hTERT‐ and hCTLA4Ig‐expressing human bone marrow‐derived mesenchymal stem cells: in vitro and in vivo characterization and osteogenic differentiation
hTERT‐ and hCTLA4Ig‐expressing human bone marrow‐derived mesenchymal stem cells: in vitro and in vivo characterization and osteogenic differentiation
复制标题
DOI：
10.1002/term.1924
复制
发表时间：
2017-02
影响因子：
3.3
通讯作者：
F. Dai;Si-si Yang;Fei Zhang;Dongwen Shi;Ze-hua Zhang;Jun Wu;Jianzhong Xu
中科院分区：
文献类型：
--
作者：
F. Dai;Si-si Yang;Fei Zhang;Dongwen Shi;Ze-hua Zhang;Jun Wu;Jianzhong Xu
多能间充质干细胞（Multipotent mesenchymal stem cells，MSCs）是组织工程和再生医学研究中常用的种子细胞，但由于自体MSCs数量不足、同种异体MSCs的免疫排斥和复制性衰老等原因，其临床应用受到限制。我们构建了人端粒酶逆转录酶（human telomerase reverse transcriptase，hTERT）和细胞毒性T淋巴细胞相关抗原4-IG（cytotoxic T lymphocyte-associated antigen 4-Ig，CTLA 4 IG）基因的两种基因表达载体，分别转染人骨髓源性干细胞（human bone marrow-derived stem cells，hBMSCs）。两种基因的成功转染产生hTERT-CTLA 4 Ig hBMSC，其表达端粒酶（通过免疫组织化学和TRAPeze测定显示）和CTLA 4 Ig（通过免疫细胞化学和蛋白质印迹证明），而没有明显的相互干扰。hTERT-CTLA 4 Ig hBMSCs（92个群体倍增）和hTERT-CTLA 4 Ig hBMSCs（60个群体倍增）与hBMSCs（18个群体倍增）相比具有延长的寿命。细胞周期分析显示，与hBMSCs相比，hTERT-hBMSCs的G 0/G1期比例较低，S期比例较高;与hTERT-hBMSCs相比，hTERT-CTLA 4 Ig hBMSCs的G 0/G1期比例较高，S期和G2/M期比例较低。hTERT-CTLA 4 Ig hBMSCs在体外仍保持其成骨分化能力，通过检测羟基磷灰石矿物沉积（标记四环素荧光染色）、钙结节（茜素红S染色）、碱性磷酸酶（钙钴法）和骨钙素（免疫细胞化学）显示。此外，在大鼠异种移植模型中皮下移植hTERT-CTLA 4 Ig hBMSC导致骨样组织的成功产生，使用放射照相术和组织学评估证实。结论：hTERT-CTLA 4 Ig hBMSCs是骨组织工程理想的种子细胞。版权所有© 2014约翰威利父子有限公司.
Multipotent mesenchymal stem cells (MSCs) are commonly used as seed cells in studies of tissue engineering and regenerative medicine but their clinical application is limited, due to insufficient numbers of autogeneic MSCs, immune rejection of allogeneic MSCs and replicative senescence. We constructed two gene expression vectors for transfection of the human telomerase reverse transcriptase (hTERT) and cytotoxic T lymphocyte‐associated antigen 4‐Ig (CTLA4Ig) genes into human bone marrow‐derived stem cells (hBMSCs). Successful transfection of both genes generated hTERT–CTLA4Ig hBMSCs that expressed both telomerase (shown by immunohistochemistry and a TRAPeze assay) and CTLA4Ig (demonstrated by immunocytochemistry and western blotting) without apparent mutual interference. Both hTERT BMSCs (92 population doublings) and hTERT–CTLA4Ig hBMSCs (60 population doublings) had an extended lifespan compared with hBMSCs (18 population doublings). Cell cycle analysis revealed that, compared with hBMSCs, a lower proportion of hTERT hBMSCs were in G0/G1 phase but a higher proportion were in S phase; compared with hTERT hBMSCs, a higher proportion of hTERT–CTLA4Ig hBMSCs were in G0/G1 phase, while a lower proportion were in S and G2/M phases. hTERT–CTLA4Ig hBMSCs retained their capacity for osteogenic differentiation in vitro, shown by the detection of hydroxyapatite mineral deposition (labelled tetracycline fluorescence staining), calcareous nodules (alizarin red S staining), alkaline phosphatase (calcium–cobalt method) and osteocalcin (immunocytochemistry). Furthermore, subcutaneous transplantation of hTERT–CTLA4Ig hBMSCs in a rat xenotransplantation model resulted in the successful generation of bone‐like tissue, confirmed using radiography and histological assessment. We propose that allogeneic hTERT–CTLA4Ig hBMSCs may be ideal seed cells for bone tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.