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Effects of All-trans Retinoic Acid on Glioma Stem Cells and It’s Genetic Regulatory Mechanism

Author: LiuZuoJing
Tutor: KeYiZuo
School: Southern Medical University,
Course: Neurosurgery
Keywords: All-trans retinoic acid Glioma stem cells Differentiation Vascularendothelial growth factor Basic Fibroblast growth factor
CLC: R739.41
Type: Master's thesis
Year: 2012
Downloads: 56
Quote: 0
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Abstract


Glioma is a most common entity of the tumors within the central nervous system (CNS). Among various histological types of CNS cancer, malignant glioma from astrocytic origin is characterized by their rapid proliferation, high neovascularization and great invasiveness. Despite advances in surgical techniques and adjuvant radiation and chemocherapies, the prognosis for patients with gliomas remains poor, with a median survival of14.6months. Recently, there is accumulating evidence that malignant solid tumors may contain their own stem cells, termed cancer stem cells(CSCs). Despite their small quantity, this subpopulation within tumor possesses the ability of infinite proliferation and multi-potency thus may play a crucial role in the initiation, progression and recurrence of cancer. Recent advances propose the existence of a small fraction of cells with features of primitive neural progenitor cells and tumor-initiating function in brain tumors. These cells, known as tumor-initiating cells with stem-like properties, are a small subset of cancer cells within a cancer that constitute a reservoir of self-sustaining cells with the exclusive ability to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumor. Glioma stem cells (GSCs) characteristically express ABC transporters (ATP-binding cassette), which can not only transport a variety of drug out of the cells, so as to produce resistant to chemotherapy; but also has a strong ability to repair DNA damage, resistance to radiotherapy. Glioma may give rise from GSCs, which maintain the growth of glioma cells and play a critical role in the recurrence of glioma.Recent studies have found that the secretion of vascular endothelial growth factor (VEGF) by GSCs more than the differentiation type of glioma cells, the GSCs’s ability of motion and invasion also higher than the differentiated cells. GSCs, which secrete large amounts of VEGF to promote tumor angiogenesis, play a critical role in tumor growth and invasion. VEGF are essential factors of angiogenesis in vivo, which is a highly specific and selective vascular endothelial cell mitogen-induced endothelial cell proliferation and migration, inhibit endothelial cell apoptosis in vitro, promote angiogenesis and increase vascular permeability. Basic fibroblast growth factor (bFGF) is a146-amino acid polypeptide heparin-binding properties, a potent broad-spectrum mitogen can stimulate endothelial cell division and proliferation, induce or promote the formation of capillaries, not only can directly activate endothelial cell proliferation and migration, but also indirectly promote angiogenesis by promoting the secretion of VEGF to promote vascular proliferation and extracellular matrix deposition. VEGF and bFGF is the two important angiogenesis regulatory factors in promote endothelial cell proliferation, it play a role in tumor cells and endothelial cells around the tumor efficacy, they are time-correlated and concentration-correlated.The current strategies (including surgery, radiotherapy and chemotherapy) difficult to cue glioma due to the survival of GSCs. GSCs can differentiate into a variety of progeny cells (including astrocytes, neurons and oligodendrocyte) in the induction of the differentiation conditions. So first, induced the GSCs within the glioma tissue to differentiate, then treated with conventional methods may be one of the effective way.All-trans-retinoic acid (ATRA) is one of the most in-depth study, the strongest effect induction differentiation agent. Whether some of the signaling pathways associated with ATRA-induced (such as VEGF or bFGF) is also associated with the development of glioma? How about ATRA in the role of these signaling pathways in glioma development process like? It is not yet known.Thus, our study takes he VEGF and bFGF gene as key point to explore the ability of ATRA on human GSCs to promote differentiation, and the expression of VEGF and bFGF gene in order to explore new avenues for the treatment of glioma at the point of view of the way from molecular