骨组织工程再血管化种子细胞来源:兔肾微血管内皮细胞体外培养及其细胞表型的研究

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,20 g/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,WALLAC BLCTOR2),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,电镜下观察到Weibel-Palade小体,以及间接免疫荧光检出CD31,CD34表达阳性,而CD44阴性.结论本实验方法可成功培养出肾微血管内皮细胞,且后者表达CD31,CD34细胞表型.

Source of the seedcell in bone tissue engineering revascularization:renal microvascular endothelial cellcultured in vitro and its cellular phenotype in rabbits

BACKGROUND: In bone tissue engineering researches, we are confronted with a problem of how to rapidly vascularization in the transition from experiments in vitro to osteogenesis in vivo. Only early establishment of blood supply can maintain the osteogenetic advantage of tissue-engineered bone. Only those cells that can rapidly proliferate in vitro in a short period with relatively high purity can be used as ideal seed cells in revascularization for tissue-engineered bone.OBJECTIVE: To explore the culture in vitro method for renal microvascular endothelial cells for its verification and phenotype study, which is prepared as ideal seed cell for revascularization in bone tissue engineering.DESIGN: Single sample study.SETTING: Department of Orthopaedics of Children' s Hospital affiliated to Soochow University, Institute of Biotechnology, Academy for Life Science,Soochow University.MATERIALS: Study was accomplished in the Laboratory of the Department of Orthopaedics of Children' s Hospital affiliated to Soochow University during May 2003 to April 2004. Experimental materials include CO2 incubator,M199, fetal calf serum, Ⅳ collagenase, 40 g/L of paraform, 20 g/L of cidex,ECGF, mouse-anti-human FⅧ monoclonal antibody(anti-FⅧ-RAg), multi-marked immune analyser(1 420, WALLAC BLCTOR2), centrifuge, inverted microscope, and immunofluorescence microscope, etc.METHODS: Three-step gradient sieve method was used. Glomerulus of relatively higher purity was obtained at first, and then rental microvascular endothelial cells were cultured in vitro with glomerulus external implantation method. The rental microvascular endothelial cells were further verified by immunohistochemical Ⅷ factor-related antigen. Cytoplasmic Weibel-Palade body was observed under tranmision microscope. The expression of CD31,CD34 and CD44 was detected with indirect immunofluorescence method.MAIN OUTCOME MEASURES: The morphology, activity and phenotype of renal microvascular endothelial cell.RESULTS: Vascular endothelial cells in very high purity were cultured in vitro with 11 passages. Besides, immunohistochemistry revealed positive reaction to Ⅷ-related antigen, Weibel-Palade body was observed under electron microscope, and the expressions of CD31 and CD34 were positive but CD44 was negative under immunofluorescence analysis.CONCLUSION: This method can successfully culture renal microvascular endothelial cells, which can express CD31 and CD34 phenotypes.