1. Cognition and thinking after the outbreak of COVID-19
Journal of Medical Postgraduates 2020;33(7):673-677
Coronavirus disease 2019 (COVID-19) has become a serious global public threat. With the progress of research, we have more comprehensive understanding of the COVID-19. Current advances on the etiology, transmission, clinical features, diagnosis and treatment of COVID-19, as well as the experience in the management of COVID-19 will be introduced in this article.
2. Overexpressed GATA-4 bone marrow-derived mesenchymal stem cell exosomes improve myocardial infarction via miRNA-673-5p/Tsc-1 molecular axis
Min LI ; Zi-hao WANG ; Dan YAN ; Qiao-li XIE ; Ya-lian SA ; Ji-gang HE
Journal of Medical Postgraduates 2020;33(7):678-683
ObjectiveExosomes secreted by BMSC overexpressing GATA-4 gene (BMSCGATA-4-exosome) can promote the differentiation of BMSC into cardiomyocyte-like cells, thereby improve cardiac function after myocardial infarction. However, the molecular mechanism of BMSCGATA-4-exosome in cardiomyocyte-like cell differentiation is unknown. The effect of the secretion of BMSCGATA-4 exosome from bone marrow mesenchymal stem cells (BMSC) in the differentiation of stem cells into cardiomyocytes was determined in miRNA-673-5p/Tsc-1 axis dependent manner.MethodsMouse models of myocardial infarction were established and divided into seven groups. Simulation group (BMSCmiR-673-5p-mimic exosome), inhibition group (BMSCmiR-673-5p-inhibitor exosome), GATA-4 group (BMSCGATA-4 exosome), empty vector group (BMSCempty vector exosome), and BMSC group (BMSC exosome) were injected into the tail vein for 48 h, and the untreated and normal mice were used as the control group. Cardiac ultrasound was used to detect cardiac function in each group. miRNA-673-5p expression in myocardial infarction was detected using real-time polymerase chain reaction (RT-PCR). The myocardial tissues were extracted from the same myocardial infarction site. Myocardial-specific molecules, such as α-actin, Desmin, cTnT, and Cx43, were detected using RT-PCR. Western blot was used to determine the expression of the corresponding target gene of miRNA-673-5p, Tsc-1, Erk1/2, and Mef2c proteins.ResultsThe simulation group wan shown the most significantly improved myocardial function (P<0.05) with an expression peak of miRNA-673-5p in cardiomyocytes (P<0.05). The highest content of myocardial-specific molecules including α-actin, Desmin, cTnT, and Cx43 was found in the simulation group. The simulation group had the lowest expression of Tsc-1 in cardiomyocytes (P<0.05).ConclusionOverexpressed BMSCGATA-4 exosomes inhibit Tsc-1 expression through miRNA-673-5p to improve cardiac function during myocardial infarction.
3. The role of parvalbumin interneuron-mediated disinhibition in the antidepressant effects of ketamine
Feng ZHOU ; Xiao-hui TANG ; Ru LIU ; Jian-jun YANG ; Zhi-qiang ZHOU ; Qing JI
Journal of Medical Postgraduates 2020;33(7):684-688
ObjectiveTo observe the role of parvalbumin interneuron-mediated disinhibition in the antidepressant effects of ketamine.MethodsForty-eight adult male C57BL/6 mice were randomly divided into the following four groups (n=12): group Saline + Saline (group SS), group LPS + Saline (group LS), group Saline + Ketamine (group SK) and group LPS + Ketamine (group LK). The depression model was established by intraperitoneal injection of LPS (1 mg/kg), and ketamine (10 mg/kg) or physiological saline (equal volume) intraperitoneally injected 20 hours later. Four hours after ketamine administration, the open field test and the forced swimming test were performed. The hippocampus was harvested after the behavioral test. PCR and Western blot were used to detect the expressions of PV and GAD67. Electrophysiology were used to detect the change of miniExcitatory post-synaptic current of pyramidal neurons in hippocampus CA1 region.ResultsCompared with the group SS, the time spent in the center zone of the arena was significantly decreased, the immobility time was significantly increased, the mRNA and protein content of PV were significantly increased, the amplitude and frequency of miniEPSC were significantly decreased in the group LS (P<0.05). Compared with the group LS, the time spent in the center zone of the arena was significantly increased, the immobility time was significantly decreased, the mRNA and protein content of PV were significantly decreased, the amplitude and frequency of miniEPSC were significantly increased in the group LK (P<0.05). In the open field test, no significantly difference was observed in the total traveled distance among the four groups (P>0.05).ConclusionKetamine can exert rapid antidepressant effects by down-regulating the expression of PV and then exerting disinhibition regulation on pyramidal neurons.
