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中华细胞与干细胞杂志(电子版)

中华细胞与干细胞杂志(电子版) ›› 2017, Vol. 07 ›› Issue (04) : 189 -194. doi: 10.3877/cma.j.issn.2095-1221.2017.04.001

所属专题： 文献；

论著

新型慢病毒转染四种细胞及在体内示踪的研究
刘菊芬1, 阮光萍1, 李自安1, 王金祥1, 庞荣清1, 潘兴华1,†()   
  1. 1. 650032　昆明，成都军区昆明总医院细胞生物治疗中心　干细胞与免疫细胞生物医药技术国家地方联合工程实验室　云南省细胞治疗技术转化医学重点实验室
  • 收稿日期:2016-08-03 出版日期:2017-08-01
  • 通信作者: 潘兴华
  • 基金资助:
    云南省科技计划重点项目(2013CA005，2017FA040); 全军实验动物专项(2014DW006)

Study on four kinds of cells transfected by novel lentivirus and tracing in vivo

Jufen Liu1, Guangping Ruan1, Zi'an Li1, Jinxiang Wang1, Rongqing Pang1, Xinghua Pan1,†()   

  1. 1. the Cell Biological Therapy Center of Kunming General Hospital of Chendu Military Commend, the Integrated Engineering laboratory of Cell Biological Medicine of State and Regions, the Transfer Medicine Key Laboratory of Cell Therapy Technology of Yunan Province, Kunming 650032, China
  • Received:2016-08-03 Published:2017-08-01
  • Corresponding author: Xinghua Pan
  • About author:
    Corresponding author:Pan Xinghua, Email:
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引用本文：

刘菊芬, 阮光萍, 李自安, 王金祥, 庞荣清, 潘兴华. 新型慢病毒转染四种细胞及在体内示踪的研究[J]. 中华细胞与干细胞杂志(电子版), 2017, 07(04): 189-194.

Jufen Liu, Guangping Ruan, Zi'an Li, Jinxiang Wang, Rongqing Pang, Xinghua Pan. Study on four kinds of cells transfected by novel lentivirus and tracing in vivo[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2017, 07(04): 189-194.

目的

探讨最佳的生物标记方法示踪细胞，更好地观察细胞移植后在体内的归巢情况。

方法

本实验室用一种新型慢病毒（eGFP+PURO Lentivirus）转染了四种细胞，确定了四种细胞的最佳感染复数，细胞转染成功后，用最佳浓度的嘌呤霉素筛选，获得四种稳定的GFP阳性表达细胞株。所用的细胞包括人脐带间充质干细胞（hUC-MSC），树鼩脐带间充质干细胞（TS-UC-MSC），人胚肾细胞（293T），C57BL小鼠骨髓间充质干细胞（C57-BMSC）。

结果

用新型慢病毒（eGFP+PURO Lentivirus）成功转染四种常用细胞，转染成功率100﹪，细胞成功标记上GFP荧光，并确定了最佳的嘌呤霉素使用浓度，确定了不同细胞的感染复数。最终确定TS-UC-MSC的最佳感染复数为240，hUC-MSC的最佳感染复数为150，293T的最佳感染复数为100，C57-BMSC的最佳感染复数为50。用带荧光的C57-BMSC细胞回输C57BL小鼠体内，冰冻切片在肝中找到标记细胞，在脾、肺、肾、心未找到标记细胞，说明外来细胞主要在肝脏中代谢。

结论

在细胞移植治疗时，细胞成功标记上GFP能在动物体内进行示踪，良好的细胞示踪方法可以很好地反映所研究细胞在体内外的动向。

关键词: 绿色荧光蛋白质类, 慢病毒属, 转染, 骨髓, 间质干细胞, 荧光抗体技术
Objective

Explore the best biomarker method to track cells and better observe the homogenization of cells after transplantation in the body.

Method

Four cells were transfected with a novel lentivirus (eGFP+PURO Lentivirus) . The optimal MOI of the four cells was determined. After the cells were successfully transfected, the cells were screened with the optimum concentration of puromycin. Four stable GFP-positive cell lines were obtained. The cells used included human umbilical cord mesenchymal stem cells (hUC-MSC) , tree shrew umbilical cord mesenchymal stem cells (TS-UC-MSC) , human embryonic kidney cells (293T) , C57BL mouse bone marrow mesenchymal stem cells (C57-BMSC) .

Results

Four kinds of common cells were successfully transfected with novel lentivirus (eGFP + PURO Lentivirus) . The success rate of transfection was 100﹪. The cell was successfully labeled with GFP fluorescence and the optimal concentration of puromycin and MOI value was determined. The optimal MOI of TS-UC-MSC was 240, the optimal MOI of hUC-MSC was 150, and the optimal MOI of 293T was 100, the optimal MOI of C57-BMSC was 50. C57BL mice were transfused with C57-BMSC cells labeled by GFP. Frozen sections showed labeled cells were in the liver. The labeled cells were not found in the spleen, lung, kidney and heart, indicating that the foreign cells were mainly metabolized in the liver.

Conclusion

In cell transplantation treatment, the cells successfully labeled GFP can be traced in animals, a good cell tracer method can be a good reflection of the study of cells in vitro and in vivo trends.

