汉族人群微卫星DXS16遗传多态性

目的获得X染色体短串联重复序列DXS16遗传多态性在河南汉族人群中的分布状况。方法应用聚合酶链反应,变性聚丙烯酰胺凝胶电泳（polyacrylamide gel ectrophoresis,PAGE）及银染色技术对110名无血缘关系的汉族个体DXS16基因座进行分型。结果观察到DXS16位点有14个等位基因片段,该STR位点的基因型分布符合Hardy—Weinberg平衡;杂合度为0．88,多态信息含量为0．885。结论DXS16在汉族人群中有较好的多态性分布;实验数据对X染色体特异性STR的群体遗传学研究及一些X连锁遗传病的基因诊断有重要意义。     

Phenotypes of complement knockouts

Although complete and partial complement deficiencies are well described in humans and several spontaneous animal models, many questions have remained regarding the exact role that these deficiency states play in the observed clinical manifestations. Likewise, many important mechanistic questions cannot be addressed using patients or spontaneously arising animal models of deficiency states. To provide additional insights and create readily manipulable experimental systems, over the last 5 years mice have been created by several groups in which specifically targeted insertional mutagenesis has resulted in complete deficiencies of complement activation proteins, receptors or regulatory proteins. Many surprising findings have already been made using mice derived from these strategies, and clinically relevant studies have begun to provide great insights into human deficiency states. This review includes an overview of these complement deficient mice and highlights some of the important findings that have resulted from their creation. A discussion of future experimental directions thought to be important by this author then follows and concludes the review.     

Major roles of prostanoid receptors IP and EP(3) in endotoxin-induced enhancement of pain perception

To know the roles of prostaglandin I (IP) and prostaglandin E (EP) receptors in pain perception, we compared the acetic acid-induced writhing response in mice deficient in prostaglandin receptors, i.e. IP, EP(1,) EP(2,) EP(3,) or EP(4,) with or without lipopolysaccharide (LPS) pretreatment. Without LPS pretreatment, IP-receptor deficient mice showed a significantly smaller number of responses, as previously reported, whereas mice deficient in any of the EP-receptor subtypes showed a number of writhings similar to those of wild-type mice. When mice were pretreated with LPS for 24 hr to induce cyclooxygenase-2 expression, the wild-type as well as EP(1)-, EP(2)-, or EP(4)-receptor-deficient mice showed a similar enhanced writhing response, whereas IP- and EP(3)-receptor-deficient mice had a significantly less enhanced number of writhings. These results indicate that IP and EP(3) are the major prostaglandin receptors mediating the enhanced acetic acid-induced writhing response in mice pre-exposed to LPS, i.e. in endotoxin-enhanced inflammatory nociception.     

Accumulation of immature B and null lymphocytes in the periphery after intraperitoneal administration of traditional Chinese medicine, xiao-chai-hu-tang (Japanese name: shosaiko-to)

Accumulation of lymphocytes after an intraperitoneal (ip) injection of a traditional Chinese herb medicine, XIAO-CHAI-HU-TANG (Japanese name: shosaiko-to), was investigated. Shosaiko-to induced marked accumulation of lymphocytes rather than macrophages in the peritoneal cavity of ICR mice, whereas various kinds of irritants, e.g. proteose-pepton, Escherichia coli lipopolysaccharide (LPS), OK-432 and Corynebacterium parvum, induced preferential accumulation of macrophages rather than lymphocytes. By means of analysis using two-color fluorescence-activated cell sorter (FACS), it was revealed that the increased lymphocyte subpopulations not only in the peritoneal cavity but also in the spleen of C3H/He mice by the injection of shosaiko-to were comprised of both immature B (IgM+ and IgD-) and null (thyl- and Ig-) cells. This effect of shosaiko-to was observed in other C5 normal strains, C3H/HeJ (LPS-nonresponder), C57BL/6, BALB/c and athymic nu/nu (ICR background) mice, but not in C5 deficient strains, AKR/J, A/J and DBA/2 mice, indicating that the accumulation of immature B and null cells in the periphery induced by shosaiko-to is closely related to the complement system.     

