人骨肉瘤细胞系MG-63细胞球形成细胞干性特征
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摘要
目的从人骨肉瘤细胞系MG-63中分离细胞球,鉴定其干性特征并探讨其与侵袭、转移的相关性及可能机制。方法无血清悬浮培养富集MG-63细胞球,PKH26染色确定细胞球中存在干细胞,Western blot检测细胞球干性相关蛋白表达。甲基纤维素半固体培养及Transwell小室检测细胞球自我更新及侵袭能力,并对上皮间质转化和侵袭、转移相关蛋白进行检测。结果 MG-63细胞无血清悬浮培养11 d后形成的细胞球中存在单个PKH26阳性细胞。与MG-63单层细胞相比,干细胞相关蛋白OCT-4、SOX2和Nanog表达显著提高(P<0. 05)。MG-63细胞球细胞成球数是单层细胞的1. 84倍(P<0. 01),侵袭能力较单层细胞提高1. 45倍(P<0. 01)。细胞球中E-cadherin低表达,snail及MMP2高表达(P<0. 05)。结论骨肉瘤细胞系MG-63细胞球形成细胞具有干性特征,通过上皮间质转化促进了细胞的侵袭与转移。
Objective To isolate and identify cancer stem like cells( Sphere formed cells) from human osteosarcoma cell line MG-63,and study it's invasion and metastasis ability. Methods Non-adherent culture was performed and PKH26 staining was used to identify cancer stem like cells in MG-63 spheres. Western blot was used to detect stemness associated protein expression in spheres cells. Self-renew and invasion of sphere formed cells were detected by methylcellulose semisolid culture and Transwell methods. The key proteins of epithelial to mesenchymal transition( EMT),invasion and metastasis in sphere cells were also detected by Western blot. Results The single PKH26 positive cell was observed in sphere formed cells after MG-63 cells were cultured for 11 days. Compared with the monolayer cells,the sphere formed cells presented higher self-renewal ability( P<0. 01),invasion ability enhanced 1. 45 times( P<0. 01) and increased expression of snail and MMP2 companied with decreased expression of E-cadherin. Conclusions Sphere formed cells in MG-63 show stemness characteristics and EMT,which are related with invasion and metastasis of osteosarcoma cells.
Objective To isolate and identify cancer stem like cells( Sphere formed cells) from human osteosarcoma cell line MG-63,and study it's invasion and metastasis ability. Methods Non-adherent culture was performed and PKH26 staining was used to identify cancer stem like cells in MG-63 spheres. Western blot was used to detect stemness associated protein expression in spheres cells. Self-renew and invasion of sphere formed cells were detected by methylcellulose semisolid culture and Transwell methods. The key proteins of epithelial to mesenchymal transition( EMT),invasion and metastasis in sphere cells were also detected by Western blot. Results The single PKH26 positive cell was observed in sphere formed cells after MG-63 cells were cultured for 11 days. Compared with the monolayer cells,the sphere formed cells presented higher self-renewal ability( P<0. 01),invasion ability enhanced 1. 45 times( P<0. 01) and increased expression of snail and MMP2 companied with decreased expression of E-cadherin. Conclusions Sphere formed cells in MG-63 show stemness characteristics and EMT,which are related with invasion and metastasis of osteosarcoma cells.
引文
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[5] Yang C,Jin K,Tong Y,et al. Therapeutic potential of cancer stem cells[J]. Med Oncol,2015,32:1-10.
[6] Yang J,Guo W,Wang L,et al. Cisplatin-resistant osteosarcoma cells possess cancer stem cell properties in a mouse model[J]. Oncol Lett,2016,12:2599-2605.
[7] Gatti M,Wurth R,Vito G,et al. Canine osteosarcoma cell lines contain stem-like cancer cells:biological and pharmacological characterization[J]. Jpn J Vet Res,2016,64:101-112.
[8] Li J,Yu Y,Wang J,et al. Establishment of a novel system for the culture and expansion of hepatic stem-like cancer cells[J]. Cancer Lett,2015,360:177-186.
[9] Pece S,Tosoni D,Confalonieri S,et al. Biological and molecular heterogeneity of breast cancers correlates with their cancer stem cell content[J]. Cell,2010,140:62-73.
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[15] Liu PF,AUID-Oho,Kang BH,et al. Vimentin is a potential prognostic factor for tongue squamous cell carcinoma among five epithelial-mesenchymal transition-related proteins[J]. PLoS One,2017,12:112-127.