IgG受体FcγRⅡB调节实验性自身免疫性脑脊髓炎致神经元损伤及Th17/Treg免疫平衡的作用研究

目的:探究IgG受体FcγRⅡB对实验性自身免疫性脑脊髓炎(EAE)模型小鼠神经元损伤与Th17/Treg失衡的作用。方法:C57BL/6小鼠随机分组为对照组、EAE组、FcγRⅡB组、EAE+FcγRⅡB组,每组15只,皮下注射MOG35-55肽诱导EAE模型,并给予FcγRⅡB慢病毒液处理;造模后每日称量小鼠体质量,进行神经功能评分,持续30 d;30 d后处死小鼠,HE染色观察脑组织病理形态学变化,LFB染色评估脊髓髓鞘结构变化,免疫荧光染色检测脊髓大脑皮质神经元核抗原(NeuN)和Caspase-3表达,TUNEL染色检测神经元细胞凋亡,ELISA检测血清IL-6、IL-17、IL-10及TGF-β水平,流式细胞术分析脾脏Th17、Treg细胞比例分布,Western blot测定脊髓组织维甲酸相关孤儿受体γt(RORγt)与Forkhead家族转录因子3(Foxp3)蛋白表达。结果:与对照组比较,EAE组小鼠体质量下降,神经功能评分升高,脑组织内炎症细胞浸润明显,脊髓中出现脱髓鞘迹象,NeuN荧光表达强度减弱而Caspase-3荧光表达强度增强,TUNEL阳性着色细胞多,细胞凋亡数增加,血清中IL-6和IL-17水平升高,IL-10和TGF-β水平降低,脾脏内Th17细胞比例升高,Treg比例降低,脊髓组织内RORγt蛋白表达上调,Foxp3蛋白表达下调(P<0.05);与EAE组比较,EAE+FcγRⅡB组小鼠体质量增加,神经功能评分降低,脑组织内炎症细胞浸润减轻,脊髓脱髓鞘现象得到改善,NeuN荧光表达强度增强,Caspase-3荧光表达强度减弱,TUNEL阳性着色细胞较少,细胞凋亡数减少,血清中IL-6和IL-17水平降低,而IL-10和TGF-β水平升高,同时脾脏内Th17细胞比例降低,Treg比例升高,脊髓组织内RORγt蛋白表达下调而Foxp3蛋白表达上调(P<0.05)。结论:FcγRⅡB对EAE小鼠具有神经保护作用,可减轻其脑组织炎症细胞浸润及脱髓鞘现象,作用机制可能与调节细胞因子水平及Th17/Treg细胞免疫平衡有关。

抑制IgE与肥大细胞FcεRⅠ受体结合防治变应性疾病的研究进展

变应性疾病是全世界影响最广泛的慢性疾病之一,肥大细胞是Ⅰ型变态反应最主要的终末效应细胞,IgE与其肥大细胞表面高亲和力受体FcεRⅠ的结合是启动肥大细胞级联反应释放炎性介质的关键步骤。阻断或抑制肥大细胞膜上IgEFcεRⅠ的交联,从而影响肥大细胞活化已成为变应性疾病防治的有效策略和研究热点。文章从阻断IgE抗体、IgE-FcεRⅠ结合以及FcεRⅠ受体三个方面就靶向肥大细胞上IgE和FcεRⅠ防治变应性疾病的策略及相关机制进行综述。

