| Background and objectiveStem cells possessing strong proliferative capacity and differentiation potential have been gradually used in clinical application recent years.Human umbilical cord mesenchymal stem cells?uMSC?are becoming the research hotspot because of their advantages of easy acquisition,strong proliferation,and low immunogenicity.Thus,the exosomes secreted by uMSC have also become one of the research focuses.Exosomes secreted by cells are vesicles with lipid bilayer structure and are 30–150nm in size.Since the discovery of exosomes,their functions have gradually been understood.There are many studies reported that exosomes can carry some biosignal molecules and participate in intercellular communication.However,most of the researches on exosomes focus on small molecules such as Long non-coding RNA?lncRNA?and microRNA?miRNA?,but the proteins in exosomes are lack of attention.In eukaryotes,more than one-fifth of the proteins belong to glycosylated proteins.They are involved in many biological functions such as cell recognition,intercellular signaling and cell adhesion.The structure of glycoproteins is complicated because the structure of its saccharide chain is complex and variable.Unlike DNA transcription,glycosylation of proteins does not have corresponding template,but relies on enzymatic reactions to covalently attach monosaccharides or polysaccharides to the protein backbone,which brings diversity and heterogeneity to glycoproteins.And the classification of glycoproteins is also related to saccharide chain.According to the linkage sites between protiens and accharide,glycoproteins can be classified into N-glycosylated proteins,O-glycosylated proteins,C-glycosylated proteins and so on.Glycoproteins on the cell surface can promote cell recognition,signal transduction,and cell adhesion.Exosomes,as a medium of intercellular communication,also involve recognition between cells and exosomes.The role of glycoproteins in the exosomes is worthy of our study.Hepatic fibrosis is a transitional stage in the development of hepatic chronic disease to cirrhosis.In recent years,there is still no particularly effective methods for the treatment of hepatic fibrosis.Conventional drugs,such as Angiotensin converting enzyme Inhibitors?ACEI?,TGF-related factor antagonists,interferon?INF?,usually have limited effect,and liver transplantation is the most effective treatment,but it faces the problem of donor shortage.It was reported that exosomes can promote curing diseases,and glycoproteins can be used to modify the drugs to enhance their efficacy.Thus,whether the glycosylation protein in uMSC exosomes contribute to exosomes to the treatment of disease,and whether the exosomes of uMSC can also play a role in the treatment of liver fibrosis is unknown.These issues are worth exploring and researching.CRISPR?clustered regular interspaced short palindromic repeats?is a cluster of regularly spaced short palindromic repeats.It is a DNA sequence presented in the bacterial genome.Cas?CRISPR associated?is a CRISPR-related gene.There are many types of this gene,and Cas9 is one of them.The CRISPR-Cas9 system originally found to be an acquired immune mechanism for bacteria can cleave invasive foreign DNA.Later,It was used for precise genetic editing,and its effect is superior to traditional genetic editing methods.In our previous study,CRISPR-Cas9 technology was used to knock out intracellular glycosyltransferases.Hoever,we found that the cells exogenously introduced Cas9 vector could proliferate faster than cells without vector.Usually,Cas9 vector can be transcribed into mRNA in the cell.Thus,whether the Cas9 mRNA,as an exogenous nucleic acid substance,can affect the intracellular environment and even affect the safety application of the technology remains unclear.The ceRNA?competing endogenous RNAs?mechanism is widely present in the biological metabolism of cells.MicroRNA can downregulate the genes expression by binding to the mRNA,but ceRNA can competitively bind microRNA through microRNA response elements?MREs?,resulting in upregulating genes expression.It was reported that mRNA can also play as a ceRNA to regulate the expression of genes.Therefore,Cas9 mRNA may also interact with some important microRNAs in the cell,and affect the entire RNA interaction network,which may thus exert an adverse influence on the cells,and even have a profound impact on the safety application of CRISPR-Cas9 technology in the future.In our research,we try to study the effect of uMSC exosomes on the repairation of liver fibrosis,and to explore the role and mechanism of glycosylated proteins in exosomes for the treatment of liver fibrosis.At the same time,we apply the CRISPR-Cas9 technology in our study and discover problems about safety of the technology from the perspective of ceRNA mechanism.Finally,an optimizing Cas9 sequence is found through our expeimints.Part?Study on the Glycosylation-related Mechanisms of uMSC Exosome in the Treatment of Hepatic FibrosisMethods1.The exosomes secreted by different cells collected by ultracentrifugation.And then they were examined by Western blot,qNano and TEM?Transmission electron microscope?to determine whether the exosomes we extracted were qualified and could be used for subsequent experiments.2.The repair effect of uMSC exosomes on liver damage was verified by hepatocytic injury model.After the addition of exosomes 24 hours,hepatocytic apoptosis induced by hydrogen peroxide was examined by the apoptosis kit and flow cytometry to determine whether the exosomes could inhibit cell apoptosis.In addition,Edu assays were used for detects the effect of exosomes on cell proliferation.The activation of hepatic stellate cells induced by TGF-?1and the expression of?