Construction Of CRISPR/Cas9 Editing C-erbb-2 Gene Ultrasound Microbubble Targeting Vector And Its Effect On Proliferation And Metastasis Of Endometrial Cancer Cells

Objective: Constructing cationic microbubbles containing CRISPR/Cas9 plasmid,optimize the preparation parameters and ultrasonic irradiation conditions,observing the in vitro and in vivo angiographic effects of cationic microbubble carrier.Using low-frequency ultrasound microbubble transfection method to detect the expression of target protein C-erbb-2 after transfection,and its effect on EC cells proliferation,invasion and metastasis at the cellular level,provide experimental basis for developing a new type of high-efficiency and small side-effect visualization therapy for endometrial cancer targeted therapy to providing experimental basis.Method: 1.Prepare neutral microbubble(NMB),cationic microbubbles(CMB) by mechanical shock method;2.Construct CRISPR/Cas9 plasmid for C-erbb-2;3.Testing NMB,CMB basic characteristics,physical properties and in vitro and in vivo contrast effects;4.Combine CRISPR/Cas9 plasmid with CMB(CMB-CRISPR/Cas9),ultrasound irradiation Cas9 plasmid was transfected into HEC-1A cells of endometrial carcinoma under low frequency.5.After successful transfection,the proliferation,migration and invasion of endometrial carcinoma HEC-1A cells were detected by CCK8,cell cloning,scratch and transwell assay.Results: 1.The binding efficiency and targetability of CRISPR/Cas9 plasmid and cationic microbubbles(CMB-CRISPR/Cas9) were observed under the microscope.2.NMB,CMB were well angiographically in vitro and in vivo.3.In vitro experiments,by continuously optimizing the transfection parameters,the transfection was successfully promoted under the conditions of transfection irradiation of 30 s and 0.75W/cm~2.4.After Successfully transfected C-erbb-2 CRISPR /Cas9 plasmid,the proliferation,migration and invasion ability of endometrial cancer HEC-1A cells were significantly weaker than those in the control group,and there were statistical differences.Conclusion: CRISPR/Cas9 plasmid and cationic microbubbles(CMB-CRISPR/Cas9) have good imaging function,which can effectively transfect endometrial cancer HEC-1A cells,and successfully target knockout C-erbb-2 genes.In the endometrial cancer HEC-1A cells,the proliferation,migration and invasion of HEC-1A cells were significantly attenuated after targeted knockout of C-erbb-2 gene.