| Cancer is currently recognized as a worldwide health problem.How to achieve early diagnosis and treatment of cancer is an issue that needs to be solved today.RNA interference(RNAi)can affect function and expression of genes.The discovery ofRNAi makes it possible to target many inaccessible genes and treat intractable diseases.RNAi is also important for personalized biomarker diagnosis and gene therapy for cancer.In this thesis,we mainly useRNAi as the research object,focusing on the expression level of miRNA and the intercellular transition of siRNA.We established an ultrasensitive biochip for circulating miRNA detection in serum,which is expected to be used for early diagnosis of cancer.We have also fabricated an efficient and low toxicity siRNA intracellular delivery system that is expected to achieve cancer treatment.miRNAs are 19-23 base single-strandedRNAs,which have been shown to be widely involved in a series of biological processes such as early development,cell differentiation,proliferation and apoptosis.Abnormal expression of miRNAs is believed to be closely associated with human diseases,genetic diseases and changing in immune system function.Recent reports indicate that miRNAs are expected to be biomarkers for cancer diagnosis and prognosis,classification,staging,progression and therapeutic response.In particular,the detection of of serum miRNAs expression profile is of great significance for the early diagnosis of diseases.We designed a photonic crystal assisted rolling circle amplification biodiagnostic biochip(PCs-RCA)for the detection of miRNAs in serum.We measured the fluorescence enhancement effect of this biodiagnostic biochip,which enables ultrasensitive detection of miRNAs with detection limits as low as 0.7 a M.This biochip has high sensitivity and good specificity.It can distinguish the single base discrimination of miRNA and can accurately quantify the concentration of miRNA in clinical serum samples.At the same time,this biochip can effectively distinguish between serum from non-small cell lung cancer(NSCLC)patients and healthy human.This PCs-RCA biochip can detect bioactive molecules with high sensitivity and good specificity,so it is of great significance in clinical diagnosis of diseases and health assessment.Gene silencing by siRNA delivery opens a new pathway for characterizing gene functions and treating various diseases.However,due to the large molecular weight,negative charged,easy RNase degradation,easy renal clearance,and lack of bio-targeting,siRNA treatment is inseparable from the assistance of vehicles.In view of the high toxicity of traditional vehicles and the low efficiency of siRNA delivery,we designed DNA nanoparticles based on rolling circle amplification and applied them to siRNA delivery.After successfully synthesizing DNA nanomaterials,we verified the efficiency of the gene silencing.Using laser confocal and flow cytometry,we found that this method has the same gene silencing efficiency as the commercial reagent lipofectamine 2000.Further cytotoxicity experiments have demonstrated that DNA nanoparticle-based transfection reagents have low toxicity and can achieve large cell viability even under high-concentration of siRNA transfection,which was consistent with the high biological compatibility of DNA nanomaterials.This transfection reagent has high transfection efficiency and low toxicity,so we expect that this strategy will provide a promising platform for siRNA delivery system and may promote the clinical application of siRNA therapy... |
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