signal.In order to further understand the effect and mechanism of ATRA in promoting differentiation and inhibition proliferation on GSCs, the cells with stem cell properties within U87were isolated in the first place, culture and identification; Then, we studied the role of ATRA on promote GSCs cell proliferation and the expression of VEGF and bFGF. The main results and conclusions are as follows:Chapter Ⅰ Isolation, culture and identification of glioma stem cells from human malignant glioma cell line U87Objective:To isolate, culture glioma stem cells from human malignant glioma cell line U87,. The identity of glioma stem cells was confirmed by their morphology, growth behavior, and expression of specific markers.Methods:Human malignant glioma cell line U87were cultured in serum-containing medium (DMEM/F12+10%FBS) and serum-free medium (DMEM/F12+20ng/mL bFGF+20ng/mL EGF). Floating U87tumor spheres were formed in serum-free medium. The morphology was observed by optic microscope, the immunophenotype and the ability for differentiation were tested by Immunocytochemistry.Results:Human malignant glioma cell line U87were cultured in serum-containing medium as adherent cells, they grow processes just like fibroblast, showing short spindle, long spindle-shape, star-shape and polygon-shape. U87tumor spheres were formed in serum-free medium, these U87tumor spheres could be digested by0.01%and passaged for at least1month. Immunofluorescence staining showed that human malignant glioma cell lines U87contains the CD133+cells, neurosphere formed by the CD133+cells express neural stem cell markers CD133and Nestin, did not express the differentiated marker of astrocyte GFAP; CD133+cells can differentiated into anchorage cells after cultured in serum-containing medium, which is similar to the parental U87cells in morphology; Fluorescence staining of the differentiated progeny of CD133+tumor sphere cells showed that most of the differentiated cells were GFAP positive, a small number of cells were β-tubulin Ⅲ positive or Galc positive.Conclusion:Glioma stem cells, which possessed the ability of stem cells for self-renew, infinite proliferation and multi-potency differentiation, existed in human malignant glioma cell line U87. Glioma stem cells were enriched in tumor spheres by neurosphere assay.Chapter Ⅱ Effect of all-trans retinoic acid on proliferation and expressions of VEGF and bFGF in Glioma stem cellsObjective:To investigate the effect of all-trans retinoic acid (ATRA) on proliferation and expressions of VEGF and bFGF in Glioma stem cells (GSCs)Methods:GSCs were isolated and identified from human malignant glioma cell line U87. The GSCs obtained were divided into control group, empty vector group, and ATRA group with corresponding treatments. The proliferation of the treated GSC was evaluated using CCK8assay. The expression of glial fibrillary acidic protein(GFAP) in the cells were detected by immunofluorescence after10days of differentiation. VEGF and bFGF level in cultured supernatant was measured by ELISA. Expressions of VEGF, bFGF mRNA were detected by RT-PCR. Statistical analyses were performed with SPSS13.0statistical package, one-way ANOVA were used. P values of<0.05were considered to be statistically significant.Results:Immunofluorescence staining showed that ATRA can significantly increase the percentage of GFAP positive in their progeny (P<0.01); CCK-8results showed that proliferation was unconspicuous in cells cultured for1-2days, the peak period of proliferation is the3-7th days; There is no significant difference between the control group and empty vector group, though cells proliferation rate in both groups are higher. The proliferation rate of the ATRA group significantly decreased compared with the previous two groups (P<0.05or P<0.01). ELISA showed that the VEGF and bFGF of GSCs in the ATRA group were significantly decreased (P<0.05or P<0.01); The expression of VEGF mRNA and bFGF mRNA in the ATRA group were significantly decreased (P<0.05or P<0.01)Conclusion:ATRA can significantly promote the GSCs differentiation, increase the percentage of GFAP positive in their progeny, and inhibit proliferation. ATRA can decrease the secretion and expression of VEGF and bFGF from GSCs at genetic level. Inducing differentiation can be used as a new target for the treatment of malignant glioma.

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CLC: > Medicine, health > Oncology > Nervous system tumors > Intracranial tumors and brain tumors
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