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背景:在骨组织工程研究中,面临从体外实验过渡到体内成骨时如何快速血管化的问题,尽早建立血运才能保留组织工程骨的成骨优势。在体外短时间内能大量扩增且纯度较高的细胞,才能成为组织工程化骨再血管化理想的种子细胞。目的:探索肾微血管内皮细胞的体外培养方法,并对其进行鉴定和表型研究,拟为骨组织工程再血管化提供理想的种子细胞。设计:单一样本研究。单位:苏州大学附属儿童医院骨科,苏州大学生命科学院生物技术研究所。材料:实验于2003-05/2004-04在苏州大学附属儿童医院骨科实验室完成。实验材料为CO2培养箱,M199,胎牛血清,Ⅳ型胶原酶,40g/L多聚甲醛,20g/L戊二醛,ECGF,鼠抗人第八因子单克隆抗体(抗FⅧ-RAg),多标记免疫分析系统(1420,WALLACBLCTOR2),离心机,倒置显微镜,免疫荧光显微镜等。方法:采用三步梯度筛网法,先获得较高纯度的肾血管球,再应用肾血管球外植法体外培养肾微血管内皮细胞。进一步对肾微血管内皮细胞进行免疫组化法的Ⅷ因子相关抗原鉴定,用透射电镜观察细胞浆Weibel-Palade小体,以及采用间接免疫荧光法检测CD31,CD34及CD44表达。主要观察指标:肾微血管内皮细胞形态、活性及表型。结果:体外培养出纯度很高的血管内皮细胞并培养了11代。此外,免疫组化显示Ⅷ因子相关抗原阳性,电镜下?
BACKGROUND: In the field of bone tissue engineering, faced with the problem of rapid vascularization during the transition from in vitro experiments to in vivo osteogenesis, the establishment of blood supply as early as possible can preserve the osteogenic advantages of tissue engineered bone. In a short time in vitro can be a large number of large-scale amplification and purity of cells in order to become tissue engineered bone revascularization ideal seed cells. OBJECTIVE: To explore the in vitro culture method of renal microvascular endothelial cells and to identify and phenotype them, and to provide ideal seed cells for revascularization of bone tissue engineering. Design: Single Sample Study. SETTING: Department of Orthopedics, Children’s Hospital Affiliated to Soochow University, Institute of Biotechnology, Suzhou University. Materials: The experiment was performed at the Orthopedic Laboratory of Children’s Hospital, Soochow University from May 2003 to April 2004. The experimental materials were CO2 incubator, M199, fetal bovine serum, type IV collagenase, 40g / L paraformaldehyde, 20g / L glutaraldehyde, ECGF and mouse anti-human factor VIII monoclonal antibody Multi-labeled immunoassay system (1420, WALLACBLCTOR2), centrifuge, inverted microscope, immunofluorescence microscopy and the like. Methods: The three-step gradient mesh method was used to obtain the renal pelvis with higher purity. Renal vascular grafts were used to culture renal microvascular endothelial cells in vitro. The renal microvascular endothelial cells were further identified by immunohistochemistry, the Weibel-Palade bodies were observed by transmission electron microscopy, and the expressions of CD31, CD34 and CD44 were detected by indirect immunofluorescence. MAIN OUTCOME MEASURES: Morphology, activity and phenotype of renal microvascular endothelial cells. Results: Vascular endothelial cells were cultured in vitro and cultured for 11 passages. In addition, immunohistochemistry showed Ⅷ factor-related antigen-positive, electron microscopy?