RESUMO
BACKGROUND AND OBJECTIVE: Recently, the theory of cancer stem cells (CSCs) has presented new targets and orientations for tumor therapy. The major difficulties in researching CSCs include their isolation and purification. The aim of this study is to identify and characterize the side population (SP) cells in small cell lung cancer (SCLC) cell line H446, which lays the foundation for the isolation and purification of CSCs. METHODS: Fluorescence-activated cell sorting (FACS) was used to sort SP and non-SP (NSP) cells from H446. Both subgroups were cultivated to survey the capacity to form into suspended tumor cell spheres. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR were used to evaluate the expression levels of the mRNA of CD133, ABCG2, and nucleostemin in both subgroups. The capacity of proliferation and the differences in drug resistance of both subgroups and unsorted cells were tested by the MTT method. The differentiation ability of both subgroups was determined by FACS. Proliferation was determined by subcutaneous tumor formation in nude mice. RESULTS: The percent of Hoechst 33342 negative cells was about (5.1 +/- 0.2)% in H446 by fluorescence microscopy. The percent of SP cells was (6.3 +/- 0.1)% by flow cytometry. SP cells had a stronger capability of forming into tumor spheres than NSP cells. The mRNA expression levels of ABCG2, CD133, and nucleostemin in SP cells were 21.60 +/- 0.26, 7.10 +/- 0.14, and 1.02 +/- 0.08 folds higher than that in NSP cells (P < 0.01, P < 0.01, and P > 0.05, respectively). In vivo, SP cells showed better proliferative ability and tougher viability when treated with drugs. SP cells can differentiate into NSP cells, but NSP cells cannot differentiate into SP cells. SP cells had a greater ability to form tumors. CONCLUSION: The H446 cell line contained some SP cells with stem cell properties. CD133 and ABCG2 may be cancer stem cell markers of SCLC.
Assuntos
Transformação Celular Neoplásica , Neoplasias Pulmonares/patologia , Células-Tronco Neoplásicas/patologia , Células da Side Population/patologia , Carcinoma de Pequenas Células do Pulmão/patologia , Antígeno AC133 , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/transplante , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , RNA Mensageiro/metabolismo , Células da Side Population/metabolismo , Células da Side Population/transplante , Carcinoma de Pequenas Células do Pulmão/metabolismoRESUMO
AIM: To obtain mouse B7-H3-Fc fusion protein and to investigate its biological function and effects on T lymphocyte activation. METHODS: The genes coding extracellular domain of mouse B7-H3 and the Fc fragment of human IgG1 were amplified from pMD19-T/mouse B7-H3 and pMD19-T/human IgG1 vectors by PCR. The two genes were combined with mouse B7-H3-Fc fragment by overlap PCR. Then the resulting gene fragment was inserted into eukaryotic vector pIRES2-EGFP after digested with EcoR I and Bgl II to construct the recombinant vector pIRES2-EGFP/B7-H3-Fc. The recombinant vector was transfected into CHO cells with LipfectAMINE™ 2000, and the cells were further selected with G418. The collected supernatant of the transfected cell line cultured in serum-free media was ultrafiltrated and concentrated, then purified by Protein G column. The expression of mouse B7-H3-Fc fusion protein was confirmed by Western blot. Effects of fusion protein on T cells proliferation and cytokine production in vitro was studied by methods of CCK8 and ELISA. RESULTS: The results showed that the transfected CHO cell line secreting mouse B7-H3-Fc fusion protein was constructed successfully. In vitro, mouse B7-H3-Fc fusion protein obviously promoted the proliferation of T cells in a dose-dependent manner. CONCLUSION: A transfected CHO cell line stably expressing mouse B7-H3-Fc fusion protein has been obtained and the B7-H3-Fc fusion protein stimulates the proliferation of T cells and cytokines production in vitro.