Objective To investigate the methods of isolation,culture,identification and labeling of mesenchymal stem cells(MSCs) in vitro and lay a foundation for further study on intervention of MSCs on immunologic rejection of organ transplantation. Methods MSCs were isolated and cultivated by adherent methods . The expressions of CD90 and CD45 of cells were analyzed by using flow cytometry in order to identify MSCs.The third generation of MSCs were labeled by DAPI,the labeling efficiency was detected.Results Primary cultured MSCs adhered to plastic surface within 48 h and reached 90% confluence within 7-10 d .Flow cytometry showed that the positive rates of CD90 and CD45 of MSCs at third generation were 99.8% and 6.8%. MSCs expressed CD90 but no CD45.All of the MSCs after labeling by DAPI showed blue fluorescence by immunofluoroscope. DAPI labeling was sensitive and highly efficient to MSCs.Conclusion Adherent method is simple and easy to isolate and cultivate MSCs and it can serve as a routine method.DAPI labeling can be used as a efficient method to label MSCs.

Background Acquired immune deficiency syndrome (AIDS) is an infectious disease caused by human immunodeficiency virus (HIV).Highly active antiretroviral therapy (HAART) is an effective treatment for AIDS,but it cannot completely eliminate the viral load in the body for the existence of HIV reservoir.Previous studies demonstrated that HIV could be detected in tears of virus load negative AIDS patients who received effective HAART,suggesting that lacrimal gland is another member of HIV reservoirs.Objective The aim of this study was to explore whether lacrimal gland has a molecular basis of HIV infection and the mechanism of lacrimal gland infection of HIV.Methods Fourteen specimens of lacrimal gland were collected during the surgery from 14 patients with lacrimal gland diseases in Peking Union Medical College Hospital from November 2013 to December 2015,including 13 non-HIV-infected patients and 1 HIV-infected patient.In 13 non-HIV infected patients,lacrimal glands prolapse was in 12 patients with the normal pathological tissue structure and dacryoadenitis was in 1 patient with the histopathological diagnosis of interstitial lymphoid tissue hyperplasia.The clinical manifestation of HIV-infected patient was dacryoadenitis with the histopathological diagnosis of interstitial lymphoid tissue hyperplasia.The paraffin sections of 12 non-HIV-infected specimens and 1 HIV-infected specimen were prepared,and the expressions of CD4,C-X-C chemokine receptor 4 (CXCR4) and C-C chemokine receptor type 5 (CCR5) in lacrimal gland specimens were detected by immunohistochemistry and verified in 1 specimen of non-HIV-infected specimen by immunofluorescence technology.Results Immunohistochemistry showed that CD4 was suspiciously positive expression in non-HIV-infected specimens with the strong background staining.CXCR4 was positively expressed in cytoplasm and nuclei of most lacrimal epithelial cells of lacrimal gland epithelial cells in each specimen,and CCR5 was focally expressed in few lacrimal gland epithelial cells in each specimen.In addition,CD4,CXCR4 and CCR5 were positively expressed in intercellular scattered lymphocytes on the specimens.Immunofluorescence assay showed that CD4,CXCR4 and CCR5 were expressed in the specimens with the red fluorescence,with the linear-and patchy-like distribution mainly in cellular membrane for CD4 or spot-like distribution for CXCR4 and CCR5 in the cytoplasm.Conclusions HIV receptor CD4 and accessory receptor CXCR4,CCR5 are positively expressed in the lacrimal gland epithelial cells,which is the molecular basis of HIV infection and become a potential HIV reservoir preventing HIV eradication.

OBJECTIVE To optimize the parameters of passive cutaneous anaphylaxis(PCA)in rats immunized by ovalbumin(OVA). METHODS 1-2 month-old Sprague-Dawley rats were immu?nized by ip injection of OVA(0.2,1.0 and 5.0 mg per rat)mixed with complete Freund′s adjuvant once every other day 3 times. Serum was collected on the 12th-16th days after final immunization. Then the rats were intracutaneously injected with sensitized serum and then stimulated by iv injection of the same dose of OVA mixed with Evans blue after a latent period of 0.5,1.5,3,6,12,24,36,48 and 60 h. Finally,the diameters of blue spots in the skin were measured at stimulation. RESULTS Serum total-IgE(T-IgE)and OVA-specific IgE(sIgE)levels increased significantly and reached the peak on the 3rd-7th days and 12th-16th days after final immunization,respectively. There was no correlation between the serum T-IgE level and OVA-sIgE level when the rats were immunized with OVA at OVA 0.2-5.0 mg per rat. The rats experienced PCA after injection of OVA 1.0 and 5.0 mg per rat. Diameters of blue spots in the skin reached the maximum value after rats were sensitized for 0.5-3 h. Moreover,the shape,color and size of blue spots were better 30-60 min after stimulation. CONCLUSION Optimized PCA is as follows:1-2 month-old rats are immunized on the 1st,3rd and 5th days by ip injection of OVA 1.0-5.0 mg. The immunizing serum is collected at 12-16 d after final immunization. The rats are stimulated by OVA and Evans blue after a latent period of 0.5-3 h. Diameters of blue spots in rats′ skin are then measured 30-60 min after stimulation.

