手术切除的结直肠癌组织中的肿瘤干细胞的培养及鉴定
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摘要
目的获得结直肠癌患者手术切除癌组织的肿瘤干细胞(CSC),培养并鉴定其干细胞特性。方法通过原代培养手术切除结直肠癌组织获得结直肠癌细胞,再通过有限稀释法和无血清培养法筛选结直肠癌CSC,软琼脂集落实验检测CSC和对照组人结直肠癌细胞株SW480的增殖能力;用低数量级细胞裸鼠皮下成瘤实验,检测结直肠癌CSC和SW480的成瘤能力;并运用Western blot及免疫荧光方法,检测CSC和SW480的免疫表型。结果临床结直肠癌标本原代培养的CSC,加入血清培养可使CSC分化。软琼脂集落形成实验结果显示,原代培养的CSC克隆形成率为56.64%和54.45%,对照组人结直肠癌细胞株SW480的克隆形成率为4.41%,CSC与SW480集落形成率的差异具有统计学意义(P<0.01)。裸鼠皮下成瘤实验中,原代培养的CSC皮下注射500个细胞可形成皮下瘤,而SW480皮下注射500个细胞不形成皮下瘤。CSC表达CD133、CD44,不表达CK7;SW480细胞不表达CD133、CD44,表达CK7。结论原代培养的手术切除结直肠癌组织标本获得的肿瘤细胞,再行无血清培养可形成CSC,鉴定具有CSC的自我更新及分化能力。
Objective To obtain cancer stem cells(CSCs) from surgically resected colorectal cancer specimens and identify their stem cell characteristics. Methods Colorectal cancer tissue specimen obtained from a patient undergoing radical resection of colorectal cancer were implanted in nude mice, and the xenograft was harvested 1 month later to obtain purified tumor cells by enzyme digestion and adherent culture. The CSCs were screened by limiting dilution method and serum-free culture to identify their phenotypes. Soft agar colony assay was used to assess the proliferative ability of the CSCs and human colorectal cancer cell line SW480. The tumorigenic ability of the isolated CSCs and SW480 cells was evaluated by observing their subcutaneous tumor formation in nude mice. Western blotting and immunofluorescence assay were used to detect the immunophenotype of the CSCs and SW480 cells. Results The primary cultured CSCs from clinical specimens of colorectal cancer underwent differentiation in the presence of serum in the culture. Soft agar colony formation assay showed that the CSCs had a colony formation rate above 50%, significantly higher than the rate of colorectal cancer SW480 cells(4.41%; P<0.01).In nude mice, subcutaneous injection of 500 CSCs was sufficient to result in subcutaneous tumor formation, while the injection of 500 SW480 cells failed to form any subcutaneous tumors. The CSCs expressed CD133 and CD44 but not CK7, while SW480 cells expressed CK7 but not CD133 or CD44. Conclusion CSCs can be derived by primary culture of cancer cells obtained from surgically resected colorectal cancer tissue followed by serum-free culture, and the CSCs obtained have self-renewal and differentiation abilities.
Objective To obtain cancer stem cells(CSCs) from surgically resected colorectal cancer specimens and identify their stem cell characteristics. Methods Colorectal cancer tissue specimen obtained from a patient undergoing radical resection of colorectal cancer were implanted in nude mice, and the xenograft was harvested 1 month later to obtain purified tumor cells by enzyme digestion and adherent culture. The CSCs were screened by limiting dilution method and serum-free culture to identify their phenotypes. Soft agar colony assay was used to assess the proliferative ability of the CSCs and human colorectal cancer cell line SW480. The tumorigenic ability of the isolated CSCs and SW480 cells was evaluated by observing their subcutaneous tumor formation in nude mice. Western blotting and immunofluorescence assay were used to detect the immunophenotype of the CSCs and SW480 cells. Results The primary cultured CSCs from clinical specimens of colorectal cancer underwent differentiation in the presence of serum in the culture. Soft agar colony formation assay showed that the CSCs had a colony formation rate above 50%, significantly higher than the rate of colorectal cancer SW480 cells(4.41%; P<0.01).In nude mice, subcutaneous injection of 500 CSCs was sufficient to result in subcutaneous tumor formation, while the injection of 500 SW480 cells failed to form any subcutaneous tumors. The CSCs expressed CD133 and CD44 but not CK7, while SW480 cells expressed CK7 but not CD133 or CD44. Conclusion CSCs can be derived by primary culture of cancer cells obtained from surgically resected colorectal cancer tissue followed by serum-free culture, and the CSCs obtained have self-renewal and differentiation abilities.
引文
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[2]Lapidot T,Sirard C,Vormoor J,et al.A cell initiating human acute myeloid leukaemia after transplantation into SCID mice[J].Nature,1994,367(6464):645-8.
[3]Visvader JE,Lindeman GJ.Cancer stem cells in solid tumours:accumulating evidence and unresolved questions[J].Nat Rev cancer,2008,8(10):755-68.
