CD71和Hoechst33258用于孕妇外周血中胎儿有核红细胞的分选

目的 比较ＣＤ７１单染和ＣＤ７１、Ｈｏｅｃｈｓｔ３３２５８（ＨＯ２５８）双染对单个核细胞的标记情况,并用于对孕妇外周血中有核红细胞（ｎｕｃｌｅａｔｅｄ ｒｅｄ ｂｌｏｏｄ ｃｅｌｌｓ,ＮＲＢＣｓ）的分选。方法 选用红细胞特异性抗体ＣＤ７１、核染料ＨＯ２５８对正常孕妇外周血、轻（或）中度妊娠高血压综合征（简称妊高症）患者外周血、初生婴儿脐血单个核细胞进行标记,并结合流式细胞术对有核红细胞进行分选。结     

Graves病患者外周血单个核细胞上催乳素受体mRNA的表达

目的:了解弥漫性毒性甲状腺肿(Graves disease,GD)患者外周血单个核细胞(peripheral blood mononuclear cells,PBMC)上催乳素受体(prolactin receptor,PRLR)mRNA表达及血清催乳素(prolactin,PRL)水平的变化。方法:选取新诊断或未治疗GD患者为病例组(GD组,19例),健康体检者为正常对照组(NC组,12例);测定入选者PBMC上PRLR mRNA表达及血清PRL水平。结果:1)GD组及NC组PBMC上均有PRLR表达,GD组患者PRLR表达量明显高于NC组(P<0.05);2)GD组患者血清PRL水平较NC组轻度增高,但差异无统计学意义(P>0.05)。结论:GD患者PBMC的PRLR mRNA表达增加,PRL可能参与了GD的自身免疫过程。     

超顺磁氧化铁标记对干细胞转铁蛋白受体和铁蛋白轻链基因及蛋白表达的影响

目的研究超顺磁氧化铁（SPIO）标记对大鼠脂肪干细胞（ADSCs）转铁蛋白受体（TfR）和铁蛋白轻链（Fn-L）基因及蛋白表达的影响。方法实验通过医院伦理委员会的批准。标记组（实验组）采用多聚赖氨酸（PLL,终质量浓度为1.5μg/mL）介导SPIO（终质量浓度为50μg/mL）标记ADSCs。在2、4、8、16、24、96、168、336、504、672 h,分别用实时荧光定量聚合酶链反应（RT-PCR）和WesternBlot实验定量检测实验组和未标记组（对照组）TfR和Fn-L基因及蛋白表达水平。结果 PLL-SPIO标记ADSCs后,实验组Fn-L mRNA（2、4、8、16、24、96、168、336 h）及蛋白（16、72、96、168 h）（P均〈0.05）表达水平会暂时性升高,至标记后一定时间,Fn-L mRNA（504、672h）及蛋白（336、504、672h）表达水平两组间差异无统计学意义（P均〉0.05）;实验组TfR mRNA（2、4、8、16、72、96、168、336 h）及蛋白（24、96 h）（P均〈0.05）表达水平会暂时性减低,至标记后一定时间,TfRmRNA（504、672 h）及蛋白（168、336、504、672 h）表达水平两组间差异无统计学意义（P均〉0.05）。结论在一定浓度内,PLL-SPIO标记ADSCs,对Fn-L和TfR基因及蛋白表达仅产生暂时性影响;从而为细胞内标记过程的安全性提供了实验依据。     

Expression of transferrin receptor and ferritin following ferumoxides-protamine sulfate labeling of cells: implications for cellular magnetic resonance imaging

Ferumoxides-protamine sulfate (FE-Pro) complexes are used for intracellular magnetic labeling of cells to non-invasively monitor cell trafficking by in vivo MRI. FE-Pro labeling is non-toxic to cells; however, the effects of FE-Pro labeling on cellular expression of transferrin receptor (TfR-1) and ferritin, proteins involved in iron transport and storage, has not been reported. FE-Pro-labeled human mesenchymal stem cells (MSCs), HeLa cells and primary macrophages were cultured from 1 week to 2 months and evaluated for TfR-1 and ferritin gene expression by RT-PCR and protein levels were determined using Western blots. MTT (proliferation assay) and reactive oxygen species (ROS) analysis were performed. FE-Pro labeling of HeLa and MSCs resulted in a transient decrease in TfR-1 mRNA and protein levels. In contrast, Fe-Pro labeling of primary macrophages resulted in an increase in TfR-1 mRNA but not in TfR-1 protein levels. Ferritin mRNA and protein levels increased transiently in labeled HeLa and macrophages but were sustained in MSCs. No changes in MTT and ROS analysis were noted. In conclusion, FE-Pro labeling elicited physiological changes of iron metabolism or storage, validating the safety of this procedure for cellular tracking by MRI.     

黑龙江省3866名健康儿童静脉血细胞参数参考范围调查

目的调查分析黑龙江省健康儿童静脉全血细胞8个参数的水平,建立各参数的儿童参考范围。方法 随机抽取黑龙江省部分地区8～16岁健康儿童3 866人(男性1 837人,女性2 029人),采集受试者清晨空腹静脉血,用Sysmex XT-1800i全血细胞分析仪进行检测。采用SPSS 15.0软件分析比较各参数在民族、地区及年龄间的差异。结果 黑龙江省健康儿童静脉血细胞参数均值(参考范围)中白细胞计数为6.9(3.9～9.9)×109/L,红细胞计数男性为5.0(4.2～5.8)×1012/L,女性为4.6(4.0～5.2)×1012/L,血红蛋白浓度男性为146(124～168)g/L,女性为135(117～153)g/L,血细胞比容男性为43.9%(37.5%～50.5%),女性为41.2%(36.4%～46.0%),红细胞平均体积为88(80～96)fl,红细胞平均血红蛋白含量为29(25～33)pg,红细胞平均血红蛋白浓度为329(311～347)g/L,血小板计数为277(161～393)×109/L。汉族、朝鲜族健康儿童的WBC、HGB、MCH、MCHC及PLT5项参数存在显著差异(P<0.05);黑...     

Overexpression of glycolysis markers in placental tissue of pregnant women with chronic venous disease: a histological study

Chronic Venous Disease (CVD) refers to a wide variety of venous disorders being the varicose veins its most common manifestation. It is well-established the link between pregnancy and the risk of suffering CVD, due to hormonal or haematological factors, especially during the third trimester. In the same manner, previous studies have demonstrated the detrimental effect of this condition in the placental tissue of pregnant women, including in the normal physiology and the metabolomic profile of this organ. In this context, the aim of this study was to evaluate the glucose homeostasis in the placental tissue of women presenting CVD. Through immunohistochemistry, we studied the protein expression of the glucose transporter 1 (GLUT-1), Phosphoglycerate kinase 1 (PGK1), aldolase (ALD), Glyceraldehyde-3-phosphate dehydrogenase (GA3PDH) and lactate dehydrogenase (LDH). Our results have reported a significative increase in the expression of GLUT-1, PGK1, ALD, GA3PDH and the isoenzyme LDHA in placentas of women with CVD. This work has proven for the first-time an altered glucose metabolism in the placental tissue of women affected by CVD, what may aid to understand the pathophysiological mechanisms of this condition in more distant organs such as placenta. Furthermore, our research also supports the basis for further studies in the metabolic phenotyping of the human placenta due to CVD, which may be considered during the late pregnancy in these women.     

Two separable T cell receptor signals reconstitute positive selection of CD4 lineage T cells in vivo

Positive selection is an obligatory step during intrathymic T cell differentiation. It is associated with rescue of short-lived, self major histocompatibility complex (MHC)-restricted thymocytes from programmed cell death, CD4/CD8 T cell lineage commitment, and induction of lineage-specific differentiation programs. T cell receptor (TCR) signaling during positive selection can be closely mimicked by targeting TCR on immature thymocytes to cortical epithelial cells in situ via hybrid antibodies. We show that selection of CD4 T cell lineage cells in mice deficient for MHC class I and MHC class II expression can be reconstituted in vivo by two separable T cell receptor signaling steps, whereas a single TCR signal leads only to induction of short-lived CD4⁺CD8^la intermediates. These intermediates remain susceptible to a second TCR signal for 12-48 h providing an estimate for the duration of positive selection in situ. While both TCR signals induce differentiation steps, only the second one confers long-term survival on immature thymocytes. In further support of the two-step model of positive selection we provide evidence that CD4 T cell lineage cells rescued by a single hybrid antibody pulse in MHC class II-deficient mice are pre-selected by MHC class 1.     

