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Central nervous system (CNS) injuries, including stroke, traumatic brain injury, and spinal cord injury, are essential causes of death and long-term disability and are difficult to cure, mainly due to the limited neuron regeneration and the glial scar formation. Herein, we apply extracellular vesicles (EVs) secreted by M2 microglia to improve the differentiation of neural stem cells (NSCs) at the injured site, and simultaneously modify them with the injured vascular targeting peptide (DA7R) and the stem cell recruiting factor (SDF-1) on their surface via copper-free click chemistry to recruit NSCs, inducing their neuronal differentiation, and serving as the nanocarriers at the injured site (Dual-EV). Results prove that the Dual-EV could target human umbilical vascular endothelial cells (HUVECs), recruit NSCs, and promote the neuronal differentiation of NSCs in vitro. Furthermore, 10 miRNAs are found to be upregulated in Dual-M2-EVs compared to Dual-M0-EVs via bioinformatic analysis, and further NSC differentiation experiment by flow cytometry reveals that among these miRNAs, miR30b-3p, miR-222-3p, miR-129-5p, and miR-155-5p may exert effect of inducing NSC to differentiate into neurons. In vivo experiments show that Dual-EV nanocarriers achieve improved accumulation in the ischemic area of stroke model mice, potentiate NSCs recruitment, and increase neurogenesis. This work provides new insights for the treatment of neuronal regeneration after CNS injuries as well as endogenous stem cells, and the click chemistry EV/peptide/chemokine and related nanocarriers for improving human health.
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Objective:To establish a fluorescent reporter human induced pluripotent stem cell line (hiPSCs) for monitoring the expression of visual system homeobox 2 ( VSX2). Methods:VSX2_small guide RNA (sgRNA) was inserted into vector PX459 to construct knockout plasmid, and the P2A-eGFP knock-in donor plasmid was conducted at the same time.The two plasmids were transfected into BC1-hiPSCs.Single cell clones were generated after treatment of puromycin.Correct insertion was confirmed by PCR and Sanger sequencing.The isogenicity of the parental and the reporter hiPSCs was confirmed by STR analysis and karyotyping.Pluripotency capacity of the reporter hiPSCs was analysed by reverse trascription PCR and immunofluorescence.Three-germ-layer formation experiment was carried out to analyse the multi-lineage differentiation ability of the reporter hiPSCs.The reporter hiPSCs were further differentiated to obtain three-dimension (3D) retinal organoids, and immunofluorescence was used to identify the co-localization of the enhanced green fluorescent protein (eGFP) and VSX2.Results:A VSX2 eGFP reporter hiPSC clone was successfully obtained by CRISPR/Cas9 technology, which was consistent with the parental hiPSCs (BC1-hiPSCs) in morphology, without any chromosomal aberrations or cell line cross-contamination.Reverse transcription PCR assay and immunofluorescence showed obvious positive expressions of iPSCs markers in BC1- VSX2 eGFP-iPSCs, including NANOG, OCT4, SOX2, DNMT3B and GDF3 mRNA as well as NANOG, OCT4, SSEA4 and TRA-1-60 protein.The α-fetoprotein (AFP), α-smooth muscle actin (α-SMA) and neuronal class Ⅲ β-tubulin (TUJ1) were expressed in endoderm, mesoderm and ectoderm, respeetively, derived from BC1- VSX2 eGFP-iPSCs, and eGFP and VSX2 were co-stained in the neural retinal layer of 3D retinal organoids derived from BC1- VSX2 eGFP-iPSCs by immunofluorescence. Conclusions:VSX2 fluorescent reporter hiPSCs is successfully generated, which can monitor the temporal and spatial expression changes of VSX2 protein in real time, providing a powerful tool for evaluation of retina development mechanism and cell therapy.
