中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (40): 7437-7440.doi: 10.3969/j.issn.1673-8225.2011.40.007

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

反转录病毒载体转染小鼠骨髓细胞

王存邦，白  海，葸  瑞，张  茜，周进茂，赵  强，潘耀柱   

  1. 解放军兰州军区总医院血液科，甘肃省兰州市730050
  • 收稿日期:2011-04-12 修回日期:2011-07-01 出版日期:2011-10-01 发布日期:2011-10-01
  • 作者简介:王存邦☆，男，1966年生，青海省西宁市人，汉族，1997年解放军第四军医大学毕业，博士，副主任医师，主要从事造血干细胞移植的研究。 wangcunbang98@163.com

Retroviral vector transfection of mouse bone marrow cells

Wang Cun-bang, Bai Hai, Xi Rui, Zhang Qian, Zhou Jin-mao, Zhao Qiang, Pan Yao-zhu   

  1. Department of Hematology, Lanzhou General Hospital of Lanzhou Military Command, Lanzhou  730050, Gansu Province, China
  • Received:2011-04-12 Revised:2011-07-01 Online:2011-10-01 Published:2011-10-01
  • About author:Wang Cun-bang☆, Doctor, Associate chief physician, Department of Hematology, Lanzhou General Hospital of Lanzhou Military Command, Lanzhou 730050, Gansu Province, China wangcunbang98@163.com

PDF

476

摘要：

背景：为方便、准确检测外源细胞在体内的存活情况，需在外源细胞转移标记基因。
目的：观察多种细胞因子联合刺激下反转录病毒载体对BALB/C×C57BL F1代小鼠骨髓细胞基因转移效率的影响。
方法：以PA317-GCGPXSN细胞制备病毒上清，NIH3T3细胞测定病毒滴度后，取经过干细胞因子、白细胞介素3、白细胞介素6预培养刺激后的BALB/C×C57BL F1代小鼠骨髓细胞，实验组实施基因转移，流式细胞仪及PCR方法测定基因转移效率。阴性对照组不做基因转移。阳性对照组为PA317-GCGPXSN细胞株。
结果与结论：①病毒滴度为1.9×108 CFU/L。②对照组BALB/C×C57BL F1代小鼠骨髓细胞测定的荧光强度数值为0.63%，实验组为76.04%，两组相比差异有显著性意义(P < 0.01)。PCR方法扩增到了NeoR基因的特异性片断，结果证实细胞因子预刺激后实施基因转移，可有效地将外源基因转移进入BALB/C×C57BL F1代小鼠骨髓细胞基因组中。

关键词: 小鼠, 骨髓细胞, 细胞因子, 反转录病毒载体, 基因转移, 绿色荧光蛋白, NeoR基因

Abstract:

BACKGROUND: Gene transfer of exogenous cells should be done for convenient and accurate detection of exogenous cell survival in the body.
OBJECTIVE: To explore the gene transfer efficiency of bone marrow cells of BALB/C×C57BL F1 mice mediated by retroviral vector and stimulated with cytokines.
METHODS: The viral supernatants were harvested with PA317-GCGPXSN and detected with NIH3T3. Then gene transfer of experimental group was made on bone marrow cells from BALB/C×C57BL F1 mice as target cells which were stimulated by stem cell factor, interleukin 3, interleukin 6. The gene transfer efficiency was detected by flow cytometry and PCR. Negative group was not treated with gene transfer. Positive group used PA317-GCGPXSN cells as target cells.
RESULTS AND CONCLUSION: ①The viral titers were 1.9×108 CFU/L. ②The gene transfer efficiency of the experiment group was 76.04%, whereas that of the control group was 0.63%, there was obviously statistical difference (P < 0.01). PCR amplification procured the specific NeoR gene fragments. Results verified that this method can transfer exogenous gene into bone marrow cell genome of BALB/C×C57BL F1 mice efficiently.

中图分类号: