系统性红斑狼疮患者外周血单个核细胞Toll样受体9的表达及血清白介素-10水平的研究

目的:研究Toll样受体9(TLR-9)在系统性红斑狼疮(SLE)患者外周血单个核细胞(PBMCs)上的表达水平及SLE患者血清白介素-10水平,探讨发病机制。方法:从23例活动期、19例缓解期SLE患者和20例正常对照组中分离PBMCs,利用反转录-聚合酶链反应(RT-PCR)法检测PBMCs中TLR9 mRNA的表达水平,利用酶联免疫吸附试验法检测其血清白介素-10水平。结果:活动期SLE患者PBMCs的TLR-9mRNA表达高于缓解组(P<0.01)及正常对照(P<0.01),缓解期和正常对照组相比,差异无统计学意义(P>0.05)。SLE活动期患者血清IL-10水平显著高于缓解期患者(P<0.01),并均高于正常对照组(P<0.01)。结论:活动期SLE患者PBMC的TLR9 mRNA的表达水平增高;并且活动期及缓解期SLE患者血清IL-10水平升高可能与TLR9 mRNA表达的上调相关。     

趋化因子CCL-18 在慢性鼻- 鼻窦炎中的表达及意义

目的:分析趋化因子CCL-18在不同组织病理特征慢性鼻-鼻窦炎和正常鼻黏膜的表达差异,探讨CCL-18在慢性鼻-鼻窦炎中的表达及意义。方法:采用苏木精-伊红染色(HE),Masson染色及过碘酸-雪夫(PAS)染色对慢性鼻-鼻窦炎组织进行病理分析。采用Western blot检测CCL-18蛋白水平在不同组织病理特征慢性鼻-鼻窦炎和正常鼻黏膜组织中的表达差异。结果:CCL-18蛋白水平在伴鼻息肉和不伴有鼻息肉慢性鼻窦炎均较正常鼻黏膜组织中显著上调(P<0.05)。CCL-18蛋白水平在嗜酸性粒细胞慢性鼻窦炎的表达水平明显高于非嗜酸性粒细胞慢性鼻窦炎(P<0.05)。腺体型,纤维炎症型及水肿型慢性鼻-鼻窦炎中CCL-18表达水平均高于正常鼻黏膜,以水肿型表达最为显著(P<0.05)。结论:CCL-18在嗜酸性粒细胞和水肿型慢性鼻窦炎中高度表达,提示CCL-18可能参与慢性鼻-鼻窦炎中嗜酸性粒细胞的浸润这一基本病理过程。     

TIM4在过敏性哮喘患者单核细胞和哮喘小鼠模型肺组织表达增高

目的探讨T细胞免疫球蛋白粘蛋白域4(T cell immunoglobulin domain and mucindomain 4,TIM4)在过敏性哮喘中的表达及与疾病之间的关系。方法随机选取了30例过敏性哮喘患者和30例健康对照的全血标本,流式细胞术检测了TIM4在单核细胞的表达水平,库尔特计数法检测了嗜酸性粒细胞、单核细胞、淋巴细胞占白细胞的比例。免疫组织化学法检测了哮喘小鼠模型和对照小鼠肺组织TIM4的表达水平。结果哮喘患者组TIM4阳性细胞占外周血单核细胞的2.51%,显著高于健康对照组的0.68%;哮喘患者组的嗜酸性粒细胞比例(3.88%)显著高于健康对照组(1.23%);但TIM4表达水平与嗜酸性粒细胞,单核细胞与淋巴细胞之间未见相关性。哮喘小鼠模型气道上皮细胞TIM4的表达水平高于小鼠对照组。结论TIM4在过敏哮喘中的表达水平升高,可能参与了过敏性哮喘的免疫失衡机制。     

