Construction of an adipose derived stem cell-based alphastatin peptide glioma targeting vector and its inhibitory effect on angiogenesis / 西安交通大学学报(医学版)

【Objective】 To construct an adipose derived stem cell-based alphastatin peptide glioma targeting vector and detect its anti-angiogenesis effect in vitro. 【Methods】 The adipose derived stem cell-based alphastatin peptide glioma targeting vector (Al-ADSCs) was constructed by transfecting the alphastatin peptide lentivirus vector into adipose derived stem cells (ADSCs). The expressions of stem cell markers on the surface of targeted vector were detected by flow cytometry. The expression of alphastatin peptide in the targeted vector and in the cell culture supernatant of the targeted vector were detected by Western blotting and ELISA, respectively. Cell migration assay was used to detect the tendency of the targeted vector toward CD133+ glioma stem cells, and lumen formation assay was used to detect the effect of the targeted vector on endothelial cell angiogenesis in vitro. 【Results】 After transfection, the surface markers of stem cells expressed by targeted vector did not significantly change compared with ordinary adipose derived stem cells. Western blotting showed that the targeted vector could successfully express alphastatin peptide. ELISA showed that the alphastatin peptide was detected in the cell culture supernatant of targeted vector \mg/L]. Cell migration test showed no significant difference in the tendency of CD133+ glioma stem cells between the targeted vector and ordinary adipose derived stem cells \. Lumen formation experiment showed that the targeted vector could inhibit endothelial cell-mediated angiogenesis in vitro [Lumen count: Control group (13.33±0.76)/HPF, ADSCs group (19.40±1.71)/HPF, Al-ADSCs group (7.27±0.31)/HPF, P<0.01]. 【Conclusion】 In the process of constructing the adipose derived stem cell-based alphastatin peptide glioma targeting vector, the stem cell biological characteristics and tumor tendency of targeted vector have no significant changes. This targeted vector can stably express and secrete alphastatin peptide and inhibit endothelial cell-mediated angiogenesis in vitro.

Brain Tumor Heterogeneity

Tumor heterogeneity is the concept that different tumor cells provide distinct biomorphological lesions, gene expressions, proliferation, microenvironment and graduated capacity of metastatic lesions. Brain tumor heterogeneity has been recently discussed about the interesting interaction of chronic inflammation, microenvironment, epigenetics and glioma steam cells. Brain tumors remain a challenge with regards to medication and disease, due to the lack of treatment options and unsatisfactory results. These results might be the result of the brain tumor heterogeneity and its multiple resistance mechanisms to chemo and radiotherapy.

Differences of biological property between glioma stem cells and glioma non-stem cells / 中华神经医学杂志

Objective To observe the differences of biological property of glioma stem cells (GSCs) and glioma non-stem cells (nGSCs), and their related protein expressions. Methods The proliferations of GSCs1, GSCs2 and nGSCs1 and nGSCs2 were detected by CCK8 after two, 4, 6, 8, 10 and 12 d of culture in vitro. The sensitivities of the cells to temozolomide (TMZ) were detected by CCK8 after 2 d of culture. The adhesion abilities of cells were tested by adhesion assay. Transwell assay was used to detect the migration and invasion abilities of cells. The activity of matrix metalloproteinase-2 (MMP-2) was detected by gelatin zymography. Western blotting and immunofluorescence staining were used to detect the protein expressions of Notchl and epidermal growth factor receptor (EGFR). Results The survival rate of nGSCs1 was significantly higher than that of GSCs1 and the survival rate of nGSCs2 was significantly higher than that of GSCs2 after 4, 6, 8, 10 and 12 d of culture (P<0.05). The inhibitory concentration (IC)50 of TMZ for GSCs1, nGSCs1, GSCs2 and nGSCs2 was (1536.0±17.67) μmol/L, (514.5±13.44) μmol/L, (2543.0±39.87) μmol/L, (889.6±17.43) μmol/L, respectively (P<0.05). Number of GSCs1 adhering to extracellular matrix proteins Fibronectin and Collagen I was significantly larger than that of nGSCs1, and that of GSCs2 was significantly larger than that of nGSCs2 (P<0.05). The number of migrated GSCs112 and 24 h of cultivation was statistically larger than that of nGSCs1, and that of GSCs2 was statistically larger than that of nGSCs2 (P<0.05). The number of invaded GSCs124 and 36 h of cultivation was larger than that of nGSCs1, and that of invaded GSCs2 was larger than that of nGSCs2, with statistical differences (P<0.05). The activity of MMP2 secreted by GSCs1 was significantly higher than that by nGSCs1, and that of MMP2 secreted by GSCs2 was significantly higher than that by nGSCs2 (P<0.05). Western blotting showed that the relative protein expression level of EGFR/Notch1 in GSCs1 was significantly lower than that in nGSCs1, and that in GSCs2 was significantly lower than that in nGSCs2 (P<0.05). The results of immunofluorescence staining were consistent with those of Western blotting; EGFR protein strongly expressed in nGSCs and weakly expressed in GSCs; Notch1 protein strongly expressed in GSCs and weakly expressed in nGSCs. Conclusion As compared with the high-EGFR-expressing and proliferative primary glioma cells, the high-Notch1-expressing glioma stem cells have higher activity level of MMP-2,stronger abilities of adhesion, migration and invasion, which may be contributed to glioma treatment resistance and its occurrence.

The effect of FSD1 protein on the invasion of glioma stem cells / 国际药学研究杂志

Objective To investigate the effect of fibronectin type Ⅲ and SPRY domain containing 1 (FSD1) protein on the invasion of glioma stem cells (GSCs), so as to probe into the new biomarkers or potential therapeutic targets for gliomas. Methods The Cancer Genome Altas (TCGA) database data were used to analyze and compare the FSD1 gene expression (the FSD1 mRNA level) in the glioblatoma (also known as glioblastoma multiforme, GBM) and normal brain tissues as well as in the different grade glioma tissues, and the correlation of the FDS1 gene expression (mRNA level) with the survival prognosis of patients was also analyzed using the TCGA database data. The lentivirus was used to overexpress the FSD1 protein in the GSCs, T4121 and D456. The effect of the overexpressed FSD1 protein on the invasive ability of the GSCs, T4121 and D456 was evaluated by Transwell invasion assay. Results The FSD1 gene expression (mRNA level) was significantly lower in GBM than in normal brain (P＜0.01). The FSD1 gene expression (the mRNA level) in gliomas significantly decreased with the increase of the gliomas grade (gradeⅡvs Ⅲ, P＜0.05;gradeⅢvs Ⅳ, P＜0.01). The survival prognosis of patients with gilomas was well associated with the level of FSD1 gene expression (the FSD1 mRNA level), as indicated by the overall survival rate of the patients, which was significantly lower in the patients with the low FSD1 mRNA level than in the patients with the high FSD1 mRNA level (P＜0.01). In the Transwell invasion assay, the count of the invasive cell numbers significantly decreased in the FSD1 protein-overexpressed T421 and D456 groups than in the corresponding control group (P＜0.01 in both T4121 and D456 cell lines). Conclusion There is a clinical relevance of the FSD1 expression for the malignant progression of gliomas (the grade of gliomas). The low level FSD1 is favorable for keeping the invasive ability in GSCs.