Objective:To explore effects of remote education based on short video on pain symptoms and rehabilitation compliance in patients with degenerative osteoarthritis.Methods:A total of 132 patients with degenerative osteoarthritis who were treated in Department of Orthopedics of Beijing Jishuitan Hospital were selected as the research objects from March 2018 to June 2020. According to the order of admission, they were divided into the control group and the observation group, with 66 cases in each group. The control group received routine health education, while the observation group received short video-based remote education. The differences of pain symptoms and rehabilitation compliance were compared between the two groups after intervention.Results:After intervention, the pain scores of the observation group during exercise [ (2.4±1.0) vs. (3.5±1.1) ]and tenderness point [ (2.7±1.0) vs. (3.9±1.2) ]were lower than those of the control group. The scores of medication [ (5.2±1.0) vs. (4.5±1.2) ], diet [ (2.2±0.6) vs. (1.6±0.8) ]and exercise [ (2.4±0.6) vs. (1.7±0.5) ]compliance were higher than those of control group. The above differences were statistically significant ( P<0.05) . Conclusions:Application of remote education based on short video in patients with degenerative osteoarthritis can reduce pain symptoms and improve rehabilitation compliance.

Objective:To investigate the application value of biological muscle flap in laparo-scopic radical proximal gastrectomy with esophagogastric anastomosis.Methods:The retrospec-tive and descriptive study was conducted. The clinicopathological data of 10 patients with adeno-carcinoma of esophagogastric junction who were admitted to The First Affiliated Hospital of Xi′an Jiaotong University from May 2023 to August 2023 were collected. All patients were males, aged (65±5)years. All patients underwent laparoscopic radical proximal gastrectomy and esophagogastric anastomosis with digestive tract reconstruction using the esophagogastric biological muscle flap. Observation indicators: (1) surgical situations and early complications; (2) follow-up and late com-plications. Measurement data with normal distribution were represented as Mean± SD, and measure-ment data with skewed distribution were represented as M(range). Count data were described as absolute numbers. Results:(1) Surgical situations and early complications. All 10 patients success-fully completed the surgery without conversion to open surgery, and the operation time was (166±18)minutes. Cases with digestive tract reconstruction as end-to-side anastomosis and Overlap anas-tomosis were 1 and 9, respectively. The time of digestive tract reconstruction, the number of lymph node dissected, volume of intraoperative blood loss, time to postoperative first anal exhaust, time to postoperative first intake of liquid food, duration of postoperative hospital stay were (40±12)minutes, 24±6, (41±9)mL, (3.4±0.5)days, (4.1±1.0)days, (8.3±0.7)days in the 10 patients. Of 4 cases with postoperative early complications, 1 case developed pulmonary infection (Clavien-Dindo grade Ⅱ) on the second day after surgery, with pulmonary infection absorbed after 5 days of antibiotic treat-ment. Two cases experienced chest distress and shortness of breath on the third day after surgery, with the diagnosis of a small to moderate amount of pleural effusion after chest B-ultrasound examination. After pleural puncture and active treatment, the symptoms of them were improved and the pleural effusion disappeared. There was 1 case with choking sensation when eating solid food, which was started from the third week after surgery. Upper gastrointestinal imaging revealed mild anastomotic stenosis of Clavien-Dindo grade Ⅰ in the patient, who was improved after conservative treatment. On the 7th day after surgery, all 10 patients underwent upper gastrointestinal angiography, and no anastomotic leakage or stenosis occurred. There was no sign of contrast agent reflux in the supine position and 30° head down position. (2) Follow-up and late complications. All 10 patients were followed up for 59.5(range, 31.0-127.0)days. The esophageal reflux scale score of 10 patients was 1.4±0.3. During the follow-up, 1 case underwent gastroscopy on 40 days after surgery, which showed reflux esophagitis with Los Angeles grade as B and the Clavien-Dindo grade as Ⅰ. There was no clinical symptom such as heartburn or acid reflux. Results of 24-hour pH monitoring showed that the patient experienced 24 instances of reflux in an upright position and 15 instances of reflux in a supine position, with no prolonged reflux. The total reflux time within 24 hours was 75 minutes. The DeMeester score was 38.3. Results of esophageal pressure measurement showed that the esophageal contraction morphology was normal, but the anastomotic opening was not well relaxed. The rest of 9 cases had no complication such as reflux esophagitis.Conclusion:Biological muscle flap applied in the laparoscopic radical proximal gastrectomy with esophagogastric anastomosis is safe and feasible, with satisfied short-term efficacy.

