泛素特异性蛋白酶42调节人脂肪干细胞成骨向分化

doi:10.19723/j.issn.1671-167X.2024.01.003

摘要/Abstract

摘要：

目的: 探索泛素特异性蛋白酶42(ubiquitin-specific protease 42，USP42)在人脂肪干细胞(human adipose-derived stem cells，hASCs)体内外成骨向分化中的作用。方法: 用慢病毒转染hASCs，构建敲低和过表达USP42的稳定转染细胞系，通过碱性磷酸酶(alkaline phosphatase，ALP)染色及活性定量、茜素红S矿化结节染色及定量，检测实验组(敲低组和过表达组)及对照组在成骨诱导下hASCs成骨向分化能力的差异，通过定量逆转录聚合酶链反应(quantitative reverse transcription polymerase chain reaction，qRT-PCR)检测实验组及对照组成骨相关基因的表达水平，通过蛋白免疫印迹实验检测实验组及对照组成骨相关蛋白的表达水平，通过裸鼠异位成骨实验评价USP42在hASCs体内成骨向分化中的作用。结果: 敲低组USP42的mRNA和蛋白表达显著低于对照组，过表达组显著高于对照组。成骨诱导7 d后，敲低组的ALP活性显著高于对照组，过表达组显著低于对照组；成骨诱导14 d后，敲低组茜素红S染色显著深于对照组，过表达组显著浅于对照组。qRT-PCR结果显示，成骨诱导14 d时，敲低组ALP、成骨细胞特异性转录因子(osterix，OSX)和Ⅰ型胶原(collagen type Ⅰ，COLⅠ)的mRNA表达水平显著高于对照组，过表达组显著低于对照组。蛋白免疫印迹实验结果显示，成骨诱导14 d时敲低组runt相关转录因子2(runt-related transcription factor 2，RUNX2)、OSX和COLⅠ蛋白表达水平显著高于对照组，过表达组显著低于对照组。裸鼠皮下移植物苏木精-伊红染色结果显示，敲低组类骨组织百分比较对照组显著增高。结论: 敲低USP42显著促进hASCs的体内外成骨向分化，过表达USP42显著抑制hASCs的体内成骨向分化，USP42可作为骨组织工程学潜在治疗靶点。

关键词: 泛素特异性蛋白酶类, 人脂肪干细胞, 细胞分化, 骨和骨组织, 再生医学

Abstract:

Objective: To explore the effect of ubiquitin-specific protease 42 (USP42) on osteogenic differentiation of human adipose-derived stem cells (hASCs) in vivo and in vitro. Methods: A combination of experiments was carried out with genetic depletion of USP42 using a lentiviral strategy. Alkaline phosphatase (ALP) staining and quantification, alizarin red S (ARS) staining and quantification were used to determine the osteogenic differentiation ability of hASCs under osteogenic induction between the experimental group (knockdown group and overexpression group) and the control group. Quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression levels of osteogenesis related genes in the experimental group and control group, and Western blotting was used to detect the expression levels of osteogenesis related proteins in the experimental group and control group. Nude mice ectopic implantation experiment was used to evaluate the effect of USP42 on the osteogenic differentiation of hASCs in vivo. Results: The mRNA and protein expressions of USP42 in knockdown group were significantly lower than those in control group, and those in overexpression group were significantly higher than those in control group. After 7 days of osteogenic induction, the ALP activity in the knockdown group was significantly higher than that in the control group, and ALP activity in overexpression group was significantly lower than that in control group. After 14 days of osteogenic induction, ARS staining was significantly deeper in the knockdown group than in the control group, and significantly lighter in overexpression group than in the control group. The results of qRT-PCR showed that the mRNA expression levels of ALP, osterix (OSX) and collagen type Ⅰ (COLⅠ) in the knockdown group were significantly higher than those in the control group after 14 days of osteogenic induction, and those in overexpression group were significantly lower than those in control group. The results of Western blotting showed that the expression levels of runt-related transcription factor 2 (RUNX2), OSX and COLⅠ in the knockout group were significantly higher than those in the control group at 14 days after osteogenic induction, while the expression levels of RUNX2, OSX and COLⅠ in the overexpression group were significantly lower than those in the control group. Hematoxylin-eosin staining of subcutaneous grafts in nude mice showed that the percentage of osteoid area in the knockdown group was significantly higher than that in the control group. Conclusion: Knockdown of USP42 can significantly promote the osteogenic differentiation of hASCs in vitro and in vivo, and overexpression of USP42 significantly inhibits in vivo osteogenic differentiation of hASCs, and USP42 can provide a potential therapeutic target for bone tissue engineering.

Key words: Ubiquitin-specific proteases, Human adipose-derived stem cells, Cell differentiation, Bone and bones, Regenerative medicine

中图分类号:

R318

潘媛,顾航,肖涵,赵笠君,汤祎熳,葛雯姝. 泛素特异性蛋白酶42调节人脂肪干细胞成骨向分化[J]. 北京大学学报（医学版）, 2024, 56(1): 9-16.

Yuan PAN,Hang GU,Han XIAO,Lijun ZHAO,Yiman TANG,Wenshu GE. Ubiquitin-specific protease 42 regulates osteogenic differentiation of human adipose-derived stem cells[J]. Journal of Peking University (Health Sciences), 2024, 56(1): 9-16.

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Gene	Forward primer (5′ to 3′)	Reverse primer (5′ to 3′)
GAPDH	GGAGCGAGATCCCTCCAAAAT	GGCTGTTGTCATACTTCTCATGG
USP42	AATCTTCAGACCCATCAGCCT	AGAACCTGCATCCATGTCTCC
ALP	ATGGGATGGGTGTCTCCACA	CCACGAAGGGGAACTTGTC
OSX	CCTCTGCGGGACTCAACAAC	TAAAGGGGGCTGGATAAGCAT
COLⅠ	TGGTCCCAAGGGTAACAGCG	AACACCAACAGGGCCAGGCT