| Fibroblast growth factor 9?FGF9?is a well-known secretory protein,which is a member of the FGFs family.FGF9 is secreted by mesothelial cells and epithelial cells,which encompasses many biological activities,such as bone development,tissue repair,and hair regeneration and so on.The FGF9 family originating from the mechanism of cancer,bone related diseases and gonadal differentiation which can lead to the treatment of osteoarthritis,promotion of hair growth and wound repair,there is an increasing need for the clinical application of FGF9 studies.The present study were undertaken as the extensive application of plant oil body fusion technology and safflower as a bioreactor towards the production of pharmaceutical protein.In this experiment,safflower plants express oleosin-rhFGF9recombinant proteins were obtained successfully with genetic stability,biological activity and preliminary pharmacodynamics analysis.Our finding suggested two main components of the FGF9 research.The main research results are as follows:Firstly the identified pOTBar-oleosin-rhFGF9 plasmid was transformed into Agrobacterium EHA105 by freeze-thaw method,the oleosin-rhFGF9 gene was transformed into safflower leaves by Agrobacterium mediated method,and transgenic safflower seedlings were obtained by tissue culture.After Basta screening,transgenic T3 safflower seeds were obtained by grafting method,PCR verification,and propagation.Secondly,the analysis of oil body bound oleosin-rhFGF9 was verified through Western blotting.The content of oleosin-rhFGF9 fusion protein was measured 0.13%with ELISA quantitative method.It was observed that NIH/3T3 cells promote higher proliferation rate treated with 60 ng·mL-1 transgenic safflower oil body bound oleosin-rhFGF9 by MTT method,which showed a certain dose-dependent manner.Thirdly,the alopecia model of C57BL/6 mice was established,the state of hair growth,HE staining and hair regrowth number showed that the hair of mice was grown well,the high dose group mice bushy hair and similar to the positive group,and hair regrowth number compared with the blank group with significant effect;The analysis of immunohistochemical staining and the WB for the skin total protein showed that the expression of?-catenin in the high dose group and the positive group was significantly different from that in the blank group,and ultimately related to the growth of hair.Fourthly,the wound model of C57BL/6 mice was established,the size of wound,HE staining,Masson staining and wound healing rate showed that the capability of wound healing were significantly determined.Positive control group,high dose group and low dose group had a significant healing effect compared to control group.Furthermore,high dose group remarkably showed a better and higher healing effect than the positive group in 5 days. |
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