Objective:To evaluate the heterogeneity in pediatric ETV6-RUNX1 acute lymphoblastic leukemia (ALL) by gene expression profile and to study clinical characteristics in different clusters.Methods:An improved advanced fragment analysis (iAFA) technique was developed to detect 57 marker genes in 264 pediatric ALL patients treated in Beijing Children’s Hospital from August 2016 to June 2019. The 56 ALL patients with ETV6-RUNX1 positive were evaluated by clinical characteristics in gene expression profile, immunophenotype and early response of chemotherapy in different clusters.Results:The 56 ETV6-RUNX1-positive patients were clustered into 2 groups of E/R-1 (45, 80.4%) and E/R-2 (11, 19.6%) . Spearman coefficient was 0.788 and 0.901 in E/R-2 and E/R-1, respectively. The median of initial platelet counts was 104 (27-644) and 50 (8-390) ( P<0.01) in E/R-2 and E/R-1, respectively. The median of proportion of initial bone marrow immature cells was 0.830 (0.270-0.975) and 0.935 (0.445-0.990) ( P<0.05) in E/R-2 and E/R-1, respectively. The most specific immunophenotype at initial diagnosis, CD22 +CD34 +CD20 -CD117 -CD56 -, mainly gathered in E/R-2 ( P<0.001) . Patients negative of minimal residual disease detected by flow cytometry (MRD-FCM) at day 33 were 5 (55.6%) and 32 (88.9%) in E/R-2 and E/R-1, respectively. There was no significant difference in the original analysis ( P=0.064) but difference in sensitivity analysis ( P=0.035) . Nevertheless, patients negative of MRD detected by polymerase chain reaction (MRD-PCR) at day 33 were 7 (77.8%) and 36 (100%) in E/R-2 and E/R-1, respectively, with significant difference ( P=0.047) . Conclusion:Gene expression profile shows heterogeneous in ETV6-RUNX1 ALL, and the E/R-2 profile indicates that these patients may have a less tendency to thrombocytopenia at the initial diagnosis but have poorer response to induction chemotherapy and may influence further outcome.

Objective To analyze the chemical constituents in the water extract of Bupleuri Radix and investigate the active ingredients of Bupleuri Radix for the treatment of depression.Methods The chemical constituents in the water extract of Bupleuri Radix were identified by Ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF/MS).CORT-induced poorly differentiated PC12 depression cell model was launched,and PC12 cells were pretreated with monomeric compounds from Bupleuri Radix for 24 h.The cell viability and LDH release rate were measured by CCK-8 assy kit and LDH assay kit,respectively.Results A total of 53 compounds were identified in the water extract of Bupleuri Radix,mainly including type Ⅰ,type Ⅱ and type Ⅲsaikosaponins.Among them,saikosaponin A,saikosaponin B2,saikosaponin C,saikosaponin E,saikosaponin F and 6″-acetyl saikosaponin A contributed the most to the metabolite profile of Bupleuri Radix,and could improve the viability of CORT-induced PC12 cells(P<0.05,P<0.01).Furthermore,saikosaponin A,saikosaponin B2,saikosaponin C,saikosaponin E and saikosaponin F could decrease the LDH release rate of CORT-induced PC12 cells(P<0.05,P<0.01).Conclusion The major anti-depression active ingredients in Bupleuri Radix may be Saikosaponin A,saikosaponin B2,saikosaponin C,saikosaponin E and saikosaponin F,which lays a foundation for the research of the quality control and pharmacodynamic material basis of Bupleuri Radix.

