再生丝素膜对转基因角膜上皮细胞表达细胞因子的影响

Impact of regenerated silk protein membrane on the cytokine expression of transfected human corneal epithelium cells

目的探讨再生丝素膜对转基因人角膜上皮细胞表达细胞因子的影响，以及转基因细胞修饰的再生丝索膜构建生物材料复合体的可能性。方法实验研究。兔角膜缘注射重组腺病毒载体血管内皮生长因子165（Ad-VEGF165）诱导新生血管，测量角膜新生血管生长面积，分析角膜组织血管内皮细胞生长因子免疫组织化学结果。用再生丝素膜培养角膜上皮细胞，观察细胞形态、生长曲线、Ad—VEGF165感染效率。收集再生丝素膜Ad—VEGF165转基因角膜上皮细胞培养上清，酶联免疫吸附试验法检测上清中血管内皮生长因子、血管生成素1、表皮生长因子、转化生长因子等细胞因子浓度。采用双因素方差分析基因感染和再生丝素膜材料两因素对HCECs细胞因子表达水平的比较。结果（1）角膜缘注射Ad-VEGF165后第1周、第1个月角膜新生血管面积分别为（7．60±1．12）mm^2、（12．28±2．54）mm^2，注射后第3天、第1周、第1个月角膜基质内可见血管内皮生长因子阳性表达。（2）再生丝素膜及无再生丝素膜两种培养条件，角膜上皮细胞细胞形态、生长曲线、Ad-VEGF165感染效率均无明显差异。（3）再生丝素膜培养Ad-VEGF165转基因角膜上皮细胞上清中各细胞因子浓度分别为血管内皮生长因子（721．67±66．97）ng／L、表皮生长因子（1042．67±315．81）ng／L、血管生成素1（2421．00±0．00）ng／L、转化生长因子（313．33±34．06）ng／L。无再生丝素膜Ad-VEGF165转基因角膜上皮细胞培养上清各细胞因子浓度分别为血管内皮生长因子（721．67±66．97）ng／L、表皮生长因子（860．33±315．81）ng／L、血管生成素1（1960．33±797．90）ng／L、转化生长因子（278．00±53．11）ng／L。Ad—VEGF165感染角膜上皮细胞培养上清中各细胞因子浓度均显著高于对照组，分别为血管内皮生长因子（F=168．16，P〈0．0001）、表皮生长因子（F=52．76，P〈0．0001）、血管生成素1（F=12．47，P=0．001）、转化生长因子（F=5．647，P=0．016）。再生丝素膜与无再生丝素膜两种培养条件下，血管内皮生长因子（F=0．071，P=0．793）、表皮生长因子（F=0．563，P=0．465）、血管生成素1（F=0．14，P=0．714）、转化生长因子（F=0．008，P=0．932）浓度比较差异无统计学意义。结论再生丝素膜与细胞培养板一样，Ad-VEGF165转基因角膜上皮细胞目的基因能获得高效表达，同时还使角膜上皮细胞自分泌的新生血管相关细胞因子表皮生长因子、血管生成素1、转化生长因子等获得高效表达。

Objective The purpose of this research was to study the influence of the regenerated silk fibroin film （SF） on cytokines expression of transfected human corneal epithelial cells （HCECs） and to investigate the possibility of constructing biomaterial complex using SF, modified by transgenic cells.Methods Empirical study. Ad-VEGF165 was injected into the limbus of a rabbit＇s cornea to induce cornea neovascularization （CNV）. CNV was evaluated by growth areas and VEGF characteristic was evaluated by immunohistochemistry. HCECs was cultivated on silk protein membrane in the cell cultivation plate. Modality of cells, activity of cell proliferation and infection efficiency of Ad-VEGF165 were monitored to evaluate the biocompatibility of silk fibroin. The angiogenesis-related cytokines in the cell cuhivation supernatant was measured using ELISA method such as vascular endothelial growth factor （VEGF）, angiogenin 1 （Angl）, epidermal growth factor （EGF） and transforming growth factor-beta （TGF-β） in the supernatant （ Two-way analysis of variance）. Results （ 1 ） The area of corneal neovascularization was observed to be （ 7.60 ± 1.12 ） mm^2 at 1 week after Ad-VEGF165 was injected and it became （ 12.28± 2. 54）mm^2 another three weeks later. Positive expression of VEGF in corneal stromal was observed by immunohistochemistry at 3 d, 1 week and 1 month after injection. （2） There was no difference noticed in morphous, growth curve and infection efficiency of Ad-VEGF165 between both cells culture conditions of silk protein membrane and plate cultivation. （3） After transfection, the concentration of VEGF, Angl, EGF and TGF-β expressions in the corneal epithelium cell cultivation supernatant with silk protein membrane as carriers was （721.67 ±66.97） ng/L,（1042. 67 ±315.81） ng/L, （2421.00 ±0.00） ng/L, and （313.33 ± 34. 06） ng/L respectively; and the concentration of each of the aforementioned expression was （721.67 ± 66. 97） ng/L, （ 860. 33 ± 315.81 ） ng/L, （ 1960. 33± 797.90） ng/L, and （278.00 ± 53.11 ） ng/L without using silk protein membrane as carriers. The increase of VEGF （F = 168. 16, P 〈 0. 0001 ）, EGF （F=52.76,P〈0.0001）, Angl（F=12.47,P=0.001）, and TGF-β（F=0.008,P=0.932）in the Ad-VEGF165 group was considered statistically significant; however, there was no evident change in the concentration of VEGF （ F = 0. 071, P = 0. 793 ）, EGF （ F = 0. 563, P = 0. 465 ）, Angl （ F = 0. 14, P = 0. 714）, and TGF-β （F = 0. 008, P = 0. 932）expressions in the corneal epithelium cell cultivation supernatant both with or without using silk protein membrane as carriers. Conclusion Same as cell HCECs culturing in the cultivation plate, through SF application, VEGF165 destination gene could be high-level expressed in the supernatant having which the HCECs is cultured on SF, and in addition, the angiogenesisrelated cytokines content of Angl, EGF, and TGF-β autocrined in the HCECS cultivation supernatant could be high-level expressed as well.