Research On Regulation And Mechanism Of BMSCs On Microglial Cells Of Inflammatory Response To Ischemic Stroke In Rats

Objectives Part 1: To construct,pack and identify CX3CL1 RNA interference lentiviral vector.Part 2: To construct the MCAO model of SD rats;detect three groups of the pro-inflammatory microglial cells(Iba+TNF-α)and anti-inflammatory microglia cells(Iofba+CD206+)in the ischemic area of stroke of rats injected with BMSCs,p LVX-sh RNA2-CX3CL1 lentivirus-infected BMSCs,and normal saline.Part 3: To determine whether the transplanted BMSCs can reverse the distribution of anti-inflammatory and pro-inflammatory microglia in the ischemic area of stroke.Part 4: To clarify whether the reversal of microglia by transplanted BMSCs is mediated by CX3CL1.MethodsPart 1: Isolate and culture rat mesenchymal stem cells by density gradient centrifugation,and select well-grown third-generation cells for subsequent experiments.The cell surface antigens CD29,CD31,CD44 and CD45 of BMSCs were detected by flow cytometry to identify the purity of BMSCs.Part 2: Refer to the target gene CX3CL1 sequence and design PCR primers to amplify the corresponding interference sequence.Digest the vector p LVX-sh RNA2,purify the digested product,and then directionally connect the interference sequence to the digested product,then transform the product into bacterial competent cells,digest and sequence the clones that have grown,and identify positive clones,which was a successfully constructed p LVX-sh RNA2-CX3CL1 lentiviral interference vector.Part 3: Remove endotoxin and highly purify the constructed lentiviral interference plasmid vector and empty vector,and then transfect the constructed RNAi lentiviral vector along with the lentiviral packaging vector to 293 T cells to collect the cell supernatant rich in lentiviral particles.Then,the supernatant was purified and concentrated to obtain p LVX-sh RNA2 and p LVX-sh RNA2-CX3CL1 lentivirus,and finally the titer of the lentiviral concentrated solution was measured by dilution.Part 4: Infect the BMSCs cells with lentivirus,observe the fluorescence expression with a fluorescence microscope 72 hours later,and estimate the efficiency of infection.Then extract RNA from BMSCs cells,reverse transcribe the RNA into c DNA with random primers,and then design specific primers and sybr green I fluorescent dye for QPCR detection.Finally,a one-way analysis of variance was used to explain the difference in the relative ratio of CX3CL1 m RNA expression between the three groups of cells in the BMSCs group,BMSCs+empty vector group,and BMSCs+ interference group,to further clarify whether the constructed interference virus significantly inhibited the expression of CX3CL1 in BMSCs cells.Part 5: Refer to the modified Zea Longa thread plug method to build a MCAO model of rat.After 24 hours of modeling,the rats were scored for neurological function according to the Zea Longa grade 5 neurological deficit scoring method,and the success rate of the model was verified by TTC staining.Part 6: Divide the constructed MCAO model into three groups,and inject normal saline,BMSCs,and BMSCs transplanted with interfering virus into the lateral ventricle.Each group was treated at three time points(1d,3d,and 7d),and the brain tissue was fixed with paraformaldehyde.All samples were made into sections by means of tissue paraffin production,paraffin section dewaxing and rehydration,and antigen heat repair.Each section was subjected to anti-Iba and anti-CD206/TNF-α double immunofluorescence staining,and then counted the number of cells with double-staining coincidence positive quality under light scanning confocal microscope(400×)and the images were collected.Part 7: Through single factor analysis of variance,statistically analyze the number of double staining positive cells at different time points(1d,3d,and 7d)in these three groups of models,and clarify that if the anti-inflammatory microglia(Iba+CD206+)and pro-inflammatory microglia(Iba+TNF-α+)have significant differences.ResultsPart 1: Flow cytometry detection of the third-generation BMSCs cell surface markers suggested that CD29,CD44 expression is positive,and CD31,CD45 expression is negative,indicating that the third-generation BMSCs highly express antigens CD29,CD44,basically do not express CD31,CD34,in line with the characteristics of BMSCs cell surface markers and can be used in subsequent experiments.Part 2: Identify the constructed recombinant plasmid by enzyme digestion and obtain a DNA fragment consistent with the expected result.Then,the recombinant plasmid was sequenced,and the sequencing results were compared with the target gene sequence.The results were completely consistent,indicating that the target gene has been directed into the target vector,which means that the p LVX-sh RNA2-CX3CL1 lentiviral interference vector was successfully constructed.Part 3: Transfect the constructed lentiviral interference vector with the lentiviral packaging vector into 293 T cells,collect the supernatant.After packaging,purification and concentration,the p LVX-sh RNA2-CX3CL1 lentivirus is obtained.Compared with white light and fluorescent photos,the infection efficiency is more than 90%,so the infected BMSCs can be used in subsequent experiments.QPCR results showed that the interference group significantly inhibited the expression of CX3CL1 m RNA of BMSCs,and the interference efficiency was above 70%.Part 4: The rat MCAO model was successfully constructed,and 24 hours after the model was established,its neurological function was scored and the rat model with a score of 1-3was selected.The model was verified by TTC staining.Compared with the normal control group,the MCAO model group had infarct lesions in the blood supply area of the middle cerebral artery.Part 5: The statistical results show that: compared with the BMSCs transplantation group,the number of anti-inflammatory microglia(Iba+CD206+)in the MCAO group and the BMSCs group transplanted with interfering virus in the ischemic area had an obvious decrease on day 1,day 3 and day 7(all p<0.01),and the number of pro-inflammatory microglia(Iba+TNF-α+)significantly increased on days 3 and 7(all p<0.01).There was no significant difference in the number of anti-inflammatory microglia and pro-inflammatory microglia in the ischemic area of MCAO group and BMSCs transplanted with interfering virus at three time points(all p>0.01).The above results indicate that the MCAO model can increase anti-inflammatory microglia in the ischemic area and reduce pro-inflammatory microglia after transplantation of BMSCs;this reversal may be related to the role of CX3CL1.ConclusionPart 1: CX3CL1 interference lentiviral vector was successfully constructed and packaged,and it is confirmed that CX3CL1 interference lentiviral vector significantly silenced the expression of CX3CL1 in BMSCs cells.Part 2: It is clear that the transplantation of BMSCs can reverse the distribution of anti-inflammatory(Iba+CD206+)and pro-inflammatory(Iba+TNF-α+)microglia cells in the ischemic area of stroke,increase anti-inflammatory(Iba+CD206+)microglia and decrease pro-inflammatory(Iba+TNF-α+)microglia and confirms that the reversal of microglia by the transplanted BMSCs cells may be mediated by CX3CL1.