人骨髓基质细胞分化为神经细胞潜力的研究

目的 研究骨髓基质细胞在含血清培养基中向神经细胞分化的潜能。方法 从健康人肋骨分离原代骨髓基质细胞 ,接种于含 10 %胎牛血清 (FBS)的DMEM中 ,分为A组 (3～ 4d换液一次 )、B组 (不换液 ) ,在相差显微镜下观察生长情况。在培养的 1,5 ,10d用免疫荧光法检测两组骨髓基质细胞的各种表面标志物巢蛋白 (nestin)、胶质纤维酸性蛋白 (GFAP)、微管相关蛋白 (MAP 2 )、S 10 0表达情况。并在培养的第 1,4 ,7,10 ,13d用酶联免疫吸附法 (ELISA)检测B组培养细胞上清中的脑源性神经营养因子 (BDNF)和神经生长因子 (NGF)的分泌水平。结果 A组细胞分裂繁殖速度明显高于B组 ,在培养的第 11～ 12d ,B组中部分细胞表现为胞体锥形并有长突起的神经元样形态 ,且形成网状。A组中随着培养时间的延长仅仅有nestin表达增加 ,B组中Nestin ,GFAP ,MAP 2的表达随时间的不同有差异 ,S 10 0在体积较小的细胞表达较强。B组细胞培养的第 10d ,检测到BDNF的表达 ,培养第 7,10 ,13d检测到NGF分泌。结论 人骨髓基质细胞在含血清培养基中能够表达神经前体细胞和神经细胞的相关表面标志物 ,并且能够分泌一定水平的神经营养因子 ,表现出可分化成神经细胞的潜能。血清和骨髓基质细胞自身分泌物质的蓄积可能在其中起到了重要的作用。

Effects of hepatitis B virus infection on human sperm chromosomes

AIM: To evaluate the level of sperm chromosome aberrations in male patients with hepatitis B, and to directly detect whether there are HBV DNA integrations in sperm chromosomes of hepatitis B patients.METHODS: Sperm chromosomes of 14 tested subjects (5healthy controls, 9 patients with HBV infection, including 1with acute hepatitis B, 2 with chronic active hepatitis B, 4with chronic persistent hepatitis B, 2 chronic HBsAg carriers with no clinical symptoms) were prepared using interspecific in vitro fertilization between zona-free golden hamster ova and human spermatozoa, and the frequencies of aberration spermatozoa were compared between subjects of HBV infection and controls. Fluorescence in situ hybridization (FISH) to sperm chromosome spreads was carried out with biotin-labeled full length HBV DNA probe to detect the specific HBV DNA sequences in the sperm chromosomes.RESULTS: The total frequency of sperm chromosome aberrations in HBV infection group (14.8%, 33/223) was significantly higher than that in the control group (4.3%,5/116). Moreover, the sperm chromosomes in HBV infection patients commonly presented stickiness, clumping, failure to staining, etc, which would affect the analysis of sperm chromosomes. Specific fluorescent signal spots for HBV DNA were seen in sperm chromosomes of one patient with chronic persistent hepatitis. In 9 (9/42) sperm chromosome complements containing fluorescent signal spots, one presented 5 obvious FISH spots, others presented 2 to 4signals. There was significant difference of fluorescence intensity among the signal spots. The distribution of signal sites among chromosomes was random.CONCLUSION: HBV infection can bring about mutagenic effects on sperm chromosomes. Integrations of viral DNA into sperm chromosomes which are multisites and nonspecific, can further increase the instability of sperm chromosomes. This study suggested that HBV infection can create extensively hereditary effects by alteration genetic constituent and/or induction chromosome aberrations, as well as the possibility of vertical transmission of HBV via the germ line to the next generation.

