Adolescent ; Child ; Female ; Fever ; etiology ; Histiocytic Necrotizing Lymphadenitis ; classification ; complications ; diagnosis ; Humans ; Lymph Nodes ; pathology ; Male

Background Establising the culture model of Müller cells for obtaining the highly putified target cells is essential for the study about the physiology and pathology of retinal Müller cells. The exsiting purifing method for culturing Müller cells is dissatisfactory. Objective This study was to establish a method to obtain high purifing Müller cells. Methods The retina from 5 clean newborn SD rats were isolated and digested by 0. 01% trypsin and cultured in DMEM containing 10% fetal bovine serum. The cellular suspension was then prepared,and the target cells were screened using flow cytometry based on the size and the quantity of cells. Cultured and passaged cells were identified by transmission electron microscope and light microscope. Immunocytochemistry was used to detecte the expression of GFAP in cultured cells for the determination of type and purity of the cells. Results The cells showed the similar shape to retinal Müller cells after primarily culture with the large volume, and some small other types of cells could been seen. The growth of cells was quickly 3 weeks later. The fibroblasts were removed using sticking-wall by steps,and neurons were eliminated following passage. Aboundent of cellular organs were seen under the transmission electron microscope. The positive response rate of the cells for CFAP was 100%. Conclution Flow cytometry offer a rapid and feasible approach for purifying Muller cell and it builds the foundation for further study about Müller cells.

The aim of this study is to establish a method to culture and purify cerebral astrocyte of tree shrew (Tupaia belangeri), a kind of new laboratorial animal which is a relative of primates. Newborn tree shrews were used in this experiment. The cortex of cerebrum was isolated and placed in 4°C for 20 min to injure neurons. The cortical tissue was disaggregated by trypsin digestion. Differential attachment method was used to remove fibroblasts. The mixed culture was rinsed by trypsin (0.005%) solution to remove neurons. Upon reaching 70% confluence, the culture was subjected to static trypsin digestion until a white slice film exfoliated from the bottom of culture bottle. This film, i.e. astrocyte layer, was taken out and cultured, and the third passage was identified by immunocytochemical staining and immunofluorescence with anti-glial fibrillary acidic protein (GFAP) antibody. The result showed the purity of tree shrew astrocytes was more than 98%. Thus the method to culture highly purified astrocyte of tree shrew was successfully established, which would contribute to further study in central nervous system physiology and diseases in this new laboratorial animal.
Animals ; Astrocytes ; cytology ; Brain ; cytology ; Cell Separation ; methods ; Primary Cell Culture ; methods ; Tupaiidae

It is the objective of this paper to study pharmacokinetics-pharmacodynamics (PK-PD) characteristics of ginsenoside Rg1 and Rb1 on the effect of inducing nitric oxide (NO) release after intravenous administration of Shengmai injection to rats with myocardial ischemia. The model of myocardial ischemia rats was produced by subcutaneous injection of isoproterenol. The serum samples were collected at different time points after intravenous administration of Shengmai injection to rats with the dose of 10.8 mL x kg(-1). The concentrations of ginsenoside Rg1 and Rb1 in serum were determined, and then the concentration-time curves were drawn. Pharmacokinetic parameters of ginsenoside Rg1 and Rb1 were calculated after the construction of pharmacokinetic models. Meanwhile, NO2- and NO3-, the metabolites of NO, in serum were determined, and then the effect-time curve was drawn. The combined PK-PD model was established based on the theory of effect compartment by Sheiner et al. Then pharmacodynamic parameters were calculated. The results indicated that the pharmacokinetics of ginsenoside Rg1 and Rb1 conformed to a two-compartment model. Ginsenoside Rg1 and Rb1 exhibited quick and slow elimination in rats respectively. The effect of Shengmai injection on inducing NO release did not relate directly with and lagged behind the concentrations of ginsenoside Rg1 and Rb1 in serum. The effect exhibited good correlation with ginsenoside Rg1 and Rb1 levels in effect compartment. The relationship between effect and serum concentration fits Sigmoid-E(max) model. This study successfully established the combined PK-PD model of ginsenoside Rg1 and Rb1 after intravenous administration of Shengmai injection to rats. The model can efficiently predict the concentration and effect of Shengmai injection in vivo.
Administration, Intravenous ; Animals ; Ginsenosides ; administration & dosage ; pharmacokinetics ; Humans ; Male ; Myocardial Ischemia ; drug therapy ; metabolism ; Nitric Oxide ; metabolism ; Rats ; Rats, Sprague-Dawley

