猪血脑屏障内皮细胞微囊的制备与鉴定

选二花脸母猪和长白母猪各 5头 ,取大脑皮质 ,制备微血管 ,分离内皮细胞微囊 ,应用倒置显微镜和透射电镜观察微囊的完整性 ;通过测定γ-谷氨酰转肽酶、5′-核苷酸酶、乙酰胆碱酯酶、乳酸脱氢酶、碱性磷酸酶等标志酶活性 ,鉴定微囊的纯度和生物学活性。结果表明 ,用该方法分离得到的大脑皮质内皮细胞微囊的完整性较好 ,纯度较高 ,生物学活性好 ,适合于对血脑屏障的转运研究     

微血管内皮细胞的体外分离培养方法概述

微血管内皮细胞在许多病理生理过程中起殴丶缘淖饔?应用体外培养的微血管内皮细胞,不仅广泛应用于微循环系统对不同生理或病理刺激的反应,也可阐明伴随微血管功能障碍和组织损伤的某些疾病的病理机制,所以分离培养动物及人各个器官组织微血管内皮细胞的报道日渐增多.微血管内皮细胞分离方法主要有3种,包括酶消化法、机械分离法和磁珠分离法,每种方法各有利弊.微血管内皮细胞一般通过其表面的血管紧张素酶、摄取乙酰基低密度脂蛋白和表达Ⅷ因子相关抗原进行鉴定.目前从不同组织器官分离培养微血管内皮细胞的方法已经比较成熟.     

猪脑微血管内细胞的原代分离培养与鉴定

探讨获取纯度较高的原代猪脑微血管内皮细胞的分离和培养方法。采用1月龄的三元杂交猪,通过两次酶消化、BSA和Percoll非连续梯度离心获得较纯的脑微血管段后,接种于涂布有鼠尾胶的培养瓶进行原代培养;相差显微镜观察细胞并进行纯化和传代,采用Ⅷ因子相关抗原免疫荧光检测法对培养的细胞进行鉴定。结果表明,培养24 h即可见细胞从贴壁的脑微血管段周围爬出,细胞呈短梭形,集落呈典型的鹅卵石样,区域性单层生长,6~7 d内皮细胞开始融合,血管内皮细胞特异性标志物Ⅷ因子相关抗原表达阳性。说明本试验成功的培养出了纯度较高的脑微血管内皮细胞,为后续的体外血脑屏障模型的建立奠定了基础。     

Enolase在鸭疫里默氏杆菌侵袭鸭脑微血管内皮细胞中的作用

旨在明确鸭疫里默氏杆菌烯醇化酶(Enolase)在其侵袭鸭脑微血管内皮细胞(DBMEC)以及血脑屏障(BBB)中的作用。本研究以鸭疫里默氏杆菌RA-LZ01株为亲本株,利用同源重组和结合转移的方法构建enolase基因缺失株ΔEnolase和回复株cΔEnolase,并测定RA-LZ01、ΔEnolase和cΔEnolase对DBMEC黏附和侵袭能力的差异;用上述菌株感染雏鸭,测定雏鸭血液和脑组织中的载菌量。结果表明,与亲本株RA-LZ01相比,缺失株ΔEnolase对DBMEC的黏附率和入侵率均极显著降低;回复株cΔEnolase恢复了对DBMEC的黏附和入侵能力。感染RA-LZ01、ΔEnolase和cΔEnolase菌株的雏鸭的血液载菌量无显著差异;与感染RA-LZ01和cΔEnolase菌株的雏鸭相比,感染ΔEnolase菌株的雏鸭脑组织中的载菌量极显著降低。以上结果说明,Enolase与鸭疫里默氏杆菌黏附和入侵DBMEC以及入侵雏鸭脑组织显著相关,可能为介导鸭疫里默氏杆菌突破鸭血脑屏障的毒力因子。     

CREM在体外共培养山羊睾丸生精细胞中的定位研究

为进一步深入研究CREM基因在雄性动物中的表达调控机制,试验成功建立体外生精细胞和支持细胞共培养体系,并应用免疫细胞化学研究CREM在体外培养山羊睾丸生精细胞的表达特点。结果显示:CREM仅表达于圆形精子细胞,其他生精细胞和支持细胞检测不到CREM的表达。表明CREM基因在圆形精子细胞发育过程中起重要调节作用,但其作用机制有待深入研究。     

PHGPx在体外共培养山羊睾丸生精细胞中的定位研究

为探讨磷脂氢谷胱甘肽过氧化物酶(PHGPx)在雄性动物生殖机能中的作用及其在体外培养山羊睾丸生精细胞的定位,在已成功构建的山羊睾丸支持细胞-生精细胞共培养的基础上,制作细胞爬片,采用免疫细胞化学技术定位PHGPx的表达。结果显示,PHGPx在支持细胞、精原细胞中不表达,在圆形精子细胞细胞质检测到阳性表达产物。表明PHGPx在精子发生后期圆形精子的变态发育中起特定的调节作用,但作用机制有待深入研究。     