4. Effects of cold induced RNA binding protein on hippocampal neurons and mitochondrial damage after mild hypothermia in a rat model of cardiac arrest
Jie-jie ZHOU ; Juan LI ; Jie ZHANG ; Hui-xian CHENG ; Zhi-qiang ZHOU ; Man-lin DUAN
Journal of Medical Postgraduates 2020;33(7):689-695
ObjectiveMild hypothermia was an effective way of cerebral resuscitation after cardiac arrest. The expression of cold-induced RNA binding protein (CIRP) was significantly enhanced when the temperature was lowered. This study was to evaluate the effects and the mechanisms of CIRP inhibition on hippocampal neurological and mitochondria function after mild hypothermia in a rat model of cardiac arrest.MethodsFive male Sprague-Dawley rats were injected with AAV9 in the hippocampus, 1 μL on each side, speeding 0.2 μL/min. The expression of GFP was observed by fluorescence microscopy after 2w. Sixty rats were randomly divided into 5 groups (n= 12 for each group): sham operation group, model group, mild hypothermia group, mild hypothermia + CIRP inhibition group and mild hypothermia + normal control group. Injection of AAV9 was performed on mild hypothermia + CIRP inhibition group, same amount of empty vector on mild hypothermia + normal control group, while normal saline on the other groups. Animal models of global cerebral IR were established by transesophageal cardiac pacing inducing cardiac arrest followed by cardiopulmonary resuscitation at 2w after injection. Cooling to 32-34℃ was initiated and the temperature was maintained for 6h on mild hypothermia groups. NDS score, HE staining and pyramidal cell counting on hippocampal CA1 area were performed at 72h after reperfusion. At 24h after reperfusion, mitochondrial structure of pyramidal cells in hippocampal CA1 was observed under electronic microscope and the expressions of CIRP, dynamin-related protein 1 (Drp1) and cytochrome C (Cyt-C) were detected by Western blot.ResultsThe NDS score of model group was decreased, the number of pyramidal cells was reduced, and the mitochondria were severely damaged. The NDS score of mild hypothermia group was increased, and the number of pyramidal cells was increased (all P<0.05), and mitochondrial damage was reduced compared with model group. In mild hypothermia + CIRP inhibition group, the NDS score was no significant difference compared with mild hypothermia + normal control group and model group, and the number of pyramidal cells was lower than that in mild hypothermia + normal control group [(27.2±4.9) vs (50.2±4.4), P<0.05], similar to model group (25.2±3.8), the damage of mitochondria was severe. After 2 weeks of AAV9 injection, GFP was widely expressed in the hippocampus. The expression of CIRP in mild hypothermia + CIRP inhibition group was respectively small compared with sham operation group [(0.14±0.03) vs (0.03±0.01),P<0.05], which was successfully inhibited by injection of AAV9. The expression of CIRP in model group (0.25±0.05) was significantly higher than that in sham operation group. The expression of CIRP in mild hypothermia group (0.37±0.08) and mild hypothermia + normal control group (0.39±0.04) were higher than that in model group (all P<0.05). The trends of Drp1 and Cyt-C expression were the same, in model group was higher than that in sham operation group, in mild hypothermia group was lower than that in model group, in mild hypothermia + CIRP inhibition group was higher than in mild hypothermia + normal control group (all P<0.05); There were no significant differences between model group and mild hypothermia + CIRP inhibition group, and between mild hypothermia group and mild hypothermia + normal control group.ConclusionInhibition of CIRP expression in hippocampus can weaken the protective effects of mild hypothermia on neurons in a rat model of cardiac arrest. The mechanism of those effects might be association with mitochondrial division.