Objective To study the damage to striatum in patients perorally addicted to compound codeine phosphate solution by using the brain dopamine transporter SPECT imaging. Methods Patients p erorally addicted to compound codeine phosphate solution ( n = 29 ) and addicted to heroin ( n = 27 ), as well as healthy volunteers (n = 31 ) were included in the study. Each of them underwent dopamine transporter (DAT) SPECT imaging with 99Tcm-2β-[N, N'-bis-( 2- mercaptoethyl ) ethylenediamino] methyl, 3β-(4-chlorophenyl)tropane (99Tcm-TRODAT-1). The striatum volume (V, cm3), mass (m, g) and radiactivity ratio (Ra) of striatum to whole brain were calculated using physio-mathematical modeling method.R esults Bilateral striatum of healthy volunteers showed typical "panda eyes" pattern and the distribution of DAT was uniform and symmetrical. Bilateral striatum of patients addicted to compound codeine phosphate showed impaired tracer uptake, similar to those addicted to heroin. The V, m and Ra of bilateral striatum of patients addicted to compound codeine phosphate were (23.68 ±4.94) cm3, (24.87 ±5.19) g and (5.01 ±0. 88 ) %, respectively, which were significantly lower than those of healthy controls: ( 35.39 ± 4.42 ) cm3,(37.16 ±4.64) g and (7.93 ±0.86)% (t = -9.69, -9.69, - 13.01, all P =0.000), but significantly higher than those addicted to heroin: ( 18.87 ± 4.66 ) cm3, ( 19.81 ± 4.90 ) g and (4.26 ± 1.02 ) % ( t =3.74, 3.74, 2.96, P = 0.000, 0.000, 0.005 ). Conclusion Long-term peroral intake of compound codeine phosphate solution may damage the function of cerebral striatum, which is someway similar to though less severe than, the impairment caused by heroin.

Objective To study the association between the plasma homocysteine level and coronary artery disease(CAD), and the methylenetetrahydrofolate reductase (MTHFR) A1298C polymorphism, the methionine synthase (MS) A2756G polymorphism and their associations to the plasma homocysteine level and CAD in the elderly . Methods One hundred and twenty-nine elderly patients with CAD documented by coronary angiogram and 48 elderly patients with normal coronary angiographic results were included in this study. Plasma homocysteine level were measured by fluorescence polarization immunoassay (FPIA) method and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to analyse the MTHFR A1298C and MS A2756G genotypes. Results The plasma homocysteine level was significantly higher in CAD group than that in the control group〔(16.2?8.6) ?mol/L vs (12.7?5.0) ?mol/L,P0.05);the prevalence of MTHFR 1298CC homozygous in the CAD patients was significantly less than that in the control group (3.1% vs 14.6%, P

Objective To explore the expression and distribution of transient receptor potential cation channel 6(TRPC6)in normal human,mice,rats' renal tissue and the mouse podocyte clone 5(MPC5)for further investigating the relationship between TRPC6 and the protei-nuria-related podocyte molecules.Methods 1.The distributions of TRPC6 in normal human,mice,rats' renal tissue and MPC5 were observed by using the immunochemistry staining.2.The mRNA expression of TRPC6 in mouse renal cortex and differentiated MPC5 was detected by using reverse transcriptase-polymerase chain reaction(RT-PCR).3.The protein expression of TRPC6 in human,mice and differentiated MPC5 was detected by using Western blotting.Results 1.In human kidney,TRPC6 showed a weak staining in glomeruli and a strong staining in renal tubules and vessels.In mice and rats' kidney,TRPC6 showed a strong staining in glomeruli and was mainly distributed along the capillary loops of glomerulus and in mesangium.The positive staining of TRPC6 was observed in MPC5,which was distributed evenly on the cell membrane in differentiated podocytes.2.The specific PCR band of TRPC6 was detected in mouse renal cortex and differentiated MPC5.3.The specific protein band of TRPC6 was detected in normal human,mice renal cortex and differentiated MPC5 with the size of 106.Conclusions The expression of TRPC6 is verified in normal human,mice and rats' kidneys,and in differentiated MPC5.These results will benefit for further exploring the relationship between TRPC6 and the proteinuria-related podocyte molecules.

