Tchorzewski M T, Duncan M D, Nass P, Qureshi F G, Gearhart P J, Winchurch R, Harmon J W
Section of Surgical Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, 21224, USA.
J Surg Res. 1998 Jul 1;77(2):99-103. doi: 10.1006/jsre.1998.5351.
Topical application of growth factors to wounds has proven to be suboptimal in achieving epithelial growth and accelerating healing. We propose transfection of fibroblasts with a gene for acidic fibroblast growth factor (aFGF) which will allow continuous, local delivery of the growth factor to wounds, ulcerative lesions, or healing tissues.
We utilized a pMEXneo vector containing the human aFGF gene with a secretory signal sequence from the hst/KS3 gene to obtain continuous secretion of therapeutic doses of aFGF. NIH 3T3 fibroblasts were transfected using a liposomal transfection reagent and grown in selective media.
Dot blot hybridization with labeled complementary DNA probes revealed the presence of plasmid DNA in transfected but not wild type fibroblasts. Intracellular concentrations of aFGF remained low in transfected cells; however, the media contained high levels (32 +/- 7 nM) of aFGF as measured by ELISA. Concentrations of aFGF capable of stimulating cell proliferation were maintained for several weeks.
The aFGF cDNA was transcribed and translated into a functional polypeptide that is secreted from NIH 3T3 cells at physiologically significant concentrations. Stable transfection with a eukaryotic vector which induces secretion of aFGF at levels promoting cell growth holds promise for clinical application in wounds or healing tissue. Transfection could be achieved by topical or endoscopic injection of this type of vector.
已证明将生长因子局部应用于伤口在促进上皮生长和加速愈合方面效果欠佳。我们提议用酸性成纤维细胞生长因子（aFGF）基因转染成纤维细胞，这将使生长因子能持续、局部地递送至伤口、溃疡性病变或愈合组织。
我们利用一种含有人类aFGF基因及来自hst/KS3基因的分泌信号序列的pMEXneo载体，以获得治疗剂量aFGF的持续分泌。使用脂质体转染试剂转染NIH 3T3成纤维细胞，并在选择性培养基中培养。
用标记的互补DNA探针进行斑点印迹杂交显示，转染的成纤维细胞中存在质粒DNA，而野生型成纤维细胞中不存在。转染细胞内aFGF的浓度保持较低水平；然而，通过酶联免疫吸附测定法测得培养基中含有高水平（32±7 nM）的aFGF。能够刺激细胞增殖的aFGF浓度持续维持了数周。
aFGF cDNA被转录并翻译成一种功能性多肽，该多肽以具有生理意义的浓度从NIH 3T3细胞中分泌出来。用真核载体进行稳定转染，可诱导aFGF以促进细胞生长的水平分泌，这为在伤口或愈合组织中的临床应用带来了希望。这种类型的载体可通过局部或内镜注射实现转染。