5. Protective effects of Rhein on cardiomyocyte induced with H2O2
Journal of Medical Postgraduates 2020;33(7):696-701
ObjectiveThe effect of Rhein on myocardial cell injury and its possible mechanism of action remains unknown. This study aims to explore the effect and mechanism of rhein on cardiomyocyte injury induced by hydrogen peroxide (H2O2).MethodsRat cardiomyocyte H9c2 was stimulated using H2O2 to establish the myocardial cell injury model. The experiment was divided into three groups including a control group, H2O2 induced group, and Rhein treated group. Fluorescence probe and biochemical methods were used to detect the oxidative stress level of H9c2 cells. RT-qPCR and immunofluorescence tests were adapted to determine the apoptosis of H9c2 cells. Besides, RT-qPCR was carried out to evaluate the inflammatory response of H9c2 cells. Finally, western blot assay was performed to evaluate the expression of MAPK and NF-κB signaling pathways.ResultsThe results of fluorescence probe showed that the expression of ROS of H9c2 cells induced by H2O2 was decreased significantly after Rhein intervention. Besides, the biochemical test showed that the expressions of GSK-Px and CAT of H9c2 cells induced by H2O2 were increased significantly after Rhein treatment. RT-qPCR data suggested that Rhein could significantly decrease the mRNA expressions of Bax, caspase-3 and caspase-9, and upregulate the mRNA expression of Bcl-2 of H9c2 cells induced by H2O2. Moreover, immunofluorescence experiments showed that Rhein could significantly inhibit the expression of caspase-3 of H9c2 cells induced with H2O2. Further, the results of RT-qPCR indicated that the mRNA expressions of IL-6, TNF-α、IL-1β and IL-1α of H9c2 cells induced by H2O2 was decreased significantly after Rhein intervention. Finally, western blot showed that Rhein can significantly inhibit the expressions of MAPK and NF-κB signaling pathways.ConclusionRhein can reduce the injury of cardiomyocytes induced by H2O2, which may be achieved by inhibiting the expressions of MAPK and NF-κB signaling pathways.
6. Effect of H2S on the expression of p38MAPK and caspase3 and apoptosis in renal tissue of urogenous sepsis
Xiong ZOU ; Xian CHEN ; Ya-chun TANG ; Bing-hui WANG ; Guo-tao LIAO ; Wu-jun XU
Journal of Medical Postgraduates 2020;33(7):702-707
ObjectiveTo study the mechanism of renal injury in urogenic sepsis and explore the effect of H2S on the expression of p38 mitogen-activated protein kinase (p38MAPK) and Cysteine protease 3(Caspase3) and apoptosis in renal tissue of urogenic sepsis. Hydrogen sulfide (H2S) has a wide range of biological effects and has a certain protective effect on the kidney. The main objective of this study is to investigate the effect of H2S on the expression of p38 mitogen-activated protein kinase (p38MAPK), cysteine proteinase 3 (Caspase3), and cell apoptosis in renal tissue of urogenic sepsis, and further to study the mechanism of urinary sepsis renal injury.MethodsNew Zealand rabbits (n=40) were divided into five groups Control, Sham, Sepsis, NaHS, and PAG, with eight rabbits in each group. The vital signs, blood routine white blood cell count, neutrophil count, and renal function (Cr, BUN) of five groups of New Zealand rabbits were recorded before the operation, 24 hrs after the operation, 48 hrs after the operation, and 72 hrs after the operation. 72 hrs after the operation, the left kidney tissue was taken for HE staining to observe the changes of cell morphology and structure of the kidney tissue. The apoptotic cells in renal tissue were labeled by Tunel assay (in situ terminal transferase labeling technique). The expression levels of p38MAPK and Caspase3 in renal tissue were tested by ELISA.Results The apoptosis indexes of renal tissue cells in group Control and group Sham were (5.65±2.43)% and (5.57±2.72)%, respectively, with no statistically significant difference (P>0.05). The apoptosis index of rabbit kidney tissue in group Sepsis was (25.26±2.70)%, which