密度梯度离心并贴壁法分离成人骨髓间充质干细胞的成骨特性

目的:观察密度梯度离心法结合贴壁法体外分离纯化培养人骨髓间充质干细胞的效果,以及诱导后的成人骨髓间充质干细胞的成骨特性。方法:实验于2005-05/09在中国协和医科大学中国医学科学院整形外科医院进行。用密度梯度离心法结合贴壁法分离纯化成人骨髓间充质干细胞。采用倒置显微镜观察,噻唑兰法测定细胞生长曲线,免疫细胞化学鉴定骨髓间充质干细胞膜抗原和细胞基质蛋白属性。取体外扩增的第3代骨髓间充质细胞,以3.0×103/cm2的浓度接种于放置有无菌处理的盖玻片的六孔板中,每孔内加2mL含10%胎牛血清的低糖型DMEM培养基培养液。当细胞贴壁生长达到60%～70%汇合时,将培养基更换为含5%胎牛血清的低糖型DMEM培养液,其中含骨诱导剂地塞米松、抗坏血酸和β-磷酸甘油,浓度分别为:100nmol/L、0.25mmol/L、10mmol/L。间充质细胞在这样的诱导体系中进行诱导培养,对照只加培养基。在第4、8、12、16天分别观察各孔中细胞的形态学和组织化学变化。体外加成骨诱导剂后分别作碱性磷酸酶活性测定、茜素红染色和VonKossa染色,观察骨髓间充质干细胞的成骨分化结果。结果:①分离的骨髓间充质干细胞培养48h后贴壁,72h后出现纺锤状细胞,贴壁的骨髓间充质细胞平均约12d后形成克隆。②第2代骨髓间充质干细胞99%表现为CD44 、Vim 、CD34-,CD29-。细胞表型稳定。③加成骨诱导剂后碱性磷酸酶活性明显增高,钙沉积在第8d就开始出现,茜素红染色、VonKossa染色阳性。结论:密度梯度离心法结合贴壁法分离纯化的成人骨髓间充质干细胞纯度高,表型稳定,体外加成骨诱导剂培养后,符合成骨细胞的形态特征和生物学特性,具有成骨活性,可为骨组织工程和基因治疗提供比较理想的种子细胞。     

大鼠卵巢组织中早期卵泡发育及卵母细胞凋亡与白藜芦醇的影响

目的:观察白藜芦醇对大鼠卵巢组织中早期卵泡发育及凋亡的影响。方法:实验于2006-07/12在汕头大学医学院细胞衰老实验室完成。成年SD大鼠合笼,所生的新生雌鼠分为3组:①对照组:正常出生者,不干预。②腹腔注射组:正常出生后12h内开始腹腔注射白藜芦醇25mg/(kg·d),1次/d。③母鼠灌胃组:正常怀孕的母鼠在孕12d(以发现阴道栓为怀孕0.5d)开始进行白藜芦醇25mg/(kg·d)灌胃,1次/d,直至分娩后生下的雌鼠。各组大鼠分别在出生2,4d处死取卵巢,应用苏木精-伊红染色观察大鼠早期卵泡发育情况(计算卵母细胞巢中卵母细胞及原始卵泡和发育卵泡的比率),应用TUNEL染色观察卵母细胞凋亡的情况。结果:①腹腔注射组和母鼠灌胃组大鼠卵母细胞巢内卵母细胞比例高于对照组[2d龄:(39.78±9.22)%,(53.20±5.08)%,(15.76±6.68)%;4d龄:(9.39±1.82)%,(5.39±2.12)%,(2.62±1.18)%;P均<0.05];2d龄母鼠灌胃组和腹腔注射组大鼠原始卵泡和发育卵泡比例低于对照组[原始卵泡:(57.64±7.23)%,(46.40±5.20)%,(74.57±9.36)%;发育卵泡:(2.58±2.41)%,(4.51±4.60)%,(9.67±4.70)%;P均<0.05]。②生后第2天是大鼠卵母细胞凋亡的高峰期,母鼠灌胃组和腹腔注射组卵泡凋亡率高于对照组,但差异不显著[(9.88±3.30)%,(12.95±3.34)%,(8.50±4.13)%,P>0.05]。结论:白藜芦醇可以延缓大鼠卵巢组织中卵母细胞巢破裂,抑制原始卵泡的发育启动;但是对于卵母细胞的凋亡没有明显影响。     

