| Objective:Objective To observe the effect of exosomes derived from BMSCs on axonal regeneration and the expression of phosphatase,tensin homologous protein(PTEN)andβ-tubulin Ⅲ in PC12 cells after oxygen glucose deprivation / reoxygenation(OGD / R),and to explore the possible effect and mechanism of exosomes derived from BMSCs on axonal regeneration in PC12 cells after OGD / R.Methods:The experiment is divided into three parts.The first part: the extraction of rat primary BMSCs and the extraction and identification of exosomes.Ten healthy and clean female SDF rats were killed by cervical dislocation,bilateral hip joints to ankle joints were exposed under aseptic conditions,bilateral lower limb muscles were removed,bilateral femurs and tibias were removed,and washed with sterile PBS for three times.The two ends of femur and tibia were cut off by ophthalmic scissors to expose the bone marrow cavity.The bone marrow cavity was repeatedly washed on both sides with 5ml sterile syringe containing basic culture medium,and the bone marrow was flushed out into the centrifuge tube until the bone marrow cavity became white.After centrifugation at 1200 R / min for 5min,the supernatant was discarded and inoculated into the culture flask with basic medium,then cultured in the incubator.After inoculation,the BMSCs were changed in the second half of 48 hours,and then changed in full every 3 days.The fusion reached 80%.After trypsin digestion,the culture medium was neutralized,and then transferred into a centrifuge tube.After centrifugation at 1200 R / min for 5 minutes,the supernatant was discarded,and the cells were resuspended in the basic culture medium and inoculated in the appropriate proportion in the culture flask.The third generation BMMSCs with 80-90% fusion were cultured in exosome free medium for 48 hours.The culture medium was collected and centrifuged at 300 × g for10 min,2000 × g for 20 min and 10000 × g for 30 min respectively.The dead cells and fragments were discarded.The supernatant was collected and centrifuged at 100000 × g for 90 min.the supernatant was discarded and resuspended with PBS to obtain exosomes.The expression of TSG101 and CD9 was detected by Western blot.The exosomes were observed and identified by transmission electron microscope.Part two: the culture of PC12 cells and the selection of typical time points after oxygen glucose deprivation / reoxygenation.PC12 cells were inoculated into the culture flask with PC12 basic medium,and the medium was changed every 3 days.When the cells fused to 80%-90%,they were passaged,and the cells passaged to 3-5 generations were used in the experiment.PC12 cells were divided into control group,oxygen glucose deprivation 6h / reoxygenation 3H group,oxygen glucose deprivation 6h / reoxygenation6 h group,oxygen glucose deprivation 6h / reoxygenation 12 h group,oxygen glucose deprivation 6h / reoxygenation 24 h group.After reoxygenation,the covered glass slides with cells were taken out from the 24 well plate.After treatment,the fluorescence intensity of cells and the length of axons were observed under the fluorescence microscope.After reoxygenation,cell protein was extracted and protein concentration was determined.The expression of β-tubulin Ⅲ was detected by Western blot.Select the appropriate time index to carry out the following experiments.Part three: after selecting typical time points,PC12 cells were divided into three groups: control group,oxygen glucose deprivation 6h / reoxygenation 24 h group(model group),oxygen glucose deprivation / reoxygenation 24 h group + exosome group(treatment group).CCK-8 method was used to determine the cell viability.After the exosomes were intervened,the cover glass slides with cells in different groups were taken out from 24 well plates.After treatment,the fluorescence intensity of cells and the length of axons were observed under the fluorescence microscope.After reoxygenation,cell protein was extracted and protein concentration was determined.Western blot was used to detect the expression of PTEN,β-tubulin Ⅲ,PI3 K and Akt.The above data were analyzed statistically.Results:The first part: 1.Morphological observation of BMSCs: the primary cells were mainly round and mixed cells with different sizes under light microscope.After one day,a small number of adherent cells were observed,and the number of adherent cells increased gradually.The morphology of adherent cells was more consistent,spindle shaped,and the arrangement of cell colonies appeared.After 7 days,the cells fused into pieces,and the arrangement of cells was more regular and vortex like.Observation and identification of exosomes derived from BMSCs: the diameter of exosomes derived from BMSCs was about 30-110 nm under transmission electron microscope,and they were round,oval or saucer like vesicles with complete membrane structure around them.The expression of TSG101 and CD9 was detected by Western blot.Part two: the results of grouping experiment showed that the axons of PC12 cells were the shortest after OGD6 h / r24 h under light microscope.The expression of β-tubulin Ⅲ in PC12 cells after OGD / R was detected by Western blot.Compared with the control group,we found that the expression of β-tubulin Ⅲ in OGD6 h / r3 h group,OGD6 h / r6 h group,OGD6 h / r12 h group and OGD6 h / r24 h group decreased gradually,and the difference was statistically significant(# P < 0.05),and the expression of β-tubulin Ⅲ in OGD6 h / r24 h group decreased most significantly.Therefore,we can draw a conclusion: compared with the control group,OGD6 h / r3 h group,OGD6 h / r6 h group and OGD6 h / r12 h group,the expression of β-tubulin Ⅲ protein in OGD6 h / r24 h group was the least,and the difference was statistically significant(* P < 0.05).Immunofluorescence staining showed that compared with the control group,the fluorescence intensity of β-tubulin Ⅲ in OGD6 h / r3 h group,OGD6 h / r6 h group,OGD6 h / r12 h group and OGD6 h / r24 h group was significantly lower than that in the control group.With the extension of reoxygenation time,the fluorescence intensity gradually decreased,and the fluorescence intensity of OGD6 h / r24 h group reached the lowest peak(P < 0.005).These results showed that the length of axon was the shortest and the expression of axon associated protein β-tubulin Ⅲ was the least after OGD6 h /r24 h in PC12 cells.This time point met the requirements of the next experiment and was designated as the model group in the following experiments.The third part: the results of grouping experiment: CCK-8 results showed that the control group had the highest cell viability,followed by OGD6 h / r24 h + exosome group,and the model group had the lowest cell viability.Compared with the control group,the cell viability of the model group and OGD6 h / r24 h + exosome group decreased,with statistical significance(# P < 0.005).Compared with the model group,the cell viability of the control group and OGD6 h / r24 h + exosome group increased,with statistical significance(* P < 0.005),which indicated that the exosome treatment could significantly improve the cell survival rate.Western blot was used to detect the expression of β-tubulin Ⅲ,PTEN and PI3 K in PC12 cells.Compared with the control group,the expression of PTEN in the treatment group and the model group was significantly increased,and the difference was statistically significant(# P < 0.05);compared with the model group,the expression of PTEN in the control group and the treatment group was significantly decreased,and the difference was statistically significant(* P < 0.05),which indicated that although the exosome could not reduce the PTEN content of PC12 cells after OGD / R to the normal level,it could inhibit the proliferation of PC12 cells The expression level of PTEN was decreased.Compared with the control group,the expression of PI3 K in the treatment group and the model group was significantly decreased,and the difference was statistically significant(# P < 0.05);compared with the model group,the expression of PI3 K in the control group and the treatment group was significantly increased,and the difference was statistically significant(* P < 0.05);compared with the control group,the expression of Akt in the treatment group and the model group was significantly decreased,and the difference was statistically significant Compared with the model group,the expression of Akt in the control group and the treatment group was significantly increased,and the difference was statistically significant(* P < 0.05).These results indicate that exosomes can promote the expression of PI3 K and Akt in PC12 cells after OGD / R.Compared with the control group,the expression of β-tubulin Ⅲ in the treatment group and the model group was significantly decreased,and the difference was statistically significant(# P < 0.05);compared with the model group,the expression of β-tubulin Ⅲ in the control group and the treatment group was significantly increased,and the difference was statistically significant(* P < 0.05).These results indicate that exosomes can promote the expression of β-tubulin Ⅲ in PC12 cells after OGD / R.The control group,model group and treatment group were stained with fluorescence.The results showed that: compared with the control group,the fluorescence intensity of the treatment group was lower,but compared with the model group,the fluorescence intensity of the treatment group was significantly enhanced,and the data of the three groups were statistically significant(P < 0.05),which proved that exosomes had a strong role in promoting the growth of axons after injury.These results suggest that exosomes can inhibit the expression of PTEN and then activate PI3K-Akt pathway,thus promoting the expression of axon associated protein β-tubulin Ⅲ,and then promoting the growth of axons and the recovery of neural function.Conclusion:Exosomes derived from BMSCs can promote the extension of axon length,the expression of axon associated protein β-tubulin Ⅲ and the expression of pathway inhibitor PTEN in PC12 cells after oxygen glucose deprivation / reoxygenation,which may promote axon regeneration and neural function recovery by inhibiting PTEN-PI3 K pathway. |
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