ObjectiveTo explore the feasibility and mechanism of inhibiting miR-429 to improve the permeability of the blood spinal cord barrier (BSCB) in vitro, and provide a new gene therapy target for enhancing the spinal cord microenvironment.MethodsFirst, the immortalized human brain microvascular endothelial cell line (hCMEC/D3) was transfected with the anti-miR-429 antagonist (antagomiR-429) and its negative control (antagomiR-429-NC), respectively. The miR-429 expression of hCMEC/D3 cells was observed by fluorescence microscopy and real-time fluorescence quantitative PCR to verify the transfection efficiency of antagomiR-429. Then the effect of miR-429 on BSCB permeability was observed in vitro. The experiment was divided into 4 groups. The blank control group (group A) was constructed of normal hCMEC/D3 cells and Ha-sc cells to prepare the BSCB model, the hypoxia-induced group (group B), the hypoxia-induced+antagomiR-429-NC group (group C), and the hypoxia-induced+antagomiR-429 group (group D) were constructed of normal, antagomiR-429-NC transfected, and antagomiR-429 transfected hCMEC/D3 cells and Ha-sc cells to prepare the BSCB models and hypoxia treatment for 12 hours. The permeability of BSCB in vitro was measured by horseradish peroxidase (HRP) permeability. Real-time fluorescence quantitative PCR, Western blot, and immunofluorescence staining were used to observe the expressions of ZO-1, Occludin, and Claudin-5.ResultsThe antagomiR-429 and antagomiR-429-NC were successfully transfected into hCMEC/D3 cells under a fluorescence microscope, and the transfection efficiency was about 90%. Real-time fluorescence quantitative PCR results showed that the relative expression of miR-429 in antagomiR-429 group was 0.109±0.013, which was significantly lower than that of antagomiR-429-NC group (0.956±0.004, P<0.05). HRP permeability measurement, real-time fluorescence quantitative PCR, and Western blot results showed that the HRP permeability of groups B and C were significantly higher than those of groups A and D (P<0.05), and the relative expressions of ZO-1, Occludin, and Claudin-5 proteins and mRNAs were significantly lower in groups B and C than in groups A and D (P<0.05) and in group D than in group A (P<0.05); there was no significant difference between groups B and C (P>0.05). Immunofluorescence staining showed that the immunofluorescence of ZO-1, Occudin, and Claudin-5 at the cell membrane boundary in group D were stronger than those in groups B and C, but not as strong as that in group A.ConclusionInhibition of miR-429 expression can promote the expressions of ZO-1, Occludin, and Claudin-5 proteins in microvascular endothelial cells, thereby improving the increased permeability of BSCB due to hypoxia.
Objective To observe the effect of BMSCs transplantation on gene and protein expression of VEGF receptor fetal l iver kinase 1 (Flk-1) after spinal cord injury (SCI), and to investigate the mechanism of repairing the SCI by BMSCs transplantation. Methods BMSCs were isolated and cultured from five 4-week-old male Wistar rats weighing100-120 g. The SCI model was made by using the modified Allen’s impactor device. Eighty-one adult female Wistar rats weighing 220-250 g were randomly divided into 3 groups: sham-operated group (group A, n=21), in which spinous process and vertebral plate of thorax 8-10 spinal cord segment were removed; DMEM group (group B, n=30), in which rats received four injections of DMEM in the peri-lesion area; and BMSCs group (group C, n=30), in which rats received four injections of BMSCs in the peri-lesion area. The changes of Flk-1 mRNA expression in rats’ spinal cord tissues were detected with RT-PCR method 1, 3 and 5 days after transplantation. The expression of Flk-1 protein was observed by using immunohistochemical technology in spinal cord 3, 7 and 14 days after transplantation. Results Morphology of the primary cultured BMSCs was various. Cell morphology tended to be uniform with the accumulation of passages, which appeared flat and spindle-shaped. RT-PCR results showed that there was no significant differences (P gt; 0.05) in Flk-1 mRNA expression between group C and group B at different time points after transplantation. But Flk-1 mRNA levels of group B and group C significantly increased and peaked 1 day after transplantation (P lt; 0.01), and then decreased 3 days after transplantation (P lt; 0.01) compared with that of group A, and were still higher than that of group A 5 days after transplantation (P lt; 0.05). Immunohistochemical staining results revealed that the expression of Flk-1 in group B was enhanced 3 and 7 days after transplantation compared with group A, which was significantly different (P lt; 0.01). There was no significant difference in the expression of Flk-1 between group B and groupp A 14 days after transplantation (P gt; 0.05). There was no significant difference in Flk-1 protein expression between group C and group B 3 days after transplantation (P gt; 0.05). The expression of Flk-1 protein in group C was significantly higher than that in group B 7 and14 days after transplantation (P lt; 0.01). Conclusion BMSCs transplantation after SCI does not have regulatary effect onthe expression of Flk-1 mRNA, but it does upregulate the Flk-1 protein expression, which may be one of the mechanisms of repairing SCI.