Update on Anti-Saccharomyces cerevisiae antibodies, anti-nuclear associated anti-neutrophil antibodies and antibodies to exocrine pancreas detected by indirect immunofluorescence as biomarkers in chronic inflammatory bowel diseases: Results of a multicent

AIM： Anti-Saccharomyces anti-nuclear associated cerevisiae antibodies （ASCA）, anti-neutrophil antibodies （NANA） and antibodies to exocrine pancreas （PAB）, are serological tools for discriminating Crohn＇s disease （CrD） and ulcerative colitis （UC）. Like CrD, coeliac disease （COD） is an inflammatory bowel disease （IBD） associated with （auto） antibodies. Performing a multicenter study we primarily aimed to determine the performance of ASCA, NANA and PAB tests for IBD diagnosis in children and adults, and secondarily to evaluate the prevalence of these markers in CoD. METHODS： Sera of 109 patients with CrD, 78 with UC, 45 with CoD and 50 healthy blood donors were retrospectively included. ASCA, NANA and PAB were detected by indirect immunofluorescence （IIF）. RESULTS： ASCA＋/NANA- profile displayed a positive predictive value of 94.2% for CrD. Detection of ASCA was correlated with a more severe clinical profile of CrD and treatment of the disease did not influence their serum levels. ASCA positivity was found in 37.9% of active CoD.PAB were found in 36.7% CrD and 13.3% CoD patients and were not correlated with clinical features of CrD, except with an early onset of the disease. Fifteen CrD patients were ASCA negative and PAB positive. CONCLUSION： ASCA and PAB detected by IIF are specific markers for CrD although their presence does not rule out a possible active CoD. The combination of ASCA, NANA and PAB tests improves the sensitivity of immunological markers for CrD. Repeating ASCA, NANA, and PAB testing during the course of CrD has no clinical value.

Blocking NF-kB nuclear translocation leads to p53-related autophagy activation and cell apoptosis

AIM: To investigate the anti-tumor effects of nuclear factor-κB (NF-κB) inhibitor SN50 and related mechanisms of SGC7901 human gastric carcinoma cells. METHODS: MTT assay was used to determine the cytotoxic effects of SN50 in gastric cancer cell line SGC7901. Hoechst 33258 staining was used to detect apoptosis morphological changes after SN50 treatment. Activation of autophagy was monitored with monodansylcadaverine (MDC) staining after SN50 treatment.Immunofluorescence staining was used to detect the expression of light chain 3 (LC3). Mitochondrial membrane potential was measured using the fluorescent probe JC-1. Western blotting analysis were used to determine the expression of proteins involved in apoptosis and autophagy including p53, p53 upregulated modulator of apoptosis (PUMA), damage-regulated autophagy modulator (DRAM), LC3 and Beclin 1. We detected the effects of p53-mediated autophagy activation on the apoptosis of SGC7901 cells with the p53 inhibitor pifithrin-α. RESULTS: The viability of SGC7901 cells was inhibited after SN50 treatment. Inductions in the expression of apoptotic protein p53 and PUMA as well as autophagic protein DRAM, LC3 and Beclin 1 were detected with Western blotting analysis. SN50-treated cells exhibited punctuate microtubule-associated protein 1 LC3 in immunoreactivity and MDC-labeled vesicles increased after treatment of SN50 by MDC staining. Collapse of mitochondrial membrane potential Δψ were detected for 6 to 24 h after SN50 treatment. SN50-induced increases in PUMA, DRAM, LC3 and Beclin 1 and cell death were blocked by the p53 specific inhibitor pifithrin-α. CONCLUSION: The anti-tumor activity of NF-κB inhibitors is associated with p53-mediated activation of autophagy.

