APPLICATION OF PKH26 LABELING COMBINED WITH IN VIVO IMAGING TECHNOLOGY IN INTERVERTEBRAL DISC TISSUE ENGINEERING_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WU Yaohong ¹ , XU Baoshan ¹ , YANG Qiang ¹ , LI Xiulan ² , ZHANG Yang ²¹ , MA Xinlong ¹ , XIA Qun ³ , ZHANG Chunqiu ¹ , XU Haiwei ¹

1Deapartment of Spinal Surgery, 2Cell Engineering Laboratory of Orthopaedic Institute, Tianjin Hospital, Tianjin, 300211, P.R.China;;
3School of Mechanical Engineering, Tianjin University of Technology. Corresponding author: XU Baoshan, E-mail: xubshn@yahoo.com.cn;;
YANG Qiang, E-mail: yangqiang1980@126.com;

Corresponding author：

Keywords：

Intervertebral disc tissue engineering; PKH26; In vivo imaging technology; Goat

DOI：

10.7507/1002-1892.20130045

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Objective To evaluate the influence of PKH26 labeling on the biological function of the goat nucleus pulposus cells and the biological function of seeded cells in nude mice by in vivo imaging techonology. Methods Primary nucleus pulposus cells were isolated by enzymatic digestion from the nucleus pulposus tissue of the 1-year-old goat disc. The nucleus pulposus cells at passage 1 were labeled with PKH26 and the fluorescent intensity was observed under the fluorescence microscopy. The labeled cells were stained with toluidine blue and collagen type II immunocytochemistry. The cells viability and proliferation characteristics were assessed by trypan blue staining and MTT assay, respectively. Real-time fluorescent quantitative PCR was used to detect the gene expressions of collagen types I and II, and aggrecan. The fluorescent intensity and scope of the nucleus pulposus cells-scaffold composite in vivo for 6 weeks after implanting into 5 6-week-old male nude mice were measured by in vivo imaging technology. Results Primary nucleus pulposus cells were ovoid in cell shape, showing cluster growth, and the cells at passage 1 showed chondrocyte-like morphology under the inverted phase contrast microscope. The results of toluidine blue and collagen type II immunocytochemistry staining for nucleus pulposus cells at passage 1 were positive. The fluorescent intensity was even after labeling, and the cell viability was more than 95% before and after PKH26 labeling. There was no significant difference in cell growth curve between before and after labeling (P gt; 0.05). The real-time fluorescent quantitative PCR showed that there was no significant difference in gene expressions of collagen types I and II, and aggrecan between before and after labeling (P gt; 0.05). Strong fluorescence in nucleus pulposus cells-scaffold composite was detected and by in vivo imaging technology. Conclusion The PKH26 labeling has no effect on the activity, proliferation, and cell phenotype gene expression of the nucleus pulposus cells. A combination of PKH26 labeling and in vivo imaging technology can track the biological behavior of the cells in vivo.

Citation： WU Yaohong,XU Baoshan,YANG Qiang,LI Xiulan,ZHANG Yang,MA Xinlong,XIA Qun,ZHANG Chunqiu,XU Haiwei,ZENG Chao. APPLICATION OF PKH26 LABELING COMBINED WITH IN VIVO IMAGING TECHNOLOGY IN INTERVERTEBRAL DISC TISSUE ENGINEERING. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(2): 207-211. doi: 10.7507/1002-1892.20130045 Copy