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Objective:To evaluate the retinal differentiation ability of human induced pluripotent stem cells (hiPSCs) from various somatic cell sources.Methods:The hiPSCs lines BC1- green fluorescent protein (GFP) and Gibco obtained by blood cell reprogramming and the hiPSCs line UE017 obtained by urine cell reprogramming were used to induce retinal differentiation.The morphogenesis and development of retina were recorded with an optical microscope, and the expression of specific molecular markers of various cell subclasses in the retina was detected by immunofluorescence, and the efficiency of retinal differentiation of different cell lines was analyzed and compared.Results:All three hiPSC lines derived from blood and urine cells were able to be induced into three-dimensional (3D) retinal organoids, including neuroretina and retinal pigment epithelial cells.Retinal organoids simulated the development process of retina in vivo and gradually differentiated into all cell subtypes of retina, including retinal ganglion cells, photoreceptor cells, amacrine cells, horizontal cells, bipolar cells, Müller cells, and even formed lamellar structures.However, in terms of the efficiency of acquiring retinal organoids, the hiPSCs derived from blood were more efficient than those derived from urine. Conclusions:hiPSCs from both blood and urine somatic cells can differentiate into 3D retinal organoids, including all subtypes of retinal cells.The differentiation efficiency among lines is different.
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OBJECTIVE： To study on the effects of prophylactic administration of Ramulus mori polysaccharides （RMP） on inflammatory response of renal ischemia reperfusion injury （RIRI） model mice and to explore its possible mechanism. METHODS： Totally 60 C57BL/6 mice were randomly divided to sham operation group， model group， atorvastatin group （positive control， 15 mg/kg）， RMP low-dose， medium-dose and high-dose groups （300， 600， 1 200 mg/kg）. Except for sham operation group， RIRI model was induced in other 5 groups. 24 h before surgery， they were given relevant medicine intragastrically， once a day， for consecutive one week. 24 h after reperfusion， the mice were sacrificed. The serum levels of Scr and BUN were detected. The morphological changes of renal tissue were observed under optical microscope. The serum levels of IL-1β， IL-6， IL-10 and TNF-α were determined by ELISA. Western blot assay was used to determine the protein expressions of Toll-like receptor 4 （TLR4）， p38 mitogen-activation protein kinase （p38MAPK） and p-p38MAPK. RESULTS： Compared with sham operation group， the serum levels of Scr and BUN were significantly elevated in model group （P＜0.01）. RIRI led to typical inflammatory response of renal tissue， widespread renal tubular epithelial cell degeneration and necrosis， and inflammatory cells infiltration. Serum levels of IL-1β， IL-6， IL-10 and TNF-α were increased significantly （P＜0.01）. The protein expressions of TLR4， p38MAPK and p-p38MAPK were increased significantly in renal cortex （P＜0.01）. Compared with model group， serum levels of Scr and BUN were decreased significantly in administration groups （P＜0.05 or P＜0.01）. The pathological damage of renal tissue was improved in varying degrees， especially in the RMP medium-dose and high-dose groups. Serum levels of IL-1β and IL-6 were decreased significantly in administration groups （P＜0.05 or P＜0.01）. Serum levels of IL-10 were further increased in atorvastatin group and RMP high-dose group （P＜0.01）， and serum level of TNF-α was decreased significantly in atorvastatin group and RMP medium-dose and high-dose groups （P＜0.05 or P＜0.01）. The protein expressions of TLR4 and p-p38MAPK in renal cortex were decreased significantly in administration groups （P＜0.05 or P＜0.01）. CONCLUSIONS： RMP prophylactic administration can improve RIRI of mice， the mechanism of which may be associated with relieving the inflammatory response through inhibition of TLR4/p38MAPK signaling pathway.
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OBJECTIVE:To study the protective effect of fingolimod on renal ischemia reperfusion injury (RIRI) model mice and its mechanism.METHODS:A total of 60 mice were randomly divided into sham operation group,model group,fingolimod group (1 mg/kg) and fingolimod+wortmannin group [fingolimod 1 mg/kg+phosphatidylinositol 3-kinase (PI3K) specific blocker wortmarmin 1.4 mg/kg],with 15 mice in each group.Except for sham operation group,RIRI model was induced in other 3 groups,and those model mice were given relevant medicine via caudal vein at once 24 h before surgery.Serum of mice were collected in each group after 24 h perfusion.Serum levels of Scr and BUN were measured by automatic biochemical analyzer.The pathological changes of renal tissue were observed under light microscope.The protein expression of intercellular cell adhesion molecule-1 (ICAM-1),monocyte chemoattractant protein-1 (MCP-1) and phosphorylated protein kinase B (p-Akt) in renal tissue were measured by Western blot assay.RESULTS:Compared with sham operation group,the serum levels of Scr and BUN in model group were increased significantly (P＜0.01).Pathological changes were found in the kidney,and RIRI led to widespread renal tubular epithelial cell injury,apoptosis and inflammatory cells infiltration.The protein expression of ICAM-1 and MCP-1 in renal tissue were increased significantly (P＜0.01),the protein expression of p-Akt was increased slightly (P＞0.05).Compared with model group,other indexes of fingolimod group were improved significantly (P＜0.01) except that the protein expression of p-Akt in renal tissue was increased significantly (P＜0.01).Compared with fingolimod group,above indexes of fingolimod+wortmannin group were reversed (P＜0.05 or P＜0.01).CONCLUSIONS:Fingolimod can obviously ameliorate renal injury induced by RIRI in mice,the mechanism of which may be associated with the activation of PI3K/Akt signaling pathway.
