AIM:To explore a method of isolation,purification,culture and identification of human microglia.METHODS:The brain tissue from abortive fetus was sterilely obtained,then chopped and triturated gently.The suspension was filtered and centrifuged to separate and isolate mixed glia cells.The cell density was adjusted to 2?1010 cells/L with DMEM/F12 complete medium and cultured in 75 cm2 flask at 37 ℃ in CO2 incubator(marked as first 0 d).After 7-10 days culture,floating cells including microglia and oligodendrocytes appeared in the flask.The first time purification was carried out to obtain highly purified microglia,for which the flasks were shaken gently.The floating cells were collected and transferred into a new flask(marked as second 0 d).4-5 days later,when microglia and oligodendrocytes grew in confluent state,the second purification was performed,for which the mixed cultured cells were digested with trypsin-EDTA and cell suspension was centrifuged,then the cell pellet was suspended by DMEM/F12 complete medium and cultured in flask(marked as 0 h).After 2 h,non-microglial cells including oligodendrocytes and cell debris were removed,and fresh medium was added into the adherent cells.In this way,the whole procedure of microglia purification was completed and the highly purified microglial cells were obtained.After purification,the expression rate of CD45,CD11b and CD68 on/in microglia were detected by laser-confocal microscopy and flow cytometry on the basis of immuno-fluorescence staining to identify the purity of microglial cells.Microglial phagocytotic function was evaluated by phagocytosis of fluorescent microspheres.The level of activation was identified by the phenomena of membrane ruffling in combination with fluorescent phalloidin staining.RESULTS:More than 98% of the microglias that we isolated and purified expressed CD45,CD11b and CD68.Almost all of the microglias could phagocytize fluorescent microbeads CONCLUSION:We succeed in isolating and purifying human microglias,which express CD45,CD11b and CD68.Almost all of the cells exhibit phagocytic function.So these cells will be useful for further functional and proteomic studies.

Objective To study the effect of overexpression of TRPC6 on Ang Ⅱ-induced apoptosis of mouse podocytes in vitro and to explore the possible mechanisms. Methods Mouse TRPC6 cDNA eukaryotie expression vector pEGFP-NI-mTRPC6 was transfected to conditionally immortalized routine podocyte cell line by liposome. The fluorescent microscopy was used to examine the expression of EGFP after 24 hours. The change of TRPC6 protein expression was observed by Western-blot. Podocytes were treated by different concentrations of Ang Ⅱ. The podocyte intracellular calcium concentration was measured with laser-scanning con_focal microscope. The expression of Bax and Bcl-2 mRNA was assessed by RT-PCR and the expression of Bax and Bcl-2 protein was measured by Western-blot. The apoptotic ratio of podocytes was monitored by flow cytometry and Hoechst staining. Results About 35% of the cells expressed EGFP. An up-regulation of protein expression of TRPC6 was detected in podocytes when transfected with pEGFP-N1-mTRPC6 (P<0.01). The overexpression of TRPC6 promoted the Ang Ⅱ-induced influx of extracellular calcium and elevated the expression of Bax but decreased the expression of Bcl-2 (P<0.01, P<0.05). The apoptotic ratio of podocyte was (2.50±0.72)% when treated by low-dose Ang Ⅱ (10-10 mol/L), and it was increased to (4.33±0.45)% when transfected with pEGFP-N1-mTRPC6 (P <0.05 ). Transfection with pEGFP-NI-mTRPC6 increased apoptosis rate from (15.46± 1.40)% to (18.33±0.87)%(P<0.01) by high-dose Ang Ⅱ (10-6 mol/L). Conclusion TRPC6 plays an important role in the Ang Ⅱ-induced apoptosis of podocytes by promoting the influx of extraeellular calcium, which leads to the apoptosis cascade initiation.

Objective To compare the effects of different doses of sufentanil on theα1?band of quantitative pharmaco?electroencephalography (QPEEG)during the induction of general anesthesia by tracheal intubation(TI). Methods Forty selected patients under general anesthesia were randomly divided into two groups,with 20 patients per group. Patients in group Ⅰ were administered 0.2μg/kg sufentanil,whereas patients in group Ⅱ were administered 0.3μg/kg sufentanil. Subsequently,the patients were administered 2 mg/kg propofol and 0.15 mg/kg cisatracurium. HR,MAP,and QPEEG were recorded before induction(T0),after induction(T1),and after insertion of the cannula(T2). Using the method of power spectrum analysis,theα1?band power percentage of QPEEG was calculated. Results In comparison with T0,the values of HR,MAP,andα1?band power percentage in most areas of the brain were both decreased at T1(P<0.05). Furthermore,in comparison with T1,the parameters were increased in group Ⅰ at T2(P<0.05),but no significant changes were observed in group Ⅱ (P>0.05). Conclusion The administration of 0.3μg/kg sufentanil during anesthesia induction can effectively depress the cardiovascular response to TI and stabilize theα1?band power per?centage. This suggests that theα1?band power percentage of QPEEG can be an effective means to monitor the depth of sedation.

