人骨髓间充质干细胞分化为软骨细胞的诱导条件

目的:分析人骨髓间充质干细胞分离、纯化、体外扩增及其向软骨细胞分化的条件,为人骨髓间充质干细胞用于修复损伤的软骨组织提供依据。方法:实验于2005-03/2006-04在南昌大学医学院生化与分子生物学教研室完成。实验材料:无菌条件下采集无血液系统疾病和家族遗传史的28～65岁成人骨髓8份,进行骨髓间充质干细胞分离与培养,骨髓采集经患者及医院同意,并签署知情同意书。实验方法:取第3代对数生长期骨髓间充质干细胞,实验组使用特定的、内含2mg/L胰岛素、3mg/L转铁蛋白、1mmol/L丙酮酸、100nmol/L地塞米松和10μg/L转化生长因子β1的低糖DMEM培养基诱导人骨髓间充质干细胞向软骨细胞分化,对照组细胞以低糖DMEM基础培养液培养。倒置显微镜下观察细胞形态,诱导7,14d采用半定量RT-PCR方法检测细胞软骨特异性Ⅱ型胶原表达,蕃红花O-亮绿染色分析蛋白多糖粘多糖含量,观察细胞分化情况。结果:①光镜观察成人骨髓间充质干细胞的形态变化:培养7d细胞集落明显,集落中央为数个至数十个圆形细胞,周围变形细胞围绕中央细胞呈放射状生长。培养14d细胞呈平行状或旋涡状排布,细胞排列紧密。②骨髓间充质干细胞向软骨细胞的诱导分化:实验组细胞生长较对照组慢,培养7d后细胞大多呈椭圆形或三角形,部分细胞伸出似触角突起,培养14d细胞变形较明显,排列较为紧密,细胞突起多见,突起间相连,呈较明显软骨细胞形态特征。③骨髓间充质干细胞的番红花-O染色:实验组骨髓间充质干细胞蕃红花O-亮绿呈深染色,对照组人骨髓间充质干细胞呈阴性染色。④RT-PCR检测软骨特异性的Ⅱ型胶原COL2A1 mRNA表达:实验组诱导培养7,14d人骨髓间充质干细胞有Ⅱ型胶原COL2A1 mRNA表达,400～500bp条带间可见明显的荧光条带,诱导培养14d时表达更明显。对照组未见Ⅱ型胶原COL2A1 mRNA表达。结论:采用直接贴壁培养法成功分离和培养出人骨髓间充质干细胞,并将人骨髓间充质干细胞诱导分化成软骨细胞,该软骨细胞具有软骨细胞的形态、生化特征。     

Ultrasonography of alcoholic liver disease with histological correlation

In alcoholic liver disease (fatty infiltration, alcoholic cirrhosis), the liver is diffusely abnormal on ultrasound. Changes in size, dilatation of the hepatic veins, and ascites may also occur. The authors conducted a histological correlation of these abnormalities in 22 alcoholic patients and 16 controls, grading the changes on a scale of 0 to 4+ for fat, fibrosis, and necrosis and noting tumor whenever present. Ultrasound detected abnormality in 21 cases (sensitivity = 95%) and correctly identified 15 controls (specificity = 94%). Of the 5 tumors seen, 4 hepatomas were detected and biopsied and 1 metastatic squamous-cell carcinoma was missed. Applications of commercially available A-scan module are considered and its limitations discussed. With the exception of minimal change (1+ fat or fibrosis), ultrasound detected many of the pathological changes seen in alcoholic liver disease.     

Sources and sequelae of bacterial contamination of hematopoietic stem cell components: implications for the safety of hematotherapy and graft engineering

Background: It is important to compare the incidence of bacterial contamination of components collected from the peripheral blood or bone marrow (BM), as well as of components processed with or without cell selection or depletion, and to evaluate the sequelae of such contamination. Study Design and Methods: Bacterial contamination rates were compared in 1380 untreated autologous peripheral blood progenitor cells (PBPCs), 291 untreated autologous BM samples, 916 monoclonal antibody (MoAb)-treated autologous and allogeneic BM samples, and in 45 autologous PBPC components from which the CD34+ cells were selected. Bacterial cultures were performed at sequential time points during the processing of MoAb-treated BM. Results: Bacterial contamination was documented in 44 of 2632 components from 1593 patients (1.67% of components, 2.76% of patients) before cryopreservation. Although only 0.65% of untreated PBPCs were contaminated before cryopreservation, each patient was more likely to have given a contaminated PBPC component than a contaminated BM component (2.41% vs. 0%, p < 0.01). Bacterial contamination of MoAb-treated BM was greater during or after manipulation than it was before (2.33% vs. 0.77%, p < 0.05). At thawing, contamination was documented in 42 (1.97%) of 2136 components cultured. Ten (13.7%) of 73 patients who received hematopoietic progenitor cells that were contaminated before cryopreservation or at thawing developed fever or positive blood cultures within 48 hours of transfusion. Fever was associated with bacteremia in two cases, but no irreversible clinical sequelae were noted. Conclusion: These studies suggest that, despite careful attention to sterile procedures, low-level contamination of hematopoietic stem cell components can be introduced before or during manipulation as well as at thawing, and that standards for monitoring of the procedures for collection, processing, cryopreservation, thawing, and transfusion of hematopoietic progenitor cells are necessary.