过敏煎通过IgE/FcεRⅠ途径抑制肥大细胞脱颗粒改善特应性皮炎的作用机制

目的通过体内外实验,研究过敏煎改善特应性皮炎的作用机制。方法采用2,4-二硝基氯苯(2,4-dimethyl sulfoxide,DNCB)反复刺激皮肤建立特应性皮炎(Atopic dermatitis,AD)模型,给予氯雷他定片及过敏煎低、中、高剂量(1.32、2.64、5.28 g/kg)进行干预,ELISA法检测血清总IgE、IL-4、IL-13水平,IHC法测定皮损组织中IL-4、IL-13的表达水平。体外运用anti-DNP-IgE/DNP-HSA诱导RBL-2H3细胞复制脱颗粒模型,MTS法检测过敏煎对RBL-2H3活力,通过ELISA法检测细胞上清中β-hex酶释放率,Histamine、LTB4、IL-4、IL-13水平,RT-PCR法检测细胞Histamine、IL-4、IL-13mRNA表达,Western blotting法检测p-ERK/ERK、p-JNK/JNK、p-p38/p-38蛋白表达。结果与模型组比较,过敏煎各剂量组显著减轻AD小鼠血清总IgE、IL-4、IL-13水平(P<0.05,P<0.01),减轻AD小鼠皮损组织中IL-4、IL-13表达。明显下调RBL-2H3细胞脱颗粒后β-Hex酶释放率(P<0.05,P<0.01)和Histamine、LTB4、IL-4、IL-13水平(P<0.05,P<0.01),显著减少细胞Histamine、IL-4、IL-13mRNA表达(P<0.01)和p-ERK/ERK、p-JNK/JNK、p-p38/p38蛋白表达(P<0.05,P<0.01)。结论过敏煎通过调控Th2型反应和血清中总IgE水平改善特应性皮炎,其作用机制可能是通过IgE/FcεRⅠ途径抑制肥大细胞脱颗粒。

IL23R single nucleotide polymorphisms could be either beneficial or harmful in ulcerative colitis

AIM To investigate the association of seven single nucleotide polymorphisms(SNPs) of the IL23 R gene with the clinical picture of ulcerative colitis(UC). METHODS Genomic DNA samples of 131 patients (66 males, 65 females, mean age 55.4 ± 15.8 years) with Caucasian origin, diagnosed with UC were investigated. The diagnosis of UC was based on the established clinical, endoscopic, radiological, and histopathological guidelines. DNA was extracted from peripheral blood leukocytes by routine salting out method. Polymerase chain reaction and restriction fragment length polymorphism were used to identify the alleles of seven SNPs of IL23 R gene(rs11209026, rs10889677, rs1004819, rs2201841, rs7517847, rs10489629, rs7530511).RESULTS Four out of seven analyzed SNPs had statistically significant influence on the clinical picture of UC. Two SNPs were associated with greater colonic extension(rs2201841 P = 0.0084; rs10489629 P = 0.0405). For two of the SNPs, there was more frequently need for operations (rs2201841 P = 0.0348, OR = 8.0; rs10889677 P = 0.0347, OR = 8.0). The rs2201841 showed to be a risk factor for the development of iron deficiency (P = 0.0388, OR = 6.1837). For patients with the rs10889677, a therapy with azathioprine was more frequently necessary(P = 0.0116, OR = 6.1707). Patients with rs10489629 SNP had a lower risk for weight loss(P = 0.0169, OR = 0.3394). Carriers of the heterozygous variant had a higher risk for an extended disease (P = 0.0284). The rs7517847 showed a protective character leading to mild bowel movements. Three SNPs demonstrated no statistically significant influence on any examined clinical features of UC.CONCLUSION We demonstrated susceptible or protective character of the investigated IL23 R SNPs on the phenotype of UC, confirming the genetic association.