was significantly higher than that in group Control and group Sham (P<0.05). The apoptosis index (16.93±2.03)% in group NaHS was significantly lower than that in group Sepsis (P<0.05). The apoptosis index of group PAG (33.09±3.70)% was significantly higher than that of group Sepsis and group NaHS (P<0.05). There was no significant difference between group Control and group Sham (P>0.05). Pairwise comparison between groups Sepsis, NaHS, and PAG showed statistically significant differences (P<0.05). The expression levels of group Sepsis and group PAG were significantly higher than that of group NaHS (P<0.05). The expression level of group PAG was significantly higher than that of group Sepsis (P<0.05).Conclusion H2S can alleviate renal damage caused by urine-derived sepsis, and its mechanism combined with H2S to suppress the expression of p38MAPK and Caspase3 in the renal tissue of urogenous sepsis, thereby reducing renal cell apoptosis Death.
7. Cytological study on aptamer selection for bladder cancer through CELL-SELEX
Qin-rong PING ; Chong LI ; Jian-wei LIANG ; Yang-wenyi LIU ; Rong LEI ; Jian-song WANG ; Ru-ping YAN
Journal of Medical Postgraduates 2020;33(7):708-714
ObjectiveThe methods based on bladder cancer markers which could be applied to early diagnosis and postoperative recurrence monitoring of bladder cancer were current research hotspots. This study aims to screen aptamers that specifically recognize human bladder cancer cell lines (EJ, T24, BIU87) through cell-based systematic evolution of ligand by exponential enrichment (CELL-SELEX).MethodsFor CELL-SELEX screening, bladder cancer cell lines EJ, T24, and BIU87 were used as positive control cells. HCV 29 (human normal urothelial cell line), 293T (human embryonic kidney cell line), huh7 (human hepatocellular carcinoma cell line) were used as negative control cells. PCR upstream primers were labeled with FITC, downstream primer was labeled with Biotin. ssDNA fragments collected from each round were amplified by PCR, and the amplified product was then purified using a DNA purification Kit. The biotin-streptavidin magnetic separation methods were used to isolate the PCR product to obtain secondary FITC-ssDNA for the next CELL-SELEX round. The screening process was monitored by flow cytometry. ssDNA pool with the highest binding rates to bladder cancer cell lines(EJ, T24, and BIU87) was selected to PCR amplification, product purification, molecular cloning, and sequencing. According to the sequencing results, the secondary structure of the aptamer was pre-simulated by Dnaman software. Aptamer labeled with FITC was synthesized in vitro, flow cytometry was used to detect the binding rate of the aptamer to bladder cancer cell lins (EJ, T24 and BIU87).ResultsWith the advance of the CELL-SELEX process, the binding rate of FITC-ssDNA to bladder cancer cell lins (EJ, T24, and BIU87) increased gradually. By the 15th round, the binding rate of FITC-ssDNA to EJ cells reached the highest level. The apt1 had the highest enrichment among the 15th round ssDNA pool. By the 18th round, the binding rate of FITC-ssDNA to T24 or BIU87 cells reached the highest level. The apt2 and apt3 had the highest enrichment among the 18th round ssDNA pool. DNA structure prediction showed that the secondary structure of apt1, apt2, and apt3 was mainly stem-loop structure. Flow cytometry showed that the highest binding rate was FITC-apt1 to EJ cells, FITC-apt2 to T24 cells, and FITC-apt3 to BIU87 cells, respectively. There is no significant combination between these aptamers with the negative cells.ConclusionIn this study, three kinds of aptamers with high specificity for bladder cancer cell lines were successfully screened by CELL-SELEX. The apt1 can specifically recognize EJ cells, apt2 can specifically recognize T24 cells and apt3 can specifically recognize BIU87 cells, all of which provide experimental evidence for early diagnosis and targeted therapy technology research of bladder cancer.