外周血单个核细胞TNF-α 基因表达与哮喘相关表型*

目的:探讨外周血单个核细胞(PBMC)TNF-α基因mRNA表达与哮喘严重程度、临床病理及相关影响因素的关系。方法:采用实时荧光定量PCR技术检测67例哮喘病人和25例健康对照PBMC中TNF-α mRNA表达水平,分析其与哮喘控制程度、血浆TNF-α浓度、嗜酸性粒细胞百分比(EOS%)、血浆总IgE浓度和哮喘相关影响因素的关系。结果:经方差分析和SNK-q检验,哮喘未控制组PBMC中TNF-α mRNA表达水平高于正常组(P<0.01)、控制组(P<0.01)和部分控制组(P<0.05)。相关分析显示哮喘病人TNF-α mRNA表达与血浆TNF-α浓度和EOS%呈正相关,相关系数分别为r=0.584(P<0.01)和r=0.29(P<0.05),有吸烟史的哮喘病人TNF-α mRNA表达水平高于非吸烟病人(P<0.05)。结论:哮喘病人PBMC中TNF-α mRNA表达水平与哮喘的控制程度呈负相关,与血浆TNF-α浓度呈正相关,PBMC中TNF-α mRNA表达水平与血浆TNF-α浓度可作为哮喘控制程度的参考指标。     

白细胞介素-33在慢性鼻-鼻窦炎伴鼻息肉患者黏膜组织中的表达及意义

目的观察慢性鼻-鼻窦炎伴鼻息肉(CRSwNP)患者鼻黏膜组织中白细胞介素-33(IL-33)的表达与嗜酸性粒细胞(Eos)的浸润情况,探讨IL-33与Eos在其发病中作用及相关性。方法采用免疫组织化学LSAB法分别检测CRSwNP患者(观察组)32例和单纯鼻中隔偏曲患者(对照组)24例的鼻黏膜组织中IL-33的表达情况;苏木精-伊红染色下观察炎性细胞浸润程度并对Eos计数;全血细胞分析法测定两组患者血液中嗜酸性粒细胞含量。结果 (1)CRSwNP组织中上皮层及间质内Eos、中心粒细胞为主的炎性细胞及部分腺体内有IL-33的表达;(2)免疫组织化学检测显示IL-33阳性细胞数、苏木精-伊红染色Eos计数均高于对照组,差异具有统计学意义(P0.05)。Eos计数与IL-33阳性细胞数在CRSwNP中存在正相关性(P0.05);(3)血液中Eos含量无统计学意义。结论 IL-33是一种新型的细胞因子,在CRSwNP患者体内高表达,可能与嗜酸性粒细胞浸润互相促进,在CRSwNP发生发展中具有重要的作用。     

内源性逆转录病毒长末端重复序列在嗜酸性粒细胞增多症的基因表达及核苷酸序列分析

探索人内源性逆转录病毒长末端重复序列(LTR)基因及表达与嗜酸性粒细胞增多症发生的关系。PCR法检测嗜酸性粒细胞增多症患者外周血中内源性逆转录病毒长末端重复序列基因,RT-PCR法检测内源性逆转录病毒基因表达。变性高效液相分析和序列测定LTR片段核苷酸序列,对不同株基因序列作同源性的比较分析。PCR结果显示:20例嗜酸性粒细胞增多症患者细胞中均获得内源性逆转录病毒长末端重复序列扩增产物,嗜酸性粒细胞增多症组中长末端重复序列基因有高的表达,而正常人表达为阴性。与HERV-K家族LTR基因相应区域核苷酸序列比较;嗜酸性粒细胞增多组长末端重复序列U3、R、U5区同源性分析有核苷酸的改变,与淋巴瘤对照比较没有大片段的缺失。人类基因组中普遍存在逆转录病毒长末端重复序列。正常人和嗜酸性粒细胞增多症患者中长末端重复序列有不同程度核苷酸碱基的变异,但是,二者比较,这种改变与嗜酸性粒细胞的增多没有明显的相关性。在嗜酸性粒细胞增多症患者中有高的基因表达而正常人中没有可检出的病毒基因的表达,嗜酸粒细胞的增多可能与逆转录病毒基因表达水平有关,其诱导嗜酸粒细胞增多的机制需进一步的研究。     