-SMA in hepatic stellate cells was detected by qPCR and Western blot3.The exosomes stained by CM-Dil can be observed the location in the cells At the same time,the exosomes derived from different groups of cell supernatants were detected by Lectin Chip,and the difference content of carbohydrate structures was discovered in the groups.The results of the chip were verified by lectin blotting experiments.Finally,qPCR was used for detecting the expression levels of glycosyltransferases that catalyze the formation of specific glycostructures in the cells.4.The glycoprotein-specific glycostructure in the exosomes was excised by the action of exogenous sialidase.Lectin blotting experiments confirmed whether the glycosidase successfully removed the glycostructure of the exosomes.Flow cytometry and qPCR experiments were then performed to compare the effects of both glycostructure resection and uncut exosomes on hepatocyte apoptosis and hepatic stellate cell activation.5.The specific glycosyltransferase was removed by CRISPR/Cas9 technology,and the effect of the specific sugar structure on exosome was investigated.A gRNA plasmid targeting?-2,6-sialylglycosyltransferase was constructed and co-transfected into cells with Cas9 vector.The PCR verified that cells genome was successfully broken.Results1.The results of Western blot showed that the exosomes can express the specific markers,CD63 and CD9.In addition,the outcome of qNano showed that the exosomes have a particle size distribution of 118.3±37.8 nm and a concentration of 9.67x1010particles/ml.Finally,the results of transmission electron microscopy showed that the exosomes were round and about 50-100 nm in size.All of above results indicate that the quality of the exosomes we extracted is qualified.2.First,the results of flow cytometry to detect apoptosis and cellular ROS showed that hydrogen peroxide can successfully induce apoptosis and increase ROS levels in normal liver cell lines WRL68 and L02.The percentage of apoptosis cells reduced in the group of uMSC-Exo.Edu's assays found that exosomes can promote the proliferation of liver cells.Western blot and qPCR experiments suggested that exosomes can inhibite the hepatic stellate cell activation induced by TGF-?1 and expression of?-SMA3.The results of immunofluorescence experiments indicated that exosomes secreted by uMSC could enter the interior of hepatocytes.The results of the lectin chip showed that the binding rate of SNA and ConA in uMSC-Exo was significantly higher than that in the control group.The results of the lectin blotting experiments were consistent with the chip.And the results of qPCR showed that among the eight enzymes that catalyze the production of?-2,6-sialic acid sugar,the expression of ST6Gal-2 was highest in uMSC.4.After excision of the exosomes,the results of the lectin blotting confirmed that the sialidase can successfully excise the?-2,6-sialylate structure in exosomes.The exosomes excised of the sugar structure could be observed,and the results of the apoptosis and qPCR experiment found that it lost of ability to inhibite hepatocyte apoptosis and to active hepatic stellate cells.5 A gRNA plasmid targeting Exon 2 of ST6Gal-2 gene was successfully constructed using the existing vector in the laboratory.After transfection of the Cas9 and gRNA plasmids in the cells,the PCR results confirmed that the ST6Gal-2 gene was successfully cleaved.ConclusionsFrom the experiment,the uMSC exosomes can promote the treatment of liver fibrosis,which can not only inhibit the apoptosis of hepatocytes,but also promote the proliferation of hepatocytes and inhibit the activation of hepatic stellate cells at the same time.Lectin microarray suggested that the glycoprotein in exosomes may play a key role in the mechanism.And the?-2,6-sialic acid sugar structure contained in glycoprotein may have a great impact on the function of exosomes.Part?CRISPR-Cas9 Technical Security Research and Improvement MethodsMethods1.Prediction of the possibility of Cas9 mRNA interaction with microRNAs by Miranda software.By transfection,the miRNA was transferred into the cell line HEK293-Cas9 which stably expressed Cas9 protein,and the impact of miRNA on Cas9mRNA was further detected by qPCR and Western blot.On the other hand,bioinformatics analysis was used for analyzing the enrichment of genes downstream of miRNA after transfection of Cas9.The Cas9 virus was transfected into different tumor cell lines?Du145,U251,SH-SY5Y?,and the expression level of miRNA downstream genes were detected by qPCR and Western blot.2.Based on the prediction results of the previous step,it was found that Cas9 mRNA