自噬在结核分枝杆菌感染中的作用及相关基因的表达

目的:探讨自噬在结核分枝杆菌(MTB)感染中的作用及相关基因的表达.方法:透射电镜观察在雷帕霉素诱导下自噬体的形成,通过克隆形成单位(CFU)检测自噬形成后对胞内感染的MTB H37Rv菌株的清除作用,以实时定量PCR方法检测自噬相关基因atg5、atS7、atg8、atg12 mRNA表达水平.结果:在宙帕霉素诱导下,RAW264.7细胞可形成自噬体,自噬体产牛后对胞内感染的H37Rv菌株具有一定的清除作用.在MTB感染过程中参与自噬形成的atg5、atg8、atg12 mRNA表达量上调,而atg7表达量无变化.结论:自噬参与抗MTB免疫应答过程,而ats5、atg8、atg12是MTB感染形成自噬体的重要调控分子.     

结核分枝杆菌小分子热休克蛋白16.3蛋白的原核表达及功能检测

目的进行结核分枝杆菌小分子热休克蛋白MTB Hsp16.3原核表达载体的构建、表达、纯化并初步观察其生物学效应。方法提取临床H37Rv分离株基因组DNA,PCR扩增Hsp16.3基因,将其重组到原核表达载体Pet28a中,构建原核表达载体Pet28a-Hsp16.3,进行双酶切及测序鉴定。将测序正确的重组质粒转化至E.coli BL21(DE3)中,经IPTG诱导表达后,对表达产物进行SDS-PAGE检测,同时通过镍柱纯化试剂盒纯化Hsp16.3,测定纯化后蛋白浓度,并进行Western blot法检测。将不同浓度纯化后蛋白作用小鼠腹腔巨噬细胞,实时定量PCR(qRT-PCR)检测巨噬细胞IL-10和IFN-γ的表达,同时设定空白对照组及阳性对照组。结果成功构建重组质粒Pet28a-Hsp16.3,并在E.coli BL21(DE3)中获得成功表达,通过镍柱纯化系统得到纯化Hsp16.3融合蛋白。qRT-PCR检测结果显示,不同浓度纯化后Hsp16.3蛋白作用小鼠腹腔巨噬细胞,可促进IFN-γ的产生而抑制IL-10的产生。结论成功克隆、表达和纯化了MTB Hsp16.3蛋白,Hsp16.3能促进小鼠腹腔巨噬细胞产生IFN-γ,抑制IL-10的产生。     

结核分枝杆菌小分子热休克蛋白16．3蛋白的原核表达及功能检测北大核心CSCD

目的进行结核分枝杆菌小分子热休克蛋白MTB Hsp16.3原核表达载体的构建、表达、纯化并初步观察其生物学效应。方法提取临床H37Rv分离株基因组DNA,PCR扩增Hsp16.3基因,将其重组到原核表达载体Pet28a中,构建原核表达载体Pet28a-Hsp16.3,进行双酶切及测序鉴定。将测序正确的重组质粒转化至E.coli BL21(DE3)中,经IPTG诱导表达后,对表达产物进行SDS-PAGE检测,同时通过镍柱纯化试剂盒纯化Hsp16.3,测定纯化后蛋白浓度,并进行Western blot法检测。将不同浓度纯化后蛋白作用小鼠腹腔巨噬细胞,实时定量PCR(qRT-PCR)检测巨噬细胞IL-10和IFN-γ的表达,同时设定空白对照组及阳性对照组。结果成功构建重组质粒Pet28a-Hsp16.3,并在E.coli BL21(DE3)中获得成功表达,通过镍柱纯化系统得到纯化Hsp16.3融合蛋白。qRT-PCR检测结果显示,不同浓度纯化后Hsp16.3蛋白作用小鼠腹腔巨噬细胞,可促进IFN-γ的产生而抑制IL-10的产生。结论成功克隆、表达和纯化了MTB Hsp16.3蛋白,Hsp16.3能促进小鼠腹腔巨噬细胞产生IFN-γ,抑制IL-10的产生。     

