实验性小鼠急性Coxsackie B-3病毒性心肌炎的研究

小鼠感染Coxsackie B-3病毒后7天,各鼠心脏均出现巨检病变,组织学检查心肌细胞示变性、坏死及炎性细胞浸润,心肌切片作图象分析示病灶总面积/所测心肌总面积为11.3％,并有明显心肌超微结构改变。心肌中分离到病毒。对照鼠心脏均无上述改变,亦未分离到病毒。本模型可作为进一步探索病毒性心肌炎药物治疗的基础。     

频谱水对BALB/c小鼠T淋巴细胞转化的影响

目的探讨频谱水对实验小鼠细胞免疫功能的影响。方法将60只SPF级BALB/c小鼠随机分成喝泡频谱水组、喝自来水泡频谱水组、喝泡自来水组（对照组）,分别检测每组经ConA处理后T淋巴细胞转率。结果前两组处理组与对照组相比较,T淋巴细胞转化率增高,差异有显著性。结论频谱水可以提高机体的细胞免疫功能。     

药物对病毒感染培养心肌细胞Ca^2＋内流及CVB3—RNA复制的影响

应用放射性同位素＾４５Ｃａ＾２＋示踪技术及光敏生物素标记ｃＤＮＡ探针杂交方法,观察了维拉帕米、黄芪、地塞米松等药物对感染柯萨奇Ｂ３病毒（ＣＶＢ３）的大鼠培养心肌细胞Ｃａ＾２＋内流及细胞中ＣＶＢ３－ＲＮＡ复制的作用。结果发现：在病毒感染４８ｈ,上述三药均可显著减少感染细胞及正常对照的心肌Ｃａ＾２＋内流;若在病毒感染后即加入上药物,经４８ｈ培养后,黄芪组细胞中的ＣＶＢ３－ＲＮＡ含量显著少于病毒对照组,     

慢病毒介导Occludin过表达影响BVDV感染BALB/c小鼠的研究

为研究紧密连接蛋白(occludin,OCLN)是否影响牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)在BALB/c小鼠体内的复制。构建慢病毒表达载体pLVML-Myc-bOCLN-linker-GFP-IRES-Puro,连同辅助质粒pSPAX2和pMD2.G共同转染至HEK-293T细胞中,转染48 h后收集OCLN-GFP慢病毒悬液并测定其滴度,同时以pLVML-Myc-Mcs-linker-GFP-IRES-Puro载体包装的慢病毒作为阴性对照(GFP);将4-5周龄BALB/c小鼠随机分为A(0 d)、B(4 d)、C(8 d)、D(10 d)、E(15 d)五组,每组6只,分别注射5×10⁷ IU OCLN-GFP和GFP慢病毒悬液,连续注射两次,间隔48 h,第二次注射后96 h时以1.68×10⁵ TCID₅₀/只BVDV攻毒BALB/c小鼠,分别于攻毒后0、4、8、10、15 d时,解剖小鼠并采集肝脏、脾脏、肺脏、肾脏、小肠等组织,使用实时荧光定量PCR(real-...     

免疫检查点分子在慢性HBV感染中对T细胞功能影响的研究进展

阻断免疫检查点分子的信号通路可以增强免疫系统功能,这在肿瘤治疗中获得了显著效果。乙型肝炎病毒(Hepatitis B virus,HBV)慢性感染的原因之一为HBV在体内通过一系列方式来减弱、抑制免疫系统的功能,从而逃避免疫识别和杀伤。HBV可以通过上调病毒特异性T细胞表面的抑制性受体来抑制T细胞活化、增殖和效应。本文总结了HBV导致的人体内T细胞上免疫检查点程序性死亡受体1(Programmed cell death protein 1,PD-1)、细胞毒T淋巴细胞相关抗原4(Cytotoxic T-lymphocyte-associated protein 4,CTLA-4)、T细胞免疫球蛋白黏蛋白3(T-cell immunoglobulin domain and mucin domain-containing molecule-3,Tim-3)、淋巴细胞活化基因3(Lymphocyte-activation gene 3,LAG-3)、T细胞免疫球蛋白和免疫受体酪氨酸抑制基序(T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain,TIGIT)的异常表达以及它们影响T细胞功能的机制,揭示了免疫检查点在治疗慢性乙肝中的良好应用前景。     

基于PI3K/PKB通路研究EGCG对CVB3感染致病毒性心肌炎小鼠细胞凋亡的影响

细胞凋亡是造成病毒性心肌炎(VMC)发病过程中心肌损伤的重要因素;表没食子儿茶素没食子酸酯(EGCG)对缺血再灌注引起的细胞凋亡具有抑制作用,但是否抑制VMC发病过程中的心肌细胞凋亡尚不明确.因此,本研究将分析EGCG对VMC小鼠细胞凋亡的影响及分子机制.BALB/c小鼠随机分为对照组、VMC组(腹腔注射CVB3悬液造模)、VMC+EGCG组(腹腔注射CVB3悬液造模后腹腔注射EGCG)、VMC+EGCG+LY组(腹腔注射CVB3悬液造模后腹腔注射EGCG及P13K抑制剂LY294002).检测血清心肌损伤标志物肌钙蛋白I(cTnI)及乳酸脱氢酶(LDH),心肌中CVB3滴度及RNA表达、HE染色、凋亡基因及p-PI3K、p-PKB表达.结果显示,与对照组比较,VMC组血清cTnI、LDH含量、心肌中CVB3滴度及RNA表达、细胞凋亡率、cleaved caspase-3表达升高,心肌中Survivin、p-PI3K、p-PKB表达降低(P＜0.05);与VMC组比较,VMC+EGCG组血清cTnI、LDH含量及心肌中细胞凋亡率、cleaved caspase-3表达降低,心肌中Survivin、p-PI3K、p-PKB表达升高(P＜0.05),心肌中CVB3滴度及RNA表达无明显变化(P＞0.05);与VMC+EGCG组比较,VMC+EGCG+LY组血清cTnI、LDH含量、心肌中细胞凋亡率、cleaved caspase-3表达升高,心肌中Survivin、p-PI3K、p-PKB表达降低(P＜0.05),心肌中CVB3滴度及RNA表达无明显变化(P＞0.05).以上结果表明EGCG对VMC小鼠心肌细胞凋亡具有抑制作用且该作用与激活PI3K/PKB通路有关.     

黄芪多糖对C3H10T1/2细胞棕色脂肪分化中长链非编码RNA表达谱的影响

本试验旨在探究黄芪多糖(APS)对小鼠间充质干细胞C3H10T1/2细胞棕色脂肪化过程中长链非编码RNA(lncRNA)表达谱的影响。试验以C3H10T1/2细胞为研究对象,在成脂诱导分化培养液中添加0.4 g/L的APS,以诱导分化1.5 d的细胞构建文库后测序,筛选差异m RNAs和差异lncRNAs,并对差异m RNAs和差异lncRNAs的顺式及共表达作用预测的靶基因进行了功能分析。通过荧光定量PCR随机分析了3个lncRNAs和3个m RNAs的表达水平,验证了测序结果的准确性。研究结果表明,本次测序共得到13 450个lncRNAs和57 776个m RNAs。通过对其表达量、长度、外显子个数和成对重复性检验分析证明了测序数据的可靠性较好。筛选共得到153个差异表达的lncRNAs和1 238个差异表达的m RNAs。结果表明,差异m RNAs主要基因本体(GO)注释富集在53个功能分类中,京都基因与基因组百科全书(KEGG)分析富集在343条通路中。差异lncRNAs顺式及共表达作用预测的靶基因GO注释分别富集在34和33个功能分类中,在分子功能中条目一致。KEGG分析显示,多个基因富集在脂肪代谢和脂肪分化的信号通路中,尤其在胰高血糖素及cAMP信号通路中富集显著。综上表明,APS导致了C3H10T1/2细胞成脂分化中lncRNA表达谱的变化。本研究结果可为进一步解析APS对干细胞的分化调节提供科学依据。     

香菇多糖对急性弓形虫感染过程中CD4+CD25+Foxp3+调节性T细胞的调节作用研究

目的 探讨香菇多糖(Lentinan,Lent)对急性弓形虫感染BALB/c小鼠CD4+ CD25+ Foxp3+调节性T细胞(Tregs)数量和功能的调节作用.方法 对RH强毒株感染的BALB/c小鼠进行不同时间点的Lent预处理,动态观察用药后各组感染小鼠的生存率;在感染后第0、3、5、8和10天提取小鼠的脾细胞,FACS检测Tregs细胞数量的动态变化,ELISA法检测脾细胞培养上清中IL-10的分泌水平.结果 感染前6 d 1 mg/kg Lent用药组与药物未处理组相比显著提高了弓形虫感染小鼠的生存率;具有免疫抑制功能的Tregs数量于感染后8d达峰值,同时免疫应答中关键细胞因子IL-10的分泌水平也于感染后8d和10d显著增加.结论 对急性弓形虫感染的BALB/c小鼠采用Lent预处理之后能有效的调节Tregs的数量和功能,从而调控Th1/Th2之间的动态平衡达到治疗弓形虫的作用,为弓形虫病的临床治疗提供的新的理论依据.     

