中华眼科杂志
中華眼科雜誌
중화안과잡지
Chinese Journal of Ophthalmology
2009年
11期
992-998
,共7页
张晓峰%刘铁连%盛伟华%杨吉成%缪竞成
張曉峰%劉鐵連%盛偉華%楊吉成%繆競成
장효봉%류철련%성위화%양길성%무경성
丝素蛋白%上皮,角膜%上皮细胞%腺病毒科%血管内皮生长因子A
絲素蛋白%上皮,角膜%上皮細胞%腺病毒科%血管內皮生長因子A
사소단백%상피,각막%상피세포%선병독과%혈관내피생장인자A
Silk%Epithelium,corneal%Epithelial cells%Adenoviridae%Vascular endothelial growth factor A
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素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-VEGF_(165))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-VEGF_(165)感染效率.收集再生丝素膜Ad-VEGF_(165)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-VEGF_(165)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-VEGF_(165)感染效率均无明显差异.(3)再生丝素膜培养Ad-VEGF_(165)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-VEGF_(165)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).g/L、血管生成素1(1960.33±797.90)ng/L、转化生长因子(278.00±53.11)ns/L.Ad-VEGF_(165)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-VEGF_(165)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.
目的 探討再生絲素膜對轉基因人角膜上皮細胞錶達細胞因子的影響,以及轉基因細胞脩飾的再生絲素膜構建生物材料複閤體的可能性.方法 實驗研究.兔角膜緣註射重組腺病毒載體血管內皮生長因子165(Ad-VEGF_(165))誘導新生血管,測量角膜新生血管生長麵積,分析角膜組織血管內皮細胞生長因子免疫組織化學結果.用再生絲素膜培養角膜上皮細胞,觀察細胞形態、生長麯線、Ad-VEGF_(165)感染效率.收集再生絲素膜Ad-VEGF_(165)轉基因角膜上皮細胞培養上清,酶聯免疫吸附試驗法檢測上清中血管內皮生長因子、血管生成素1、錶皮生長因子、轉化生長因子等細胞因子濃度.採用雙因素方差分析基因感染和再生絲素膜材料兩因素對HCECs細胞因子錶達水平的比較.結果 (1)角膜緣註射Ad-VEGF_(165)後第1週、第1箇月角膜新生血管麵積分彆為(7.60±1.12)mm~2、(12.28±2.54)mm~2,註射後第3天、第1週、第1箇月角膜基質內可見血管內皮生長因子暘性錶達.(2)再生絲素膜及無再生絲素膜兩種培養條件,角膜上皮細胞細胞形態、生長麯線、Ad-VEGF_(165)感染效率均無明顯差異.(3)再生絲素膜培養Ad-VEGF_(165)轉基因角膜上皮細胞上清中各細胞因子濃度分彆為血管內皮生長因子(721.67±66.97)ng/L、錶皮生長因子(1042.67±315.81)ng/L、血管生成素1(2421.00±0.00)ng/L、轉化生長因子(313.33±34.06)ng/L.無再生絲素膜Ad-VEGF_(165)轉基因角膜上皮細胞培養上清各細胞因子濃度分彆為血管內皮生長因子(721.67±66.97)ng/L、錶皮生長因子(860.33±315.81).g/L、血管生成素1(1960.33±797.90)ng/L、轉化生長因子(278.00±53.11)ns/L.Ad-VEGF_(165)感染角膜上皮細胞培養上清中各細胞因子濃度均顯著高于對照組,分彆為血管內皮生長因子(F=168.16,P<0.0001)、錶皮生長因子(F=52.76,P<0.0001)、血管生成素1(F=12.47,P=0.001)、轉化生長因子(F=5.647,P=0.016).再生絲素膜與無再生絲素膜兩種培養條件下,血管內皮生長因子(F=0.071,P=0.793)、錶皮生長因子(F=0.563,P=0.465)、血管生成素1(F=0.14,P=0.714)、轉化生長因子(F=0.008,P=0.932)濃度比較差異無統計學意義.結論 再生絲素膜與細胞培養闆一樣,Ad-VEGF_(165)轉基因角膜上皮細胞目的 基因能穫得高效錶達,同時還使角膜上皮細胞自分泌的新生血管相關細胞因子錶皮生長因子、血管生成素1、轉化生長因子等穫得高效錶達.
목적 탐토재생사소막대전기인인각막상피세포표체세포인자적영향,이급전기인세포수식적재생사소막구건생물재료복합체적가능성.방법 실험연구.토각막연주사중조선병독재체혈관내피생장인자165(Ad-VEGF_(165))유도신생혈관,측량각막신생혈관생장면적,분석각막조직혈관내피세포생장인자면역조직화학결과.용재생사소막배양각막상피세포,관찰세포형태、생장곡선、Ad-VEGF_(165)감염효솔.수집재생사소막Ad-VEGF_(165)전기인각막상피세포배양상청,매련면역흡부시험법검측상청중혈관내피생장인자、혈관생성소1、표피생장인자、전화생장인자등세포인자농도.채용쌍인소방차분석기인감염화재생사소막재료량인소대HCECs세포인자표체수평적비교.결과 (1)각막연주사Ad-VEGF_(165)후제1주、제1개월각막신생혈관면적분별위(7.60±1.12)mm~2、(12.28±2.54)mm~2,주사후제3천、제1주、제1개월각막기질내가견혈관내피생장인자양성표체.(2)재생사소막급무재생사소막량충배양조건,각막상피세포세포형태、생장곡선、Ad-VEGF_(165)감염효솔균무명현차이.(3)재생사소막배양Ad-VEGF_(165)전기인각막상피세포상청중각세포인자농도분별위혈관내피생장인자(721.67±66.97)ng/L、표피생장인자(1042.67±315.81)ng/L、혈관생성소1(2421.00±0.00)ng/L、전화생장인자(313.33±34.06)ng/L.무재생사소막Ad-VEGF_(165)전기인각막상피세포배양상청각세포인자농도분별위혈관내피생장인자(721.67±66.97)ng/L、표피생장인자(860.33±315.81).g/L、혈관생성소1(1960.33±797.90)ng/L、전화생장인자(278.00±53.11)ns/L.Ad-VEGF_(165)감염각막상피세포배양상청중각세포인자농도균현저고우대조조,분별위혈관내피생장인자(F=168.16,P<0.0001)、표피생장인자(F=52.76,P<0.0001)、혈관생성소1(F=12.47,P=0.001)、전화생장인자(F=5.647,P=0.016).재생사소막여무재생사소막량충배양조건하,혈관내피생장인자(F=0.071,P=0.793)、표피생장인자(F=0.563,P=0.465)、혈관생성소1(F=0.14,P=0.714)、전화생장인자(F=0.008,P=0.932)농도비교차이무통계학의의.결론 재생사소막여세포배양판일양,Ad-VEGF_(165)전기인각막상피세포목적 기인능획득고효표체,동시환사각막상피세포자분비적신생혈관상관세포인자표피생장인자、혈관생성소1、전화생장인자등획득고효표체.
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-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 (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-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 morphous, 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, 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-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), 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, 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 Angl, EGF, and TGF-β autocrined in the HCECS cultivation supernatant could be high-level expressed as well.
