新生大鼠肝脏间充质干细胞的体外培养扩增及其生物学表征

doi:10.12122/j.issn.1673-4254.2026.04.20

南方医科大学学报 ›› 2026, Vol. 46 ›› Issue (4): 923-928.doi: 10.12122/j.issn.1673-4254.2026.04.20

• • 上一篇

新生大鼠肝脏间充质干细胞的体外培养扩增及其生物学表征

池茗¹(), 李柏霖², 吴晶¹, 葛剑云³, 唐元瑜²()

^1.福建中医药大学，中西医结合学院中西医结合研究院，福建福州 350122
^2.福建中医药大学，中医学院，福建福州 350122
^3.福建中医药大学，科技创新与转化中心，福建福州 350122

收稿日期:2025-09-05 出版日期:2026-04-20 发布日期:2026-04-24
通讯作者: 唐元瑜 E-mail:1609695786@ qq.com;2422198977@ qq.com
作者简介:池茗，在读本科生，E-mail: 1609695786@ qq.com
基金资助:
国家自然科学基金(81072714);福建中医药大学校管课题(X2024012)

In vitro culture, expansion, and biological characterization of mesenchymal stem cells derived from neonatal rat liver

Ming CHI¹(), Boling LI², Jing WU¹, Jianyun GE³, Yuanyu TANG²()

^1.College and Institute of Integrated Traditional Chinese and Western Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China
^2.College of TCM, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China
^3.Center for Scientific Innovation and Translation, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China

Received:2025-09-05 Online:2026-04-20 Published:2026-04-24
Contact: Yuanyu TANG E-mail:1609695786@ qq.com;2422198977@ qq.com
Supported by:
National Natural Science Foundation of China(81072714)

摘要/Abstract

摘要：

目的采用“组织块联合酶解法”提取新生大鼠肝脏间充质干细胞（L-MSCs），为开展脂肪肝、肝硬化等慢性肝病治疗及肝脏再生医学研究提供理想的种子细胞。方法无菌分离1周龄SD新生大鼠肝脏，经漂洗、剪碎、0.1% II型胶原酶/0.05% IV型胶原酶消化15~20 min、100目筛网过滤后，进行L-MSCs原代培养。通过细胞形态学观察、细胞表面标志物CD分子及成脂、成骨诱导分化潜能检测，多维度鉴定所培养的目的细胞。结果原代接种培养6~8 d后，短梭形贴壁生长的细胞呈漩涡状铺满皿底。流式细胞仪检测分析第4代细胞表面标志物，高表达CD90（99.33%±0.06%）、CD73（99.60%±0.10%）、CD44（99.50%±0.10%）、CD29（97.60%±0.17%），平均阳性率为（99.01%±0.86%）；低表达CD45（0.87%±0.12%）、CD34（0.95%±0.22%）、CD11b/c（1.71%±0.28%），平均阳性率为（1.18%±0.44%），显著低于前者（P<0.01），符合MSCs的免疫表型特征。诱导成脂、成骨分化实验均为阳性。结论 “组织块联合酶解法”能够成功提取新生大鼠L-MSCs。

关键词: 新生大鼠, 肝脏间充质干细胞, 组织块联合酶解法, 细胞形态, 细胞免疫标志物, 三系分化潜能

Abstract:

Objective To isolate liver-derived mesenchymal stem cells (L-MSCs) from neonatal rats using tissue explant method combined with enzymatic digestion. Methods Liver tissues aseptically harvested from 1-week-old SD rats were rinsed, minced, and digested with D-Hanks solution containing 0.1% type-II collagenase and 0.05% type-IV collagenase for 15-20 min. After filtration through a 100-mesh screen, the cells were cultured for primary L-MSC isolation. The target cells were identified by morphological observation, flow cytometric analysis of surface CD markers, and adipogenic/osteogenic differentiation assays. Results Six to eight days after primary seeding, short spindle-shaped adherent cells formed a confluent monolayer arranged in a swirling pattern. Flow cytometry of passage-4 cells showed high expressions of CD90 [(99.33±0.06)%], CD73 [(99.60±0.10)%], CD44 [(99.50±0.10)%], and CD29 [(97.60±0.17)%] with an average positive rate of (99.01±0.86)% and low expressions of CD45 [(0.87±0.12)%], CD34 [(0.95±0.22)%], and CD11b/c [(1.71±0.28)%] with an average positive rate of (1.18±0.44)%, which was significantly lower than that of CD90, CD73, CD44, and CD29 (P<0.01), consistent with the immunophenotypic characteristics of MSCs. Both adipogenic and osteogenic induction tests of the cells yielded positive results. Conclusion L-MSCs can be successfully isolated from neonatal rat liver using tissue explant method combined with enzymatic digestion.

Key words: newborn rats, liver mesenchymal stem cells, tissue explant-enzymatic digestion combined method, cell mophorlogy, surface markers of mesenchymal stem cells, trilineage differentiation

池茗, 李柏霖, 吴晶, 葛剑云, 唐元瑜. 新生大鼠肝脏间充质干细胞的体外培养扩增及其生物学表征[J]. 南方医科大学学报, 2026, 46(4): 923-928.

Ming CHI, Boling LI, Jing WU, Jianyun GE, Yuanyu TANG. In vitro culture, expansion, and biological characterization of mesenchymal stem cells derived from neonatal rat liver[J]. Journal of Southern Medical University, 2026, 46(4): 923-928.

图/表 5

图1 原代细胞生长情况观察

Fig.1 Observation of primary cell growth. A-C： On day 3 of primary culture， short spindle cells budded and migrated out from the liver tissue explant. D-F： After 4-8 days, these cells assembled into island-like clusters that gradually fused.

图2 第1-6代细胞生长情况观察

Fig.2 Observation of cell growth from passage 1 to passage 6. A-F: P1-P6 cells formed fibroblast-like whorls after confluence, without age-related thinning, widening or needle-like stretching.

图3 流式细胞仪检测和分析第4代细胞表面抗原标志物

Fig.3 Analysis of surface antigen markers of passage-4 cells by flow cytometry. The cells highly expressed CD90 [(99.33±0.06)%], CD73 [(99.60±0.10)%], CD44 [(99.50±0.10)%], and CD29 [(97.60±0.17)%] with a mean of (99.01±0.86)%. The cells show low expressions of CD45 [(0.87±0.12)%], CD34 [(0.95±0.22)%], and CD11b/c [(1.71±0.28)%] with a significantly lower mean rate of (1.18±0.44)% (P<0.01), matching MSC phenotype.

图4 第4代细胞诱导成脂及油红O染色

Fig.4 Adipogenic induction of passage 4 cells and Oil Red O staining. A: After 10 days of adipogenic induction, perinuclear "ring-like" early lipid granules appeared. B, C: The cells were stained red with Oil Red O. D: By 20 days, granules fused into large unilocular spherical droplets in the cells. E, F: The mature droplets showed deep red Oil Red O staining.

图5 第4代细胞诱导成骨及茜素红染色

Fig.5 Osteogenic induction of passage 4 cells and Alizarin Red staining. A: After 21 days osteogenic induction, P4 cells formed clusters with scattered black mineralized crystals of varying sizes. B, C: Alizarin Red staining showed deep red in the mushroom-like nodules in the cells.

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新生大鼠肝脏间充质干细胞的体外培养扩增及其生物学表征

In vitro culture, expansion, and biological characterization of mesenchymal stem cells derived from neonatal rat liver

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