Effect Of Mesenchymal Stem Cells (MSCs-Exosome) On The Differentiation Of Fibroblasts Into My Ofibroblast And Its Mechanism Study

Fibroblast (fibroblasts) is an important cell in wound repair. It is mainly derived from the connective tissue, which can synthesize and secrete collagen fibers, elastic fibers, reticular fibers and organic matrix. Myofibroblast is a highly differentiated cell derived from fibroblasts. Its function is to generate power and change the tissue tension. It is mainly responsible for tissue contraction in the wound healing or the extracellular matrix in fibrosis organse. These contractile cells can up regulate extracellular matrix proteins such as type I collagen (Collagen I), and highly expressed alpha smooth muscle actin (aSMA). Under physiological conditions, this is a reliable marker of wound healing, and is also the key to the formation of connective tissue remodeling and fibrosis. However, in pathological conditions, such as a huge trama or the presence of abnormal inflammatory response, myofibroblast differentiation was out of control. The persistance of this contidition will cause excessive contraction of wound and lead to hypertrophic scar. If happened at the activitie parts such as joint, it could lead to contracture and seriously impact the quality of patients’life. Morever, the literature indicates that it has a direct relationship with the presence of organ fibrosis and the promotion of tumor growth.Mesenchymal stem cells (MSCs) are widely used in the field of tissue injury repair based on their highly potential of proliferation and differentiation. However, there still exists a lot of problem such as lack of cell sources, a weak capabitly of proliferation and differentiation after subcultured. Compared with the preparation, the effects are not significant. It requires people to further clarify the mechanism of MSCs from a new perspective in order to provide a new target for improving the optimization of stem cell treatment strategies.Exosome as an important part of paracrine is secreted by various alive cells. Its size is about 50-150 nm, and fused with cellular membrane and released into the external environment. When the Exosome contact with the target cell, the biological molecules it carried were received in the form of endocytosis and produce effect.MicroRNA (miRNAs) which carried by the Exosome is widely concerned because of its important role in the regulation of gene expression in the biological molecules. Recent studies have confirmed that the miRNAs of the donor cells can be received by the target cells in the form of Exosome, and the related target genes will be "reprogramming". In additional, the Exosome also protects the miRNAs which carried remaining stable from high temperature, acid or alkali environment, repeated freezing and thawing or other harsh conditions. The transport mechanism of miRNAs in cell to cell has opened a new chapter in the field of miRNAs. Combined with the precise and efficient mechanism and structure for easy storage, the target treatment of Exosome-miRNAs process a high potential commercialization and conversion applications.At the present stage, the research of MSCs is in the ascendant around world. In view of the fact that the MSCs can be used to repair wound tissue defect and accelerate wound healing at the same time reduce scar formation. Considering Exosome containing the intracellular source of the active substance, we hypothesized that Exosomes are important media in the treatment of MSCs for repairing wound and mediated by miRNAs transport may play an important role in some phases.This research mainly studies the following contents:The First Part:The Identification and Abstraction for Exosome derived from umbilical cord mesenchymal stem cellsMethods:isolated exosme with graded centrifugation and filtration method. By gradually increased centrifugal force, the cells, cellular debris and larger particles were successively removed, then filterred with a pore size of 200 m of the membrane. At last the Exosome was precipitated at a speed of 100000 x g centrifugation for 2 hours. Using BCA, Western Blot, Nanosight and other techniques to identify the extraction of the Exosome is pure.Results:Western Blot showed that the expressions of Exosome’s specific marker, CD63 and CD81, were significantly increased. The Nanosight showed that the diameter of the Exosome was around 100nm, and the concentration was 2000 μg/ml.The Second Part The function of uMSC-Exosome for differentiation from fibroblasts to myofibroblastMethod:firstly constructing full thickness defect model on mouse back. uMSC group, uMSC-Exo group, PBS group and no treatment group were designed for the experiment. Measuring the wound area at 7、14、21 day to verify the wound healing rate and detected a-SMA expression to quantify the myofibroblasts’ count. Then we used TGFβ to induced fibroblast into myofibroblastfiber and intervent uMSC-Exo to analyse the