黄芪多糖对体外培养大鼠肠黏膜微血管内皮细胞增殖的影响

研究黄芪多糖对体外培养的大鼠肠黏膜微血管内皮细胞（rat intestinal mucosa microvascular endothelial cells,RIMMVECs）增殖的影响。应用不同浓度的黄芪多糖作用于体外培养的RIMMVECs。通过MTT法检测黄芪多糖对RIMMVECs增殖的影响。不同浓度的黄芪多糖对细胞增殖的影响不同:黄芪多糖浓度在25～800 mg/L时可明显促进细胞的增殖（P<0.05或0.01）;浓度为1.6×10³ mg/L时对细胞的增殖无影响;浓度在3.2×10³～25.6×10³ mg/L时对细胞生长具有抑制作用（P<0.05或0.01）。黄芪多糖有促进体外培养RIMMVECs的增殖作用,此作用对保护内皮损伤、加速内皮修复有重要作用。     

肉鸡肺动脉内皮细胞的体外培养和鉴定

分别采用组织贴块法和胶原酶消化法培养肉鸡肺动脉内皮细胞,并对其进行传代。采用形态学和凝血因子相关抗原免疫组化染色法对所培养的细胞进行鉴定。结果表明,2种方法均能够获取原代肉鸡肺动脉内皮细胞,采用贴块法培养2周左右可获得单层生长的细胞,而采用胶原酶消化法3~5d即可获得单层生长的细胞。原代肉鸡肺动脉内皮细胞在体外可传5~6代。倒置显微镜和HE染色观察培养细胞的形态符合血管内皮细胞的特征,因子抗原免疫组化染色结果呈阳性进一步证明培养的细胞是肺动脉内皮细胞。     

体外血脑屏障模型的构建及致脑膜炎粪肠球菌对其的影响

试验旨在探索致脑膜炎粪肠球菌对体外血脑屏障功能的影响,寻求稳定的构建体外血脑屏障损伤模型的方法。选用1周龄ICR小鼠进行原代脑微血管内皮细胞（BMEC）分离并通过BMEC特有的凝血因子Ⅷ（coagulation factor Ⅷ,Factor Ⅷ）进行荧光标记,鉴定分离的细胞并判定分离率。选用1~3日龄ICR小鼠分离星形胶质细胞并通过其特有的胶质纤维酸性蛋白（glial fibrillary acidic protein,GFAP）对进行荧光标记,鉴定分离的细胞并判定分离率。将原代细胞传至3代,用Transwell二维小孔分别构建单层BMEC和BMEC与星形胶质细胞共培养的血脑屏障模型。选用致脑膜炎粪肠球菌、非致脑膜炎粪肠球菌和大肠杆菌DH5α感受态细胞分别与构建的体外血脑屏障模型共培养,评估致脑膜炎粪肠球菌对体外血脑屏障模型的穿透性。通过4 h渗透试验、荧光素钠通透性试验和血脑屏障损伤相关标志基因基质金属蛋白酶2（MMP-2）表达量的检测,评估致脑膜炎粪肠球菌对体外血脑屏障模型的影响。结果显示,试验成功分离到BMEC和星形胶质细胞,纯度分别达90%和95%以上。细菌穿透试验结果显示,所选的3株细菌只有致脑膜炎粪肠球菌可穿越体外血脑屏障模型。4 h渗透试验和荧光素钠通透性试验结果显示,共培养模型优于单层BMEC模型。与其他组相比,构建的体外血脑屏障与致脑膜炎粪肠球菌共培养组荧光素钠的穿透力更高,MMP-2基因的表达量明显上升。综上,BMEC与星形胶质细胞共培养的模型优于单层BMEC模型,致脑膜炎粪肠球菌可穿越体外血脑屏障模型,并介导体外血脑屏障损伤,本试验结果为揭示致脑膜炎粪肠球菌对体外血脑屏障损伤机制提供了理论依据。     