Evaluation of iNOS-dependent and independent mechanisms of the microvascular permeability change induced by lipopolysaccharide

1. Subcutaneous injection of lipopolysaccharide (LPS) increases plasma leakage in mouse skin. Pretreatment with LPS conditions mice tolerant to the LPS-induced plasma leakage. Nitric oxide (NO) has been suggested to be involved in these LPS effects. A specific role of inducible NO synthase (iNOS) was investigated in the LPS-induced plasma leakage using iNOS deficient mice. 2. Plasma leakage in mouse skin was measured by the local accumulation of Pontamine sky blue at the site of subcutaneous injection of LPS (Sal. typhimurium). LPS (100 - 400 microg site(-1)) produced a dose-related increase in dye leakage in both iNOS deficient and wild-type mice with about 40% less dye leakage in iNOS deficient mice. 3. Indomethacin (5 mg kg(-1)), N-[-2-cyclohexyloxy]-4-nitrophenyl methanesulphonamide (NS-398) (1 mg kg(-1)), diphenhydramine (10 mg kg(-1)) and anti-TNF-alpha antibody (dilution 1 : 400, 10 ml kg(-1)) inhibited the LPS-induced dye leakage in both iNOS deficient and wild-type mice, whereas N(G)-nitro-L-arginine methyl ester (L-NAME) (10 mg kg(-1)) or aminoguanidine (10 mg kg(-1)) inhibited that in wild-type but not in iNOS deficient mice. 4. Pretreatment with LPS (0.15 mg kg(-1) i.p.) 4 h before decreased the LPS-induced dye leakage in wild-type but not in iNOS deficient mice. LPS pretreatment increased serum corticosterone levels in both mice, while it increased the serum nitrate/nitrite levels in wild-type but not in iNOS deficient mice. 5. These studies indicate that an increase in vascular permeability induced by LPS is mediated by NO produced by iNOS, eicosanoids, histamine and TNF-alpha. The tolerance against LPS-induced vascular permeability change may be mediated by iNOS induction but not by an increased release of endogenous corticosteroids.     

Sex-dependent compensated oxidative stress in the mouse liver upon deletion of catechol O-methyltransferase

Catechol-O-methyl transferase (COMT) methylates catechols, such as l-dopa and dopamine, and COMT deficient mice show dramatic shifts in the metabolite levels of catechols. Increase in catechol metabolite levels can, in principle, lead to oxidative stress but no indices of oxidative stress have been reported in COMT-knockout (KO) mice [Forsberg MM, Juvonen RO, Helisalmi P, Leppanen J, Gogos JA, Karayiorgou M, et al. Lack of increased oxidative stress in catechol-O-methyltransferase (COMT)-deficient mice. Naunyn Schmiedebergs Arch Pharmacol 2004;370:279-89.]. Here we perform a proteomic based analysis of the livers of COMT-KO mice in search for potential compensatory mechanisms developed to cope with the effects of disrupted catechol metabolism. We found sex specific changes in proteins connected to stress response. Our results show that alterations in protein levels contribute to the homeostatic regulation in the liver of COMT deficient mice.     

X染色体STR基因座在法医鉴定中的应用

目的通过探讨太原地区汉族人群位于X染色体上的短串联重复序列DXS6804的遗传多态性,分析X染色体STR基因座在法医鉴定中的利与弊,以促进X染色体STR基因座在法医学实践中的应用。方法随机抽取太原无血缘关系汉族个体静脉血500μL,采集50例两代家系血进行突变观察;采集同一例健康男性尸体的心脏、肝脏、肌肉组织等进行同一性测定。乙二胺四乙酸抗凝,酚-氯仿法提取DNA,聚合酶链反应扩增,8%非变性聚丙烯酰胺凝胶电泳分型。基因座的等位基因频率采用直接计算法,与其他人群比较采用χ2检验。结果共检出5种不同的等位基因,基因多样性为0.7024。与不同地区及不同民族的等位基因频率分布进行了比较均有明显的差异。同一尸体血液,器官组织检测结果分型一致。50例两代家系观察未见突变。结论DXS6804基因座在太原地区汉族人群中有较好的多态性,X染色体STR基因座在法医鉴定中具有重要价值。     

