The Study On Different Diameter Of PLLA Fiber And Their Macrophage Polarization Affecting Neural Differentiation Derived From Rat Bone Marrow Mesenchymal Stem Cells

Part One Isolation and identification of rat bone marrow mesenchymal stemcellsObjective: To isolate and purify bone marrow mesenchymal stem cells from rat femur and tibia.The cells were cultured and purified to observe their morphology and immunophenotyping.Bone marrow mesenchymal stem cells were loaded into electrospun fiber membrane to prepare neuronal differentiation research.Results: After cultured for 24 h,the bone marrow mesenchymal stem cells were cultured for a long time.The cells were adherent under light microscope.The morphology of the cells was spherical,spindle-shaped and spindle-shaped,the rest of the cells was red blood cells.After incubation on day 7,the fusion rate of adherent cells reached 80%-90%,and the pancreatic trypsin was centrifuged and resuspended for 1: 2 passages.After 3-4 hours,the cells began to adhere,and on the third day,Rate of up to 80% or more,there "cell edge phenomenon".Culture to the third generation,its flow cytometry,CD29 and CD90 positive results,and CD34 and CD45 was negative.After incubation on day 7,the fusion rate of adherent cells reached 80%-90%,and the pancreatic trypsin was centrifuged and resuspended for 1: 2 passages.After 3-4 hours,the cells began to adhere,and on the third day,Rate of up to 80% or more,there "cell edge phenomenon".Culture to the third generation,its flow cytometry,CD29 and CD90 positive results,and CD34 and CD45 was negative.Conclusion: The rat bone marrow mesenchymal stem cells with high purity and uniformity were extracted by adherent separation method.Part two Preparation and Characterization of Electrospinning Diameter PLLAFiber Films with Different DiametersObjective: To prepare different polylactic acid fiber membranes with different diameters of polylactic acid by electrospinning,and to analyze the fiber membranes of different diameters for preparation of bone marrow mesenchymal stem cells and macrophage biopsies.Methods: different quality,1 g,1.5 g and 0.75 g 2.0 g polylactic acid solid particles in dichloromethane(DCM)and dimethyl formamide(DMF)in dissolved,preparation quality volume ratio(w/v)into a concentration of 6.25wt%,8.33wt%,12.5wt%,16.67wt% mixed solution,application of electrostatic spinning equipment preparation into different diameter levorotatory polylactic acid fiber membrane,The microstructures of L-lactic acid were studied by X-ray diffraction(XRD),scanning electron microscopy(SEM)Water contact angle(WCA)to detect the biological scaffold material.Results: The diameter of the dipped polylactic acid fiber was measured by scanning electron microscopy and particle size analysis.The average diameters of the fiber membranes were 710 nm,1000nm,1560 nm and 2060 nm respectively.The concentration of the polylactic acid mixed solution was proportional to the diameter of the fiber membrane relationship.The contact angle of four different diameters of fiber membrane was less than 90 °,all of which belonged to the hydrophilic bio-scaffold material,which was suitable for the follow-up test.Conclusion: Four different diameter L-polylactic acid fiber membrane scaffolds were successfully prepared by electrospinning.Part Three Effect of electrospinning on the polarization of macrophages withdifferent diameter PLLA fiber membraneObjective: To investigate the changes of macrophages after inoculation with different diameter L-lactic acid fiber membranes,and to prepare the effects of subsequent macrophage polarization on the differentiation of rat bone marrow mesenchymal stem cells into neurons.Methods: The cell phenotype was measured by flow cytometry.The supernatants of RAW264.7 cells cultured in different diameter fibrous membranes were collected for 1,3 and 5 days respectively.q RT-PCR was performed and the cells of RAW264.7 cells Factor expression.Methods: using flow cytometry detection technology to determine cell phenotypic changes;Collected respectively 1,3,5 days training on different diameter fiber membrane RAW264.7 cell supernatant q RT-PCR test,The levels of cytokines IL-1?,TGF-?in the supernatant were determined by ELISA.Results: Flow cytometry was used to detect the expression of RAW264.7 cells on different diameter fibrous membranes prepared at a concentration of 710 nm,1000nm,1560 nm,2060nm(W/W)CDF4/80?CD11b?CD16/32 ?CD206 phenotype,and the positive rate of CDF4 / 80(91.5%,78.2%,86.0%,87.6%)and CD11b(95.2%,95.2%,95.6%,91.3% The positive rate of CD16 / 32 in macrophages was 31000 nm,35.0%,48.8% and 44.1% respectively,while that of M2 macrophages was 76.2%,72.0% and 88.2% respectively.88.5%.Collect 1,3,5 days training in different diameter fiber membrane RAW264.7 cells supernatant real-time quantitative PCR detection(q RT-PCR analysis),M1 subtype of IL-1 high levels of inflammatory factors and gene expression of TNF alpha respectively in 3 days concentration of 1000 nm and the first day of the concentration is 2060 nm of fiber membrane expression peak;Cytotoxic factor i NOS gene in 5 days on the fiber membrane concentration of 1000 nm to express the highest;Effect on macrophage polarization to the M1 and M2 IFN-gamma and IL-4 genes respectively in day 1 concentration is 1000 nm and 2060 nm high expression on fiber