8. Analysis of macrophage marker genes and their functions in triple negative breast cancer based on single cell sequencing
Tao XU ; Jun YANG ; Yong HAN ; Pei-yao ZHU ; Yong YANG ; Ya-dong HAN ; Lei ZHUANG ; Ren-hao WANG ; Jun SONG ; Bin ZHANG
Journal of Medical Postgraduates 2020;33(7):715-719
ObjectiveAs the search for effective tumor markers has been the focus of current research, this paper aims to identify the macrophage marker genes in triple negative breast cancer (TNBC) tissue by single cell sequencing, and to analyze its biological processes and the key genes involved.MethodsFirstly, the GEO database was used to download the data set GSE118389 to obtain the required single cell data, and the dimension reduction of PCA and T-SNE was used to obtain 15 subsets of cells, and the marker genes of different subsets of cells were obtained. Then, gene ontology (GO) and KEGG enrichment analysis was performed on the macrophage marker genes to analyze the biological processes in which these genes may be involved. Finally, a protein-protein interaction (PPI) network was constructed to find key genes in each network, and the Kaplan-Meier Plotter database was used to analyze the prognosis of key genes, and the tumor immune assessment (TIMER) database was used to analyze the correlation between the expression level of key genes and the infiltration state of macrophages in TNBC cancer tissues.ResultsA total of 244 macrophage marker genes were obtained, mainly in the processes of neutrophil degranulation, neutrophil activation, amide binding, peptide binding and cell adhesion molecule binding, and four key genes CSF-1R, HLA-DQA1, OLR1 and LAPTM5 were further obtained. The overall survival rate of TNBC patients with high expression of CSF-1R, HLA-DQA1 and OLR1 was higher than those with low expression. Moreover, the expression levels of CSF-1R, LAPTM5 and OLR1 were positively correlated with the immune infiltration of macrophages.ConclusionMarkers may be an important target for conquering tumors in the future. In addition, CSF-1R, HLA-DQA1 and OLR1 can be used as an effective biomarker to predict the prognosis of TNBC patients and provide ideas for targeted therapy and immunotherapy for them.
9. Inhibitory effect of ursolic acid on proliferation of TPC-1 cells in thyroid carcinoma
Dong-sheng HOU ; Jing ZHANG ; Li FENG ; Jing-jing ZHANG ; Ying WANG
Journal of Medical Postgraduates 2020;33(7):720-725
ObjectiveAt present, there are relatively few studies on the inhibitory effect of ursolic acid (UA) on the proliferation of thyroid cancer cells. This paper intends to explore the inhibitory effect and mechanism of ursolic acid on the proliferation of TPC-1 cells in thyroid papillary carcinoma.MethodsAfter adhering TPC-1 cells to the wall, the original medium was discarded and added ursolic acid medium without fetal bovine serum (0, 2, 4, 8, 16, 32 μmol/L, respectively, with 0 μmol/L as the control), and then the culture medium without cells was used as blank. The proliferation inhibition rate of TPC-1 cells was detected by CCK8 reagent at different times (24 h, 48 h); Flow cytometry was used to detect the apoptosis rate; JC1 kit was used to detect the changes of mitochondrial membrane potential (MMP) of TPC-1 cells after ursolic acid was applied; Fluorescent probe DCFH-DA was used to detect reactive oxygen species in TPC-1 cells after ursolic acid intervention; Flow cytometry was used to detect the protein expression of survivin and vascular endothelial growth factor (VEGF) in cells. RT-PCR assay detected the expression of survivin and VEGF mRNA in TPC-1 cells after the intervention of ursolic acid at different concentrations.ResultsThe inhibitory rate of 2, 4, 8, 16 and 32 mol/L ursolic acid on TPC-1 cells was significantly higher than