改良全骨髓贴壁法培养SD大鼠骨髓间充质干细胞及其生物学特性

doi:10.3969/j.issn.1006-5725.2025.24.009

摘要/Abstract

摘要：

目的建立大鼠骨髓间充质干细胞（BMSCs）高效的分离、培养和纯化的方法，同时探究BMSCs的生物学特性。方法取3 ~ 4周龄的SD雄性大鼠脱臼处死，在无菌条件下分离双侧股骨、胫骨，分别采用传统冲髓法和改良全骨髓贴壁法分离培养大鼠BMSCs，待细胞融合率达80%时，进行消化传代及纯化培养。倒置显微镜下观察细胞形态；吉姆萨染色后观察BMSCs克隆形成情况及克隆形成率；流式细胞仪检测细胞表面特异抗原（CD29和CD45）；利用CCK-8检测细胞增殖能力，同时绘制细胞传代后第1 ~ 8天的生长曲线；诱导BMSCs向成骨细胞分化，分别利用碱性磷酸酶（ALP）染色、定量及茜素红染色、定量方法，对两组BMSCs的成骨分化能力进行检测；另一方面，诱导BMSCs向脂肪细胞分化，利用油红O染色及定量方法，对两组BMSCs的成脂分化能力进行检测。结果相较于对照组，应用改良全骨髓贴壁培养法体外分离、纯化培养的SD大鼠BMSCs形态好、纯度高、增殖能力强。同时，在细胞生物学特性方面，改良组BMSCs较对照组BMSCs的成骨分化能力轻微增强，成脂分化能力轻微减弱。结论改良全骨髓贴壁法分离培养的SD大鼠BMSCs，其细胞形态好、增殖能力强、分化能力优，可以选择作为组织工程的种子细胞。

关键词: 骨髓间充质干细胞, 改良全骨髓贴壁法, 生物学特性, 分化

Abstract:

Objective To establish an efficient method for the isolation， culture， and purification of rat bone marrow mesenchymal stem cells （BMSCs）， while simultaneously investigating the biological characteristics of these cells. Methods The 3 ~ 4 weeks old male SD rats were sacrificed via cervical dislocation， followed by the sterile dissection of both femurs and tibiae. BMSCs from rats were separated and cultivated employing two techniques： the traditional marrow flush-out method and the optimized whole marrow adhesion method. When cell confluence reached 80%， the cells were digested for subculture and purified. Cellular morphology was observed under an inverted microscope； Colony formation and the colony-forming rate of BMSCs were assessed following Giemsa staining； Flow cytometric analysis was performed to identify specific surface markers （CD29 and CD45）； Cell proliferation capacity was measured using the CCK-8 assay， and growth curves from day 1 to day 8 after passaging were plotted； To assess osteogenic differentiation capability， BMSCs were directed toward the osteoblast lineage， and the differentiation efficacy was analyzed in the two groups through ALP staining with quantitative analysis and Alizarin Red staining followed by quantification； Additionally， adipogenic differentiation was induced in BMSCs， and the lipid accumulation capacity of the two groups was detected via Oil Red O staining and subsequent quantitative measurement. Results Compared to the control group， SD rat BMSCs isolated and purified in vitro using the modified and optimized whole bone marrow adherence method exhibited superior morphology， higher purity， and enhanced proliferation capacity. Furthermore， regarding cellular biological properties， BMSCs from the modified group showed a slight enhancement in osteogenic differentiation potential and a mild reduction in adipogenic differentiation capacity compared to those from the control group. Conclusion BMSCs derived from SD rats using the optimized whole marrow adhesion technique demonstrate superior morphological characteristics， enhanced proliferation ability， and optimal differentiation capability， thus qualifying as viable progenitor cells for tissue engineering purposes.

Key words: bone marrow mesenchymal stem cells, modified and optimized whole marrow adhesion method, biological characteristics, differentiation

中图分类号:

R-331

黄玉圣,刘心雨,周穗珊. 改良全骨髓贴壁法培养SD大鼠骨髓间充质干细胞及其生物学特性[J]. 实用医学杂志, 2025, 41(24): 3860-3866.

