| ObjectiveIsolated and cultured U251glioma stem cells, and the application of ordinary U251glioma cells and U251glioma stem cells were transplanted mice, comparing the two cell lines tumor formation rate; Immune radiotherapy technology based on application-CD133antibody labeled Na131I. CD133antigen targeting, targeted radiotherapy for malignant glioma in vivo research objective experimental basis for further clinical treatment of malignant glioma.Methods1、Cultured normal U251cells, EGF, bFGF, LIF, B27factor under serum-free conditions, obtain U251stem cells.2、Concentration of3.5×107/mL were prepared U251and U251stem cell suspension, And BALB/c Nude mice were planted in the left and right hips subcutaneous, Observe and measure the volume of the tumor-bearing, Two cell seeding rate of tumor formation in the skin as well as the growth rate. By HE staining,immunohistochemical CD133factor, Comparing the two cell lines into the pathology of tumor morphology and express CD133antigen differences.3. use the NBS labeling method, Take200ul containing the400ug CD133antibody join lm10.1M pH7.4phosphate buffer solution and mix, thereto was added at a concentration10GBq/ml1.4ml Na131I solution, and human blood albumin reaction was terminated, Final purification, filtered, and131I-CD133antibody conjugates.4、131I-CD133antibody conjugates with marked good(Group A)、No mark of Na131I(Group B)、And saline(Group C), Is divided into three experimental groups, Tumor-bearing mice were injected through a vein.5、Each experimental group were injection24h,48h,72h propofol after phenol anesthetized by intraperitoneal injection dual-head coincidence SPECT coincidence the iodine tracking imaging and CT scans, the active iodine tracking imaging.6、At the same time Each group at24h,72h3collection lung, liver, kidney, spleen, brain and tumor tissue were killced,48h and half-life (8days) were also killed6collection lung, liver, kidney, spleen, brain and tumor tissue. those Organ and tumor tissue flushed with saline after the filter paper the tissue surface water priming, Flushed with saline after the filter paper suction the tissue surface water, radioactive Activity Meter detect tissue nuclide activity and weighed organizations.7、48h, the half-life of materials in groups of six tumor specimens were fixed in10%formalin for24h, Dehydration, embedding, made of wax block and slice CD133immunohistochemical detection by conventional method, Each specimen in each group make immunohistochemistry, and then the application of Image-Pro Plus software to analyze the gray value for statistical analysis.Results1、Join the EGF, bFGF, B27, LIF factor in serum-free medium, to train ordinary U251glioma cells, Can be obtained U251glioma stem cells, glioma stem cells have features suspension and huddle growth.2、Plus nutritious factor by serum-free medium and cultured U251stem cells, and identified by immunohistochemistry, which shows a lot of CD133antigen expression is found in the cell clones ball.3、Planting U251stem cells in mice4-5days late, see the subcutaneous the tiny tissue blocks growth, about1.5×1.5×1cm size tumor can be seen in the planting site about three and a half weeks late, Tumor growth rate after transplantation of the tumor cells2weeks after long fastest.4、Column chromatography method separation and purification for the determination of the labeled product of131I-CD133antibody labeling rate of75.8%. 5^Three experimental groups at24h,48h and72h iodine SPECT scan tracking imaging,24h scan saline group(Group C) did not find any active iodine imaging. Simple Na131I solution injection group(Group B), found in the thyroid strong active iodine imaging in various organs of the tumor tissue, bladder and other weak activity iodine imaging; Injection of I-CD133antibody conjugates group(Group A) has a strong active iodine in the thyroid tumor imaging, bladder and other organs also have weak activity iodine distribution.48h,72h the blank control group(Group C) still does not have any active point; The second group(Group B), in the thyroid always have a strong activity of iodine, and the very small amount in the tumor tissue and other organs active iodine significantly lifetime; The third group(GroupA), the experimental group, and in the tumor tissue and thyroid still strong active iodine retention. Na131I,131I-CD133antibody conjugates are mainly distributed in the thyroid, lung, liver, kidney, spleen, blood, brain and tumor tissue.6、After the targeting radiotherapy,CD133factor immunohistochemical detection, microscope observation, and application of the Image-pro plus software gray value analysis shows that the blank control group(Group C) basically no change in the amount of the entire time period CD133antigen expression. The control group(Group B) in the whole period is slightly reduced, but not statistically significant compared with the blank control group; Experimental group(Group A) in48h hours decreased compared to the previous two groups, with the two groups in front of statistical differences is not obvious, but statistically significant difference in the half-life after.(P<0.05)Conclusion1、Join the EGF, bFGF, B27, LIF factor in serum-free medium, to train ordinary U251glioma cells, Can be obtained U251glioma stem cells.2、U251malignant glioma stem cells than ordinary U251glioma cells with a high growth rate, high invasiveness, and high expression of CD133antigen, Application U251malignant glioma stem cell to establish tumor model more than ordinary U251glioma cells. 3、The CD133antibody131I labeled experimental group, radiation therapy more effective than no labeled131I experimental group significant effect. |
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