Effects of human amniotic homogenates supernatant on rat keratinocytes vitro culture

doi:10.3877/cma.j.issn.1673-9450.2016.02.004

Abstract

Abstract:

Objective

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

Methods

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.

Results

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).

Conclusion

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.

Key words: Amnion, Rat keratino cytes, Cell culture, Growth factors

Lu Lyu, Wenxian Yang, Yalin Tong, Lei Wang, Yongliang Mo, Liang Liu, Qiu Zhan, Yong Li, Fujun Zhu, Haiming Xin, Zhenyu Gong. Effects of human amniotic homogenates supernatant on rat keratinocytes vitro culture[J]. Chinese Journal of Injury Repair and Wound Healing(Electronic Edition), 2016, 11(02): 96-100.

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References 29

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组别	培养24 h	培养48 h	培养96 h
对照组	1.156±0.156	1.033±0.200	0.773±0.129
10% hAHS组	1.057±0.141	1.495±0.243	1.259±0.092
15% hAHS组	1.120±0.125	1.385±0.121	1.424±0.281
20% hAHS组	1.280±0.142	1.521±0.233	1.394±0.180
25% hAHS组	1.082±0.119	0.858±0.156	0.488±0.118
t₁值	2.055	4.644	9.694
P₁值	>0.05	<0.01	<0.01
t₂值	0.471	4.766	6.648
P₂值	>0.05	<0.01	<0.01
t₃值	2.272	5.027	8.861
P₃值	<0.05	<0.01	<0.01
t₄值	1.057	2.188	5.147
P₄值	>0.05	<0.05	<0.01

组别	培养24 h	培养48 h	培养96 h
对照组	1.156±0.156	1.033±0.200	0.773±0.129
10% hAHS组	1.057±0.141	1.495±0.243	1.259±0.092
15% hAHS组	1.120±0.125	1.385±0.121	1.424±0.281
20% hAHS组	1.280±0.142	1.521±0.233	1.394±0.180
25% hAHS组	1.082±0.119	0.858±0.156	0.488±0.118
t₁值	2.055	4.644	9.694
P₁值	>0.05	<0.01	<0.01
t₂值	0.471	4.766	6.648
P₂值	>0.05	<0.01	<0.01
t₃值	2.272	5.027	8.861
P₃值	<0.05	<0.01	<0.01
t₄值	1.057	2.188	5.147
P₄值	>0.05	<0.05	<0.01

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