社区获得性肺炎痰菌群特征与嗜酸性粒细胞水平的相关性

目的探究社区获得性肺炎(CAP)患者痰液菌群特征及其与嗜酸性粒细胞水平的相关性。方法对2018年9月-2018年11月间于我院就诊的63例患者(CAP组)和63例同时期、同年龄段体检者(对照组)的痰液菌群进行16S rDNA检测,比较两组患者痰液菌群特征、痰液和血液嗜酸性粒细胞百分比(Eos%)水平差异,应用Pearson模型和多因素线性回归模型探究患者痰液菌群与嗜酸性粒细胞水平的相关性。结果 CAP组患者的外周血和痰液的Eos%水平明显低于对照组(t=9.486,6.436,Ps0.001)。两组的Chao1和ACE指数差异无统计学意义(Ps0.05),CAP组的Shannon指数明显低于对照组,Simpson指数显著高于对照组,差异有统计学意义(Ps0.05)。16S rDNA分析结果显示,两组患者的痰液菌群分布存在明显差异。CAP组中,肺炎链球菌、金黄色葡萄球菌、流感嗜血杆菌、肺炎克雷伯菌、铜绿假单胞菌、大肠杆菌、嗜肺军团菌为主要优势菌(LDA4log10),而对照组中优势菌为链球菌属、厌氧菌、奈瑟菌属和韦荣球菌属等。Pearson相关分析显示,肺炎链球菌、金黄色葡萄球菌、流感嗜血杆菌、肺炎克雷伯菌、铜绿假单胞菌、大肠杆菌和嗜肺军团菌等病原菌相对丰度与外周血和痰液的Eos%水平均呈负相关(Ps0.05)。多因素线性回归分析显示,肺炎链球菌水平与痰液和血液Eos%水平呈明显的独立负相关(β=-0.633,-0.525,P=0.000)。结论 CAP患者痰液菌群特征与健康人群存在明显差异,其中肺炎链球菌水平与痰嗜酸性粒细胞百分比呈明显的负相关。     

鼻息肉患者前列腺素E2受体的表达及意义

摘要 目的：研究EP受体在慢性鼻-鼻窦炎伴鼻息肉（chronic rhinosinusitis with nasal polyps, CRSwNP）中的表达及意义。方法：收集20例嗜酸粒细胞性CRSwNP(eosinophilic CRSwNP,ECRSwNP )、20例非嗜酸粒细胞性CRSwNP（noneosinophilic CRSwNP,non-ECRSwNP)患者息肉和14例正常对照组鼻腔钩突黏膜。免疫组织化学和Western blot技术检测各组鼻组织中四种EP受体亚型蛋白的表达;对连续切片行免疫组化染色,检测EP受体与活化的嗜酸粒细胞之间的关系;用Real-time PCR检测各组EP受体和IL-5/IL-13 mRNA的表达水平。结果：EP受体主要表达于鼻黏膜上皮、腺体和上皮下炎症细胞,EP1受体选择性表达于上皮下炎症细胞。与对照组和non-ECRSwNP相比较,ECRSwNP组中EP1 mRNA和蛋白表达均上调,而三组间EP2、EP3和EP4受体的表达无明显差异。连续切片免疫组化染色示,EP1阳性的嗜酸粒细胞占EP1阳性总细胞数的50%。息肉组织EP1 mRNA与IL-5（r=0.55; P <0.001）、IL-13（r=0.69; P<0.001）mRNA的表达水平呈正相关。结论：ECRSwNP中EP1的表达上调与大量的嗜酸粒细胞等浸润有关。EP1受体可能通过趋化和活化嗜酸粒细胞参与ECRSwNP组织炎症的发生和发展。     

