Isolation And Verification Of Osteosarcoma Exosomes Targeting Lung Metastasis And Establishment Of Signal For Homogeneous Detection Of Exosomes

The exosomes are extracellular vesicles with a diameter of 30-150 nm,which originate from the endosomes.During the late mature stage,late endosomes(also called multivesicular bodies)membrane merges with the cell membrane to secrete exosomes into extracellular and peripheral blood.There are many types of molecules in exosomes,including proteins,lipids,RNA,and DNA.Exosomes are involved in intercellular substance exchange and information transmission,playing important biological functions.Many studies have shown that exosomes are closely related to tumor progression and metastasis,and certain specific proteins expressed in exosomes can serve as specific markers for certain tumors to target specific organ metastases.Exosomes that have been verified to express specific proteins are expected to develop into blood markers for tumor metastasis.Osteosarcoma is a primary malignant tumor of bone tissue.Its basic diagnostic feature is that malignant tumor cells produce tumorous bone and bone-like tissue.It is the most common malignant tumor with a high degree of malignancy.It mostly occurs in adolescents and is prone to tumor metastasis targeting the lung in the early stage.Due to lacking early diagnosis techniques,it seriously affects the prognosis of patients,endangering the physical and mental health of adolescents.Thus,exploring and establishing an effective detection way of early diagnosis of osteosarcoma and early warning of lung metastasis are particularly important.Considering the important role of exosomes secreted by tumor cells in the blood on the occurrence,development and metastasis of tumors,close correlation between exosome specific protein expression and metastatic target organs,the characteristic of osteosarcoma prone to lung metastasis,in order to explore and verify the role and mechanism of osteosarcoma-derived exosomes in lung metastasis,and to establish a new technique for early diagnosis of osteosarcoma lung metastasis with exosomes as markers,the following studies were conducted.CHAPTER ONE ISOLATION AND IDENTIFICATION OF EXOSOMES DERIVED FROM OSTEOSARCOMA CELLObjective: Culturing a variety of osteosarcoma cell lines,isolating and characterizing exosomes from osteosarcoma cell supernatants,and verifying CD63 is localized on the membrane surface of osteosarcoma exosomes.Method: Selecting some kinds of osteosarcoma cell lines(HOS,MG63,KHOS-240 S,Saos-2)to culture and collecting cell supernatants.Exosomes were isolated by ultracentrifugation and exosome isolation kits;Using transmission electron microscope to observe the morphology of negatively stained exosomes,nanoparticle tracking analysis technique to measure the size and concentration of exosomes and western blotting to verify the characteristic protein markers in exosomes.The secondary antibody labeled with nanogold was specifically bound to the anti-CD63 first antibody,and the surface of the exosomal membrane was observed by TEM to see if gold particles were bound.Results: Exosomes isolated by ultracentrifugation revealed a milky white precipitate at the bottom of the centrifuge tube.Under the transmission electron microscope,a typical extravesicular structure with a circular,elliptical shape and a diameter of about 50-150 nm can be observed,and a cup-shaped structure can be seen.Through nanoparticle tracking analysis,we can acknowledge that the exosomes diameter is mainly distributed in the range of 70-200 nm,its concentration is 9.55×108/m L,the peak diameter is about 127 nm,and the average diameter is about 150 nm.Western blotting confirmed the expression of CD63 and TSG 101 in osteosarcoma cells and their exosomes.By immunogold labelling and TEM,a typical extravesicular structure with a circular,elliptical shape and a diameter of about 50-150 nm can be observed.There are several small black dots with a diameter of about 10 nm on the surface of the vesicle membrane.Conclusions: Osteosarcoma cells are capable of secreting exosomes and exosomes derived from osteosarcoma cells have been successfully isolated.CD63 is localized on the exosomal membrane rather than inside exosome.CHAPTER TWO ISOLATION AND IDENTIFICATION OF EXOSOMES DERIVED FROM OSTEOSARCOMA CELLObjective: To very the expression of integrin in osteosarcoma exosomes.Exploring the mechanism of exosomes mediating lung metastasis of osteosarcoma in vivo.Method: The expression