睾酮对结核分枝杆菌感染的RAW264.7细胞自噬的影响

目的探讨睾酮对结核分枝杆菌(Mycobacterium tuberculosis,MTB)感染的鼠源巨噬细胞RAW264.7细胞自噬的影响及其分子机制。方法 MTB感染RAW264.7细胞24 h后,给予10~(-8)mol/L睾酮处理24 h;采用透射电镜观察细胞自噬情况,Western blot法检测细胞自噬相关蛋白以及MAPKs信号通路蛋白。结果睾酮干预MTB感染的RAW264.7细胞后自噬体、自噬特异标志物LC3Ⅱ均明显增加(P0.01),通路蛋白JNK磷酸化水平明显上调(P0.001)。结论睾酮可能通过激活JNK信号通路诱导MTB感染的RAW264.7细胞自噬。     

结核分枝杆菌国际标准强毒株H37Rv菌株Hsp16.3基因打靶载体的构建与鉴定

结核分枝杆菌感染人体后主要寄生在宿主巨噬细胞内,结核分枝杆菌小分子热休克蛋白( small heat shock proteins,sHSPs) Hsp16.3是其在宿主巨噬细胞内生存繁殖所必需的蛋白质,已有研究表明Hsp16.3与结核分枝杆菌的潜伏感染关系密切,本研究利用基因敲除技术构建了结核分枝杆菌国际标准强毒株H37Rv菌株Hsp16.3基因打靶载体,为进一步敲除结核分枝杆菌Hsp16.3基因( hspX,Rv2031C),并为研究Hsp16.3基因的功能及探讨结核病的防治提供可行的研究方法.     

维生素D诱导自噬对巨噬细胞清除结核分枝杆菌的作用

目的:探讨维生素D诱导巨噬细胞产生自噬并清除巨噬细胞内结核分枝杆菌(Mtb)的作用。方法:使用豆蔻酰佛波醇乙酯(PMA)诱导U937细胞分化使之具有吞噬能力,分化后的U937细胞随机分为阴性对照组、维生素D组、自噬抑制剂(3-MA)+维生素D组、阳性对照(雷帕霉素)组,以Mtb感染U937细胞6 h。感染后第4天,利用半定量RT-PCR检测自噬相关基因ATG5、Beclin-1及LL-37、LC3B mRNA的表达,流式细胞术检测LC3B-Ⅱ+和/或结核分枝杆菌抗原85A+(Ag85A+)的细胞。结果:与对照组相比,维生素D组ATG5、Beclin-1、LL-37、LC3B mRNA的表达增强(P<0.01),LC3B-Ⅱ+-细胞增多,Ag85A+-细胞减少,且LC3B-Ⅱ+-Ag85A--细胞增加(维生素D组38.0%比阴性对照组1.08%)。与维生素D组相比,在自噬抑制剂3-MA+维生素D组中,不但ATG5、Beclin-1、LC3B的mRNA表达受到抑制,LL-37的mRNA表达较维生素D组减少,而且3-MA抑制了细胞LC3B-Ⅱ的表达,同时抑制了1,25(OH)2D3对LC3B-Ⅱ+-Ag85A--细胞增加的作用。结论:维生素D能够诱导巨噬细胞产生自噬作用,并进一步有助于巨噬细胞清除结核分枝杆菌。     

自噬基因多态性与机体对结核病的敏感性

结核分枝杆菌是胞内病原体,主要感染巨噬细胞.最近的研究显示,自噬参与了胞内杀死结核分枝杆菌的过程.胞内结核分枝杆菌的存活与自噬的抑制有关,而自噬和自噬相关基因缺失鼠更容易受结核分枝杆菌感染.据报道,在一些地区,自噬基因IRGM的高多态性与保护人们抵抗结核分枝杆菌的感染有关.此外,R2RX7也与结核病相关.     

巨噬细胞的自噬及其在抗结核分枝杆菌感染中的作用

自噬是广泛存在于真核细胞内的一种溶酶体依赖性的自降解途径,可通过降解长寿蛋白和受损细胞器维持细胞内的平衡.近年来的研究发现,巨噬细胞的自噬还是固有免疫和适应性免疫的重要组成部分,可参与胞内感染病原体的清除.目前已发现有多种途径参与自噬的诱导和调节.在感染的巨噬细胞内,诱导自噬的发生能促进吞噬体和溶酶体的融合,抑制胞内结核分枝杆菌(Mtb)的存活.但同时Mtb也可通过某些机制抑制巨噬细胞自噬的发生以逃避巨噬细胞的杀伤,进而长期持留于巨噬细胞内.深入了解巨噬细胞自噬与胞内Mtb相互作用的机制,有助于人类更好的预防和控制结核病.     