淡水石斑外周血细胞显微结构观察

淡水石斑(Cichlasoma managuense)外周血细胞可区分出红血细胞、嗜中性粒细胞、嗜酸性粒细胞、单核细胞、淋巴细胞和血栓细胞,未发现嗜碱性粒细胞。外周血液中还存在少量未成熟的和正在分裂的红血细胞。白细胞中,血栓细胞体积最小,嗜中性粒细胞体积最大;数量上,血栓细胞最多,而嗜酸性粒细胞则最少。     

似刺鳊鮈外周血细胞显微结构和血清生化指标的初步研究

似刺鳊(鮈)(Paracanthobrama guichenoti)外周血细胞可分为红血细胞、嗜中性粒细胞、单核细胞及大、小淋巴细胞和血栓细胞,未发现嗜酸性和嗜碱性粒细胞.外周血液中可观察到少量未成熟的及正在分裂的红血细胞.白细胞中,血栓细胞体积最小,嗜中性粒细胞体积最大;数量上,血栓细胞最多,而大淋巴细胞则最少.似刺鳊(鮈)血清生化指标测定结果显示,谷丙转氨酶(ALT)、胆固醇(CHOL)和总蛋白(TP)变化范围较大,且谷草转氨酶(AST)的水平较高,似刺鳊(鮈)雄鱼的血液生化指标相应值均显著低于雌鱼(P<0.05).     

Enhanced Tissue Factor Expression by Blood Eosinophils from Patients with Hypereosinophilia: A Possible Link with Thrombosis

Thrombotic risk is increased in eosinophil-mediated disorders, and several hypotheses have been proposed to link eosinophilia and thrombosis. In particular, eosinophils have been described as source of tissue factor (TF), the main initiator of blood coagulation; however, this aspect is still controversial. This study was aimed to evaluate whether TF expression varies in eosinophils isolated from normal subjects and patients with different hypereosinophilic conditions. Eosinophils were immunologically purified from peripheral blood samples of 9 patients with different hypereosinophilic conditions and 9 normal subjects. Western blot analysis and real-time polymerase chain reaction (RT-PCR) were performed to test eosinophil TF expression. For comparison, TF expression was evaluated in monocytes from blood donors and in human endothelial (ECV304) and fibroblast (IMR90) cell lines. Western blot analysis revealed a major band of 47,000 corresponding to native TF in homogenates of purified eosinophils with a higher intensity in the 9 patients than in the 9 controls (p<0.0001). According to RT-PCR cycle threshold (Ct), TF gene expression was higher in eosinophils from patients than in those from controls, median (range) 35.10 (19.45–36.50) vs 37.17 (35.33–37.87) (p = 0.002), and was particularly abundant in one patient with idiopathic hypereosinophilic syndrome and ischemic heart attacks (Ct: 19.45). TF gene expression was moderate in monocytes, Ct: 31.32 (29.82–33.49) and abundant in endothelial cells, Ct: 28.70 (27.79–29.57) and fibroblasts, Ct: 22.77 (19.22–25.05). Our results indicate that human blood eosinophils contain variable amounts of TF. The higher TF expression in patients with hypereosinophilic disorders may contribute to increase the thrombotic risk.     

Expression of membrane type-4 matrix metalloproteinase (metalloproteinase-17) by human eosinophils

Circulating eosinophils need proteinases to mediate a spatially limited and orientated digestion of the extracellular matrix and to migrate into tissue. Moreover, proteinases are likely involved in tissue remodeling, a crucial feature of chronic diseases including asthma. Eosinophils express matrix metalloproteinase (MMP)-9, which is increased upon stimulation with TNF-. Other MMPs, the membrane type (MT)-MMPs, likely play a major role in cell invasion and tissue remodeling. MT4-MMP was identified in peripheral blood leukocyte preparations, but it is not known whether eosinophils express MT4-MMP. We investigated the expression of MT4-MMP and its modulation by TNF- in purified human blood eosinophils. The constitutive expression of MT4-MMP mRNA was detected by RT-PCR in unstimulated eosinophils, lymphocytes, and monocytes, but not neutrophils. Stimulation of eosinophils with TNF- increased MT4-MMP mRNA expression. This effect appeared at 4 h and reached a maximum at 8 h of incubation. MT4-MMP protein was detected in freshly isolated blood eosinophils by Western blotting and immunocytochemistry. TNF- increased expression of the MT4-MMP protein. MT4-MMP protein was also detected in nasal polyp eosinophils by immunohistochemistry. In conclusion, eosinophils constitutively express MT4-MMP, which is increased upon stimulation with TNF-. Consequently, MT4-MMP may be directly involved in the degradation of extracellular matrix components and/or modulate the activity of other proteins implicated in eosinophil migration and tissue remodeling.     