Abstract: Objective　To compare the mosquito trapping effect of BG-trap mosquito trap using carbon dioxide versus BG-lure attractant under filed conditions. Methods　In August and September 2020, two areas were set with a distance of 100 m. Two sites were set at each area, and one mosquito trap BG trap was set with a distance of 5 m. Each site was set with different flow of CO2 and different amount of BG-lure attractants. The BG-trap mosquito traps on the same area would exchange positions every other day. The mosquitoes captured by each mosquito trap was collected and classified. and the species, sex and number of mosquitoes captured were recorded and counted. Results　The densities of Aedes albopictus captured by BG+/CO2-and BG-/CO2+were 14 and 31, and that of Culex pipiens pallens were 2 and 16, respectively. The differences were statistically significant (Aedes albopictus, t=-2.675, P<0.05; Culex pipiens pallens, t=-4.873, P<0.05). With BG-lure attractant, the females of Aedes albopictus and Culex pipiens pallens in the CO2+group were 2.6 (25/9.5) and 12.0 (12 /1) times higher than those in the CO2-group, and the differences were statistically significant (female Aedes albopictus, t=-4.119, P<0.01; female Culex pipiens pallens, t=-4.592, P<0.01), suggesting that the most important attractant to female mosquitoes is CO2. With BG-lure attractant, the male Aedes albopictus in the CO2+ group was 3.0 (12/4) times higher than that in the CO2-group, and the difference was statistically significant (male Aedes albopictus, t=-3.284, P<0.01). Without BG-lure attractant, female Aedes albopictus and female Culex pipiens pallens in the CO2 + group were 1.8 (18 / 10) and 15.5 (15.5/1.0) times higher than those in the CO2-group, and the difference was statistically significant (female Aedes albopictus, t=-2.868, P<0.05; female Culex pipiens pallens, t=-5.259, P<0.05). Without BG-lure attractant, the male Aedes albopictus in the CO2+group was 2.0 (9.0/4.5) times higher than that in the CO2-group, with a statistically significant difference (t=-2.508, P<0.05). With CO2, Aedes albopictus and Culex pipiens pallens in the BG + attractant group were 1.4 (43.5/31) and 0.78 (12.5/16.0) times higher than those in the BG-attractant group, and the differences were not statistically significant (Aedes albopictus, t=-0.943, P>0.05 ; Culex pipiens pallens, t=0.709, P>0.05). Without CO2, Aedes albopictus and Culex pipiens pallens in the BG + attractant group were 1.0 (14/14) and 2.0 (2.0/1.0) times higher than those in the BG + attractant group, and the differences were not statistically significant (Aedes albopictus, t=-0.500, P>0.05; Culex pipiens pallens, t=-1.000, P>0.05). Without BG-lure attractant, the densities of female Aedes albopictus captured by adding 0, 1 and 2 parts of dry ice were 10, 17.5 and 18 respectively, and the difference was statistically significant among the three groups (F=3.942, P<0.05). The densities of female Culex pipiens pallens captured were 1, 13 and 18 respectively, and the difference was statistically significant among the three groups (F=13.881, P<0.05). However, there was no significant difference between the capture of female Aedes albopictus and female Culex pipiens pallens by adding 1 part of dry ice and 2 parts of dry ice (female Aedes albopictus, t=0.112, P>0.05; female Culex pipiens pallens, t=-0.540, P>0.05). Without CO2, 10, 10, 9.5 and 1, 1 and 1.5 female Aedes albopictus and Culex pipiens pallens were captured by adding 0, 1 and 2 portions of BG-lure attractants, respectively. There were no significant differences between the three groups (female Aedes albopictus, F=0.120, P>0.05; female Culex pipiens pallens, F=0.477, P>0.05). Conclusions　In the monitoring of BG-trap mosquito trap, the mosquito trapping effect of CO2 is better than that of BG-lure attractant. When the same monitoring effect is obtained, the use of CO2 (100 mL/min) can save the use cost.

