Cai Jing-Bo, Li Wen-Qing, Wen Rui-Cheng, Jiang Chao, Li Xiao-Kun, Li Hai-Yan
Engineering Research Center of Bioreactor and Drug Development, Ministry of Education, Jilin Agricultural University, Changchun 130118, China.
Pharmaceutical College, Wenzhou Medical University, Wenzhou 325035, China.
Zhongguo Zhong Yao Za Zhi. 2018 Jul;43(13):2758-2763. doi: 10.19540/j.cnki.cjcmm.20180207.001.
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 pOTBar-oleosin-rhFGF9 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/3T3 cells proliferation in a certain dose-dependent manner. Sixty C57BL/6 mice were used to establish alopecia model and wound model respectively, and then 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-rhFGF9 was successfully transformed into safflower, and T₃ transgenic safflower oil bodies expressed oleosin-rhFGF9 fusion protein were obtained, with the role of promoting hair regeneration and wound repair in mice.
研究利用红花表达成纤维细胞生长因子9（FGF9）重组融合蛋白，以鉴定其对小鼠毛发生长和伤口修复系统的影响，为红花作为植物生物反应器提供依据，并为FGF9融合蛋白在毛发生长和伤口修复方面的商业应用奠定基础。将鉴定后的pOTBar-油质蛋白-rhFGF9质粒通过冻融法转化到根癌农杆菌EHA105中，采用农杆菌介导法将油质蛋白-rhFGF9基因导入红花叶片，然后通过组织培养获得转基因红花幼苗。经草丁膦筛选后，通过嫁接法、PCR验证及扩繁获得转基因T₃红花种子。通过蛋白质免疫印迹法检测油质蛋白-rhFGF9的表达，采用酶联免疫吸附测定（ELISA）定量法测得油质蛋白-rhFGF9融合蛋白含量为0.09%。结果表明，60 μg·L⁻¹转基因红花油对促进NIH/3T3细胞增殖具有一定的剂量依赖性，效果较好。选用60只C57BL/6小鼠分别建立脱毛模型和伤口模型，然后随机分为对照组（用PBS或生理盐水处理）、阴性组（用野生型红花种子油体处理，60 g·L⁻¹）、阳性组（用FGF9处理，0.054 g·L⁻¹）、低剂量组（用转基因红花油体处理，10 g·L⁻¹）和高剂量组（用转基因红花油体处理，60 g·L⁻¹）。上述所有小鼠模型的皮肤每隔一天以轻柔粘贴的方式给药，每次100 μL。15天后，将小鼠皮肤剪下并包埋进行组织学分析。毛发生长实验结果显示，小鼠毛发长得很好，高剂量组小鼠毛发浓密，与阳性组相比，对再生毛发数量的影响显著。伤口实验中愈合明显，与空白对照组相比，阳性组、高剂量组和低剂量组均有显著的愈合效果。此外，在第5天时，高剂量组的愈合效果明显优于阳性组。油质蛋白-rhFGF9成功转化到红花中，获得了表达油质蛋白-rhFGF9融合蛋白的T₃转基因红花油体，其具有促进小鼠毛发生长和伤口修复的作用。