ObjectiveUnder hypoxic conditions, the survival and apoptosis of human amniotic mesenchymal stem cells (hAMSCs) were observed by transient transfection of hypoxia-inducible factor 1α (HIF-1α) gene, to investigate the effect of HIF-1α on hypoxic tolerance of hAMSCs.MethodsThe hAMSCs were isolated and cultured from amniotic membrane tissue from voluntary donors who were treated with cesarean section. And the morphological observation by inverted phase contrast microscope and immunofluorescence detection of the expressions of stem cell markers OCT-4 and NANOG were performed to identify the cultured cells. The third generation hAMSCs were treated with 200 μmol/L CoCl2, and transient transfection of plasmids were added according to the following grouping: group A was hAMSCs blank group; group B was pcDNA3.1 negative control group; group C was short hairpin RNA (shRNA) negative control group; group D was shRNA-HIF-1α interference group; group E was pcDNA3.1-HIF-1α over expression group. Cell survival rate of each group was measured by cell counting kit 8 (CCK-8) at 12, 24, 48 hours after hypoxia treatment. Flow cytometry was used to detect apoptosis rate of each group at 24 hours after hypoxia treatment. The expression levels of HIF-1α, vascular endothelial growth factor (VEGF), B-cell lymphoma 2 (Bcl-2), Bax, and cleaved Caspase-3 (C-Caspase-3) proteins were detected by Western blot at 24 hours after hypoxia treatment.ResultsCCK-8 assay showed that the cell survival rate of group D was significantly lower than those of groups A and C at all time points after hypoxia treatment; while the cell survival rate in group E was significantly increased than those in groups A and B, and the diffrences at 24 hours were significant (P<0.05). In group E, the cell survival rate at 24 hours was significantly higher than those at 12 and 48 hours (P<0.05). The results of flow cytometry showed that the apoptosis rate in group D was significantly higher than those in groups A and C (P<0.05), and the apoptosis rate in group E was significantly lower than those in groups A and B (P<0.05). Western blot showed that the expressions of HIF-1α, VEGF, and Bcl-2 proteins in group D were significantly decreased when compared with those in groups A and C, and the expressions of Bax and C-Caspase-3 proteins were significantly increased (P<0.05). On the contrary, the expressions of HIF-1α, VEGF, and Bcl-2 proteins in group E were significantly higher than those in groups A and B, and the expressions of Bax and C-Caspase-3 proteins were significantly decreased (P<0.05).ConclusionOverexpression of HIF-1α gene can significantly improve hAMSCs tolerance to hypoxia, the mechanism may be related to up-regulation of VEGF and Bcl-2 expressions, and down-regulation of Bax and C-Caspase-3 expressions.
ObjectiveTo analyze the efficacy of promoting the development of day surgery in a municipal third-level public hospital under the guidance of national hospital performance appraisal system. MethodThe annual data relevant day surgery in the Yibin Second People’s Hospital were collected from 2016 to 2022, which were divided into three stages: 2016–2018, 2019–2020, and 2021–2022. The trend and correlation of the performance appraisal indicators were analyzed. ResultsThe day surgery in the Yibin Second People’s Hospital started in 2018, and its proportion in the elective surgery was only 1.2% in 2018, then increased continuously after the implementation of performance appraisal system, and was up to 34.7% in 2022. From 2016 to 2022, the proportion of discharged patients underwent surgery in the entire hospital increased continuously from 22.4% in 2016 to 35.7% in 2022, and the average hospital stay in the entire hospital gradually decreased from 10.9 d to 8.1 d, which both had a significant linear correlation with the proportion of day surgery in the elective surgery (rs=0.93, P=0.002; rs=–0.99, P<0.001, respectively). In the recent implementation of performance appraisal system, the re-operation rate after day surgery was less than 0.1%, the readmission rate of day surgery after discharge was 0%, and the satisfaction rate of day surgery patients was more than 95.0%, which reached 97.0% by 2022, higher than the average level of inpatient satisfaction in the entire hospital. Taking laparoscopic cholecystectomy, cataract phacoemulsification and intraocular lens implantation, internal fixation extraction, vocal cord polypectomy, and endoscopic gastric polypectomy as example, the average total hospitalization cost and average cost excluding drug and medical materials consumption of the day surgery all decreased compared to non-day-surgery mode, respectively. ConclusionUnder the guidance of national hospital performance appraisal system, day surgery has entered a rapid developing stage, but it is still necessary to promote the medical quality by standardized, precise, and informationized day surgery management.
