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目的:评价应用99Tcm-rh-Annexin Ⅴ显像检测单次放疗后早期肿瘤细胞凋亡的可行性,并进一步探讨肿瘤组织内99Tcm-rh-Annexin Ⅴ的分布与凋亡调控蛋白bcl-2、bax表达的相关性。方法:采用直接标记法标记rh-Annexin Ⅴ作为核素凋亡显像示踪剂。将小鼠肝癌细胞(Hca-F25)接种于小鼠右腋下部位;随机分为A(n=9,对照组)、B(n=10,放疗组)两组,B组另分为3个亚组(分别为B1组,n=4;B2组n=3;B3组,n=3)。8d后肿瘤生长至直径约1cm时,B1、B2、B3组分别接受2Gy、5Gy、10Gy不同剂量直线加速器照射;1h后两组同时由鼠尾静脉注入99Tcm-rh-Annexin Ⅴ;4h后行SPECT显像并处死、取材,应用井型闪烁探测器检测荷瘤小鼠肿瘤组织内示踪剂分布,以%ID/g(每克组织百分注射剂量率)表示。采用TUNEL法检测肿瘤细胞凋亡;采用免疫组化SP法检测标本中bcl-2、bax蛋白表达。结果:B组肿瘤组织的%ID/g、TUNEL检测阳性细胞数及bax蛋白表达均明显多于A组(P<0.01);A组bcl-2蛋白表达及bcl-2/bax明显高于B组(P<0.05)。相关性研究表明,在A、B组中肿瘤组织的%ID/g与TUNEL阳性细胞数均呈明显正相关(r=0.801、0.846),而bcl-2蛋白表达与bax蛋白表达间无明确相关性;A组肿瘤组织内bcl-2、bax蛋白表达与肿瘤组织的%ID/g及TUNEL阳性细胞数均无明确相关性;B组肿瘤组织内,bcl-2蛋白表达与%ID/g及TUNEL阳性细胞数均无明确相关性,bax蛋白表达与%ID/g及TUNEL阳性细胞数均呈明显正相关(r=0.921、0.723),而bcl-2;bax比值与%ID/g及TUNEL阳性细胞数均呈明显负相关(r=-0.828、-0.687)。结论:肿瘤组织内99Tcm-rh-Annexin Ⅴ的分布可以反映放疗后早期肿瘤组织细胞凋亡的状况以及凋亡调控蛋白bcl-2、bax表达水平的变化。
OBJECTIVE: To evaluate the feasibility of 99Tcm-rh-Annexin Ⅴ imaging in the detection of early apoptosis of tumor cells after single-dose radiotherapy and to further investigate the distribution of 99Tcm-rh-Annexin Ⅴ and the expression of apoptosis regulatory proteins bcl-2, bax Correlation of expression. Methods: rh-Annexin Ⅴ labeled by direct labeling was used as radionuclide imaging tracer. The mouse hepatoma cells (Hca-F25) were inoculated to the right axillary region of mice. The mice were randomly divided into two groups: A (n = 9, control group), B (n = 10, radiotherapy group) Subgroups (group B1, n = 4; group B2 n = 3; group B3, n = 3). After 8 days of tumor growth to a diameter of about 1cm, B1, B2 and B3 groups were irradiated with different doses of 2 Gy, 5 Gy and 10 Gy linear accelerator respectively. After 1 h both groups were injected with 99Tcm-rh-Annexin Ⅴ via tail vein of rats. Imaging and sacrificed, drawn, the application of well-type scintigraphy detection of tumor-bearing mice bearing tumor tracer distribution in% ID / g (per gram tissue dose rate) said. The apoptosis of tumor cells was detected by TUNEL method. The expression of bcl-2 and bax protein in specimens was detected by immunohistochemical SP method. Results: The percentages of% ID / g, the number of TUNEL positive cells and the expression of bax in group B were significantly higher than those in group A (P <0.01). The expression of bcl-2 and bcl-2 / bax in group B were significantly higher than those in group B Group (P <0.05). Correlation studies showed that there was a significant positive correlation between% ID / g and TUNEL positive cells in group A and group B (r = 0.801,0.846), but there was no clear correlation between bcl-2 protein and bax protein expression There was no significant correlation between the expression of bcl-2 and bax in group A and the% ID / g and TUNEL positive cells in group A. The expression of bcl-2 protein in group B was significantly correlated with% ID / g and There was no significant correlation between the number of TUNEL positive cells and the expression of bax protein (P <0.05). The expression of bax protein was positively correlated with% ID / g and TUNEL positive cells (r = 0.921,0.723) The number of positive cells showed a significant negative correlation (r = -0.828, -0.687). CONCLUSION: The distribution of 99Tcm-rh-Annexin Ⅴ in tumor tissue can reflect the apoptosis of tumor cells and the expression of bcl-2 and bax in early stage after radiotherapy.