参芪蛭龙汤对膜性肾病模型小鼠肾组织中FcγRⅠ、FcγRⅡB、FcγRⅢA表达的影响

目的 探讨参芪蛭龙汤对膜性肾病(MN)模型小鼠肾组织中FcγRⅠ、FcγRⅡB、FcγRⅢA表达的影响。方法 60只SPF级ICR小鼠,6周龄,体重(23±4)g,雌雄各半,按照随机数字表法分为空白组(10只)和造模组(50只)。造模组小鼠注射阳离子化牛血清白蛋白,复制MN小鼠模型。取其中造模成功的40只小鼠按照随机数字表法分为模型组,参芪蛭龙汤高、低剂量组(24、12 g/kg)和雷公藤多苷片组(14 mg/kg),每组10只。给药4周后,处死小鼠。HE、Masson染色观察肾脏组织病理变化;免疫荧光法观察肾组织中FcγRⅠ、FcγRⅡB、FcγRⅢA蛋白荧光强度;Western blot检测肾脏组织内FcγRⅠ、FcγRⅡB、FcγRⅢA蛋白表达;RT-PCR检测肾脏组织中FcγRⅠ、FcγRⅡB、FcγRⅢA mRNA表达。结果 HE染色显示空白组小鼠上皮细胞、基底膜、肾小管及肾间质形态结构均未见明显异常。模型组小鼠肾小球体积增大,肾小球毛细血管袢扩张,血细胞溢出,肾小球系膜增厚,肾小囊腔增大,局部肾小囊腔壁上皮严重破损。与模型组比较,参芪蛭龙汤高剂量组小鼠可见肾小球体积减小,基底膜增厚减轻,肾小囊壁上皮未破损或局部破损。Masson染色显示空白组小鼠肾小球基底膜正常。模型组小鼠上皮下大量嗜复红蛋白沉积,可见基底膜基质反应,肾小管空泡样脂肪变性,肾间质淋巴大量增生。与模型组比较,各给药组小鼠病变减轻,以参芪蛭龙汤高剂量组改善更加明显。与空白组比较,模型组FcγRⅠ、FcγRⅢA蛋白荧光增强,FcγRⅡB蛋白荧光减弱(P<0.05);与模型组比较,参芪蛭龙汤高剂量组FcγRⅠ、FcγRⅢA蛋白荧光减弱(P<0.05),参芪蛭龙汤高、低剂量组FcγRⅡB蛋白荧光增强(P<0.05);与参芪蛭龙汤低剂量组比较,参芪蛭龙汤高剂量组FcγRⅠ、FcγRⅢA蛋白荧光减弱,FcγRⅡB蛋白荧光增强(P<0.05)。与空白组比较,模型组FcγRⅠ、FcγRⅢA蛋白表达升高,FcγRⅡB蛋白表达降低(P<0.05);与模型组比较,参芪蛭龙汤高剂量组FcγRⅠ、FcγRⅢA蛋白表达降低,参芪蛭龙汤高、低剂量组FcγRⅡB蛋白表达升高(P<0.05);与参芪蛭龙汤低剂量组比较,参芪蛭龙汤高剂量组FcγRⅡB蛋白表达升高,FcγRⅢA蛋白表达降低(P<0.05)。与空白组比较,模型组FcγRⅠ、FcγRⅢA mRNA表达升高,FcγRⅡB mRNA表达降低(P<0.05);与模型组比较,参芪蛭龙汤高、低剂量组FcγRⅠ、FcγRⅢAmRNA表达降低,FcγRⅡB mRNA表达升高(P<0.05);与参芪蛭龙汤低剂量组比较,参芪蛭龙汤高剂量组FcγRⅠ、FcγRⅢA mRNA表达降低,FcγRⅡB mRNA表达升高(P<0.05)。结论 参芪蛭龙汤对MN小鼠的治疗作用与改善肾组织病理改变,减少FcγRⅠ、FcγRⅢA蛋白的表达,增加FcγRⅡB蛋白表达有关,其中高浓度的参芪蛭龙汤效果更好。

Lethal-7-related polymorphisms are associated with susceptibility to and prognosis of gastric cancer

BACKGROUND The lethal-7(let-7)family members and their targets are involved in the development and progression of tumors.Let-7-related polymorphisms have been reported to be associated with tumorigenesis and prognosis.In gastric cancer,however,the related studies are limited.AIM To investigate the role of let-7-related microRNA polymorphisms in the tumorigenesis and prognosis of gastric cancer in a Chinese population.METHODS A total of 898 gastric cancer patients and 992 tumor-free controls were recruited into this study from 2008 to 2013.Gastric cancer patients were followed periodically.Ten single nucleotide polymorphisms(SNPs)in the let-7 gene region or their target mRNAs were genotyped using the MassARRAY system and their associations with the risk for or overall survival of gastric cancer were analyzed.RESULTS All the ten SNPs were in Hardy-Weinberg equilibrium.The C allele of the rs3811463 polymorphism in the 3’-untranslated region(UTR)of LIN28A was associated with a lower risk of gastric cancer[odds ratio(OR)=0.74,95%confidence interval(CI):0.61-0.88,P=0.001]after adjustment for age and Helicobacter pylori status.Seven hundred and thirty-five gastric cancer patients who had undergone radical tumorectomy were included in the survival analysis and their 5-year survival rate was 53.9%(95%CI:50.1%-57.6%).The rs10889677 in the 3’-UTR of IL23R was corresponded to the prognosis of gastric cancer in a dose-response manner,in which the death risk increased by 25%[hazard ratio(HR)=1.25,95%CI:1.04-1.45,P=0.011]with each increase in the number of C alleles after controlling for other potential clinicopathological parameters.CONCLUSION The let-7-related polymorphism rs3811463 in LIN28A is associated with the susceptibility to gastric cancer and the let-7-related polymorphism rs10889677 in IL23R is associated with the prognosis of gastric cancer.