can bind to miRNAs from let-7 family members,and the majority of let-7 downstream target genes are oncogenes.Therefore,we transfected the Cas9 lentivirus into the tumor cell line Du145,and the effect of cell proliferation was detected by Cell Counting Kit-8?CCK8?,Ki-67 cell immunofluorescence assay and PI staining cell cycle assay.Next,we established a Du145 cell line stably expressing Cas9 to further study the effect of Cas9in vivo.3.Similarly,we transfected Cas9 vector into normal cells,and detected the expression of let-7 target genes by qPCR and Western blot.The proliferation of Hacat cells was detected by Edu assays and clone formation experiments.In addition,we performed high-throughput sequencing of transcriptomes from bMSC transduced with Cas9 and the control vector to gain a more systematic understanding of the effect of Cas9 on endogenous molecules.Finally,we also analyzed the expression of let7 target genes by Western blot and immunohistochemistry in Cas9 transgenic mice.4.To confirm the presence of this mechanism and optimize the current Cas9 sequence,we constructed the synonymous mutant plasmid Cas9-mut,which can be translated into the same amino acid sequence without the functional target sequence for let-7 binding.The effects of Cas9-mut on cell proliferation were detected by CCK8 assays,colony formation assay and cell cycle assay.5.To determine whether the optimized Cas9-mut still has the gene editing function,we tested the ability of Cas9-mut to knock out the GFP reporter gene.We introduced Cas9-mut and the designed GFP-gRNA into a GFP-stable 293 cell line,and the results was observed by the fluorescence microscope..Results1.Using miRanda we found that Cas9 mRNA can bind to miRNAs from let-7 family members with high affinity at multiple binding sites.The expression level of Cas9 was significantly decreased when let-7mimics were overexpressed in HEK293-Cas9 cells.We could observed that let7 target gene were positive enriched after introducing of Cas9.The results of qPCR and Western blot showed that some let7 target gene were up-regulated after transfection of Cas9 in different tumor cell lines.2.After transfection of Cas9 vector into Du145 cells,CCK8 assays,Ki-67 cell immunofluorescence assays and PI staining cell cycle assays all indicate that Cas9 could promote the malignancy in tumor cells.In vivo,we found that the tumor growth rate was higher and the tumor volume was larger in the group with stable Cas9 expression3 We introduced the Cas9 expression vector or control vector into HaCat and found let-7 target genes also changed in real-time PCR and Western blot experiments.The Edu assay showed that Cas9 promoted cell proliferation in Hacat cells.Increased colony formation of Hacat cells was also observed after the expression of Cas9 Therefore,Cas9does affect proliferation in this normal cell line.And we also found let-7 target genes have similar changes in real-time PCR and Western blot experiments in bMSC transfected into Cas9.GSEA analysis of all the let-7 target genes showed positive enriched?NES:1.12?after introducing of Cas9.GO-analysis found that differe ntially expressed genes were enriched in the RAS pathway.Next,we found Cas9 could also mildly increase representative let-7 target genes,such as Cdk6 and Kras,in brain and spinal cord tissues of Cas9-transgenic mice compared to control mice4.The cell proliferation ability of the Cas9-mut transduced cells was found to be weaker than that of cells overexpressing Cas9 in the CCK8 assay in HepG2.Although Cas9-mut can partially promote colony formation in Du145 cells compared with the control group,its activity was weaker than that of Cas9.The percentage of G2 phase cells decreased in the Cas9-mut group compared with the Cas9 group.At the molecular level,we found that Cas9-mut mildly upregulated downstream genes of let-7 at both the RNA and protein levels.5.After introducing Cas9-mut and the designed GFP-gRNA into a GFP-stable 293 cell line,some GFP-negative cells appeared,indicating that Cas9-mut still has a gene editing functionConclusionsCas9 mRNA can bind let7 through the ceRNA mechanism,thus upregulating the let-7target gene,which create the“side effect”of proliferation promotion by Cas9 mRNA in some cells.The synonymous mutation with a modified let-7 binding site of Cas9 produced weaken“side effect”of promotion of cell proliferation and oncogene expression.Our work not only provides a new aspect in understanding the mechanism of CRISPR-Cas9technology from the RNA level but also provides an improved and secure form of Cas9,which may have application potentialSummaryStem cell exosomes and CRISPR-Cas9 technology have great potential for development in medical research and are likely to be used in clinical treatment.The exosomes of uMSC can promote the repair of liver fibrosis by inhibiting apoptosis of hepatocyte and activation of hepatic stellate cell,and promoting hepatocyte proliferation at the same time.In this process,the?-2,6-sialic acid sugar structure in the exosomes may play a key role.Cas9 mRNA can bind let7 through the"miRNA sponge"mechanism,causing cell proliferation.Cas9-mut can reduce this"side effect."... |
PDF Full Text Request