Experimental Study On Specific Imaging Of Recombinant Surface Antigen EPC1 Based On Hepatic Alveolar Echinococcosis

Objective: To prepare,extract and purify the recombinant surface antibody EPC1 against liver hydatid,and then couple EPC1 with superparamagnetic iron oxide nanoparticles(SPION)to obtain SPION-EPC1 complex tracer,and fluorescein isothiocyanate(FITC)to obtain FITC-EPC1 complex tracer.The characterization study and related toxicity test of two different complex tracers were carried out,and then the two complex tracers were verified at the cell level in vitro and animal model.In vitro cell model,we mainly observed the position of the two tracers in the cell before and after coupling and their related toxicity at the cell level,and whether the tracers had influence on cell proliferation,apoptosis and cell growth cycle changes before and after coupling.In the part of animal model experiment,the membrane of hepatic alveolar echinococcus was first made,and the nuclear magnetic resonance imaging and fluorescence imaging detection of liver lesions were performed on the mice with hepatic alveolar echinococcus successfully made the membrane.The simple tracer and complex tracer were used to observe and compare the development effects and differences of the two kinds of tracers before and after coupling.It was proved that the complex tracer combined with EPC1 could target to combine with the recombinant surface antigen EPC1 of hydatid in hepatic alveolar echinococcus,Realize targeted imaging,make the development effect of liver lesions in mice better,make it easier to find lesions with diameter less than 1 cm in the early stage,and provide reliable scientific basis for early diagnosis and treatment.Methods: 1.In the first part,(1)first prepare the recombinant surface antibody EPC1 of liver hydatid,and then couple it with superparamagnetic iron oxide nanoparticles(SPION)to form the SPION-EPC1 complex tracer,and combine it with fluorescein isothiocyanate(FITC)to form the FITC-EPC1 complex tracer,Two different complex tracers were obtained(2)The morphological characteristics,particle size,surface potential and particle distribution of SPION tracers before and after coupling EPC1 were observed using dynamic laser light scatterometer(DLS),nanoparticle size Zeta analyzer,transmission electron microscope(TEM)and other detection instruments.The fluorescence spectrum range of FITC and the coupled EPC1-FITC complex tracer was measured by Tecan multifunctional enzyme marker.2.In the second part,this part is mainly the experiment of cells in vitro.(1)First,it is envisaged to verify the combination of the recombinant surface EPC1 antigen and antibody of hydatid at the cell level to achieve targeted imaging.Therefore,the overexpression vector of liver hydatid EPC1 is constructed first,and then the overexpression vector of EPC1 is transferred into three different cells,namely,human non-small cell lung cancer cell NCI-H1299,human liver cancer cell Hep G2 Human normal hepatocyte HL7702.(2)QPCR was used to detect whether the EPC1 vector was transferred into three cells and whether the gene level was expressed.WB was used to detect whether the EPC1 vector was transferred into three cells and whether the protein level was expressed.If the cells can express the recombinant surface EPC1 antigen of hydatid disease after being transferred into the over-expression vector,then the targeted imaging effect of SPION-EPC1 and FITC-EPC1 complex tracer will be further verified at the cell level.If the cells do not express the recombinant surface EPC1 antigen of liver hydatid disease after being transferred into the over-expression vector,then the human normal hepatocyte HL7702 will be finally selected for subsequent relevant verification at the cell level,namely(3).(3)Use CCK8 kit to test the toxicity of five different concentration groups of SPION,SPION-EPC1,FITC and FITC-EPC1.The effects of SPION,SPION-EPC1,FITC and FITC-EPC1 on cell proliferation,apoptosis and cell cycle were detected by flow cytometry.Finally,the confocal observation was used to analyze whether FITC and FITC-EPC1 entered the cell and the position of adsorption in the cell,and Prussian staining was used to observe the situation of SPION and SPION-EPC1 entering the cell and the position of distribution in the cell.3.The third part,this part is mainly the animal model experiment in vitro:(1)The C57 mouse model of hepatic alveolar echinococcosis(HAE)was established by the method of hepatic portal vein infection.After 9～10 weeks of modeling,the mice of hepatic alveolar echinococcosis with successful membrane formation were selected for subsequent relevant experimental detection and verification.