|本期目录/Table of Contents|

[1]文玉军,秦毅,吴均,等.骨髓基质细胞的分离培养及转基因标记[J].宁夏医科大学学报,2007,(01):4-7.
 WEN Yu-jun,QIN Yi,WU Jun,et al.The Culture and Trangenetic Mark of Bone Marrow Stromal Cells in Vitro[J].Ningxia Medical University,2007,(01):4-7.
点击复制

骨髓基质细胞的分离培养及转基因标记(PDF)
分享到:

《宁夏医科大学学报》[ISSN:1005-8486/CN:64-1029/R]

卷:
期数:
2007年01期
页码:
4-7
栏目:
论著
出版日期:
2007-01-20

文章信息/Info

Title:
The Culture and Trangenetic Mark of Bone Marrow Stromal Cells in Vitro
作者:
文玉军1秦毅1吴均2张敏2魏灵荣2
1.宁夏医学院人体解剖学教研室,银川 750004; 2.首都医科大学,北京 100054
Author(s):
WEN Yu-jun1QIN Yi1WU Jun2et al
1.The Dept. of Anatomy, Ningxia Med. Coll., Yinchuan 750004; 2.Capital University of Medical Sciences, Beijing 100054
关键词:
骨髓基质细胞 增强型绿色荧光蛋白 流式细胞术
Keywords:
bone marrow stromal cells enhanced green fluorescent protein flow cytometry
分类号:
R329.4
DOI:
-
文献标志码:
A
摘要:
目的 采用密度梯度离心法进行骨髓基质细胞(MSCs)的分离培养,结合体外转入 增 强型绿色荧光蛋白(EGFP)基因标记方法,可提供大量纯度高、便于观察的种子细胞。方法 取成年SD大鼠四肢骨及胸骨获取骨髓细胞冲洗液,密度梯度离心法分离后培养,HE染色观 察细胞核浆比,流式细胞术测定细胞纯度; 用逆转录病毒载体转染EGFP标记基因,G418筛选 后传代培养,流式细胞术检测EGFP的表达率。结果 原代培养的MSCs形态 多样,增殖迅速, 随着培养时间的延长,细胞增殖变慢,形态趋于同一,以梭形细胞为主,核浆比高; 流式细 胞术检测发现CD90+/CD45-/CD11b-细胞达到75%,细胞在体外能够稳定地表达EGFP ,表达 率达到55.16%。结论 运用密度梯度离心法分离培养的MSCs纯度高,增 殖能力强; 体外转 入EGFP基因是一种方便、特异性高的标记MSCs的方法,为脑损伤修复研究提供了依据。
Abstract:
Objective To separate and culture the Bone Marrow Stromal Cell s(MSCs) by density gradient centrifugation method and use the method of enhanced green fluorescent protein(EGFP)gene mark in vitro, and to offer abundant pure se ed cells that easy to be observed in study of brain repairing. Methods MSCs was collected from adult Sprague-Dawley(SD)rats by density gradient centrifugation method. Then Hematoxylin-Eosin(HE)staining was performed to observe the ratio of karyon and cytoplasm. Cell surface antigen CD11b, CD45 and CD90 were measure d in order to estimate the purity of MSCs. After being expanded, transduction of MSCs with a retroviral vector containing the EGFP cDNA was performed, and second ary culture was done after being filtered with G418. Expression ratio of EGFP w a s examined with flow cytometry. Results The results showed that primary cu ltured MSCs had a multiple morphological appearance, and proliferated rapidly. A s time goes by culture, proliferation of MSCs became slow. Morphological appeara nce of MSCs tended to similar.Most of MSCs presented shuttle shape, the ratio o f karyon and cytoplasm is high. Cell surface antigen test showed that CD90+/CD4 5 -/CD11b-cells added up to 75%. MSCs expressed EGFP steadily in vitro and exp r ession ratio increased to 55.16%. Conclusion The cultured MSCs that was collect ed by density gradient centrifugation method had a high purity, strong ability t o proliferate. Transduction of MSCs with a retroviral vector containing the EGF P cDNA in vitro was a convenient and specific method in marking MSCs. It was use ful for brain repairing.