淋巴细胞趋化因子增强柯萨奇病毒DNA疫苗黏膜免疫及预防小鼠心肌炎的作用

本研究在已建立的柯萨奇病毒B3型(CVB3)黏膜疫苗chitosan-pVP1基础上,引入C家族趋化因子,即淋巴细胞趋化因子(LTN),以期诱导更强的黏膜免疫应答,获得更有效的免疫保护作用。将pLTN与pVP1各50μg混合后,与chitosan形成共聚复合物,隔周滴鼻免疫小鼠,共4次;末次免疫后2周,检测血清IgG、粪便IgA及肠系膜淋巴结细胞毒性T淋巴细胞(CTL)活性。同时,以3LD50/0.1mlCVB3腹腔感染小鼠,7d后检测血清肌酸激酶(CK)活性及心肌病理学改变。结果显示,与对照组相比,chi-(pVP1+pLTN)可显著提高CVB3特异性血清IgG水平、粪便IgA水平以及增强肠系膜淋巴结特异性CTL应答。病毒攻击后,chi-(pVP1+pLTN)组心肌炎发病率仅为16.7%,显著低于chi-(pVP1+pcDNA3.1)组的33.3%。心肌组织病理显示,chi-(pVP1+pLTN)组心外膜下仅有轻微炎症,而chi-(pVP1+pcDNA3.1)组除心外膜下有较多淋巴细胞聚集外,心肌内尚有少量炎症浸润和坏死灶。结果提示,LTN与VP1质粒经chitosan共包装后进行滴鼻免疫,可增强CVB3特异性黏膜免疫应答,更有效地预防病毒性心肌炎的发生。     

Antiviral Effect of Interferon-Induced Guanylate Binding Protein-1 against Coxsackie Virus and Hepatitis B Virus B3 in Vitro

Guanylate binding protein-1(GBP-1) is an interferon-induced protein. To observe its antiviral effect against Hepatitis B virus (HBV) and Coxsackie virus B3 (CVB3), we constructed an eukaryotic expression vector of human GBP-1(hGBP-1). Full-length encoding sequence of hGBP-1 was amplified by long chain RT-PCR and inserted into a pCR2.1 vector, then subcloned into a pCDNA3.1(-) vector. Recombinant hGBP-1 plasmids and pHBV1.3 carrying 1.3-fold genome of HBV were contransfected into HepG2 cells, and inhibition effect of hGBP-1 against HBV replication was observed. Hela cells transfected with recombinant hGBP-1 plasmids were challenged with CVB3, and viral yield in cultures were detected. The results indicated that recombinant eukaryotic expression plasmid of hGBP-1 was constructed successfully and the hGBP-1 gene carried in this plasmid could be efficiently expressed in HepG2 cells and Hela cells. hGBP-1 inhibit CVB3 but not HBV replication in vitro. These results demonstrate that hGBP-1 mediates an antiviral effect against CVB3 but not HBV and perhaps plays an important role in the interferon-mediated antiviral response against CVB3.     

Antiviral Effect of Interferon-Induced Guanylate Binding Protein-1 against Coxsackie Virus and Hepatitis B Virus B3 in Vitro

Guanylate binding protein-1(GBP-1)is an interferon-induced protein.To observe its antiviral effect against Hepatitis B virus(HBV)and Coxsackie virus B3(CVB3),we constructed an eukaryotic expression vector of human GBP-1(hGBP-1).Full-length encoding sequence of hGBP-1 was amplified by long chain RT-PCR and inserted into a pCR2.1 vector,then subcloned into a pCDNA3.1(-)vector.Recombinant hGBP-1 plasmids and pHBV1.3 carrying 1.3-fold genome of HBV were contransfected into HepG2 cells,and inhibition effect of hGBP-1 against HBV replication was observed.Hela cells transfected with recombinant hGBP-1 plasmids were challenged with CVB3,and viral yield in cultures were detected.The results indicated that recombinant eukaryotic expression plasmid of hGBP-1 was constructed successfully and the hGBP-1 gene carried in this plasmid could be efficiently expressed in HepG2 cells and Hela cells.hGBP-1 inhibit CVB3 but not HBV replication in vitro.These results demonstrate that hGBP-1 mediates an antiviral effect against CVB3 but not HBV and perhaps plays an important role in the interferon-mediated antiviral response against CVB3.     