Characterization and application of monoclonal antibodies against Shewanella marisflavi, a novel pathogen of Apostichopus japonicus

Shewanella marisflavi strain AP629 was certified as a novel pathogen of the sea cucumber Apostichopusjaponicus. In this study, four monoclonal antibodies （MAbs） （3C1, 3D9, 2F2, 2A8） against strain AP629 were developed by immunizing Balb/C mice. 3C1 and 3D9 recognized S. marisflavi only, showing no cross reactivity to other gram-negative bacteria, However, 2F2 and 2A8 showed cross reactivity to all tested bacteria. Indirect immunofluorescence, and immunogold electron microscopy, showed the binding antigens of 3C1 and 3D9 were located at the secretion on the surface of strain AP629. The binding antigens of 2F2 and 2A8 were noted on the membrane of the cells. MAbs 3C1 and 3D9 recognized the lipopolysaccharide fraction of strain AP629, and 2F2 and 2A8 recognized in western-blotting protein antigens with molecular weights of 113 and 128 kDa respectively. MAbs 3C1 and 3D9 have the potential for use in pathogen diagnosis, epidemiology and studies on the mechanism of how S. marisflavi infects A. japonicus. Imrnunohistochemistry with 3C1 or 3D9 identified strain AP629 in the body wall of infected A. japonicus. In the adult sea cucumbers that were infected via body wall injection, positive signals were observed at the site of skin ulceration, and at the connective tissue of the non-ulcerated body wall. In addition, some large blue-stained cells aggregated at the connective tissue colonized by large numbers of bacteria. In juveniles infected via immersion infection, positive signals were observed at the cuticle of the body wall only. Our results suggest that 3C1 and 3D9 could be used in various immunological assays to study the invasion mechanism of strain AP629 in A. japonicus, the law of bacterial colonization, proliferation in different tissues of A. japonicus, and correlation between secretion on the surface of strain AP629 and its pathogenesis to A. japonicus.

Liraglutide directly protects cardiomyocytes against reperfusion injury possibly via modulation of intracellular calcium homeostasis

Background Liraglutide is glucagon-like peptide-1 receptor agonist for treating patients with type 2 diabetes mellitus. Our previous studies have demonstrated that liraglutide protects cardiac function through improving endothelial function in patients with acute myocardial infarction undergoing percutaneous coronary intervention. The present study will investigate whether liraglntide can perform direct protective effects on cardiomyocytes against reperfusion injury. Methods In vitro experiments were performed using H9C2 cells and neonatal rat ventricular cadiomyocytes undergoing simulative hypoxia/reoxygenation （H/R） induction. Cardiomyocytes apoptosis was detected by fluorescence TUNEL. Mitochondrial membrane potential （AWm） and intracellular reactive oxygen species （ROS） was assessed by JC-1 and DHE, respectively. Fura-2/AM was used to measure intracellular Ca2＋ concentration and calcium transient. Immtmofluorescence staining was used to assess the expression level of sarcoplasmic reticulum Ca2＋-ATPase （SERCA2a）. In vivo experiments, myocardial apoptosis and expression of SERCA2a were detected by colorimetric TUNEL and by immunofluorescence staining, respectively. Results In vitro liraglutide inhibited cardiomyotes apoptosis against H/R. △mψ of cardiomyocytes was higher in liraglntide group than H/R group. H/R increased ROS production in H9C2 cells which was attenuated by liraglutide. Liraglutide significantly lowered Ca2＋ overload and improved calcium transient compared with H/R group, lmmunofluorescence staining results showed liraglutide promoted SERCA2a expression which was decreased in H/R group. In ischemia/reperfusion rat hearts, apoptosis was significantly attenuated and SERCA2a expression was increased by liraglutide compared with H/R group. Conclusions Liraglutide can directly protect cardiomyocytes against reperfusion injury which is possibly through modulation of intracellular calcium homeostasis.

