| IntroductionMeningiomas are extra-axial brain tumors and represent 18-20% of all intracranial tumors. Overall, 90% of meningiomas are benign, 6% atypical, and 2% are malignant. Meningiomas, which invade intracranial bone structures and the adjacent connective tissue, are frequently unresectable because of their aggressive and recalcitrant growth behavior. Complete surgical resection is usually curative. For incompletely resected or recurrent tumors, radiotherapy or chemotherapy is administered. Meningiomas have a high recurrence rate, hi addition, many tumors are not amenable to surgery due to their deep location or proximity to delicate structures. Meningiomas in eloquent or surgically inaccessible locations is difficult to radical resect. Radiotherapy may be administered as either conventional external beam irradiation or stereotactically. Stereotactic radiotherapy (SRT) either as LINAC or gamma knife radiosurgery is increasingly utilized. When the meningioma is unresectable or all other treatments (surgery, radiotherapy) have failed, immunochemotherapy may be considered Alpha interferon, tamoxifen, and mifepristone (RU-486) have been modestly successful in patients with recurrent meningiomas whereas cyclophosphamide, Adriamycin and vincnstine(CAV), ifosfamide/Mesna or Adriamycin/dacarbazine (DTIC) have been administered to patients with aggressive or malignant meningiomas. Chemotherapy is being explored as another potential treatment option for unresectable or refractory meningiomas Hydroxyurea is an agent that inhibits ribonucleotide reductase and can induce apoptosis in memngioma cell cultures and animal models. Schrell M.D. and accompaniers treat meningiomas with hydroxyurea taking into account that meningiomas are slowly growing tumors with a low mitotic index. Unfortunately, there is no advanced investigation inland being reported.Objective: To investigate a method to construct the model of meningiomas in vitro To determine the tumor-suppression effect of hydroxyurea and it's possible mechanism.Materials and Methods:1. Acquiring of meningioma cellFresh meningioma tissue of 23 patients was obtained during surgery. Tumor tissue was divided and used for cell culture and routine pathological study. Separating meningioma tissue into cells with mechanical methods.2.Cell cultureThe meningioma cells were cultured with RPMI 1640 as the culture medium, substituted with 10% vol/vol fatal bovine serum(FBS),100U/ml penicillin, and lOOug/ml streptomycin. The cells were grown in a nonhumidified incubator at 37癈 with 5% CO2 for several days, with periodic medium changes. Once the cells reached confluence, they were removed by trypsin (0.25%). Growing for 48 hours, the culture medium was completely removed fresh growth medium containing varying doses of hydroxyurea(5x 10"3M> 5x 1O^M^ 5><10" M )was added. The experiments were stopped when cells in the control cultures were almost confluent.3. routine pathological and immunohistochemical studiesTumor tissue was send for routine pathological and immunohistochemical studies(S100 ^ EMA -. Vimentin).The primary culture cells were also send for immunohistochemical studies(S 100 > EMA^ Vimentin).4.Cell morphologyWe can observe the cells of different phases and all groups with invert microscope.5.Cell countCultures were stopped when cells in the control cultures were almost confluent On the final day of culture ,the number of cells was determined by haemacytometer.6.Detection of apoptosis with flow cytometry(FCM)Culture the cells in 24 well-plates with 500ul medium/well for 7~9 days. Add 5ul of diluted annexin V FITC, Incubate the cells for at least 10 minutes(0~37癈).Remove the supernatant containing the cetached cells from the well Wash the cells twice with DMEM Rinse the adherent cells and add PI solution to the cell sample. Incubate for 15 minutes on ice before flow cytometric analysis.7.MTTCulture the cells in 96 well-plates for 7-9 days. Add MTT solution 20ul per well and incubate the cells for at least 4 hours(37...
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