微囊化NGF基因修饰NIH3T3细胞对体外培养的组织工程皮肤功能的影响

神经生长因子(Nerve growth factor,NGF)能促进周围神经再生,还能加速创面收缩和再上皮化。本实验采用微囊和组织工程研究技术,探讨构建微囊化转NGF基因NIH3T3细胞复合组织工程真皮的可行性及其生物效应。我们将NGF基因修饰的NIH3T3细胞包裹在海藻酸钠-多聚赖氨酸-海藻酸钠微囊内体外培养,夹心ELISA法测定其上清NGF含量,将此微囊与人表皮细胞、成纤维细胞共培养一周后行MTT、羟脯氨酸(HP)测定、超微结构及组织形态学观察。用组织工程皮肤培养系统构建复合微囊的组织工程真皮,HE染色、羟脯氨酸测定和扫描电镜观察其生物功能及活性。我们发现:转NGF基因微囊体外培养8周,仍稳定分泌NGF,能促进人表皮细胞增殖,促进人成纤维细胞分泌胶原,复合组织工程真皮后仍保持此功能。因此,将微囊和转基因技术结合构建组织工程皮肤是可行的。

The Effect of Microencapsulated NGF-expressing NIH-3T3 Cells on Bioengineered Dermis Function In Vitro

Nerve growth factor(NGF) can promote the regeneration of peripheral nerve as well as contraction and reepithelization of wound.We constructed a bioengineered dermis containing microencapsulated NGF-expressing NIH-3T3 cells and study the effect of the microencapsule to the bioengineered dermis and seed cells.NGF gene was transfected into NIH-3T3 cells and enclosed into alginate-poly-L-lysine-alginate(APA) microencapsulation and cultivated in vitro.Content of NGF in microencapsules culturing supernatant was measured by enzyme linked immunosorbent assay(ELISA) method.These microencapsules were co-cultured with epidermic cells and fibroblast cells.Bioengineered dermis was constructed with NGF-expressing micorencapsules as seed cells using tissue engineering method.NIH-3T3 microencapsules,empty microencapsules,normal culture media were control groups.After one week culture,the characteristics of the dermis were described by MTT test,the content of hydroxyproline(HP),HE staining and ultrastructure photograph.We found the NGF-expressing microencapsulates can secret NGF steadly after cultured 8w in vitro,promot the proliferation of epidermic cells and secret collagen of fibroblast cells.These functions can maintaine in bioengineered dermis.So NGF-expressing NIH-3T3 microencapsulates can promote the quality of bioengineered dermis.