membrane;Macrophage inflammation suppression of M2 factor IL-10 and Arg-1 in the same day 1 concentration of 2060 nm high expression in the fiber membrane.Inflammatory factor beta IL-1 ELISA test result and the content of TNF-? on 1000 nm fiber membrane cleaning the highest 5 days,and on the fiber membrane from 1,3,5 days in a state of high content;Suppression of inflammatory factor TGF-beta 2060 nm of fiber membrane after 3 days in the supernatant content increased sharply,reached the highest content of 2060 nm in 5 days.Conclusion: RAW264.7 cells can be successfully loaded with L-lactic acid fiber membranes with different diameters,and the low expression of phenotype CD16 / 32 and the high expression of M2 type macrophage phenotype CD206 in M1 macrophages were detected by flow cytometry.It was confirmed that the fiber membrane had a general trend towards M2 type polarization in RAW264.7 cells.q RT-PCR analysis various inflammatory factor(IL-1,i NOS,TNF alpha)and inflammation suppression factor(IL-10 and Arg-1)gene and the macrophages to M1 and M2 subtype polarization ability of IFN-gamma and IL-4 genes the peak in the concentration of 1000 nm and 2060 nm of electrostatic spinning fiber membrane,so the selection and collection of the concentration of two kinds of fiber membrane cultivate RAW264.7 cell supernatant 1?3?5 heaven for subsequent research macrophage polarization effect on bone marrow mesenchymal stem cells between neural differentiation.Part Four Effect of Electrospinning Different Diameter L-polylactic Acid FiberMembrane on Neuronal Differentiation of Rat Bone Marrow Mesenchymal StemCellsObjective: To investigate the effects of different diameter fibroblasts on the proliferation of BMSCs into neurons in Neuron-like cells by using electrospinning different diameter L-polylactic acid fiber membrane as the biological scaffolds of rat bone marrow mesenchymal stem cells(BMSCs).Methods: BMSCs were isolated and purified by whole bone marrow adherence method.The BMSCs were inoculated into different diameter L-lactic acid fiber membranes on the basis of the amount of cells per 100 000 cells.There are three groups,respectively for the experimental group(experimental group)and Positive control group(Positive control)and the Negative control group(Negative control).When the cell fusion rate was 80%,the preconducting solution was added and replaced with the induction solution after 24 hours.And after 5 hours,the cells were treated with Neuron-specific enolase(Neuron-specific enolase)by immunohistochemi-cal staining NSE),Neurofilament-200(NF-200),microtubule-associated protein 2(MAP-2)staining.q RT-PCR was used to detect neuronal cell marker gene expression after induction of neuron differentiation.Results: NF-200,MAP-2,Nestin,NSE and DAPI staining showed that neurons were stained on bone marrow mesenchymal stem cells on pre-induced 24 hours and 5 hours after induction of different diameter diastolic polylactic acid electrospun fiber membranes.The expression of NF-200,MAP-2,Nestin and NSE in the experimental group was higher than that in the positive control group and the negative control group.The expression level of fibrous membrane at 1000 nm in experimental group was significantly higher than that in other concentrations,and the expression of gene was higher than that of 1560 nm and 2060 nm.710nm of fibrous membrane.Conclusion: Bone marrow mesenchymal stem cells(MSCs)can differentiate into neuron-like cells after induced by electrospinning on different diameter L-lactic acid fiber membrane scaffolds.Part Five Effects of macrophage polarized supernatant on neuraldifferentiation of bone marrow mesenchymal stem cellsObjective: To investigate the effect of different diameter diastolic polylactic acid electrospun fiber membrane on the differentiation of bone marrow mesenchymal stem cells into macrophages.Methods: bone marrow mesenchymal stem cells per 40000 / cm2 between amount of vaccination in 24 orifice,fusion rate above 80%,add the induced liquid induced after 24 hours,with the collection of RAW264.7 cells supernatant liquid joint induced by 1:4 ratio on bone marrow mesenchymal stem cells into neural induction between 5 hours,and then induced cells were detected by inverted microscope,immunofluorescence staining and q RT-PCR.Results: After 24 hours of pre-induction of bone marrow mesenchymal stem cells,microscopic observation showed that the cytoplasm was contracted to the nucleus and had short antennal angles.After induction with RAW264.7 supernatant,most of the cells were neuron-like cells,The cell body is round or oval,cell body around the long protrusions,the end of the branch and the surrounding cells connected into a network.Immunofluorescence staining showed that the positive rates of NSE,NF-200,MAP-2 were 79%,64%and 81%.The results of q RT-PCR showed that the fibroblast supernatant had the strongest gene expression on BMSCs after 2060 nm fibroblast supernatant on the first day.The fibroblast supernatant of the fibrous membrane with the concentration of 1000 nm was induced to differentiate into BMSCs on the 1st,3rd and 5th days.Conclusion: The cells of RAW264.7 with different diameters of L-polylactic acid fiber membrane were obtained by electrospinning,and their effects on M2 were induced by IL-1?,TGF-? and Arg-1 Factor can assist BMSCs to differentiate into nerves.