Chinese Journal of Tissue Engineering Research ›› 2010, Vol. 14 ›› Issue (29): 5339-5343.doi: 10.3969/j.issn.1673-8225.2010.29.005

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Transfecting human transformation growth factor beta 3 gene into precartilaginous stem cells cultured in three-dimensional self-assembled peptide nanofiber scaffold of KLD-12

Ding Ran, Zhang Yong, You Hong-bo, Li Feng, Sun Kai   

  1. Department of Orthopedic Surgery, Tongji Hospital, Huazhong University of Science and Technology, Wuhan   430030, Hubei Province, China
  • Online:2010-07-16 Published:2010-07-16
  • Contact: You Hong-bo, Doctor, Associate professor, Department of Orthopedic Surgery, Tongji Hospital, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China youhongbo003@yahoo.com.cn
  • About author:Ding Ran☆, Studying for doctorate, Department of Orthopedic Surgery, Tongji Hospital, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China dana_doctor@hotmail.com
  • Supported by:

     the Natural Science Foundation of China, No. 30571873*

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Abstract:

BACKGROUND: Previous tests of our research group have proved that, exogenous cytokines human transforming growth factor-β3 (hTGF-β3) has a strong capacity of inducing precartilaginous stem cells (PSCs) differentiate to mature cartilage and of promoting the proliferation. Abroad studies also confirmed a beneficial effect on the proliferation and differentiation of cartilage-derived stem cells in three-dimensional culture of the self-assembled peptide nanofiber scaffold.
OBJECTIVE: To transfect the hTGF-β3 gene in the PSCs cultured in three-dimensional self-assembled peptide nanofiber scaffold of KLD-12, and to compare the transfection efficiency with PSCs cultured in two-dimensional medium.
METHODS: The PSCs were obtained and purified using magnetic-activated cell sorting method, then transplanted into the self-assembled peptide nanofiber scaffold of KLD-12. The gene of hTGF-β3 was respectively transfected into the PSCs cultured in self-assembled scaffold and common two-dimension medium by means of xfectTM stem nano-transfection reagent. After 48 hours, the gene expression levels of hTGF-β3 were detected by immunofluorescence and RT-PCR, and the concentration of TGF-β3 in the supernatant of medium was determined in different groups and different time points after transfection by the method of ELISA.
RESULTS AND CONCLUSION: Immunofluorescence results showed that the rate of TGF-β3-positive cells in the group of PSCs cultured in three-dimensional self-assembled scaffold of KLD-12 was higher than those cultured in common two-dimensional medium (P < 0.05), which was consistent with the test results of RT-PCR. By the method of ELISA, the concentration of TGF-β3 in the supernatant of medium increased at 24 hours after transected, and reached peak after 72 hours, then decreased a little and entered the plateau stage. It’s indicated that the transfection efficiency of hTGF-β3 gene in PSCs cultured in self-assembled peptide nanofiber scaffold is higher than the group in common two-dimensional medium.

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