Loss of locomotor sensitisation in response to morphine in D1 receptor deficient mice

Mice lacking D1 receptors were used to study the role of these receptors in morphine-induced antinociception and locomotor sensitisation. In the hot-plate test D1 receptor deficient (-/-) and wild-type (+/+) mice showed similar reaction times under basal conditions. A single injection of 1.25 mg/kg and 2.5 mg/kg morphine resulted in a stronger antinociceptive response in D1 receptor deficient mice than in wild-type animals. Tolerance to the analgesic effect did not develop in both groups of animals when 12.5 mg/kg morphine was chronically applied twice daily for 13 days. There was no change in basal locomotor activity between saline-injected wild-type and D1 receptor deficient mice. After chronic treatment wild-type mice showed a continuous increase in locomotor activity, indicating the development of sensitisation. In contrast, a subchronic administration of morphine did not change locomotor activity in mutant mice. The lack of the development of locomotor sensitisation in D1 deficient mice was associated with reduced levels of immunoreactive mu opioid receptors in dorsal striatal patches as compared to wild-type mice. In contrast, no change in the distribution of immunoreactive mu receptors could be detected in areas related to pain pathways such as the spinal cord. Taken together, these results suggest an involvement of D1 receptors in morphine-induced locomotor activity and analgesia.     

Augmentation of allergic inflammation in prostanoid IP receptor deficient mice

1 To evaluate the role of prostaglandin I(2) (PGI(2)) in allergic inflammation, allergic responses in the airway, skin and T cells were studied in mice lacking the receptor for PGI(2) (the prostanoid IP receptor) through gene disruption. 2 Three inhalations of antigen caused an increase in plasma extravasation, leukocyte accumulation and cytokine (interleukin (IL)-4 and IL-5) production in the airway of sensitized mice. These airway inflammatory responses were significantly greater in IP receptor deficient mice than in wild-type mice. 3 The vascular leakage caused by passive cutaneous anaphylaxis, substance P and 5-hydroxytryptamine was markedly increased in the skin of IP receptor deficient mice, compared with comparably treated wild-type mice. 4 The inhalation of antigen in sensitized mice resulted in increased serum antigen specific IgE, total IgE and IgG levels. The magnitude of the elevations of each immunoglobulin level in IP receptor deficient mice is notably higher than that in wild-type mice. To elucidate the mechanism of an enhancement of immunoglobulin production, the activity of T cells in sensitized and non-sensitized mice was studied by means of the production of cytokines. The antigen-induced IL-4 production by spleen cells from sensitized IP receptor deficient mice was almost three times greater than that in wild-type mice. On the contrary, the anti-CD3 antibody-induced interferon-gamma production by CD4(+) T cells from non-sensitized IP receptor deficient mice was significantly lower than that in wild-type mice. 5 The present data indicate that IP receptor deficiency reinforced an allergic airway and skin inflammation by augmentation of vascular permeability increase and the T helper 2 cell function. These findings suggest a regulatory role of PGI(2) in allergic inflammation.     

Chemotactic activity of venom from the brown recluse spider (Loxoscelles reclusa).

The incubation of whole Loxosceles reclusa venom with complement generated a chemotactic activity for guinea pig neutrophils which had been harvested from peritoneal exudate cells. Venom-guinea pig complement mixtures were 42% more chemotactic than the complement alone and venom-human complement mixtures were 55% more chemotactic than human complement alone. Venom plus diluent was no more chemotactic than diluent alone, although venom plus heat inactivated guinea pig serum was slightly chemotactic. Purified guinea pig or human C3 or C5 incubated with recluse spider venom was not chemotactic nor did venom alter the serologically active quantity of C3 or C5 in human serum. Both C4 deficient and properdin deficient sera were suitable substrates for the chemotaxigen in recluse spider venom.     

Role of complement in endotoxin initiated lethality in mice.