(2)The mice with alveolar hydatid cyst of the liver were divided into control group and experimental group by random number table,with 10 mice in each group.The two groups were respectively treated with SPION tracer and SPION-EPC1 complex tracer,and then the intrahepatic lesions were observed by magnetic resonance scanning at two time points,one hour and four hours after the injection of the tracer,The T2 signal intensity and signal noise ratio(SNR)of hepatic alveolar hydatid lesions at two time points after injection of contrast agent were compared with those of plain scan.(3)The isolated liver fluorescence imaging method was used to observe and detect the liver alveolar echinococcus in mice with successful membrane formation.The control group used FITC tracer,and the experimental group used FITC-EPC1 complex tracer.The effect and difference of the liver lesions in vitro development were observed at two time points,30 minutes and 60 minutes after the injection of the tracer.(4)HE staining was used to detect the liver pathology of mice with hepatic alveolar echinococcus,immunohistochemistry was used to detect the expression of recombinant surface antigen protein EPC1 of hydatid,Prussian blue staining was used to detect the deposition of SPION and SPION-EPC1 in liver lesions.Results: 1.The first part:(1)The EPC1 gene was screened from the c DNA library of liver hydatid disease,and cloned into p ET-41b(+)vector.The glutathione S-transferase fusion EPC1 protein(r EPC1-GST)was expressed.It was used for immunoblotting to detect the serum of mice with liver hydatid disease.The sensitivity of 92.2%and specificity of 95.6%were obtained,which was higher than the positive rate of natural Ag B(84.5%),Therefore,the recombinant surface antigen EPC1 of hydatidosis can be used as a specific diagnostic antigen for liver hydatidosis,and the concentration of EPC1 monoclonal antibody CP30 prepared is 1mg/ml.(2)Dynamic light scattering: the hydrodynamic size of carboxylated Fe3O4 nanoparticles,the average particle diameter of Z-average is 49.26 nm,the average particle size Intensity calculated by the proportion of light intensity distribution is61.63 nm,the average particle size Number calculated by the quantity distribution is30.09 nm,and the polydispersity coefficient is 0.23.(3)The hydrodynamic size of carboxylated Fe3O4 nano-coupled with EPC1 antibody has an average particle diameter of 54.53 nm,an average particle size of 51.04 nm calculated by the proportion of light intensity distribution,an average particle size of 32.47 nm calculated by the number distribution,and a polydispersity coefficient of 0.29.(4)The Zeta potential of carboxylated Fe3O4 nanoparticles is-24.91 m V.The Zeta potential of carboxylated Fe3O4 nanoparticles coupled with EPC1 antibody was-11.96 m V,and the surface carboxyl group consumption was used to couple the antibody,and the potential shifted forward.In this experiment,the potential value of the nanoparticles after coupling is higher than that before coupling,which indicates that the antibody coupling is successful and effective,and the antibody on the coupling has a significant impact on the surface potential of the particles.(5)The fluorescence spectrum of EPC1-FITC antibody tested by Tecan multi-function enzyme marker is 488 nm,the emission wavelength is515nm～650nm,and the maximum emission wavelength is 520 nm.2.The second part:(1)The EPC1 overexpression vector was transferred into three different cells,and the EPC1 of the three cells transfected with the plasmid was detected by QPCR at the gene level.WB detection showed that EPC1 vector was transferred into three cell lines,and the protein level was not expressed.Therefore,it is impossible to verify the targeted binding imaging of recombinant EPC1 antigen and antibody on the surface of hepatic alveolar hydatid at the cellular level.(2)In the five groups of different concentrations in this study,when the concentration of SPION and SPION-EPC1 is 10ug/ml,the cell survival rate is the highest.The OD values of SPION and SPION-EPC1 groups at 24 h and 48 h are compared respectively.Only when the concentration is 100ug/ml,the OD values of the two groups are different.The OD values of SPION-EPC1 group at the two time points are slightly higher than that of SPION.When the concentration of FITC and FITC-EPC1 is0.1ug/ml,the cell survival rate is the highest.With the continuous increase of the concentration,the cell survival rate will gradually decrease.However,comparing the OD values of FITC and FITC-EPC1 at the two time points of 24 h and 48 h,there is no difference between the two groups in five different concentration groups.