参考文献/References:

[1] Pirrenger MF,Mackay AM,Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells[J]. Science,1999,284(1):143-147.
[2] Woodbury D,Schwarz EJ,Prockop DJ, et al. Adult rat and human bone mar row stromal cells differentiate into neurons[J]. J Neurosci Res,2000,61(4):364-3 70.
[3] Ivan M, Venkantram PS, Robert FP, et al. Selective differentiation of ma mmal ian bone marrow stromal cells cultured on three dimensioned polymerfoams[J]. Biomed Mater Res,2001,55(2):229-235.
[4] Quirici N, Soligo D, Bossolasco P, et al. Isolation of bone marrow mesen chym al stem cells by anti-nerve growth factor receptor antibodies[J]. Exp Hematol , 2002, 30(7): 783-791.
[5] Beresford JN,Bennett JH,Devlin C,et al.Evidence for aniverse relationshi p be tween the differentiation of adipocytic and osteogenic cells in rat marrow strom al cell cultrues[J]. J Cell Sci,1992,102(3):341.
[6] Haynesworth SE,Goshima J,Goldberg VM,et al. Characterazition of cells wi th osteogenic potential from human marrow[J].Bone,1992,13(4):69.
[7] 徐群渊. 脑内细胞移植[M]. 福州:福建科学技术出版社,2000. 33-35.
[8] Lee J, Kuroda S, Shichinohe H, et al. Migration and differentiation of n ucle ar fluorescence-labeled bone marrow stromal cells after transplantation into cer ebral infarct and spinal cord injury in mice[J]. Neuropathology,2003,23(3):16 9-180.
[9] Devine SM. Mesenchymal stem cells: will they have a role in the clinic? [J]. J Cell Biochem Suppl, 2002, 38(2): 73-79.
[10] Mezey E, Chandross KJ. Bone marrow: a possible alternative source of ce lls in the adult nervous system[J]. Eur J Pharmacol, 2000, 405(3):297-302.
[11] Weimann JM, Charlton CA, Brazelton TR, et al. Contribution of transplan ted bone marrow cells to Purkinje neurons in human adult brains[J]. Proc Natl Aca d Sci USA, 2003, 100(4): 2088-2093.
[12] Alvarez-Dolado M, Pardal R, Garcia-Verdugo JM, et al. Fusion of bone-ma rrow -derived cells with Purkinje neurons, cardiomyocytes and hepatocytes[J].Natur e,2003,425(6961):968-973.
[13] Chalfie M,Tu Y,Euskirchen G,et al.Green fluorescent protein as a marker for gene expression[J]. Science, 1994,263(5148):802-805.
[14] Shioda T, Munn LL, Fenner MH, et al. Early events of metastasis in the micr ocirculation in volve changes in gene expressin of cancer cell[J].Am J Pathol ,1997,150(6):2099-2112.

相似文献/References:

[1]张春,李清漪,兰彦平,等.莪术醇诱导胶质瘤U87细胞凋亡的实验研究[J].宁夏医科大学学报,2016,(05):494.[doi:10.16050/j.cnki.issn1674-6309.2016.05.004]
 ZHANG Chun,LI Qingyi,LAN Yanping,et al.Curcumol Inducing the Apoptosis of Human Glioma Cell Line U87 Cells[J].Ningxia Medical University,2016,(01):494.[doi:10.16050/j.cnki.issn1674-6309.2016.05.004]
[2]贾咏存,李芹,朴文花,等.血小板相关IgG与网织血小板联合检测在原发免疫性血小板减少症诊断中的应用[J].宁夏医科大学学报,2015,(08):947.[doi:10.16050/j.cnki.issn1674-6309.2015.08.022]
[3]文玉军,何玲,秦毅,等.骨髓基质细胞的生物学特性及其神经分化潜能[J].宁夏医科大学学报,2006,(06):554.
[4]文玉军,吴均,张敏,等.骨髓基质细胞在大鼠纹状体出血模型脑内的存活及迁移[J].宁夏医科大学学报,2007,(03):234.
 WEN Yu-jun,WU Jun,ZHANG Ming,et al.Surviving and Migration of Bone Marrow Stromal Cells in Brain of the Striatal Hemorrhage Model of Rat[J].Ningxia Medical University,2007,(01):234.
[5]王立斌,刘 婷,马晓娜,等.人胎盘间充质干细胞的分离与鉴定[J].宁夏医科大学学报,2010,(08):864.
 WANG Li-bin,LIU Ting,MA Xiao-na,et al.Isolation and Characterization of Mesenchymal Stem Cells from Human Placenta[J].Ningxia Medical University,2010,(01):864.

备注/Memo

备注/Memo:
收稿日期:2006-06-23 基金项目:宁夏自然科学基金项目(A4004) 作者简介:文玉军(1974-),男(回族),宁夏人,讲师,硕士,从事神经生物 学研究。 通讯作者:秦毅,男,教授,硕士研究生导师,从事神经移植和神经干细胞研究。
更新日期/Last Update: 2007-01-20