Quantification of all fetal nucleated cells in maternal blood between the 18th and 22nd weeks of pregnancy using molecular cytogenetic techniques

Different types of nucleated fetal cells (trophoblasts, erythroblasts, lymphocytes, and granulocytes) have been recovered in maternal peripheral blood. In spite of many attempts to estimate the number of fetal cells in maternal circulation, there is still much controversy concerning this aspect. The numbers obtained vary widely, ranging from 1 nucleated cell per 104 to 1 per 109 nucleated maternal cells. The purpose of our project was to determine the absolute number of all different types of male fetal nucleated cells per unit volume of peripheral maternal blood. Peripheral blood samples were obtained from 12 normal pregnant women known to carry a male fetus between 18 and 22 weeks of pregnancy. Three milliliters (3 ml) of maternal blood has been processed without any enrichment procedures. Fluorescence in situ hybridization (FISH) and primed in situ labeling (PRINS) were performed, and fetal XY cells were identified (among maternal XX cells) and scored by fluorescent microscopy screening. The total number of male fetal nucleated cells per milliliter of maternal blood was consistent in each woman studied and varied from 2 to 6 cells per milliliter within the group of normal pregnancies. The number of fetal cells in maternal blood, at a given period, is reproducible and can therefore be assessed by cytogenetic methods. This confirms the possibility of developing a non-invasive prenatal diagnosis test for aneuploidies. Furthermore, we demonstrate that it is possible to repeatedly identify an extremely small number of fetal cells among millions of maternal cells.     

Appearance of large scavenger receptor A-positive cells and increased small scavenger receptor A positive cells in peripheral blood is associated with mortality in systemic inflammatory response syndrome and multiple organ dysfunction syndrome

We investigated the association of scavenger receptor A-positive (SRA(+) ) cells in peripheral blood (PB) with mortality in subjects with systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS). A total of 467 subjects with SIRS (62 of 467 satisfied the diagnostic criteria of MODS) were prospectively examined. The subjects were classified into three groups according to the SRA index (number of small SRA(+) cells in 10 high power field, normal upper limit < 30) and the appearance of large SRA(+) cells as follows: group A, large SRA(+) cells were not detected; group B, large SRA(+) cells were detected but SRA index did not exceed 30; group C, the two factors (appearance of large SRA(+) cells and SRA index > 30) coincided. The duration from the diagnosis of SIRS to death in groups A and B was significantly shorter than in group C. The mortality rate in group C was significantly higher than in groups A and B. Kaplan-Meier curves of group C showed significantly worse survival than groups A and B. These results indicate that the coincidence of two factors (appearance of large SRA(+) cells and SRA index > 30) may be useful to predict the outcome in patients with SIRS or MODS.     

HBV阳性孕妇DC—SIGN／DC—SIGNR基因多态性与宫内感染关系的研究

目的分析HBV阳性孕妇DC—SIGN与DC—SIGNR的表达水平及其编码基因的多态性与HBV宫内感染相关性。方法应用PCR技术、琼脂糖凝胶电泳检测HBV阳性孕妇DC—SIGN与DC—SIGNR基因的颈区重复序列分型,然后用卡方检验分析DC—SIGN与DC—SIGNR基因各亚型出现的频率在两组中的差异。结果①29例宫内感染组DC—SIGN基因型全部为7／7型的纯合子基因,54例非宫内感染组中出现2例7／5型的基因,其他52例均为7／7型的基因,两组之间差异无统计学意义（P=0．54）。②29例宫内感染组发现4种DC—SIGNR基因型,分别为7／7型、7／5型、9／7型、6／5型,基因型频率分别为0．3793、0．3448、0．2414、0．0345。54例非宫内感染组发现6种基因型,为7／7型、7／5型、9／5型、9／7型、7／6型、6／5型,基因型频率分别为0．5186、0．1481、0．0926、0．1852、0．0370、O．0185。7／5型基因型在宫内感染组（29例）和非宫内感染组（54例）中的分布的差异有统计学意义（P=0．038）,其他基因型两组之间差异无统计学意义（P〉0．05）。结论①DC—SIGN编码基因存在个体基因变异,但变异相对较少,在HBV阳性产妇宫内感染组和非宫内感染组中DC—SIGN基因型分布无明显差异。②DC—SIGNR编码基因存在个体基因变异,变异相对较大,DC—SIGNR基因型“7／5型”在HBV阳性产妇宫内感染组和非宫内感染组中分布差异有统计学意义（P=0．038）,可能是影响宫内感染的易感基因。     

