小鼠骨髓和脂肪间充质干细胞定向分化能力的比较研究

doi:10.11958/20230437

天津医药 ›› 2024, Vol. 52 ›› Issue (2): 129-135.doi: 10.11958/20230437

小鼠骨髓和脂肪间充质干细胞定向分化能力的比较研究

钟家帅(), 冯玉梅^△()

天津医科大学肿瘤医院，国家恶性肿瘤临床医学研究中心，天津市恶性肿瘤临床医学研究中心，天津市肿瘤防治重点实验室，天津医科大学肿瘤医院肿瘤研究所生物化学与分子生物学研究室（邮编300060）

收稿日期:2023-03-27 修回日期:2023-05-08 出版日期:2024-02-15 发布日期:2024-01-26
通讯作者: ^△ E-mail：ymfeng@tmu.edu.cn E-mail:whutzjs@163.com;ymfeng@tmu.edu.cn
作者简介:钟家帅（1999），男，硕士在读，主要从事肿瘤分子生物学方面研究。E-mail：whutzjs@163.com
基金资助:
天津市医学重点学科（专科）建设项目(TJYXZDXK-009A)

Comparative study on the directed differentiation ability of mouse bone marrow and adipose-derived mesenchymal stem cells

ZHONG Jiashuai(), FENG Yumei^△()

Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer;Tianjin’s Clinical Research Center for Cancer;Key Laboratory of Cancer Prevention and Therapy, Tianjin;Department of Biochemistry and Molecular Biology, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China

Received:2023-03-27 Revised:2023-05-08 Published:2024-02-15 Online:2024-01-26
Contact: ^△ E-mail：ymfeng@tmu.edu.cn E-mail:whutzjs@163.com;ymfeng@tmu.edu.cn

钟家帅, 冯玉梅. 小鼠骨髓和脂肪间充质干细胞定向分化能力的比较研究[J]. 天津医药, 2024, 52(2): 129-135.

ZHONG Jiashuai, FENG Yumei. Comparative study on the directed differentiation ability of mouse bone marrow and adipose-derived mesenchymal stem cells[J]. Tianjin Medical Journal, 2024, 52(2): 129-135.

摘要/Abstract

摘要：

目的探讨小鼠骨髓源性间充质干细胞（BM-MSCs）和脂肪源性间充质干细胞（AD-MSCs）的定向分化能力。方法从C57BL/6J小鼠股骨骨髓和腹股沟白色脂肪组织中分别分离和培养BM-MSCs和AD-MSCs，分别使用成骨、成软骨和成脂诱导分化培养基诱导两种细胞定向分化。采用茜素红、阿利新蓝和油红O染色检测成骨、成软骨和成脂分化程度；实时荧光定量PCR（qPCR）鉴定MSCs并检测定向分化相关基因Runx2、Sp7（成骨），Sox9、Col2a1（成软骨），Pparg和Cebpa（成脂）表达水平，确定细胞的定向分化能力。基于GEO数据库中GSE43804和GSE122778数据集的小鼠和人类BM-MSCs和AD-MSCs基因表达谱数据，分析差异表达基因及其富集的信号通路。结果分离培养得到的BM-MSCs和AD-MSCs细胞形态不同，AD-MSCs梭形形态更明显；两种细胞均表达CD29、CD44和CD90，不表达CD34和CD45。定向诱导后AD-MSCs的成骨和成脂分化程度高于BM-MSCs，而成软骨分化程度低于BM-MSCs（P<0.05）；定向诱导后AD-MSCs中Runx2、Pparg和Cebpa mRNA表达水平高于BM-MSCs，Sox9 mRNA表达水平低于BM-MSCs（P<0.05）。小鼠和人的AD-MSCs高表达的基因富集于PPAR和WNT信号通路，BM-MSCs高表达的基因富集于软骨和骨发育信号通路。结论小鼠AD-MSCs成骨和成脂分化能力强于BM-MSCs，而成软骨分化能力弱于BM-MSCs，PPAR、WNT、软骨和骨发育信号通路的活化状态在决定BM-MSCs和AD-MSCs不同定向分化潜能中起重要调节作用。

关键词: 间质干细胞, 骨髓, 脂肪类, PPARγ, Wnt信号通路, 成骨分化, 成软骨分化, 成脂分化

Abstract:

Objective To investigate the targeted differentiation ability of mouse bone marrow derived mesenchymal stem cells (BM-MSCs) and adipose-derived mesenchymal stem cells (AD-MSCs). Methods BM-MSCs and AD-MSCs were isolated and cultured from bone marrow of femur and white adipose tissue of groin of C57BL/6J mice respectively, and the two types of cells were induced by osteogenic, chondrogenic and adipogenic differentiation medium respectively. Alizarin red, alcian blue and oil red O staining were used to detect the differentiated degree of osteogenic, chondrogenic and lipogenic differentiation. Real-time fluorescence quantitative PCR (qPCR) was used to identify MSCs and detected expression levels of directed differentiation-related genes Runx2, Sp7 (osteoblast), Sox9, Col2a1 (chondroblast), Pparg and Cebpa (lipogenesis) to determine the directed differentiation ability of cells. Based on gene expression profiles of mouse and human BM-MSCs and AD-MSCs in GEO database GSE43804 and GSE122778, the differentially expressed genes and their enrichment signal pathways were analyzed. Results The cell morphology of BM-MSCs and AD-MSCs obtained by isolation and culture was different, and spindle-shaped morphology was more obvious in AD-MSCs. Both cells expressed CD29, CD44 and CD90, but did not express CD34 and CD45. AD-MSCs showed higher osteogenic and lipogenic differentiation than those of BM-MSCs after directed induction, while chondrogenic differentiation was lower in AD-MSCs than that of BM-MSCs (P<0.05). After directional induction, expression levels of Runx2, Pparg and Cebpa mRNA were higher in AD-MSCs than those in BM-MSCs, and Sox9 mRNA expression levels were lower than those in BM-MSCs (P<0.05). Highly expressed genes of AD-MSCs in mice and human were enriched in PPAR and WNT signaling pathways. Highly expressed genes of BM-MSCs were enriched in cartilage and bone developmental signaling pathways. Conclusion The osteogenic and adipogenic differentiation ability of mouse AD-MSCs is stronger than those of BM-MSCs, while the chondrogenic differentiation ability AD-MSCs is weaker than that of BM-MSCs. The activation status of PPAR, WNT, cartilages and skeletal system development signaling pathways plays an important regulatory role in determining the different directional differentiation potential of AD-MSCs and BM-MSCs.

Key words: mesenchymal stem cells, bone marrow, fats, PPAR gamma, Wnt signaling pathway, osteogenic differentiation, chondrogenic differentiation, adipogenic differentiation

中图分类号:

R329.24

图/表 13

图1 BM-MSCs和AD-MSCs的分离和培养

Fig.1 Isolation and culture of BM-MSCs and AD-MSCs

表1 引物序列

Tab.1 Primer sequences

基因名称	引物序列（5′→3′）	产物大小/bp
Gapdh	上游：ATTGTCAGCAATGCATCCTG	102
Gapdh	下游：ATGGACTGTGGTCATGAGCC	102
CD29	上游：CCTGTAACTCCGACGCCTTT	137
CD29	下游：AAGGTCCCCACTCAGCAATG	137
CD34	上游：TTTCCTGATGAACCGTCGCA	165
CD34	下游：GCAGGGTTGTGAGGTACTGT	165
CD44	上游：ACCTTGGCCACCACTCCTAAT	118
CD44	下游：TCACATGGGAGTCTTCACTTGG	118
CD45	上游：CTTTGCTTATGTGGCGTGTGT	139
CD45	下游：TTATCCCCTTCTGATGCGCC	139
CD90	上游：TCCAAGTCGGAACTCTTGGC	133
CD90	下游：TCCAGGCGAAGGTTTTGGTT	133
Runx2	上游：CTCTGCACCAAGTCCTTTTAATC	104
Runx2	下游：AGGAGGGGTAAGACTGGTCATAG	104
Sp7	上游：CTGAGAGAGGAGCAGATCCC	135
Sp7	下游：GTGAGCTTCTTCCTGGGTAGG	135
Sox9	上游：AGGAAGTCGGTGAAGAACGG	167
Sox9	下游：GGACCCTGAGATTGCCCAGA	167
Col2a1	上游：ATGAGGGAGCGGTAGAGACC	190
Col2a1	下游：GCCCTAATTTTCGGGCATCC	190
Pparg	上游：GAGCACTTCACAAGAAATTACC	192
Pparg	下游：GAACTCCATAGTGGAAGCCT	192
Cebpa	上游：CAAGAACAGCAACGAGTACCG	120
Cebpa	下游：AGGCGGTCATTGTCACTGGT	120