布鲁菌干扰MΦ泛素依赖型自噬通路的研究

目的 探讨布鲁菌减毒株S1330对于小鼠巨噬细胞(MΦ)泛素依赖型自噬通路的影响.方法 应用布鲁菌S1330株作用于小鼠MΦ构建体外感染模型.观察MΦ内的吞噬过程、泛素化水平及自噬水平.实验设立巨噬细胞正常组、感染组、自噬诱导对照组、自噬诱导感染组,应用吉姆萨染色法、免疫荧光法、Western blot法分别观察不同时间点、不同组MΦ内泛素化蛋白含量及自噬水平变化.结果 布鲁菌感染MΦ0.5 h胞内出现泛素化菌体蛋白,随着感染时间的延长,胞内泛素化蛋白聚集持续增多,12 h MΦ死亡;与之相对应的自噬体标记物LC3B蛋白表达严重不足,而自噬诱导的感染组MΦ内泛素化的菌体蛋白比例减少.结论 布鲁菌S1330株感染MΦ启动胞内泛素化机制,却干扰泛素依赖型自噬通路的成熟,使泛素化菌体蛋白大量聚集不能有效清除,最终导致了MΦ功能障碍而死亡.     

巨噬细胞的自噬及其在抗结核分枝杆菌感染中的作用

白噬是广泛存在于真核细胞内的一种溶酶体依赖性的自降解途径,可通过降解长寿蛋白和受损细胞器维持细胞内的平衡。近年来的研究发现,巨噬细胞的自噬还是固有免疫和适应性免疫的重要组成部分,可参与胞内感染病原体的清除。目前已发现有多种途径参与自噬的诱导和调节。在感染的巨噬细胞内,诱导自噬的发生能促进吞噬体和溶酶体的融合,抑制胞内结核分枝杆菌（Mtb）的存活。但同时Mtb也可通过某些机制抑制巨噬细胞自噬的发生以逃避巨噬细胞的杀伤,进而长期持留于巨噬细胞内。深入了解巨噬细胞自噬与胞内Mtb相互作用的机制,有助于人类更好的预防和控制结核病。     

Insensitivity of PI3K/Akt/GSK3 signaling in peripheral blood mononuclear cells of age-related macular degeneration patients

Our recent studies with cultured retinal pigment epithelium cells suggested that overexpression of interleukin 17 receptor C (IL-17RC), a phenomenon observed in peripheral blood and chorioretinal tissues with age-related macular degeneration (AMD), was associated with altered activation of phosphatidylinositide 3-kinase (PI3K), Akt, and glycogen synthase kinase 3 (GSK3). We wondered whether or not altered PI3K, Akt, and GSK3 activities could be detected in peripheral blood mononuclear cells (PBMC) obtained from AMD patients. In the patients' PBMC, absentor reduced serine-phosphorylation of GSK3α or GSK3β was observed, which was accompanied with increased phosphorylation of GSK3 substrates (e.g. CCAAT enhancer binding protein α, insulin receptor substrate 1, and TAU), indicative of enhanced GSK3 activation. In addition, decreased protein mass of PI3K85α and tyrosinephosphorylation of PI3K50α was present in PBMC of the AMD patients, suggesting impaired PI3K activation. Moreover, abnormally lowered molecular weight forms of Akt and GSK3 were detected in PBMC of the AMD patients. These data demonstrate that despite the presence of high levels of IL-17RC, Wnt-3a and vascular endothelial growth factor, the PI3K/Akt/GSK3 signaling pathway is insensitive to these stimuli in PBMC of the AMD patients. Thus, measurement of PI3K/Akt/GSK3 expression and activity in PBMC may serve as a surrogate biomarker for AMD.     

Deletion of chromosome 3 and a 3;20 reciprocal translocation demonstrated by chromosome painting

The combined use of high resolution banding and chromosome painting techniques allowed us to identify a reciprocal translocation involving chromosomes 3 and 20 and simultaneous interstitial deletion of chromosome 3 in a patient with several minor anomalies of the face and hands. His karyotype is described as 46,XY,t(3;20) (p14.2;p12.2),del(3)(p11-p14.1). © 1995 Wiley-Liss, Inc.     