Study on nonspecific immunity in pregnant women: II. Effect of hormones on chemiluminescence response of peripheral blood phagocytes.

To analyze the mechanisms of increased nonspecific immunity in pregnant women, the effect of various hormones on the phagocytic activity was estimated by a luminol-dependent chemiluminescence (CL) response during phagocytosing opsonized zymosan. The CL response of whole blood supplemented with exogenous human chorionic gonadotropin (hCG) increased significantly in all the male and female subjects and pregnant women. An approximate two- to fourfold increase was observed in comparison with the unsupplemented control in each subject at concentrations ranging from 1 to 1,000 IU/ml after 48 h of incubation (P less than 0.05). Progesterone slightly stimulated the CL response in female subjects only, but had no effect on male and pregnant women. Estradiol (E2) did not stimulate the CL response in any subject. The expression of Fc and C3b receptors on the surface of polymorphonuclear leucocytes (PMNL) in pregnant women was also investigated by measuring the immunofluorescence stained with monoclonal antibody to Fc and C3b receptors, respectively. The relative numbers of Fc receptors increased significantly in the third trimester compared to those of female control (P less than 0.05). Those of C3b receptor also increased in the second and third trimester (P less than 0.005). These results suggested that the nonspecific immunity represented by phagocytic activity in pregnant women increased with both oxidative metabolic responsiveness and the expression of membrane receptors. Besides, the increased phagocytic activity of the maternal host is probably due to the stimulatory effect of both endogenous and exogenous hCG on their peripheral blood phagocytes.     

A requirement for native (undenatured) IgG for detection of species specificity of the IgG-Fc receptor on lymphocytes

The species specificity of the Fc receptor involved in binding of preformed antibody-antigen complexes was examined using complexes of deaggregated 7S antibodies and albumin antigens. Complexes prepared with mouse antibodies bound to 3–4 times as many cells as complexes prepared with rabbit, guinea pig, or goat antibodies. Using lymphocytes from each species, it was shown that homologous complexes consistently labeled a higher frequency of cells than heterologous complexes. The binding of heterologous complexes seemed to be generally restricted to monocytic populations which were also capable of binding monomeric 7S immunoglobulins. It is concluded that the lymphocytic Fc receptor for antigen-complexed 7S immunoglobulin displays strict species specificity. Heat aggregation of the heterologoud immunoglobulins eliminated the species restriction on their binding to the lymphocytic Fc receptor. It is therefore suggested that artificially aggregated immunoglobulins may not be reliable probes for the specificity of the Fc receptors.     

Frequency and cell specificity of T-cell receptor interlocus recombination in human cells

