Lei Pedro, Ogunade Adebimpe, Kirkwood Keith L, Laychock Suzanne G, Andreadis Stelios T
Bioengineering Laboratory, Department of Chemical and Biological Engineering, University at Buffalo, The State University of New York, Amherst, New York 14260, USA.
Tissue Eng. 2007 Aug;13(8):2119-31. doi: 10.1089/ten.2006.0210.
Despite many years of research, daily insulin injections remain the gold standard for diabetes treatment. Gene therapy may provide an alternative strategy by imparting the ability to secrete insulin from an ectopic site. The epidermis is a self-renewing tissue that is easily accessible and can provide large numbers of autologous cells to generate insulin-secreting skin substitutes. Here we used a recombinant retrovirus to modify human epidermal keratinocytes with a gene encoding for human proinsulin containing the furin recognition sequences at the A-C and B-C junctions. Keratinocytes were able to process proinsulin and secrete active insulin that promoted glucose uptake. Primary epidermal cells produced higher amounts of insulin than cell lines, suggesting that insulin secretion may depend on the physiological state of the producer cells. Modified cells maintained the ability to stratify into 3-dimensional skin equivalents that expressed insulin at the basal and suprabasal layers. Modifications at the furin recognition sites did not improve proinsulin processing, but a single amino acid substitution in the proinsulin B chain enhanced C-peptide secretion from cultured cells and bioengineered skin substitutes 10- and 28-fold, respectively. These results suggest that gene-modified bioengineered skin may provide an alternative means of insulin delivery for treatment of diabetes.
尽管经过多年研究，但每日注射胰岛素仍是糖尿病治疗的金标准。基因治疗或许能提供一种替代策略，即赋予异位位点分泌胰岛素的能力。表皮是一种易于获取的自我更新组织，能够提供大量自体细胞以生成分泌胰岛素的皮肤替代物。在此，我们使用重组逆转录病毒，用编码人胰岛素原的基因修饰人表皮角质形成细胞，该胰岛素原在A - C和B - C连接处含有弗林蛋白酶识别序列。角质形成细胞能够加工胰岛素原并分泌促进葡萄糖摄取的活性胰岛素。原代表皮细胞比细胞系产生的胰岛素量更高，这表明胰岛素分泌可能取决于产生细胞的生理状态。经过修饰的细胞保持了分层形成三维皮肤等效物的能力，这些等效物在基底层和基底上层表达胰岛素。在弗林蛋白酶识别位点的修饰并未改善胰岛素原的加工，但胰岛素原B链中的单个氨基酸替换分别使培养细胞和生物工程皮肤替代物的C肽分泌增加了10倍和28倍。这些结果表明，基因修饰的生物工程皮肤可能为糖尿病治疗提供一种替代的胰岛素递送方式。