We have examined the role of complement in eliciting a lethal response in C3H/HeJ and C3H/St mice. The results reported here indicate that endotoxin-initiated complement activation, leading to significant drops in circulating C3 levels, is not sufficient to cause lethality. The complement system in both strains was demonstrated to be responsive in vitro to activation both by E. coli 0111:B4 LPS I, an alternative pathway activator in other systems, as well as S. minnesota R595 LPS, which activates almost exclusively the classical pathway. In vivo injection of high (lethal) doses of E. coli 0111:B4 LPS I and S. minnesota R595 LPS causes a significant decrease in the circulating C3 levels of both strains after 4 hr. In contrast, circulating C3 levels were not significantly different from normal values in either strain following injection with minimal (lethal) amounts of E. coli 0111:B4 LPS II, a weakly anticomplementary LPS preparation. In all cases, lethality was observed in only the C3H/St mice, indicating that neither complement activation, nor the lack of it, is responsible for lethality in mice.     

Role of 5-lipoxygenase in myocardial ischemia-reperfusion injury in mice

Myocardial ischemia induces 5-lipoxygenase (LOX) translocation and leukotriene production in the heart. Leukotrienes increase inflammatory responses and could thereby aggravate ischemic injury. However, the role of lipoxygenase and leukotrienes in cardiac ischemia/reperfusion damage has not been well defined. Therefore, we tested the effect of ischemia reperfusion in mice with targeted deletion of 5-lipoxygenase, the enzyme converting arachidonic acid in leukotrienes. 5-LOX deficient (KO) and wild-type (WT) mice underwent 30 min of coronary artery ligation and 24 h of reperfusion in vivo. In mice with equivalent area at risk, infarct size was not significantly different between WT and KO mice (infarct/area at risk 61.7+/-3.9 vs. 55.8+/-6.6%, WT vs. KO, P=n.s.). However, neutrophil infiltration as well as tumor necrosis factor expression were increased in 5-lipoxygenase deficient mice. In summary, inhibition of 5-lipoxygenase does not affect cardiac ischemia-reperfusion injury but the post-ischemic inflammatory response.     

Effects of dextran sulphate on lymphoblast extravasation into inflammatory skin sites

The effects of high molecular weight dextran sulphate (DXS) on the migration of 125I-labelled deoxyuridine-labelled peripheral lymphoblasts (stimulated by oxazolone) were studied in vivo by injecting the drug (50 mg/kg) subcutaneously in recipients, and by following the fate of oxazolone-stimulated peripheral lymphocytes treated in vitro with DXS in non-treated syngeneic recipients. Both types of experiments demonstrate that DXS considerably reduces lymphoblast extravasation in skin sites inflamed either by non-immune causes or by DTH. Our results also demonstrate that oxazolone-stimulated peripheral lymphocytes, after in vitro treatment with DXS, are unable to transfer antigen-specific contact hypersensitivity to unsensitized recipients. The results obtained suggest that the drug acts on both T effector cells and lymphoblasts.     

Prevention of fentanyl-induced delayed pronociceptive effects in mice lacking the protein kinase Cgamma gene

It has recently been reported in several nociceptive models of rats that the antinociceptive effect of fentanyl, an opioid analgesic widely used in the management of per-operative pain, was followed by paradoxical delayed hyperalgesia dependent on N-methyl-D-aspartate (NMDA) mechanisms. Events upstream of the NMDA receptor, especially the activation of the protein kinase Cgamma (PKCgamma), have been involved in the persistence of pain states associated with central sensitisation. In order to evaluate the contribution of the PKCgamma in early and delayed fentanyl nociceptive responses, we studied these effects in knock-out mice deficient in such a protein. We found that fentanyl antinociception was followed by the spontaneous appearance of prolonged hyperalgesia in the paw pressure and formalin tests, and allodynia in the Von Frey paradigm. In PKCgamma deficient mice, an enhancement of the early fentanyl antinociceptive effects was observed, as well as a complete prevention of the fentanyl delayed hyperalgesic/allodynic effects. Finally, naloxone administration in mice that had recovered their pre-fentanyl nociceptive threshold, precipitated hyperalgesia/allodynia in wild-type but not in mutant mice. This study identifies the PKCgamma as a key element that links opioid receptor activation with the recruitment of opposite systems to opioid analgesia involved in a physiological compensatory pain enhancement.     