Phenotype change of glioma stem cells is related with the RNA binding proteins in hypoxia / 基础医学与临床

Objective To explore the relationship between the change of phenotype of glioma stem cells and expres-sion of RNA binding proteins in hypoxia. Methods Glioma stem cells(U87MG-SLC and GSC5) were cultured un-der hypoxia (1% O2) and normoxia(20% O2). Cell proliferation was measured by MTS assay and self-renewal ability was determined by tumorsphere formation assay. The expression of RNA binding protein and stemness mark-ers protein were examined by Western blot and statistics was carried out. Results The proliferation of glioma stem cells was inhibited and the self-renewal ability was promoted in hypoxia. Meanwhile,hypoxia significantly promoted the expression of HIF-1α and stemness markers.Under hypoxia, the expression of RNA binding protein was changed. The expression of hnRNPF, UNRIP and HuD increased. Meanwhile the expression of PCBP2 and UNR was downregulated. But,other RNA binding proteins(hnRNPK,ADAR1,PCBP1,CIRP,EBP1,eEF1A,PTBP1,PTBP2) had no significant change. Conclusions The change of phenotype of glioma stem cells in hypoxia is relat-ed with the RNA binding proteins (hnRNPF,UNRIP,HuD,PCBP2 and UNR).

TGF-β2 enhances invasion ability of glioma stem cell through matrix metalloproteinase pathway / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To study the effect and possible mechanism of TGF-β2 on the invasion of glioma stem cells (GSCs). Methods: Tumor tissues of 8 patients with glioblastoma multiforme, who underwent resection at Department of Neurosurgery of the FirstAffiliated Hospital of China Medical University duringApril 2016 toApril 2017, were collected. The primary culture of glioma cells were conducted with trypsin digestion. Partial primary glioma cells were seeded into serum-free DMEM/F12 culture medium containing EGF, bFGF and B27 to obtain suspension of tumor spheres. Immunoflurenscent staining and differentiation assay were used to detect whether the tumor spheres were GSCs. TGF-β2 secretion ability of GSCs was determined by ELISAassay.After transfection of TGF-β2 siRNA, the invasion ability of glioma stem cells was determined by Transwell assay. Western blotting was used to examine the effect of TGF-β2 on expression of matrix metalloproteinases (MMP) in glioma stem cells. Results: The suspended tumor spheres were proved to be GSCs by immunofluorescent staining and differentiation assay; the tumor spheres expressed the marker of GSCs（CD133）and had the ability to multi-differentiate (glia and neuronal cells). Compared with the primary glioma cells, Glioma stem cells exerted significantly improved TGF-β2 secretion ability ([74.13±3.63] vs [46.13±2.61] pg/ml, P<0.05); and TGF-β2 silencing significantly reduced the invasion ability of glioma stem cells ([105.71±8.69] vs [63.67±5.93], P<0.05) and inhibited MMP-2 and MMP-9 expressions. Conclusion: TGF-β2 can promote the invasiveness of glioma stem cells through MMP-2 and MMP-9 pathway.

Effect of corilagin on IκB-α and nuclear factor-κB P65 in U251 glioma cells and glioma stem cells / 中华神经医学杂志

Objective To explore the effect of corilagin on proliferation of glioma U251 cells and glioma stem cells and IκB-α and nuclear factor (NF)-κB P65 protein expressions in these cells.Methods The glioma stem cells were isolated from glioma U251 cells by using immune magnetic beads.The cells were intervened by different corilagin concentrations (0,25,50 and 100 μg/mL) for 48 h,respectively.Cell morphology changes were observed by microscope;cell counting kit (CCK)-8 assay was used to detect the cell proliferation;dual-luciferase reporter assay was employed to detect the P65 gene promoter expression;Western blotting was used to investigate the protein expressions of Iκ B-α in cytoplasm and NF-κB P65 in nucleus.Results (1) Cell morphology observation results showed that the cells became shrunken,cell density was decreased,and cell structure was destroyed with a great deal of cell debris.(2) CCK-8 assay results showed that as compared with those in the 0 μg/mL corilagin group,the survival rates ofU251 glioma cells and glioma stem cells were significantly decreased in the 25,50 and 100 μg/mL corilagin groups (P＜0.05);while in the presence of the same corilagin concentration,the survival rate of U251 glioma cells was significantly higher than that of glioma stem cells (P＜0.05).(3) Dual-luciferase reporter assay results showed that as compared with the 0μg/mL group,the P65 gene promoter expressions of U251 glioma cells and glioma stem cells in the 25 μg/mL corilagin group were significantly increased (P＜0.05),but with increasing concentrations of corilagin,the expressions were gradually decreased.(4) Western blotting results showed that the IκB-α expressions in cytoplasm of U251 cells and U251 stem cells were significantly increased,but the NF-κB P65 expression in nucleus of U251 cells and U251 stem cells was significantly decreased with increasing concentrations of corilagin (0,25,50 and 100 μg/mL),with signficant differences between each two groups (P＜0.05).Conclusion Corilagin could inhibit the expression of P65 gene promoter,promote the IκB-α protein expression in cytoplasm,reduce NF-κB P65 protein into the nucleus,thereby to inhibit the NF-κB signaling pathway,and it is likely to be one of the important mechanisms to inhibit the proliferation of glioma cells and glioma stem cells.

