Study Of Ultrasound Targeted Nano Microbubbles With STEAP-1 Antibody To Prostate Cancer In Vitro

PART? Culture of prostate cancer cells and Xenotransplanted tumors in nude miceObjective: To culture two kinds of prostate cancer cell lines,PC3 and LNCaP,observe the cell growth everyday.Then implant the two kinds of tumor cells into subcutaneous tissue of BALB/c nude mice,observe the size of the transplanted tumor.Methods:Remove the PC3 and LNCaP cell lines from-80 ? refrigerator,transfer to cell culture bottle after rapid rewarming in water bath pot and place in constant temperature incubator for culture,transfer the cells to a larger culture flask for continuous culture when it is in logarithmic growth and go down to the next generation,count the cell number and calculate the cell concentration when the cell density is high in the large cell culture bottle;plant a certain concentration of cell suspension on the back of the naked mouse neck which is fed in advance,observe the planting position daily,and measure the size of the subcutaneous nodule once a week,four weeks later,record the size of all transplanted tumors in nude mice,weigh the weight of each nude mouse,and calculate the mean and standard deviation of Xenotransplanted tumors.After anaesthesia,two-dimensional and color Doppler examination were performed on nude mice with ultrasonic diagnostic instrument.Results:The two kinds of prostate cancer cells grew well,and could be planted on the back of the neck of the nude mouse and forms a nodule after four weeks.The diameter of PC3 and LNCaP cell lines were 15.7mm ±0.6mm and 14.6mm ±0.7mm,respectively.After four weeks,the weight of xenotransplanted tumor nude mice was about 17.6 g ±1.2 g(The body weight of unplanted nude mice was about 22.5 g ±1.7 g,and the difference was significant,P < 0.05).The boundary of tumor displayed by ultrasound is still clear,internal echo was imbalance,blood flow signal could be showed by CDFI.Conclusion:The cultured prostate cancer cells PC3 and LNCaP can grow in vitro and can be implanted subcutaneously into nude mice,and the growth of the tumor is good.Quality ultrasound imaging could be obtained.PART ? Experimental study of preparation of Common Nanobubbles and STEAP-1 targeting NanobubblesObjective: to prepare blank lipid nano microbubbles and STEAP-1 antibody targeted microbubbles,observation of targeted Nanobubbles combined with Fluorescent second Antibody labeled with FITC under fluorescence microscope,detection of stability of targeted microbubbles.Methods:Preparation of blank lipid nano microbubbles and biotinylated blank lipid nano microbubbles by rotating evaporation method,adding avidin to biotin nano microbubbles,and preparation of targeted nano microbubbles by ligation of biotinylated antibodies,observe of the characters of two kinds of nano microbubbles under microscopes;Blood cell counter to calculate concentration of microbubbles;Using Ma Erwen Laser Particle size Meter to examine the particle size and surface potential of two different kinds of nano microbubbles,the microbubbles were observed under microscope for 1 day,2 days,3 days,5 days and 1 week later,measurement of microbubbles size and comparison with each other;FITC labeled second antibodies were added to the blank and targeted nano microbubbles,respectively,and observed under fluorescence microscope;The stability of the targeted microbubbles was measured by flow cytometry before and after oscillation and compared with each other.Results:Under the optical microscope,the blank nano microbubbles and the targeted nano microbubbles were all slightly circular,the particle size was relatively uniform,and the distribution was uniform;Concentrations were about 4 × 107 and 3.6 × 106,respectively;The particle size is about 422.6nm + 53.4nm and 587.6nm + 53.5nm,respectively,There is a statistical difference between the two kinds of microbubbles(P<0.05),The surface potentials of the two microbubbles are about-14.8m V ±1.4m V and-19.4m V ±1.8m V,respectively;The average diameter of two kinds of microbubbles measured by continuous observation and after one week were about 460.5nm + 48.6nm and 618.5nm + 47.2nm,respectively,compared with the original preparation,there was no significant difference between the two groups(P > 0.05);The FITC labeled second antibody was added to the blank group and the target group,and then observed under the fluorescence microscope,in the blank group,the visual field of microbubble was lost,and green fluorescence was observed in the periphery of the target group;The binding rates of targeted nanobubbles before and after oscillation were(95.1 ± 2.7)% and(93.5 ± 2.1)% by flow cytometry,respectively.There was no significant difference in binding rates before and after oscillation(P > 0.05).Conclusion:Ordinary and targeted nano-lipid microbubbles were successfully prepared,the diameter of targeted microbubbles was larger than that of ordinary microbubbles,the stability of the two groups of microbubbles is better,targeted nano microbubbles can also specifically bind to antigens,it provides a basis for further in vitro experiments.PART ? Study on targeting ability of STEAP-1 targeted Nano microbubbles to Prostate Cancer cells in VitroObjective: to identify the expression of STEAP-1 antibody in two kinds of prostate cancer cells,to observe the binding of targeted nano-microbubbles to prostate cancer cells in vitro under microscope,and to observe the binding of targeted microbubbles to cancer cells again after antibody saturation test.Methods:Two kinds of prostate cancer cells were cultured,and cell climbing tablets were prepared.In each group of cancer cells,5 copies of crawling tablets were made,and then the cells were fixed with 4% paraformaldehyde and then blocked with 1% BSA block solution,and then a certain amount of STEAP-1 antibody was added(1:1000).Then a certain amount of FITC labeled second antibody of sheep anti-mouse fluorescence was added(1:200),and the reaction was observed under fluorescence microscope for 30 minutes at room temperature.To prepare blank nano-microbubbles and targeted nano-microbubbles to be used,10 cell climbing slices of prostate cancer cell line PC3 and LNCaP were prepared respectively,divided into groups after 4% paraformaldehyde fixation and 1%BSA closure,Blank nano-bubble group(A)and targeted nano-bubble group(B),ten PC3 cell climbing slices(A1,B1)and ten LNCaP cell climbing slices(A2,B2)were randomly divided into the two groups.The blank and targeted nano-microbubbles were dripped into them,and the adhesion between the microbubbles and the cells was observed under microscope.Three pieces of PC3 crawler and three pieces of LNCaP crawler were prepared.After adding enough STEAP-1 antibody,then added STEAP-1 targeting nano-microbubbles again.The binding of microbubbles to cells was observed under microscope.Results:After adding STEAP-1 antibody and FITC labeled sheep anti-mouse fluorescent second antibody,green fluorescence was observed under fluorescence microscope.There was no obvious aggregation of microbubbles around A1 and A2 cells in blank nano-bubble group,however,the aggregation of microbubbles was observed around the A1 and A2 cells in the targeted nano-bubble group.Adding sufficient amount of STEAP-1 antibody to cell climbing tablet and then adding STEAP-1 targeting nano-bubble for reaction,only a few of microbubbles combined with the cells in the two groups by microscopic.Conclusion:Both PC3 and LNCaP can express STEAP-1 antigen in prostate cancer cells.The STEAP-1 antibody targeting nano-microbubbles can bind to prostate cancer cell line PC3 and LNCaP in vitro.