that of 0 mol/L (P<0.01), and the inhibitory rate of 48 h ursolic acid on TPC-1 cells was significantly higher than that of 24 h (P<0.05). Therefore, the TPC-1 cell inhibition rate was positively correlated with ursolic acid concentration and the time (P<0.05). The apoptosis rates of 0 mol/L, 4 mol/L and 8 mol/L ursolic acid were (4.13±0.61)%, (6.53±0.65)% and (13.13±1.59)%, respectively. With the increase of the concentration, the apoptosis rate of TPC-1 cells increased gradually (P<0.05). The relative expression levels of survivin, VEGF protein and mRNA of 4 and 8 mol/L ursolic acid were significantly lower than those of 0 mol/L (P<0.05), and the expression levels of 8 mol/L ursolic acid was significantly lower than that of 4 mol/L (P<0.05).ConclusionUrsolic acid can effectively inhibit the proliferation and induce the apoptosis of TPC-1 cells, and its inhibitory induction pathway is related to the expression of survivin and VEGF in cells.
10. Expression of tRF 31 in breast cancer tissues and its effect on cancer cell proliferation
Journal of Medical Postgraduates 2020;33(7):726-731
ObjectiveAt present, there are few reports on the effect of tRF 31 on breast cancer. This study aims to detect and analyze the expression level of tRF 31 in breast cancer tissues and to explore its effect on the proliferation of breast cancer cells.Methods32 tumor tissue samples and the corresponding para-cancer tissues from breast cancer patients in The Affiliated Cancer Hospital of Nanjing Medical University from November 2017 to February 2019 were collected. Six out of the thirty two samples were selected for high-throughput sequencing and at last tRF 31 (FC=6.5781, P=0.023) was selected as the research object. The expression of tRF-31 in cancer and para-cancer tissues was measured by RT-PCR. The sensitivity and specificity of tRF-31 expression in the breast cancer diagnosis were analyzed by ROC curve. Two kinds of human breast cancer cell lines (MDA-MB-231 and MCF-7) were divided into two groups: control group (transfected with negative control) and tRF-31 group (transfected with tRF-31 inhibitor). The proliferation of transfected breast cancer cells was detected by CCK-8 and clonal formation assay. The expression changes of threonine kinase (AKT) and mammalian target of rapamycin (mTOR) in breast cancer cells after transfection were measured to explore the relationship between TRF-31 and AKT/mTOR signaling pathway.ResultsThe expression of tRF-31 in cancer tissues (0.103±0.207) was significantly higher than that in para-cancerous ones (0.028±0.039). ROC curve showed that the sensitivity and specificity of tRF-31 in detecting breast cancer were 90.63% and 53.13%, respectively. In MDA MB 231 cells, the expression of tRF-31 in the tRF-31 group was significantly lower than that in the control group [(0.267±0.012) vs (1±0.040), P<0.01)], and in MCF-7 cells, the expression of tRF-31 in the tRF-31 group was also significantly lower than that in the control group. In MDA-MB-231 and MCF-7 cells, the proliferation ability of the tRF-31 group was lower than that of the control group at 0h, 24h, 48h and 72h. In MDA-MB-231 cells, the clonal formation rate of tRF-31 group [(43.67±3.29)%] was significantly lower than that of the control group [(100±3.74)%] (P<0.01). In MCF-7 cells, the clonal formation rate of tRF-31 group [(49±2.94)%] was significantly lower than that of the control group [(100±4.89)%] (P<0.01). In MDA-MB-231 and MCF-7 cells, the protein levels of AKT and mTOR in tRF-31 group were significantly lower than those in the control group.ConclusiontRF-31 is highly expressed in breast cancer tissues and can promote the proliferation of breast cancer cells. This result is expected to provide a new target for the treatment of breast cancer.