The coronavirus disease 2019 (COVID-19) is an acute infectious disease caused by the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which has led to serious worldwide economic burden. Due to the continuous emergence of variants, vaccines and monoclonal antibodies are only partial effective against infections caused by distinct strains of SARS-CoV-2. Therefore, it is still of great importance to call for the development of broad-spectrum and effective small molecule drugs to combat both current and future outbreaks triggered by SARS-CoV-2. Cathepsin L (CatL) cleaves the spike glycoprotein (S) of SARS-CoV-2, playing an indispensable role in enhancing virus entry into host cells. Therefore CatL is one of the ideal targets for the development of pan-coronavirus inhibitor-based drugs. In this study, a CatL enzyme inhibitor screening model was established based on fluorescein labeled substrate. Two CatL inhibitors IMB 6290 and IMB 8014 with low cytotoxicity were obtained through high-throughput screening, the half inhibition concentrations (IC₅₀) of which were 11.53 ± 0.68 and 1.56 ± 1.10 μmol·L^-1, respectively. SDS-PAGE and cell-cell fusion experiments confirmed that the compounds inhibited the hydrolysis of S protein by CatL in a concentration-dependent manner. Surface plasmon resonance (SPR) detection showed that both compounds exhibited moderate binding affinity with CatL. Molecular docking revealed the binding mode between the compound and the CatL active pocket. The pseudovirus experiment further confirmed the inhibitory effects of IMB 8014 on the S protein mediated entry process. In vitro pharmacokinetic evaluation indicated that the compounds had relatively good drug-likeness properties. Our research suggested that these two compounds have the potential to be further developed as antiviral drugs for COVID-19 treatment.

OBJECTIVETo investigate the influence of down-regulating Smoothened (SMO) gene expression through short hairpin RNA (shRNA) on the proliferation of breast cancer stem cells.

METHODSHuman SMO shRNA was designed, synthesized chemically, and transfected into MCF-7 cells to down-regulate SMO gene. By using G418, stable cells with down-regulated SMO were selected. In vitro proliferation of these cells was measured by CCK8 assay. The proportion of CD44(+)/CD24(-) cells was detected by flow cytometry and the mammospheres formation was determined by suspension sphere culture. The expression of SMO, GLI1 and Oct4 was detected by Western blot. In vivo, the volume of tumor was measured every 3 days and the expression of SMO, GLI1 and Oct4 detected by Western blot.

RESULTSIn vitro, the cells were transfected with SMO-shRNA and selected by G418 after 21 days. SMO-shRNA effectively down-regulated the expression of SMO gene and protein, and inhibited the proliferation of MCF-7 and markedly reduced the proportion of CD44(+)/CD24(-) cells and mammospheres. In vivo, SMO-shRNA treatment of MCF-7 significantly inhibited the volume of tumor. The positive rate of SMO in negative control and SMO-shRNA group was 5/5 and 2/5, respectively. The expression of SMO, GLI1 and Oct4 in different groups were 0.72 ± 0.17 and 0.21 ± 0.09, 1.21 ± 0.21 and 0.47 ± 0.12, 0.83 ± 0.13 and 0.25 ± 0.07. SMO, GLI1 and Oct4 down-regulation significantly suppressed at protein levels (P < 0.05).

CONCLUSIONThe shRNA by chemical synthesis can effectively down-regulate SMO gene expression and inhibit the proliferation of breast cancer stem cells.

Animals ; Cell Proliferation ; Down-Regulation ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Hyaluronan Receptors ; metabolism ; MCF-7 Cells ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Neoplastic Stem Cells ; pathology ; Octamer Transcription Factor-3 ; metabolism ; RNA, Small Interfering ; genetics ; Receptors, G-Protein-Coupled ; genetics ; metabolism ; Smoothened Receptor ; Transcription Factors ; metabolism ; Transfection ; Tumor Burden ; Zinc Finger Protein GLI1