Experimental Study Of The Killing Effect On Human Brain Glioma Stem Cells By Brachytherapy With ～(125)I Seeds

Objective To evaluate the cytocidal effect of ¹²⁵I seeds brachytherapy on human glioma stem cell, approach the mechanism of action and radiation dose. Compare to neural stem cells, observe the sensible difference of ¹²⁵I seeds brachytherapy. It will provide the experiment rationale for the therapy of gliomas, meanwhile so dose the occurrence mechanism of the gliomas. It can provide experiment rationale for the discrimination between gliomas stem cells and neural stem ceils.Methods The tumor from patients undergoing tumor resections were acutely dissociated, triturate into single cell and then filter, the cells were seeded into the serum-free medium, make the gliomas stem cells of gliomas cells proliferating. We perform immunocytochemistry of gliomas stem cell for CD133 express, then calculate the proportion of CD133 positive cells. Use a part of gilomas stem cells serial subcultivation, after 3-5 days there are a lot of cell sphere forming. Make single cell suspension which density is 13×104. Separate the cells into 4 groups and culture in 96 shadow mask. Each group set three time stage(20,40,60hours) according to irradiation dose, each stage have four hole, each hole has 200μl. The number of cells are 2.6×104. Use three of four holes to calculate the dead cells and the one hole for growth inhibiting. Meanwhile take neural stem cells (provided by kunming medical college institute of neuroscience) to culture 3-5 days, after forming cell sphere, make single cell suspension. Setting control group in the same dose as gliomas stem cells. Then use different dose of ¹²⁵I seeds in plate and culture together for 20,40, 60 hours. Count the CD 133 positive cells and observe the change of cells; determine the growth inhibiting of the glioma stem cells by brachytherapy of ¹²⁵I seeds by the measure of cell clone formation., the same time, compare to neural stem cells, observe the sensibility difference by brachytherapy of ¹²⁵I seeds.Results The dead cells in radiation group are obviously higher than in the control group. So does growth inhibition. The proportion of gliomas stem cell(CD133 positive) is 0.25,0.14,0.10,0.04 in different irradiation dose. The radiation group compare to the control group, P<0.05, which is statistics significance. In the radiation group 20 hours compare to 40hours, which isn't statistics significance; 20 hours compare to 60 hours, P<0.05. which is statistics significance. The growth curve of clone inhibition in radiation group is obviously lower than in the control group, the growth fraction in different time stage is 1,0.75,0.52,0.25.the survival curve of gliomas stem cells is a straight line, linear regression equation is Y=40.979-38.205X, equations matching grade is F=51.713,P<0.001,which indicate that the survival rate of gliomas stem cells have regression relationship with the radiation activity of ¹²⁵I seeds, furthermore, there are line negative correlation between the two ones(r=-0.981,P<0.05); compare to neural stem cells, brain tumor stem cells have more sensible and are statistics significance.Conclusion1. The result of experiment indicate we can culture gliomas stem cell from the human medulloblastoma, which is correspond with the theory of tumor stem cell.2. we prove that CD133, which is specificness marker of nerve stem cell, can mark gliomas stem cell.3. Use ¹²⁵I seeds to irradiate gliomas stem cell in a short distance. The result indicate that the dead rate of gliomas stem cells is very obviously difference with different radiation dose and time.4. Use ¹²⁵I seeds to irradiate nerve stem cells and gliomas stem cells. The result indicate that in the same radiation dose, gilomas stem cell is more sensitive than nerve stem cell. The difference of dead cell is obviously significance, which can provid some evidence for the identification of the two kinds of cell. 5. the result indicate in 30Gy the dead rate of gliomas stem cell is the highest in all radiation dose, the cytocidal effect of ¹²⁵I seeds brachytherapy is significance, however, in vitro and in vivo there are a lot of influencing factor, thus a lot of things still are to do. In a shot, brachytherapy of ¹²⁵I seeds can kill the glioma stem cell and inhibit the proliferation in two groups, it should play an important role in the therapy of gliomas and have great perspective.