家兔TIMP1基因的克隆表达及其对毛乳头细胞增殖的影响

doi:10.11843/j.issn.0366-6964.2025.10.019

摘要/Abstract

摘要：

旨在对基质金属蛋白酶组织抑制剂(tissue inhibitor of matrix metalloproteinases，TIMPs)家族成员中的TIMP1进行克隆和分析，通过过表达与敲减TIMP1，探究TIMP1对毛囊毛乳头细胞(dermal papilla cells，DPCs)毛囊生长相关基因的调控作用。本研究通过分子克隆TIMP1编码区(coding sequence，CDS)序列，并对其进行生物信息学功能分析。之后，构建过表达载体pcDNA 3.1-TIMP1并设计合成siRNA，在DPCs中过表达和敲低TIMP1，检测毛囊生长发育相关基因的表达，并通过EdU和CCK-8检测DPCs的增殖水平。结果显示，兔TIMP1基因的CDS区全长624 bp，共编码207个氨基酸。生物信息学分析表明TIMP1蛋白存在信号肽，不包含跨膜结构域，在不同哺乳动物中存在同源性。在DPCs中过表达TIMP1能够极显著上调BMP2(bone morphogenetic protein 2)、SFRP2(secreted frizzled-related protein 2)、TGFβ1(tranforming growth factorβ1)基因的mRNA表达水平(P < 0.01)，极显著下调WNT2(wnt family member 2)基因的mRNA表达水平(P < 0.01)，敲减TIMP1能显著下调BMP2、SFRP2基因的mRNA的表达(P < 0.05)，极显著下调TGFβ1基因的mRNA表达(P < 0.01)。此外，EdU和CCK-8结果显示过表达TIMP1能够抑制DPCs的增殖，敲低TIMP1能够促进DPCs的增殖。本研究成功克隆家兔TIMP1基因CDS序列，并对其生物信息学功能进行预测，初步分析TIMP1对毛囊生长相关基因的调控作用，验证了其抑制DPCs增殖的作用，为阐明家兔毛囊生长发育的理论研究提供参考。

关键词: TIMP1, 毛囊, 家兔, 毛乳头细胞

Abstract:

The aim of this paper was to clone and analyse TIMP1, a member of the tissue inhibitor of matrix metalloproteinases (TIMPs) family. The regulatory role of TIMP1 on hair follicle growth-related genes in hair follicle papilla cells (DPCs) was explored by overexpression and knockdown of TIMP1. In this study, we cloned the coding sequence (CDS) of TIMP1 and analysed its bioinformatics function. After that, we constructed the overexpression vector pcDNA3.1-TIMP1 and designed and synthesized siRNA. We overexpressed and knocked down TIMP1 in DPCs to probe the expression of genes related to hair follicle growth and development, and detected the proliferation level of DPCs by EdU and CCK-8. The results showed that the CDS region of the rabbit TIMP1 gene was 624 bp in length, encoding a total of 207 amino acids. Bioinformatics analysis indicated the presence of a signal peptide in the TIMP1 protein, which did not contain a transmembrane structural domain and was homologous in different mammals. Overexpression of TIMP1 in DPCs was able to highly significantly up-regulate the mRNA expression levels of BMP2, SFRP2, and TGFβ1 genes (P < 0.01), and highly significantly down-regulate the mRNA expression level of WNT2 gene (P < 0.01), and knockdown of TIMP1 was able to significantly down-regulate the expression of mRNA of BMP2, SFRP2 genes (P < 0.05), and highly significantly down-regulated the mRNA expression of TGFβ1 gene (P < 0.01). In addition, EdU and CCK-8 results showed that overexpression of TIMP1 could inhibit the proliferation of DPCs, and knockdown of TIMP1 could promote the proliferation of DPCs. In this study, we successfully cloned the CDS sequence of rabbit TIMP1 gene and preliminarily predicted its bioinformatics function, analysed the regulatory effect of TIMP1 on hair follicle growth-related genes. We verified its effect on inhibiting DPCs proliferation, and provided a reference for elucidating the theoretical research of rabbit hair follicle growth and development.

