探索人Ⅰ型胶原包被培养板不同时间对培养的人原代表皮细胞形态、黏附、增殖、迁移率及细胞周期的影响。

取来源于解放军总医院第四医学中心泌尿外科新鲜人体皮肤组织标本，配置体积分数为1%的人Ⅰ型胶原蛋白溶液并包被培养板，按包被时间不同分为10 s组、1 min组、5 min组、15 min组、30 min组及0 s组，采用胰蛋白酶动态消化法消化分离表皮细胞，接种于不同胶原包被时间的培养板中，加入表皮细胞培养基(CnT-Pr培养基)于37 ℃、含5%CO₂的细胞培养箱中培养。倒置相差显微镜下观察细胞形态，细胞计数试剂盒8(CCK-8)法检测细胞黏附及增殖，划痕实验观察细胞迁移，流式细胞法检测细胞周期。对数据行方差分析与LSD-t检验。

(1)细胞数量及形态：随时间推移，各包被组细胞形态未见明显差异，大小较均匀，呈球形或眼型，细胞数量逐渐增多。0 s组细胞数量较其他5组少且未贴壁细胞比例多。(2)细胞黏附：人原代表皮细胞接种后24 h，10 s组、1 min组、5 min组、15 min组、30 min组、0 s组的吸光度值分别为0.28±0.07、0.30±0.05、0.33±0.06、0.34±0.07、0.36±0.05、0.16±0.02，6组间比较，差异有统计学意义(F＝4.640，P＝0.014)；10 s组、1 min组、5 min组、15 min组、30 min组组间两两比较，差异均无统计学意义(P值均大于0.05)。0 s组分别与10 s组、1 min组、5 min组、15 min组、30 min组比较，差异均有统计学意义(t＝2.640、3.059、3.584、3.889、4.187，P＝0.021、0.010、0.004、0.002、0.001)。(3)细胞增殖：人原代表皮细胞增殖曲线显示胶原包被的各组细胞增殖情况相似，明显高于0 s组细胞增殖情况。细胞接种后2 d，10 s组、1 min组、5 min组、15 min组、30 min组及0 s组的细胞吸光度值分别为0.41±0.05、0.41±0.02、0.46±0.06、0.49±0.08、0.53±0.12、0.09±0.04，6组间比较，差异有统计学意义(F＝16.050，P<0.05)。组间两两比较可以发现，10 s组、1 min组、5 min组、15 min组、30 min组间差异均无统计学意义(P值均大于0.05)。0 s组分别与10 s组、1 min组、5 min组、15 min组、30 min组比较，差异均有统计学意义(t＝0.323、0.323、0.374、0.401、0.440，P值均小于0.05)。细胞接种后4 d，6组间吸光度值比较，差异均有统计学意义(F＝9.816，P＝0.001)；组间两两比较可以发现，10 s组、1 min组、5 min组、15 min组、30 min组间差异均无统计学意义(P值均大于0.05)。0 s组分别与10 s组、1 min组、5 min组、15 min组、30 min组比较，差异均有统计学意义(P值均小于0.05)。细胞接种后6 d，10 s组、1 min组、5 min组、15 min组、30 min组及0 s组的细胞吸光度值分别为2.76±0.20、3.03±0.17、3.03±0.16、3.18±0.17、3.33±0.26、0.53±0.25。(4)细胞迁移划痕后12 h，10 s组、1 min组、5 min组、15 min组、30 min组及0 s组细胞迁移率分别为(15.60±4.11)%，(18.26±6.79)%，(18.09±6.97)%，(18.00±4.70)%，(17.40±5.97)%，(4.05±1.71)%；划痕后24 h，10 s组、1 min组、5 min组、15 min组、30 min组及0 s组细胞迁移率分别为(36.33±5.63)%，(38.45±11.97)%，(42.36±14.40)%，(41.96±10.78)%，(44.04±12.28)%，(9.17±3.28)%；划痕后36 h，10 s组、1 min组、5 min组、15 min组、30 min组及0 s组细胞迁移率分别为(73.71±17.90)%，(62.33±12.45)%，(69.79±20.82)%，(81.89±18.05)%，(73.49±22.89)%，(11.62±2.64)%。各组间整体比较，差异有统计学意义(F＝9.914，P<0.05)；不同包被时间各组间进一步比较，可以发现10 s组、1 min组、5 min组、15 min组、30 min组间两两比较，差异均无统计学意义(P值均大于0.05)，0 s组分别与10 s组、1 min组、5 min组、15 min组、30 min组比较，差异均有统计学意义(P值均小于0.05)。(5)细胞周期：细胞周期检测结果显示，接种后5 d，各组人原代表皮细胞G1、G2、S期所占比例比较，差异均无统计学意义(P值均大于0.05)。

人Ⅰ型胶原包被对人原代表皮细胞贴壁培养非常必要，胶原包被培养板不同时间对人原代表皮细胞培养无显著影响。

To explore the effects of different culture plates coating time with human type Ⅰ collagen on the cell morphology, adhesion, proliferation, migration rate and cell cycle of cultured human primary epidermal cells.

