Cell fractionation and isolation of plasma membrane enriched fractions of normal granulocytes and leukemic myeloblasts

Cell fractionation studies were done on normal human granulocytes and leukemic myeloblasts. Methods were developed to prepare plasma membrane enriched fractions from these cells.

Homogenization could be performed with a Potter Elvehjem tube or a Dounce homogenizer in hypotonic sucrose. Short sonication in isotomic sucrose also gave good disruption. More reproducible fractionation results were obtained after sonication.

As the classical plasma membrane marker enzymes (5′-nucleotidase and alkaline phosphatase) cannot be used for human granulocytes and no plasma membrane marker was known for leukemic myeloblasts we introduced a radioactive marker. ¹²⁵I-labeled Fab, prepared from an antiserum against human leucocytes was found to be a reliable plasma membrane marker both for normal granulocytes and leukemic myeloblasts.

Using sonication for disruption of cells and [¹²⁵I] Fab as plasma membrane marker, plasma membrane enriched fractions could be prepared with high yield (60%) and 10 fold enrichment.

It was concluded from fractionation studies that enzyme +5 like β-glucuronidase, peroxidase and lysozyme that are confined to granules in granulocytes have other localization in leukemic myeloblasts.     

Immunophenotypic characterization and distribution of dendritic cells in odontogenic cystic lesions

Oral Diseases (2012) 19 , 85–91 Objective: To analyze the expression and distribution patterns of mature dendritic cells (mDCs) and immature DCs (imDCs) in radicular cysts (RCs), dentigerous cysts (DtCs), and keratocystic odontogenic tumors (KCOTs). Materials and methods: Forty‐nine odontogenic cystic lesions (OCLs) (RCs, n = 20; DtCs, n = 15; KCOTs, n = 14) were assessed using the following markers: S100, CD1a and CD207 for imDCs; and CD83 for mDCs. Results: Almost all cases were S100, CD1a, and CD207 positive, whereas 63% were CD83 positive. RCs presented greater number of immunostained cells, followed by DtCs, and KCOTs. The number of S100+ cells was greater than both CD1a+ and CD207+ cells (P < 0.001), which showed approximately similar amounts, followed by lower number of CD83+ cells (P < 0.001) in each OCL type. Different from S100+ cells, both CD1a+ and CD207+ cells on the epithelium (P < 0.05) and CD83+ cells on the capsule (P < 0.05) were preferentially observed. In RCs, significant correlation was found between the thickness epithelium with S100+ and CD1a+ cells, and between the degree of inflammation with CD83+ cells. Conclusions: Dendritic cell populations in OCLs can be phenotypically heterogeneous, and it could represent distinct lineages and/or functional stages. It is suggested that besides DC‐mediated immune cell interactions, DC‐mediated tissue differentiation and maintenance in OCLs should also be considered.     

New variable-number tandem-repeat markers for typing Mycobacterium avium subsp. paratuberculosis and M. avium strains: comparison with IS900 and IS1245 restriction fragment length polymorphism typing

Mycobacterium avium subsp. paratuberculosis, the etiological agent of paratuberculosis, affects a wide range of domestic ruminants and has been suggested to be involved in Crohn's disease in humans. Most available methods for identifying and differentiating strains of this difficult species are technically demanding and have limited discriminatory power. Here, we report the identification of novel PCR-based typing markers consisting of variable-number tandem repeats (VNTRs) of genetic elements called mycobacterial interspersed repetitive units (MIRUs). Eight markers were applied to 183 M. avium subsp. paratuberculosis isolates from bovine, caprine, ovine, cervine, leporine, and human origins from 10 different countries and to 82 human isolates of the closely related species M. avium from France. Among the M. avium subsp. paratuberculosis isolates, 21 patterns were found by MIRU-VNTR typing, with a discriminatory index of 0.751. The predominant R01 IS900 restriction fragment length polymorphism type, comprising 131 isolates, was divided into 15 MIRU-VNTR types. Among the 82 M. avium isolates, the eight MIRU-VNTR loci distinguished 30 types, none of which was shared by M. avium subsp. paratuberculosis isolates, resulting in a discriminatory index of 0.889. Our results suggest that MIRU-VNTR typing is a fast typing method that, in combination with other methods, might prove to be optimal for PCR-based molecular epidemiological studies of M. avium/M. avium subsp. paratuberculosis pathogens. In addition, presumably identical M. avium subsp. paratuberculosis 316F vaccine strains originating from the Weybridge laboratory and from different commercial batches from Mérial actually differed by one or both typing methods. These results indicate a substantial degree of genetic drift among different vaccine preparations, which has important implications for prophylactic approaches.     

