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Objective To observe the expression of neuropilin-1 (NRP-1) in glioma cells of different grades,and evaluate the application value of a novel molecular probe(USPIO-PEG-tLyP-1)in the grading diagnosis of heterotopic glioma in nude mice by magnetic resonance imaging (MRI).Methods Expression levels of NRP-1 in glioma cell lines of different grades were detected by Western-Blot.USPIO-PEG-tLyP-1 was synthesized by carbon diimine method.The U87-MG tumor-bearing mice model (U87-MG group) and CHG-5 tumor-bearing mice model(CHG-5 group) were established with 10 mice in each group.Six tumorbearing mice with a tumor volume about 0.6 cm3 were selected from each group,and they were given with 2mg/kg molecular probes via tail vein respectively and was detected by MRI at 0 h,6 h,12 h and 24 h,then R2 values were calculated.After the imaging,tumor-bearing mice were sacrificed,and tumor tissue sections were made.The iron particles in the sections was detected by Prussian blue staining.The binding ability of molecular probes and tumor tissues in the two groups was compared.Results The expression of NRP-1 in U87-MG and CHG-5 cell lines was significantly higher than that in HA.In addition,the expression of NRP-1 in U87-MG was higher than that in CHG-5 cell(P＜0.01).MRI results showed that R2 values of tumor tissues in the two groups were compared,and the difference was not statistically significant before the injection of molecular probe(U87-MG group(10.35±0.52)vs CHG-5 group(9.86±0.43),t=1.779,P=0.106).The R2 value of tumor tissue in the U87-MG group was higher than that in the CHG-5 group after the injection of molecular probe (6 h:U87-MG group (11.63±0.85)vs CHG-5 group (10.51 ±0.49),t=2.796,P=0.019;12h:U87-MG group(14.23±0.68)vs CHG-5 group(12.29±0.28),t=6.462,P=0.000;24 h:U87-MG group (13.36±0.92) vs CHG-5 group(11.32±0.64),t=4.459,P=0.001).The results of Prussian blue staining showed that there were significantly more blue staining particles in tumor tissues of the U87-MG group than that of the CHG-5 group,and the difference was statistically significant(P＜0.01).Conclusion The NRP-1 targeted molecular probe can be used for grading diagnosis of high and low grade heterotopic brain glioma in nude mice.
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Objective To synthetize a novel MR molecular imaging probe named USPIO?PEG?tLyP?1,and to evaluate its value in detecting U87 cells by MR imaging. Methods USPIO?PEG?tLyP?1 was synthetized by conjugating USPIO?PEG with tLyP?1. Neuropilin?1 expression levels of glioma cell lines were detected by Western blot. The cytotoxicity of USPIO?PEG and USPIO?PEG?tLyP?1 were assessed by MTT colorimetric assay. The uptake efficiency of USPIO?PEG?tLyP?1 was measured by Prussian blue staining, transmission electron microscope and MR imaging in vitro. Results The novel MR molecular imaging probe was synthetized with an average diameter of 43.84 nm. U87 glioma cell line was screened as test subject for the highly expression of NRP?1(P<0.05). USPIO?PEG?tLyP?1 group showed much more intracellular blue particles than USPIO?PEG group after Prussian blue staining. After incubation,USPIO?PEG?tLyP?1 mainly existed in lysosme,endoplasmic reticulum and mitochondria. In vitro MRI showed that USPIO?PEG?tLyP?1 significantly enhanced the negative contrast effect compared with USPIO?PEG(P<0.01). Conclusion The decoration of tLyP?1 enhanced targeting ability of USPIO?PEG to glioma cells and MR molecular imaging can be a promising method for early diagnosis of gliomas.
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Objective To study the effect of PcG member NSPc1 on proliferation of HeLa cells.Methods Using bioinfomatic analysis to design the siRNA sequence to knockdown NSPc1.Detecting the expression level of NSPc1 in HeLa cell line using semi-quantitative RT-PCR,Real-time PCR and Western blot after transfection of the designed siRNA.Transient transfecting pSUPER-NSPc1 into Hela cells and performing BrdU incorporation assay.Establishing NSPc1 stably knockdown cell line,comparing proliferation abilities with the control cells.Results(1)The designed siRNA did efficiently knockdown the expression of NSPc1;(2)Transient knockdown of NSPc1 could repress BrdU incorporation;(3) The established NSPc1-knockdown cell lines had a significantly lower proliferation rate than that of control cells.Conclusion The expression of NSPc1 is necessary for the normal proliferation of HeLa cells.The NSPc1 stably knockdown cell pool is a useful model for further study of pathway related to NSPc1.
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Objective To screen the proteins interact with cytoplasmic tail of NECL1.Methods The cytoplasmic tail sequence of human NECL1 gene was inserted into the "bait" vector pGBKT7 in frame.The recombinant pGBKT7-NECL1C was then transformed into yeast cells and followed by screening of human fetal brain cDNA library.Then the GST pull down assay was used to confirm the interactions between the proteins.Results Nine different sequences by sequencing the positive clones and five alternative proteins were obtained by the amino acid sequence blast.Using the GST pull down analysis,we confirmed that two of them can interact with the cytoplasmic tail of NECL1 in vitro.Conclusion The alternative proteins obtained with the yeast dual-system may interact with the cytoplasmic tail of NECL1.
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Objective To express the human recombinant SUMO1 protein and prepare monoclonal antibody(mAb) against it.Methods The recombinant expression plasmid pET32a-HIS-SUMO1 was made and transformed into E.coli(BL21),then the recombinant fusion protein HIS-SUMO1 was expressed and purified.The BALB/c mice were immuned with pure protein HIS-SUMO1 as antigen.Monoclonal antibody against SUMO1 was prepared with standard hybridoma technology.The hybridoma cell lines were obtained by ELISA and Western blot screening procedure,the isotype of the mAbs were further identified by immune-double diffusion.Ascites were collected from one propagated hybridoma cell line and mAbs were purified by using the Kit of Millipore.The valence of mAb was detected by Western Blot.Results The recombinant protein HIS-SUMO1 is expressed and purified.Three hybfidmas producing antibodies against SUMO1 were obtained,the isotypes of three mAbs are IgG1,Western blot showed that the antibodies were specific for SUMO1.The antibody purified from the ascites has better specificity.Conclusion The SUMO1 mAb prepared by using recombinant SUMO1 protein as antigen can be used for detectingthe protein sumoylation.