兔脂肪干细胞的分离培养及定向诱导为软骨细胞的实验观察

目的:探索从脂肪组织中分离、培养干细胞的方法;研究脂肪干细胞向软骨方向定向诱导的可行性。方法:从成年兔颈后部脂肪组织取材,酶消化法分离、培养脂肪干细胞;免疫荧光测定CD34抗原的表达;诱导培养基定向软骨细胞方向诱导;组化及免疫组化鉴定软骨细胞特性。结果:从兔脂肪组织中能够分离出生长旺盛的干细胞,CD34表达阳性,定向诱导后表现出软骨细胞的特性。结论:从兔颈后部脂肪组织中能够分离出干细胞,并能定向软骨细胞方向诱导,说明脂肪干细胞作为软骨组织工程种子细胞有一定的可行性。     

Lymphoid tissues from patients with infectious mononucleosis lack monoclonal B and T cells

In typical cases of infectious mononucleosis (IM), lymphoid tissue is rarely submitted for pathological examination. When lymphoid tissues from IM cases are examined, the histological appearance of IM may be difficult to distinguish from malignant lymphoma. The purpose of this study was to address the utility of clinical molecular assays for T and B cell clonality in distinguishing IM from lymphoid malignancy. DNA was recovered from paraffin-embedded archival lymphoid tissues of 18 cases of IM and 13 control cases representing other reactive lymphoid hyperplasias. T cell receptor γ (TCR-γ) and immunoglobulin heavy chain (IgH) gene rearrangements were assayed using our standard clinical polymerase chain reaction procedures targeting each of the four functional variable (V) families and the three joining (J) families of the TCR-γ gene, and framework III of the IgH gene, respectively. In 17 of 18 cases of IM, no monoclonal T or B cell populations were detectable. One case, the only spleen specimen in the study, had an oligoclonal pattern of TCR-γ rearrangements. The control cases representing other reactive hyperplasias also lacked monoclonality. The assays used were sensitive to clonal populations as small as 5% of cells. In this case series, no monoclonal lymphoid populations were identified in any case of IM. This finding suggests that molecular studies are useful in distinguishing IM from lymphoid neoplasms.     

Carcinogenesis in tissue culture 24: tumorigenicity and aggregate-forming capacity of mammalian cells in culture.

Twenty two kinds of strains and substrains of mammalian cells were cultivated on a gyratory shaker to obtain cell aggregates. The relationship between the morphology of aggregates and tumorigenicity of the cells was investigated. In some groups of cells, the size of aggregates corresponded with their tumorigenicity, e.g. among rat liver cells untreated, hepatoma cells produced by back-transplantation of rat liver cells after the treatment with a chemical carcinogen in tissue culture, and the hepatoma cells passaged serially through rats. The correspondence, however, was generalized to not all the cell strains. It was impossible to identify the tumorigenicity of cells by their aggregate-forming capacity.     

Immunoelectrophoretic identification and purification of herpes simplex virus antigens released from infected cells in tissue culture.

Proteins released from herpes simplex virus type 1 (HSV-1)-and type 2 (HSV-2)-infected HEp-2 cells have been characterized by the crossed immunoelectrophoretic technique. Both HSV type-common and type-specific antigens were found in the tissue culture medium 24 h after infection. Antigen Ag-6, an HSV-1-specific antigen, was found in high concentration in the medium as compared to other HSV antigens released from HSV-1-infected cells. The HSV-2-specific antigens, Ag-4 and Ag-1, were released in molecular modifications with altered electrophoretic mobility as compared to their cellular counterparts. Purification of HSV antigens was performed by ion-exchange chromatography, and an HSV type-common antigen, Ag-11, and an HSV-2-specific antigen, Ag-4A, were isolated.     

Carcinogenesis in tissue culture 30: malignant transformation of normal rat liver cells treated with diethylnitrosamine in tissue culture with special reference to the differential effects of cytochalasin B on various cells with and without tumorigenicity.

