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Part I: malignant glioma tumor fractionated irradiation of subclinical Optimization Experimental Study Objective: To compare different programs fractionated irradiation after irradiation of subclinical malignant glioma tumor formation rate differences in tumor growth and tumor screening for malignant reasonable subclinical glioma tumor fractionated irradiation optimization program, to better carry out clinical glioma measured postoperative radiotherapy provide experimental basis. MATERIALS AND METHODS: The human brain glioblastoma subclinical tumors in nude mice as experimental models for different partitioning scheme irradiation (conventional fractionated irradiation program 2Gy / d × 5 / w; unconventional fractionated irradiation scheme: 1.6Gy × 2 / dx5 / w, 4Gy × 3 / w, 3Gy / d × 5 / w, total doses of 40Gy and 60Gy) and targeted drug Taixinsheng (0.5mg / only / week, 7 weeks) combined irradiation (conventional split, total dose 60Gy). OUTCOME MEASURES: 1) the end of treatment irradiated tumor formation rate, 2) the average time to recurrence of tumor, 3) the final rate of tumor formation (18-24 weeks end of the experiment). Results: (1) the number of inoculated cells 2.2 × 105, the control tumor formation rate was 87.5% (7/8) in the case, when the total dose of 40Gy irradiation split ends the routine group (5 weeks), 1.6Gy × 2 / d × 5 / w group (4 weeks) and 4Gy × 3 / w groups (4 weeks) tumor formation rate was 75%, 25% and 0, tumor recurrence were 96 days, 109 days and 102 days, until the end of observation (18 weeks) tumors into three groups of tumor rates were 100%, 100% and 100%, regardless of the segmentation scheme a total dose of 40Gy irradiation have failed to effectively control subclinical tumor continue to grow; (2) inoculation was 3.1 × 105 cells in the control was 100% tumor formation case, the total radiation dose of 60Gy group at the end of normal division (week 7), 1.6Gy × 2 / d × 5 / w group (5 weeks), 4Gy × 3 / w group (6 weeks) and 3Gy × 5 / w group (5 weeks) of tumor formation rate was 100%, 0,0 and 0, respectively, tumor recurrence 103 days, 131 days, 110 days and 124 days, at the end of the observation (24 weeks) of the tumor formation rates were 100%, 75%, 100% and 100%; (3) Taixinsheng alone administration group ( 0.5mg / only / week and 1mg / only / week), irradiation group (conventional fractionated radiotherapy, total dose 60Gy) and put the end of the chemotherapy group irradiated tumor formation rate in each group were 100%, 100%, 100% and 75%, the mean tumor recurrence were: 31 days, 46 days, 103 days and 97 days. To observe the end (week 24) of each tumor formation rate was 100%. Conclusions: (1) on the short-term effect, in the blank control tumor formation was 87.5% of the conditions, 40Gy irradiation total dose to achieve a certain recent subclinical tumor control rate, of which split group (4Gy × 3 / w) the best, but long-term effects are not ideal. Experimental results show that the end of the observation time (18 weeks) in each experimental group of tumor formation rate was 100%, indicating that the total dose of 40Gy can only delay the time of tumor recurrence, but did not obtain the effect of cure, so the control of tumor growth purposes, 40Gy total dose is not enough. (2) The total dose up to 60Gy, glioma forward control have some increase. 1.6Gy × 2 / d × 5 / w group forward control rate was 25%. (3) the total radiation dose the same circumstances, targeted drug Taixinsheng conventional fractionated irradiation combined short-term effect is better than pure and simple administration of conventional fractionated radiotherapy [end of treatment (Week 7) into the tumor were Table Mingtai Xin Sang United conventional fractionated radiation therapy glioma ineffective, may be negative for EGFR expression. Part II: Determination of stem cell markers in vitro brain glioblastoma (BT325) cell line stem cells preliminary study Objective: To identify the stem cells with the classic method - cell colony formation analysis comparing the results to explore the use of modern molecular biology Determination of stem cell markers learning glioma cell population number of stem cells is feasible. Materials and Methods: The experiment exponential phase of human brain glioblastoma cell line BT325 (Beijing Neurosurgical Institute established), with the classic single-cell colony formation analysis determination of cell colony formation rate; using modern molecular biology stem cell markers technology (flow cytometry and immunohistochemistry) Determination BT325 cells stem cell markers (CD133)-positive cells were labeled rate and xenografts in nude mice tissue stem cell markers (CD133, Nestin) and classic and modern expression of stem cell The measurement results of identification techniques were analyzed and compared. Results: The experimental results showed that: (1) When the reaction system CD133 antibody levels were 0.45μg/100μl, 0.9μg/100μl, 1.8μg/200μl when the human brain glioblastoma cell line BT325 CD133 positive cells were 0.62 %, 2.55%, 0.067%, PBS group was 0.1%, 0.1%, and 0%, PE group was 0.95%, 0.3% and 0.1%, the difference between the groups was not significant (P = 0.311 and P = 0.473); (2) Immunohistochemical analysis showed BT325 xenografts in nude mice tissues was no expression of CD133, a little expression of nestin-positive cells; (3) and stem cell molecular markers simultaneously measured single-cell colony formation analysis of measurement results Show cultured human brain glioblastoma cells (BT325) colony formation was 39.25% ± 11.35. Conclusion: simultaneous expression of molecular markers and single-cell colony formation was measured analysis of experimental results show that (1) the human brain glioblastoma cell line BT325 stem cell markers CD133 expression was negative, a few cells were Nestin positive; (2) with the classical clonogenic analysis compared to modern stem cell identification techniques have failed to effectively identify the clonogenic activity of glioma stem cells.
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