观察不同浓度人羊膜匀浆上清液(hAHS)对体外培养的SD大鼠角朊细胞生长的影响。

取SD大鼠背部皮肤，采用中性蛋白酶与胰蛋白酶复合消化的方法，分离第一代角朊细胞。将新鲜人羊膜制备成hAHS，采用考马斯亮蓝法、酶联免疫吸附试验分别测定hAHS中总蛋白含量和表皮生长因子(EGF)、碱性成纤维生长因子(bFGF)、血管内皮生长因子(VEGF)的浓度。用不同浓度hAHS对第一代角朊细胞进行体外培养：将细胞以2.5×10⁴个/mL密度接种于96孔板中(每孔100 μL)，随机数字表法将其分为体积分数0(对照组)及10%、15%、20%、25% hAHS组，用含10%胎牛血清低糖培养基培养24 h后，根据hAHS在低糖DMEM培养基中的不同体积分数进行换液，分别于培养即刻和培养24、48、96 h，用噻唑兰法检测各孔的吸光度值并绘制角朊细胞的生长曲线和计算各组细胞增殖率。采用SPSS13.0统计软件进行分析，各不同体积分数hAHS组的数据与对照组比较采用Dunnett－t检验。

hAHS中总蛋白含量为(675.435±9.215)×10^－3 g/L，EGF、bFGF和VEGF的浓度分别为(470.625±2.546)×10^－6、(4.121±0.026)×10^－6和(0.172±0.002)×10^－6 g/L。与对照组比较，10% hAHS组培养48、96 h角朊细胞增殖率明显高于对照组，差异均有显著性统计学意义(t＝4.644、9.694，P值均小于0.01)，15% hAHS组培养48、96 h角朊细胞增殖率均明显高于对照组，差异均有显著性统计学意义(t＝4.766、6.648，P值均小于0.01)；20% hAHS组培养24 h角朊细胞增殖率高于对照组，差异有统计学意义(t＝2.272，P<0.05)，培养48、96 h增殖率均明显高于对照组，差异有显著性统计学意义(t＝5.027、8.861，P值均小于0.01)；25% hAHS组培养48 h增殖率低于对照组，差异有统计学意义(t＝2.188，P<0.05)，培养96 h增殖率明显低于对照组，差异有显著性统计学意义(t＝5.147，P<0.01)。

体积分数10%、15%、20% hAHS对体外培养的角朊细胞增殖有明显促进作用，且促增殖的效果随hAHS体积分数的增加而增强，但hAHS的体积分数在25%时角朊细胞的增殖受到抑制，平稳期相对缩短。

To observe the effects of different concentrations of human amniotic homogenates supernatant on the growth of SD rat keratinocytes in vitro.

The skin was taken from back of young SD rats. The skin was digested by dispase and trypsin to seperate the keratinocytes. Fresh human amniotic were made into human amniotic homogenates supernatant. Coomassie Brilliant Blue method was used to determinate the total protein in human amniotic homogenates supernatant. The concentrations of epidermal growth factor, basic fibroblast growth factor and vascular endothelial growth factor were detected by enzyme linked immunosorbent assay. Different concentrations of human amniotic homogenates supernatant was used to culture the first generation of rat keratinocytes in vitro: the cells were seeded in 96-well culture plates at density of 2.5×10⁴/mL. After 24 h incubation, cells were divided into five groups (0, 10%, 15%, 20%, 25% hAHS in low sugar DMEM medium), At 0 (the right day), 24 h, 48 h, 96 h, thiazolyl blue assay was used to detect absorbance values in each well and calculate the rate of each group cell proliferation after incubation. By using SPSS13.0 statistical software for analysis, the data of different volume fraction hAHS group was compared with the control group by Dunnett-t.

The total protein concentration of hAHS was (675.435±9.215)×10^-3 g/L, in which the concentration of EGF, bFGF and VEGF were(470.625±2.546), (4.121±0.026), (0.172±0.002)×10^-6 g/L. 10%, 15%, 20%, 25% hAHS group was separately compared with the control group in proliferation rate. The proliferation rate of 10% hAHS group had no statistical difference at 24 h compared with control group (P>0.05) and was greater than the control group with a statistically significant difference at 48, 96 h(t=4.644, 9.694, all P values were less than 0.01); The proliferation rate of 15% hAHS group had no statistical difference at 24 h compared with control group (P>0.05)and was greater than the control group with a statistically significant difference at 48, 96 h(t=4.766, 6.648, all P values were less than 0.01). The proliferation rate of 20% hAHS group was greater than the control group with a statistically significant difference at 24, 48, 96 h(t=2.272, 5.027, 8.861, all P values were less than 0.05). The proliferation rate of 25% hAHS group had no statistical difference at 24 h compared with control group(P>0.05) and was smaller than control group with a statistically significant difference at 48, 96 h(t=2.188, 5.147, all P values were less than 0.05).

hAHS plays a significant role in promoting the SD rat KCs vitro culture at 10%, 15%, 20% concentration. The effect of promoting proliferation gets better with the increasing concentration. But hAHS inhibits rat keratinocytes proliferation at 25% concentration and the stable period gets shorter.