oleosin-rhFGF9融合蛋白的红花表达及其毛发再生、创伤修复研究

Expression of oleosin-rhFGF9 fusion protein in Carthamus tinctorius and determination of hair regeneration and wound repair potential in mice

利用红花表达成纤维细胞生长因子9（fibroblast growth factor 9,FGF9）重组融合蛋白,研究其对毛发生长及创伤修复的作用,为红花作为植物反应器提供基础并对FGF9融合蛋白毛发及创伤商业化应用奠定基础。通过冻融法将鉴定的p OTBar-oleosin-rhFGF9质粒转入农杆菌EHA105中,农杆菌介导法将oleosin-rhFGF9基因转入红花子叶中,经红花组织培养、Basta筛选获得转基因再生苗,嫁接法,PCR试验验证,扩繁,获得转基因T3红花种子。Western blot法分析及ELISA定量融合蛋白含量为0.09%。在质量浓度为60μg·L-1时,转基因红花油体促NIH/3T3细胞增殖较好,且有一定剂量依赖性。C57BL/6小鼠60只,分别建立脱发模型和创伤模型,分别为空白组（PBS或生理盐水）、阴性组（野生型红花种子油体,60 g·L-1）、阳性组（FGF9,0.054 g·L-1）、低剂量组（转基因红花油体,10 g·L-1）和高剂量组（转基因红花油体,60 g·L-1）,隔天涂抹1次,每次100μL,15 d后取皮,固定包埋进行组织学分析。小鼠毛发实验结果表明各组小鼠毛发长势良好,其中高剂量组小鼠毛发浓密,再生毛发数均较阳性组有显著效果;创伤实验愈合明显,阳性组、高剂量组、低剂量组愈合效果较空白组均显著,且在第5天,高剂量组愈合效果显著优于阳性组。该实验成功转化红花,获得了oleosin-rhFGF9融合蛋白表达的T3转基因红花,其具有促进毛发再生和创伤修复的作用。

The expression of fibroblast growth factor 9（FGF9） recombinant fusion protein in Carthamus tinctorius was used to identify its effect on hair regrowth and wound repair system in mice,providing a basis for C. tinctorius as a plant bioreactor,and establishing a foundation for commercial applications of FGF9 fusion protein in hair regrowth and wound repair. The identified p OTBar-oleosinrhFGF9 plasmid was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method,and the oleosin-rhFGF9 gene was transformed into safflower leaves by A. tumefaciens mediated method. Transgenic safflower seedlings were then obtained by tissue culture. After basta screening,transgenic T3 safflower seeds were obtained by grafting method,PCR verification and propagation. The expression of oleosin-rhFGF9 was detected by Western blot,and the content of oleosin-rhFGF9 fusion protein was 0. 09% by using ELISA quantitative method. It was observed that 60 μg·L-1 transgenic safflower oil had better effect on promoting NIH/3 T3 cells proliferation in a certain dose-dependent manner. Sixty C57 BL/6 mice were used to establish alopecia model and wound model respectively,andthen were randomly divided into control group（treated with PBS or saline）,negative group（treated with wild type safflower seed oil bodies,60 g·L-1）,positive group（treated with FGF9,0. 054 g·L-1）,low dose group（treated with transgenic safflower oil bodies,10 g·L-1） and high dose group（treated with transgenic safflower oil bodies,60 g·L-1）. The skin of all above-mentioned mice models were coated with soft adhesive manner every other day,100 μL/time. After 15 days,the mice skin was cut and embedded for histological analysis. The hair regrowth experimental results showed that the hair of mice grew well,and the mice in high dose group had bushy hair,with significant effect on regeneration hair number as compared with the positive group. The healing was obvious in wound experiment,with significant healing effect in positive group,high dose group and low dose group as compared to blank control group. Furthermore,high dose group remarkably showed a better and higher healing effect than the positive group at day 5. Oleosin-rhF GF9 was successfully transformed into safflower,and T3 transgenic safflower oil bodies expressed oleosin-rhF GF9 fusion protein were obtained,with the role of promoting hair regeneration and wound repair in mice.