病毒受体及其研究方法概况

病毒受体在介导病毒与靶细胞的特异性结合过程中起着非常重要的作用。病毒受体的特性与病毒的宿主范围和组织特异性密切相关,是病毒致病性的关键因素之一。研究病毒受体的方法很多,笔者综述了病毒受体特征及其几种主要研究方法,以期为有效控制、治疗病毒感染提供理论参考。     

Effects of extracellular potassium on expression of HERG gene nonsense mutant L539fs/47

AIM: To study the effects of extracellular potassium on the protein expression of wild- type HERG and its mutant L539fs/47. METHODS: Wild-type HERG (WT) or its mutant HERG-L539fs/47 (MT) were transfected into HEK293 cells for 36 h. The cells were incubated in different media containing 0.8, 4.3 or 10 mmol/L potassium. After 6 h of incubation, the protein expression of HERG was detected by flow cytometry.After 12 h of incubation, the localization and quantity of the proteins were detected by laser confocal imaging and Western blot. RESULTS: Different from the retention of mutant protein in cytoplasm, wild-type HERG protein was mainly distributed in the cell membrane. The 2 proteins both increased with the changes of extracellular potassium. Flow cytometry showed that the fluorescence in the 2 groups both increased with the changes of extracellular potassium (P < 0.01). The fluorescence in WT group was significantly higher than that in MT group (P < 0.01). Western blot showed that mutant HERG protein included only one 60 kD band, different from the 135 kD and 155 kD bands in wild-type HERG, which were affected by the changes of extracellular potassium (P < 0.05). CONCLUSION: The retention of HERG mutant L539fs/47 protein in the cytoplasm is more than wild-type HERG. Chronic high extracellular potassium keeps the stability of wild-type and mutant HERG proteins on the cell membrane. Chronic low potassium reduces the expression of HERG channel proteins in a time-dependent manner.     

武安国家森林公园蕨类植物叶表皮细胞的显微观察

利用光学显微镜对武安国家森林公园14种观赏蕨类植物叶表皮细胞的显微结构进行了观察.结果表明:几种蕨类植物不同种属间具有一定的共性,表现为气孔器全部分布在叶的下表皮,即全部为下生气孔.但在叶表皮细胞的大小、形态、垂周壁式样及气孔器的类型、保卫细胞的长宽、气孔指数等特征上存在一定的差异,基于14个分类群和35项表型性状特征相似性的聚类分析结果,表明蕨类植物各个种之间的亲缘关系和传统分类方法基本相同.     

加味柴胡汤对子宫内膜炎奶牛血液白细胞的影响

本试验选子宫内膜炎患牛10头,随机分为中药治疗组和患病对照组（每组5头）,5头健康奶牛作为正常对照组.中药治疗组子宫灌注复方中药剂加味柴胡汤,连用6d,其他两组给予生理盐水.在用药前1d和用药2d、4d、6d采血,用血液自动分析仪检测.结果：（1）治疗前,中药治疗组和患病对照组白细胞数量显著（P＜0.05）高于正常对照组,治疗6d后,中药治疗组显著（P＜0.05）低于治疗前和患病对照纽.（2）治疗前,中药治疗组和患病对照组中间细胞数量和比率均显著（P＜0.05）高于正常对照组,用药6d后,中药治疗组显著（P＜0.05）或极显著（P＜0.01）低于患病对照组和治疗前.（3）治疗前,中药治疗组和患病对照组嗜中性粒细胞数量比率显著（p＜0.05）高于正常对照组.用药6d后,中药治疗组与正常对照组均显著（P＜0.05）低于患病对照组,低于治疗前.（4）治疗前,中药治疗组和患病对照组淋巴细胞数量显著（P＜0.05）高于正常对照组,比率显著（P＜0.05）低于正常对照组.用药6d后,中药治疗组和正常对照组淋巴细胞数量和比率显著（P＜0.05）高于患病对照纽.结论：加味柴胡汤可降低子宫内膜炎奶牛的炎症反应,提高免疫力.     

