Li Yong-Jing, An Xi-Zhou, Bao Bin-Xia, Wang Han-Yi, Tang Xue, Yao Xin-Yuan, Liu Qi-Hui, Zhang Lu-Ying, Liang Shao-Yan, Yu Jie
Department of Hematology & Oncology, Children's Hospital Affiliated to Chongqing Medical University,Key Laboratory of Child Development and Disorders of Ministry of Education,National Center for Clinical Medical Research on Child Health and Diseases (Chongqing),National International Scientific and Technological Cooperation Base for Major Diseases of Child Development, Chongqing Key Laboratory of Pediatrics,Chongqing 400014, China.
Institute of Pediatrics, Children's Hospital affiliated to Chongqing Medical University; Chongqing 400014, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2020 Apr;28(2):385-393. doi: 10.19746/j.cnki.issn.1009-2137.2020.02.005.
To investigate the correlation of the Netrin-1 expression level with the clinical characteristics in children with acute lymphoblastic leukemia (ALL) and to explore its possible regulatory mechanism.
ELISA was used to detect the expression level of Netrin-1 in peripheral blood serum from 48 child ALL patients (newly diagnosed, recurrent), and its relevance with clinical indicators was statistically analyzed. The blood serum samples from 27 children with non malignant hematological diseases were choosen as controls. Leukemia cell lines of Jurkat,Molt-4,SUP-B15 and Raji were cultivated in vitro, after treated with different concentrations of recombinant human Netrin-1 protein， the invasive ability of the cells was detected by Transwell method； the effect of Netrin-1 to the proli feration of cells was detected by CCK-8 method； The expression and phosphorylation level of key molecules, such as FAK,Erk1/2,PI3K and Akt signaling pathway were detected by Western blot.
The expression of Netrin-1 in child patients was significantly higher than that of the control group (P<0.05). With the increasing of Netrin-1 level, the level of Plt (r=0.483, P<0.05) increased, while the level of WBC (r=-0.290, P<0.05) decreased, and there were no significant correlation with age, Hb level and the proportion of immature cells in bone marrow. When the concentration of Netrin-1 was 25-50 ng/ml, the level of Netrin-1 positively correlated with WBC (r=0.886, P<0.05) ; the level of Netrin-1 significantly decreased when the patient's WBC was ＞50×10/L and Plt ＞20×10/L(P=0.042，P=0.001); The expression level of Netrin-1 was significantly different in the risk group(P=0.017), and level of Netrin-1 in high-risk group was significantly higher than that in low risk group and middle risk group, but there was no significant difference of Netrin-1 expression in sex, hepatosplenomegaly, MRD, recurrence and chromosome abnormality. Netrin-1 could promote the invasiveness of the four kinds of cells (P<0.05). With the increase of Netrin-1 concentration, the number of cells increased at first and then decreased, and the number of cells in the invading chamber was the highest when the concentration of Netrin-1 was 100 ng/ml; the survival rate of the four kinds of cells significantly increased when the concentration of Netrin-1 was 25 ng/ml(P<0.05), and SUP-B15 cells showed the highest cell survival rate at a concentration of 100 ng/ml; The survival rate of the four kinds of cells showed a tendency : survival of cells increased at low concentration of Netrin-1 and survival of cells decreased at high concentration of Netrin-1. The results of Western blot showed that Netrin-1 activated the phosphorylation level of key molecules such as FAK,Erk1/2,PI3K,Akt signaling pathway (P<0.05).
There is abnormal expression of Netrin-1 in serum of children with ALL. Netrin-1 may affect the occurrence and development of ALL by increasing the proliferation and invasiveness of leukemia cells, and may become a risk factor of ALL or a potential target in biotherapy.
探讨Netrin-1表达水平与急性淋巴细胞白血病（ALL）患儿临床特征的相关性，并探讨其可能的调控机制。
采用ELISA法检测48例ALL患儿（初诊、复发）外周血血清中Netrin-1的表达水平，并对其与临床指标的相关性进行统计学分析。选取27例非恶性血液病患儿的血清样本作为对照。体外培养Jurkat、Molt-4、SUP-B15和Raji白血病细胞系，用不同浓度的重组人Netrin-1蛋白处理后，采用Transwell法检测细胞的侵袭能力；采用CCK-8法检测Netrin-1对细胞增殖的影响；采用Western blot检测FAK、Erk1/2、PI3K和Akt信号通路等关键分子的表达及磷酸化水平。
ALL患儿Netrin-1的表达明显高于对照组（P<0.05）。随着Netrin-1水平的升高，Plt水平升高（r=0.483，P<0.05），而WBC水平降低（r=-0.290，P<0.05），且与年龄、Hb水平及骨髓中幼稚细胞比例无明显相关性。当Netrin-1浓度为25~50 ng/ml时，Netrin-1水平与WBC呈正相关（r=0.886，P<0.05）；当患者WBC＞50×10⁹/L且Plt＞20×10¹²/L时，Netrin-1水平明显降低（P=0.042，P=0.001）；Netrin-1表达水平在危险组中有明显差异（P=0.017），高危组Netrin-1水平明显高于低危组和中危组，但Netrin-1表达在性别、肝脾肿大、微小残留病、复发及染色体异常方面无明显差异。Netrin-1可促进4种细胞的侵袭能力（P<0.05）。随着Netrin-1浓度的增加，细胞数量先增加后减少，当Netrin-1浓度为100 ng/ml时，侵袭小室中的细胞数量最多；当Netrin-1浓度为25 ng/ml时，4种细胞的存活率明显增加（P<0.05），SUP-B15细胞在浓度为100 ng/ml时细胞存活率最高；4种细胞的存活率呈趋势：Netrin-1低浓度时细胞存活率增加，Netrin-1高浓度时细胞存活率降低。Western blot结果显示，Netrin-1激活了FAK、Erk1/2、PI3K、Akt信号通路等关键分子的磷酸化水平（P<0.05）。
ALL患儿血清中Netrin-1表达异常。Netrin-1可能通过增加白血病细胞的增殖和侵袭能力影响ALL的发生发展，可能成为ALL的危险因素或生物治疗的潜在靶点。