Protective function of complement against alcohol-induced rat liver damage

The complement system can promote tissue damage or play a homeostatic role in the clearance and disposal of damaged tissue. We assessed the role of the terminal complement pathway in alcohol-induced liver damage in complement C6 (C6-/-) genetically deficient rats. C6-/- and corresponding C6+/+ rats were continuously exposed to ethanol by feeding ethanol-supplemented liquid diet for six weeks. Liver samples were analyzed for histopathology and complement component deposition by immunofluorescence microscopy. Prostaglandin E receptors and cytokine mRNA levels were analyzed by RT-PCR and plasma cytokines by ELISA. Deposition of complement components C1, C3, C8 and C9 was observed in C6+/+ rats, but not in C6-/- animals. The histopathological changes, the liver weight increase and the elevation of the plasma pro-/anti-inflammatory TNF-alpha/IL-10 ratio were, on the other hand, more marked in C6-/- rats. Furthermore, ethanol enhanced the hepatic mRNA expression of the prostaglandin E receptors EP2R and EP4R exclusively in the C6-/- rats. Our results indicate that a deficient terminal complement pathway predisposes to tissue injury and promotes a pro-inflammatory cytokine response. This suggests that an intact complement system has a protective function in the development of alcoholic liver damage.     

Mast cell deficient W/W(v) mice lack stress-induced increase in serum IL-6 levels,as well as in peripheral CRH and vascular permeability,a model of rheumatoid arthritis

Corticotropin releasing hormone (CRH) and interleukin-6 (IL-6) are implicated in inflammatory diseases triggered by stress. Acute restraint stress increases serum IL-6 in the blood, but its source is not known. Our current study was carried out in order to determine the contribution of mast cells to stress-induced IL-6 release and to investigate skin CRH and vascular permeability in mice. W/W(v) mast cell deficient and their wild type control +/+ mice were stressed in a plexiglass restraint chamber for 60 or 120 min. Serum corticosterone and IL-6 levels were measured. Other mice were injected with (99)Tchnetium gluceptate ((99)Tc) and its extravastion, indicating vascular permeability, was determined along with CRH levels in the skin and knee joints. Acute stress increased serum IL-6 in mice, but was greatly inhibited in W/W(v) mast cell deficient mice. Vascular permeability to (99)Tc, as well as local CRH levels, were also increased by stress, but not in W/W(v) mice. Findings from our current study suggest a link between mast cells and stress-related skin and joint inflammation and may explain initial events in psoriatic and rheumatoid arthritis.     

Cannabinoid 2 (CB2) Receptor Involvement in the Down-regulation but not Up-regulation of Serum IgE Levels in Immunized Mice

Marijuana cannabinoids such as Δ(9)-tetrahydrocannabinol (THC) have been shown in experimental systems to bias T helper immunity towards Th2 and away from Th1. This effect if broadly applicable to humans could have important implications in Th2-mediated diseases such as allergy. In the current study, we examined the effect of cannabinoids on serum immunoglobulin IgE levels in immunized mice and also examined the role of cannabinoid receptors in the response. The method involved pre-injecting mice with cannabinoid receptor agonists and antagonists followed 18-24?h later with an immunizing injection with two different antigen/adjuvant combinations. This treatment was followed 2-3?weeks later with a booster injection of antigen and the subsequent bleeding of mice 1-2?weeks later for serum immunoglobulin analysis by ELISA. Our results showed that THC injection enhanced total IgE serum levels in response to antigen immunization even under conditions of deficient cannabinoid receptor 2 (CB2) and cannabinoid receptor 1 (CB1) activity and furthermore the increase in IgE was accompanied by a decrease in serum IgG2a. In addition, we observed that l-α-lysophosphatidyliniositol (LPI) increased serum IgE levels and that IgE levels were higher in CB2 deficient mice and suppressed by the CB2 agonist, Gp1a. These results suggest that in this IgE induction model in mice, non-selective cannabinoids such as THC increase IgE through receptors other than CB1 and CB2 but that CB2 receptors do play a suppressive role in the control of serum IgE levels.