NOD2/CARD15 , ATG16L1 and IL23R gene polymorphisms and childhood-onset of Crohn’s disease

AIM: To assess whether the polymorphisms of NOD2/ CARD15 , autophagy-related 16-like 1 (ATG16L1 ), and interleukin-23 receptor (IL23R ) genes play a more critical role in the susceptibility of childhood-onset than in adult-onset Crohn’s disease (CD). METHODS: Polymorphisms R702W, G908R, and 3020insC of NOD2/CARD15 ; rs2241880 A/G of ATG16L1 , and rs11209026 (R381Q) of IL23R gene were assessed in 110 childhood-onset CD, 364 adult-onset CD, and 539 healthy individuals. Analysis of polymorphisms R702W, G908R, and 3020insC of NOD2/CARD15 genotyping was performed by allele specific polymerase chain reaction (PCR) or by PCR-restriction fragment length polymor-phism analysis. The polymorphisms rs2241880 A/G of the ATG16L1 , and rs11209026 (R381Q) of the IL23R gene in the children’s cohort were genotyped by PCR and melting curve analysis whereas adult group genotyping was performed using the Affymetrix Genome-Wide Human SNP Array 5.0 (500K). RESULTS: The 3020insC allele in NOD2/CARD15 was significantly higher in childhood than in adult-onset CD (P = 0.0067). Association with at least 1 NOD2/CARD15 variant was specific for ileal disease (with or without co- lonic involvement). Even if the frequency of G allele of the rs2241880 ATG16L1 polymorphism was increased in both paediatric and adult CD patients compared to con- trols (P = 0.017 and P = 0.001, respectively), no difference was observed between the childhood and the adult cohort. The rare Q allele of IL23R rs11209026 polymorphism was underrepresented in both paediatric and adult CD cases (P = 0.0018 and P = 0.04, respectively) and no difference was observed between the childhood and the adult cohort. The presence of the rs2241880 ATG16L1 and rs11209026 IL23R polymorphisms did not influence disease phenotype. CONCLUSION: Polymorphism 3020insC in NOD2/ CARD15 occurs statistically significantly more often in patients with childhood-onset CD than in patients with adult-onset CD. The ATG16L1 and IL23R variants are associated with susceptibility to CD, but not earlyonset disease.

NOD2,IL23R and ATG16L1 polymorphisms in Lithuanian patients with inflammatory bowel disease

AIM:To investigate the frequency of NOD2, IL23R and ATG16L1 genetic variants in a case-control panel for inflammatory bowel disease (IBD) from Lithuania.METHODS: One hundred and eighty unrelated IBD pa- tients [57 Crohn's disease (CD) and 123 ulcerative colitis (UC)] and 186 healthy controls were genotyped for the following known genetic susceptibility variants:NOD2-Arg702Trp (rs2066844), Gly908Arg (rs2066845) and Leu1007insC (rs2066847), as well as IL23R-Arg381Gln (rs11209026) and ATG16L1-Thr300Ala (rs2241880).RESULTS:The effect that carriership of at least one NOD2 risk allele predisposes to CD was replicated in the Lithuanian population (41.1% CD vs 16.9% controls, P=2×10-4, OR=3.48,95% CI:1.81-6.72). In the allelic single marker analysis, Leu1007insC was strongly associated with CD (21.4% CD vs 4.7% controls, P=3.687×10-8, OR=5.54, 95% CI:2.85-10.75). Neither the other two NOD2 variants, nor the known variants in IL23R and ATG16L1 were found to be risk factors for CD, UC or IBD. However, our relatively small study population was underpowered to demonstrate such weak to moderate disease associations.CONCLUSION: The results support a strong association between CD susceptibility and the Leu1007insC variant in NOD2 in the Lithuanian study population.