Key words: Green fluorescent protein, Lentivirus, Transfection, Bone marrow, Mesenchymal stem cells, Fluorescent antibody technique
图1 荧光显微镜下观察四种细胞转染GFP慢病毒（×200）
图2 不同感染复数下，eGFP+PUROLentivirus转染TS-UC-MSC后流式分析
图3 GFP阳性细胞在肝、脾、肺、肾、心的分布（×200）
[1]
Wang X, Smith GM, Xu XM. Preferential and bidirectional labeling of the rubrospinal tract with adenovirus-GFP for monitoring normal and injured axons[J]. J Neurotrauma, 2011, 28(4):635-647.
[2]
Chen CH, Kuo SM, Liu GS, et al. Enhancement of neurite outgrowth in neuron cancer stem cells by growth on 3-D collagen scaffolds[J]. Biochem Biophys Res Commun, 2012, 428(1):68-73.
[3]
Rebersek M, Kanduser M, Miklavcic D. Pipette tip with integrated electrodes for gene electrotransfer of cells in suspension:a feasibility study in CHO cells[J]. Radiol Oncol, 2011, 45(3):204-208.
[4]
Zuo Q, Cui W, Liu F, et al. Co-cultivated mesenchymal stem cells support chondrocytic differentiation of articular chondrocytes[J]. Int Orthop, 2013, 37(4):747-752.
[5]
姚志成，刘波. 间充质干细胞促进肝再生的研究进展[J]. 中华肝脏外科手术学电子杂志, 2016, 5(3):198-200.
[6]
邓陶然，肖毅. 慢性移植物抗宿主病的治疗进展[J]. 器官移植, 2016, 7(1):67-71.
[7]
Wang Z, Tang X, Xu W, et al. The different immunoregulatory functions on dendritic cells betwe mesenchymal stem cells derived from bone marrow of patients with low-risk or high-risk myelodysplastic syndromes[J]. PLoS One, 2013, 8(3):e57470.
[8]
Xu L, Wang Q, Xu F, et al. Mesenchymal stem cells downregulate articular chondrocyte differentiation in noncontact coculture systems: implications in cartilage tissue regeneration[J]. Stem Cells Dev, 2013, 22(11):1657-1669.
[9]
Yan J, Tie G, Xu TY, et al. Mesenchymal stem cells as a treatment for peripheral arterial disease: current status and potential impact of type Ⅱ diabetes on their therapeutic efficacy[J]. Stem Cell Rev, 2013, 9(3):360-372.
[10]
Chalfie M, Tu Y, Euskirchen G, et al. Green fluorescent protein as a marker for gene expression[J]. Science, 1994, 263(5148):802-805.
[11]
Lippincott-Schwartz J, Patterson GH. Development and use of fluorescent protein markers in living cells[J]. Science, 2003, 300(5616):87-91.
[12]
Salamon A, Toldy E, Nagy L, et al. The role of adult bone marrow derived mesenchymal stem cells in the repair of tissue injuries[J]. Orv Hetil, 2012, 153(46):1807-1815.
[13]
Stepanenko OV, Verkhusha VV, Kuznetsova IM, et al. Fluorescent proteins as biomarkers and biosensors: throwing color lights on molecular and cellular processes[J]. Curr Protein Pept Sci, 2008, 9(4):338-369.
[14]
Perez-Cunningham J, Boyer SW, Landon M, et al. Hematopoietic stem cell-specific GFP-expressing transgenic mice generated by genetic excision of a pan-hematopoietic reporter gene[J]. Exp Hematol, 2016, 44(8):755-764.
[15]
王伟，陈超，刘延明, 等. 应用小动物活体成像追踪观察EGFP标记的间充质干细胞[J]. 中国细胞生物学学报, 2016, 3(2):185-192.
[16]
Rajabathar JR, Munusamy MA, Al-Lohedan HA. Preparation, textural and photoluminescence characterization of green fluorescence protein-immobilised Ga-ZnO(GZO)-nanocomposites[J]. J Photochem Photobiol B, 2016, 165:202-212.
[17]
Park JW, Rhee YM. Electric field keeps chromophore planar and produces high yield fluorescence in green fluorescent protein[J]. J Am Chem Soc, 2016. [Epub ahead of print].
[18]
Kataoka C, Tomiyama H, Kashiwada S. Three-dimensional visualization of green fluorescence protein-labelled Edwardsiella tarda in whole Medaka larvae[J]. J Fish Dis, 2017, 40(4):479-484.
[19]
Nagayama K, Onuma T, Ueno R, et al. Cathodoluminescence and Electron-Induced fluorescence enhancement of enhanced green fluorescent protein[J]. J Phys Chem B, 2016, 120(6):1169-1174.
[20]
Kubo S, Kataoka M, Tateno C, et al. In vivo stable transduction of humanized liver tissue in chimeric mice via high-capacity adenovirus-lentivirus hybrid vector[J]. Hum Gene Ther, 2010, 21(1):40-50.
[21]
Montini E, Cesana D, Schmidt M, et al. Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration[J]. Nat Biotechnol, 2006, 24(6):687-696.
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