Correlation between multi -voxel magnetic resonance spectroscopy parameter choline/creatine and distribution of glioma stem cells / 中华神经医学杂志

Objective To investigate the correlation between mutiple-voxel magnetic resonance spectroscopy (1H-MRS) parameter choline/creatine (Cho/Cr) and distribution of glioma stem cells (GSCs). Methods Sixteen patients with high-grade glioma approved by pathology, admitted to our hospital form August 2012 and March 2015, were enrolled in our study. They were performed 1H-MRS before surgery, and apparently different regions of Cho/Cr were identified. With the help of intraoperative neuronavigation, different Cho/Cr tissue samples were gained accurately (Cho/Cr hypermetabolism group and Cho/Cr hypometabolism group). The different distribution of glioma stem cells in glioma tissues of the two groups was detected via neurosphere culture; immunohistochemistry and Western blotting were employed to detect the CD133 and nestin expressions. Results Neurospheres were successfully cultured from different glioma tissues, and the sphere formation rate from Cho/Cr hypermetabolism group was significantly higher as compared with that from Cho/Cr hypometabolism group (13.94±3.55 vs. 8.04± 1.47, P<0.05). The immunohistochemistry results indicated that the expressions of CD133 and nestin in the Cho/Cr hypermetabolism group were significantly higher as compared with those in the Cho/Cr hypometabolism group ([22.96±2.28]% vs. [18.04±1.36]%, [25.47±2.43]% vs. [19.74±1.66]%, P<0.05). Western blotting showed that the relative protein expressions of CD133 and nestin in the Cho/Cr hypermetabolism group were significantly higher as compared with those in the Cho/Cr hypometabolism group (0.50±0.17 vs. 0.30±0.08, 0.45±0.13 vs. 0.27±0.07, P<0.05); and the protein expressions of CD133 and nestin were positively correlated with Cho/Cr (r=0.972, P=0.000; r=0.762, P=0.000). Conclusion 1H-MRS parameter Cho/Cr reveals the distribution differences of cancer stem cells in high-grade gliomas, which can assist in finding and resecting the glioma stem cells-rich region.

Effect of Pirin on the proliferation and self-renew of glioma stem cell / 国际药学研究杂志

Objective To study the effect of Pirin(an iron-binding nuclear protein)on the proliferation and self-renewal of glioma stem cell(GSC)so as to provide a potential therapy target for malignant glioma.Methods PLKO.1-shPirin lentiviral plasmids were constructed to stably knock down Pirin in GSC by using lentivirus infection system.The interference efficiency of Pirin short hair?pin RNA(shRNA)was detected by Western blotting. The capacity of GSC was examined by the assessment of cell viability. Tumor sphere formation assay was used to detect the effect of Pirin on GSC self-renewal capacity. Results Pirin was highly expressed in GSC.Consistently,the protein level of Pirin in the conditioned medium from GSC was much higher than that from the corresponding non-stem tumor cell(NSTC).Gene-sequencing analysis demonstrated that PLKO.1-shPirin lentiviral plasmids were successfully con?structed.Pirin shRNA transfection significantly inhibited the expression of Pirin in GSC and suppressed the cell viability and ability of tumorsphere formation.Conclusion Knocking down Pirin significantly inhibites the cell proliferation and self-renewal of GSC.

Glioma stem cell differentiation induced by micro RNA 124 overexpression / 中华神经医学杂志

Objective Glioma stem cells (GSCs) are the root causes ofglioblastoma recurrence.Glioma expresses low level of micro RNA 124 (miR-124), which plays an important role in the development of glioma.In this study, we explore the mechanisms miR-124 by overexpressing miR-124 in GSCs.Methods Human GSCs were transfected with lentivirus mediated miR-124 overexpression vector (pGC-miR-124-GSCs group), and GSCs in normal culture were used as control group.Cell proliferation was accessed by MTT assay, and stem cell phenotype was analyzed by flow cytometry.Quantitative real time PCR was employed to detect the miR-124 expression and its target genes (A kt and RelA) expressions;and ELISA was carried out to detect the secretions of downstream inflammatory cytokines interleukin (IL)-1 and IL-8.Results The lentiviral miR-124 expression vector was constructed successfully and transfected into human GSCs (pGC-miR-124-GSCs).The proliferative capacity of cells in the pGC-miR-124-GSCs group was significantly lower than that in the control cells (P＜0.05).CD133 positive rate was statistically decreased, the miR-124 expression was significantly increased, the A kt and RelA expressions were significantly decreased, and correspondingly the secretions of IL-1and IL-8 were significantly reduced in the pGC-miR-124-GSCs group as compared with those in the control group (P＜0.05).Conclusion MiR-124 overexpression induces GSCs differentiation and activates a strong anti-tumor ability, whose mechanism may be related to inhibition of inflammatory cytokine production.

Proliferation and apoptosis of glioma stem cells after Gamma knife treatment / 中华神经医学杂志

Objective To research the proliferation and apoptosis of glioma stem cells after Gamma knife treatment.Methods The glioma stem cells were cultured in serum-free suspension; real time-PCR was used to detect the CD133 expression; fluorescence staining was employed to observe the expressions of nestin,glial fibrillary acidic protein (GFAP) and [3-tubulin after stem cell differentiation.After 10 Gy Gamma knife treatment for 48 h,the glioma U87 cell and stem cell survival was detected under microscope; after 15 Gy Gamma knife treatments for 8-10 h,immunofluorescent staining was performed to detect the 5-bromodeoxyuridine (BrdU)-positive cells; flow cytometry was employed to compare the changes of cell apoptosis before and after 10 and 15 Gy Gamma knife treatment.Results Under the culture conditions of serum-free medium,glioma stem cells became spherical suspended growth and had proliferation and self-renewal capacity,expressing CD133 and nestin,and containing the ability to differentiate into astrocytes and neural elements.After 10 Gy gamma knife treatments,the cell survival in the glioma stem cells was significantly higher than that in the U87 cells (86±3 vs.22±2,P＜0.05).About 35％ glioma stem cells showed positive BrdU staining before 15 Gy gamma knife treatment and it was about 22％ after treatment with statistical difference (P＜0.05).The apoptosis rate in the glioma stem cells was low; that in cells with 15 Gy gamma knife treatment (0.312±0.011) was significantly higher than that in cells with 10 Gy gamma knife treatment (0.112±0.014,P＜0.05).Conclusions Under the culture conditions of serum-free medium,glioma stem cells can be derived from human glioma tissue.Treatment of gamma knife could inhibit the proliferation of glioma stem cells,causing their apoptosis.As compared with glioma cells,glioma stem cells are not sensitive to gamma knife radiotherapy and radio-resistant.