Liver tissue of a suckling rat was cultured. After 3 weeks of cultivation, the cultures consisting of epithelial cells were treated with 50 micrograms/ml or 100 micrograms/ml DEN for 7 days. 5 months after the treatment, the mode of chromosome number was found decreased from 42 to 40 in the 100 micrograms/ml DEN-treated group and shifted to triploid range after 21 months. The mode in the 50 micrograms/ml DEN-treated group maintained the diploid number until the 5th month but was found reduced to 40 in 21 months. On subcutaneous backtransplantation into young rats at the 22nd month, the treated cells produced tumors at the site inoculated in all the rats. Metastatic foci were also detected in lungs. These tumors were histologically diagnosed as hepatomas. Untreated control cells did not produce tumors. The differential effects of cytochalasine B on the cells with and without tumorigenicity were examined by the use of these cells and other cells, and it revealed that the capacity of multinucleated cell-formation by cytochalasin B fairly corresponds with the backtransplantability of the cells. Binucleated cell formation, not more than 2 nuclei, in the culture of normal cells was found by time-lapse cinemicrography to be not due to the non-capacity of multinucleation but to the destruction of multinucleated cells.     

人脂肪组织分离培养基质干细胞的方法及其表型鉴定

目的：分离培养人脂肪基质干细胞，鉴定其表型。 方法：实验于2005—03／2005-12在华中科技大学同济医学院血研所实验室完成。脂肪组织取自华中科技大学同济医学院附属协和医院普通外科阑尾炎及腹股沟斜疝的14例手术患者，排除了冠心病、高血压病及糖尿病等。手术前均与本人谈话并征得其同意获取脂肪组织进行实验。分离人脂肪组织，胶原酶消化后制备细胞悬液并培养。消化传代以纯化细胞。传代的脂肪基质干细胞进行细胞生长曲线绘制。流式细胞术及免疫荧光法检测其表型。 结果：14份标本均成功培养出脂肪基质干细胞并生长良好。平均传至20-25代。①原代细胞培养72h后可见细胞贴壁，5d后可见细胞呈纺锤形、多角形改变，9d后细胞生长明显增加，排列密集并呈涡旋状生长，随着培养时间的延长，梭形细胞逐渐占据优势。到第12天后可见细胞铺满瓶底80％。传代后细胞增殖速度明显加快，于第3天开始数量明显增多，五六天后细胞即已长满培养瓶底，融合成片状＋细胞排列更加整齐有序。多次传代后细胞呈更均匀有序的分布。②脂肪基质干细胞倍增时间约为48h。③第5代的脂肪基质干细胞细胞表型。呈CD31^-，CD34^-，CD45^-，HLA-DR^-，CD2^＋,CD105^＋。④免疫荧光法鉴定脂肪基质干细胞表达CD13和CD44。 结论：本实验成功从人脂肪组织中分离培养出基质干细胞，通过流式细胞术和免疫荧光检测，培养后的细胞表达CD13、CD29、CD44、CD105．而不表达CD31、CD34、CD45、HLA—DR。     

人脂肪组织分离培养基质干细胞的方法及其表型鉴定

目的:分离培养人脂肪基质干细胞,鉴定其表型。方法:实验于2005-03/2005-12在华中科技大学同济医学院血研所实验室完成。脂肪组织取自华中科技大学同济医学院附属协和医院普通外科阑尾炎及腹股沟斜疝的14例手术患者,排除了冠心病、高血压病及糖尿病等。手术前均与本人谈话并征得其同意获取脂肪组织进行实验。分离人脂肪组织,胶原酶消化后制备细胞悬液并培养,消化传代以纯化细胞。传代的脂肪基质干细胞进行细胞生长曲线绘制,流式细胞术及免疫荧光法检测其表型。结果:14份标本均成功培养出脂肪基质干细胞并生长良好,平均传至20～25代。①原代细胞培养72h后可见细胞贴壁,5d后可见细胞呈纺锤形、多角形改变,9d后细胞生长明显增加,排列密集并呈涡旋状生长,随着培养时间的延长,梭形细胞逐渐占据优势。到第12天后可见细胞铺满瓶底80%。传代后细胞增殖速度明显加快,于第3天开始数量明显增多,五六天后细胞即已长满培养瓶底,融合成片状,细胞排列更加整齐有序。多次传代后细胞呈更均匀有序的分布。②脂肪基质干细胞倍增时间约为48h。③第5代的脂肪基质干细胞细胞表型,呈CD31-,CD34-,CD45-,HLA-DR-,CD29 ,CD105 。④免疫荧光法鉴定脂肪基质干细胞表达CD13和CD44。结论:本实验成功从人脂肪组织中分离培养出基质干细胞,通过流式细胞术和免疫荧光检测,培养后的细胞表达CD13、CD29、CD44、CD105,而不表达CD31、CD34、CD45、HLA-DR。     