牛原代肌肉卫星细胞和前体脂肪细胞不同混合比例共培养细胞活性的比较

为探索不同细胞混合比例对牛原代肌肉卫星细胞和前体脂肪细胞共培养体系细胞活性的影响,本研究采集新生秦川犊牛背最长肌和肾周脂肪,分离提取肌卫星细胞和前体脂肪细胞,采用DMEM/F12培养基,建立不同血清浓度(5%、10%、15%、20%的胎牛血清,FBS)的牛原代肌肉卫星细胞和前体脂肪细胞的共培养体系,然后通过分别调整各共培养体系中肌卫星细胞和前体脂肪细胞的混合比例(肌肉细胞∶脂肪细胞=10∶1、5∶1、2∶1),来研究细胞混合比例对各共培养体系的影响。共培养14d,每两天更换培养基,并采用MTT染色法测定细胞活性。统计分析后发现∶随着共培养体系中前体脂肪细胞比例增加,共培养细胞活性增加;共培养10d以后不同混合比例细胞活性有着显著差异(P<0.05);混合比例为2∶1时共培养细胞活性最高。综上,为了获得最好的牛肌卫星细胞和前体脂肪细胞共培养效果,达到较高的细胞培养活性,建议牛肌卫星细胞和前体脂肪细胞按5∶1或2∶1的混合比例进行共培养。     

猪胎盘屏障的研究进展

妊娠是一个微妙而复杂的生理过程,胎盘的建立为母胎之间的物质交换奠定了基础.对于猪来说,胎盘功能障碍会降低胎盘效率,引起猪宫内发育迟缓、窝内体重变异增大,给养猪业的经济效益带来巨大损失.胎盘是母胎之间的一道天然屏障,其功能较为复杂,有物质交换、排泄、免疫保护、合成和分泌等多种功能.鉴于胎盘屏障的重要作用,本文综述了猪胎盘...     

Oviduct epithelial cell co-culture of early porcine embryos.

One- to 16-cell porcine embryos were cultured in either Whittens medium supplemented with bovine serum albumin and fetal calf serum (WM) or in the same medium with porcine oviduct epithelial cell co-culture (WM-Poec). All stages of embryos cultured in WM-POEC had higher cell counts after 144-168 h of development than did embryos in WM. There was however, no significant difference in blastocyst formation rate of embryos cultured in WM-POEC over those cultured in WM. A high proportion of the embryos entering culture at the 1-2-cell were able to pass the 4-cell block stage in both WM and WM-POEC, 81% and 77%, respectively. In both media, most of the 1-2-cell embryos arrested their development at the compacted morula stage and failed to blastulate while embryos initiating culture at the 4- and 8-16-cell embryos formed blastocysts in culture at a rate of 80-90%.     

A bovine mammary endothelial/epithelial cell culture model of the blood/milk barrier.

The complex nature of the mammary gland has hampered in-depth studies of the relationship of the circulatory system to cells lining the teat ducts and alveoli of the gland. This study reports an in vitro model of endothelial and epithelial cells separated by a subcellular matrix that simulates the blood milk barrier of the bovine mammary gland. Dual chamber culture dishes with a porous membrane separating the upper and lower chamber were used. Endothelial and epithelial cells were cultured on opposite sides of the porous membrane. A collagen and fibroblast subcellular matrix, separating the 2 cell layers, simulated the in vivo interstitial tissue. Changes in surface binding of anti-bodies to polymorphonuclear neutrophils (PMN) following their migration from the upper to the lower chamber simulated the passage of PMN from blood to milk. Changes in the binding of antibodies to PMN agreed with results observed following the migration of PMN from blood to milk in vivo. This gives credence to the model's potential value for studies where more direct observation of the blood/milk barrier is required. The model will be further tested for its usefulness as an assay for determining: 1) antibiotic diffusion from milk to blood and from blood to milk, 2) cytotoxicity of prophylactic and therapeutic mammary infusion products, 3) factors affecting bacterial adhesion and penetration of mammary epithelial tissue, 4) effectiveness of antibodies present in lacteal secretions in preventing bacterial adhesion, and 5) the feasibility of gene constructs to induce synthesis and secretion of mastitis-preventing compounds and prophylactic and therapeutic compounds for treatment of human disorders.     

Disruption of blood-brain barrier by an Escherichia coli isolated from canine septicemia and meningoencephalitis

Escherichia coli (E. coli) is one of the common pathogenic bacteria in veterinary clinical infection. As an opportunistic microorganism, E. coli normally does not cause diseases. However, it causes infections under certain circumstance to domesticated animal and poultry, resulting in severe diarrhea, septicemia, and respiratory infections. Although there are increasing reports regarding the infections of E. coli to domestic animals and poultry, the infection of E. coli in dogs is relatively less reported, especially on septicemia and meningoencephalitis. Here, we reported the isolation and identification of an E. coli isolate named CEC-GZL17 from dogs characterized by septicemia and sudden death, and found that CEC-GZL17 is able to cause meningoencephalitis. Exploration on the potential mechanism underlying meningoencephalitis demonstrated that CEC-GZL17 infection significantly increases TNF-α expression and inhibits ZO-1 and occludin expressions in brain tissue, indicating that the E coli likely use the mechanism to penetrate the blood-brain barrier via disrupting tight junction architecture, thus leading to the invasion to brain tissue.     