Different Nrf2 expressions in glioblastoma cell lines and glioma stem cells from xenografts / 实用医学杂志

Objective To compare different Nrf2 expressions in glioblastoma cell lines and glioma stem cells (GSCs) from xenografts and to study the concentration of Nrf2 in nuclear. Methods GSCs were analyzed by immunofluorescence and different expressions of Nrf2 in glioblastoma cell lines and GSCs from xenografts were detected with real-time RCR and Western. Results GSCs were successfully isolated from xenografts of U251 and U87 cell lines. The percentage of tumor stem cells in total cells was 1.24%, and that was 1.63% in xenografts. Immunofluorescence indicated that Nrf2 was overexpressed in GSCs as compared with that in glioblastoma cell lines. Conclusion Nrf2 may be a potential biomarker and rational therapeutic target for GSCs.

Effect of histone deacetylase inhibitor apicidin on biological activity of glioma stem cells via down-regulating Nanog expression / 中华神经医学杂志

Objective To investigate the effect ofhistone deacetylase inhibitor apicidin induced Nanog repression on proliferation,migration and invasion of glioma stem cells (GSCs).Methods GSCs were isolated from glioma cell line U87 and cultured in simplified serum-free neural stem cell medium by nanosphere suspension culture method,and purified continuously through the monoclonal formation experiment.The immunofluorescence staining of cells was employed to detect the CD133 and Nestin expressions to identify GSCs.Apicidin treatment group (GSCs treated with 0.5 μmol/L apicidin for 48 h) and blank control group were employed.Nanog mRNA and protein expressions were detected by real time-PCR and Western blotting,respectively.Double immunofluorescence staining was used to detect the co-expressions ofNanog/CD133.MTT assay was used to detect the proliferation modification of GSCs,while migration and invasion of GSCs were detected by Transwell migration and invasion assay after 0.2,0.5,1.0 and 2.0 μg/mL apicidin of treatment.Results GSCs were isolated,cultured and purified from glioblastoma cell line U87; as compared with those in the blank control group,the mRNA and protein expressions of Nanog were significantly repressed and Nanog+,CD133+ and Nanog+/CD133+ cells were obviously reduced in the apicidin treatment group (P＜0.05).The migration and invasion in the apicidin treatment group were inhibited dramatically as compared with those in the blank control group (cell number of migration:[87.50±4.65]/field vs.[128.50±6.14]/field; cell number of transmembrane:[55.75±4.79]/field vs.[81.50±5.45]/field,P＜0.05).MTT assay indicated that the absorbance value of the apicidin treatment group was significantly lower as compared with that in the blank control group (P＜0.05); the higher the apicidin concentration,the lower the absorbance value and the more obvious the proliferation inhibition.Conclusion Histone deacetylase inhibitor apicidin could inhibit the mRNA and protein expressions of Nanog,and sequentially repress the proliferation,migration and invasion of GSCs.

Transforming growth factor β2 mRNA expression in human brain glioma stem cells and its significance / 中华神经医学杂志

Objective To determine the mRNA expression of transforming growth factor β2 (TGF-β2) in human brain glioma stem cells (BGSCs),and investigate its effect onimmunotherapy of glioma. Methods BGSCs were grown from 8 specimens resected from glioma from October 2008 to January 2009,then,they were isolated and identified.The expressions ofCD133,GFAP and TU-20 in the tumor cell spheres were detected by immunofluorescence staining; the TGF-β2 mRNA expression in BGSCs was detected by RT-PCR,and then,this result was compared with that of respective differentiated glioma cells. Results Positive expression of CD133 was noted in the tumor cell spheres,and GFAP and TU-20 could be expressed in the tumor cell spheres. A quantitative determination exhibited higher TGF-β2 mRNA expression in BGSCs than that in their counterparts of primary cultured glioma cells,which was statistically significant (t=3.619,P=0.009). Conclusion High TGF-β2 mRNA expression is noted in BGSCs,which might be the main producer of overproduction of TGF-β2 in gliomas leading to immune escape and resistance to current therapies.

