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目的研究高迁移率蛋白(HMGB1)在多形性成胶质细胞瘤(GBM)中的作用及其机制。方法用9 L胶质瘤细胞颅内接种Wistar鼠构建同源GBM模型。建立GBM模型鼠后将其随机分为3组:GBM+Saline(n=20),GBM+Ad(n=20),GBM+Ad+Gly(n=20)。10 d后对GBM+Ad组鼠进行Ad-TRAIL感染(8×107pfu/5μl),GBM+Ad+Gly组鼠进行Ad-TRAIL感染和甘草酸(Glycyrrhizin)喂养(稀释于氢氧化钠,100 mg腹腔注射,每天两次连续喂养10d),GBM+Saline组和对照组用生理盐水处理。采用ELISA测定体内和体外HMGB1的含量水平,TUNEL法测定细胞的凋亡,免疫细胞组化(ICC)检测HMGB1的表达。结果 HMGB1在GBM+Ad组中的血清含量为5.540±0.054 ng,显著高于GBM+saline组的0.0060±0.004 ng(P=0.0001);GBM+Ad+Gly组中HMGB1血清含量(0.07±0.016 ng)显著低于GBM+Ad组(P=0.04);GBM+saline组的HMGB1血清含量高于对照组,但差异无统计学意义(P=0.923)。免疫细胞组化实验中,HMGB1在GBM-Ad组中呈高阳性表达。体外检测中,GBM+Ad组中HMGB1浓度(77.505±1.196 ng)高于GBM+saline组(8.623±0.052 ng)(P=0.0003);GBM+Ad0组中HMGB1浓度低于GBM+saline组,但差异无统计学意义(P>0.05)。TUNEL法检测Ad-TRAIL感染GBM原代细胞后的细胞凋亡发现,GBM+Ad组的细胞死亡率为(78.91±0.17)%,显著高于GBM+saline组(18.29±0.73)%(P=0.04)。相关性分析结果显示HMGB1含量水平与细胞死亡率呈正相关,R2=0.9976,P<0.0001。GBM+saline和GBM+Ad+Gly组鼠在第12天开始出现死亡,在第24天和第25天全部死亡;而GBM+Ad组在第16天开始出现死亡,在第40天时有50%(5/10)长期存活。结论HMGB1在GBM治疗中高释放,且其浓度与细胞凋亡正相关。HMGB1是GBM的治疗靶点,并且是检测腺病毒治疗或免疫治疗GBM疗效的潜在生物标记。
Objective To investigate the role and mechanism of HMGB1 in glioblastoma multiforme (GBM). Methods Ninety-nine glioma cells were intracranially inoculated with Wistar rats to construct a homologous GBM model. GBM model rats were established and randomly divided into 3 groups: GBM + Saline (n = 20), GBM + Ad (n = 20), GBM + Ad + Gly (n = 20). After 10 days, Ad-TRAIL infection and Glycyrrhizin (GBM + Ad + Gly) mice were inoculated with Ad-TRAIL (8 × 107pfu / 5μl) Intraperitoneal injection, two consecutive days of feeding 10d), GBM Saline group and control group with saline treatment. The levels of HMGB1 in vivo and in vitro were determined by ELISA. The apoptosis of cells was detected by TUNEL method. The expression of HMGB1 was detected by immunocytochemistry (ICC). Results The serum level of HMGB1 in GBM + Ad group was 5.540 ± 0.054 ng, significantly higher than that in GBM + saline group (0.0060 ± 0.004 ng, P = 0.0001). The serum levels of HMGB1 in GBM + Ad + Gly group were 0.07 ± 0.016 ng ) Was significantly lower than that of GBM + Ad group (P = 0.04). The serum level of HMGB1 in GBM + saline group was higher than that in control group, but the difference was not statistically significant (P = 0.923). In immunocytochemistry, HMGB1 was highly positive in GBM-Ad group. In vitro, the concentration of HMGB1 in GBM + Ad group (77.505 ± 1.196 ng) was higher than that in GBM + saline group (8.623 ± 0.052 ng) (P = 0.0003). The concentration of HMGB1 in GBM + Ad0 group was lower than that in GBM + saline group The difference was not statistically significant (P> 0.05). TUNEL assay showed that the cell death rate of GBM + Ad group was (78.91 ± 0.17)%, significantly higher than that of GBM + saline group (18.29 ± 0.73)% (P = 0.04). Correlation analysis showed that the level of HMGB1 was positively correlated with cell death rate, R2 = 0.9976, P <0.0001. The GBM + saline and GBM + Ad + Gly mice died on the 12th day and all died on the 24th and 25th day, while the GBM + Ad group began to die on the 16th day and 50% (5/10) Long-term survival. Conclusion HMGB1 is highly released during GBM treatment and its concentration is positively correlated with apoptosis. HMGB1 is a therapeutic target for GBM and is a potential biomarker to detect the efficacy of adenovirus or immunotherapy GBM.