(3)The results of flow cytometry showed that there were differences between SPION and SPION-EPC1 in the G2/M phase of cell growth,and the proportion of cells in SPION-EPC1 group was higher;Compared with the apoptosis rate of SPION and SPION-EPC1 cells,there was a statistical difference between the two groups only in the proportion of early apoptotic cells,and the proportion of SPION-EPC1 cells was smaller.There was no statistical difference between FITC and FITC-EPC1 on cell cycle growth.Compared with the apoptosis rate of FITC and FITC-EPC1,the apoptosis rate of late,early and early+late apoptosis in the two groups were statistically different,and the apoptosis rate of FITC-EPC1 group was lower.(4)Confocal observation: FITC-EPC1 complex tracer with small molecule before coupling can enter the cell through the cell membrane,while FITC-EPC1 complex tracer with large molecule after coupling cannot enter the cell through the cell membrane.(5)Prussian blue staining was used to observe that SPION was small molecule that could enter the cell before coupling,and SPION-EPC1 complex tracer after coupling was large component,which could not enter the cell through the cell membrane.3.Part III:(1)A total of 30 C57 mice were selected for this study to infect hepatic alveolar hydatid through hepatic portal vein.Among them,20 mice were successfully screened by abdominal ultrasound for membrane formation.(2)The control group was injected with SPION tracer,and the experimental group was injected with SPION-EPC1 complex tracer.The results showed that the T2 signal intensity and the SNR value of the lesions in the SPION-EPC1 experimental group were lower than those in the plain scan at 1 hour and 4 hours after the injection of the tracer.Compared with the plain scan,the T2 signal intensity and the SNR value of the lesions in the SPION control group decreased only at 1 hour after the injection of the tracer,while the T2 signal intensity gradually recovered at 4 hours,It shows that complex tracer can make the development of intrahepatic lesions more effective and lasting.(3)Fluorescent imaging part: the control group used FITC fluorescent developer,and the experimental group used FITC-EPC1 complex fluorescent developer.The results showed that the complex tracer could combine with the EPC1 antibody of the hydatid recombined surface in the hepatic alveolar hydatid,accurately display the focus,and increase the fluorescence imaging of the focus.(4)HE staining showed that the fibrous wall of hepatic alveolar hydatid cyst was enlarged,the structure of hepatic lobule was unclear,and there was obvious inflammatory cell reaction zone between normal hepatocytes and alveolar hydatid cyst wall.Immunohistochemical examination showed that there was no clear expression of EPC1 in the liver tissue of normal mice group,but there was significant expression of EPC1 in the liver tissue of mice model group of hepatic alveolar hydatid disease.Prussian blue staining showed that SPION-EPC1 could appear and concentrate in the lesions,while SPION only appeared in a small amount in the lesions.Conclusion: 1.The recombinant surface EPC1 overexpression vector was constructed and transferred into human non-small cell lung cancer cell NCI-H1299,human liver cancer cell Hep G2,and human normal liver cell HL7702.The results of QPCR showed that the three groups of cells EPC1 were transcribed after transfection of the plasmid,but the WB test showed that the EPC1 vector was transferred into three cells,and the protein level was not expressed,Therefore,the feasibility and reason of realizing the overexpression of recombinant surface antibody EPC1 against liver hydatid in vitro need further study.2.It is mainly aimed at the specific recombinant surface antibody EPC1 of liver hydatid,which is coupled with SPION to obtain SPION-EPC1 complex tracer with negative potential and good magnetic sensitivity.It can be combined with EPC1 in liver hydatid to achieve targeted imaging,generate stable nuclear magnetic signal,improve sensitivity,effectively reduce T2 signal intensity,reduce SNR value of lesions,and last for a long time,so that smaller lesions can be more easily displayed,so as to achieve early diagnosis Early treatment.3.FITC-EPC1 can realize the targeted imaging of recombinant surface antigen antibody EPC1 of hydatid cyst,increase the fluorescence development of mouse lesions,which is better than FITC development,and has good biological safety.