摘要
本研究以体外培养的人成骨肉瘤细胞系—MG63细胞为对象,应用有限稀释法进行单细胞分离和单克隆细胞株的培养,成功建立MG63骨肉瘤单克隆细胞株20余个。根据细胞形成的克隆形态,选取其中的完全克隆进行ALP、OCN、OPN等一系列成骨分化标志物基因表达的检测,同时检测各组细胞生物学行为指标,包括肿瘤细胞成瘤、侵袭、迁移、铺展粘附等,发现具有高低不同转移能力和成瘤能力的单克隆细胞株之间在部分成骨细胞标志物、肿瘤转移及促血管形成因子等基因表达具有显著差异。说明检测骨肉瘤细胞成骨分化标志物的基因表达差异可以判断肿瘤的分化程度。进一步通过RT-PCR、RealTime-PCR及Western blot等方法对它们的侵袭能力进行检测,发现高低不同转移能力的细胞株的Cofilin、Limk1基因及蛋白表达存在显著差异,说明Cofilin介导的信号通路在人成骨肉瘤细胞的侵袭、转移等行为中发挥极为重要的作用。最后本实验选取集落形成能力最强的Holoclone细胞株,利用无血清悬浮成球的方式分离出类肿瘤干细胞亚系---MG63-M,并在无血清培养环境中加入低浓度长春新碱,进一步富集、纯化。经RT-PCR、免疫荧光及流式细胞术等检测发现其表达CD133、Oct4、nestin等干细胞表面标志物,同时高表达Mdr1、ABCG2等多药耐药基因。另外,体外实验和动物实验都表明MG63-M不但具有与正常干细胞相似的自我更新和多项分化能力,同时具有极强的致瘤能力和极为显著的多药耐药性。
本实验进一步验证了骨肉瘤异质性学说和肿瘤干细胞学说,为早期判断骨肉瘤侵袭及转移能力,克服骨肉瘤的多药耐药提供了新的理论基础和实验依据。
Oseteosarcoma is a kind of very common primary malignant neoplasms of bone. Oseteosarcoma can attack any age, Most of Oseteosarcoma attacks the adolescent, and causes great harm and high mortality. At the outset the disease is latent and in high-degree malignancy. Its early clinical symptoms may be confused with trauma of bone and pulmonary metastasis may occur in the early stage of the disease.
At present, the biological mechanism behind osteosarcoma formation is unclear. However, many researchers accept the‘cancer stem cell’hypothesis that there is a small number of self-renewing cancer cells with properties reminiscent of normal stem cells within a tumor. Cancer stem cells divide asymmetrically. The vast majority of cells derived from cancer stem cells are ordinary cells. A few, however, possess drug resistance and high self-renewal and proliferative capacity, which may explain the poor prognosis, recurrence, and metastasis of most tumors even after chemotherapy.
Previous studies have shown that MG-63 cells are not monoclonal but are heterogeneous in origin. Many researchers believe that purification of monoclonal cell strains from cell lines is essential for the study of cells. To obtain monoclonal stem-like cell strains from MG-63 cells, we first identified several subpopulations of holoclones. Each holoclone originated from a round- or oval-shaped dense colony of cells. Holoclone cells from breast cancers, gliomas, and other solid tumors tend to form small colonies and have high clonogenicity and strong adhesion abilities. Our findings suggest that the osteosarcoma cells that formed holoclones were tumorigenic. We also found that cultured monoclonal cells did not always retain their unique shape and characteristics, but underwent re-differentiation or automatic subcloning after passage over several generations. For this reason, cells of the same generation were used in each experiment in the present study (all within 12 passages or less).
Actin cytoskeletal reorganization is essential for numerous cell activities, including migration, morphological change, and vesicle transport. Cofilin plays an essential role in regulating actin filament dynamics. Its actin-depolymerizing and actin -severing activities are inhibited by phosphorylation by LIM kinases and testicular protein kinases and reactivated by dephosphorylation by SSH family phosphatases. In the early stage of cell responses, LIMK1 can stimulate the cell-foot formation, therefore it is necessary in cell migration. Boydern Chamber assay showed that cell invasion significantly in Subline A3, and compared with the control group it was significantly different. By Real Time-PCR and Western blot, Limk1 showed higher expression in Subline A3.
In the present study, we isolated tumor stem-like cells from the human MG63 osteosarcoma cell line. Limiting dilution of MG63 cells was first performed to isolate holoclones with high clonogenicity. Several osteosarcoma cell sublines with high tumorigenicity were identified. The sublines were then selected in serum-free medium containing a low concentration of vincristine to identify cells that were capable of forming suspended sarcospheres. A highly enriched subpopulation, designated as MG63-M, with the capacity of self-renewal, multilineage differentiation, high tumorigenicity and significant multi-drug resistance was characterized.
To isolate stem-like cells from the human MG63 osteosarcoma cell line, different subpopulations of MG63 cells were cloned by limiting dilution and passaged to obtain different sublines. The subline with highest clonogenicity was identified using a proliferation assay, cell-cycle analysis, and soft-agar colony-forming assay; sublines were further selected in serum-free medium containing a low concentration of vincristine to identify cells that could form suspended sarcospheres. Identified cells were then characterized based on morphology, cell surface markers, adipogenic and osteogenic differentiation, and tumorigenicity in nude mice. A total of 19 holoclones that could be stably passaged were obtained. Sublines A1, A3, and D1 were markedly different from other sublines and the parental cell line. Subline D1 not only had a higher colony-forming efficiency and formed larger colonies, but also possessed a shorter latency of tumorigenesis in vivo. After subline D1 was cultured in suspension in medium containing vincristine, a highly enriched subpopulation of cells that could form sarcospheres and be stably passaged were obtained. These cells, designated as MG63-M, expressed multiple markers of multipotent or embryonic stem cells and possessed the capacity for self-renewal, multilineage differentiation, and significant multi-drug resistance. Thus, our results suggest that a subpopulation of stem-like cells can be isolated from human MG63 osteosarcoma cell line.
The cancer stem cell hypothesis provides an explanation for the etiology of many heterogeneous and highly malignant tumors. In this study, we successfully isolated a subpopulation of tumor stem-like cells from a human osteosarcoma cell line by harvesting sarcospheres formed in serum-free culture medium containing a low concentration of vincristine. Based on our results, we believe that initial identification of holoclones with high tumorigenicity followed by further enrichment is an effective approach for isolation of tumor stem cells. The tumor stem-like cells obtained in this study not only possess the capacity of self-renewal and multilineage differentiation, but also had higher tumorigenicity and more significant drug resistance than those obtained using the conventional method. Thus, it is plausible to speculate that this subpopulation of cells plays a crucial role in the recurrence and chemotherapy resistance in osteosarcomas and are therefore important targets for treatment of the disease.
引文
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