雷帕霉素-替罗非班复合药物涂层支架的血液及组织相容性

目的:观察雷帕霉素-替罗非班复合药物涂层支架置入后再狭窄、血栓、生化指标和重要器官组织病理学变化情况,评估其血液及组织相容性。方法:实验于2005-03/11在解放军第四军医大学实验外科实验室完成。以聚合高分子材料为药物载体,采用微喷法制备雷帕霉素支架及雷帕霉素-替罗非班复合药物涂层支架。实验分组:8只小型猪按随机数字表法分为两组,复合药物组和雷帕霉素组,每组4只。复合药物组应用雷帕霉素-替罗非班复合药物涂层支架,雷帕霉素组应用雷帕霉素支架。再狭窄模型采用国际公认的小型猪冠状动脉过度扩张再狭窄模型。每只猪于冠状动脉中成功置入上述支架各2枚。术后两组小型猪服用硫酸氯吡格雷75mg/d抗血小板治疗,雷帕霉素组服用3个月,复合药物组服用1个月。实验评估:①观察术后动物行为状态。②支架置入术前、术后2,7d,4,12周分析全血细胞计数、生化指标。③麻醉后处死动物,取出支架置入段血管,行组织病理学观察,计算管腔面积丢失率。管腔面积丢失率(%)=(支架围绕面积-管腔面积)/支架围绕面积×100%。④取肺、肝、肾组织及支架血管段供血的心室壁行组织学观察。结果:8只小型猪均进入结果分析。①术后动物行为学观察:两组术前、术中及术后3个月造影时,动物心率、血压、体质量的变化差异无显著性意义,术后也无明显行为异常及饮食、排尿便异常。②血细胞计数和生化检测结果:复合药物组、雷帕霉素组术后2d白细胞计数均高于术前[分别为(13.1±2.8)×109,(7.2±2.1)×109L-1;(12.5±3.3)×109,(6.3±2.2)×109L-1],差异有显著性意义(t=3.37,3.08,P<0.05);其余各时段两组外周全血红细胞、白细胞、血小板计数差异无显著性意义(P>0.05)。γ-血清谷丙转氨酶、谷氨酰转肽酶及肌酐、尿素氮未见明显改变(P>0.05)。③血管组织病理学检测:两组血管均未见附壁血栓。复合药物组血管腔面积、新生内膜面积高于雷帕霉素组[分别为(4.96±0.35),(4.89±0.46)mm2;(1.12±0.10),(1.05±0.09)mm2],管腔面积丢失率低于雷帕霉素组[(19±9)%,(21±7)%],差异均无显著性意义(P>0.05)。④重要脏器大体形态及病理学检查:证实细胞形态结构正常,无炎性细胞浸泡、出血和坏死。结论:雷帕霉素-替罗非班复合药物涂层支架置入小型猪冠状动脉后3个月的血液相容性及组织相容性良好,并可减少术后抗血小板药物的全身用药量。     

The affinity and stoichiometry of binding of human factor VIII to von Willebrand factor

To study the interaction between factor VIII and von Willebrand factor (vWF), binding experiments were performed using immobilized plasma vWF. Plasma was obtained from healthy donors and from patients with severe hemophilia A. For normal and hemophilic vWF, the dissociation constants (kd) for binding of factor VIII to vWF were 0.21 +/- 0.04 and 0.22 +/- 0.05 nmol/L, respectively. At saturation, the stoichiometry was one factor VIII molecule per 50 vWF monomers. In gel-filtration experiments, vWF was saturated by 23 times more factor VIII. However, when this FVIII-vWF complex was immobilized on microtiter plates, the ratio of factor VIII/vWF decreased to the same ratio as in the solid- phase binding assay. To exclude any effect of antibody binding, colloidal gold particles with a diameter of 15 nm were coupled to purified vWF. This vWF-gold complex remained immunoreactive toward polyclonal and monoclonal antibodies, and was able to bind factor VIII, specifically, saturably, and reversibly. After incubation of vWF-gold with factor VIII, unbound and bound factor VIII were separated by centrifugation. Binding isotherms of these fluid-phase binding experiments indicated a kd of 0.32 +/- 0.09 nmol/L and a stoichiometry of approximately 0.5 factor VIII molecule per vWF monomer. We conclude that vWF-binding to a surface, with or without an antibody, may induce a conformational change causing a dissociation of bound factor VIII from vWF.     

Spectroscopic studies of rat mast cells, mouse mastocytoma cells, and compound 48/80-I. A spin-label study of membrane fluidities and the effect of 48/80

Purified rat mast cells, mixed peritoneal leukocytes and mouse mastocytoma cells take up spin-labeled membrane probes (5-doxyl, 12-doxyl and 16-doxyl stearic acids and their methyl stearate counterparts) in a manner similar to that of other cell types. The electron spin resonance spectra of these probes suggest that the membrane fluidities in the regions probed are similar in both mast cells and other normal leukocytes of the rat peritoneal cavity. However, the membranes of the mastocytoma cells were significantly more fluid in all of the regions probed. Membrane fluidity differences reported by the methyl stearate probes were greater than those reported by the corresponding fatty acid probes. This suggests that the chemical nature of the probe may contribute significantly to the apparent reported fluidity. The histamine liberator, compound 48/80, did not affect the spectra of any of the probes at concentrations that were magnetically dilute; however, it reduced the degree of spin exchange at high probe concentrations in mastocytoma cells. This suggests that 48/80 may increase the volume of the mastocytoma cell membranes, resulting in reduced intermolecular interactions among the probe molecules.     

Tropical illness profiles: the psychology of illness perception in Malawi

Psychological and social investigations of tropical diseases can make a significant contribution to understanding and managing many illnesses. One-hundred and seventy-five Malawian university students rated peopled who suffered from AIDS, malaria, schistosomiasis and the ‘common cold’, on 11 psycho-social dimensions related to illness. Analysis of variance and factor analysis was used to distinguish distinctive illness profiles reflecting perceptions of (people with) these illnesses. Results suggested that infirmity was associated with AIDS and that the seriousness of malaria was minimized. Within a context of many threats to health the importance of distinguishing between serious illnesses is emphasized for effective health promotion interventions.