Polymorphisms in CYP2R1 Gene Associated with Serum Vitamin D Levels and Status in a Chinese Rural Population

Vitamin D, a fat-soluble vitamin and endocrine horm one, and it impacts various bone and extra-bone health, such as osteoporosis, diabetes, and cancer. The main circulating form of vitamin D is 25-hydroxyvitamin D [25(OH)D] and it is a useful clinical biomarker of vitamin D status. The Institute of Medicine (IOM) defines as vitamin D deficiency (VDD) when serum 25(OH)D concentration is less than 20 ng/mL⑴.Worldwide, VDD is recognized as a severe public health problem. In 2007, Holick estimated that globally over one billion people suffered from VDD or vitamin D insufficiency (VDI). In China, it has bee n reported that the prevale nee of VDD ranged from 38.8% to 91.2% in different regions.

Relationship between R219K polymorphism of adenosine triphosphate-binding cassette transporter 1 gene and cerebral infarction: A case-controlled analysis

BACKGROUND： Studies have shown that adenosine triphosphate-binding cassette transporter 1 （ABCA1） gene influences atherosclerosis. Studies have also demonstrated that cerebral infarction does not occur often in pre-menopausal women. It has been, therefore, assumed that sex plays a role in R219K polymorphism of ABCA1 gene and cerebral infarction. OBJECTIVE： To explore the relationship between lipid metabolism-correlated R219K polymorphism of ABCA1 gene, risk factors of cerebral infarction and lipid level, and to determine whether there were significant differences in gender between R219K polymorphism of ABCA1 gene and cerebral infarction. DESIGN, TIME AND SETTING： A multicentral and non-randomized, controlled study based on gene polymorphism was performed at the Chinese National Human Genome Center, and lipid concentrations were measured at Beijing Xuanwu Hospital. Patients with cerebral infarction and healthy subjects were enrolled from eight hospitals of six provinces of China between October 2002 and December 2004. PARTICIPANTS： There were 177 patients in the cerebral infarction group, including 119 males and 58 females, with a mean age of （60 -＋ 13） years, and 234 healthy subjects in the normal control group, including 79 males and 155 females, with a mean age of （58 ± 12） years. METHODS： R219K polymorphism of the ABCA1 gene was detected using polymerase chain reaction-restriction fragment length polymorphism, and blood lipid concentrations were simultaneously measured. MAIN OUTCOME MEASURES： Genotype and allele frequency of R219K polymorphic site, and blood lipid concentrations. RESULTS： RR genotype and R allele frequency of males in the cerebral infarction were significantly greater than males in the normal control group [RR genotype： x2 = 5.305, OR （95% CO, 2.326 （1.120 4.828）, P〈 0.05; R allele： x2= 4.219, OR （95% CO, 1.528 （1.019 2.292）, P〈 0.05]. In addition, RR genotype and R allele frequency of males were significantly greater than females in the cerebral infarction group [RR genotype： x2= 5.172, OR （95% C/）, 2.604 （1.120-6.057）, P〈 0.05; R allele： x2= 4.818, OR （95% CO, 1.652 （1.053 2.589）, P〈 0.05]. There were no significant differences between genotype and lipid concentrations between the two groups （P〉 0.05）. CONCLUSION： The RR genotype of ABCA1 R219K might be associated with onset of cerebral infarction in males, but blood lipid concentrations do not relate to R219K polymorphism.

