神经肽P物质对放射损伤皮肤成纤维细胞分泌功能及VEGF、bFGF、PDGF表达的影响

doi:10.11958/20220599

摘要/Abstract

摘要：

目的探讨神经肽P物质（SP）对放射损伤皮肤成纤维细胞分泌功能及血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）、血小板衍生生长因子（PDGF）表达的影响。方法将对数生长期皮肤成纤维细胞HFF-1按照射剂量分为未照射组（0 Gy）,照射组（2 Gy、6 Gy、12 Gy、18 Gy）,SP干预组（0 Gy+SP、2 Gy+SP、6 Gy+SP、12 Gy+SP、18 Gy+SP）。照射后24 h,酶联免疫吸附试验检测培养基及细胞裂解液Ⅰ型胶原（Col-Ⅰ）、Ⅲ型胶原（Col-Ⅲ）、降钙素基因相关肽（CGRP）、VEGF、bFGF、PDGF表达情况;实时荧光定量PCR检测细胞VEGF、bFGF、PDGF mRNA表达。结果与0 Gy组比较,培养基Col-Ⅰ、Col-Ⅰ/Col-Ⅲ及细胞内CGRP、VEGF蛋白在各照射组均升高;bFGF、PDGF mRNA在照射组（6 Gy、12 Gy、18 Gy）升高;Col-Ⅰ在6 Gy组培养基与细胞内一致高表达;Col-Ⅰ、Col-Ⅲ、Col-Ⅰ/Col-Ⅲ、CGRP在18 Gy组培养基与细胞内均一致高表达。bFGF在照射组（12 Gy、18 Gy）呈现蛋白和mRNA水平高表达。照射前给予SP干预,与未干预相应组相比,CGRP在0 Gy+SP组培养基与细胞内一致高表达;Col-Ⅰ和Col-Ⅰ/Col-Ⅲ分别在干预组（2 Gy+SP、6 Gy+SP）和（2 Gy+SP、6 Gy+SP、18 Gy+SP）培养基与细胞内一致低表达;VEGF和PDGF分别在干预组（2 Gy+SP、12 Gy+SP、18 Gy+SP）和（12 Gy+SP、18 Gy+SP）呈现蛋白和mRNA水平一致高表达。结论 SP可调节放射损伤皮肤成纤维细胞Col-Ⅰ、Col-Ⅲ表达与分泌,降低Col-Ⅰ/Col-Ⅲ比值,协同成纤维细胞对电子束照射的应激反应,促进放射损伤皮肤成纤维细胞中VEGF、PDGF的高表达。

关键词: 辐射损伤, 皮肤疾病, 成纤维细胞, 血管内皮生长因子类, 成纤维细胞生长因子, 神经肽P物质

Abstract:

Objective To explore the the effects of neuropeptide substance P (SP) on the secretory function and the expression of endogenous vascular endothelial cell growth factor (VEGF), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) in skin fibroblasts after radiation injured. Methods HFF-1 cells in logarithmic growth phase were divided into 10 experimental groups according to the irradiation dose: the 0 Gy, 2 Gy, 6 Gy, 12 Gy, 18 Gy, 0 Gy+SP, 2 Gy+SP, 6 Gy+SP, 12 Gy+SP and 18 Gy+SP. Exogenous SP intervention was performed before irradiation with 0 Gy+SP, 2 Gy+SP, 6 Gy+SP, 12 Gy+SP and 18 Gy+SP. The enzyme linked immunosorbent assay (ELISA) was used to detect the expression levels of typeⅠcollagen (Col-Ⅰ), type Ⅲ collagen (Col-Ⅲ), calcitonin gene related peptide (CGRP) in cell culture medium and Col-Ⅰ, Col-Ⅲ, CGRP, VEGF, bFGF and PDGF in cell lysate 24 h after irradiation. Quantificational real-time polymerase chain reaction (qPCR) was used to detect the expression levels of VEGF, bFGF and PDGF mRNA. Results Compared with the 0 Gy experimental group, Col-Ⅰ, Col-Ⅰ/Col-Ⅲ in culture medium and CGRP and VEGF proteins in cell lysate were increased in the irradiated groups, bFGF and PDGF mRNA were increased in the irradiated groups (6 Gy, 12 Gy and 18 Gy). The Col-Ⅰ was highly expressed in the culture medium and cell lysate of the 6 Gy group. The Col-Ⅰ, Col-Ⅲ, Col-Ⅰ/Col-Ⅲ and CGRP were highly expressed in the culture medium and cell lysate of the 18 Gy group. The bFGF was highly expressed at protein and mRNA levels in irradiated groups (12 Gy and 18 Gy). When SP intervention was given before irradiation, the CGRP expression was consistently high in the culture medium and lysate of the 0 Gy+SP group than that of the corresponding group without intervention. The Col-Ⅰ and Col-Ⅰ/Col-Ⅲ were consistently low expressed in the culture medium and lysate of the intervention groups (2 Gy+SP, 6 Gy+SP) and (2 Gy+SP, 6 Gy+SP and 18 Gy+SP), respectively. VEGF and PDGF were highly expressed at protein and mRNA levels in the intervention groups (2 Gy+SP, 12 Gy+SP and 18 Gy+SP) and (12 Gy+SP, 18 Gy+SP), respectively. Conclusion Exogenous SP could regulate the expression and secretion of Col-Ⅰ and Col-Ⅲ, decrease the ratio of Col-Ⅰ/Col-Ⅲ and promote the high expression of VEGF and PDGF in radiation injured skin fibroblasts, so as to coordinate the stress response of fibroblasts to electron beam irradiation.