Yusheng HUANG,Xinyu LIU,Suishan. ZHOU. Study on the culture and biological characteristics of SD rat bone marrow mesenchymal stem cells using optimized whole marrow adhesion method[J]. The Journal of Practical Medicine, 2025, 41(24): 3860-3866.

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参考文献 26

[1]	HUANG Y S， GAO J W， AO R F， et al. Accumulation of advanced oxidation protein products aggravates bone-fat imbalance during skeletal aging［J］. J Orthop Translat， 2025， 51： 24-36. doi:10.1016/j.jot.2024.12.010 doi: 10.1016/j.jot.2024.12.010
[2]	SUN H H， DAI J， CHEN M Z， et al. miR-139-5p Was Identified as Biomarker of Different Molecular Subtypes of Breast Carcinoma［J］. Front Oncol， 2022，12：857714. doi:10.3389/fonc.2022.857714 doi: 10.3389/fonc.2022.857714
[3]	LI W J， SONG L， ZHANG F X， et al. Cigarette smoke disrupts osteogenic-adipogenic balance via Nrf2/HERC2 axis-driven ferroptosis［J］. Free Radic Biol Med， 2025， 240：704-716. doi:10.1016/j.freeradbiomed.2025.09.014 doi: 10.1016/j.freeradbiomed.2025.09.014
[4]	巢素珍，周年，石心怡，等. 芒果苷对同型半胱氨酸诱导骨髓问充质干细胞分化的影响［J］. 实用医学杂志， 2024，40 （23）：3284-3290.
[5]	HUANG X X， CHEN W K， GU C， et al. Melatonin suppresses bone marrow adiposity in ovariectomized rats by rescuing the imbalance between osteogenesis and adipogenesis through SIRT1 activation［J］. J Orthop Translat， 2022，38：84–97. doi:10.1016/j.jot.2022.10.002 doi: 10.1016/j.jot.2022.10.002
[6]	WU S X， OHBA S， MATSUSHITA Y. Single-Cell RNA-Sequencing Reveals the Skeletal Cellular Dynamics in Bone Repair and Osteoporosis［J］. Int J Mol Sci， 2023，24（12）：9814. doi:10.3390/ijms24129814 doi: 10.3390/ijms24129814
[7]	FU X R， LIU G， HALIM A， et al. Mesenchymal Stem Cell Migration and Tissue Repair［J］. Cells， 2019，8（8）：784. doi:10.3390/cells8080784 doi: 10.3390/cells8080784
[8]	MARGIANA R， MARKOV A， ZEKIY A O， et al. Clinical application of mesenchymal stem cell in regenerative medicine： A narrative review［J］. Stem Cell Res Ther， 2022，13（1）：366. doi:10.1186/s13287-022-03054-0 doi: 10.1186/s13287-022-03054-0
[9]	刘慧珍，卢晓南，柳歌，等. 肿节风总黄酮影响骨髓间充质干细胞及其外泌体促进巨核细胞分化的作用及机制［J］. 实用医学杂志， 2025，41 （11）：1618-1626.
[10]	SHEN C Q， TAO C B， ZHANG A J， et al. Exosomal microRNA⁃93⁃3p secreted by bone marrow mesenchymal stem cells downregulates apoptotic peptidase activating factor 1 to promote wound healing［J］. Bioengineered， 2022，13（1）：27-37. doi:10.1080/21655979.2021.1997077 doi: 10.1080/21655979.2021.1997077
[11]	ZHONG L L， YAO L T， HOLDREITH N， et al. Transient expansion and myofibroblast conversion of adipogenic lineage precursors mediate bone marrow repair after radiation［J］. JCI Insight， 2022，7（7）：e150323. doi:10.1172/jci.insight.150323 doi: 10.1172/jci.insight.150323
[12]	PURWANINGRUM M， JAMILAH N S， PURBANTORO S D， et al. Comparative characteristic study from bone marrow-derived mesenchymal stem cells［J］. J Vet Sci， 2021，22（6）：e74. doi:10.4142/jvs.2021.22.e74 doi: 10.4142/jvs.2021.22.e74
