中国组织工程研究

中国组织工程研究 ›› 2014, Vol. 18 ›› Issue (15): 2351-2356.doi: 10.3969/j.issn.2095-4344.2014.15.010

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

慢病毒携带增强型绿色荧光蛋白转染的人脐静脉内皮细胞

丁  强1，杨  波2，王  簕3，尹  飚2，唐  龙2，章  波2   

  1. 广州医科大学附属第三医院，1骨外一区，2骨科，广东省广州市  510150；3广州医科大学荔湾医院，广东省广州市  510150
  • 出版日期:2014-04-09 发布日期:2014-04-09
  • 通讯作者: 杨波，主任医师，教授，广州医科大学附属第三医院骨科，广东省广州市 510150
  • 作者简介:丁强，男，1984年生，山西省大同市人，汉族，广州医科大学在读硕士，主要从事骨外科学方面的研究。
  • 基金资助:

    国家青年科学基金项目(31200726)；2011年广州市科技计划项目(2011J4100052)

Transfection of human umbilical vein endothelial cells with lentivirus containing enhanced green fluorescent protein

Ding Qiang1, Yang Bo2, Wang Le3, Yin Biao2, Tang Long2, Zhang Bo2   

  1. 1 First Ward, Department of Orthopedic Surgery, 2 Department of Orthopedics, Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, Guangdong Province, China; 3 Liwan Hospital of Guangzhou Medical University, Guangzhou 510150, Guangdong Province, China
  • Online:2014-04-09 Published:2014-04-09
  • Contact: Yang Bo, Chief physician, Professor, Department of Orthopedics, Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, Guangdong Province, China
  • About author:Ding Qiang, Studying for master’s degree, First Ward, Department of Orthopedic Surgery, Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, Guangdong Province, China
  • Supported by:

    the National Science Foundation for Distinguished Young Scholars of China, No. 31200726; the Scientific Plan of Guangzhou City in 2011, No. 2011J4100052

PDF

415

被引次数

3

摘要：

背景：人脐静脉内皮细胞转染慢病毒携带增强型绿色荧光蛋白方便后期示踪该细胞，掌握转染的最佳感染复数(MOI)和产生较强荧光强度的时间，可为后期观察人脐静脉内皮细胞在动物模型体内的示踪奠定基础。
目的：观察慢病毒携带增强型绿色荧光蛋白在人脐静脉内皮细胞中的表达，找到稳定标记人脐静脉内皮细胞的方法。
方法：采用胶原酶灌注消化分离人脐静脉内皮细胞，将其置于含有体积分数20%胎牛血清、内皮细胞生长因子的培养基内培养，倒置显微镜观察内皮细胞生长情况，消化离心重悬后进行计数，平均分成4组，每组细胞数5.0×105个，接种于24孔培养皿，空白组加入10 μL的DMEM无血清培养基，剩余3组加入慢病毒携带增强型绿色荧光蛋白进行转染，MOI分别为2，3，4，每组3个皿。
结果与结论：分离的人脐静脉内皮细胞在体外培养5-7 d可长成单层，光镜下胞体为单层鹅卵石状排列，第Ⅷ因子相关抗原的检测为阳性。转染后24 h可见绿色荧光，72 h荧光达到最强，细胞转染率与MOI值呈线性增长关系，至21 d 时仍可见绿色荧光。转染后0，7，14，21 d，MOI=3 组的人脐静脉内皮细胞数量均与空白组差异无显著性意义，表明转染后细胞增殖能力不受慢病毒携带增强型绿色荧光蛋白影响。提示慢病毒携带增强型绿色荧光蛋白转染人脐静脉内皮细胞效率高，绿色荧光蛋白基因可持续表达21 d，且标记后不影响人脐静脉内皮细胞的增殖活性。



中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程


全文链接：

关键词: 组织构建, 血管内皮细胞, 慢病毒, 增强型绿色荧光蛋白, 人脐静脉内皮细胞, 国家自然科学基金

Abstract:

BACKGROUND: Human umbilical vein endothelial cells transfected with lentivirus containing enhanced green fluorescent protein can be easily traced. The optimal multiplicity of infection and time for producing strong fluorescence intensity can lay the foundation of tracing human umbilical vein endothelial cells in animal models.
OBJECTIVE: To observe expression of lentivirus containing enhanced green fluorescent protein in human umbilical vein endothelial cells, and thereby to find a stable method to label human umbilical vein endothelial cells.
METHODS: Using 0.1% collagenase perfusion digestion, we isolated human umbilical vein endothelial cells, which then were placed into a culture medium containing 20% fetal bovine serum and endothelial cell growth factor and observed under an inverted microscope. Following digestion, centrifugation and suspension, the cells were counted and divided into four groups, 5.0×105 cells in each group. After cells were seededonto 24-well plates, 10 μL serum-free Dulbecco’s modified Eagle’s medium was added into the blank group, and lentiviruses containing enhanced green fluorescent protein were added into another three groups for cell transfection respectively at multiplicities of infection of 2, 3, 4. There were three dishes in each group.
RESULTS AND CONCLUSION: After cultured for 5-7 days, isolated cells grew into a single layer and exhibited a cobblestone-like arrangement under a light microscope. In addition, factor VIII related antigen test was positive. A green fluorescence was visible at 24 hours of transfection, and peaked at 72 hours. Transfection efficiency was in a linear growth with the multiplicity of infection. Up to the 21st day of transfection, the green fluorescence was still visible. After 0, 7, 14, 21 days of transfection, the number of human umbilical vein endothelial cells showed no difference between the transfection group with the multiplicity of infection=3 and blank group, suggesting the proliferative ability of cells has no changes after transfection with lentivirus containing enhanced green fluorescent protein. These findings indicate that the lentivirus containing enhanced green fluorescent protein can highly transfect human umbilical vein endothelial cells, and green fluorescent protein can sustainably express for 21 days but cannot impact the cell proliferation.



中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程


全文链接：

Key words: lentivirus, green fluorescent proteins, umbilical veins, endothelial cells, transfection

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