Genetic Engineering of Human Skin Substitutes

描述(由申请人提供)：烧伤创面感染是导致患者发病率和死亡率的主要并发症。局部抗菌剂对角质形成细胞有细胞毒性，并影响培养的皮肤替代物的植入。这项提议的目标是产生一种具有抗菌活性的治疗性人体皮肤替代品，用于治疗严重烧伤患者。在过去的十年里，人们对人类衍生的内源性抗菌肽产生了新的兴趣。人β防御素是多肽家族中的一员，对革兰氏阳性和革兰氏阴性细菌具有广泛的抗菌特性。NIKS人角质形成细胞系是遗传统一、无肿瘤、无病原体的人角质形成细胞的始终如一的来源，这些细胞将经过基因工程，表达增强水平的广谱防御素家族成员HBD-3。我们的具体目标是：1)建立稳定的转基因NIKS细胞克隆，并在单层培养中评估HBD-3的表达水平和抗菌活性。2)使用稳定的HBD-3克隆产生器官类型培养，并根据表达水平、生长特性、形成分层表皮的能力和抗菌活性进行表征。3)安全有效地进行动物移植。

DESCRIPTION (provided by applicant): Burn wound infection is a major complication leading to patient morbidity and mortality. Topical antimicrobials are cytotoxic to keratinocytes and compromise engraftment of cultured skin substitutes. The goal of this proposal is to generate a therapeutic human skin substitute with antimicrobial activity for use in the treatment of severely burned patients. In the past decade there has been emerging excitement regarding human derived endogenous antimicrobial peptides. One family of peptides, human beta defensins has broad antimicrobial properties against gram positive and gram negative bacteria. The NIKS human keratinocyte cell line is a consistent source of genetically uniform, non-tumorigenic, pathogen free human keratinocytes that will be genetically engineered to express enhanced levels of the broad spectrum defensin family member, hBD-3. Our specific aims are to 1) Develop stable genetically modified NIKS cell clones and evaluate expression levels of hBD-3 and antimicrobial activity in monolayer culture. 2) Produce organotypic cultures using stable hBD-3 clones and characterize based on expression levels, growth properties, ability to form stratified epidermis, and antimicrobial activity. 3) Perform animal grafting for safety and efficacy.