Isolation and identification of osteosarcoma stem cells

doi:10.3969/j.issn.2095-4344.2013.14.025

Abstract

Abstract:

BACKGROUND: Osteosarcoma is the most common bone primary malignant tumors, and the occurrence of osteosarcoma may relate with osteosarcoma stem cells.
OBJECTIVE: To evaluate the isolation, culture and identification method of osteosarcoma stem cells, to investigate the expression of relative tumor markers.
METHODS: The osteosarcoma stem cells were isolated and cultured with magnetic activated cell sorting method under serum-free condition and serum-free joint antineoplastic condition, in order to sort Stro-1 positive and CD133 positive osteosarcoma stem cells. The expression level and tumorigenic properties of CD133, Oct3 / 4 and Nanog markers of osteosarcoma cancer stem cells were tested with immunofluorescence staining and Western blotting methods.
RESULTS AND CONCLUSION: Suspended cell condensation could be seen in the osteosarcoma stem cells after culture for 2-10 days, the proliferation incubation period was about 24 hours, and the Stro-1 positive stem cells could form the suspended cell condensation, while the Stro-1 negative stem cells could not form the suspended cell condensation. In addition, osteosarcoma stem cells could highly express Oct3/4, Nanog and CD133, the CD133-positive osteosarcoma stem cells could highly express CD133 molecule with strong invasiveness, while the CD133 negative cells could not express CD133 molecule and the invasiveness was weak. Celecoxib could inhibit the formation of osteosarcoma stem cells to some extent, and could reduce the expression of tumor angiogenic blood vessels and vascular endothelial growth factor.

Key words: stem cells, stem cell academic discussion, bone tumors, osteosarcoma, tumor stem cells, doxorubicin, vincristine, CD133, Nanog, Stro-1, suspended cells

CLC Number:

R318

Qu Shao-zheng, Li Shu-zhong, Gao Jia-ke, Zhang Jin-feng. Isolation and identification of osteosarcoma stem cells [J]. Chinese Journal of Tissue Engineering Research, 2013, 17(14): 2641-2648.

Figures/Tables 1

2.1 人骨肉瘤细胞株U2-OS中分离的肿瘤干细胞欧阳振等+人骨肉瘤细胞株U2-OS中分离肿瘤干细胞进行了实验研究。首先进行细胞培养，选取原代培养的骨肉瘤细胞，采用胰蛋白酶进行消化降解，充分吹打成单细胞悬液后丢弃上清液，用无血清DMEM/F12培养基重悬细胞，培养基内含表皮生长因子、碱性成纤维细胞生长因子、L-谷氨酰胺、胰岛素、青霉素和链霉素，pH值为7.2-7.5。调整细胞浓度为2×106/L进行细胞接种，置入37 ℃、体积分数5%CO2培养箱中培养。隔日加1 mL培养液，培养5-7 d，待培养基中悬浮的肉瘤细胞球体积变大后，吸取培养基并离心，用无血清培养基重新吹打成单细胞悬液，按1∶2或1∶3比例传代。将无血清培养基培养的骨肉瘤细胞球接种于96孔板，置于培养箱内分别培养0，1，2，3，4，5，6，7 d，结束前4 h每孔加入噻唑蓝溶液，继续培养4 h后，用二甲基亚砜震荡溶解蓝紫色结晶，并应用全自动荧光酶标仪在490 nm波长下测定吸光度值。将骨肉瘤干细胞球与小鼠抗人Stro-1单克隆抗体在4 ℃条件下反应15 min，磷酸盐缓冲液洗涤2次后，与大鼠抗小鼠IgM免疫磁珠4 ℃条件下反应15 min，将免疫标记好的干细胞放入细胞分离系统的细胞分离柱内，无免疫磁珠标记的细胞流出分离柱，而免疫标记的细胞则由于磁场的作用滞留于分离柱中，获取免疫磁珠标记的细胞。将免疫标记阳性和阴性的细胞计数后调整细胞浓度，并接种于培养瓶内，观察细胞增殖情况。提取细胞内RNA，置于PCR扩增仪后进行反转录、变性和延伸处理。PCR扩增产物行琼脂糖凝胶电泳，并进行染色观察。收集干细胞球第3次传代5 d后所形成的细胞球，离心后丢弃上清液，用含血清培养液重悬，接种于预先包被多聚赖氨酸的玻片上，置于6孔板中，在37 ℃、体积分数5%CO2培养箱中培养，倒置显微镜下定期动态观察细胞的分化和生长情况。当干细胞球在含血清培养基中分化14 d后，取出有分化细胞贴壁的玻片，进行骨形态蛋白和Ⅰ型胶原酶的免疫细胞化学染色观察。结果观察发现原代骨肉瘤细胞离心后置于无血清培养基中，3 d后数个细胞能够形成大小不等的圆形类细胞球，悬浮生长，大部分细胞不能形成细胞球。细胞球传代后2 d即可见到次代肿瘤细胞球的形成，以后体积逐渐增大，形态规则。肿瘤干细胞的增殖潜伏期约为24 h，传代后1-2 d即可见肿瘤干细胞形成，以后体积逐渐增大。肿瘤干细胞对数倍增时间约为传代后第3-5天。用免疫磁珠法分选的Stro-1阳性细胞，接种于无血清培养基后24-48 h即可形成和原代肿瘤干细胞球形态一样的干细胞球，Stro-1阴性细胞却不能形成肿瘤细胞球。RT-PCR检测结果可见Stro-1阳性细胞和Stro-1阴性细胞mRNA扩增产物的吸光度值分别为1.58±0.24和0.37±0.68。用含血清培养基重悬肿瘤细胞球后二三天开始贴壁，7 d后小干细胞球中的细胞几乎全部贴壁分化。之后可以见到部分细胞形态开始发生变化，形态变圆并有部分突起。对分化2周的细胞进行免疫细胞化学染色，部分细胞呈圆形或不规则形状，胞膜染色为棕黄色或棕褐色，表现为骨形态蛋白阳性。部分细胞胞浆染色为棕黄色，核深染，Ⅰ型胶原酶呈阳性表达。 2.2 三维无血清条件培养结合抗癌药物分离的人骨肉瘤肿瘤干细胞周松等[19]和娄楠等[20]对三维无血清培养基结合抗肿瘤药物分离骨肉瘤干细胞进行了研究，2组实验同样应用肿瘤细胞的培养传代，并应用免疫化学染色等方法对检测细胞进行观察，结果见表2。"

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