of integrin gene in exosomes of osteosarcoma cells was analyzed by RT-PCR.Western blotting was used to analyze the expression of integrin associated with lung metastasis in exosomes of osteosarcoma.Using the isolated exosomes as a medium,an animal model of osteosarcoma lung metastasis was established in nude mice to study the effect of osteosarcoma exosomes on the local microenvironment of lung tissue.Exosomes from osteosarcoma patients serum were isolated with kit,and blood samples were detected by TEM and western blotting.Results: RT-PCR confirmed that the expression levels of integrin ?6 and ?1 in osteosarcoma exosomes were significantly higher than those in normal osteoblast exosomes.It was also confirmed that integrin protein was expressed in exosomes of osteosarcoma by western blotting.The immunohistochemical results of lung tissue in nude mice showed that the expression of ITG ?6,MMP-2 and MMP-9 in lung tissues of nude mice treated with osteosarcoma exosomes was significantly higher than that of the blank control group.In the blood of osteosarcoma patients,exosomes also express the specific protein marker of CD63 and integrin ?6.Conclusions: Integrins are elevated in osteosarcoma exosomes compared to normal osteoblast exosomes.Exosomes secreted by osteosarcoma cells can change the local microenvironment of lung tissue,which is conducive to the colonization and metastasis of osteosarcoma.CHAPTER THREE THE ESTABLISHMENTOF HOMOGENEOUS DETECTIONSIGNALS for EXOSOMES DERIVED FROM OSTEOSARCOMA CELLObjective: Obtaining a stable and reliable nitrogen-doped carbon quantum dots(N-CDs),clarifying the relationship between fluorescence changes of N-CDs and the value of p H under a homogeneous system,and establishing a fluorescent signal system for detecting exosomes derived from osteosarcoma based on N-CDs.Method: Using citric acid,glutathione and polyethene polyamine as materials to synthesize a nitrogen-doped carbon quantum dots by simple two-step thermal decomposition method.The morphology of N-CDs was characterized by transmission electron microscopy.The functional groups,elemental structures and compositions of N-CDs were studied by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy.The optical properties of N-CDs were analyzed by UV-visible absorption spectroscopy and fluorescence spectroscopy.A series of different concentrations of salt solution were set up to detect ionic strength tolerance of N-CDs.Continuously irradiating N-CDs at the optimum excitation wavelength to detect the photobleaching resistance of N-CDs.Setting a series of different p H reaction systems to detect the change of fluorescence intensity of N-CDs and analyzing the relationship between the fluorescence intensity of N-CDs and the value of p H.Introducing the p H variable system of urease-catalyzed urea decomposition,and the urease concentration was determined by the established relationship between the fluorescence intensity of N-CDs and p H.Results: The synthesized N-CDs are nearly uniform spherical particles with an average particle diameter of 5.6 nm.The typical lattice space distance is 0.22 nm.N-CDs are mainly composed of C,N,O and S,and their surfaces are rich in-NH2 and-OH.The water-soluble N-CDs are yellow in sunlight and emit blue fluorescence under ultraviolet light.Under the excitation of the optimum excitation wavelength of 350 nm,N-CDs have the maximum emission fluorescence intensity at 450 nm.It can withstand the interference of high-intensity ions and has a good anti-light bleaching property.The maximum fluorescence intensity of N-CDs at 450 nm gradually decreased with the increase of the value of p H.When the p H ranged from 3.0 to 9.0,the fluorescence intensity of N-CDs has a good linear relationship with the p H value.The linear equation is Y =-97.924 [p H] + 1043.85,R2 = 0.9888.When the urease concentration range is 5-800 U/L,the fluorescence intensity of N-CDs has a good linear relationship with the urea concentration.The linear equation is Y=-0.2809Curease(U/L)+ 487.276,R2 = 0.9960,and the detection limit is calculated to be 4.5 U/L.Glucose,cysteine and other interfering substances hardly affect the detection of urease by N-CDs.Conclusions: The nitrogen-doped carbon quantum dots whose fluorescence intensity can be sensitively adjusted with p H were successfully obtained.By introducing urease and urea reaction system,a sensitive and reliable homogeneous signal response system for the detection of osteosarcoma exosomes in peripheral blood was provided.