Properties of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) films modified with polyvinylpyrrolidone and behavior of MC3T3-E1 osteoblasts cultured on the blended films

A series of composite films of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) modified with polyvinylpyrrolidone (PVP) was prepared by varying the ratio of constituents, and their properties and cytocompatibility were evaluated. The hydrophilicity of the blended materials surfaces increased and the amounts of fibronectin and laminin adsorbed on the materials surface increased remarkably compared with PHBHHx. FT-IR spectra of the blended films showed a new band, implying that a surface physical interpenetrating network structure had formed. Scanning electron microscopy showed that there were dense pits and holes on the blended films surface. For the films of PHBHHx with 20 wt% and 40 wt% PVP, MTT assay indicated that PVP enhanced cell adhesion and proliferation, but that the effects were impaired by excessive PVP. The results suggested that proper addition of PVP increased the cytocompatibility of PHBHHx because the material surface had increased hydrophilicity and presented an appropriate morphology.     

A novel pathogenic variant in TNPO3 in a Hungarian family with limb-girdle muscular dystrophy 1F

Limb-girdle muscular dystrophies (LGMDs) are a group of genetically heterogeneous muscular diseases that predominantly affect the proximal muscles. Pathogenic variants in TNPO3 have been associated with a rare, autosomal dominant limb-girdle muscular dystrophy 1F (LGMD1F) in a large Italian-Spanish family and an isolated LGMD1F case. Here we present two individuals from a Hungarian family with an early-onset, slowly progressive muscular dystrophy. Both the female proband and her affected son had delayed early motor milestones including first walking at 14 months and 18 months, respectively. Both present with progressive weakness of facial, bulbar, axial, and distal muscles especially of the lower extremities. Electromyography indicated myogenic damage and muscle biopsy from the proband showed myopathic alterations with sarcoplasmic masses and signs of mitochondrial dysfunction. Exome sequencing of the female proband identified a novel c.2767delC p.(Arg923AspfsTer17) variant in TNPO3. Sanger sequencing confirmed the presence of the TNPO3 variant in the affected son; the unaffected son did not have the variant. The identification of the c.2767delC variant further supports the clinical significance of TNPO3 and expands the clinical spectrum of TNPO3-associated LGMD1F.     

苦参总碱体外抗柯萨奇B病毒3型作用测定及其机理的初步研究

先用酸水浸泡、氯仿萃取等处理提取苦参总碱，然后通过微量细胞培养法，病毒蛋白质Ｗｅｓｔｅｒｎｂｌｏｔ，分析了不同浓度苦参总碱对病毒增殖和衣壳蛋白含量的影响，表明苦参总碱在体外试验中能抑制柯萨奇Ｂ病毒３型（ＣＶＢ３）的增殖，并呈一定的剂量依赖性。通过病毒吸附和穿入细胞前后苦参总碱作用的比较，显示苦参总碱能进入细胞内发挥抗病毒作用，它可能不影响ＣＶＢ３吸附、穿入等环节，而是影响ＣＶＢ３侵入细胞后某环节，特别是病毒的生物合成。     

Preparation and characterization of poly(hydroxyalkanoate) from the fermentation of Haloferax mediterranei

In this study, fed-batch fermentation of Haloferax mediterranei using glucose and yeast extract as carbon and nitrogen source, respectively, was carried out to produce poly(hydroxyalkanoate) (PHA). After fermentation for 117 h, the concentration of H. mediterranei and PHA content reached 85.8 g/l and 48.6%, respectively. ¹H- and ¹³C-NMR spectra proved that the produced PHA was poly(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV) co-polymer. However, further fractionation using chloroform/acetone revealed that the produced PHA consisted of at least two compositionally different co-polymers (P1 and P2). One P(3HB-co-3HV) co-polymer (P1, 93.4 wt%) contains 10.7 mol% of 3-HV unit in the chain structure and has a high molecular weight of 569.5 kg/mol. The other one (P2, 6.6 wt%) has a slightly higher 3-HV content, ca. 12.3 mol%, but its molecular weight is relatively low, 78.2 kg/mol. Both fractions exhibit two overlapped melting peaks measured by differential scanning calorimetry when the heating rate is at and below 20°C/min. For example, at a heating rate of 10°C/min, the two melting peaks occur at 134.8°C and 144.3°C for P1, and 131.1°C and 140.6°C for P2. Through observing the variation of relative intensity of these two melting peaks by changing the heating rate, it was proven that the phenomenon is caused by a melt/recrystallization process. Glass-transition temperature, crystallization temperature and thermal degradation behavior of these co-polymers were also discussed.     