Objective To investigate the effect of bone marrow mesenchymal stem cells (BMSCs) transplantation on the motor function recovery, the expression of vascular endothel ial growth factor (VEGF) gene, and angiogenesis after spinal cord injury (SCI) in rats, and to explore the treatment mechanism of BMSCs in SCI. Methods BMSCs were isolated and cultured from the marrow of 5 Wistar rats (4 weeks old) and the 3rd-4th passage cells were prepared for the experiment. Atotal of 87 adult female Wistar rats (weighing 220-250 g) were randomly divided into 3 groups: sham-operated group (group A, n=21), DMEM group (group B, n=33), BMSCs group (group C, n=33). A laminectomy was only performed at T8-10 levels in group A. The SCI models were establ ished by modified Nystrom’s compression method in groups B and C, and BMSCs and DMEM were injected in groups B and C respectively at 30 minutes after SCI. Basso-Beattie-Bresnahan (BBB) score was used for the motor function recovery at 3, 7, 14, and 28 days, RT-PCR for the VEGF mRNA at 1, 3, and 5 days, and immunohistochemical staining for angiogenesis at 3, 7, 14, and 28 days. Results In groups B and C, the hindl imb locomotor function was improved at different degrees with time, showing significant difference in BBB score between groups B, C and group A (P lt; 0.05). At 28 days, the BBB score in group C was significantly higher than that in group B (P lt; 0.05) and there was no significant difference between groups B and C (P gt; 0.05) at 3, 7, and 14 days after transplantation. The numbers of microvessels in the ventral horns of gray matter around SCI in groups B and C were significantly lower than that in group C (P lt; 0.05) at 3 days, but there was no significant difference at 7, 14, and 28 days after transplantation (P gt; 0.05). There was no significant difference in the number of microvessels between group C and group B (P gt; 0.05) at 3 and 7 days, but the number of microvessels in group C was significantly higher than that in group B (P lt; 0.05) at 14 and 28 days after transplantation. However, there was no significant difference in the number of microvessels in the white matter around SCI in 3 groups at different time points after transplantation (P gt; 0.05). The RT-PCR results showed that VEGF mRNA expressed at a low level in group A. Compared with group A, the expression level of VEGF mRNA in groups B and C increased at 1 day and reached the peak at 3 days, then decreased at 5 days after transplantation; and the expression of VEGF mRNA was significantly higher in groups B and C than in group A (P lt; 0.05),and in group C than in group B (P lt; 0.05) at 1, 3, and 5 days. Conclusion BMSCs may promote the motor function recoveryby up-regulating VEGF mRNA expression and increasing angiogenesis in the spinal cord after SCI in rats.
Objective Aminoguanidine (AG) can reduce brain edema and increase the recovery of neuron functions in surgical brain injury and stroke. To investigate the effect of AG on spinal cord injury (SCI) in rats and its mechanism. Methods A total of 150 adult male Sprague Dawley rats (weighing, 230-255 g) were divided into control group (group A, 25 rats without treatment), the sham-operated group (group B, 25 rats undergoing laminectomy), SCI group (group C, 25 SCI rats with injection of 5%DMSO), SCI + AG groups (groups D, E, and F, 25 SCI rats and AG injection of 75, 150, and 300 mg/kg, respectively). The optimal dosage of AG was screened by dry-wet weight method with the percentage of water content at 0, 12, 24, and 48 hours after injury. The blood-spinal cord barriar permeability was further detected by Evans blue (EB) method, aquaporins 4 (AQP4) mRNA expression by RT-PCR, AQP4 protein expression by immunohistochemistry and Western blot. Results AG injection at dosage of 150 mg/kg can significantly reduce edema of spinal cords at 12, 24, and 48 hours after SCI (P lt; 0.05), so 150 mg/kg was the optimal dosage. The EB content in group E was significantly lower than that in group C at 12, 24, and 48 hours after SCI, and the permeability of blood-spinal cord barrier was significantly decreased compared with group C (P lt; 0.05). The AQP4 mRNA expressions in groups B and E were significantly lower than that in group C at 12, 24, and 48 hours after SCI (P lt; 0.05). AQP4 protein expressions in groups B and E were significantly lower than that in group C at 24 and 48 hours after SCI (P lt; 0.05) by Western blot. Immunohistochemical staining revealed that AQP4 protein expression in group C was significantly higher than that in groups B and E (P lt; 0.05) at 48 hours after SCI, but no significant difference was found between group B and group E (P gt; 0.05). Conclusion AG injection at dosage of 150 mg/kg can induce spinal cord edema and injury in rats, which could be correlated with the down-regulation of AQP4 expression.