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Objective To optimize the detection test of Pig-a gene mutation in peripheral blood of rats by enriching and detecting mutant erythrocytes, using immunomagnetic separation technique in combination with flow cytometry. Methods SD rats were administered with 20,40 and 80 mg/(kg·bw)doses of N-ethyl-N-nitrosourea(ENU) continually for 3 days. The peripheral blood samples of rats were collected on the 7th,14th and 28th days, respectively, after treatment. Immunomagnetic separation columns were used to enrich RETCD59- and RBCCD59- cells, and then flow cytometry was used to count the number of pre-column and post-column peripheral erythrocytes. Results Compared with the control group,the frequencies of RETCD59- and RBCCD59- were significantly increased in each ENU group(P<0.05). With immunomagnetic separation technique, the test of Pig-a gene mutation of a sample could be completed within 3 minutes,and the number of detected RETCD59- or RBCCD59-cells was up to 2×104or 9×104, respectively. Conclusions In this study,immunomagnetic separation in combination with flow cytometry is used to establish and optimize the Pig-a gene mutation test in rat peripheral blood,showing a high-throughput detection and improved accuracy and efficiency of the experiment.
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Objective·To analyze high-risk factors of infection of multidrug resistance Klebsiellapneumonia (MDR-Kpn) and difference of therapeutic effects for different treatments.Methods·One hundred and ten MDR-Kpn strains were collected from a hospital.K-B slip diffusion method was utilized to detect the drug susceptibility of all the strains.Based on electronic medical records system,MDR-Kpn infected group included 51 patients and control group was picked out,including 51 patients as well (byl:1 ratio basing on the infected group according to the rules of under the same department,similar basic disease and all the patients in the control group isolated with the strain of Kpn).Thirty-nine clinical information of MDR-Kpn infected and control groups are collected to make single-factor analysis of high risk factors of the infection with MDR-Kpn.Multi-factor analysis was utilized between MDR-Kpn infected and control groups.The lasting time of different antibiotics used in MDR-Kpn infected patients were made statistics between effective and inefficacy patients.Results·Drug susceptibility test showed that sulfonamide,phosphonomycin and amikacin,were the three most sensitive antibiotics for 110 MDR-Kpn strains.12 clinical information,such as blood transfusion、sputum suction、invasive ventilation were all high-risk factors for the infection of MDR-Kpn (P＜0.05).The lasting time during with carbapenems (P=0.025) was statistically different between effective (n=28)and non-effective group (n=23) of MDR-Kpn infection patients.Conclusion·Controlling and eliminating high-risk factors do help to protect and decrease the infection of MDR-Kpn.Using carbapenems correctly has great influence on prognosis.