Fresh human skin tissue was taken from Department of Urology Surgery, Fourth Medical Center of PLA General Hospital, then the human type Ⅰ collagen solution with a volume fraction of 1% was placed. Dishes were coating with collagen solution and divided into 10 s group, 1 min group, 5 min group, 15 min group, 30 min group, and 0 s group according to the different coating time. Then epidermal cells were dissociated from skin tissue by dynamic digestion with trypsin, inoculated in culture plates with different collagen coating time and cultured in a cell culture incubator with 5% CO₂ at 37 ℃ with epidermal culture medicum (CnT-Pr medium). Then cell morphology was observed under interted phase contrast microscope. Cell adhesion and proliferation were detected using cell counting kit-8 (CCK-8). Cell migration was observed by scratch assay, and cell cycle was detected using flow cytometry. Data were processed with analysis of variance and LSD-t test.

(1)Cell quantity and morphology: there was no significant difference in the morphology of epidermal cells within each group over time. The size of cells in each group were relatively uniform, spherical or eye-shaped, and the number of cells gradually was increasing. The number of cells in 0 s group was less than that of the other 5 groups and more unattached cells were found in the 0 s group than in the other groups. (2)Cell adhesion: 24 hours after human primary epidermal cells were inoculated, the absorbance value of epidermal cells in the 10 s group, 1 min group, 5 min group, 15 min group, 30 min group and 0 s group was 0.28±0.07, 0.30±0.05, 0.33±0.06, 0.34±0.07, 0.36±0.05, and 0.16±0.02. The difference in absorbance among the 6 groups was statistically significant (F=4.640, P=0.014). There were no significant differences between any 2 of 5 groups (the 10 s group, the 1 min group, the 5 min group, the 15 min group and the 30 min group) (with P values above 0.05). The 0 s group was compared with the 10 s group, the 1 min group, the 5 min group, the 15 min group, the 30 min group respectively, and the difference were statistically significant (t=2.640, 3.059, 3.584, 3.889, 4.187; P=0.021, 0.010, 0.004, 0.002, 0.001). (3) Cell proliferation: the human primary epidermal cells proliferation curve showed that the proliferation of cells in each collagen-coated group was similar, which was significantly higher than that of cells in the 0 s group. Two days after human primary epidermal cells were inoculated, the absorbance value of epidermal cells in the 10 s group, the 1 min group, the 5 min group, the 15 min group, the 30 min group and the 0 s group was 0.41±0.05, 0.41±0.02, 0.46±0.06, 0.49±0.08, 0.53±0.12 and 0.09±0.04. The difference in absorbance among the 6 groups was statistically significant (F=16.050, P<0.05). There were no significant differences between any 2 of 5 groups (the 10 s group, the 1 min group, the 5 min group, the 15 min group and the 30 min group) (with P values above 0.05). The 0 s group was compared with the 10 s group, the 1 min group, the 5 min group, the 15 min group, the 30 min group respectively, and the differences were statistically significant (t=0.323, 0.323, 0.374, 0.401, 0.44; with P values below 0.05). Four days after human primary epidermal cells were inoculated, the difference in absorbance among the 6 groups was statistically significant (F=9.816, P=0.001). There were no statistical differences among the 10 s group, the 1 min group, the 5 min group, the 15 min group and the 30 min group (with P values above 0.05). The 0 s group was compared with the 10 s group, the 1 min group, the 5 min group, the 15 min group, the 30 min group respectively, and the difference were statistically significant (with P values below 0.05). Six days after primary human epidermal cells were inoculated, the absorbance value of epidermal cells in the 10 s group, the 1 min group, the 5 min group, the 15 min group, the 30 min group and the 0 s group was 2.76±0.20, 3.03±0.17, 3.03±0.16, 3.18±0.17, 3.33±0.26 and 0.53±0.25. (4) Cell migration: 12 hours after scratching, the cell migration rates of the 10 s group, the 1 min group, the 5 min group, the 15 min group, the 30 min group and the 0 s group were (15.60±4.11)%, (18.26±6.79)%, (18.09±6.97)%, (18.00±4.70)%, (17.40±5.97)%, (4.05±1.71)% respectively. Twenty-four hours after scratching the cell migration rates of the 10 s group, the 1 min group, the 5 min group, the 15 min group, the 30 min group and the 0 s group were (36.33±5.63)%, (38.45±11.97)%, (42.36±14.40)%, (41.96±10.78)%, (44.04±12.28)%, (9.17±3.28)%. Thirty-six hours after scratching the cell migration rates of the 10 s group, the 1 min group, the 5 min group, the 15 min group, the 30 min group and the 0 s group were (73.71±17.90)%, (62.33±12.45)%, (69.79±20.82)%, (81.89±18.05)%, (73.49±22.89)%, (11.62±2.64)%. Overall comparison among groups showed statistically significicant difference (F=9.914, P<0.05). Further comparison between the groups showed that there were no statistical differences between the 10 s group, the 1 min group, the 5 min group, the 15 min group and the 30 min group (with P values above 0.05). The 0 s group was compared with the 10 s group, the 1 min group, the 5 min group, the 15 min group, the 30 min group respectively, and the differences were statistically significant (with P values below 0.05). (5) Cell cycle: the results of cell cycle test showed that 5 days after human primary epidermal cells were inoculated , there were no significant differences of the cell proportions of G1, G2 and S phase in cell cycle among the 6 groups (with P values above 0.05).

Type Ⅰ collagen coating culture plates play an important role on the culture of human epidermal cells and different culture plates coating time showed no significant effects on that.