Immunoglobulin and T-cell receptor (TCR) genes are assembled by a site-specific rearrangement known as V(D)J [variable-(diversity)-joining] recombination. These rearrangements occur normally in pre-B- and pre-T-cells using signal sequences adjacent to coding exons for immunoglobulin and TCR genes, respectively. However, aberrant recombination may result in the generation of hybrid TCR genes by joining of TCR-β with TCR-γ specific sequences. Such hybrid TCR genes occur at a low frequency in peripheral blood lymphocytes (PBL) of healthy individuals, and can be detected by PCR amplification. We have determined the in vivo frequency of hybrid Vγ-Jβ1 TCR (hybrid TCR) genes in lymphocyte DNA from 12 healthy individuals. The average frequency was found to be 5.83 in 0.75 × 10⁶ PBL, with a threefold difference between the highest and lowest individual value. The presence of similar TCR gene rearrangements in individual samples suggests that T-cells with a hybrid TCR gene are capable of clonal expansion in vivo. The individual hybrid TCR gene frequency remained relatively constant during 72 hours of in vitro cultivation. In long-term culture, the frequency gradually decreased, and after 28 days no hybrid TCR genes were detectable in lymphocyte DNA. These results show that T-cells with a hybrid TCR gene are able to respond to mitogen stimulation in vitro, and may have a proliferative disadvantage or are selected against during prolonged in vitro cultivation. No hybrid TCR genes were detected in ten proliferating T-cell clones, indicating that the rate of hybrid TCR gene formation is <2.0 × 10⁻⁸ per cell per cell division. No hybrid TCR genes were detected in DNA from B-lymphocytes, sperm, granulocytes, fibroblasts, keratinocytes, and three B-lymphoblastoid ataxia telangiectasia cell lines. In agreement with previous reports, the frequency of hybrid TCR genes in peripheral blood DNA from two ataxia telangiectasia patients was found to be more than 15-fold higher than in lymphocytes from normal individuals. These data show that formation of hybrid TCR genes is restricted to T-cells in vivo, and occurs at a very low frequency, if at all, in proliferating T-cells in vitro, and with an increased frequency in patients with ataxia telangiectasia. Environ. Mol. Mutagen. 30:245–253, 1997 © 1997 Wiley-Liss, Inc.     

723例O型血孕妇产前IgG抗-A（B）效价对新生儿红细胞致敏的研究

目的研究O型血孕妇产前IgG抗-A（B）血型抗体的效价对新生儿红细胞致敏情况,以预防新生儿溶血病的发生。方法采用抗人球蛋白法检测723例丈夫为非O型的O型血孕妇IgG抗-A或抗-B的ABO血型抗体效价,分娩时取脐静脉血做红细胞四项试验：血型、直接抗人球蛋白试验、游离抗体检测、释放试验。结果 723份血清中IgG抗-A（B）效价≥1∶64者有168例,异常检出率为23.24%;丈夫为A型、B型、AB型者,IgG抗-A（B）异常检出率分别为26.01%、20.13%、23.53%。当孕妇IgG抗-A（B）效价≤32时,新生儿血清学检验结果均为阴性,而效价为64、128、256、≥512新生儿脐血血清学检验结果阳性率分别为7.69%、64.29%、94.12%、100.00%。结论孕妇IgG抗-A（B）效价与新生儿红细胞致敏呈正相关,是产前预报母子血型不合而引起HDN的有效方法。可及早发现,及时治疗,减少由于母婴血型不合引起的溶血病发生。     

Inhibitory effect of pentobarbital anesthesia on venous stasis induced arteriolar vasoconstriction in the dog hindleg

The effect of close intrarterial administration of pentobarbital in the concentration of about 2times10^-4 mol/1 on the venous stasis induced arteriolar constriction in the dog hindleg was studied in 6 neurolept anesthetized dogs. It was found that the blood flows and vascular resistances in the legs before pentobarbital infusion were equal and the vasoconstrictor responses to venous stasis were the same. Pentobarbital infusion into the femoral artery in one of the legs increased the total leg blood flow compared to the control leg and abolished the increase in vascular resistance during venous stasis. In another experimental series the effect of general pentobarbital anesthesia on the vasoconstrictor activity in response to venous stasis locally in subcutaneous and muscle tissue in the hind limb was examined in 6 dogs. It was found that during the first 2–3 h of anesthesia the vasoconstrictor response was present in both tissues although the response in muscle tissue exhibited a great variation between the dogs during this period. However, after 4–5 h of anesthesia the response was abolished in both tissues. During neurolept anesthesia with fentanyl/N₂O the same vasoconstrictor response was demonstrated in the hindleg 1 h and 5 h after induction of the anesthesia. It is concluded that pentobarbital anesthesia abolishes the arteriolar constriction induced by venous stasis. The mechanism may be blockade of the local sympathetic vasoconstrictor fibres or interference with myogenic vasoconstrictor mechanism of the vascular smooth muscle cells or both. It is suggested that fentanyl/N₂O anesthesia is better suited for this kind of studies.     