Key words: radiation damage, skin diseases, fibroblasts, vascular endothelial growth factors, fibroblast growth factors, neuropeptide substance P

中图分类号:

图/表 5

参考文献 13

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基因名称	引物序列（5'→3'）	产物大小（bp）
VEGF	上游：CGAGACCCTGGTGGACATCTT	180
VEGF	下游：TGGCCTTGGTGAGGTTTGATC	180
bFGF	上游：AGAAGAGCGACCCTCACATCA	226
bFGF	下游：CGTTTCAGTGCCACATACCAA	226
PDGF	上游：ACCAGGACGGTCATTTACGA	136
PDGF	下游：AGGGCTGGCACTTGACACT	136
GAPDH	上游：TGGGTGTGAACCATGAGAAGT	126
GAPDH	下游：TGAGTCCTTCCACGATACCAA	126

基因名称	引物序列（5'→3'）	产物大小（bp）
VEGF	上游：CGAGACCCTGGTGGACATCTT	180
VEGF	下游：TGGCCTTGGTGAGGTTTGATC	180
bFGF	上游：AGAAGAGCGACCCTCACATCA	226
bFGF	下游：CGTTTCAGTGCCACATACCAA	226
PDGF	上游：ACCAGGACGGTCATTTACGA	136
PDGF	下游：AGGGCTGGCACTTGACACT	136
GAPDH	上游：TGGGTGTGAACCATGAGAAGT	126
GAPDH	下游：TGAGTCCTTCCACGATACCAA	126

组别	Col-Ⅰ （μg/L）	Col-Ⅲ （μg/L）	Col-Ⅰ/ Col-Ⅲ	CGRP （ng/L）
0 Gy组	14.34±0.25	31.50±0.69	0.46±0.02	52.48±2.04
2 Gy组	20.50±0.19^a	30.14±0.35	0.68±0.01^a	53.07±3.50
6 Gy组	19.31±0.51^a	32.52±1.18	0.59±0.03^a	61.48±1.86^a
12 Gy组	21.07±0.84^a	33.37±0.74^a	0.63±0.02^a	99.91±4.94^a
18 Gy组	26.33±0.63^a	33.50±0.74^a	0.79±0.03^a	103.80±2.82^a
0 Gy+SP组	21.86±0.93^a	28.16±1.26^a	0.78±0.01^a	62.57±4.17^a
2 Gy+SP组	18.22±0.63^b	31.97±0.57^b	0.57±0.01^b	76.89±4.11^b
6 Gy+SP组	16.10±0.57^c	33.35±1.01	0.48±0.03^c	98.92±2.78^c
12 Gy+SP组	23.31±1.25^d	42.24±1.15^d	0.55±0.02^d	102.29±3.64
18 Gy+SP组	25.07±0.99	41.37±0.74^e	0.61±0.02^e	108.88±3.13
F	76.684^＊＊	77.852^＊＊	80.440^＊＊	135.039^＊＊

组别	Col-Ⅰ （μg/L）	Col-Ⅲ （μg/L）	Col-Ⅰ/ Col-Ⅲ	CGRP （ng/L）
0 Gy组	14.34±0.25	31.50±0.69	0.46±0.02	52.48±2.04
2 Gy组	20.50±0.19^a	30.14±0.35	0.68±0.01^a	53.07±3.50
6 Gy组	19.31±0.51^a	32.52±1.18	0.59±0.03^a	61.48±1.86^a
12 Gy组	21.07±0.84^a	33.37±0.74^a	0.63±0.02^a	99.91±4.94^a
18 Gy组	26.33±0.63^a	33.50±0.74^a	0.79±0.03^a	103.80±2.82^a
0 Gy+SP组	21.86±0.93^a	28.16±1.26^a	0.78±0.01^a	62.57±4.17^a
2 Gy+SP组	18.22±0.63^b	31.97±0.57^b	0.57±0.01^b	76.89±4.11^b
6 Gy+SP组	16.10±0.57^c	33.35±1.01	0.48±0.03^c	98.92±2.78^c
12 Gy+SP组	23.31±1.25^d	42.24±1.15^d	0.55±0.02^d	102.29±3.64
18 Gy+SP组	25.07±0.99	41.37±0.74^e	0.61±0.02^e	108.88±3.13
F	76.684^＊＊	77.852^＊＊	80.440^＊＊	135.039^＊＊

组别	Col-Ⅰ （μg/L）	Col-Ⅲ （μg/L）	Col-Ⅰ/ Col-Ⅲ	CGRP （ng/L）
0 Gy组	38.78±0.53	73.59±0.66	0.53±0.01	26.56±0.61
2 Gy组	39.10±0.62	72.29±0.90	0.54±0.01	33.53±0.71^a
6 Gy组	42.98±1.28^a	78.70±1.94^a	0.55±0.00	31.10±0.58^a
12 Gy组	39.69±1.12	72.84±0.62	0.54±0.02	32.55±0.73^a
18 Gy组	43.86±0.71^a	78.05±1.83^a	0.56±0.01^a	31.48±0.09^a
0 Gy+SP组	37.81±0.49	72.13±0.90	0.52±0.01	29.52±1.25^a
2 Gy+SP组	33.64±0.42^b	65.40±1.88^b	0.51±0.02^b	28.81±0.41^b
6 Gy+SP组	37.25±0.31^c	79.14±1.70	0.47±0.01^c	32.70±0.96^c
12 Gy+SP组	38.19±0.37^d	68.44±0.77^d	0.56±0.01	31.07±0.95^d
18 Gy+SP组	37.19±0.44^e	70.83±0.84^e	0.53±0.00^e	38.15±0.58^e
F	52.361^＊＊	34.876^＊＊	14.151^＊＊	51.309^＊＊