1.	王兴忠, 许文荣, 朱伟, 等. 人骨髓间质干细胞PKH26荧光标记技术. 中华检验医学杂志, 2006, 29(9): 834-837.
2.	Morimoto N, Saso Y, Tomihata K, et al. Viability and function of autologous and allogeneic fibroblasts seeded in dermal substitutes after implantation. J Surg Res, 2005, 125(1): 56-67.
3.	张珏, 智伟, 谭美云, 等. PKH26与BrdU体外双重标记兔BMSCs在心肌补片中的实验研究. 中国修复重建外科杂志, 2010, 24(7): 828-833.
4.	张乃丽, 马洪强, 张育敏, 等. PKH26标记BMSCs示踪技术在构建组织工程骨中的应用. 中国修复重建外科杂志, 2009, 23(10): 1246-1249.
5.	Lee KH, Yoon DH, Park YG, et al. Effects of glial transplantation on functional recovery following acute spinal cord injury. J Neurotrauma, 2005, 22(5): 575-589.
6.	Haas SJ, Bauer P, Rolfs A, et al. Immunocytochemical characterization of in vitro PKH26-labelled and intracerebrally transplanted neonatal cells. Acta Histochem, 2000, 102(3): 273-280.
7.	Oostendorp RA, Audet J, Eaves CJ. High-resolution tracking of cell division suggests similar cell cycle kinetics of hematopoietic stem cells stimulated in vitro and in vivo. Blood, 2000, 95(3): 855-862.
8.	Lee HP, Chen YL, Shen HC, et al. Baculovirus transduction of rat articular chondrocytes: roles of cell cycle. J Gene Med, 2007, 9(1): 33-43.
9.	Fischell TA, Carter AJ, Laird JR. The beta-particle-emitting radioisotope stent (isostent): animal studies and planned clinical trials. Am J Cardiol, 1996, 78(3A): 45-50.
10.	张雪梅, 杜芳, 杨丹, 等. PKH26和DAPI联合示踪骨髓干细胞在脑缺血模型小鼠脑内迁移的实验研究. 中风与神经疾病杂志, 2011, 28(4): 296-299.
11.	胡卫华, 王净, 窦骏, 等. PKH26标记UMSC示踪技术在细胞移植治疗卵巢癌中的应用. 中国免疫学杂志, 2010, 26(9): 828-831.
12.	Medarova Z, Pham W, Kim Y, et al. In vivo imaging of tumor response to therapy using a dual-modality imaging strategy. Int J Cancer, 118(11): 2796-2802.
13.	Wetterwald A, van der Pluijm G, Que I, et al. Optical imaging of cancer metastasis to bone marrow: a mouse model of minimal residual disease. Am J Pathol, 2002, 160(3): 1143-1153.
14.	彭秀华, 沈艳, 徐春华, 等. 采用小动物活体荧光成像对胃癌皮下和原位移植肿瘤生长的动态观察. 实验动物与比较医学, 2011, 31(5): 371-375.
15.	张胜华, 程昕, 钟根深, 等. 活体成像分析异硫氰酸荧光素标记Rituximab在荷淋巴瘤裸鼠体内的生物分布. 中华医学杂志, 2010, 90(33): 2367-2370.
16.	Wu JC, Inubushi M, Sundaresan G, et al. Optical imaging of cardiac reporter gene expression in living rats. Circulation, 2002, 105(14): 1631-1634.
17.	Iyer M, Berenji M, Templeton NS, et al. Noninvasive imaging of cationic lipid-mediated delivery of optical and PET reporter genes in living mice. Mol Ther, 2002, 6(4): 555-562.
18.	管小俊, 宋琳, 郭雪君, 等. 肺内输入骨髓间充质干细胞对肺气肿大鼠慢性炎症的调控. 中国呼吸与危重监护杂志, 2011, 10(6): 520-526.
19.	李珂, 赵光, 高春芳, 等. 小动物活体成像技术的应用进展. 实用医药杂志, 2012, 29(1): 81-82.
20.	Yang Q, Peng J, Guo QY, et al. A cartilage ECM-derived 3-D porous acellular matrix scaffold for in vivo cartilage tissue engineering with PKH26-labeled chondrogenic bone marrow-derived mesenchymal stem cells. Biomaterials, 2008, 29(15): 2378-2387.