Growth and characterisation of a cell culture model of the feline blood-brain barrier

An in vitro model of the feline blood-brain barrier was developed using primary cultures of brain capillary endothelial cells derived from adult cats. They were grown in the presence of astrocytes obtained from newborn kittens. Feline endothelial cell cultures were characterised by uptake of DiI-acetylated low-density lipoprotein (DiI-Ac-LDL) and expression of von Willebrand factor. Astrocytes were characterised based on their expression of glial fibrillary acidic protein (GFAP). Electron microscopy revealed junctional specialisation between endothelial cells. Occludin and ZO-1 expression by the endothelial cell cultures was detected by Western blot analysis. Barrier function of co-cultured endothelial cells and astrocytes was confirmed by a transendothelial electrical resistance (TEER) value of 30-35 Omegacm2 and apparent permeability coefficients (Papp) for FD-40 (FITC-dextran, 40 kDa) of 4x10(-6) cm/s and for FD-4 (4kDa) of 1.92x10(-5) cm/s. In endothelial cell monolayers grown with astrocyte-conditioned medium, the TEER value was lower (20-25 Omegacm2), and Papp of FD-40 and FD-4 was higher at 6.27x10(-6) and 3.96x10(-5) cm/s, respectively. This model should have useful applications in the examination of events occurring at the BBB early in FIV infection, and may provide knowledge applicable to HIV infection.     

肠道黏膜屏障机能及其调控研究进展

肠道不仅仅是机体消化、吸收营养物质的主要场所,也是机体的一道防御屏障。肠道黏膜屏障包括黏膜生物屏障、机械屏障、化学屏障和免疫屏障,不论是哪一种肠道黏膜屏障受到损伤,都会导致机体不同程度的伤害,与一些疾病的发生密切相关。本文就肠道黏膜屏障机能及其调控研究进展进行综述,为进一步认识肠道黏膜屏障及治疗由其损伤引起的疾病提供参考和帮助。     

药物对不同品种犬的影响

伊(阿)维菌素是属于新型大环内酯类高效生物抗生素类杀虫、杀螨剂,其中B la为主要活性成分。伊(阿)维菌素通过剌激神经传递介质y-氨基丁酸的释放,干扰节肢动物正常的神经生理活动起到杀虫作用,而人和其他哺乳动物外周神经传递过程无此物质参与,因而对人及哺乳动物很安全。伊(阿)维菌素是大分子药物,一般不能通过血脑屏障进入脑细胞中,但柯利犬或幼犬因为血脑屏障发育不完善,药物得以进入脑细胞,从而造成中毒(这也是柯利犬容易对其他药物产生过敏中毒的原因)。虽然伊维菌素驱绦虫效果非常好,但副作用也比较大,仅限于成犬严格按照剂量使用,柯利牧羊犬禁用。     

Detection of blood‐brain barrier dysfunction using advanced imaging methods to predict seizures in dogs with meningoencephalitis of unknown origin

BackgroundThe blood‐brain barrier (BBB), which separates the intravascular and neuropil compartments, characterizes the vascular bed of the brain and is essential for its proper function. Recent advances in imaging techniques have driven the development of methods for quantitative assessment of BBB permeability.Hypothesis/ObjectivesPermeability of the BBB can be assessed quantitatively in dogs with meningoencephalitis of unknown origin (MUO) and its status is associated with the occurrence of seizures.AnimalsForty dogs with MUO and 12 dogs without MUO.MethodsRetrospective, prospective cohort study. Both dynamic contrast enhancement (DCE) and subtraction enhancement analysis (SEA) methods were used to evaluate of BBB permeability in affected (DCE, n = 8; SEA, n = 32) and control dogs (DCE, n = 6; SEA, n = 6). Association between BBB dysfunction (BBBD) score and clinical characteristics was examined. In brain regions where BBBD was identified by DCE or SEA magnetic resonance imaging (MRI) analysis, immunofluorescent staining for albumin, glial fibrillary acidic protein, ionized calcium binding adaptor molecule, and phosphorylated mothers against decapentaplegic homolog 2 were performed to detect albumin extravasation, reactive astrocytes, activated microglia, and transforming growth factor beta signaling, respectively.ResultsDogs with BBBD had significantly higher seizure prevalence (72% vs 19%; P = .01) when compared to MUO dogs with no BBBD. The addition of SEA to routine MRI evaluation increased the identification rate of brain pathology in dogs with MUO from 50% to 72%.Conclusions and Clinical ImportanceImaging‐based assessment of BBB integrity has the potential to predict risk of seizures in dogs with MUO.