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

Abstract

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

CLC Number:

S829.1

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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Cloning and Expression of TIMP1 in Oryctolagus Cunicilus and Its Impact on the Proliferation of Dermal Papilla Cells

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