猪骨髓间充质干细胞分离培养及生物学特性

采集健康猪股骨骨髓,利用percoll梯度离心法分离单个核细胞,进行体外原代和传代培养,并用不同代数细胞进行了细胞生长能力、膜表面抗原(CD105、CD90、CD45)及诱导分化能力的检测.结果显示分离培养的单个核细胞贴壁生长,形态呈成纤维细胞样及涡旋状克隆团；细胞传代至第17代生长状况仍然良好；用F3代细胞进行膜表面抗原标记显示,CD90和CD105阳性表达率分别为(97.4±1.8)％和(99.6±0.9)％,而CD45阳性表达率仅为(1.8±0.55)％,证实这些细胞具有猪骨髓间充质干细胞(Mesenchymal stem Cells,MSCs)表面抗原；经地塞米松、维生素C和β-磷酸甘油等诱导F3代细胞,21d后出现钙物质沉积细胞群,用茜素红和Von kossa染色呈阳性,证实这些细胞已分化形成成骨细胞；经地塞米松、IBMX、胰岛素及吲哚美辛等诱导F3代细胞,21d后细胞质出现脂滴样结构,用油红O染色呈阳性,证实已分化形成成脂细胞；冷冻-解冻细胞的膜表面抗原及多能分化能力与未冻存细胞的检测结果基本一致.结果证实,从猪骨髓中分离培养及经传代扩增获得的贴壁细胞是纯化的MSCs.     

Effects of L-carnosine on NIT-1 islet cells

AIM: To investigate the protective effect of L-carnosine on insulin secretion, proliferation and apoptosis of β-cells impaired by high glucose. METHODS: NIT-1 cells were pre-treated with glucose at concentrations of 11.1 mmol/L (control level) and 33.3 mmol/L (high level) for 72 h, and then the cells were stimulated with various concentrations of glucose (0, 5 and 25 mmol/L) and/or L-carnosine (0, 1 and 20 mmol/L). The level of insulin in the medium was measured by radioimmunoassay. To detect the effect of L-carnosine on proliferation and apoptosis, NIT-1 cells were divided into 4 groups according to different culture conditions for 72 h: group C (with 11.1 mmol/L glucose), group H (with 33.3 mmol/L glucose), group H+A (with 33.3 mmol/L glucose+ 1 mmol/L L-carnosine) and group H+B (with 33.3 mmol/L glucose +20 mmol/L L-carnosine). Proliferous or apoptotic cells were identified by BrdU labeling and flow cytometry (labeling with annexin V-FITC/PI),respectively. Total RNA was extracted and the mRNA expression of caspase-3 and bcl-2 was measured by RT-PCR. The caspase-3 activity was also checked by fluorometric assay kit. RESULTS: The insulin in high-level glucose group was lower than that in control-level glucose group. L-carnosine at concentration of 20 mmol/L notably increased the insulin secretion of the cells pre-treated with glucose at control level or high level. The proliferation and apoptosis were both increased in group H compared with group C, but the total cell counts declined because the apoptotic rate was higher than the proliferation rate. L-carnosine at concentration of 1 mmol/L significantly increased the proliferation rate and decreased the apoptotic rate. The mRNA level of caspase-3 was decreased and the mRNA level of bcl-2 was increased after the cells were treated with L-carnosine at concentration of 1 mmol/L. L-carnosine at concentrations of both 1 mmol/L and 20 mmol/L significantly decreased the caspase-3 activity. CONCLUSION: L-carnosine at high level directly stimulates insulin secretion in NIT-1 cells, and L-carnosine at normal level promotes the cell proliferation and inhibits apoptosis induced by high concentration of glucose. Caspsase-3 and Bcl-2 may be partly involved in this process.     

