Construction of self microbe-protective transplantable epithelium by gene engineering

为了构建自身微生物保护的可移植上皮，我们研究了上皮细胞或粘膜细胞的转染方法、抗微生物肽基因质粒的构建及转染后的活性。(1)研究口腔黏膜细胞的非病毒转染方法。测试了六种商业转染试剂。CellFECTIN表达效果最好。并确定了转染的最佳条件。当在最佳条件下转染粘膜细胞时，报告蛋白的产量比其他转染试剂高10倍。(2)构建了sapecin表达质粒pSecSape。沙培素是一种抗菌肽。通过我们的转染方法将质粒pSecSape转染角质形成细胞。通过使用大肠杆菌、金黄色葡萄球菌和铜绿假单胞菌的抗微生物试验评估转染的角质形成细胞上清液的抗微生物活性。上清液强烈地使这些微生物消退。

In order to construct self microbe-protective transplantable epithelium, we investigated transfection method for epitherial cells or mucosal cells, plasmid construction of antimicrovial peptide gene, and its activity after transfection.(1) Non-viral transfection method for oral mucosal cell was investigated. Six commercial transfection reagents were tested. The CellFECTIN performed best expression of transfected gene. Furthermore, the optimal condition of transfection was determined. When the mucosal cells were transfected at the optimal condition, the production of reporter protein increased up to ten times higher than other transfection reagents.(2) A sapecin expression plasmid, pSecSape was constructed. Sapecin is one of antimicrobial peptide. The plasmid pSecSape was transfected in keratinocyte cells by our transfection method. Antimicrobial activity of supernatant of transfected keratinocyte cells was assessed by antimicrobial tests using E.coli, S.aureus, and P.aeruginosa. The supernatant strongly regressed these microbes.