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目的本研究拟建立成人骨髓间充质干细胞(mesenchymal stem cell,MSCs)体外培养和扩增的方法,探讨其生物学特性,为进一步将其应用于临床奠定理论和实验基础。方法取正常成人骨髓液5 mL,用Percoll分离液(密度1．073g/mL)经密度梯度离心法分离得到单个核细胞,以2×108个细胞／cm2的密度接种于含10％新生牛血清的LG-DMEM培养液。经培养、扩增后,进行倒置相差显微镜观察其形态,MTT法测定生长曲线,流式细胞仪行细胞表面抗原及细胞周期的检测,并在透射电镜下观察其超微结构。MtT法观察MSCs的免疫调节功能,观察MSCs对K562细胞生长的影响。检测MSCs培养上清中HA、IV-C、LN浓度的动态变化。结果培养扩增获取的成人骨髓MSCs形态均一,为梭形或纺锤形的成纤维细胞样外观,生长曲线示其增殖能力强。流式细胞仪检测显示有90％以上的细胞处于G0／G1期,表面标记物中CD4表达阳性,而造血细胞表面标志CD3、CD4、CD7、CD13、CD14、CD15、CD19、CD22、CD33、CD34、CD45和与移植排斥发生密切相关的HLA-DR表达阴性。超微结构显示细胞内有丰富的细胞器。成人骨髓MSCs抑制PHA诱导的异体淋巴细胞增殖转化,其增殖转化抑制率为60．68％(P<0．01)。抑制作用同样存在于培养上清中,其增殖转化抑制率为9．00％(P<0．05)。在有PHA刺激淋巴细胞增殖的情况下,培养上清中增殖转化抑制率达20．91％(P<0．01)。和单独的K562细胞生长曲线相比,与骨髓MSCs共孵育的K562细胞生长缓慢,无明显的指数生长期。由浓度变化曲线图可以看出,随着天数的延长,HA升高较迅速,而IV-C、LN则浓度变化不大。结论所建立的分离和培养方法可获取骨髓粘附细胞中一组独特的细胞群,具有MSCs的生物学特性。初步的生物学特性研究显示其具有免疫调节、抗肿瘤、造血支持等作用,可作为组织工程中的种子细胞。
OBJECTIVE: To establish a method for culturing and amplifying adult human mesenchymal stem cells (MSCs) in vitro and to explore the biological characteristics of MSCs in order to lay a theoretical and experimental basis for their further application in clinical practice. Methods 5 mL normal adult bone marrow fluid was obtained. Single nucleated cells were isolated by density gradient centrifugation with Percoll (density: 1.073 g / mL) and inoculated into 10% neonatal calf serum at a density of 2 × 10 8 cells / cm 2 LG-DMEM broth. After being cultured and expanded, the morphology was observed by inverted phase contrast microscope. The growth curve was measured by MTT assay. The cell surface antigens and cell cycle were detected by flow cytometry. The ultrastructure of the cells was observed under transmission electron microscope. MtT method was used to observe the immunoregulatory function of MSCs to observe the effect of MSCs on the growth of K562 cells. The dynamic changes of HA, IV-C and LN concentrations in the culture supernatant of MSCs were detected. Results The bone marrow MSCs obtained by culture and amplification were uniform in morphology and spindle-shaped or spindle-shaped fibroblast-like appearance. The growth curve showed that their proliferation ability was strong. Flow cytometry showed that more than 90% of the cells were in G0 / G1 phase, and CD4 was positive in surface markers, while hematopoietic cell surface markers CD3, CD4, CD7, CD13, CD14, CD15, CD19, CD22, CD33, CD34 , CD45 and HLA-DR expression negatively associated with graft rejection. Ultrastructure shows abundant organelles in cells. Adult bone marrow MSCs inhibited PHA-induced proliferation and transformation of allogeneic lymphocytes, and the inhibition rate of proliferation and transformation was 60.68% (P <0.01). Inhibition also existed in the culture supernatant, the proliferation inhibition rate was 9.00% (P <0.05). In the presence of PHA stimulated lymphocyte proliferation, the proliferation inhibition rate in culture supernatant reached 20.91% (P <0.01). Compared with the K562 cell growth curve alone, K562 cells co-incubated with bone marrow MSCs grew slowly with no apparent exponential growth phase. As can be seen from the concentration curve, with the number of days, HA increased rapidly, while IV-C, LN concentration was not changed. Conclusion The established isolation and culture methods can obtain a unique cell population of bone marrow adherent cells with the biological characteristics of MSCs. Preliminary biological studies have shown that it has immunomodulatory, anti-tumor, hematopoietic support role, can be used as seed cells in tissue engineering.