| There are still some difficulties to be solved in the diagnosis and treatment of renal cell carcinoma(RCC).Ultrasound microbubbles not only enable contrast enhancement of organizations,but also as targeted drug carriers which can be directed to release the drugs under ultrasonic irradiation.So it has the dual effect of diagnosis and treatment,and offers possibility for solving the difficulties of kidney cancer diagnosis and treatment.We have pre-prepared nanoscale microbubbles(nanobubble,NB)which could pass through blood vessels into the tumor tissue gap.They were confirmed to have the ability to enhance organization contrast imaging and to carry drugs and gene for therapy.On the basis of this,this study intends to prepare nanoscale microbubbles connected with renal cell carcinoma associated antigen G250 monoclonal antibodies(Mc Ab)which can target renal cell carcinoma.We aimed to provide a new method for the diagnosis and treatment of RCC.This study was divided into three parts.Part I: Preparation of stable blank,targeted nanoscale microbubblesObjective To prepare stable blank nanoscale microbubbles and targeted nanoscale microbubbles which would lay foundation for the following in vitro and in vivo experiments.Methods Blank liposome nanoscale microbubbles were prepared by mechanical oscillation.G250 monoclonal antibodies were connected to blank nanoscale microbubbles by biotin-avidin connection technology to make targeted nanosacle microbubbles.Immunofluorescence was done to verify whether the targeted nanoparticles were successfully constructed.The blood counting instrument and Malvern laser particle diameter were used to measure the concentration and particle size change of the two kinds of microbubbles after 1,3,5,10 days.Results Blank NBs were successfully prepared.And on the basis of the above,the targeted NBs were prepared.Immunofluorescence technique confirmed that G250 Mc Ab were closely connected to the nanoscale microbubbles.The prepared NBs could maintain stable for 5 days.Part II:Validation of targeting capability of the targeted nanoscale microbubbles for RCC cells in vitroObjective To verify whether the targeted nanoscale microbubbles carrying G250 monoclonal antibody have specific targeting capability.Methods Immunofluorescence technique was used to identify expression of G250 antigen in two kinds of renal cell carcinoma cells: G250 antigen-positive 786-O cells and G250 antigen-negative ACHN cells.Specific binding capacity of the two kinds of nanoscale microbubbles to the two kinds of renal cell carcinoma cells was tested by targeted cell binding assay.Resuls The targeted NBs with G250 monoclonal antibody could bind to G250 antigen-positive 786-O cells specifically,and could not bind to G250 antigen-negative ACHN cells.No combination was happened between the blank NBs and the two kinds of renal cell carcinoma cells.Part III: Experiments of the targeted NBs enhancing ultrasound molecular imaging of RCC xenografts in nude mice in vivoObjective To verify if the targeted nanoscale microbubbles have the ability of enhancing ultrasound imaging when under ultrasound irradiation on the two kinds of RCC transplanted tumors in nude mice.Methods Eight BLBA/c nude mice were divided into two groups.Four were implanted subcutaneously with 786-O cells,the other 4 were implanted with ACHN cells.Nude mice were implemented the ultrasound contrast experiment when the tumors grew up to the size about 1cm.Iu22 ultrasound diagnostic instrument and 50 mm ultra wideband linear array probe IL12-5 were used to find the largest tumor section in B mode.1.0x107 blank NBs were injected into the body from the tail vein.Then the image information was collected.After rinsing vessels with ultrapure water,1.0x107 targeted nanoscale microbubbles were injected into the tail vein and image information was collected again.Image information data was processed by QLab 8.1 software.Results There was significant difference in the average and maximum value of the ultrasound contrast imaging effect from 786-O renal cell carcinoma in nude mice between targeted NBs and blank NBs(ultrasound mean:(11.74 ± 0.52)vs(16.34 ± 0.40),p=0.001;ultrasound max:(13.07 ± 0.94)vs(18.09 ± 0.82),p=0.003).In ACHN renal cell carcinoma model,there was no statistically significant between targeted nanoscale microbubbles and blank nanoscale microbubbles(ultrasound mean 9.01 ± 0.99 vs 9.82 ± 0.67(p=0.204);ultrasound maximum 13.04 ± 0.42 vs 13.05 ± 0.30(p=0.866)).Conclusions The targeted nanoscale microbubbles with G250 monoclonal antibody were successfully prepared in this study.The targeted NBs were confirmed to have the ability of binding to G250 positive expressing renal cell carcinoma cells in vitro specifically and enhancing ultrasound imaging significantly in vivo. |
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