Key words: TIMP1, hair follicle, domestic rabbit, dermal papilla cells

中图分类号:

S829.1

李云鹏, 韩小曼, 俞咏麒, 蔡佳炜, 赵博昊, 陈阳, 吴信生. 家兔TIMP1基因的克隆表达及其对毛乳头细胞增殖的影响[J]. 畜牧兽医学报, 2025, 56(10): 4988-4997.

LI Yunpeng, HAN Xiaoman, YU Yongqi, CAI Jiawei, ZHAO Bohao, CHEN Yang, WU Xinsheng. Cloning and Expression of TIMP1 in Oryctolagus Cunicilus and Its Impact on the Proliferation of Dermal Papilla Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(10): 4988-4997.

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试剂名称Reagent name	公司名称Company name	所在地Location
间充质干细胞培养基Mesenchymal stem cell medium	Sciencell^Ⓡ公司	美国
TaKaRa胶回收试剂盒	TaKaRa公司	日本
MiniBEST Agarose Gel DNA Extraction Kit Ver 4.0
SteadyPure通用型RNA提取试剂盒	艾科瑞生物工程有限公司	中国长沙
SteadyPure Universal RNA Extraction Kit
SYBR Green Pro Taq HS预混型qPCR试剂盒Ⅱ	艾科瑞生物工程有限公司	中国长沙
SYBR Green Premix Pro Taq HS qPCR Kit Ⅱ
琼脂糖Agarose	BioFroxx公司	德国
BeyoClick^TMEdU-555细胞增殖检测试剂盒	碧云天生物技术有限公司	中国上海
BeyoClick^TM EdU Cell Proliferation Kit with AF555
免疫染色固定液P0098 Immunol Staining Fix Solution	碧云天生物技术有限公司	中国上海
免疫染色封闭液P0102 Immunol Staining Blocking Buffer	碧云天生物技术有限公司	中国上海
免疫染色强力通透液	碧云天生物技术有限公司	中国上海
P20097 Enhanced Immunostaining Permeabilization Buffer
CCK-8 CCK-8 Cell Counting Kit	诺唯赞生物科技股份有限公司	中国南京
Lipofectamine^TM2000转染试剂	Invitrogen公司	美国
Lipofectamine^TM 2000 Transfection Reagent
无内毒素大提质粒盒EndoFree Maxi Plasmid Kit	天根生化科技有限公司	中国北京
干扰RNA si-RNA	苏州吉玛基因股份有限公司	中国苏州

基因名称 Gene name	序列(5′→3′) Sequence
siRNA-TIMP1	F: GGAACGGGCUCUUGCACAUTT
	R: AUGUGCAAGAGCCCGUUCCTT
siRNA-NC	F: UUCUCCGAACGUGUCACGUTT
	R: ACGUGACACGUUCGGAGAATT

基因名称Gene	引物序列(5′→3′) Sequence	退火温度/℃ Tm	产物长度/bp Products size
TIMP1	F: CTGCGGGTACTCCCACAAAT	60.40	157
	R: AGCGTAGGTCTTGGTGAAGC
GAPDH	F: TGGAATCCACTGGCGTCTTC	57.45	241
	R: GTCATGAGCCCCTCCACAAT
SFRP2	F: GGCCACGAGACCATGAAAGA	57.45	239
	R: CGATCGCACTCGAGCATGT
BMP2	F: TCGTTGGAAGAACTGCCAGAA	55.40	159
	R: ATGATGGAAACCGCTGTCGT
FGF2	F: GGCTGTACTGCAAAAACGGG	57.45	161
	R: AGCAAGGTAACGGTTTGCAC
TGFβ1	F: TCTGGAACGGGCTCAACATC	57.45	174
	R: CAGAAGTTGGCGTGGTAGCC
WNT2	F: CTCTCGGTGGAATCTGGCTG	55.40	224
	R: ATACTGGCACTCTGCTGTCC

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