Objective: To explore the gene transduction method of chimeric antigen receptor (CAR) mediated by novel cationic polymer nanocarrier mPEG-P (Asp-AED-g-HFB) (PAEF) and PigyBac transposon system to modify natural killer (NK) cells, providing a new strategy for immunotherapy of cancer cells. Methods: PAEF/DNA (transposase+transposon) complex were prepared. The particle size distribution and surface potential of PAEF/DNA complexes were measured with Nano-ZSE Dynamic Light Scattering System (Malvern Instruments). The DNA encapsulation rate, release and stability of PAEF were evaluated by DNA gel electrophoresis, and then by combiningwithparticlesizeandsurfacepotentialtodeterminethepreferentialN/PratiotoenterNKcells.Thecell cytotoxicity of PAEF/DNA complexes under different N/P ratios was analyzed by CCK-8 cytotoxicity test. Transduction efficiency of NK cells was evaluated by Fluorescence microscopy and Flow cytometry, and the feasibility of PAEF gene transfection vectors was assessed. Results: PAEF could encapsulate DNA to form nano-complexes with the diameter of 100-150 nm, which was suitable to mediate DNA entering into cells. PAEF could completely encapsulate DNA with N/P ratio of 20. In the presence of reducing agent dithiothreitol (DTT), PAEF had a good ability to release DNA. NK-92 cells transfected with PAEF/DNA complex, which was formed at the N/P ratio of 80, attained a significantly higher cell viability than cells of lipofectamine transfection group [(72.50±3.9)% vs (64.03±1.8)%, P<0.05]; Fluorescence microscopic observation showed more fluorescence and higher fluorescence intensity in cells of PAEF/DNA group; Flow cytometry showed the highest transfection efficiency of 83.4%. Conclusions: Nanocarrier PAEF can encapsulate DNA well by electrostatic adsorption, and has good biocompatibility and high efficiency for gene transduction. It provides a good experimental basis for adoptive immunotherapy.

ObjectiveTaking the rat model of spleen-stomach damp-heat syndrome（SSDHS） as the research object， this study aimed to investigate the potential biomarkers of SSDHS and the related metabolic pathways based on urine metabolomics， and tried to reveal the essence of SSDHS at the level of endogenous small molecular metabolites. MethodSixteen SD rats were randomly divided into normal and model groups. The normal group was fed normal chow and the model group was fed with 200 g·L^-1 honey water daily， and lard and Chinese Baijiu alternately on alternate days for 17 days. The SSDHS model rats were exposed to external dampness-heat environment with temperature at 30-34 ℃， relative humidity of 95% for 2 h at the same time every day from the 10^th day for 7 d. Then， the model was evaluated by observing the general conditions of the rats， measuring the contents of motilin（MTL） and gastrin（GT） in plasma by enzyme-linked immunosorbent assay（ELISA）， and examining the histopathology of gastronitestinal tissues. In additon， the urine metabolomics analysis was performed by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS）， and the detection conditions was as follows：ACQUITY™ UPLC BEH C₁₈ column（2.1 mm×100 mm， 1.7 μm）， mobile phase of 0.1% formic acid aqueous solution（A）-0.1% formic acid acetonitrile solution（B） for gradient elution （0-3 min， 1%-18%B； 3-8 min， 18%-40%B； 8-10 min， 40%-100%B）， the flow rate of 0.4 mL·min^-1， electrospray ionization（ESI） in positive and negative ion modes， scanning range of m/z 50-1 000. The univariate and multivariate statistical analysis were constructed for screening inter-group differential ions， the element composition was calculated according to the precise relative molecular weight， and ion information was matched with databases such as Human Metabolome Database（HMDB） to identify biomarkers. Kyoto Encyclopedia of Genes and Genomes（KEGG） database was used to obtain the biological information of metabolites， and their associated metabolic pathways were analyzed by MetaboAnalyst 5.0. ResultCompared with the normal group， the rectal temperature of the model group increased significantly（P<0.01）， the levels of plasma MTL and GT decreased significantly（P<0.05， P<0.01）， and pathological changes such as bleeding， congestion and inflammatory infiltration in the gastric and colonic tissues. A total of 25 differential metabolites such as L-histidine， citric acid and isocitric acid were found to be the potential biomarker of SSDHS by urine metabolomics， 13 of which were phase Ⅱ metabolites of endogenous substances（glucuronic acid conjugates， sulfuric acid conjugates and acetyl conjugates）， involving the metabolic pathways of histidine metabolism， tricarboxylic acid cycle， glyoxylate and dicarboxylate metabolism. ConclusionSSDHS primarily causes disorders of histidine metabolism， tricarboxylic acid cycle， glyoxylate and dicarboxylate metabolism， as well as the imbalance of the activation/inactivation of endogenous metabolites， which may involve the immune response， material and energy metabolism， inflammatory response and intestinal flora， and may provide a basis for the establishment and application of SSDHS model.