Construction Of Novel Methods For Exosomes Detection And Their Applications In Monitoring Regulation Of Active Ingredients Of Traditional Chinese Medicine On Exosomal MiRNA

Tumor is a kind of disease that seriously threatens human health and life.The pathogenesis is complex,so it is not easy for tumor to be detected in the early stage.Once cell spreads and metastasis occurs,it is difficult for patients to gain the best opportunity for treatment.Therefore,early detection and early treatment are particularly important in the treatment of tumor.In recent years,researchers have found that exosomes are closely related to tumors.Exosome is a kind of nano-scale vesicles with a particle size of 30?150 nm,which exists widely in blood,urine,saliva and other body fluids and has high homology with its source cells.Both normal cells and cancer cells can secrete exosomes,but cancer patients have more exosomes in their blood,making exosome ideal as a tumor marker.In addition,the exosomes secreted by tumor cells have the ability to promote tumor cell invasion and metastasis.Exosomes can be secreted in large quantities under the stimulation of some radiotherapy and chemotherapy drugs,inducing tumor cell diffusion and metastasis,leading to chemotherapy failure and bringing great pain to patients.Therefore,it is very important to find safe,low-toxic and effective antineoplastic drugs and to pay attention to the regulation of drug on exosome.Traditional Chinese medicine(TCM)is the treasure of the Chinese nation,which has gradually formed its own unique theoretical system and application methods in thousands of years of development.Some TCM and their active ingredients can effectively inhibit the growth of tumor cells through the regulation of exosome,and the side effects are small,safe and effective,so they have become the hotspot of research.The exosomes secreted by malignant tumor cells can transform normal cells into tumor cells or further improve the malignant degree of tumor cells through intercellular communication,which is a potential therapeutic target.It is reported that the active ingredients of TCM can intervene in all aspects of tumor therapy through the intervention of exosome,change the microenvironment of tumor cells and reduce the ability of tumor cell proliferation by inhibiting the release of exosome.Exosomes regulate cells mainly through exomal mi RNA.Exosomal mi RNA is protected from degradation by various biological enzymes in body fluids due to phospholipid bilayer membrane structure of exosome.Compared with peripheral blood mi RNA,exomal mi RNA is more stable and abundant.Its content is closely related to tumor stage,tumor metastasis and therapeutic effect,and it is a potential non-invasive biomarker in the diagnosis of malignant tumor.Therefore,by detecting the exosome and monitoring the regulation of the active ingredients in TCM on exomal mi RNA,it is of great significance to realize the early diagnosis of tumor,carry out the efficacy evaluation and guide the rational use of drugs,and further clarify the anti-tumor mechanism of TCM.Ultracentrifugation and polyethylene glycol(PEG)precipitation are often used in the separation and purification of exosomes.Ultracentrifugation can separate exosomes with high purity,but the separation process is tedious and time-consuming,and the equipment required is expensive.The exosome can be separated by PEG precipitation under low speed centrifugation,but the isolated exosome shows low purity and contains more impure proteins.At present,Nanoparticle Tracking Analysis(NTA)and flow cytometry can realize the quantification of exosome bodies,but their clinical application is limited because of the tedious operation,high cost and limited detection sensitivity.The development of reduced graphene oxide-field effect transistor(RGO FET)biosensor provides a new detection platform for biomolecular detection.As an important branch of biosensor,FET biosensor combines the characteristics of high sensitivity and high selectivity of biosensor with the advantages of easy integration and miniaturization of FET,showing great advantages in analysis and detection.Combined with nano-material RGO,the biocompatibility,carrier mobility and thermoelectric conductivity of the sensor are improved,and the detection performance is greatly improved.Label-free and highly sensitive detection can be achieved without using signal amplification strategy.In this study,a highly sensitive and label-free method for the detection of exosome is established by using RGO FET biosensor.On this basis,a detection microsystem is established,which integrates exosome separation,enrichment and exosomal mi RNA detection.Antibody-functionalized magnetic beads can be used to isolate tumor-specific exosomes efficiently and quickly.Moreover,the PNA-functionalized RGO FET is used to specifically detect exosomal mi RNA.Finally,the constructed magnetic separation-FET microsystem is used to monitor the regulation of active ingredients of TCM on exosomal mi RNA,which provides theoretical and technical support for further exploring the anti-tumor mechanism of active ingredients of TCM.This subject mainly includes the following three parts.Chapter 1:Highly sensitive label-free detection of exosome based on reduced graphene oxide field effect transistorThe FET chip was fabricated by the standard semi-conductor technology..The prepared reduced graphene oxide(RGO)was fixed on the sensing area of the FET chip by drop casting,and the RGO FET biosensor was constructed.Exosomes were isolated and purified from liver cancer cells(Hep G2)by multiple steps of ultracentrifugation.Standard characterization of exosomes was performed using TEM,NTA,and Western blot.The morphology,particle size,and conventional protein markers showed that exosomes were successfully purified.The antibody is then covalently immobilized on the FET surface.BSA is used for blocking the unreacted aldehyde groups to prevent nonspecific adsorption.Finally,when the exosomes are captured by the specific antibody,the net carrier density on the chip surface changes due to a contribution of the negative charges