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不同年龄小鼠骨来源间充质干细胞衰老相关特性与成骨分化能力的比较研究
Comparison on senescence-related properties and osteogenic differentiation capacity of bone-derived mesenchymal stem cells from mice in different ages
- 陆军军医大学学报 2024年46卷第13期 页码：1512-1522
- 作者机构：
400014 重庆，重庆医科大学附属儿童医院儿科研究所，儿童发育疾病研究教育部重点实验室，儿科学重庆市重点实验室
- 作者简介：
[李娅莎，E-mail: liyasha@hospital.cqmu.edu.cnLI Yasha, E-mail: liyasha@hospital.cqmu.edu.cn
- 基金信息：国家自然科学基金青年科学基金(81800786)
DOI：10.16016/j.2097-0927.202310032
中图分类号： R322.71;R329.25;R339.38收稿：2023-10-10，
修回：2023-12-06，
纸质出版：2024-07-15
- 稿件说明：
移动端阅览
李媛, 钟海英, 董世访, 等. 不同年龄小鼠骨来源间充质干细胞衰老相关特性与成骨分化能力的比较研究[J]. 陆军军医大学学报, 2024,46(13):1512-1522.
Yuan LI, Haiying ZHONG, Shifang DONG, et al. Comparison on senescence-related properties and osteogenic differentiation capacity of bone-derived mesenchymal stem cells from mice in different ages[J]. Journal of Army Medical University, 2024, 46(13): 1512-1522.
李媛, 钟海英, 董世访, 等. 不同年龄小鼠骨来源间充质干细胞衰老相关特性与成骨分化能力的比较研究[J]. 陆军军医大学学报, 2024,46(13):1512-1522. DOI： 10.16016/j.2097-0927.202310032.
Yuan LI, Haiying ZHONG, Shifang DONG, et al. Comparison on senescence-related properties and osteogenic differentiation capacity of bone-derived mesenchymal stem cells from mice in different ages[J]. Journal of Army Medical University, 2024, 46(13): 1512-1522. DOI： 10.16016/j.2097-0927.202310032.
摘要
目的
2
探索不同年龄组小鼠骨来源的间充质干细胞(bone derived mesenchymal stem cells
BMSCs)衰老相关的生物学特性及骨形态发生蛋白2(bone morphogenetic protein 2，BMP2)诱导成骨分化能力的改变。
方法
2
将8只C57BL/6J小鼠分为青年组(4周龄，体质量约为10~15 g
n
=4)和老年组(12月龄，体质量约为20 g~25 g，
n
=4)，每组雌雄各半。分别从2组小鼠的整骨中提取间充质干细胞(mesenchymal stem cells
MSCs)，通过流式细胞术鉴定2组BMSCs的表面标志物，通过β-半乳糖苷酶染色比较2组BMSCs的衰老程度，通过MTT和EdU掺入实验比较2组BMSCs的增殖及自我更新能力。通过Western blot分析2组BMSCs中周期蛋白CyclinD1、P21的表达情况。采用ALP染色和茜素红S染色及RT-qPCR评估2组BMSCs的体外成骨分化能力，使用RNA-seq分析比较2组BMSCs经BMP2诱导后的差异基因表达，并用流式分析验证测序结果。
结果
2
流式细胞术分析结果显示，分离培养的小鼠BMSCs符合间充质干细胞判定标准。β-半乳糖苷酶染色结果显示，老年组BMSCs的衰老水平显著高于青年组(
P
＜0.05)。而MTT和EdU掺入实验结果表明，老年组BMSCs的细胞活力和增殖能力显著低于青年组(
P
＜0.05)。Western blot结果显示，与青年组比较，老年组BMSCs的细胞周期蛋白CyclinD1表达水平较低，而细胞周期阻抑因子P21蛋白表达水平显著增高(
P
＜0.05)。ALP染色和茜素红S染色及RT-qPCR检测结果显示，BMP2诱导后青年组BMSCs的成骨分化能力显著高于老年组(
P
＜0.05)。RNA-seq结果显示，BMP2诱导后，整合素αV重组蛋白(cluster of differentiation 51，CD51)在青年组和老年组中的表达趋势相反。而流式细胞术分析结果显示，青年组CD51
+
细胞在CD45
-
细胞中占比明显高于老年组(
P
＜0.01)。
结论
2
老年组BMSCs中CD51
+
细胞
在CD45
-
细胞中占比下降与CD51
+
细胞对BMP2的成骨诱导响应性下降密切相关。
Abstract
Objective
2
To explore the age-related biological properties of bone-derived mesenchymal stem cells (BMSCs) from mice of different age groups and their osteogenic differentiation induced by bone morphogenetic protein 2 (BMP2).
Methods
2
Eight C57BL/6J mice were divided into a young group (4 weeks old
weighing 10~15 g
n
=4) and an old group (12 months old
weighing 20~25 g
n
=4)
with half male and half female. MSCs were extracted from the whole bones of the 2 groups of mice. After the obtained cells were identified with flow cytometry for the surface markers
β-galactosidase staining was employed to compare the senescence level of BMSCs
MTT and EdU incorporation assays were conducted to compare the proliferation and self-renewal abilities of between the 2 groups. Western blotting was employed to analyze the expression of CyclinD1 and P21 in BMSCs. Then ALP staining
Alizarin Red staining and RT-qPCR were used to evaluate the osteogenic differentiation ability of the cells. RNA sequencing was performed to compare the differential gene expression in BMP2-induced BMSCs. Lastly
the sequencing results were re-confirmed by using flow cytometry.
Results
2
Flow cytometry showed that the sorted and cultured mouse BMSCs met the criteria for MSCs. The results of β-galactosidase staining indicated that the senescence level of BMSCs in the old group was significantly higher than that in the young group (
P
<
0.05). MTT and EdU doping experiments revealed that the cell viability and proliferation ability of BMSCs were significantly lower in the old group than the young group (
P
<
0.05). Western blotting displayed that the expression level of cell cycle protein CyclinD1 was lower
whereas that of cell cycle inhibitory factor P
21 was significantly higher in the BMSCs from the old group than the cells from the young group (
P
<
0.05). ALP/Alizarin Red staining and RT-qPCR demonstrated that the BMSCs from the young group had stronger osteogenic differentiation capacity after BMP2 treatment when compared the cells of the old group (
P
<
0.05). RNA sequencing results displayed that the changing profile of CD51 expression was in opposite trends in the young and old BMSCs after BMP2 treatment. Finally
flow cytometry revealed that the percentage of CD51
+
cells within the CD45
-
cells was significantly higher in the young group than the old group.
Conclusion
2
The decrease in the percentage of CD51
+
cells among CD45
-
cells in aged BMSCs is closely associated with their decreased responsiveness to BMP2-induced osteogenic differentiation.
关键词
Keywords
references
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