重组弹性蛋白的表征及在紫外诱导皮肤光老化过程中的作用

doi:10.3969/j.issn.2097-2806.2024.09.012

摘要/Abstract

摘要：

采用基因工程技术构建重组弹性蛋白工程菌，再利用发酵纯化技术得到重组弹性蛋白，并经氨基酸N端测序、分子量测定、氨基酸组成、蛋白纯度分析、内毒素含量测定及其红外光谱和圆二色谱分析对其进行表征鉴定。通过测定重组弹性蛋白对L929小鼠胚胎成纤维细胞粘附能力和细胞活力影响，以及在中波紫外线（UVB）诱导L929细胞光老化模型中，考察重组弹性蛋白对Ⅰ型胶原蛋白（Collagen Ⅰ）、Ⅲ型胶原蛋白（Collagen Ⅲ）、弹性蛋白（Elastin）以及基质金属蛋白酶1（MMP-1）mRNA转录水平的影响，探究重组弹性蛋白在UVB诱导皮肤光老化过程中的作用。结果表明，获得的重组弹性蛋白与天然弹性蛋白二级结构相似；在0.1 mg/mL质量浓度下无细胞毒性且具有一定的细胞活力，显著促进L929细胞的粘附和增殖。此外，重组弹性蛋白可通过下调UVB辐射后L929细胞内MMP-1转录水平，提高细胞中的Collagen Ⅰ，Collagen Ⅲ和Elastin表达，对UVB诱导的L929细胞光损伤具有良好的保护作用，显著提升L929细胞的存活率。

关键词: 重组弹性蛋白, 小鼠胚胎成纤维细胞, 细胞外基质蛋白, 光老化, 紫外线（UVB）

Abstract:

The recombinant-elastin engineering bacteria was constructed by genetic engineering technology, and then recombinant elastin was obtained by fermentation purification technology. The recombinant elastin was characterized and identified by N-terminal sequencing of amino acids, molecular weight determination, analysis of amino acid composition, protein purity analysis, endotoxicity determination, infrared spectroscopy and circular dichroism analysis. To explore the roles of recombinant elastin in UVB-induced skin photoaging, the effects of recombinant elastin on the adhesion ability and cell viability of L929 mouse embryonic fibroblasts, as well as the effects of recombinant elastin on the expression levels of Collagen Ⅰ, Collagen Ⅲ, Elastin and matrix metalloproteinase 1 （MMP-1）, were investigated in the model of UVB-induced L929 cell photoaging. The results showed that the secondary structure of recombinant elastin was similar to that of natural elastin. The recombinant elastin showed certain cell viability and no cytotoxicity at 0.1 mg/mL, which significantly promoted the adhesion and proliferation of L929 cells. Additionally, recombinant elastin could also improve the mRNA transcription levels of Collagen Ⅰ, Collagen Ⅲ and Elastin in UVB irradiated cells by down-regulating the expression of MMP-1 in UVB irradiated cells, thus protecting L929 cells from light damage. That was why recombinant elastin could improve the survival rate of photodamaged fibroblasts.

Key words: recombinant elastin, mouse embryonic fibroblast, extracellular matrix protein, photoaging, ultraviolet light

中图分类号:

TQ658

张娅, 周浩, 王蓉, 侯增淼, 黄文涛, 杨鹭. 重组弹性蛋白的表征及在紫外诱导皮肤光老化过程中的作用[J]. 日用化学工业（中英文）, 2024, 54(9): 1106-1116.

Ya Zhang, Hao Zhou, Rong Wang, Zengmiao Hou, Wentao Huang, Lu Yang. Characterization of recombinant elastin and its roles in ultraviolet-induced skin photoaging[J]. China Surfactant Detergent & Cosmetics, 2024, 54(9): 1106-1116.

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氨基酸名称	理论氨基酸个数	样品考察
氨基酸名称	理论氨基酸个数	实测摩尔数	实测氨基酸个数	误差百分比/%	实测摩尔百分比/%
Asp（天冬氨酸）	0	—	—	—	—
Glu（谷氨酸）	0	—	—	—	—
Ser（色氨酸）	0	—	—	—	—
Gly（甘氨酸）	95	4 325.46	92.45	-2.67	32.78
His（组氨酸）	0	—	—	—	—
Arg（精氨酸）	0	—	—	—	—
Thr（苏氨酸）	0	—	—	—	—
Ala（丙氨酸）	46	1 872.70	40.03	-12.99	14.19
Pro（脯氨酸）	49	2 277.15	48.67	-0.67	17.26
Tyr（酪氨酸）	0	—	—	—	—
Val（缬氨酸）	92	4 718.73	100.85	9.62	35.76
Met（蛋氨酸）	0	—	—	—	—
Cys-Cys（半胱氨酸）	0	—	—	—	—
Lle（异亮氨酸）	0	—	—	—	—
Leu（亮氨酸）	0	—	—	—	—
Phe（苯丙氨酸）	0	—	—	—	—
Trp（色氨酸）	0	—	—	—	—
Lys（赖氨酸）	0	—	—	—	—

泳道	纯度/%
泳道2	97.9
泳道3	97.5
泳道4	97.0
泳道5	97.5
泳道6	98.0
泳道7	98.5
平均值	97.7

样品名称	重组弹性蛋白	2λ/重组弹性蛋白	2λ/ 检查用水	检查用水
1	-/-	+/+	+/+	-/-
2	-/-	+/+
3	-/-	+/+

组别	照射时间/min	A值	存活率/%
对照	0	0.66±0.031	100
UVB造模	1	0.65±0.019	98
	3	0.59±0.011	89
	5	0.48±0.017 ^*	73
	10	0.30±0.012 ^**	45
	15	0.24±0.014 ^**	36
	30	0.21±0.005 ^**	32

组别	A值	存活率/%
对照	0.31±0.028	100
UVB模型组	0.23±0.008 ^**	74
UVB+0.01 mg/mL重组弹性蛋白	0.25±0.016 ^*	81
UVB+0.05 mg/mL重组弹性蛋白	0.26±0.008 ^*	84
UVB+0.1 mg/mL重组弹性蛋白	0.29±0.011	94
UVB+0.25 mg/mL重组弹性蛋白	0.25±0.019 ^*	81