猪Fc_γRⅡb亚型(Fc_γRⅡb1)基因的分子克隆及鉴定

IgG Fc受体(FcγRs)的一个主要效应机制是可结合IgG免疫复合物发生反应。目前已鉴定出的FcγRs有4类:高亲和力的FcγRⅠ(CD64),中等亲和力的FcγRⅠ、FcγRⅡ(CD32),以及低亲和力的FcγRⅢ(CD16)。人FcγRⅡ主要有FcγRⅡa、FcγRⅡb、FcγRⅡc 3个亚型;小鼠有1个亚型FcγRⅡb;牛有2个亚型FcγRⅡb、FcγRⅡc。人、小鼠、牛中FcγRⅡb又包括2个亚型:FcγRⅡb1(b1)和FcγRⅡb2(b2),但有关猪FcγRⅡb亚型的研究鲜有报道。本研究对猪FcγRⅡb亚型(FcγRⅡb1)进行了分子克隆、测序及鉴定,并运用RT-PCR从猪外周血白细胞RNA中扩增出猪FcγRⅡb1cDNA序列。结果发现,猪FcγRⅡb1cDNA序列包含一个951bp的开放阅读框(ORF),可编码316个氨基酸跨膜糖蛋白,包括两个免疫球蛋白(Ig)样胞外结构域,一个跨膜区域,一个带有免疫受体酪氨酸抑制基序(ITIM)的胞质尾区。猪FcγRⅡb1cDNA序列与DQ026064同源性为98.3%,其在胞质尾区有一19个氨基酸的插入框。荧光免疫试验结果发现,猪FcγRⅡb1cDNA序列编码的糖蛋白可稳定转染COS-7细胞,结合并激活IgG免疫复合物。猪FcγRⅡb1序列的鉴定为进一步了解猪免疫反应中IgG-FcγR相互作用的分子基础提供参考。

Polymorphisms of interleukin-1R receptor antagonist genes in patients with chronic hepatitis B in Iran

AIM： To investigate the relationships between polymorphisms of interleukin-1R receptor antagonist genes and susceptibility to chronic hepatitis B in Iran population. METHODS： Genomic DNA was extracted from the peripheral blood of 80 patients with chronic hepatitis B （57 males, 23 females） aged 12-77 years （mean 36.1 ± 13.8 years） and 147 normal controls （96 males, 51 females） aged 6-75 years （mean 41 ± 18.7 years） who referred to a liver clinic of Tehran and then subjected to polymerase chain reaction （PCR） amplification. PCR products were resolved on a 3% agarose gel and stained with ethidium bromide. RESULTS： Only three of the five kinds of polymorphism （2/2, 2/4, and 4/4） were found in this study. The frequencies of 2/2, 2/4, and 4/4 were 12.5%, 17.5%, 70% respectively in chronic hepatitis B patients and 6.8%, 24.5%, and 68.7% respectively in controls. IL-1 R allele 2 was detected in 30% of chronic hepatitis B patients and in 31.3% of controls, while IL-1 R allele 4 was detected in 87.5% of chronic hepatitis B patients and in 93.2% of controls. The frequency of IL-1R alleles 2 and 4 was detected in 21.25% and 78.75% of the patients and 19.04% and 80.96% of the controls, respectively. CONCLUSION： Our results suggest that the carriage of IL-1R receptor antagonist alleles 2, 4, 6 may not play any role in the development of HBV infection. Large population-based studies are needed to investigate the role of IL-1 polymorphisms in the pathogenesis of developing chronic hepatitis B.

Relationship between IL-23R rs11209026 Gene Polymorphism and Susceptibility to Acute Coronary Syndrome

Background: Several studies illustrated that IL-23/Th17 axis could be an important pro-inflammatory pathway in atherosclerosis. As a key element in this inflammatory mechanism, interleukin-23 receptor (IL-23R) may play a critical role in the pathological process of atherosclerosis. Single nucleotide polymorphisms (SNPs) in IL-23R have recently been consistently found to be associated with atherosclerosis diseases. However, its association with acute coronary syndrome (ACS) is still indistinct. Here, we discussed whether genetic polymorphisms in IL-23R (rs11209026 G/A) were associated with susceptibility to ACS. Methods: Among 160 patients with ACS, it includes 80 patients with unstable angina pectoris (UAP), 80 patients with myocardial infarction (MI), and 80 control subjects were selected randomly. The polymorphisms of IL-23R (rs11209026 G/A) were analyzed by the Sanger method. Results: Data showed that percentages of rs11209026 AG genotypes were significantly lower in ACS group (including: UAP and MI) than in controls (odds ratio [OR] = 0.324, 95% confidence interval [CI]: 0.148 - 0.712, p = 0.005;OR = 0.351, 95% CI: 0.135 - 0.910, p = 0.031;OR = 0.303, 95% CI: 0.112 - 0.817, p = 0.018, respectively). Furthermore, there was no correlation between rs11209026 G/A SNP and dyslipidemia-associated ACS. Conclusions: The variant of the rs11209026 polymorphism in IL-23R gene might decrease the risk of ACS, and these data suggest that AG genotype of the rs11209026 G/A polymorphism may act as a protective factor for acute coronary syndrome and subtype of ACS.