Decreased expression of matrix metalloproteinase-9 and increased expression of tissue inhibitors of matrix metalloproteinase-1 in paratuberculosis-infected cattle in the ELISA-negative subclinical stage

We investigated the gene expression of matrix metalloproteinases-9 (MMP-9) and tissue inhibitors of matrix metalloproteinases-1 (TIMP-1) in peripheral blood cells from infected cattle with Mycobacterium avium subsp. paratuberculosis (Map) in the ELISA-negative subclinical stage compared with uninfected control cattle. Significant decreased MMP-9 expression and increased TIMP-1 expression were found in peripheral blood cells from Map-infected cattle after stimulation with Map lysate and Map purified protein derivative (PPD) than in control cattle by real-time RT-PCR analysis. In contrast to the uninfected controls, the activity of MMP-9 was also decreased in peripheral blood cell culture supernatants from Map-infected cattle at 24?hr after Map lysate and MapPPD stimulation by gelatin zymography analysis. As a result, the MMP-9 may play an important role in the development of Mycobacterium avium subsp. paratuberculosis disease.     

DcR3在甲状腺乳头状癌中的表达及临床意义

应用RT-PCR、Westem blot、免疫组化分别检测甲状腺乳头状癌组织与癌旁正常甲状腺组织标本中DcR3mRNA及蛋白的表达情况,探讨DcR3在甲状腺乳头状癌组织中的表达及,临床意义。RT-PCR检测显示,甲状腺乳头状癌中DcR3 mRNA的表达明显高于正常甲状腺组织（P〈0．05）：Western blot提示,DcR3蛋白在甲状腺乳头状癌中表达比正常甲状腺组织高（P〈0．05）;免疫组化显示,DcR3蛋白在甲状腺乳头状癌中高表达（P〈0．05）。DcR3mRNA及蛋白质在甲状腺乳头状癌及正常甲状腺组织间的表达差异有统计学意义（P〈0．05）。DcR3基因及蛋白在甲状腺乳头状癌中高表达,提示DcR3可能促进了甲状腺乳头状癌的发生发展。     

RNAi 介导的组织因子基因沉默抑制裸鼠移植性结肠癌的生长

组织因子(tissue factor,TF) 是机体外源性凝血途径的启动因子,发挥生理性止血的重要作用.近来研究表明,TF 除凝血功能外尚与多种恶性肿瘤的血管生成,侵袭转移及预后密切相关.为探讨 TF 对人结肠癌细胞（LoVo 细胞）生长能力的影响,构建带有 TFsiRNA 的重组腺病毒Ad pTF 和不带有 TFsiRNA 的对照病毒 Ad pDC,分别感染 LoVo 细胞,采用RT PCR 和 Western 免疫印迹法检测被感染 LoVo 细胞的 TF mRNA 和蛋白表达水平,并通过体内成瘤实验,进一步分析对 LoVo 细胞生长能力的影响.结果显示,与感染 Ad pDC 的 LoVo 细胞相比,感染 Ad pTF 的 LoVo 细胞的 TF mRNA 和蛋白表达水平更低,皮下种植瘤的体积更小,局部侵袭范围也更局限.研究表明,腺病毒介导的 RNAi 能特异而有效地沉默 LoVo 细胞中 TF 基因的表达,进而抑制 LoVo 细胞体内种植瘤的生长侵袭能力.     