图2 MSCs细胞形态的比较明场拍摄，×200，标尺=50 μm。

Fig.2 Comparison of cell morphology of MSCs

表2 2种细胞MSCs标志基因表达水平（n=3，$\bar{x}±s$）

Tab.2 The gene expression levels of two types of MSCs

细胞	CD29/×10^-3	CD44/×10^-3	CD90/×10^-3	CD34	CD45
BM-MSCs	1.861±0.288	0.562±0.120	0.456±0.017	0	0
AD-MSCs	0.805±0.208	0.433±0.158	0.851±0.261	0	0

图3 2组MSCs成骨分化程度（茜素红染色，×200）

Fig.3 Osteogenic differentiation degree of MSCs in two groups (Alizarin red staining, ×200)

图4 2组MSCs成软骨分化程度（阿利新蓝染色，×200）

Fig.4 Degree of chondroblast differentiation of MSCs in two groups (Alcian Blue staining, ×200)

图5 2组MSCs成脂分化程度（油红O染色，×200）

Fig.5 Degree of adipogenic differentiation of MSCs in two groups (Oil Red O staining, ×200)

表3 诱导成骨分化前后2组MSCs成骨标志基因表达水平的比较（n=3，$\bar{x}±s$）

Tab.3 Comparison of expression levels of MSCs marker genes before and after osteogenic differentiation between the two groups

细胞	诱导分化前		诱导分化后
细胞	Runx2/×10^-5	Sp7/×10^-6	Runx2/×10^-4	Sp7/×10^-5
BM-MSCs	4.85±0.99	6.11±1.17	2.42±0.44	4.66±3.73
AD-MSCs	4.36±1.13	2.01±1.22	4.61±0.92	4.17±3.54
t	0.510	3.024^＊	3.923^＊	1.171

表4 诱导成软骨分化前后2组MSCs成软骨标志基因表达水平的比较（n=3，$\bar{x}±s$）

Tab.4 Comparison of expression levels of MSCs marker genes before and after induction of chondroblast differentiation between two groups

细胞	诱导分化前		诱导分化后
细胞	Sox9/×10^-5	Col2a1/×10^-6	Sox9/×10^-3	Col2a1/×10^-5
BM-MSCs	9.22±1.67	2.12±1.88	1.71±0.08	2.35±1.48
AD-MSCs	4.24±0.99	1.58±1.16	0.98±0.15	1.80±1.38
t	4.282^＊	0.326	4.786^＊	0.365

表5 诱导成脂分化前后2组MSCs成脂标志基因表达水平的比较（n=3，$\bar{x}±s$）

Tab.5 Comparison of expression levels of MSCs marker genes before and after induction of adipogenic differentiation between the two groups

细胞	诱导分化前		诱导分化后
细胞	Pparg/×10^-5	Cebpa/×10^-4	Pparg/×10^-3	Cebpa/×10^-2
BM-MSCs	4.92±1.48	6.91±0.95	1.70±0.87	0.72±0.05
AD-MSCs	4.86±2.54	7.47±1.96	10.50±6.52	2.00±0.35
t	0.032	0.406	2.123^＊	5.593^＊

图6 BM-MSCs与AD-MSCs显著差异基因的火山图

Fig.6 Volcanic map of significant difference genes between BM-MSCs and AD-MSCs

图7 GO富集分析mBM-MSCs和hBM-MSCs中高表达基因集

Fig.7 GO enrichment analysis of high expression gene sets in mBM-MSCs and hBM-MSCs

图8 GO富集分析mAD-MSCs和hAD-MSCs中高表达基因集

Fig.8 GO enrichment analysis of high-expression gene sets in mAD-MSCs and hAD-MSCs

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小鼠骨髓和脂肪间充质干细胞定向分化能力的比较研究

Comparative study on the directed differentiation ability of mouse bone marrow and adipose-derived mesenchymal stem cells

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