HPPH光动力治疗对鼠G422脑胶质瘤各部位突变型p53蛋白和p16蛋白表达及与HpD-PDT对比

Research on Expression of Mutant Type P53 Protein and P16 Protein of Mice G422 Glioma of Different Parts in Response to HPPH-based Photodynamic Therapy

目的:探讨HPPH光动力治疗对鼠G422脑胶质瘤脑及腋部皮下移植瘤各部位mtp53蛋白和p16蛋白表达的影响并和HpD-PDT做比较。方法:建立鼠G422脑胶质瘤脑及腋部皮下移植瘤模型,设立空白对照组、单注射HPPH 0.45mg/kg组、单注射HPD 5 mg/kg组、单665 nm激光照射组、单630n m激光照射组、HPPH-PDT各组(0.15 mg/kg组、0.3 mg/kg组、0.45 mg/kg组)、HpD-PDT 5 mg/kg组。单注射HPPH组、HPPH-PDT组和单注射HPD组、HpD-PDT组自尾静脉注入光敏剂,24 h后以波长665 nm的半导体激光照射HPPH-PDT组和单665 nm激光组肿瘤,功率密度200 mW/cm^2,每光斑照射17 min,能量密度为204 J/cm^2;以波长630 nm的半导体激光照射HpD-PDT组及单630 nm激光组肿瘤,功率密度200 mW/cm^2,每光斑照射20 min,能量密度为240 J/cm^2。于PDT后9 d处死鼠行酶联免疫吸附法(ELISA)的双抗体夹心法检测HPPH-PDT和HpD-PDT后肿瘤及对照组各部位(肿瘤、肿瘤边缘、正常脑组织)、血液突变型p53蛋白(mutant type p53 protein,mtp53)和p16蛋白表达变化。结果:空白、单注药和单照光三组对照组之间mtp53蛋白和p16蛋白表达值两两比较,P>0.05,差别无统计学意义。HPPH-PDT各剂量组及HPD-PDT组与空白对照组比较,mtp53蛋白表达值明显降低,p16蛋白表达值明显升高,P<0.01,差别有统计学意义。且接近正常脑组织值,或p16蛋白表达稍低于正常脑值,mtp53蛋白值表达稍高于正常脑组织值。0.3mg/kg和0.45mg/kg HPPH-PDT组与0.15 mg/kg HPPH-PDT组及HPD-PDT相比,mtp53蛋白表达值降低,p16蛋白表达值升高,P<005,差别有统计学意义,0.45 mg/kgHPPH-PDT组mtp53蛋白表达值稍低于0.3 mg/kgHPPH-PDT组,p16蛋白表达值稍高于0.3 mg/kgHPPH-PDT组,P>0.05,差别无统计学意义。HPPH-PDT0.3 mg/kg组的mtp53蛋白表达值低于HpD-PDT5 mg/kg组,p16蛋白表达值高于HpD-PDT 5 mg/kg组,P<0.05,差别有统计学意义。肿瘤边缘mtp53蛋白值稍高于肿瘤值,血液低于肿瘤值;p16蛋白值肿瘤边缘稍低于肿瘤值,血液高于肿瘤值。结论:HPPH-PDT能诱导mtp53蛋白表达降低,p16蛋白表达升高。与HpD-PDT相比,HPPH-PDTmtp53蛋白表达更低,p16蛋白表达更高。本次实验条件下HPPH 0.3 mg/kg是适宜的HPPH-PDT剂量。

Objective： The expression of mutant type p53 protein and p16 protein of mice g422 glioma in brain and subcutaneous axillary tumors in response to HPPH-PDT was evaluated. The comparison of effect of HPPH-PDT and HpD-PDT was assessed. Methods： The mice G422 glioma model in brain and subcutaneous axillary tumors was established. Mice suffering G422 glioma were randomly divided into seven groups： blank control,HPPH0.45mg/kg aIone,665nm laser alone,HPPH-PDT（0.15mg/kg,0.3mg/kg, 0.45mg/kg） and HpD-PDT5mg/kg. Mice were injected intravenously with 0.15,0.3,0.45mg/kg of HPPH utilizing 665nm diode laser doses of 204J/cm2 at 200mW/cm2 delivered at 24 hours post drug injection for HPPH-PDT groups. For HpD-PDT group, 24 hours after intravenous injection of HpD5mg/kg, 630nm diode laser doses of 240J/cm： at 200mW/cm2 was given. The expression of mutant type p53 protein and p16 protein of mice g422 glioma （ in tumor , edge of tumor ,blood）was tested on the ninth day post PDT by enzyme-linked immunosorbent assay （ELISA）. Results： No significant difference of expression of mtp53 and p16 protein among blank control group,HPPH0.45mg/kg alone group and 665nm laser alone group. Compared with blank control group, expression of mtp53 protein of HPPH-PDT groups significantly decreased, p16 protein significantly increased, P〈0.01. Expression of mtp53 protein of HPPH-PDT 0.3mg/kg and 0.45mg/kg group was lower than that of 0.15mg/kg group obviously, expression of p16 protein was higher, P〈0.05. No significant difference between 0.45mg/kg group and 0.3mg/kg group, P〉0.05. Expression of mtp53 protein of HPPH- PDT 0.3mg/kg group decreased in comparison with HpD-PDT group. Expression of p16 protein was on the contrary, P 〈0.05. Expression of mtp53 protein of HPPH-PDT was higher edge of tumor than that of tumor; blood was lower than that of tumor; Expression of p16 protein of HPPH-PDT was lower edge of tumor than that of tumor; blood was higher than that of tumor. Conclusion： HPPH-PDT induced decrease of mtp53 protein expression and increase of p16 protein expression. Appropriate dose was 0.3mg/kg. Expression of mtp53 protein of HPPH-PDT was lower than that of HpD-PDT. Expression of p16 protein of HPPH-PDT was higher than that of HpD-PDT.