| Starting from nuclear transfer,cell reprogramming went through cell fusion reprogramming,extraction-induced reprogramming,transcription factor-mediated reprogramming,and small-molecule-induced reprogramming.At present,direct reprogramming induced by small molecule compounds has received extensive attention and in-depth research,and considerable progress has been made in revealing the mechanism of drug action.Using small molecule direct reprogramming induced myocardial cells,neural stem cells,neurons,and liver cells also have great progress.Small molecule compounds have simple structure,clear target,easy synthesis,small molecular weight,can directly replace reprogramming factors,easy to penetrate into cells,non-immunogenicity,easy to control the concentration and time of action,no tumorigenicity,relatively safe,controllable action.Based on the research so far,induction system of medium and small molecular quantity is more,the joint mechanism is difficult to explore,and some small molecular water-soluble liquid of caused some difficulties for further research.Aiming at this problem,this paper selects fewer small molecular compounds combination(K,F,A,T)and the best concentration,preparation into small molecular compound micelle,the anthropogenic fibroblasts in vitro directly transdifferentiation of functional neurons.The efficiency of induction is improved,avoid the transcription factors bring risks and embryonic stem cells into a tumor facing ethical issues.The induction of mature neurons to treat spinal cord injury in rats by cell transplantation also provides a source of functional cells for regenerative medicine cell transplantation therapy.The full text is expounded from the following six chapters.Chapter one:Reviews This chapter begins with an overview of small molecule compounds.The concept,action result and action path of small molecule compounds were introduced.The development of cell reprogramming is introduced in detail,from the earliest research of nuclear transfer,cell fusion,cell extract induced cell reprogramming,gradually developed to the transcription factors mediating cell reprogramming,development to the present small molecule compounds mediated all chemical induction of cell reprogramming,for the current research is still in the upsurge of small molecule compounds induced directly reprogrammed cells(i.e.transdifferentiation)for different types of functional cells research provides the reference and the new way of thinking,Reprogrammed cells and the relationship between transdifferentiation and explanation,the full chemical induction of somatic cell differentiation into different functional cells in detail,including the heart muscle cells,liver cells,nerve cells,and finally,the small molecules involved in cell transdifferentiation and their functional properties and the introduction of spinal cord injury were introduced,which laid a solid theoretical foundation for the following research.Chapter two:Preparation and characterization of small molecule micelles The solubility of small molecule K is very low in the induction system of neuronal cells in vitro,and it has a great influence on the induction process of transdifferentiation of fibroblasts into neuronal cells.In this chapter,based on the solubilization of glycyrrhizic acid with amphiphilic structure,small molecule K micelles were prepared by thin film dispersion method,and a high-performance liquid chromatography(HPLC)method was established for the determination of small molecule K concentration in vitro.Through the preparation of micellar particle size,Zeta potential,multiple scattering coefficient(PDI),packet encapsulation efficiency and drug loadings of the investigation,determined the weight ratio of 1:10,hydration time of 2 hours,can obtain the best particle size of 57.88±3.01 nm,minimum Zeta potential-23.53±1.37 m V,more dispersed coefficient(PDI)was 0.104±0.006,the highest rate of coating 90.24±1.19% and 9.22±0.14%,the drug was the most suitable micelles.Chapter three:Small molecules induce the transdifferentiation of somatic cells into neurons This chapter first introduces the sources of somatic cells used in this paper,and introduces the separation and culture process of somatic cells in detail,which provides sufficient and high-quality cell sources for the study of this paper.The best small molecule combinations of KE,Fo,A and Th were selected by orthogonal experiment.After induction for 4 days,human fibroblasts could be directly transformed into functional neurons with typical neuronal characteristics: the cell body became smaller,the refraction became stronger,and the number of processes increased.With the increase of induction time,the cell transmittance increased and the axial mutation was longer.The induction efficiency can reach 81.5% by flow cytometry.Immunofluorescence(IF),q RT-PCR,and Western Blot were used to verify the expression of neuron-related genes and proteins by induced neurons,which proved that the induced cells were functional neurons.Chapter four: Study on small molecule induced neuron therapy for spinal cord injury(SCI)repair In this study,a rat spinal cord injury model was established by cross-section method,and the induced mature nerve cells were labeled with fluorescence(CFSE).Fibrin scaffolds were directly transplanted into the injured spinal cord of rats in situ to mediate the repair of the injured spinal cord.BBB score,open field test and footprint test were used to analyze the recovery of hind limb motor function.Biotinylated dextran amine(BDA)was used for anterograde nerve tracing to observe whether the tracer could be transmitted from the brain down the spinal cord and whether the anterograde conduction function of the spinal cord was repaired after treatment.Fluoro-gold(FG)was used to trace the nerve retrograde to observe whether the tracer could be transferred retrograde along the spinal cord to the brain,and whether the retrograde conduction function of the spinal cord was repaired after treatment.Ed U kit was used to detect cell proliferation at the spinal cord injury site.In vivo imaging was used to detect the migration of transplanted nerve cells in the spinal cord.Finally,immunofluorescence staining was performed on the spinal cord tissue sections to identify the expression of neuro related proteins,such as TUJ1,MAP2,Tau,Syn Gap,Synpsin 1,NH-H,GAP43,and GFAP in glial cells.The results confirmed that the small molecule induced neurons migrate to injury,the rat hind legs movement function to get a good recovery,can be realized in the process of the short distance movement of the front and hind legs coordination walking gait,and other groups cannot load the hind legs stand,is still a drag and drop movement,that nerve cells induced by small molecular compound micelle Ke,A,Fo,Th,joint fibrin scaffold can promote the injury nerve connected up and down,have a better therapeutic effect on spinal cord injury However,the hind limbs of the other groups were still dragging and unable to stand in the weight-bearing position.These results indicated that nerve cells induced by small molecular compounds Ke micelle,A,Fo and Th combined with fibrin scaffold could promote the upper and lower connectivity of neurons in the injured area and form a neural circuit,which had a good therapeutic effect on spinal cord injury. |
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