Inhibitory effect of anti-human NRP-1 monoclonal antibody on hepatocellular carcinoma cell line HepG2 and its mechanism in vitro

目的研究抗人神经纤毛蛋白-1(Neuropilin1,NRP-1)单克隆抗体对肝癌Hep G2细胞的生长抑制作用及其机制。方法小鼠腹水法制备抗NRP-1单克隆抗体(NRP-1 m Ab)并用r Protein A亲和柱纯化抗体,间接ELISA检测抗体的滴度水平。Western blot检测NRP-1 m Ab对Hep G2细胞的特异性,细胞免疫荧光和流式细胞术检测NRP-1蛋白在肝癌细胞株Hep G2上的表达,MTT法检测NRP-1 m Ab对Hep G2的生长抑制作用,Western blot检测ERK1/2、P-ERK1/2、Akt、P-Akt蛋白的表达水平。结果 SDS-PAGE和间接ELISA检测纯化的NRP-1 m Ab纯度为95%以上,效价为1×10~(-6);Western blot检测结果显示NRP-1 m Ab可与Hep G2细胞膜上的NRP-1蛋白特异性结合。细胞免疫荧光染色结果显示NRP-1定位于Hep G2细胞膜,流式细胞术结果显示NRP-1蛋白在Hep G2细胞上表达水平较高;MTT法检测结果显示NRP-1 m Ab对Hep G2细胞有生长抑制作用。Western blot检测到在不同浓度NRP-1 m Ab作用下,Hep G2细胞裂解液P-ERK1/2、P-Akt蛋白的条带信号逐渐减弱。结论纯化的NRP-1m Ab能抑制Hep G2细胞的生长,其抑制作用是通过EGF和HGF信号通路实现的。The aim of the experimental is to investigate the inhibitory effect of anti-human nerve cilia protein1(Neuropilin-1,NRP-1) monoclonal antibody(NRP-1 mAb) on hepatocellular carcinoma cell line HepG2 and its mechanism in vitro.Anti-human NRP-1 monoclonal antibody(NRP-1 mAb) was prepared from mouse ascites and purified by rProteinA affinity column assay.The titer of antibody was determined using indirect ELISA assay;the characteristic of NRP-1 mAb binding to NRP-1 was determined using Western blotting;the expression of NRP-1protein in hepatocellular carcinoma cell line HepG2 was determined using immunofluorescence assay and flow cytometry assay.Growth inhibition of HepG2 cells treated with different concentrations of NRP-1 mAb was determined using MTT assay,while Western blotting was used to detect the expression levels of ERK1/2,P-ERK1/2,Akt and P-Akt proteins.The results of SDS-PAGE and indirect ELISA showed that the purity of purified NRP-1mAb was more than 95%and the titer was 1×10~(-6).Western blotting analysis suggested that NRP-1 mAb could bind specifically to NRP-1 on HepG2 cell;immunofluorescence staining showed that NRP-1 was located in the membrane of HepG2 cells.Flow cytometry analysis showed that the expression level of NRP-1 on HepG2 cell was relatively high.Western blotting analysis suggested that P-ERK1/2 and P-Akt expression levels were down-regulated after having incubated HepG2 cells with different concentrations of NRP-1 mAb.In conclusion,NRP-1 mAb could inhibit the growth of HepG2 cells(P<0.05),and its inhibitory effect is achieved by reducing the P-ERK1/2 and P-Akt expression.南京军区医学科技创新项目(12MA061,15MS104

重组人血管内皮生长抑制因子对人肺腺癌裸鼠移植瘤的放射效应影响

目的:探讨重组人血管内皮抑制因子(recombinant human vascular endothelial growth inhiloitor-192,rhVEGI-192)对人肺腺癌裸鼠移植瘤的放射增敏作用。方法:采用原核表达rhVEGI-192,获得目的蛋白。通过肿瘤倍增时间,计算药物的增敏系数。通过建立人肺腺癌裸鼠移植瘤模型,荷瘤裸鼠随机分为4组:对照组、10Gy、rhVEGI-192、rhVEGI-192+10Gy。采用6MV-X线进行照射,照射剂量为10Gy。获得移植瘤标本,利用免疫印迹法检测移植瘤中VEGF(vascular endothelial growth factor)的表达变化。结果:SDS电泳结果显示,目的蛋白位于22k D左右。10Gy照射时,重组人血管内皮抑制因子的EF(enhancement factor)值为1.5。和空白对照组相比,rhVEGI-192组和10Gy组移植瘤的生长受到抑制(P<0.001),rhVEGI-192+10Gy组移植瘤生长显著抑制(P<0.001),rhVEGI-192+10Gy组移植瘤较10Gy组有明显生长抑制。和空白组相比,rhVEGI-192组VEGF表达减少,而10Gy组VEGF表达变化不明显,rhVEGI-192+10Gy组VEGF表达明显减少。rhVEGI-192+10Gy和rhVEGI-192组相比,VEGF表达减少。结论:rhVEGI-192联合照射能够减少VEGF的表达。这可能是rhVEGI-192的增敏机制之一。中国人民解放军南京军区医学科技创新项目(编号:No.12MA061

JUNO Sensitivity on Proton Decay p→νˉK+p\to \bar\nu K^+ Searches

The Jiangmen Underground Neutrino Observatory (JUNO) is a large liquid scintillator detector designed to explore many topics in fundamental physics. In this paper, the potential on searching for proton decay in $p\to \bar\nu K^+$ mode with JUNO is investigated.The kaon and its decay particles feature a clear three-fold coincidence signature that results in a high efficiency for identification. Moreover, the excellent energy resolution of JUNO permits to suppress the sizable background caused by other delayed signals. Based on these advantages, the detection efficiency for the proton decay via $p\to \bar\nu K^+$ is 36.9% with a background level of 0.2 events after 10 years of data taking. The estimated sensitivity based on 200 kton-years exposure is $9.6 \times 10^{33}$ years, competitive with the current best limits on the proton lifetime in this channel

JUNO sensitivity on proton decay p → ν K + searches*

The Jiangmen Underground Neutrino Observatory (JUNO) is a large liquid scintillator detector designed to explore many topics in fundamental physics. In this study, the potential of searching for proton decay in the $p\to \bar{\nu} K^+$ mode with JUNO is investigated. The kaon and its decay particles feature a clear three-fold coincidence signature that results in a high efficiency for identification. Moreover, the excellent energy resolution of JUNO permits suppression of the sizable background caused by other delayed signals. Based on these advantages, the detection efficiency for the proton decay via $p\to \bar{\nu} K^+$ is 36.9% ± 4.9% with a background level of $0.2\pm 0.05({\rm syst})\pm$$0.2({\rm stat})$ events after 10 years of data collection. The estimated sensitivity based on 200 kton-years of exposure is $9.6 \times 10^{33}$ years, which is competitive with the current best limits on the proton lifetime in this channel and complements the use of different detection technologies