change of αSMA protein expression by qRT-PCR, Western Blot and flow cytometry. The difference of cell proliferation ability was detected by scratch and cell cycle test.Results:The vivo animal model showed that there was no significant difference in wound healing rate between the uMSC group and the uMSC-Exo group, and the content of the myofibroblast was basically consistent. This indicated that uMSC-Exo could have the function like MSCs. From the in vitro cell model, qRT-PCR and Western blot were detected in uMSC-Exo can reduce the expression ofaSMA and Collagen I protains. The scratch and cell cycle test showed that uMSC-Exo could accelerate the migration and proliferation of fibroblasts.The Third Part Components identification of Exosomes and components on fibroblast proliferation and differentiationMethod:Using the enzyme protein or nucleic acid enzyme to remove the protein or nucleic acid components of Exosome and making it become to protein free Exosome (uMSC-Exo-PROnase) or nucleic acid free Exosome (uMSC-Exo-RNAse). Next we tested the difference in fibroblast proliferation ability through the cell cycle assay, and through Western Blot and qRT-PCR detecting a-SMA protein expression level differences between two groups. Last we used the 3D collagen contraction assay to test the myofibroblast contractility in both groups.Results:The gel electrophoresis showed that protains were removed using protainase and the majority nucleic acid s were smaller than 100bp, which showed that most of the uMSC-Exo nucleic acid is for microRNA. Followed by Western Blot and qRT-PCR technology show thatcafter intervention of uMSC-Exo-PROnase cells, aSMA expression was significantly decreased, while the uMSC-Exo-RNAse didn’t change significantly. This indicated that the microRNAs in uMSC-Exo mainly inhibitted myofibroblast differentiation. The cell cycle experiment showed the cell activity was significantly faster in uMSC-Exo-RNAse group compared to uMSC-Exo-PROnase group which indicated uMSC-Exo mighet accelerate fibroblast proliferation through the protein component. It is also a good proof of the first part of the vivo experiment that uMSC-Exo can accelerate the healing of the wound and also reduce the scar contracture.The Fourth Part Detection for miRNA in uMSC-Exo and prediction for its pathwayMethods:by high throughput sequencing comprehensive detection for miRNA in uMSC-Exo and the HEK293-Exosome was set as the control. Using existing GEO data (gse46989 and gse56862) to analyze the miRNA abundance of uMSC and HEK293 cells. Filterring the candidate target genes on differentiation and regulation of myofibroblast. Next through Targetscan, GO and Pathway prediction software to predicte the specific microRNAs regulating in the pathway. Last qRT-PCR and Western Blot werw applied to verify the conclusion.Results:The high throughput sequencing displayed miRNAs in uMSC-Exo. The top ten abundance were miR-21-5p, miR-125-5p, miR-23-3p, miR-100-5p, let-7f-5p, let-7a-5p, miR-145-5p, miR-1260b, miR-1260a, miR-199a-3p. MiR-21-5p. The GEO found that except miR-21-5p, the content of the other miRNAs in uMSC cells was little indicated that uMSC secrete miRNA in an activity way. Then the GO, Pathway predicted the target genes and pathways and found that the miR-21-5p, miR-125-5p, miR-23-3p, miR-145-5p were targetted to SMAD22, TGFβ2 and TGFβR2 which were important in myofibroblast formation. The TGFβ2/SMAD2 is the specific pathway.The Fifth Part The mechanism of uMSC-Exosome-miRNA on the myofibroblast differentiationMethod:First of all, we construct the plasmid of TGFβ2 and TGFβR2 and SMAD2 3 ’UTR and conducted dual luciferase report genethe with miR-21-5p, miR-125-5p, miR-23-3p, miR-145-5p mimics. After over expressing or inhibiting the specific micorRNAs in uMSC-Exo, we use qRT-PCR and Western Blot to test aSMA and SMAD2 phosphate activity. Finally in animal models, we used fluorescence in situ hybridization (FISH) to localize the exosmal miR-21-5p, miR-125-5p, miR-23-3p and miR-145-5p distribution and p-SMAD2 activation conditions.Results:The dual luciferase report gene showed miR-21-5p, miR-125-5p, miR-23-3p, miR-145-5p can directly bind with 3’UTR of TGFβ2, TGFβR2 or SMAD2, indicating that miRNAs can direct effect the protein translation level. qRT-PCR and Western Blot showed that the expression of SMAD2 and aSMA were down regulated after over expressing the miRNAs whereas after the inhibiting these miRNAs, the target genes were not statistically different from the control group. These datas indicated that miR-21-5p, miR-125-5p, miR-23-3p, miR-145-5p can downregulation aSMA via inhibiting TGFβ2, TGFβR2 and SMAD2. In vivo study, FISH experiment proved after being injected uMSC-Exo into the skin around the wound, the specific miRNAs were mainly distributed around the nucleus of cells, and could inhibit the p-SMAD2 activity so as to supress the myofibroblast formation. However the rate of wound healing was not affected.