Clinicopathologic features of 112 cases with mantle cell lymphoma

Objective： This study aims to explore the clinicopathologic features of 112 patients with mantle cell lymphoma （MCL）. Methods： Data from 112 MCL cases were collected, and immunohistochemical assay was conducted. Fluorescence in situ hybridization （FISH） detected a break in the CCND 1 gene. The t-test was used in the statistical analysis. Results： All tumor cells in the 112 cases expressed B cell-related antigen, including 1 blastoid subtype and 1 polymorphic subtype. Among all cases, 106 expressed CD5 and 104 expressed cyclin D1. A break in the CCND1 gene was not found in 3 cases with CDS-MCL. IgH/CCND 1 polyploid was observed in 2 classic cases. Conclusion： MCL is a type of special immunophenotypic B-cell lymphoma, The prognoses ofblastoid and polymorphic subtypes are poor. Special subtypes should be classified during diagnosis.

Relationship between Chlamydia pneumoniae infection and occurrence of bronchial asthma

Objective: To study the relationship between Chlamydia pneumoniae (C. pneumoniae) infection and asthma exacerbation. Methods: A prospective study of C. pneumoniae infection was conducted in 75 patients with asthma and 63 patients with respiratory tract infection, and 100 blood donors served as controls. The presence of infection was convinced by the polymerase chain reaction and direct immunofluorescence assay for C. pneumoniae DNA from throat swab specimens and micro-immunofluorescence testing for C. pneu-moniae-specific IgG, IgM and IgA antibodies. Results: Prevalence of specific IgG in asthma patients (81. 3%) was higher than that of the blood donors (68. 0%, P<0. 05) and was not significantly different from respiratory tract infection patients (68. 0%, P>0. 05). The acute C. pneumoniae infection rate of symptomatic asthma patients (59. 4%) was markedly higher than that of respiratory tract infection patients (34. 9% , P<0. 05). The average titer of C. pneumoniae IgG instead of IgA in asthma patients (48. 38±6. 94) was significantly higher than respiratory tract infection patients (24. 70±8. 77, P<0. 05). Other pathogens were identified in 12 of 21 (57. 1%) asthma patients with C. pneumoniae. The symptoms of 7 asthma patients with C. pneumoniae infection were improved through antibiotic treatment. Conclusion: The findings suggest a possible role of C. pneumoniae infection in asthma.

Distribution and localization of microfilament cytoskeleton is regulated by EBP50

Objective:To explore the influence of EBP50(ezrin-radixin-moesin-binding phospho-protein-50) on microfilament cytoskeleton content and distribution in cultured Hela cells, and to investigate the relationship between the changes in microfilament cytoskeleton localization and EBP50 after PDGF(platelet-derived growth factor) stimulation, and to further clarify the molecular mechanism by which EBP50 suppresses tumor cell proliferation and migration.Methods:pBK-CMV-HAEBP50 wild type recombinant plasmid and pBK-CMV-HA empty vector were transfected into Hela cells.G418 at 350 mg/L was used to screen for cell clones stably expressing EBP50.Western blot was carried out to detect EBP50 expression.Similarities and differences in microfilament cytoskeleton content and distribution in Hela cells transfected with pBK-CMV-HA-EBP50 wild type recombinant plasmid and pBK-CMV-HA empty vector were also treated with PDGF(10 ng/mL and 20 ng/mL, 37 ℃, 15 min) and stained by rhodamine-labeled phalloidin to observe the distribution of microfilament cytoskeleton in the two groups.EBP50 protein distribution in PDGF-stimulated Hela cells was detected by immunofluorescence.Results:Western blot results confirmed that the EBP50 cDNA fragment could express EBP50 in cultured Hela cell lines and that cell lines stably expressing EBP50 were successfully obtained.Western blot and fluorescence results showed that in the cell line transfected with empty vector, the microfilament cytoskeleton was thick, loose, multidirectional and displayed crossing arrangements.The content of microfilament cytoskeleton in the cell line transfected with pBK-CMV-HA-EBP50 was different from that found in the cell line transfected with empty vector.EBP50 expression enhanced microfilament cytoskeleton polymerization into compact thin filaments.Under the stimulation of PDGF, EBP50 migrated to the cell membrane from the cytosol together with microfilament cytoskeleton and co-localized there.Conclusion:EBP50 can change the distribution of microfilament cytoskeleton in cultured Hela cells and can also bind the microfilament cytoskeleton to the cell membrane under the stimulation of PDGF.EBP50 may play a role in the proliferation and migration of tumor cells by influencing the distribution and localization of microfilament cytoskeleton.

