为感染模型，用微量蚀斑法测定病毒滴度，用ＥＬＩＳＡ法测定ＩＬ－６含量。结果：重组ＩＬ－６能增强Ｄ２Ｖ的复制，Ｄ２Ｖ感染能引起人单核细胞产生ＩＬ－６，它们的水平与Ｄ２Ｖ感染量正相关。结论：ＩＬ－２在登革病毒感染中起重要的作用。     

登革病毒对人血管内皮细胞分泌ET-1及PGI2的影响

目的　研究登革病毒感染对人血管内皮细胞分泌重要的血管活性物质ＥＴ１ 及ＰＧＩ２ 的影响,以了解登革出血热及登革休克综合征（ＤＨＦＤＳＳ）的发病机制。方法　用登革病毒Ⅱ型,感染人脐静脉内皮细胞（ＨＵＶＥＣ） ,于感染后４ 、２４ 、４８ 、７２ 及９６ 小时,分别收集病毒感染上清液,用放射免疫检测法测定ＥＴ１ 及ＰＧＩ２ 的含量。结果　登革病毒感染可使ＨＵＶＥＣ分泌ＥＴ１ 及ＰＧＩ２ 的能力受到明显抑制。在病毒感染早期（４ 小时）,ＨＵＶＥＣ分泌ＥＴ１ 及ＰＧＩ２ 的能力即受到明显抑制。登革病毒对ＨＵＶＥＣ分泌ＥＴ１ 抑制作用强烈而持久,至感染后９６ 小时,ＨＵＶＥＣ分泌ＥＴ１ 的能力与未受感染的阴性对照组比较,差异仍有显著性。然而,登革病毒对ＨＵＶＥＣ 分泌ＰＧＩ２ 的抑制作用,可随时间的推移而减弱,至感染后９６ 小时,ＨＵＶＥＣ分泌ＰＧＩ２ 的能力已达正常水平。结论　登革病毒感染可影响血管内皮细胞分泌血管活性物质ＥＴ１ 及ＰＧＩ２ 的功能,导致血管通透性增加和凝血、止血功能障碍。因此,登革病毒所致的血管内皮细胞功能障碍,可能是ＤＨＦＤＳＳ重要的发病机制     

人IL-6、TNF-α对登革病毒感染人树突状细胞的影响

目的:探讨IL-6和TNF-α对登革病毒感染人树突状细胞(DC)的影响。方法:从人外周血中常规分离单核细胞,以GM-CSF和IL-4诱导成DC并进行形态学特征、细胞表型和淋巴细胞刺激能力的鉴定。用不同浓度的IL-6、TNF-α作用于Ⅱ型登革病毒(DV2)感染的DCs,于感染后6、24、48、72、96h收集上清,采用甲基纤维素微量病毒空斑法检测病毒的滴度,以MTT比色法测定IL-6和TNF-α对DV感染DC及正常DC数量的变化。结果:中、低浓度的IL-6对DC中DV2的增殖均有增强作用;高、中浓度的TNF-α对DC中DV2的增殖具有抑制作用。IL-6和TNF-α对DV感染DC及正常DC数量无明显影响。结论:IL-6和TNF-α通过对DC的影响在DV2感染中具有重要作用。     

重组登革病毒E蛋白结构域Ⅲ抑制登革病毒感染的初步研究

目的 了解原核表达的登革病毒(dengue virus, DV)的E蛋白结构域Ⅲ直接抑制登革病毒感染及其抗体的中和作用.方法 在大肠埃希菌中表达1~4型登革病毒E蛋白结构域Ⅲ(EⅢ).重组蛋白纯化后,进行阻断DV-2感染BHK~21细胞试验.用重组蛋白制备免疫血清,检测抗体中和作用.结果 在大肠埃希菌中成功表达了1-4型登革病毒E蛋白结构域埃希菌,4型重组E蛋白结构域Ⅲ均能够阻断2型DV感染,4型重组蛋白的免疫血清均能中和2型DV,但中和抗体效价不同.结论 原核表达的登革病毒结构域Ⅲ可以直接抑制病毒感染,所产生的抗体具有中和作用.直接抑制和中和抗体均对同型病毒作用较强.     

Depletion of macrophages in mice results in higher dengue virus titers and highlights the role of macrophages for virus control

Monocytes and macrophages are target cells for dengue infection. Besides their potential role for virus replication, activated monocytes/macrophages produce cytokines that may be critical for dengue pathology. To study the in vivo role of monocytes and macrophages for virus replication, we depleted monocytes and macrophages in IFN‐αβγR knockout mice with clodronate liposomes before dengue infection. Although less virus was first recovered in the draining LN in the absence of macrophages, monocyte/macrophage depletion eventually resulted in a ten‐fold higher systemic viral titer. A massive infiltration of CD11b⁺CD11c^lowLy6C^low monocytes into infected organs was observed in parallel with increasing virus titers before viremia was controlled. Depletion of monocytes in the blood before or after local infection had no impact on virus titers, suggesting that monocytes are not required as “virus‐shuttles”. Our data provide evidence that systemic viremia is established independently of tissue macrophages present at the site of infection and blood monocytes. Instead, we demonstrate the importance of monocytes/macrophages for the control of dengue virus.     

