Preliminary Study Of The Effect Of Exosomes Derived From Cervical Squamous Cancer Cell On Angiogenesis In Tumor Microenvironment

BACKGROUDCervical cancer,that derived from cervical epithelial tissue,is one of the most common malignancy of women's reproductive systems.Among all kinds of gynecologic malignancies,the morbidity of cervical cancer is only second to breast carcinoma.It is reported that the mortality rate of cervical cancer tend to be younger in average age in recent years,which is a huge danger to female life and fitness.It has been confirmed that the long-term chronic infection of HPVH(human papillomavirus high)is closely related to the progress of cervical cancer.While at present the pathogenesis of cervical cancer are still not completely clarified.So further study of the specific of the molecular mechanism of the pathogenesis and useful therapy of cervical cancer is the research direction of present clinical prevention and treatment of cervical cancer.Tumor microenvironment is external environment surrounding tumor cells.It plays key role in the development of tumor through interaction with tumor cells.Angiogenesis in tumor microenvironment,the basis of tumor development and metastasis,is definitely the key event of the development of malignancy.Endothelial cells,the key cells of microenvironment,are effective target of tumor angiogenesis.The functional change of endothelial cells is basis of tumor angiogenesis.Exosomes are biological functional extracellular vesicles(EVs)shed from the budding of the plasma membrane,which can mediate intercellular communication of materials and bioinformation through a variety of ways in microenvironment.After released to microenvironment,exosomes interact with target cells through endocytosis or membrane fusion,which is a new intercellular communication way.A growing number of research indicate that exosomes derived from cancer cells play important roles in the tumor angiogenesis.Exosomes contain a variety of protein?lipid?nucleic acid and other biological functional elements.Extensive studies indicate that microRNAs abundant in exosomes are important exosome molecules mediate intercellular communication.RESEARCH CONTENTS:1.Identification and biological activity evaluation of exosomes secreted by cervical squamous cancer cells1.1 Extraction and identification of Siha-Exo and Ectl-Exo Produced non-exosomes FBS(Fetal Bovine Serum)and prepared DMEM medium including 10%non-exosomes FBS to cultivate cervical squamous cancer cells(Siha)and the immortalized cervical squamous epithelial cells(Ect1),collected cell culture supernatant,then extracted exosomes secreted by the Siha and Ectl through a series of different ultracentrifugation.After ultracentrifugation,white briquette,the size of a rice grain,was detected adhere to the shell of pipe.Typical cup-shaped vesicles with lipid bilayer and a size range of 30-100nm of Siha-Exo and Ectl-Exo were detected by transmission electron microscopy.And the expression of marker protein of exosomes were identified by western blot,the result indicated that both Siha-Exo and Ectl-Exo expressed exosomal marker protein CD63 and CD81.1.2 Biological activity evaluation of Siha-Exo and Ectl-Exo Green fluorescent PKH67-labeled Siha-Exo and Ectl-Exo were respectively co-cultured with HUVECs in the same concentration.48h later,both Siha-Exo and Ectl-Exo were uptaked by HUVECs,shown as green fluorescent PKH67-labeled exosomes distributing in the cytoplasm,detected by confocal images.2.Identification of the effect of exosomes secreted by cervical squamous cancer cells on angiogenesis of HUVECs2.1 Identification of the effect of Siha-Exo on angiogenesis of HUVECs through cell proliferation,cell migration and tube formation in vitro experiments Siha-Exo and Ectl-Exo extracted by different ultracentrifugation are respectively co-cultured with HUVECs in the same concentration.48h later,the effect of Siha-Exo and Ectl-Exo on proliferation,migration and tube formation ability of HUVECs were detected through CCK8,Transwell Migration assay and In-vitro Matrigel tube formation assay.The results showed that the in vitro proliferation,migration and tube formation ability of HUVECs in Siha-Exo group was significantly higher than Ectl-Exo group and blank group,the difference between Ectl-Exo group and blank group was insignificant.2.2 Identification of the effect of Siha-Exo on angiogenesis of HUVECs through In-vivo Matrigel Plug Angiogenesis assay Siha-Exo and Ectl-Exo extracted by different ultracentrifugation were respectively injected with HUVECs in the same concentration to the inguinal region of nude mice,HUVECs in the blank group were co-injected with PBS.7 days later,matrigel bolt were taken out,embedded and cut into slices,further detected the effect of Siha-Exo and Ectl-Exo on tube formation of HUVECs through hematoxylin eosin staining(HE staining).The result showed that the number of tube of matrigel bolt in Siha-Exo group was significantly higher than Ectl-Exo group and blank group,the difference between Ect1-Exo group and blank group was insignificant.3.Extraction and component analysis of Siha-Exo RNA and Ectl-Exo RNA3.1 Extraction and quality detection of Siha-Exo RNA and Ectl-Exo RNA RNA were extracted from Siha-Exo?Ectl-Exo?Siha and Ectl follow the instruction of miRNeasy Kit and the quality of RNA were analyzed through Agilent2100 Detector.The results showed that the main ingredients of Siha-Exo RNA and Ectl-Exo RNA were sRNAs.3.2 Component annotation of Siha-Exo RNA and Ectl-Exo RNA The result of High throughput sequencing showed that microRNAs accounted for considerable proportion in both non-coding RNA of Siha-Exo and Ectl-Exo.