免疫学因素对重症肌无力患者外周血单个核细胞糖皮质激素受体的影响

目的　探讨重症肌无力 (MG)患者外周血单个核细胞 (PBMC)糖皮质激素受体 (GR)数改变的可能机制。方法　 13例MG患者及 11例正常对照PBMC在体外与乙酰胆碱受体 (AChR)、IFN γ或AChR +IFN γAb孵育前后同时测定PBMCGR数及GRmRNA含量。3H 地塞米松 (3H Dex)放射配体法测定GR数 ,狭缝印迹杂交法测定GRmRNA量 ,以 β actincDNA为内对照。同时 ,ELISA法测定患者血浆中的IFN γ含量及AChRAb水平。结果　正常对照PBMC分别在AChR、IFN γ、AChR +IFN γAb中培养后其GR数虽有降低 ,但未达到统计学意义 (P >0 .0 5 )。MG患者PBMC的GR数在AChR及IFN γ中培养后明显降低 (与培养前相比 ,前者P <0 .0 5 ,后者P <0 .0 1) ,而在AChR +IFN γAb中培养后则无明显改变 (P >0 .0 5 )。正常对照PBMC分别在AChR、IFN γ、AChR +IFN γAb中培养后其GRmRNA无显著降低 (P >0 .0 5 )。MG患者PBMC的GRmRNA在AChR及IFN γ中培养后与培养前相比明显降低 (前者P <0 .0 5 ,后者P <0 .0 1) ,而在AChR +IFN γAb中培养后则无明显改变 (P >0 .0 5 )。MG患者的血浆中IFN γ及AChRAb水平均显著高于正常对照 (前者P <0 .0 1;后者P <0 .0 5 )。MG患者的IFN γ含量与AChRAb水平间具有良好的正相关关系 (r=0 .92 ,P <0 .0 1)。MG患者的PBMC在体     

The role of natural antibodies and ABO (H) blood groups in transplantation of human lymphoid cells into mice

Recently, evidence was presented that natural antibodies (NAb) are a crucial barrier to human cellular engraftment in severely immunosuppressed normal mice (Eur. J. Immunol. 1992.22:197.). In this report we show that normal mouse serum contains low titers of NAb against human cells of blood groups type O (H) and B and high titers against human cells of blood group A. Accordingly, human peripheral blood leukocytes (PBL) of group O (H) and B donors could be grafted successfully into normal BCBA mice (H-2^h/k) following irradiation with high dose total body irradiation (TBI). PBL of blood group type A donors did not engraft in normal mice but could be transplanted without difficulty in B cell-deficient CBA/N mice which lack NAb, after conditioning with high dose TBI. Treatment of lethally irradiated normal BCBA mice with cobra venom factor (COF), which eliminates the third factor of complement, and liposomes containing dichloromethylene diphosphonate (C1₂DMP), which eliminates macrophages, resulted in engraftment of human blood group type A PBL. This implies that the NAb barrier for discordant xenogeneic cell transplantation can be abrogated. A method utilizing directly labeled probes and flow cytometry is described for the quantitation in mouse serum of NAb, reacting with human cells. Using sera of H-2^b/k mice we show that murine NAb react with human stem cells, granulocytes, lymphocytes and monocytes of blood group A and only weakly with similiar cells from blood group O (H) and B donors. Sera of H-2^b, H-2^d and H-2^k mice of different ages and microflora possess NAb against human erythrocytes of blood group type A and occasionally demonstrate weak titers against erythrocytes of blood groups B and O (H) and the Rhesus factor.     

Expression profiles of 10 circadian clock genes in human peripheral blood mononuclear cells

The circadian clock system regulates daily rhythms of physiology and behavior. The mammalian master clock in the suprachiasmatic nuclei orchestrates these biological rhythms in peripheral tissues. Since blood is the most accessible tissue source, we sought to dissect the human circadian clock system by characterizing clock gene expression in human peripheral blood mononuclear cells (PBMCs) isolated from eight young, healthy subjects. By evaluating the temporal expression profiles of 10 circadian clock genes, we found that Period 1 (Per1), Per2, and Per3 are rhythmically expressed in human blood samples. Our results suggest that evaluating the rhythmic expression of human Per genes could reveal an individual's circadian phenotype.