Objective:To investigate the effects of acupuncture therapy on acute cerebral infarction. Methods: Eighty patients with acute cerebral infarction were randomly divided into a treatment group of 40 cases and a control group of 40 cases. Xuanzhong(GB 39)-through-Sanyinjiao(SP 6) acupuncture was performed as a main treatment. The curative effects were compared between the two groups and the sizes of cerebral infarct, between pretreatment and posttreatment after one course of treatment. Results: The total recovery rate was 88.5% in the treatment group and 57.5% in the control group after one course of treatment. There was a significant difference between the two(P<0.05). The rate of change in the infarct for the better was significantly higher in the treatment group than in the control group. There was also a significant difference(P<0.05). Conclusion: This treatment is an effective method for lowering the rate of apoplectic disability and raising the cure rate.

Objective To investigate the effects of transcutaneous electrical acupoint stimulation (TEAS) on electroencephalography (EEG) in piglets anesthetized with sevoflurane.Methods Twelve piglets,aged three to seven days,weighing 1.5 to 3.5 kg,were randomly divided into 2 groups:TEAS (group T,n =6) and control (group C,n =6).Group T received continuous TEAS at points baihui and tianmen for 30 minutes.Anesthesia was induced with 8.0％ sevoflurane over 3 minutes and maintained with 3.5％ sevoflurane in both groups.The changes were observed on EEG.Results The heart rates (HR) at intubation and extubation were lower in group T than group C (P ＜ 0.05).Compared with group C,the EEG spike frequency was lower in group T during anesthesia induction and maintenance (P ＜ 0.05).Conclusion Sevoflurane can induce EEG spikes in piglets,which can be reduced by TEAS.

An increasing number of studies have shown that neuroimaging techniques, including CT- and MRI-related imaging biomarkers, are associated with the clinical outcome after intravenous thrombolysis in patients with acute ischemic stroke. Therefore, as a necessary diagnostic item for acute ischemic stroke, imaging examinations and related biomarkers have important value in predicting the outcome after intravenous thrombolysis in patients with acute ischemic stroke.

Objective:To discuss the promotion effect of chemokine C-C motif ligand 19(CCL19)induced macrophage M1 polarization on chronic pancreatitis of the mice,and to clarify its related mechanism.Methods:Ten male C57BL/6N mice were selected,and the pancreatic acinar cells and peritoneal macrophages were extracted from these mice to construct the macrophage-acinar cell co-culture system.The co-culture system cells were divided into control group,model group,and small interfering RNA CCL19(si-CCL19)group.The morphology of the acinar cells in various groups were observed under microscope.Forty mice were randomly selected and divided into normal group and chronic pancreatitis group,and there were 20 mice in each group.HE staining was used to observe the pathomorphology of pancreatic tissue of the mice in two groups;immunofluorescence staining was used to observe the expressions of cytokeratin 19(CK19),amylase,M1 macrophage-related markers inducible nitric oxide synthase(iNOS),and F4/80 in pancreatic tissue of the mice in two groups and morphology of follicular cells and the expressions of CK19,amylase in the co-culture system cells in various groups;enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6,and IL-1β in serum of the mice in two groups and in the co-culture system cells in various groups;immunohistochemistry was used to observe the expression of CCL19 protein in pancreatic tissue of the mice in two groups;Western blotting method was used to detect the expression levels of CCL19 protein and two nuclear factor-κB(NF-κB)signaling pathway-related proteins P65,phosphorylate P65(p-P65),kappa B inhibitor of kinase α/β(IKKα/β),phosphorylated IKKα/β(p-IKKα/β),IkBα,phosphorylated IκBα(p-IκBα)in pancreatic tissue of the mice in two groups and in the co-culture system cells in various groups.Results:The HE staining results showed that the acinar cells in pancreatic tissue of the mice in normal group were tightly arranged;compared with normal group,the acinar cells of the mice in chronic pancreatitis group showed obvious vacuolation and acinar cell ductal metaplasia,indicating successful preparation of the mouse pancreatitis model.The immunofluorescence staining results showed that compared with control group,the acinar cells in model group exhibited severe vacuolation,the CK19 expression was significantly increased,and the amylase expression was significantly decreased;compared with model group,the acinar cell ductal metaplasia in si-CCL19 group was decreased,the CK19 expression was significantly decreased,and the amylase expression was significantly increased;compared with normal group,the expression of amylase in pancreatic tissue of the mice in chronic pancreatitis group was significantly decreased,while the expressions of CK19 and M1 macrophage markers iNOS and F4/80 were significantly increased.The ELISA results showed that compared with normal group,the serum levels of TNF-α,IL-6,and IL-1β of the mice in chronic pancreatitis group were significantly increased(P<0.05);compared with control group,the levels of TNF-α,IL-6,and IL-1β in the cells in model group were significantly increased(P<0.05);compared with model group,the levels of TNF-α,IL-6,and IL-1β in the cells in si-CCL19 group were significantly decreased(P<0.05).The immunohistochemistry results showed that compared with normal group,the expression of CCL19 protein in pancreatic tissue of the mice in chronic pancreatitis group was significantly increased.The Western blotting results showed that compared with normal group,the expression levels of CCL19 protein and NF-κB signaling pathway-related proteins p-P65,p-IKKα/β,and p-IκBα in pancreatic tissue of the mice in chronic pancreatitis group were significantly increased(P<0.05);compared with control group,the expression levels of CCL19,p-IKKα/β,p-P65,and p-IκBα proteins in the cells in model group were significantly increased(P<0.05);compared with model group,the expression levels of CCL19,p-IKKα/β,p-P65,and p-IκBα proteins in the cells in si-CCL19 group were decreased(P<0.05).Conclusion:CCL19 promotes the macrophage M1 polarization through the NF-κB signaling pathway,induces the formation of inflammatory microenvironment,and promotes the occurrence and development of pancreatitis.