[4]O'brien CA,Pollett A,Gallinger S,et al.A human colon cancer cell capable of initiating tumour growth in immunodeficient mice[J].Nature,2007,445(7123):106-10.
[5]Ricci-Vitiani L,Lombardi DG,Pilozzi E,et al.Identification and expansion of human colon-cancer-initiating cells[J].Nature,2007,445(7123):111-5.
[6]Dalerba P,Dylla SJ,Park IK,et al.Phenotypic characterization of human colorectal cancer stem cells[J].Proc Natl Acad Sci USA,2007,104(24):10158-63.
[7]Singh SK,Clarke ID,Terasaki M,et al.Identification of a cancer stem cell in human brain tumors[J].Cancer Res,2003,53(2):487-8.
[8]Vermeulen L,Todaro M,de Sousa Mello F,et al.Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity[J].Proc Natl Acad Sci USA,2008,105(36):13427-32.
[9]Heidt DG,Li C,Mollenberg N,et al.Identification of pancreatic cancer stem cells[J].cancer Res,2006,130(2):194-5.
[10]Li C,Lee CJ,Simeone DM.Identification of human pancreatic cancer stem cells[J].Methods Mol Biol,2009,568(6):161-73.
[11]Kondo T,Setoguchi T,Taga T.Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line[J].Proc Natl Acad Sci USA,2004,101(3):781-6.
[12]Al-Hajj M,Wicha MS,Benito-Hernandez A,et al.Prospective identification of tumorigenic breast cancer cells[J].Proc Natl Acad Sci USA,2003,100(7):3983-8.
[13]Feng L,Wu JB,Yi FM.Isolation and phenotypic characterization of cancer stem-like side population cells in colon cancer[J].Mol Med Rep,2015,12(3):3531-6.
[14]Mahkamova K,Latar N,Aspinall S,et al.Hypoxia increases thyroid cancer stem cell-enriched side population[J].World J Surg,2018,42(2):350-7.
[15]Fanali C,Lucchetti D,Farina M,et al.Cancer stem cells in colorectal cancer from pathogenesis to therapy:controversies and perspectives[J].World J Gastroenterol,2014,20(4):923-42.
[16]贾健锋,贺长林.结直肠肿瘤干细胞体外培养形态、标志物及转移动力学特征[J].检验医学与临床,2014(z1):290-3.
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[20]Zhou Y,Xia L,Wang H,et al.Cancer stem cells in progression of colorectal cancer[J].Oncotarget,2018,9(70):33403-15.
[21]Ramesh P,Kirov AB,Huels DJ,et al.Isolation,propagation,and clonogenicity of intestinal stem cells[J].Methods Mol Biol,2018,11:1-13.
[22]Zhou SL,Ochalek A,Szczesna K,et al.The positional identity of iPSC-derived neural progenitor cells along the anterior-posterior axis is controlled in a dosage-dependent manner by bFGF and EGF[J].Differentiation,2016,92(4):183-94.
[23]Ye Q,Feng B,Yf P,et al.Expression ofγ-synuclein in colorectal cancer tissues and its role on colorectal cancer cell line HCT116[J].World J Gastroenterol,2009,15(40):5035-43.
[24]Yang K,Tang Y,Habermehl GK,et al.Stable alterations of CD44isoform expression in prostate cancer cells decrease invasion and growth and alter ligand binding and chemosensitivity[J].BMCcancer,2010,10(1):16.
[25]Takemasa I,Ishii H,Haraguchi N,et al.Perspectives on current status and future directions for cancer stem cells theory in gastrointestinal cancer[J].Nihon Geka Gakkai Zasshi,2009,110(4):207-12.
[26]Wahab S,Islam F,Gopalan V,et al.The identifications and clinical implications of cancer stem cells in colorectal cancer[J].Clin Colorectal cancer,2017,16(2):93-102.
[27]Cherciu I,B?rb?lan A,Pirici D,et al.Stem cells,colorectal cancer and cancer stem cell markers correlations[J].Current health sciences journal,2015,40(3):153-61.
[28]Lugli A,Iezzi G,Hostettler I,et al.Prognostic impact of the expression of putative cancer stem cell markers CD133,CD166,CD44s,EpCAM,and ALDH1 in colorectal cancer[J].Br J cancer,2010,103(3):382-90.
[29]Fedyanin M,Anna P,Elizaveta P,et al.Role of stem cells in colorectal cancer progression and prognostic and predictive characteristics of stem cell markers in colorectal cancer[J].Curr Stem Cell Res Ther,2017,12(1):19-30.
[30]Park SY,Kim HS,Hong EK,et al.Expression of cytokeratins 7 and20 in primary carcinomas of the stomach and colorectum and their value in the differential diagnosis of metastatic carcinomas to the ovary[J].Hum Pathol,2002,33(11):1078-85.