Effects of human bone morphogenetic protein 2 and 9 on proliferation,apoptosis and migration of human gastric carcinoma MNK-45 cells

AIM: To investigate the effects of human bone morphogenetic protein 2 (BMP2) and BMP9 on the proliferation, apoptosis and migration of human gastric carcinoma cell line MNK-45. METHODS: Immunocytochemical staining, MTT assay, wound-healing test, Transwells migration test, Hoechst 33258 staining and flow cytometry (FCM) were used to determine the infection of AdBMP2 and AdBMP9 on the proliferation, apoptosis and migration of MNK-45 cells. The expression of GSK-3β (including p-GSK-3β and total GSK-3β) and β-catenin in MNK-45 cells was also detected by Western blotting. RESULTS: The proliferation of MNK-45 cells was inhibited from the third day on and in a time-dependent manner after infected with AdBMP2 and AdBMP9. The results of Hoechst 33258 staining and FCM proved that apoptosis rates in BMP2 group and BMP9 group were higher than that in GFP group. Both wound-healing test and Transwell experiment indicated that up-regulating the expression of BMP2 and BMP9 inhibited the migration of MNK-45 cells. The phosphorylation levels of GSK-3β in BMP2 group and BMP9 group were higher than that in GFP group. However, no significant change of β-catenin among groups was observed. CONCLUSION: Up-regulation of BMP2 and BMP9 expression inhibits the proliferation of MNK-45 cells.     

PPARδ agonist GW501516 inhibits ox-LDL-or high glucose-induced apoptosis in human umbilical vein endothelial cells

AIM: To investigate the inhibitory effect of peroxisome proliferator-activated receptor δ (PPARδ) agonist GW501516 on the apoptosis induced by oxidized low-density lipoprotein(ox-LDL) or high concentration of glucose in human umbilical vein endothelial cells (HUVECs). METHODS: Cell apoptosis was induced by ox-LDL or high concentration of glucose in HUVECs and was examined by flow cytometry.The HUVECs were treated with GW501516 at different concentrations. The viability of HUVECs was analyzed by MTT assay. RESULTS: The apoptosis rate of HUVECs treated with ox-LDL was 21.3%, while those of HUVECs treated with ox-LDL combined with low, medium and high concentrations of GW501516 were 17.47%, 9.72% and 3.94%, respectively. The apoptosis rate of HUVECs treated with glucose was 22.60%, while those of HUVECs treated with glucose combined with different concentrations of GW501516 were 20.23%, 17.01% and 9.38%, respectively.The results indicated that ox-LDL or glucose induced apoptosis of HUVECs and GW501516 decreased the apoptotic rates induced by ox-LDL or glucose in a dose-dependent manner. The results of MTT assay showed that glucose or ox-LDL decreased the viability of HUVECs and GW501516 attenuated the effect of glucose or ox-LDL on HUVECs. CONCLUSION: GW501516 inhibits the apoptotic effects of ox-LDL and glucose on HUVECs and increases the viability of the cells.     

Effects of high glucose on proliferation,migration, adhesion and secretion of rat late endothelial progenitor cells

AIM: To investigate the effects of high glucose on the proliferation, adhesion, migration and secretion potentials of late endothelial progenitor cells (EPCs) from bone marrow. METHODS: Mononuclear cells were collected from rat bone marrow by density gradient centrifugation and cultured with M199 medium. The early EPCs were identified by DiI-ac-LDL and FITC-UEA-1 double staining. The late EPCs were identified by RT-PCR to detect the expression of von Willebrand factor (vWF) and VE-cadherin. Moreover, the cells were identified by FACS to detect the expression of CD133 and vascular endothelial growth factor receptor-2(VEGFR-2). The 3rd generation of EPCs was harvested and incubated with glucose in a series of concentrations (5, 10, 20 or 40 mmol/L). The cell proliferation, adhesion, migration and the secretion of chemokines such as monocyte chemoattractant protein-1(MCP-1) and interleukin-8 (IL-8) were assayed with MTT, adhesion test, modified Boyden chamber assay and ELISA, respectively. RESULTS: Compared with normal glucose (5 mmol/L)treatment, high glucose (10, 20, 40 mmol/L) dose-dependently degraded the proliferation and migration of late EPCs (P<0.05 or P<0.01). At the same time, treatment with glucose at the concentration of 40 mmol/L decreased the adhesion of EPCs (P<0.05) and increased the release of MCP-1 and IL-8 by late EPCs. CONCLUSION: High glucose inhibits proliferation, adhesion and migration of late EPCs, and enhances the secretion of inflammatory factors, indicating that the high glucose correlates with the vascular complications of patients with diabetes.