Interferon Regulatory Factor 5 and Renin-Angiotensin-Aldosterone System Polymorphisms in Coronary Artery Disease: An Overview of Experimental and Clinical Studies

Heart diseases are the main cause of mortality in Mexico, being coronary heart disease the most frequent in the country. Its high prevalence makes important the study of the pathophysiology and the search for prognostic factors. Different genes and polymorphisms promote atherogenesis and coronary artery disease, they affect inflammatory and vascular pathological processes. Interferon regulatory factor 5 (IRF5) is associated with coronary heart disease, it promotes chronic inflammation and cytokines release;it could trigger immune reactions and its activating receptors express in the vascular endothelium. Besides, polymorphisms in the renin-angiotensin-aldosterone system (RAAS) are implied with coronary disease, they are found in angiotensinogen (AGT), angiotensin II type 1 receptor (AT1R), angiotensin II type 2 receptor (AT2R), and angiotensin-converting enzyme (ACE) genes. These genetic polymorphisms are associated with a prothrombotic state, endothelial dysfunction, and immune activation. Multiple experimental studies showed that chronic activation of RAAS and chronic expression of IRF5 generates an environment prone to the development of atherosclerosis, and autoimmune and cardiovascular diseases. Studying these specific genes and their relationship with coronary heart disease will allow a better understanding of the pathological process and possibly the quest for new treatments.

猪肺巨噬细胞FcγR Ⅲ受体基因的克隆与序列分析

为研究猪肺巨噬细胞FcγRⅢ的生物学功能,本研究应用RT-PCR技术从猪肺巨噬细胞总RNA中克隆出猪FcγRⅢ的cDNA序列,并对其进行了分析。结果表明,克隆到的序列长820 bp,包含有1个771 bp完整开放阅读框(ORF),与Gen-Bank中登录的猪FcγRⅢ序列(AF237453)的核苷酸同源性为99.9%;与人、牛、马、绵羊、猕猴、狗、猫、小鼠氨基酸同源性分别为61.6%、62.9%、55.3%、62.2%、63.0%、59.0%、61.8%和53.2%;蛋白质分子结构预测结果表明,该分子由信号肽(20个氨基酸)、胞外区(185个氨基酸)、跨膜区(23个氨基酸)和胞内区(28个氨基酸)组成,在胞外区存在2个Ig样结构域。猪肺巨噬细胞FcγRⅢ基因的成功克隆,为进一步研究其结构与功能奠定基础。

PRRSV感染PAM中猪FcγRⅢ介导的免疫抑制反应

目的：为研究猪FcγRⅢ介导猪繁殖与呼吸综合征病毒（ Porcine reproductive and respiratory syndrome virus ， PRRSV）的免疫抑制反应。方法：本研究将含有200个TCID50的PRRSV、脂多糖（Lipopolysaccharide，LPS）（100 ng/ml）和纯化鼠抗猪FcγRⅢIgG（550μg/ml）分别处理猪肺泡巨噬细胞（ Pulmonary alveolar macrophages cell ，PAM），同时用纯化鼠抗猪FcγRⅢIgG处理PAM细胞后接种PRRSV，并设PAM细胞对照组。各组分别培养12、24、36、48、60、72 h后收集细胞及上清，用已经建立的绝对荧光定量PCR方法检测接毒组不同时间段的PRRSV复制水平，并用相对荧光定量PCR方法检测各组PAM细胞中IFN-α、TNF-α的mRNA转录水平。结果：PRRSV在感染PAM细胞后12～24 h期间可促进IFN-αmRNA转录水平，36～72 h期间抑制IFN-αmRNA转录水平，之后IFN-α的mRNA转录水平恢复正常；TNF-αmRNA转录水平在感染后12～72 h均略微上调。 LPS处理PAM细胞后，IFN-α、TNF-αmRNA转录水平均上调。用纯化鼠抗猪FcγRⅢIgG处理猪PAM细胞后，选择性激活猪PAM细胞表面FcγRⅢ，IFN-α、TNF-αmRNA转录水平显著下调，用PRRSV感染选择性激活猪PAM细胞表面FcγRⅢ后显著抑制抗病毒因子IFN-α、TNF-αmRNA转录水平。结论：FcγRⅢ的选择性激活抑制了宿主细胞的抗病毒因子水平及在PRRSV感染过程中的天然抗病毒免疫反应。

