Gao K, Lu Y R, Wei L L, Lu X F, Li S F, Wan L, Li Y P, Cheng J Q
Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, China.
Transplant Proc. 2008 Mar;40(2):634-7. doi: 10.1016/j.transproceed.2008.01.053.
Our previous experiments indicated that bone marrow mesenchymal stem cells of rhesus monkey (RhBMSCs) have a low proliferative ability with a finite life span, which will hamper their application in biomedical research. Establishing an immortalized RhBMSC lineage might solve the problem.
RhBMSCs isolated from the bone marrow of rhesus monkeys using density gradient centrifugation were purified using adherence separation. Then, the cells were steadily transfected by plasmid containing human telomerase reverse transcriptase gene (pCI-neo-hTERT). We analyzed expression of hTERT, proliferation, phenotype (SH-2, SH-3, SB-10, CD29, CD34, CD45, and HLA-DR), differentiation toward osteogenic lineage, karyotype, and tumorigenesis of transfected cells.
After transfection, the RhBMSCs proliferated vigorously, undergoing more than 50 population doublings (PDs). Apoptotic rate of transfected RhBMSCs at PD40 was only 4.5%, versus untransfected RhBMSCs at PD15, which was more than 33.5%. Compared with normal RhBMSC, the life span of transfected RhBMSCs was prolonged, retaining similar morphology, karyotype, and potential to differentiate into an osteogenic lineage. More than 99% of transfected RhBMSCs were positive for stem cell markers, including SH-2, SH-3, SB-10, and CD29, and negative for CD34, CD45, and HLA-DR. Furthermore, the transfected cell line was benign in nude mice tumor formation.
Our results demonstrated that hTERT gene had been transfected into RhBMSCs. The transfected RhBMSCs proliferated vigorously. Phenotype, differentiation, and karyotype of transfected RhBMSC showed no significant difference from untransfected cells. The transfected RhBMSCs are a potential cell source for transplantation as well as tissue engineering.
我们之前的实验表明，恒河猴骨髓间充质干细胞（RhBMSCs）增殖能力低，寿命有限，这将阻碍它们在生物医学研究中的应用。建立永生化的RhBMSC细胞系可能会解决这个问题。
使用密度梯度离心法从恒河猴骨髓中分离出RhBMSCs，通过贴壁分离法进行纯化。然后，用含人端粒酶逆转录酶基因的质粒（pCI-neo-hTERT）稳定转染细胞。我们分析了转染细胞中hTERT的表达、增殖、表型（SH-2、SH-3、SB-10、CD29、CD34、CD45和HLA-DR）、向成骨谱系的分化、核型以及致瘤性。
转染后，RhBMSCs增殖旺盛，经历了超过50次群体倍增（PDs）。转染的RhBMSCs在PD40时的凋亡率仅为4.5%，而未转染的RhBMSCs在PD15时凋亡率超过33.5%。与正常RhBMSC相比，转染的RhBMSCs寿命延长，保留了相似的形态、核型以及向成骨谱系分化的潜能。超过99%的转染RhBMSCs干细胞标志物呈阳性，包括SH-2、SH-3、SB-10和CD29，而CD34、CD45和HLA-DR呈阴性。此外，转染细胞系在裸鼠肿瘤形成实验中表现为良性。
我们的结果表明hTERT基因已转染到RhBMSCs中。转染的RhBMSCs增殖旺盛。转染的RhBMSC的表型、分化和核型与未转染细胞相比无显著差异。转染的RhBMSCs是移植以及组织工程的潜在细胞来源。
