Experimental Study Of The Coefficient In EGFR、HER2、CXCR4and Targeting Specific Human Anti-EGFR ScFv Molecular Probe In NSCLC

Objective:1. To detect EGFR, HER2and CXCR4correlation and significance in patients with NSCLC tissues;2. To construct human anti-EGFR single chain antibody (EGFR-scFv) fusion protein in E.coli. and study its binding activity and internalization ability.3. To study the binding specificity between EGFR-scFv nano molecular probe (scFv@GoldMag) and EGFR-positive cells in vitro and in vivo.Methods:1. We select untreated75cases of primary tumor tissues of NSCLC,which were fixed in4%formaldehyde, paraffin-embedded and sliced.The staining of EGFR, HER2and CXCR4were according to PV-step immunohistochemical method with PBS as negative control,the known positive sample as positive control. Immunohistochemistry results were determined by microscope.Immunohistochemical analysis:We used semi-quantitative integration counted positive cells,Selected five horizons field and counted500cells in high-power optical microscope (×20)randomly.The scoring criteria were in according with positive cells proportion and staining intensity:positive cells are determined0(<5%),1(5%-24%),2(25%-50%),3(51%-74%),4(≥75%) points respectively. Staining intensity were judged0,1,2,3points with no coloring, yellow, light brown and brown respectively.We were in according with the result of the determination of two points,0into the negative (-),1-2into weakly positive (+),3-5into moderately positive (++),6-7into strongly positive (+++). The judgement result are in according with the two points,0is negative (-),1-2is weakly positive (+),3-5is moderately positive (++),6-7is strongly positive (+++).Statistical analysis:Data analysis were used SPSS21.0statistical software.Count data use two (or more) sample rate comparison test and correlation analysis use Spearman rank correlation analysis. Test level P=0.05.2Construct anti-EGFR single chain antibody (scFv) fusion:The plasmid with correct sequence was cloned into the expression vector pGEX-4T-1,After being induced in E.coli.BL21(DE3) by IPTG and purified by Ni-NTA.The EGFR-scFv expression was used SDS-PAGE.The expression of6×his tagged protein (EGFR-scFv-His) was used Western blot.Detection of EGFR-scFv fusion protein activity in vitro:In vitro,EGFR-positive NSCLC (SPC-A1) and EGFR-negativeSCLC H69) cells were cultured in RPMI1640with10%FBS.The internalization ability of purified products was analyzed by indirect immunofluorescent and flow cytometry respectively within these cells.3. Construct the EGFR specific molecular probe:The EGFR specific molecular probe scFv@GoldMag was constructed by conjugating anti-human EGFR scFv with GoldMag TM-CS nanoparticles.In vitror: The specific binding capability for scFv@GoldMag to SPC-A1and H69cells cell in vitro was detected by using laser confocal microscopy, flow cytometry and transmission electron microscope.In vivo:SPC-Al and H69cells were cultured in RPMI1640with10%FBS In vitro.We construct nude mice model by injected SPC-A1and H69cells suspension, The specific binding capability for scFv@GoldMag to NSCLC cells in vivo was detected by using laser confocal microscopy, flow cytometry and transmission electron microscope. Results:1. The expression rate of EGFR, HER2and CXCR4in NSCLC tissues were58.66%(44/75),28%(21/75),60%(45/75) respectively. The EGFR, HER2and CXCR4expression were irrelevant to age, sex and Pathologic type in NSCLC(P>0.05), but relevant to lymph node metastasis and distant metastasis (P<0.05).The correlation coefficients of EGFR and HER2, EGFR and CXCR4, HER2and CXCR4were r=0.296(P<0.01), r=0.578(P<0.01), r=0.426(P<0.01respectively. In partcular,the correlation of EGFR and CXCR4was the highest.The median survival time were18.3months(no expression of three genes),16.3months(single gene expression),6.3months(two gene expression) and3.6months (three gene expression)respectively.2. The successful expression of the EGFR-scFv-His fusion protein was confirmed by SDS-PAGE.Western blot confirmed the expressed products was the aimed protein with6-His tag.And the fusion protein can specific bind with SPC-A1cells,can not bind with H69cells.3. The specific molecular probe (scFv@GoldMag)was successfully constructed. ScFv@GoldMag can specific combine with SPC-A1cells,can not combine with H69cells.The results of laser confocal microscopy, flow cytometry and transmission electron microscope proved that scFv@GoldMag can specific bind with EGFR-positive SPC-A1cells.Conclusion:EGFR, HER2and CXCR4positively expressed in NSCLC,which were irrelevant to age, sex and Pathologic type in NSCLC(P>0.05), but relevant to poor prognosis. The correlation of EGFR, HER2and CXCR4expression were positively.The three genes simultaneous expression was the worst prognosis.EGFR-scFv fusion protein can bind with EGFR antigen and internalized into EGFR-positive tumor cells.The specific molecular probe(scFv@GoldMag)was successf-ully constructed.It could specificly bind with EGFR positive NSCLC cancer cells and internalized into EGFR-positive tumor cells in vitro and in vivo.It may therefore be a new noninvasive specific MR molecular probe in vivo.