Role of monocytes and eosinophils in human respiratory syncytial virus infection in vitro

RSV infection in airway epithelial cells (EC) results in production of the chemokines RANTES and MIP1alpha and the leukocyte differentiation factor GM-CSF. The chemokines attract monocytes and eosinophils to the site of infection, where GM-CSF may influence their function and differentiation. In turn, these inflammatory cells may limit the progression of RSV infection, as well as initiate immune responses. In the present study, the effect of monocytes and eosinophils on viral replication and infection-dependent release of EC-derived cytokines was investigated. The modulation of immune cell costimulatory molecules, CD80, CD86, CD40, and HLA-DR, and the release of the CD4(+) T cell chemoattractant IL-16 were also investigated. Employing immunofluorescence techniques, monocytes and eosinophils in cocultures with infected EC were found to inhibit the spread of RSV to uninfected cells. Monocytes also had a significant effect on replication of RSV. Monocytes phagocytized the virus, while eosinophils inhibited reinfection mainly by extracellular means. The release of G-CSF and GM-CSF in the infected cultures was not significantly affected by either monocytes or eosinophils, while RANTES release was significantly decreased. The expression of CD40, CD80, CD86, and HLA-DR on monocytes, but not on eosinophils, increased in an RSV-dose-dependent manner. IL-16 release was not induced in RSV-infected EC, but was significantly increased in coculture with monocytes. These results suggest that both monocytes and eosinophils attracted to the site of RSV infection play an important role in confining infection, while RSV-exposed monocytes may be involved in promoting/polarizing immune responses to RSV.     

Galectin-9 plasma levels reflect adverse hematological and immunological features in acute dengue virus infection

BackgroundDengue virus (DENV) infection remains a major public health burden worldwide. Soluble mediators may play a critical role in the pathogenesis of acute DENV infection. Galectin-9 (Gal-9) is a soluble β-galactoside-binding lectin, with multiple immunoregulatory and inflammatory properties.ObjectiveTo investigate plasma Gal-9 levels as a biomarker for DENV infection.Study designWe enrolled 65 DENV infected patients during the 2010 epidemic in the Philippines and measured their plasma Gal-9 and cytokine/chemokine levels, DENV genotypes, and copy number during the critical and recovery phases of illness.ResultsDuring the critical phase, Gal-9 levels were significantly higher in DENV infected patients compared to healthy or those with non-dengue febrile illness. The highest Gal-9 levels were observed in dengue hemorrhagic fever (DHF) patients (DHF: 2464 pg/ml; dengue fever patients (DF): 1407 pg/ml; non-dengue febrile illness: 616 pg/ml; healthy: 196 pg/ml). In the recovery phase, Gal-9 levels significantly declined from peak levels in DF and DHF patients. Gal-9 levels tracked viral load, and were associated with multiple cytokines and chemokines (IL-1α, IL-8, IP-10, and VEGF), including monocyte frequencies and hematologic variables of coagulation. Further discriminant analyses showed that eotaxin, Gal-9, IFN-α2, and MCP-1 could detect 92% of DHF and 79.3% of DF, specifically (P < 0.01).ConclusionGal-9 appears to track DENV inflammatory responses, and therefore, it could serve as an important novel biomarker of acute DENV infection and disease severity.     

分泌粒细胞-巨噬细胞集落刺激因子的重组痘苗病毒转染的瘤苗裂解物抗肿瘤作用

将小鼠粒细胞－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）基因经过同源重组得到表达ＧＭ－ＣＳＦ的重组痘苗病毒，用此痘苗病毒转染小鼠黑色素瘤细胞，制备黑色素瘤瘤苗裂解物（ＧＭ－ＣＳＦＶＭＯ），Ｃ５７ＢＬ／６小鼠皮下接种Ｂ１６－Ｆ１０细胞３天后在注射部位注射瘤苗裂解物，一周后再注射一次。结果发现ＧＭ－ＣＳＦＶＭＯ能够显著地抑制荷瘤小鼠肿瘤结节的生长并明显延长荷瘤小鼠的存活期。用此瘤苗裂解物免疫小鼠两次，间隔一周，免疫一周后给Ｃ５７ＢＬ／６小鼠皮下接种Ｂ１６－Ｆ１０细胞，结果肿瘤结节出现时间明显延长，部分小鼠肿瘤不再生长。经ＧＭ－ＣＳＦＶＭＯ治疗或免疫后小鼠的外周血和脾淋巴细胞对肿瘤细胞杀伤活性明显升高，ＮＫ活性变化不明显。本结果提示，诱导机体特异性细胞免疫可能是瘤苗裂解物的抗肿瘤作用机理之一。