| The bone defects caused by trauma or tumor is a common disease in the orthopedic department,plastic repair department,oral and maxillofacial surgery department,and so on.Bone cement or other artificial materials can promote the healing of mild bone defects,while larger bone defects need to be repaired by vascularized bone grafts.Common methods include iliac or fibula flap grafts,which can repair most bone defects but can lead to greater damage to the donor area.Therefore,the rapid development of tissue engineering and regenerative medicine in recent years has provided new ideas for repairing or replacing damaged tissues,seedcells are the preferred materials for researchers.Mesenchymal stem cells(MSCs)are one kind of stem cell which have the pluripotent,they can be differentiated into several tissues under a specific condition in vitro.The ability of self-renew is the basis of their differentiation for their whole life.MSCs have the potential of multilineage differentiation,such as nerve,heart,liver,bone,cartilage,tendon,fat,epithelial and other types of cells,which provides important raw material for the treatment of a variety of diseases.Mesenchymal stem cells can be obtained from fat tissue,bone marrow,periosteum,bone,umbilical cord and dental pulp.There are many advantages for MSCs,such as easy isolation,culture,proliferation and purification.They can also maintain pluripotent after long time passages,which are no immune rejection characteristics.MSCs are widely applied in the field of regenerative medicine and the treatment of autoimmune diseases.Also,mesenchymal stem cells(MSCs)have become the most promising pluripotent stem cells in clinical application due to their advantages,because oflow morale and ethical issues and easy to be prepared industrially.Mesenchymal stem cells can be divided into bone marrow mesenchymal stem cells,adipose mesenchymal stem cells(adipose-derived stem cells,ASCs),placenta mesenchymal stem cells,umbilical cord mesenchymal stem cells,and so on.Adipose-derived stem cells(ASCs)have been widely used as seed cells in clinical treatment owe to their sufficient source,obtain convenient,small trauma and high output,low risk-tumor,genetic stability,low immunogenicity,various cytokines secretion,promotion of blood vessel formation,and recruit for reconstruction,and so on.Therefore,this study selected as research materials.It has been reported that MSCs can differentiate into three lineages in vitro,includingadipogenic differentiation,chondrogenic differentiation and osteogenic differentiation,which are the major characteristic of MSCs.The ability of osteogenic differentiation of MSCs has been widely used in the repair of bone injury.As before,undifferentiated MSCswere mostly applied in the bone injury repair of MSCs.At present,several reports have shown that there is more specificity of differentiated for the replacement and repair.Therefore,the differentiated mesenchymal stem cells by specific methods have far-reaching significance in clinical application.There are many methods to induce stem cell differentiation in vitro,including compound,transcription factor induction and small-molecule induction.Although these methods can induce the differentiation of mesenchymal stem cells effectively,the specificity needs to be improved.Therefore,control of induced differentiation of MSCsrapidly and accurately is of great significance for clinical research on MSCs.Clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9 system is of interest in genomic edit owing to its convenience and operability of clinical application.Furthermore,the catalytically inactivated Cas9(dCas9)has been used for the regulation of gene expression quickly and specifically.Compared with general exogenous overexpression,the CRISPR system can be used to control multiple endogenous gene expression simultaneously,which can change the fate of MSCs,and allow MSCs to be used for more kinds of clinical applications.The osteogenic and adipogenic differentiation process isregulated by several cytokines and hormones,which are involved in complex signaling pathways.It has been reported that there is a complex link between these two differentiation processes.Several studies have shown that the imbalances of osteogenic and adipogenic differentiation will cause osteoporosis,which will generate more fat cells.Therefore,it play an important role in the MSCs’ clinical therapy.This study is mainly divided into three parts:Part Ⅰ isolation,culture and identification of adipose mesenchymal stem cellsIn this part,we isolated,cultured,and identified mouse ASCs by using enzyme and optimized tissue-isolation methods.The high purity ASCscan be obtained by these methods,which have the potential of osteogenesis,adipogenesis and chondrogenic differentiation.The morphology of ASCscan help us to understand the biological characteristics of MSCs.The results showed that ASCs had a strong proliferation and CFU(Colony-Forming Units)ability in vitro,and the proliferation ability did not change significantly after long-term passage.The surface markers of CD44 and CD90 were positive.The staining results showed that ASCs can be differentiated into osteoblasts,chondrocytes,and adipocytes.A stable system of isolation,culture,identification and induction of differentiation was established.Part Ⅱ Control of osteogenic differentiation in adipose-derived stem cells via endogenous gene activation using the CRISPR systemTo establish a method of osteogenic differentiation rapidly,CRISPR-dCas9 was used to endogenous promote the transcription of osteogenic progenitor genes(Runx2 and Osterix)in adipose-derived stem cells.The cells were identified by transcription levels and morphology analysis by RT-qPCR and Alizarin red staining.The results show that osteogenic progenitor genes could be upregulated,then the cells could differentiate towards the osteogenic lineage rapidly.The results of alizarin red staining showed that endogenous transient-overexpression of two osteogenic precursor genes significantly improved the efficiency of osteogenic differentiation.Also,detection of OPN,OCN,Coll-I and other genes during osteogenic differentiation revealed that endogenous overexpression of Runx2 and Osterix up-regulated the expression levels of these genes rapidly,which then accelerated into osteogenic differentiation.This method of rapidly changing the fate of MSCs by genetic engineering has laid a solid foundation for clinical application and pointed out the direction for the specificity of osteogenic induction differentiation of MSCs.Part Ⅲ Stimulation of osteogenic differentiation and inhibition of adipogenic differentiation in adipose-derived stem cells via the CRISPR systemIn this part,we endogenous overexpression of osteogenesis precursor gene Runx2 and Osterix by using the CRISPR system,which can regulate the fate of MSCs.And then we investigated the effect function of adipogenic differentiation.The results showed that the upregulation of Runx2 and Osteric can upregulation,which decrease the expression level the influence of the experimental results show that the osteogenesis Runx2 and Osterix precursor gene expression levels rise,PPAR gamma,LPL cut into adipocyte-related gene expression level.This result further confirmed that the CRISPR system endogenous overexpression of osteogenesis precursor gene Runx2 and Osterix can promote osteogenesis of mesenchymal stem cells to differentiate between fat and curb its differentiation,the osteogenetic differentiation and differentiation into fat has a specific role,to explore between differentiation of osteogenesis of mesenchymal stem cells and fat into the theoretical basis for the complex relationship between,between for mesenchymal stem cells in clinical treatment of the bony disease.In conclusion,our research modified the method of isolating mouse adipose-derived stem cells(ASCs),and investigate the regulation functions of Runx2 and Osterix in osteogenic differentiation process by using molecular biology,genetics,cell biology,and biochemistry methods.The results showed that the connection of osteogenic differentiation and adipogenic differentiation,which have a potential function for clinical application. |
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