牛IgG2Fc受体(boFcγ2R)胞外区基因原核表达载体的构建及其表达

目的:构建牛Fcγ2R胞外区基因的原核表达载体,并在E.coliBL21(DE3)中表达。方法:提取健康成年牛的白细胞总RNA,经RT-PCR扩增牛Fcγ2R胞外区基因片段,并将其克隆到载体pGEM-TEasy,经限制性内切酶EcoRI和NotI双酶切鉴定及序列测定后,再将其亚克隆入原核表达载体pET28a中,构建重组质粒pET-2R,转化E.coliBL21(DE3)经IPTG诱导表达组氨酸融合蛋白。结果:获得682bp的编码牛Fcγ2R胞外区基因片段。以构建的重组质粒pET-2R转化E.coliBL21(DE3)后,经IPTG诱导,表达出相对分子质量(Mr)约为30000的重组蛋白。SDS-PAGE分析显示,表达的蛋白主要以不溶性包涵体的形式存在于E.coliBL21(DE3)的胞质中,Western blot检测表明该蛋白能和牛IgG2结合。结论:成功地构建了原核表达载体pET-2R,并表达出重组蛋白。为研究牛IgG和受体的相互作用机制及其介导的免疫反应打下了基础。

FcγR基因在成人牙周炎中的分布

目的 探讨FcγR 基因多态性与成人牙周炎(AP) 易感性的关系。方法 提取60 例AP 和36 例健康对照者的静脉血白细胞DNA,分别用PCR+ BstUI酶切和PCR+ 测序检测FcγRIIA 和FcγRIIIB 的基因型,比较各基因型检出率的差别。结果 FcγRIIA 和FcγRIIIB 基因型在重度AP 吸烟者与不吸烟者间的分布无差别,在不吸烟的重度、轻中度AP 和健康组三组间的分布也无差别,FcγRIIA H131 、R131 等位基因频率和FcγRIIIB NA1 、NA2的分布亦无差别。结论 本研究提示中国人中FcγRIIA 和FcγRIIIB 基因型与AP 的遗传易感性无关。

FcγRⅡB_1介导的信号传导异常与SLE患者B细胞的过度活化

目的:观察系统性红斑狼疮(SLE)患者B细胞表面功能分子表达的特征及其功能状态,评价以FcγRⅡB1(CD32)为代表的B细胞自身抑制调节机制在SLE发病中的作用。方法:采用Ficoll密度梯度离心法分离出人外周血单个核细胞(PBMC),并以免疫磁珠法(MACS)分离纯化B细胞。采用荧光分光光度法检测B细胞受不同激活物刺激后细胞内钙([Ca2+]i)的反应。用ELISA法检测B细胞与刺激物共同培养后所分泌IgG的量。采用流式细胞术及间接免疫荧光染色法,检测B细胞膜表面CD32、CD19及IgM的表达水平。结果:(1)以羊抗人μ链的F(ab′)2片段及完整IgG分别刺激SLE患者B细胞时,其[Ca2+]i反应的比值显著低于类风湿性关节炎(RA)患者(P<0.05)及正常人对照(P<0.01)。(2)分别用葡萄球菌A蛋白(SPA)单独刺激与SPA和羊抗人μ链的完整IgG抗体共同刺激SLE患者的B细胞所分泌的IgG的比值,明显低于RA患者及正常人对照组(P<0.05)。(3)SLE患者与RA患者及正常对照组B细胞上CD19、CD32及IgM的表达无统计学意义(P>0.05)。结论:SLE患者B细胞上CD32抑制性信号传导的异常,可能是导致B细胞过度活化的重要机制。