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Objective To prospectively determine the feasibility of high-resolution in vivo MR imaging in the evaluation of esophageal carcinoma invasion at 3.0 T.Methods One hundred and eighteen patients with esophageal carcinoma,proven by the gastroscopic biopsy,were prospectively studied using 3.0 T MR.The esophageal specimens were sectioned transversely to keep consistent in the orientation with the MR images,the histopathological stage was made and the thickness of the tumor on the largest diameter of the slice were measured.The MR images were reviewed in the transverse plane.According to the seventh American joint committee on cancer,the MR stage was made and the tumor's thickness was measured.The MR images and the histopathological slices were matched.The staging diagnostic efficacy of the MR imaging was evaluated with the histopathological results as the standard reference,Kappa test was used to compare the stage of MR imaging with that at the histopathological analysis.Bland-Altman scatterplots were used to compare the thickness of tumor measured on the MR images with that at the histopathological measurement.Results Ninety seven cases(82.2％,97/118) of MR stage were accurately made,including 7 T1a,15 T1b,18 T2,25 T3 and 32 T4a cases,furthermore,14 cases were over staged and 7 cased were underestimated.The MR stage was highly consistent with the histopathological stage (Kappa=0.772).The sensitivity for the staging of high-resolution MR imaging at 3.0 T was 58.3％(7/12) to 100.0％(32/32),the specificity was 95.3％ (82/86) to 98.1％ (104/106),and the accuracy was 91.5％ (108/118) to 96.6％ (114/118),respectively.Bland-Altman scatterplots demonstrated that the discrepancy of the mean thickness between the value obtained by three radiologists respectively and the histopathological analysis were 2.0,2.6 and 2.1 mm,which demonstrated a good consistency.Conclusion High-resolution MR images obtained at 3.0 T can be used to evaluate the depth of carcinoma invasion and provide excellent diagnostic accuracy for preoperative staging.
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Objective To investigate the antimicrobial resistance of clinical isolates from Renji Hospital,Shanghai Jiao Tong University School of Medicine during the period from 2005 to 2015.Methods Antimicrobial susceptibility testing was carried out according to Kirby-Bauer method.Results were analyzed according to CLSI 2015 breakpoints.Results A total of 55 155 nonduplicate clinical isolates were collected from 2005 to 2015.The top 5 most frequently isolated bacterial species were E.coli (15.0％),P.aeruginosa (14.0％),A.baumannii (11.9％),K.pneumoniae (11.8％) and S.aureus (10.2％).Gram positive cocci and gram negative organisms accounted for 35.8％ and 64.2％,respectively.The prevalence of methicillin-resistant strains in S.aureus (MRSA) and coagulase negative Staphylococcus (MRCNS) was 70.2％ (3 967/5 650) and 83.2％ (4 997/6 004).No staphylococcal strain was resistant to vancomycin,teicoplanin or linezolid.Fifteen strains of Enterococcus were found resistant to vancomycin.The average prevalence of ESBLs-producing strains was 70.4％ (5 843/8 300) in E.coli,53.5％ (3 500/6 539) in Klebsiella spp.and 44.1％ (557/1 263) in P mirabilis.A few carbapenemaseproducing K.pneumoniae strains were identified for the first time in 2012 with the prevalence of 0.6％ (4/656),and the prevalence hit high (30.1％,142/472) in 2015.The prevalence of carbapenemase-producing E.coli was 2.0％ (16/787) in 2015,and almost zero in the other years.The prevalence of extensively drug-resistant A.baumannii and P.aeruginosa was 39.1％ (2 566/6 556) and 4.0％ (308/7 704),respectively.Extensively drug-resistant strain was identified in 9 of the strains of 189 E.aerogenes isolates.Conclusions Bacterial resistance is still on the rise,which poses a major challenge to clinical antimicrobial therapy,especially the multi-drug resistant and extensively drug resistant bacteria.