Fibroblast culture on surface-modified poly (glycolide-co-ε-caprolactone) scaffold for soft tissue regeneration

Novel porous matrices made of a copolymer of glycolide (G) and ε-caprolactone (CL) (51 : 49, M_w 103 000) was prepared for tissue engineering using a solvent-casting particulate leaching method. Poly(glycolide-co-ε-caprolactone) (PGCL) copolymer showed a rubber-like elastic characteristic, in addition to an amorphous property and fast biodegradability. In order to investigate the effect on the fibroblast culture, PGCL scaffolds of varying porosity and pore size, in addition to surfacehydrolysis or collagen coating, were studied. The large pore-sized scaffold (pore size >150 μm) demonstrated a much greater cell adhesion and proliferation than the small pore-sized one. In addition, the higher porosity, the better the cell adhesion and proliferation. The surface-hydrolyzed PGCL scaffold showed enhanced cell adhesion and proliferation compared with the unmodified one. Type I collagen coating revealed a more pronounced contribution for increased cell interactions than the surface-hydrolyzed one. These results demonstrate that surface-modified PGCL scaffold can provide a suitable substrate for fibroblast culture, especially in the case of soft tissue regenerations.     

泛素羧基末端水解酶L3及其与疾病和肿瘤的相关性

去泛素化酶(deubiquitinating enzymes,DUBs)在调节细胞内稳态、细胞分化和凋亡等过程中,均扮演着重要角色。泛素羧基末端水解酶L3(ubiquitin C-terminal hydrolases L3,UCH-L3)是去泛素化酶家族中,半胱氨酸蛋白酶类的一种,可以催化靶蛋白短肽链上附着的多余的完整泛素分子的脱落,从而实现对靶蛋白的调控以及泛素分子的循环再利用。目前对该水解酶的研究还不充分,但其在多种疾病以及肿瘤中的高表达情况,说明在疾病、肿瘤发生发展中起着重要的作用。本文将针对该水解酶及其与疾病和肿瘤的相关性作一综述。     

The Influence of Different Cultivating Conditions on Polymorphonuclear Leukocyte Apoptotic Process In Vitro,II: Ultrastructural Characteristics of PMN Populations Incubated with Proteinase 3 Anti-neutrophil Autoantibodies

The present study shows the effects of proteinase 3 anti-neutrophil cytoplasmic autoantibodies (PR3 ANCA) on polymorphonuclear leukocytes (PMN) apoptotic processes in vitro. The results are part of a generalized morphological analysis of 3 identical experiments on the influence of different cultivating conditions on the apoptotic processes. As controls, the authors use the results on spontaneous PMN apoptosis (Guejes L, Zurgil N, Deutsch M, Gilburd B, Shoenfeld Y. Ultrastruct Pathol. 2003;27:23–32) and PMN populations incubated with normal human IgG. Interaction of PR3 ANCA with the target antigen proteinase 3 (PR3) is one of the crucial pathogenic factors in Wegener granulomatosis (systemic autoimmune vasculitis). Following 40 min and 12 h incubation, PMN populations were evaluated by light microscopy, transmission electron microscopy, and immunogold electron microscopy. Twelve-hour cultures, either control or incubated with PR3 ANCA, contained different cell forms ranging from normal cells to cells at the final stages of apoptosis. Neutrophils at the state of complete manifestation of apoptotic phenotype were analyzed and compared. Three morphologically distinct apoptotic cell lines were characteristic for all PMN populations studied, regardless of cultivating conditions. As in spontaneous apoptosis, these cell lines are code-named “first,” “second,” and “third.” The present study has shown, firstly, that in the presence of PR3 ANCA, all 3 apoptotic lines were modified or altered. Secondly, the modifications or alterations of apoptotic cell lines effected by PR3 ANCA are specific for each cell line: the “first” line is characterized by intensification and modification of activation; the “second” by vacuolized cell forms; and the “third” by pronounced lytic alterations of the nuclei, while the cytoplasm is fully identical to that of control cell lines.