单纯肥胖者与代谢综合征患者血管内皮功能的比较

目的:比较单纯肥胖者与代谢综合征患者的血管内皮功能。方法:观察对象来自石家庄市2000-09/2001-06参加社区健康体检者。①体质量指数≥25kg/m2的超重/肥胖者中,单纯肥胖组295例,代谢综合征组219例,另选172例体质量指数<25kg/m2的健康人作为对照。②将肥胖者按体质量指数分为轻度肥胖(25～29.9kg/m2)和重度肥胖(≥30kg/m2),按腰围分为腹部脂肪轻度堆积(男<104cm,女<88cm)和重度堆积(男≥104cm,女≥88cm)。③血管内皮舒张功能测定:所有受试者采用NAS-1000HF彩超诊断系统测定反应性充血时及含服硝酸甘油后肱动脉内径的变化。结果:686例受试者全部进入结果分析。①单纯肥胖组和代谢综合征组体质量指数、腰围、臀围和腰臀比均高于对照组(P<0.01～0.001),其中体质量指数和臀围在男女间比较差异不显著,腰围和腰臀比男性高于女性(P<0.05),而男性代谢综合征组又高于男性单纯肥胖组(P<0.05)。②血管内皮舒张功能检测显示,反应性充血时,单纯肥胖组和代谢综合征组相对于基础血管内径变化的百分率均低于对照组[(7.79±3.93)%,(5.54±4.46)%,(11.25±3.98)%,P<0.05～0.01];代谢综合征组男性低于单纯肥胖组男性(P<0.05);男性代谢综合征组重度肥胖者低于轻度肥胖者(P<0.05);单纯肥胖组男性重度脂肪堆积低于男性轻度脂肪堆积者(P<0.05),代谢综合征组无论男女,重度脂肪堆积均低于轻度脂肪堆积者(P<0.05)。结论:①单纯肥胖者已存在血管内皮功能异常,而代谢综合征患者中内皮功能损伤程度更显著,并且与腹部脂肪堆积密切相关。②代谢综合征患者中男性血管内皮功能损伤较女性更明显。     

人骨髓间充质干细胞的贴壁分离与体外培养

目的:验证贴壁方式分离人骨髓间充质干细胞,并进行体外扩增培养的可行性。方法:实验于2006-02/12在解放军济南军区总医院脊髓修复科完成。①实验材料:骨髓来源于解放军济南军区总医院脊髓修复科收治的脊髓完全性损伤患者,对本实验知情同意。基础培养液由含体积分数为0.15胎牛血清和低糖α-MEM配置。②实验方法:无菌条件下髂后上棘穿刺抽取骨髓组织6mL,进行细胞培养,观察细胞生长情况,待细胞融合成片、长满培养瓶底部后,用质量浓度为2.5g/L的胰蛋白酶流过所有细胞表面。倒置显微镜下观察细胞变圆、部分脱壁后,立即加入有血清培养液终止消化。③实验评估:取第3代生长状态良好的细胞,胰蛋白酶消化制成细胞悬液,接种,以细胞数为纵坐标,时间为横坐标,绘制细胞生长曲线。同时每隔2h进行细胞贴壁率检测。结果:①骨髓间充质干细胞的形态学观察:倒置显微镜下,骨髓间充质干细胞接种1d即贴壁,去除悬浮细胞后继续培养3d贴壁细胞开始增殖,伸展为椭圆型、短梭型、多角型及不规则型等。至14d细胞密集在集落中心,基本铺满瓶底,第3～5代细胞呈均匀一致的长梭型,排列成旋涡状或放射状。②骨髓间充质干细胞的生长曲线:细胞传代后3d内处于潜伏期,3d后进入生长期,7d后进入平台期。③骨髓间充质干细胞的贴壁率:随着培养时间的延长,骨髓间充质干细胞贴壁率逐渐升高。传代后2,4,6,8,10,12,14,16,18,20h细胞贴壁率分别为(20.20±0.25)%,(33.00±0.29)%,(46.50±0.32)%,(69.20±0.30)%,(76.60±0.34)%,(86.50±0.27)%,(90.30±0.20)%,(96.10±0.28)%,(98.50±0.12)%,(99.00±0.07)%。结论:贴壁法分离骨髓间充质干细胞操作简便,经体外扩增培养后细胞增殖活性强,传代周期为7d,是比较理想的骨髓间充质干细胞培养方法。