重组人干细胞生长因子规模化生产分析及其应用-analysis of large - scale production of recombinant human stem cell growth factor and its application.docxVIP

万方数据 万方数据 摘 要 干细胞生长因子（Stem Cell Growth Factors，SCGF）主要是由基质细胞产生 的一类刺激早期造血干细胞及祖细胞增殖、定居的造血生长因子，是造血干细胞 增殖分化关键因子，具有多种生物学功能，可广泛应用于生物医药、精细化工等 领域。SCGF 在天然组织中含量极少，本文利用基因工程技术生产重组人干细胞 生长因子（Recombinant Human Stem Cell Growth Factors，rhSCGF），并对其在 医学美容方面的应用进行初步探索。 首先通过构建 pET-3c-Ulp1p、pET-3c-SUMO-SCGF 重组质粒表达、纯化获 得 SUMO（Small ubiquitin-related modifier）蛋白酶 Ulp1p 和 SUMO-SCGF 重组 蛋白。Ulp1p 酶切 SUMO-SCGF 重组蛋白得到去除 SUMO 标签的 rhSCGF。结果： 得到与天然结构具有相同生物学 活性的 rhSCGF，当 rhSCGF 浓度为 5.7ng/ml 时，促人脐带间充质干细胞增殖率达到 93%。 为提高 rhSCGF 生物稳定性，我们制备了 rhSCGF 冻干制剂并进行了大规模 生产工艺优化。结果：rhSCGF 冻干制剂外观为白色粉末、水分含量低于 3.0% ， 并具有明显促人脐带间充质干细胞增殖效果。质量检测结果符合国家相关法规标 准要求。 最后对 rhSCGF 在医学美容方面的应用进行初步研究。开发出系列 rhSCGF 护肤品，并结合微针技术检测了 rhSCGF 产品效果。结果：微针结合 rhSCGF 护 肤品，能达到去痘印、淡斑美白、祛皱等护肤美容效果。 关键词： 重组人干细胞生长因子；Ulp1p；表达；纯化；细胞增殖；冻干 I ABSTRACT Stem cell growth factor (SCGF) is key factor for hematopoietic stem cell differentiation and hyperproliferation, which is produced by stroma cells,and can be widely used in biomedical, fine chemicals and other fields because of a variety of biological functions . The content of SCGF in natural tissues is rare. In this study, to get a large number of rhSCGF by gene engineering, and preliminary study on its application in the aspects of medical beauty. Fristly, the recombinant plasmid pET-3c-Ulp1p and pET-3c-SUMO-SCGF were constructed to express and purify the recombinant proteins of SUMO Ulp1p and SUMO-SCGF. rhSCGF was obtained through the SUMO label of SUMO-SCGF recombinant proteins were cut by UIp1p. As the result, rhSCGF which is with the same biological activity as the natural structure was obtained . Human umbilical cord mesenchymal stem cells proliferation was significantly, up to 93%, when rhSCGF was added 5.7ng/ml. Secondly, in order to improve the stability of rhSCGF, we prepared the rhSCGF freeze-drying preparation and optimized the mass production process. As the result, the white freeze-dried powder of rhSCGF was obtained, and its moisture content was less than 3% , and human umbilical cord mesenchym