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OBJECTIVE To evaluate the protective effect of Agaricus bisporus intracellular polysaccharides(IPS) and exopolysaccharides (EPS) on immunological liver injury induced by concanavalin A (Con A). METHODS Mice were pretreated with IPS and EPS (100, 200 and 400 mg kg- 1, ig) daily for 12 d. Immunological liver injury was induced by Con A 25 mg·kg-1 byinjection via the tail vein of mice.Eight hours after injection of Con A, the indexes of the liver, spleen and thymus, serum level of glutamicpyruvic transaminase (GPT), glutamic-oxalacetic transaminase (GOT), tumor necrosis factor-α (TNF-α) and interferon- γ (IFN- γ), splenic lymphocyte percentages of CD4 + and CD8 + , and liver homogenate content of superoxide dismutase (SOD) and malondialdehyde (MDA) were measured. Liver pathological changes were observed by HE staining. RESULTS Compared with normal group, the autoimmune liver injury in mice induced by Con A resulted in an increase in the liver index (P<0.01) , spleen index (P<0.01), the activity of GPT (P<0.01) and GOT (P<0.01), the content of TNF- α (P<0.01) and IFN- γ (P< 0.01), and the level of MDA (P<0.01), but a decrease in the thymus index (P<0.01), the percentage of CD4+ (P<0.01) , the ratio of CD4+/CD8+ (P<0.01), and SOD activity (P<0.01). Compared with model group, treatment with IPS (200 and 400 mg·kg-1) and EPS (200 and 400 mg·kg-1 ) respectively resulted in an increase in the thymus index (P<0.01) but in a decrease in the liver index and spleen index (P<0.01). Similarly, the activity of GOT and GPT was decreased obviously (P<0.01), and the content of TNF-α and IFN-γ in IPS and EPS 200 and 400 mg·kg-1 groups was decreased. Compared with model group, the activity of SOD in IPS and EPS (200 and 400 mg·kg- 1) group was increased (P<0.01) while MDA was decreased (P<0.01). Moreover, the percentage of CD4 + Iymphocytes decreased (P<0.01), whereas no significant difference was found in the ratio of CD4 +/CD8 + .Pathological changes of the liver were observed under a microscope. Pretreatment with IPS and EPS could effectively reduce the liver injury induced by Con A. CONCLUSION IPS and EPS have certain protective effect on immunological liver injury, which may be related to their ability to clean up free radicals, control lipid peroxidation and regulate the balance of the immune system.
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PurposeIt is well known hypobaric hypoxia occurs with acute exposure to high altitude, with commonly associated change of cerebral blood flow (CBF). In this work, three-dimensional arterial spin-labeling (3D ASL) was used to monitor the change of CBF to further extend our understanding of hypobaric hypoxia.Materials and Methods Six healthy subjects were recruited for this study, they were asked to stay at high altitude areas for 5 days, and then returned to the plain. All subjects received MRI examination in both plain and high altitude areas using exactly the same 3.0T MR scanner. A total of 8 MR scans were performed, and all the parameters were kept the same, the changes of cerebral blood flow were observed.ResultsCBF increased obviously and reached its peak after acute exposure to high altitude, at the first day at high altitude, CBF measurements in global brain, grey matter and white matter increased signiifcantly compared to the plain, the difference was statistically significant (P<0.05); after that, the CBF measurements started to gradually decrease in the second day and a small climb on the third day at high altitude, then the CBF continued to drop after returning to sea level, even below that at sea level prior to departure. After 1 week back to the plain area, CBF measurements in global brain, grey matter and white matter were still lower than those before departure for high altitude areas, with a statistically signiifcant difference (P<0.05).ConclusionCBF measurements had obvious increase upon initial arrival at high altitude, and then the CBF continued to drop even below that at sea level prior to departure.
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Objective:To optimize the extraction process of total flavonoids in Gynostemma pentaphyllum by the method of response surface analysis ( RSM) . Methods: Based on the choice of factors and levels according to the results of single factor test, Box-Be-hnken response surface experiment was designed with the extraction rate of total flavonoids as the index to obtaln the best operation con-ditions and two equations of the response surface model. Results: The optimal extraction conditions of total flavonoids in Gynostemma pentaphyllum were as follows:the volume fraction of ethanol was 71%, the ratio of solid to liquid was 1∶14, and the ultrasonic time was 32 min. Under the conditions, the model predictive value of extraction rate of total flavonoids in Gynostemma pentaphyllum was 4. 676%, and the extraction rate in the verification test was 4. 641%. Conclusion:The fit of the regression model is good, and the ex-traction technology is feasible and reliable.
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OBJECTIVE To use the software for hospital infection control in our hospital to improve monitoring of nosocomial infection in invasive nursing procedure and reduce incidence rate of nosocomial infections.METHODS The software for hospital infection control was used in this prospective investigation to collect common information of invasive nursing procedure,institute intervention measure to invasive nursing procedure,and observe the(occurrence) of nosocomial infection.RESULTS Element administration,process administration,and monitoring(administration) were used to real-time control in invasive nursing procedure,thus fasten information transfer and optimize(performance) flow-sheet of nosocomial infections administration.The software usage could accurately(provide) the(information) of nosocomial infection in-time,and feed-back rapidly.CONCLUSIONS Whole process(control) in invasive nursing procedure can discover and solve problems,thus improve efficiency and effectiveness for preventing and controlling nosocomial infection.