高渗盐水雾化吸入治疗儿童支原体肺炎继发哮喘发作及对其T淋巴细胞亚群及Th1、Th2型细胞因子的影响

目的:探讨高渗盐水雾化吸入治疗儿童支原体肺炎继发哮喘发作的临床疗效及对其T淋巴细胞亚群及Th1、Th2型细胞因子的影响。方法:选取我院2015年6月到2017年6月间收治的支原体肺炎继发哮喘发作患儿100例为研究对象。随机分为对照组和观察组,各50例。两组均给予吸氧、抗生素、维持酸碱度、电解质平衡及相应症状等对症治疗,对照组给予生理盐水结合沙丁胺醇进行雾化治疗,观察组采用3%高渗盐水结合沙丁胺醇进行雾化治疗,比较两组患儿发热、咳嗽、肺内啰音、咽部肿痒等症状消失时间及临床疗效;比较两组治疗前后CD3~+、CD4~+、CD8~+、CD4~+/CD8~+、IFN-γ及IL-4水平情况。结果:观察组患儿发热、咳嗽、肺内啰音、咽部肿痒等临床症状消失时间显著早于对照组,差异有统计学意义(P0.05);观察组总有效率96.00%明显高于对照组80.00%,差异有统计学意义(P0.05);两组患儿治疗前CD3~+、CD4~+、CD8~+、CD4~+/CD8~+、IFN-γ及IL-4水平比较差异无统计学意义(P0.05);治疗后CD3~+、CD4~+、CD4~+/CD8~+及IFN-γ水平均明显升高,CD8~+及IL-4水平明显降低,差异均有统计学意义(均P0.05);且观察组治疗后CD3~+、CD4~+、CD4~+/CD8~+及IFN-γ水平均明显高于对照组,CD8~+及IL-4水平明显低于对照组,差异均有统计学意义(均P0.05)。结论:高渗盐水雾化吸入能够显著改善支原体肺炎继发哮喘发作患儿临床症状,提高CD3~+、CD4~+、CD4~+/CD8~+及Th1水平,抑制CD8~+及Th2水平,临床疗效确切,值得临床推广应用。     

Inhibitory Effect of 8-(N,N-Diethylamino)octyl-3, 4, 5-Trimethoxybenzoate Hydrochloride (TMB-8) on Germination of Bacillus cereus T Spores

Effect of 8-(N,N-diethylamino)octyl-3, 4, 5 - trimethoxybenzoate hydrochloride (TMB-8), a calcium antagonist, on germination of Bacillus cereus T spores induced by L -alanine and inosine was investigated. TMB-8 had no effect on the germination of heat-activated spores, whereas it inhibited that of nonactivated spores. The TMB-8 inhibitory effect was antagonized competitively by inosine, but not by L -alanine. Addition of Ca²⁺ reversed the inhibitory effect of TMB-8 in a dose-related fashion. Based on the results, a role of inosine and a site(s) for inhibitory action of TMB-8 in the process leading to the germination of nonactivated spores were discussed.     

The role of calcium ions in the permeability changes produced by external ATP in transformed 3T3 cells

External ATP causes a rapid increase in passive permeability to nucleotides and phosphate esters in transformed cell lines, such as 3T6 mouse fibroblasts. However, untransformed lines, such as 3T3, do not show a similar sensitivity to external ATP. Ca²⁺ inhibits permeabilization, but only at concentrations approaching those of external ATP. In contrast, La³⁺ and Tb³⁺ inhibit ATP-dependent permeabilization at one-fifth the concentration of external ATP. Considering reports that lanthanides can substitute for calcium ion in many enzymatic reactions, often with a higher affinity, it would appear that Ca²⁺ plays a specific role in the maintenance of a passive membrane permeability barrier and in opposing the effects of external ATP.Other data suggest a regulatory role for the Ca²⁺-calmodulin complex in the permeabilization process. Trifluoperazine, chlorpromazine and W-7, compounds which inhibit cellular functions dependent on the Ca²⁺-calmodulin complex, are able to enhance the effect of external ATP. Thus, a dramatic stimulation of nucleotide permeability occurs with concentrations of external ATP and inhibitor that are ineffective when added alone. Calmodulin antagonists and low concentrations of external ATP increased membrane permeability to Na⁺ and K⁺ as was previously shown for permeabilization with ATP alone. Earlier studies have shown that energy inhibitors which reduce intracellular ATP levels greatly increase the sensitivity of transformed cells to external ATP. However, the Ca²⁺-calmodulin antagonists used in the present study exert their effects at concentrations which do not alter intracellular ATP levels.     

Autophagy inhibition in early but not in later stages prevents 3T3-L1 differentiation: Effect on mitochondrial remodeling

Autophagy is essential for successful white adipocyte differentiation but the data regarding the timing and relevance of autophagy action during different phases of adipogenesis are limited.