[Adherent culture of CD133+ cells in glioblastomas and expression of ADLH1].

OBJECTIVE: To investigate the influence of adherent culture on the acquisition of CD133+ cells in glioblastomas and the expressions of acetaldehyde dehydrogenase 1 (ALDH1) in the undifferentiated and differentiated cells. METHODS: Adherent culture was performed with immunomagnetic bead technique in 7 glioblastoma samples to gain CD133+ cells. The cell differentiation was induced via serum medium culture. Immunocytochemistry staining was used to detect the expressions of ALDH1 and Sox2 in the undifferentiated and differentiated cells. RESULTS: CD133+ cells were obtained in 6 samples and typical tumor spheres were observed. Positive expressions of ALDH1 and Sox2 were detected in the undifferentiated cells and co-localization was found. No expressions of those two markers in the differentiated cells were observed. CONCLUSION: CD133+ cells can be efficiently obtained via adherent culture method. ALDH1 expression appears only in the undifferentiated cells, which can be a new marker for glioma stem cells.

[The CD133 polyclonal antibody generation and cancer stem cells identification].

OBJECTIVE: To generate the cancer stem cells (CSCs) specific protein CD133 polyclonal antibody for the study of the biological characteristics of CSCs in tumor tissues and CSCs screening for the mouse model. METHODS: The extracellular peptide of the human CD133 was injected into rabbits to generate polyclonal antibody which was used for glioblastoma(GBM) Western blot and immunohistochemistry. RESULTS: The CD133 antiserum we made could detect both overexpressed myc-CD133 and endogenous CD133 efficiently by Western blot. Immunohistochemistry indicated that the CD133 polyclonal antibody can label CSCs in GBM sections. CONCLUSION: High efficient and specific CD133 antibody was generated successfully and could be used to label CSCs in tumor sections and screen CSCs for the mouse model.

[AQP4 expression in the brains of patients with glioblastoma and its association with brain edema].

OBJECTIVE: To investigate the expression of aquaporin-4 (AQP4) in the brains of patients with glioblastoma and its association with brain edema. METHODS: Immunofluorescence cytochemistry and western blot tests were performed to detect the expression of AQP4 in the brain tumors and the adjacent tissues in 30 patients with glioblastoma. The association between AQP4 and the extent of brain edema was analysed. RESULTS: The AQP4 immunoreactive cells were mainly astrocytes in the brains, which were extensively distributed in the intracytoplasm. Higher expressions of AQP4 were found in the brain tumors and adjacent tissues in the patients with glioblastoma than in the normal controls (P<0.05). More AQP4 were distributed in the tumor adjacent tissues than in the tumors (P<0.05). The AQP4 was positively correlated with the extent of brain edema. CONCLUSION: AQP4 overexpress in the brain tumors and adjacent tissues, which is associated with the extent of brain edema. Cytotoxic and vasogenic edemas may coexist in the cerebral edema induced by glioblastoma.

[Preparation and identification of the Syntenin1 polyclonal antibody, a cancer metastasis related gene].

OBJECTIVE: To express the GST fusion protein, GST-Syntenin1 in E. coli, and to prepare the polyclonal antibody of Syntenin1. METHODS: CDS fragment of Syntenin1 was obtained by RT-PCR from normal mouse brain and subcloned into pGEX-4T-2 to generate pGEX-4T-2-Syntenin1 recombinant. The confirmed recombinant was transformed into the BL21 competent cells and induced with IPTG. The recombinant fusion protein was purified with immobilized Glutathione Sepharose and confirmed by SDS-PAGE. The purified fusion protein was mixed with the Freund's adjuvant, and then injected into New Zealand white rabbits by hypodermic injection. The polyclonal antibody titer and specification were identified by Western blot. RESULTS: Syntenin1 polyclonal antibody bind Sytenin1 protein specifically and the antiserum tiger reached to 1 : 20 000. CONCLUSION: The Syntenin1 polyclonal antibody with high titer and high specificity was prepared successfully. This will be very helpful for the further study on Syntenin1 function and molecule mechanism of cancer metastasis.

[Methylation and expression analysis of p16(INK4a) and RB genes in meningiomas].

OBJECTIVE: To investigate the methylation of p16(INK4a) and RB gene, and the expression of p16(INK4a) in meningiomas. METHODS: Methylation-specific polymerase chain reaction (MSP) was used to detect the methylation of p16(INK4a) and RB in 50 cases of meningiomas, and immunostaining was performed to analyze the protein expression of p16(INK4a) in 25 of those cases. RESULTS: No methylation was found in the benign meningiomas, whereas methylation of p16(INK4a)or RB occurred in 6(37.5%) cases of grade II tumors and 4(28.6%) cases of grade III tumors, and among these cases, an atypical meningioma showed methylation of both genes. Thirteen cases showed p16(INK4a) positive expression, but none of them was methylated. CONCLUSION: The methylation of p16(INK4a) or RB is related with the tumorigenesis and progression of atypical and anaplastic meningiomas, and a probable mechanism is that methylation causes the loss of expression and leads to dysfuncation of the p16(INK4a)/cyclin D1/CDK4/RB pathway.