腺体组织损伤后体外分离下颌下腺干/祖细胞后的克隆化培养

背景:体外构建组织工程化人工涎腺需获得分化增殖良好、数量充足的种子细胞,但正常下颌下腺中很难分离出成体干细胞.目的:利用腺体组织损伤动物模型体外分离下颌下腺干/祖细胞,并进行克隆化培养.设计、时间和地点:细胞学体外观察,于2006-03/2007-01在遵义医学院贵州省细胞工程实验室完成.材料:8周龄雄性SD大鼠10只,由解放军第三军医大学动物中心提供.方法:10只大鼠采用结扎下颌下腺主导管、抑制腺体分泌来建立组织损伤模型,1周后切取腺体组织,酶消化法体外分离下颌下腺干,祖细胞,原代培养10-14 d后,挑取培养皿中形成的小的类圆形、类上皮细胞集落予以纯化,传代后进行单克隆培养.主要观察指标:下颌下腺干,祖细胞免疫细胞化学染色及免疫荧光染色结果,通过绘制生长曲线分析下颌下腺干,祖细胞体外增殖能力.结果:实验获得表达层粘蛋白的细胞具有干细胞特征,CD29呈阳性表达表明其具有高黏附、高增殖等组织干细胞特性,角蛋白19的阳性表达提示下颌下腺干,祖细胞呈上皮源性.其生长曲线近似"S"形,体外培养增殖活跃.结论:实验结果显示,下颌下腺干,祖细胞具有组织干细胞的特征,有望成为组织工程化人工涎腺构建的一类种子细胞来源.     

The risk of occupational exposure and infection with infectious disease.

The diversity of potentially infectious agents that frequent the health care environment continues to increase. As a result, healthcare workers are at some degree of risk, for exposure to, and infection by, a variety of infectious diseases or conditions. This article is devoted to the epidemiology of major infectious diseases and conditions known to be transmitted in health care settings. In addition, the relative risk of occupational exposure to and infection by these diseases is also discussed as are general preventive measures associated with Standard and Transmission-based Precautions.     

Studies on collagenase from rheumatoid synovium in tissue culture

Fragments of synovium from patients with rheumatoid arthritis survive in defined tissue culture medium in the absence of added serum and, after 3-4 days, release into the medium enzyme capable of degrading undenatured collagen. Maximal activity is observed at pH 7-9 but the enzyme is inactive at pH 5. At temperatures of 20 degrees and 27 degrees C, collagen molecules in solution are cleaved into 3/4 and 1/4 length fragments with minimal loss of negative optical rotation, but with loss in specific viscosity of approximately 60%. Above 30 degrees C the fragments begin to denature and denaturation is complete at 37 degrees C. If the enzyme is not inhibited at this stage the large fragments are broken down further to polypeptides of low molecular weight. Reconstituted collagen fibrils and native fibers at 37 degrees C are cleaved to the low molecular weight fragments, although the fibrils are resistant to breakdown at lower temperatures (20 degrees -27 degrees C). It is proposed that the production of such an enzyme by inflamed and proliferating rheumatoid synovium may be responsible for some of the destruction of collagenous structures that accompanies rheumatoid arthritis.