of the exosomes,resulting in the left shift of the Dirac point.It shows a good linear relationship between 3.3×10 ⁴ particles/m L-3.3×10 ⁹ particles/m L and the linear equation is:-?V _CNP=11.6g C-40(R ²=0.997).The detection limit of 33 particles/?L was obtained.In order to further verify the application ability of the FET biosensor in real samples,we collected the blood serum samples of six prostate cancer patients as the experimental group and the blood serum samples of eight normal patients as the control group and applied these serum samples to the anti-CD63-modified RGO FET sensor.The level of exosomes in patients with prostate cancer was higher than that in normal people.Statistically,significant differences were found between the two groups(P<0.01).Thus,this reveals that the fabricated RGO FET biosensor is able to differentiate exosome levels in cancer patients and normal people,indicating its potential as a label-free sensing tool for cancer diagnosis.Chapter 2:Integrated microsystem of FET-magnetic separation for detection of exosomal mi RNAThe FET-magnetic separation microsystem was constructed to capture the exosome directly from the plasma and detect the exosomal mi RNA.When the collected plasma was added to the microsystem,the magnetic beads functionalized by double antibodies(Ep CAM antibody and GPC-1 antibody)would bind to the exosome of pancreatic cancer plasma.After enrichment,separation and lysis,the secreted exosomal mi RNA 10b was detected by PNA-functionalized RGO FET biosensor.In order to investigate the effect of the magnetic separation system on the separation and enrichment of the exosome,the fluorescence labeled double antibodies(FITC-Ep CAM and cy5-GPC1)were combined with the magnetic beads after the exosome was captured.Compared with the control group without the exosome,obvious fluorescence signals could be seen under different excitation light,indicating that the exosome could be successfully enriched by this method.It was found that the separation and enrichment efficiency of the exosome was 73%after optimization.For mi RNA detection,the PNA functionalized RGO FET biosensor was used to detect the mi RNA10b released from the exosomes.It shows a good linear relationship between 1n M-10 f M and the linear equation is:-?V_CNP=9.77lg C+142.03(R²=0.989).The detection limit of 100 f M was obtained.In addition,the biosensor shows a satisfactory specificiy,which could effectively distinguish the complementary sequence from single base mismatched and non-complementary sequence.By using this microsystem to detect exosomal mi RNA10b from pancreatic cancer cells and normal pancreatic cells,respectively,it was found that the level of exomal mi RNA10b from pancreatic cancer cells was significantly higher than that of normal controls.In order to investigate the clinical application ability of the microsystem,the plasma samples of 10 healthy volunteers and 10 patients with pancreatic cancer were collected.The plasma exosomal mi RNA10b level of the two groups was detected by integrated FET-magnetic separation microsystem.It was found that the plasma exosomal mi RNA10b level of pancreatic cancer patients was significantly higher than that of healthy controls.The receiver operator characteristic curve(ROC)of the subjects showed that this method had high sensitivity and specificity in the diagnosis of pancreatic cancer,indicating that this method has the potential of clinical application.Chapter 3:Monitoring the regulation of active ingredients of TCM on tumor exosomal mi RNA by FET-magnetic separation microsystemIn order to investigate the regulation of active ingredients of TCM on exosomal mi RNA of pancreatic cancer cells,four active ingredients of TCM,triptolide,evodiamine,luteolin and emodin,were selected to interact with pancreatic cancer cells(PANC-1),respectively,and the exomal mi RNA was detected by FET-magnetic separation microsystem constructed in Chapter 2.Different concentrations of triptolide,evodiamine,luteolin and emodin were added during the culture of PANC-1 cells.CCK-8 method and flow cytometry were used to detect the effects of drugs on tumor cell inhibition and apoptosis,respectively.The results showed that the four active ingredients of TCM could effectively inhibit the growth of PANC-1 cells,and the apoptosis rate increased with the increase of drug concentration.In order to further study the effect of the drug on exosomes,exosomes were purified from PANC-1 cells.NTA and TEM analysis showed that the four drugs had different degrees of inhibition on the tumor exosome,and the TEM results showed that the exosome was intact after the interaction of the drug.The detection results showed that the exosomal mi RNA10b level of PANC-1 cells was down-regulated after drug treatment,and the results were also verified by quantitative real-time PCR.In the analysis of cell protein expression,it was found that after drug treatment,the expression of anti-apoptotic protein bcl2 decreased,the expression of pro-apoptotic protein bax was up-regulated,and the expression of caspase3(17kd)protein was up-regulated.The above results suggest that the drug can inhibit the growth of PANC-1cells,down-regulate the exosomal mi RNA10b,and induce tumor cell apoptosis,which may be resulted from the regulation of mitochondrial pathway to promote tumor cell apoptosis.This part of the work lays a foundation for further exploring the anti-tumor mechanism of TCM.Conclusions:1.A CD63 functionalized RGO FET biosensor was constructed to achieve highly sensitive and label-free detection of exosome.2.A FET-magnetic separation microsystem for exosome separation,enrichment and detection was constructed,which was capable of realizing the rapid,effective and specific separation of exosome and the sensitive and specific detection of exosomal mi RNA.3.The established FET-magnetic separation microsystem was used to monitor the regulation of the active ingredients of TCM on the tumor-derived exosomal mi RNA level released from exosomes,providing a new detection method and laiding a theoretical foundation for further elucidating the anti-tumor mechanism of TCM.