Tissue factor expression on monocytes induced by anti-phospholipid antibodies as a strong risk factor for thromboembolic complications in SLE patients

Our aim was to clarify the role of anti-phospholipid antibodies in the pathogenesis of monocyte tissue factor (TF) expression and thromboembolic complications (TE) in patients with SLE. We examined cell surface expression of TF on monocytes in 93 SLE patients. Monocyte TF expression was significantly higher in SLE patients who had TE than in other SLE patients, and confirmed that the high expression of monocyte TF was a strong risk factor for TE. Furthermore, the presence of anti-cardiolipin/β2-glycoprotein I antibodies (anti-CL/β2-GPI) was strongly associated with the high expression of monocyte TF. We therefore studied the in vitro effect of IgG anti-CL/β2-GPI on lipopolysaccharide (LPS)-induced expression of TF on monocytes in healthy peripheral blood and found that purified IgG containing anti-CL/β2-GPI significantly enhanced LPS-induced monocyte TF expression. These results suggest that anti-CL/β2-GPI cause persistently high TF expression on monocyte, which may contribute to the risk of thromboembolic events in SLE patients.     

Inducible heat shock protein 70 expression as a potential predictive marker of metastasis in breast tumors

Heat shock protein (Hsp)-peptide complexes purified from tumors can prime the immune system against tumor antigens, but how they contribute to the generation of immune responses against naturally occurring tumors is unknown. Murine tumors expressing high amounts of Hsp70 are preferentially rejected by the immune system, suggesting that low Hsp70 expression is advantageous for tumor growth in the host. To determine whether Hsp70 was differentially expressed in human tumors, inducible Hsp70 expression was quantitatively (by Western blot) and qualitatively (by immunohistology) analyzed in 53 biopsies of tumor and normal breast tissue. The mean expression of inducible Hsp70 was significantly higher in tumor compared with normal tissue (U = 899.0; P = 0.0033). However, a significant negative association of the amount of Hsp70 expressed by tumor tissue was found with metastasis (r = -0.309; P = 0.05). After 3 years, follow-up analysis determined that 7 of the 53 patients relapsed, and 5 died. Hsp70 expression in tumor (but not normal) cells was significantly lower in relapse patients and patients with metastatic disease than in patients with no relapse or metastasis. Together, these observations support the hypothesis that Hsp70 plays a role in tumor expansion in vivo, and tumors that downregulate it may be able to evade immunosurveillance and grow.     

CD25 T-lymphocytes induce CD11b on eosinophils in allergic nasal mucosa

In the allergic mucosa, there is a significant increase in numbers of CD25(+) cells and activated eosinophils. To determine whether a link exists between the activated T-lymphocytes and tissue eosinophils in nasal allergy, we studied CD25(+) cells in the nasal mucosa and compared the levels of soluble IL-2 receptor (sIL-2R) both in the serum and the nasal secretions, and further investigated expression of CD11b on eosinophils in the nasal lavage fluids and peripheral blood of patients with nasal allergy. We also examined the effects of the culture supernatant of Con A- and IL-2-activated T-lymphocytes on CD11b expression on eosinophils in the present study. The concentration of sIL-2R in the nasal secretions from patients with Japanese cedar pollinosis (JCP) was significantly higher than that from normal subjects (p < 0.01). The sIL-2R level was significantly higher in the nasal secretions than in the sera in patients (p < 0.01), and CD11b expression on eosinophils from nasal hvage fluid was significandy higher than that of eosinophils from peripheral blood of the same individuals (p < 0.01). The activated T-lymphocytes promoted eosinophil activation with upregulation of CD11b in vitro, and eosinophils in the nasal secretions from patients significantly expressed more CD11b in vivo. These results indicate that activation of T-lymphocytes is linked to eosinophil activation in nasal allergy.