Effects of transfection of human epidermal growth factor gene with adenovirus vector on biological characteristics of human epidermal cells
-
摘要: 目的 观察腺病毒介导的人EGF(Ad-hEGF)基因转染人表皮细胞的合适条件及转染对该细胞生物学特性的影响。 方法 取包皮环切术后弃用的新鲜人体包皮组织,通过酶消化法分离人表皮细胞,传代培养,采用第3代细胞进行以下实验。(1)按随机数字表法(分组方法下同)将细胞分为未转染组及5、20、50、100、150、200倍转染组,每组3孔。未转染组不转染Ad-hEGF基因,后6组分别按感染复数(MOI)5、20、50、100、150、200转染Ad-hEGF基因。转染24、48、72 h,倒置相差显微镜下观察细胞形态,倒置荧光显微镜下观察细胞绿色荧光蛋白表达。(2)另取3个批次细胞,分别同前分组处理,分别于转染24、48、72 h,流式细胞仪检测Ad-hEGF基因转染率(样本数为3),ELISA法检测收集的细胞培养上清液中EGF质量浓度(样本数为6),细胞计数试剂盒8(CCK8)及酶标仪检测细胞增殖活性(样本数为6)。(3)另取细胞分为未转染组和转染组,每组6孔。未转染组用未转染细胞的细胞培养上清液孵育,转染组用以最佳MOI(50)转染Ad-hEGF基因细胞的细胞培养上清液孵育,分别于孵育1、3、5 d,CCK8及酶标仪检测细胞分泌的EGF生物活性。(4)另取细胞分为未转染组和转染组,每组12孔。未转染组不转染Ad-hEGF基因,转染组以最佳MOI(50)转染Ad-hEGF基因。转染24 h,免疫荧光染色法检测细胞角蛋白14(CK14)、CK19表达。(5)另取细胞,同(4)分组(每组6孔)处理,于划痕后0(即刻)、12、24、48 h,倒置相差显微镜下观测细胞迁移距离。对数据行析因设计方差分析、重复测量方差分析、LSD检验。 结果 (1)转染24、48 h,各转染组细胞形态与未转染组比较,无明显改变;转染72 h,200倍转染组的细胞碎片较未转染组明显增多。转染24、48、72 h,未转染组细胞未见绿色荧光蛋白表达,各转染组绿色荧光蛋白阳性细胞数随转染时间延长逐渐增多。(2)各转染组细胞Ad-hEGF基因转染率随转染时间延长逐渐增高。转染72 h,50~200倍转染组细胞Ad-hEGF基因转染率均大于90%;未转染组及5、20倍转染组细胞Ad-hEGF基因转染率则分别为(0.51±0.20)%、(62.44±6.23)%、(75.00±5.43)%,明显低于50倍转染组的(93.12±2.55)%,
P 值均小于0.01。随着转染时间的延长,各转染组细胞培养上清液中EGF质量浓度逐渐增高;转染72 h,50倍转染组细胞培养上清液中EGF质量浓度明显高于其余各组(P 值均小于0.01)。转染24、48 h,各组细胞增殖活性相近(P 值均大于0.05);转染72 h,200倍转染组细胞增殖活性明显低于其余各组(P 值均小于0.05)。(3)孵育1 d,2组细胞分泌的EGF生物活性相近(P >0.05);孵育3、5 d,转染组细胞分泌的EGF生物活性明显高于未转染组(P 值均小于0.01)。(4)转染24 h,转染组细胞CK14、CK19表达较未转染组增强。(5)划痕后0 h,2组细胞划痕宽度基本一致。划痕后12~48 h,转染组细胞迁移距离明显长于未转染组(P 值均小于0.01)。 结论 Ad-hEGF基因转染人表皮细胞的合适MOI为50~150,以50为最佳,以此MOI转染既能高效表达目的基因,又可保持细胞良好的增殖、分化和迁移能力。Abstract: Objective To investigate the suitable transfection condition of human epidermal cells (hECs) with human epidermal growth factor (EGF) gene by adenovirus vector (Ad-hEGF) and its effects on the biological characteristics of hECs. Methods hECs were isolated from deprecated human fresh prepuce tissue of circumcision by enzyme digestion method and then sub-cultured. hECs of the third passage were used in the following experiments. (1) Cells were divided into non-transfection group and 5, 20, 50, 100, 150, and 200 fold transfection groups according to the random number table (the same grouping method below), with 3 wells in each group. Cells in non-transfection group were not transfected with Ad-hEGF gene, while cells in the latter six groups were transfected with Ad-hEGF gene in multiplicities of infection (MOI) of 5, 20, 50, 100, 150, and 200 respectively. The morphology of the cells was observed with inverted phase contrast microscope, and expression of green fluorescent protein of the cells was observed with inverted fluorescence microscope at transfection hour (TH) 24, 48, and 72. (2) Another three batches of cells were collected, grouped, and treated as above, respectively. Then the transfection rate of Ad-hEGF gene was detected by flow cytometer (n =3), the mass concentration of EGF in culture supernatant of cells was detected by enzyme-linked immunosorbent assay (n =6), and the proliferation activity of cells was detected by cell counting kit 8 (CCK8) and microplate reader (n =6) at TH 24, 48, and 72, respectively. (3) Cells were collected and divided into non-transfection group and transfection group, with 6 wells in each group. Cells in non-transfection group were cultured with culture supernatant of cells without transfection, while cells in transfection group were cultured with culture supernatant of cells which were transfected with Ad-hEGF