Zhang Xiao-Feng, Liu Tie-Lian, Sheng Wei-Hua, Yang Ji-Cheng, Miao Jing-Cheng
Department of Ophthalmology, Affiliated First Hospital, Soochow University, Suzhou 215006, China.
Zhonghua Yan Ke Za Zhi. 2009 Nov;45(11):992-8.
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.
Empirical study.Ad-VEGF(165) 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-VEGF(165) were monitored to evaluate the biocompatibility of silk fibroin. The angiogenesis-related cytokines in the cell cultivation supernatant was measured using ELISA method such as vascular endothelial growth factor (VEGF), angiogenin 1 (Ang1), epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta) in the supernatant (Two-way analysis of variance).
(1) The area of corneal neovascularization was observed to be (7.60 +/- 1.12) mm(2) at 1 week after Ad-VEGF(165) 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 amorphous, growth curve and infection efficiency of Ad-VEGF(165) between both cells culture conditions of silk protein membrane and plate cultivation. (3) After transfection, the concentration of VEGF, Ang1, EGF and TGF-beta 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), Ang1 (F = 12.47, P = 0.001), and TGF-beta (F = 0.008, P = 0.932) in the Ad-VEGF(165) 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), Ang1 (F = 0.14, P = 0.714), and TGF-beta (F = 0.008, P = 0.932)expressions in the corneal epithelium cell cultivation supernatant both with or without using silk protein membrane as carriers.
Same as cell HCECs culturing in the cultivation plate, through SF application, VEGF(165) destination gene could be high-level expressed in the supernatant having which the HCECs is cultured on SF, and in addition, the angiogenesis-related cytokines content of Ang1, EGF, and TGF-beta autocrine in the HCECS cultivation supernatant could be high-level expressed as well.
本研究旨在探讨再生丝素蛋白膜（SF）对转染人角膜上皮细胞（HCECs）细胞因子表达的影响，并研究利用转基因细胞修饰的SF构建生物材料复合物的可能性。
实证研究。将Ad-VEGF(165)注入兔角膜缘诱导角膜新生血管化（CNV）。通过生长面积评估CNV，通过免疫组织化学评估VEGF特征。将HCECs接种于细胞培养板中的丝蛋白膜上。监测细胞形态、细胞增殖活性及Ad-VEGF(165)的感染效率，以评估丝素蛋白的生物相容性。采用ELISA法检测细胞培养上清液中血管内皮生长因子（VEGF）、血管生成素1（Ang1）、表皮生长因子（EGF）和转化生长因子-β（TGF-β）等血管生成相关细胞因子（双向方差分析）。
（1）注射Ad-VEGF(165)后1周，角膜新生血管面积为（7.60±1.12）mm²，再过3周变为（12.28±2.54）mm²。注射后3天、1周和1个月，免疫组织化学观察到角膜基质中VEGF呈阳性表达。（2）丝蛋白膜培养和板培养两种细胞培养条件下，Ad-VEGF(165)的形态、生长曲线和感染效率均无差异。（3）转染后，以丝蛋白膜为载体的角膜上皮细胞培养上清液中VEGF、Ang1、EGF和TGF-β的表达浓度分别为（721.67±66.97）ng/L、（1042.67±315.81）ng/L、（2421.00±0.00）ng/L和（313.33±34.06）ng/L；未以丝蛋白膜为载体时，上述各表达浓度分别为（721.67±66.97）ng/L、（860.33±315.81）ng/L、（1960.33±797.90）ng/L和（278.00±53.11）ng/L。Ad-VEGF(165)组中VEGF（F = 168.16，P < 0.0001）、EGF（F = 52.76，P < 0.0001）、Ang1（F = 12.47，P = 0.001）和TGF-β（F = 0.008，P = 0.932）的增加具有统计学意义；然而，无论是否以丝蛋白膜为载体，角膜上皮细胞培养上清液中VEGF（F = 0.071，P = 0.793）、EGF（F = 0.563，P = 0.465）、Ang1（F = 0.14，P = 0.714）和TGF-β（F = 0.008，P = 0.932）表达浓度均无明显变化。
与在培养板中培养HCECs细胞一样，通过应用SF，VEGF(165)目的基因可在HCECs培养于其上的上清液中高水平表达，此外，HCECS培养上清液中Ang1、EGF和TGF-β自分泌的血管生成相关细胞因子含量也可高水平表达。