1. 王兴忠, 许文荣, 朱伟, 等. 人骨髓间质干细胞PKH26荧光标记技术. 中华检验医学杂志, 2006, 29(9): 834-837.
2. Morimoto N, Saso Y, Tomihata K, et al. Viability and function of autologous and allogeneic fibroblasts seeded in dermal substitutes after implantation. J Surg Res, 2005, 125(1): 56-67.
3. 张珏, 智伟, 谭美云, 等. PKH26与BrdU体外双重标记兔BMSCs在心肌补片中的实验研究. 中国修复重建外科杂志, 2010, 24(7): 828-833.
4. 张乃丽, 马洪强, 张育敏, 等. PKH26标记BMSCs示踪技术在构建组织工程骨中的应用. 中国修复重建外科杂志, 2009, 23(10): 1246-1249.
5. Lee KH, Yoon DH, Park YG, et al. Effects of glial transplantation on functional recovery following acute spinal cord injury. J Neurotrauma, 2005, 22(5): 575-589.
6. Haas SJ, Bauer P, Rolfs A, et al. Immunocytochemical characterization of in vitro PKH26-labelled and intracerebrally transplanted neonatal cells. Acta Histochem, 2000, 102(3): 273-280.
7. Oostendorp RA, Audet J, Eaves CJ. High-resolution tracking of cell division suggests similar cell cycle kinetics of hematopoietic stem cells stimulated in vitro and in vivo. Blood, 2000, 95(3): 855-862.
8. Lee HP, Chen YL, Shen HC, et al. Baculovirus transduction of rat articular chondrocytes: roles of cell cycle. J Gene Med, 2007, 9(1): 33-43.
9. Fischell TA, Carter AJ, Laird JR. The beta-particle-emitting radioisotope stent (isostent): animal studies and planned clinical trials. Am J Cardiol, 1996, 78(3A): 45-50.
10. 张雪梅, 杜芳, 杨丹, 等. PKH26和DAPI联合示踪骨髓干细胞在脑缺血模型小鼠脑内迁移的实验研究. 中风与神经疾病杂志, 2011, 28(4): 296-299.
11. 胡卫华, 王净, 窦骏, 等. PKH26标记UMSC示踪技术在细胞移植治疗卵巢癌中的应用. 中国免疫学杂志, 2010, 26(9): 828-831.
12. Medarova Z, Pham W, Kim Y, et al. In vivo imaging of tumor response to therapy using a dual-modality imaging strategy. Int J Cancer, 118(11): 2796-2802.
13. Wetterwald A, van der Pluijm G, Que I, et al. Optical imaging of cancer metastasis to bone marrow: a mouse model of minimal residual disease. Am J Pathol, 2002, 160(3): 1143-1153.
14. 彭秀华, 沈艳, 徐春华, 等. 采用小动物活体荧光成像对胃癌皮下和原位移植肿瘤生长的动态观察. 实验动物与比较医学, 2011, 31(5): 371-375.
15. 张胜华, 程昕, 钟根深, 等. 活体成像分析异硫氰酸荧光素标记Rituximab在荷淋巴瘤裸鼠体内的生物分布. 中华医学杂志, 2010, 90(33): 2367-2370.
16. Wu JC, Inubushi M, Sundaresan G, et al. Optical imaging of cardiac reporter gene expression in living rats. Circulation, 2002, 105(14): 1631-1634.
17. Iyer M, Berenji M, Templeton NS, et al. Noninvasive imaging of cationic lipid-mediated delivery of optical and PET reporter genes in living mice. Mol Ther, 2002, 6(4): 555-562.
18. 管小俊, 宋琳, 郭雪君, 等. 肺内输入骨髓间充质干细胞对肺气肿大鼠慢性炎症的调控. 中国呼吸与危重监护杂志, 2011, 10(6): 520-526.
19. 李珂, 赵光, 高春芳, 等. 小动物活体成像技术的应用进展. 实用医药杂志, 2012, 29(1): 81-82.
20. Yang Q, Peng J, Guo QY, et al. A cartilage ECM-derived 3-D porous acellular matrix scaffold for in vivo cartilage tissue engineering with PKH26-labeled chondrogenic bone marrow-derived mesenchymal stem cells. Biomaterials, 2008, 29(15): 2378-2387.

Chinese Journal of Reparative and Reconstructive Surgery

APPLICATION OF PKH26 LABELING COMBINED WITH IN VIVO IMAGING TECHNOLOGY IN INTERVERTEBRAL DISC TISSUE ENGINEERING

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