急性淋巴细胞性白血病患儿血清高迁移率族蛋白1检测及其诱导白血病细胞分泌肿瘤坏死因子α的实验研究

目的探讨血清高迁移率族蛋白1（HMGB1）水平与儿童急性淋巴细胞性白血病（ALL）发病的关系;研究HMGB1对白血病细胞分泌肿瘤坏死因子α（TNF-α）的影响及其与丝裂原活化蛋白激酶（MAPK）信号通路的关系。方法采用Westernblot方法检测正常、ALL患儿初治期、完全缓解期各15例血清HMGB1水平;制备HMGB1重组蛋白刺激人K562白血病细胞损伤模型,采用ELISA和Westernblot方法检测HMGB1对TNF-Or．分泌及其p38、c-Jun氨基末端激酶（JNK）、细胞外信号调节激酶（ERK）MAPK信号通路活化的影响;采用ELISA方法检测MAPK抑制剂对HMGB1所致TNF-α．分泌的影响。结果初治ALL患者血清HMGB1（43．78±4．62）μg／L]显著高于正常对照组（0．60±0．48）μg／L,P〈0．01]和缓解组（0．89±0．62）μg／L,P〈0．01],正常对照组与缓解组比较差异无统计学意义（P〉0．05）;HMGB1以时效和量效依赖性方式诱导K562细胞分泌TNF-α．并且促进了p38、JNK、ERK蛋白发生磷酸化;p38抑制剂（SB203580）、JNK抑制剂（SP600125）和MEK抑制剂（PD98059）抑制了HMGB1诱导K562细胞TNF-α．的分泌。结论血清HMGB1水平与儿童ALL发病、发展密切相关;HMGB1通过活化MAPK信号通路促进白血病细胞分泌TNF-α．参与肿瘤细胞免疫调节。

High mobility group box 1 is increased in children with acute lymphocytic leukemia and stimulates the release of tumor necrosis factor-alpha in leukemic cell

OBJECTIVE: Cytokine mediated cell immunity is the main mode of anti-tumor immunity in organism, and the disequilibrium of cytokine network is the main cause of tumor cells escaping immunologic surveillance. High mobility group box 1 (HMGB1), a nuclear protein, has recently been identified as an important mediator of local and systemic inflammatory diseases when released into the extracellular milieu. In the present study, the investigators explored the clinical significance of alteration in the serum levels of HMGB1 in childhood acute lymphocytic leukemia (ALL) and the mechanism of HMGB1-induced tumor necrosis factor (TNF)-alpha secretion in leukemic cells. METHODS: The serum levels of HMGB1 in healthy children and childhood ALL were assayed by Western blotting. K562 leukemic cells were stimulated with recombinant HMGB1 protein in vitro, and the secretion of TNF-alpha was determined by using ELISA. The effects of HMGB1 on activation of p38, c-Jun amino-terminal kinase (JNK), and extracellular-signal regulated protein kinase (ERK) and mitogen-activated protein kinase (MAPK) in K562 cells were assayed by using Western blotting. The effects of inhibitors specific for the MAPK on HMGB1-induced TNF-alpha secretion were assayed by using ELISA. RESULTS: The serum levels of HMGB1 were significantly higher in ALL initial treatment group (n = 15, 43.78 +/- 4.62 microg/ml) than those in healthy control group (n = 15, 0.60 +/- 0.48 microg/ml, P < 0.01) and ALL complete remission group (n = 15, 0.89 +/- 0.62 microg/ml, P < 0.01). No significant difference was found between the healthy control group and ALL complete remission group in HMGB1 levels (P > 0.05). TNF-alpha started to become detectable at 2 h and was still increasing at 16 h after HMGB1 (1 microg/ml) treatment in K562 cell culture. TNF-alpha was also secreted from K562 cells in a dose-dependent manner after HMGB1 (1 ng/ml-1 microg/ml) exposure. HMGB1 induced the phosphorylation of p38, JNK and ERK in k562 cells. Inhibitors specific for the JNK (SP600125), MEK (PD98059), and p38 MAPK (SB203580), abrogated HMGB1-induced TNF-alpha secretion. CONCLUSIONS: The measurement of serum HMGB1 is helpful to evaluate the prognosis of the childhood ALL. HMGB1 stimulates leukemic cells to secrete TNF-alpha through a MAPK-dependent mechanism.