家兔MSX2基因的克隆表达及生物信息学分析

doi:10.11843/j.issn.0366-6964.2025.06.017

摘要/Abstract

摘要：

旨在探究Msh同源框2(Msh-homebox 2，MSX2)在长毛兔毛囊毛乳头细胞(dermal papilla cell，DPC)增殖中的调控模式。本试验选取12只健康6月龄长毛兔，采集背部皮肤分离培养DPCs, 通过克隆MSX2基因的编码序列(coding sequence，CDS)，利用生物信息学对MSX2的生物学特性进行初步分析, 分别探究过表达和敲低MSX2对DPC增殖的影响，及相关基因对其调控作用。结果显示，MSX2基因的编码序列全长807 bp，可编码268个氨基酸。通过在DPCs中过表达与敲低MSX2，检测毛囊发育相关基因的表达水平变化。过表达MSX2后，CCND1、EGF、Wnt2、Hoxc13基因的mRNA表达量极显著升高(P＜0.01)，STAT1、SFRP2和Wnt5a基因的mRNA表达量显著或极显著下降(P < 0.05或P < 0.01)；而敲低MSX2后，则显示出相反的趋势。同时，MSX2能够显著促进DPC增殖。本研究结果为进一步研究MSX2在家兔毛囊发育中的作用提供了理论支持。

关键词: 长毛兔, MSX2, 毛乳头细胞, 毛囊发育

Abstract:

This study aimed to investigate the regulatory role of Msh-homeobox 2 (MSX2) in the proliferation of dermal papilla cells (DPCs) in long-haired rabbits. Skin samples were collected from the backs of 12 healthy 6-month-old long-haired rabbits, and DPCs were isolated and cultured. The coding sequence (CDS) of the MSX2 gene was cloned, and its biological characteristics were preliminarily analyzed using bioinformatics tools. The effects of MSX2 overexpression and knockdown on DPCs proliferation were explored, along with the regulatory roles of related genes. The results showed that the CDS of the MSX2 gene was 807 bp in length, encoding 268 amino acids. Overexpression of MSX2 in DPCs significantly increased the mRNA expression levels of hair follicle development-related genes, including CCND1, EGF, Wnt2, and Hoxc13 (P < 0.01), while significantly or extremely significantly decreased the expression of STAT1, SFRP2, and Wnt5a (P < 0.05 or P < 0.01). Conversely, knockdown of MSX2 exhibited the opposite trend. Additionally, MSX2 significantly promoted DPC proliferation. These findings provide a theoretical foundation for further research on the role of MSX2 in hair follicle development in rabbits.

Key words: long-haired rabbits, MSX2, dermal papilla cell, hair follicle development

中图分类号:

S829.1

韩小曼, 孙少宁, 杨洁, 沈宁, 鲍志远, 蔡佳炜, 赵博昊, 陈阳, 吴信生. 家兔MSX2基因的克隆表达及生物信息学分析[J]. 畜牧兽医学报, 2025, 56(6): 2724-2732.

HAN Xiaoman, SUN Shaoning, YANG Jie, SHEN Ning, BAO Zhiyuan, CAI Jiawei, ZHAO Bohao, CHEN Yang, WU Xinsheng. Cloning Expression and Bioinformatics Analysis of Rabbit MSX2 Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(6): 2724-2732.

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项目Projects	用途Purpose	网址/软件Software
ProtParam^[17]	蛋白质的氨基酸序列	https://web.expasy.org/cgi-bin/protparam/
ProtScale^[17]	亲疏水性	https://web.expasy.org/cgi-bin/protscale/
SOPMA^[18]	蛋白质二级结构	https://npsa-prabi.ibcp.fr/
Swiss-Model^[19]	蛋白质三级结构	https://swissmodel.expasy.org/
NetPhos	蛋白的磷酸化位点	http://www.cbs.dtu.dk/services/NetPhos/
	蛋白的N-糖基化位点	http://www.cbs.dtu.dk/services/NetNGlyc/
STRING数据库^[20]	蛋白交联分析	https://string-db.org/cgi/input.pl
STRING data base	MSX2进化分析	MEGA 5.1

siRNAs	引物序列(5′→3′) Primers sequence
siRNA-MSX2	F：GCUUCUCCGUCCAAAGGCATT; R：UGCCUUUGGACGGAGAAGCTT
siRNA-NC	F：UUCUCCGAACGUGUCACGUTT; R：ACGUGACACGUUCGGAGAATT

基因 Gene	引物序列(5′→3′) Primers sequence	产物长度/bp Products length	退火温度/℃ Tm
MSX2	F: CTGCAAGCCACGTCCCTTTA; R: AGGACAAGTGGTACATGCCG	115	60.6
CCND1	F: GAACGCTACCTTCCCCAGTGCTC; R: CCTCACAGACCTCCAGCATCCAG	103	57.2
EGF	F: TGGAGTAGGCATGGAGCAAG; R: GCCCCGTAAGATGGTCGATG	102	59.4
SFRP2	F: CCAGCCCGACTTCTCCTACAAGC; R: TCCAGCACCTCTTTCATGGTCT	135	57.5
STAT1	F: CCAGAGGCCAGTCTTGTCAC; R: ACGGAAACACTACCTGGGAC	112	60.3
Wnt5a	F: CGCAGGACCTGGTCTACATC; R: CGCTCTGTCTGTACCGTCTT	175	59.9
HOXC13	F: AGAAAGAGTACGCAGCCAGC; R: TCTTCTCTTTGACTCGGCGG	123	60.3
Wnt2	F: AGCCATCCAGGTCGTCATGAACCAG; R: TGCACACACGACCTGCTGTACCC	164	56.3
GAPDH	F: CACCAGGGCTGCTTTTAACTCT; R: CTTCCCGTTCTCAGCCTTGACC	145	53.9

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