The morbidity of inflammatory bowel diseases (IBD) is rising rapidly but no curative therapies to prevent its recurrence. Cell death is crucial to maintaining homeostasis. Necroptosis is a newly identified programmed cell death and its roles played in IBD need to be explored. Necroptosis is mediated by receptor interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL), which resulted in cell swelling, plasma membrane rupture, intracellular content leaking, and eventually cell death as well as the promotion of inflammation. Studies have found that inhibiting necroptosis alleviated IBD in animal models and IBD patients with an increased level of necroptosis in inflammatory tissues, indicating that necroptosis is related to the pathogenesis of IBD. However, due to the complexity in regulation of necroptosis and the involvement of multiple functions of relevant signaling molecules, the specific mechanism remains elusive. Necroptosis may play a vital regulatory role in the pathogenesis of IBD, which provides a new idea and method for further exploring the therapeutic target of IBD.

Objective To explore a method to improve the accuracy and generalization ability of medical diagnostic neural network models under conditions of small data volumes,and to address the issue of poor neural network model performance in computer-aided diagnosis of nail diseases due to limited training data.Methods A dual-model strategy integrating instance segmentation with fine-grained feature classification was proposed.The neural network model based on dual-model strategy was trained using the dataset of Image-Based Intelligent Diagnosis of Nail Disease Model task of the first National Digital Health Innovation Application Competition & Health and Medical Big Data Theme Competition.This dataset covered 8 types of nail diseases,including nail matrix nevi,paronychia,nail psoriasis,onychomycosis,subungual hemorrhage,melanonychia,periungual warts,and nail melanoma,with class imbalance present.The diagnostic performance of the dual-model strategy was evaluated and compared with single-model strategies(image classification models[ResNet50 and Swin Transformer]and target detection model based on faster region-based convolutional neural network[Faster R-CNN])under the same hardware and software training conditions.Results The dataset included 1 048 samples,including 210 cases of nail matrix nevi,186 cases of paronychia,69 cases of nail psoriasis,203 cases of onychomycosis,149 cases of subungual hemorrhage,71 cases of melanonychia,93 cases of periungual warts,and 67 cases of nail melanoma,with 90％used for training various models and 10％for evaluation.The micro F1 score was 0.324 in the image classification model based on ResNet50,0.381 in the image classification model based on Swin Transformer,0.572 in the target detection model based on Faster R-CNN,and 0.714 in the dual-model strategy model Mask R-CNN+Swin Transformer.The accuracy rates for diagnosing different nail diseases in the dual-model strategy were:nail matrix nevi 80.95％(17/21),paronychia 89.47％(17/19),nail psoriasis 100.00％(7/7),onychomycosis 70.00％(14/20),subungual hemorrhage 73.33％(11/15),melanonychia 14.29％(1/7),periungual warts 55.56％(5/9),and nail melanoma 42.86％(3/7).The micro F1 score for evaluating the dual-model strategy on a test set of 1 000 cases was 0.844.Conclusion The dual-model strategy can effectively combine models with different functions to well accomplish the task of intelligent diagnosis of nail diseases under small data volume training conditions.

Objective The nursing treatment ability scale of patients with nuclear radiation damagein the hospital was developed to provide an evaluation basis for improving the nursing ability of nurses with nuclear radiation damage. Methods The scale was prepared by literature review, expert interview and expert consultation, and a total of 330 clinical nurses from a third-class hospital was randomly selected as the research objects. The scales were issued for item analysis and reliability and validity test. Results The scales were divided into 6 dimensions, including basic knowledge of nuclear radiation damage, specialized equipment use ability, specialized ward management ability, basic nursing ability, specialized nursing ability and self-ability recognition, with 51 items. After exploratory factor analysis, there were 6 principal components, and the cumulative interpreted variance was 70.757%. The χ², df, χ²/df, CFI, IFI, TLI, NFI, PNFI, PCFI, RMSEA fitting indexes of confirmatory factor analysis were all acceptable. Cronbach's α coefficient was 0.976, the retest reliability was 0.823, and the S-CVI (S-CVI/UA) was 0.84. The evaluation content validityS-CVI (S-CVI/AVE) was 0.98, and the content validity I-CVI of the item level was 0.78～1.00. Conclusion The items and dimension Settings of this scale have been tested, and all indicators met the requirements. The reliability and validity test results were good. It can be used as a scale for preliminary evaluation of hospital nursing ability of patients with nuclear radiation damage.