Bone Marrow Urokinase Plasminogen Activator Receptor Levels are Associated with the Progress of Multiple Myeloma

Objective To determine the mRNA and protein levels of urokinase plasminogen activator receptors(u PAR) in bone marrow fluid and bone marrow tissue from multiple myeloma(MM) patients and assess association of u PAR level with prognosis of MM.Methods u PAR levels in bone marrow fluid of 22 MM patients at the stable and progressive stages and 18 iron deficiency anemia patients with normal bone marrow(control) were examined by ELISA.Furthermore,u PAR expression in bone marrow tissue was investigated by RT-PCR and Western blot,respectively.The distribution of u PAR in MM cells was examined using immunofluorescence staining.The pathological changes in different stages of MM patients were studied by HE staining.Results u PAR level in bone marrow fluid of MM patients(1.52±0.32 μg/ml) was found to be higher than that in the control group(0.98±0.15 μg/ml).Interestingly,u PAR protein(0.686±0.075 vs.0.372±0.043,P<0.05) and m RNA(2.51±0.46 vs.4.46±1.15,P<0.05) expression levels of MM patients at the progressive stage were significantly higher than those at the stable stage.The expression of u PAR in MM bone marrow was confirmed by immunofluorescence staining.Moreover,HE staining revealed a great increased number of nucleated cells and severe impairment of hematopoietic function in the bone marrow of patients with progressive-stage myeloma.Conclusion Our study reveals that u PAR expression is positively correlated with the development and progress of MM.

Detection of Intracellular Cytokine Production in Peripheral Blood CD_^+ T Cells of Patients with Recurrent Genital Herpes

Objective: To study the role of Th1/Th2 cytokines in thepathogenesis of recurrent genital herpes (RGH), and tobetter understand the relationship between them. Methods: A two-color immunofluorescent staining of cellsurface antigen and intracellular cytokines for flowcytometric analysis was used for CD_3, IL-2, IL-10, IL-12,IFN-γ and TNF-α in CD_3^+ T-lymphocytes in activatedperipheral blood mononuclear cells of patients with RGH. Results: Compared to controls, patients with RGHshowed fewer CD_3^+ T cells (P<0.05) and IL-2 producing andIFN-γ producing T cells (P<0.02 and P<0.001, respectively)after in vitro stimulation with PMA and ionomycin in thepresence of a protein transport inhibitor. More IL-10producing and IL-12 producing T cells were found inpatients with RGH (P<0.01). There was no significantdifference in the number of TNF-α producing cells betweenRGH patients and controls (P<0.05). Conclusion: RGH patients showed relatively more Th2cytokines. The imbalance between Th1 and Th2 cytokinesresults in inhibitory effects on a series of cell-immuneresponses, which may play an important role in thepathogenesis of RGH.

A novel human coronavirus:Middle East respiratory syndrome human coronavirus

In 2012,a novel coronavirus,initially named as human coronavirus EMC(HCoV-EMC) but recently renamed as Middle East respiratory syndrome human coronavirus(MERS-CoV),was identified in patients who suffered severe acute respiratory infection and subsequent renal failure that resulted in death.Ongoing epidemiological investigations together with retrospective studies have found 61 laboratory-confirmed cases of infection with this novel coronavirus,including 34 deaths to date.This novel coronavirus is culturable and two complete genome sequences are now available.Furthermore,molecular detection and indirect immunofluorescence assay have been developed.The present paper summarises the limited recent advances of this novel human coronavirus,including its discovery,genomic characterisation and detection.