兔血管内皮细胞和诱导成骨细胞在可注射纳米材料上的共培养

目的：将可注射纳米骨材料与共培养的兔肾血管内皮细胞和兔骨髓间充质干细胞诱导的成骨细胞复合,并构建可注射细胞型纳米组织工程骨,观察它们体外培养的相容性. 方法：实验于2003-09/2004-11在苏州大学附属儿童医院完成.①实验材料：取16周龄雄性新西兰大耳白兔,体质量1.5 kg左右.②实验方法：麻醉后抽取兔骨髓,用淋巴细胞分离液分离出其中的间充质干细胞,在含体积分数为0.15牛血清的RPMI1640液中培养.骨髓间充质干细胞在条件培养基中,7 d后可见细胞变为多边形,碱性磷酸酶和Ⅰ型胶原染色阳性,形成钙结节,表现成骨细胞分化特点.采用三步梯度筛网法,获得肾血管球,5 g/L胶原酶Ⅳ37℃消化15～20 min,离心沉淀获取血管内皮细胞,在含体积分数为0.15小牛血清的M199中培养.③实验评估：免疫组织化学法进行第Ⅷ因子相关抗原鉴定,透射电镜观察细胞浆Weibel-Palade小体,间接免疫荧光法检测CD31、CD34及CD44的表达.将共培养的兔肾血管内皮细胞、成骨细胞与可注射纳米骨材料体外复合培养,进行形态学观察和功能测定. 结果：①在一定培养条件下成功诱导兔骨髓间充质干细胞向成骨细胞分化.②培养的血管内皮细胞免疫组织化学法检测第Ⅷ因子相关抗原阳性,透射电镜观察到细胞浆中的Weibel-Palade小体,间接免疫荧光法检测CD31、CD34表达阳性,CD44阴性.③共培养的兔肾血管内皮细胞、成骨细胞在可注射纳米骨材料上生长、增殖良好,细胞活性和碱性磷酸酶活性未受到影响. 结论：可注射纳米骨材料具有良好的细胞相容性,可作为骨组织工程理想的可注射载体材料.     

Cyclosporin nephrotoxicity in heart and lung transplant patients

Twenty-two patients with heart, lung or heart and lung transplants maintained on cyclosporin for periods ranging from 3 months to 10 years developed renal insufficiency which was investigated by renal biopsy. The histopathological changes were: (i) severe vascular and glomerular damage due to thrombotic microangiopathy (TM); (ii) a form of focal segmental glomerulosclerosis (FSGS); (iii) glomerular ischaemia. Rather than being separate entities, these changes appeared to represent a spectrum of pathology, some biopsies showing all three forms of glomerular injury. In all cases the glomerular changes were accompanied by arteriolar and arterial pathology, and we identified novel ultrastructural changes in the arteriolar endothelial basal lamina. Tubular atrophy was a consistent feature, the severity of which reflected the severity of the glomerular sclerosis, and which appeared to be a consequence of glomerular loss. Our findings are consistent with the nephrotoxic effects of cyclosporin being mediated chiefly via damage to preglomerular vessels and glomerular capillary endothelium. From an analysis of the clinical aspects of these cases, the effects of cyclosporin appear to be to some extent idiosyncratic, and therefore not entirely preventable, but strict monitoring of blood cyclosporin levels is essential to minimize the risk of permanent renal damage. Monitoring urinary protein in addition to plasma creatinine may detect the onset of FSGS, as proteinuria precedes creatinine elevation.      

Autoantibodies against bactericidal/permeability-increasing protein in patients with cystic fibrosis

Cystic fibrosis (CF), a genetic disorder, is characterized by chronic pulmonary infection/inflammation which leads to respiratory failure. The presence of anti-neutrophil cytoplasmic autoantibodies (ANCA) has previously been observed in the sera of patients with CF. In view of the known relationship of ANCA with primary vasculitis and of their putative pathogenetic role in these disorders, we studied the presence, specificity and isotype of ANCA and their clinical associations in 66 adult CF patients. None of the 66 CF samples had autoantibodies to the major ANCA antigens, proteinase 3 or myeloperoxidase. However, 60/66 (91%) CF samples contained IgG and 55/66 (83%) IgA, autoantibodies to bactericidal/permeability increasing protein (BPI), a recently characterized ANCA specificity. All the IgA anti-BPI-positive samples were also IgG anti-BPI-positive. The autoantibody specificity was confirmed by inhibition assay and immunoblotting of CF sera against a neutrophil granule preparation. Furthermore, in this cross-sectional study, anti-BPI levels were inversely correlated with the observed reductions in FEV1 and FVC (IgA anti-BPI and FEV1: r = 0.508, <it>p</it> &lt; 0.0001), and both IgG and IgA anti-BPI levels were higher in CF patients with secondary vasculitis (<it>n</it> = 6) than in those without (<it>p</it> &lt; 0.05). ANCA with specificity for BPI were present in the majority of CF sera in this study and autoimmune processes may be associated with the development of pulmonary injury in CF.      

Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis

Apoptosis, or programmed cell death, is a general mechanism for removal of unwanted cells from the immune system. It is characterized by chromatin condensation, a reduction in cell volume, and endonuclease cleavage of DNA into oligonucleosomal length fragments. Apoptosis is also accompanied by a loss of membrane phospholipid asymmetry, resulting in the exposure of phosphatidylserine at the surface of the cell. Expression of phosphatidylserine at the cell surface plays an important role in the recognition and removal of apoptotic cells by macrophages. Here we describe a new method for the detection of apoptotic cells by flow cytometry, using the binding of fluorescein isothiocyanate-labeled annexin V to phosphatidylserine. When Burkitt lymphoma cell lines and freshly isolated germinal center B cells are cultured under apoptosis inducing conditions, all cells showing chromatin condensation strongly stain with annexin V, whereas normal cells are annexin V negative. Moreover, DNA fragmentation is only found in the annexin V-positive cells. The nonvital dye ethidium bromide was found to stain a subpopulation of the annexin V-positive apoptotic cells, increasing with time. Our results indicate that the phase in apoptosis that is characterized by chromatin condensation coincides with phosphatidylserine exposure. Importantly, it precedes membrane damage that might lead to release from the cells of enzymes that are harmful to the surrounding tissues. Annexin V may prove important in further unravelling the regulation of apoptosis.     

参三七皂苷Rg1预适应对5-氮胞苷诱导大鼠骨髓间充质干细胞向心肌细胞转化的干预

目的:用药物预适应方法进行干细胞诱导已有报道,本实验观察中药参三七皂苷Rg1对5-氮胞苷诱导大鼠骨髓间充质干细胞向心肌细胞转化中的作用。方法:实验于2003-01/05在南京医科大学药理教研室完成。①实验材料:清洁级SD大鼠8只。参三七皂苷Rg18mg,批号20021017,由云南省长春花生物制剂公司提供,加入不含胎牛血清的IMDM培养液10mL,调配成10-4mol/L溶液,4℃保存。5-氮胞苷(Sigma公司,批号021209)。②实验方法:贴壁法体外培养大鼠骨髓间充质干细胞。设立4组:空白对照组常规培养后进行无血清处理,每3d换液1次;5-氮胞苷单用组单纯以10μmol/L的5-氮胞苷进行处理,其终浓度为1×10-8moL/L,连续诱导15d;5-氮胞苷 参三七皂苷Rg1预适应组分别加入0.1,1μmol/L参三七皂苷Rg1培养液处理24h,再各以10μmol/L的5-氮胞苷进行诱导,其终浓度为1×10-8moL/L,连续诱导15d。③实验评估:取第2代骨髓间充质干细胞,绘制生长曲线并计算群体倍增时间。观察诱导后骨髓间充质干细胞的生长形态学特征和细胞超微结构变化。激光共聚焦显微镜测定细胞表面积变化和细胞内钙离子浓度。结果:①5-氮胞苷诱导后骨髓间充质干细胞的生长形态学特征和细胞超微结构变化:骨髓间质干细胞胞体逐渐增大并伸出细长突起,在突起末端出现分支,部分相邻细胞的突起连接成网,形态学上表现出向心肌细胞方向转化的特征。其超微结构呈梭形,有明显的肌丝,细胞核呈单椭圆形,位于细胞中央,间质干细胞形似心肌细胞。②参三七皂苷Rg1预适应对5-氮胞苷诱导的骨髓间充质干细胞增殖特性的影响:与5-氮胞苷单用组比较,5-氮胞苷 参三七皂苷Rg1预适应组从第3天开始细胞数明显增加,细胞生长曲线均无明显的生长平台期,达到高峰后细胞数开始减少。③参三七皂苷Rg1预适应对5-氮胞苷诱导的骨髓间充质干细胞表面积的影响:与空白对照组骨髓间充质干细胞表面积比较,5-氮胞苷单用组明显降低,0.1,1μmol/L参三七皂苷Rg1预适应则能显著升高5-氮胞苷诱导的骨髓间充质干细胞表面积(P<0.01)。④参三七皂苷Rg1对5-氮胞苷诱导的骨髓间充质干细胞内游离钙水平的影响:与空白对照组比较,5-氮胞苷诱导4周后骨髓间充质干细胞内游离Ca2 相对荧光强度均明显升高(t=6.72,P<0.01),且5-氮胞苷 1μmol/L参三七皂苷Rg1预适应组升高幅度大于5-氮胞苷单用组(t=3.13,P<0.05)。结论:①参三七皂苷Rg1预适应在体外可显著刺激5-氮胞苷诱导的鼠骨髓间充质干细胞向心肌细胞转化和增殖,改善细胞形态,刺激细胞内钙离子增加。②参三七皂苷Rg1与5-氮胞苷对骨髓间充质干细胞向心肌细胞定向分化产生协同效应。     

健康人群隐神经传导中远端和近端方法的比较

隐神经传导对隐、股神经损伤,腰丛病变和L3、L4神经根损伤具有诊断价值.在通过表面电极评估隐神经逆向传导中,有远端和近端两种方法.[收稿日期 2005-09-20