gene in the optimum MOI (50). CCK8 and microplate reader were used to measure the biological activity of EGF secreted by cells on culture day 1, 3, and 5. (4) Cells were collected and divided into non-transfection group and transfection group, with 12 wells in each group. Cells in non-transfection group were not transfected with Ad-hEGF gene, while cells in transfection group were transfected with Ad-hEGF gene in the optimum MOI (50). The expression levels of cytokeratin 14 (CK14) and CK19 of cells were measured by immunofluorescence staining at TH 24. (5) Cells were collected, grouped, and treated as in (4), with 6 wells in each group. At post scratch hour (PSH) 0 (immediately after scratch), 12, 24, and 48, the migration distance of cells was observed and measured with inverted phase contrast microscope. Data were processed with analysis of variance of factorial design, analysis of variance for repeated measurement, and LSD test. Results (1) At TH 24 and 48, morphology of cells in each transfection group and non-transfection group were similar. Compared with that in non-transfection group, the cell debris increased significantly in 200 fold transfection group at TH 72. At TH 24, 48, and 72, the expression of green fluorescent protein was not seen in cells of non-transfection group, whereas it increased in cells of transfection group over transfection time. (2) The transfection rate of Ad-hEGF gene of cells in each transfection group increased gradually over transfection time. At TH 72, the transfection rates of Ad-hEGF gene of cells in 50-200 fold transfection groups were all above 90%, while the transfection rates of Ad-hEGF gene of cells in non-transfection group, 5, and 20 fold transfection groups were (0.51±0.20)%, (62.44±6.23)%, and (75.00±5.43)% respectively, which were obviously lower than the rate in 50 fold transfection group [(93.12±2.55)%, withP values below 0.01]. The mass concentration of EGF in culture supernatant of cells in each transfection group increased gradually over transfection time. At TH 72, the mass concentration of EGF in culture supernatant of cells in 50 fold transfection group was obviously higher than that in each of the other groups (withP values below 0.01). The proliferation activity of cells in each group at TH 24 and 48 was similar (withP values above 0.05). At TH 72, the proliferation activity of cells in 200 fold transfection group was obviously lower than that in other groups (withP values below 0.05). (3) On culture day 1, the biological activity of EGF secreted by cells in two groups was similar (P >0.05). On culture day 3 and 5, the biological activity of EGF secreted by cells in transfection group were obviously higher than that in non-transfection group (withP values below 0.01). (4) At TH 24, the expression levels of CK14 and CK19 of cells in transfection group were higher than those in non-transfection group. (5) The width of scratch in two groups was nearly the same at PSH 0. At PSH 12-48, the migration distance of cells in transfection group was obviously longer than that in non-transfection group (withP values below 0.01). Conclusions The suitable range of MOI of hECs transfected with Ad-hEGF gene is 50-150, and 50 is the optimum. hECs transfected with Ad-hEGF gene with MOI 50 can effectively express the EGF gene and keep its good abilities of proliferation, differentiation, and migration, as well.
点击查看大图
计量
- 文章访问数: 48
- HTML全文浏览量: 1
- PDF下载量: 2
- 被引次数: 0