[13]	鲁燕妮，龙雯，常晓峰，等. 改良全骨髓贴壁法分离及培养大鼠骨髓间充质干细胞［J］. 山西医科大学学报， 2023， 54（3）：364-369.
[14]	GU J M， WANG M Y， WANG X F， et al. Exosomal miR-483-5p in bone marrow mesenchymal stem cells promotes malignant progression of multiple myeloma by targeting TIMP2［J］. Front Cell Dev Biol， 2022，10：862524. doi:10.3389/fcell.2022.862524 doi: 10.3389/fcell.2022.862524
[15]	陈斌伟，刘圣曜，黄帅，等. 辛伐他汀联合机械牵拉激活Wnt/β-catenin信号通路促进大鼠骨髓间充质干细胞成骨分化［J］. 实用医学杂志， 2021，37（1）：41-45.
[16]	DUAN P， WANG H Y， YI X Z Y， et al. C/EBPα regulates the fate of bone marrow mesenchymal stem cells and steroid-induced avascular necrosis of the femoral head by targeting the PPARγ signalling pathway［J］. Stem Cell Res Ther， 2022，13（1）：342. doi:10.1186/s13287-022-03027-3 doi: 10.1186/s13287-022-03027-3
[17]	SUN W， LIU X S， YANG X Y， et al. SENP1 regulates the transformation of lung resident mesenchymal stem cells and is associated with idiopathic pulmonary fibrosis progression［J］. Cell Commun Signal， 2022，20（1）：104. doi:10.1186/s12964-022-00921-4 doi: 10.1186/s12964-022-00921-4
[18]	LI X Y， ZHANG Y， QI G X， et al. Evaluation of isolation methods and culture conditions for rat bone marrow mesenchymal stem cells［J］. Cytotechnology， 2013，65（3）：323-334. doi:10.1007/s10616-012-9497-3 doi: 10.1007/s10616-012-9497-3
[19]	LI T Q， MENG X B， LI S C， et al. Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells［J］. J Vis Exp， 2024，207：e66765. doi:10.3791/66765 doi: 10.3791/66765
[20]	ZHANG H， GAO L， ZHANG W， et al. Differentiation of rat bone marrow mesenchymal stem cells into neurons induced by bone morphogenetic protein 7 in vitro［J］. Neurol Res， 2023，45（5）：440-448. doi:10.1080/01616412.2022.2154487 doi: 10.1080/01616412.2022.2154487
[21]	HU H， HU X W， LI L， et al. Exosomes derived from bone marrow mesenchymal stem cells promote angiogenesis in ischemic stroke mice via upregulation of MiR-21-5p［J］. Biomolecules， 2022，12（7）：883. doi:10.3390/biom12070883 doi: 10.3390/biom12070883
[22]	WU R P， SOLAND M， LIU G H， et al. Functional characterization of the immunomodulatory properties of human urine-derived stem cells［J］. Transl Androl Urol， 2021，10（9）：3566-3578. doi:10.21037/tau-21-506 doi: 10.21037/tau-21-506
[23]	黄玉圣，吕辰菲，庄景燊，等. 细胞传代对SD大鼠骨髓间充质干细胞衰老的影响［J］. 中国老年学杂志， 2019，30（9）：2189-2192.
[24]	YAN C T， SUN W， CHEN Z， et al. ECM Protein CYR61 Promotes Migration and Osteoblastic Differentiation of Irradiation BMSCs via Migrasomes［J］. Stem Cells Int， 2025， 2025：8825935. doi:10.1155/sci/8825935 doi: 10.1155/sci/8825935
[25]	GAO J W， SONG M K， WU D Z， et al. CircRBM23 regulates the switch between osteogenesis and adipogenesis of mesenchymal stem cells via sponging miR-338-3p［J］. Clin Sci （Lond）， 2023，137（6）：495-510. doi:10.1042/cs20220833 doi: 10.1042/cs20220833
[26]	QIN W C， SHANG Q， SHEN G Y， et al. Restoring bone-fat equilibrium： Baicalin's impact on P38 MAPK pathway for treating diabetic osteoporosis［J］. Biomed Pharmacother， 2022，175：116571.