Effect of all-trans retinoic acid on expressions of vascular endothelial growth factor and basic fibroblast growth factor in glioma stem cells / 中华神经医学杂志

Objective To investigate the effect of all-trans retinoic acid (ATRA) on expressions of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in glioma stem cells (GSCs). Methods GSCs were isolated from human glioblastoma cell line U87 and identified by detecting the expressions of CD133 and nestin with immunofluorescence staining. The obtained GSCs were divided into control group,empty vector group (cultured with dimethyl sulfoxide [DMSO]) and ATRA treatment group (cultured with 10 nmool/L ATRA).After 10 d of differentiation; the proliferation of the treated GSCs was evaluated using CCK8 assay; the expressions of glial fibrillary acidic protein (GFAP),β-tubulin Ⅲ and galactoeerebroside (GralC) in the cells were detected by immunofluorescence.VEGF and bFGF levels in cultured supernatant were measured by ELISA; the mRNA expressions of VEGF and bFGF were detected by RT-PCR. Results The target antibodies of neural stem cells (NSCs), CD133 and nestin,positively expressed in the GSCs; differentiated GSCs can differentiate several kinds of homologous daughter cells,which expressed the cell markers of astrocytes,neurons and oligodendrocytes: GFAP, β-tubulin LⅢ and GalC, respectively. The percentage of GFAP-positive differentiated GSC s in the ATRA treatment group was significantly higher as compared with that in the other 2 groups after 10 d of differentiation (P＜0.05); the speed of proliferation of GSCs in ATRA treatment group was obviously slower than that in the other 2 groups 3-7 d after differentiation (P＜0.05).The VEGF and bFGF levels and the mRNA expression levels of VEGF and bFGF in GSCs of the ATRA treatment group 24 h after differentiation were also significantly lower than those in the other 2 groups (P＜0.05). Conclusion ATRA can induce the differentiation of GSCs and inhibit its proliferation.It may exerts its anti-glioblastoma effect through the VEGF and bFGF signaling pathways.

Labeling of stable glioma stem cells with red fluorescent protein transfected by lentiviral vector / 中华神经医学杂志

[Objective]To establish stable glioma stem cells with a high expression level ofred fluorescent protein (RFP) in vitro.[Methods]The glioma stem cells SU2 were transfected with lentivirus vector containing RFP gene;cell expression of RFP was observed by fluorescent microscopy;RFP-positive glioma stem cells were sorted out by fluorescence-activated cell sort.The transfection efficiency of SU2 cell before transfection and 20-passaged RFP-SU2 cells after transfection were assayed by flow cytometry.Dynamic viewing was performed to observe the cell division and cloning of RFP-SU2 single cell;RFP -positive cells were collected and immunostained with antibodies against CD133 and nestin.[Results] PFP-SU2 cells grew as levitated sphere with high RFP expression;the transfection efficiency of SU2 cell before transfection was only 1.5％,while that of20-passaged RFP-SU2 cells after transfection reached to 75％.RFP-SU2 single cell could proliferate into brain tumor stem cell spheres,having the abilities of self-renewing and clonal proliferation.Immunofluorescence showed positiveCDt 33 and nestin expressions in RFP-SU2 cells.[Conclusion] A SU2/RFP cell line marked by RFP is established;and it can serve as a promising tool for further basic research.

Expression and significance of ABCG2 in U251cells and U251 stem cells / 重庆医科大学学报

Objective:To investigate the expression and significance of ABCG2(the G2 member of ATP-binding cassette family)in U251and its stem cell.Methods:The U251 were cultured in serum-free medium with EGF,bFGF,LIF and B27.The tumor spheres formed and cultured continuously in the serum-free medium through the monoclonal formation experiment.The immunofluorescence staining was employed to identify the U251 stem cell.The expression of ABCG2 was detected by confocal microscopy and Western blot.Results:The stem cells from malignant glioma cell line U251 have been successfully isolated.The cells expressed CD 133.The ABCG2 protein positive stain was located in the cell membrane in both U251 cells and its stem cell with confocal microscopy method.Western blotting showABCG2 was expressed in U251 cells and in its stem cells.The expression level in the stem cells was significantly higher than U251 cells.Conclusion:ABCG2 is expressed in U251 cells and in U251 stem cells.The expression level in the latter was higher.