Cell biology is a highly experimental discipline, and is a discipline with theoretical knowledge closely combined with practical operation.Most of the medical graduate students should master the cell biology theory in the graduate stage, and get on a series of cell biology researches.Therefore, it is important to master the most basic cell culture techniques.As a technical instructor in the cell culture laboratory, I have been summarizing and optimizing the teaching content and teaching mode during the past few years.The aim of the teaching work is to train the students' scientific attitude of seeking truth from facts so as to develop good experimental habits and to master the basic skills of cell culture.

AIM:To evaluate the clinical effects of femto-LASIK using new Ziemer LDV Z6 femtosecond laser machine (Z6).
METHODS: Two - hundred cases ( 400 eyes ) was randomly separated into two groups: Group A included 200 eyes which corneal flaps were made by Z6, and Group B included rest of 200 eyes which corneal flaps were examined by a traditional Ziemer LDV CrystalLine femtosecond laser machine (CrystalLine). Visual acuity, slit lamp, refraction, Sim-K average, intraocular pressure (IOP), non-invasive tear break-up time (NIAvg-BUT), operation difficulty and complications were compared between two groups preoperatively and postoperatively.RESULTS:There was no significant differences between two groups in visual acuity, refraction, Sim-K average, IOP and NIAvg - BUT either preoperatively or 6mo postoperatively (P>0. 05). Although there were significant differences in operation difficulty and complications were found between two groups ( P< 0. 05 ), there was no serious complication in any group. Furthermore, a clear demarcation line could be observed in the cornea of Group A postoperatively.
CONCLUSION:More careful and strict requirements are needed when using the new Z6 femtosecond laser for corneal flaps.

OBJECTIVETo construct and characterize the TGF-β1, induced epithelial-mesenchymal transition (EMT) model of keloid epithelial cells in vitro, and to investigate the expression of epithelial stem cells related surface markers in keloid epithelial cells during EMT induction.

METHODSThe epithelial cells from 3 keloid samples of ears were cultured in vitro and induced by transforming growth factor betal (TGF-β1, 1 ng/ml) for 5 days, which was the experimental group, the same cells untreated were considered as the negative control group. The expressions of EMT-associated markers and regulative genes were detected using immunofluorescence staining, real-time PCR and western blot analysis. Then the surface markers of epithelial stem cells were detected using real-time PCR. Statistical significance was determined using Independent-Samples t Test, a p value less than 0. 05 was considered statistically significant.

RESULTSThe mRNA expression of transcription factor snail2 and mesenchymal-specific marker vimentin increased significantly in TGF-β1, induced keloid epithelial cells (P < 0. 05), in which snail2 increasing from 0. 91 ± 0. 23 to 1. 69 ± 0. 10, and vimentin from 5. 86 ± 2. 07 to 24. 29 ± 5. 39. Whereas the mRNA expression of epithelial-specific marker E-cadherin decreased from 1. 06 ± 0. 19 to 0. 65 ± 0. 09. The mRNA expression of CD29 and Lgr6, two surface markers of epithelial stem cells, significantly increased after induction of the TGF-β1, (P < 0. 05), from 0. 55 ± 0. 14 and 1. 61 ± 0. 31 to 1. 19 ± 0. 12 and 3. 84 t 0. 62 respectively. In induced cells, the immunofluorescence results showed staining of E- cadherin became faint, but the number of positive staining cells of vimentin increased. Western blot confirmed the protein expression of E-cadherin weakened, and the vimentin and p-Smad3 enhanced (P < 0. 05).

CONCLUSIONSTGF-β1, initiated EMT in keloid epithelial cells by inducing the up-regulation of snail2, and TGF-β1,/Smad3 signaling pathway was involved in EMT. EMT could change the phenotype of epithelial stem cells in keloid.

Biomarkers ; metabolism ; Cadherins ; genetics ; metabolism ; Epithelial Cells ; drug effects ; physiology ; Epithelial-Mesenchymal Transition ; drug effects ; physiology ; Humans ; In Vitro Techniques ; Keloid ; pathology ; RNA, Messenger ; metabolism ; Signal Transduction ; Smad3 Protein ; genetics ; metabolism ; Snail Family Transcription Factors ; Transcription Factors ; genetics ; metabolism ; Transforming Growth Factor beta1 ; metabolism ; pharmacology ; Up-Regulation ; Vimentin ; genetics ; metabolism

BACKGROUND:Mesenchymal stem cel s have an immunoregulatory capacity to suppress the airway inflammation of asthmatic mice, but the mechanism remains unclear. Our preliminary studies indicated that mesenchymal stem cel s can upregulate the CD4+CD25+regulatory T cel s (Treg) of the asthmatic mice. OBJECTIVE:To study the effect of mesenchymal stem cel s on the airway inflammation in asthmatic BALB/c mice depleted of CD4+CD25+Treg. METHODS:Forty BALB/c mice were randomly divided into five groups:normal control group (group A), asthmatic group (group B), asthmatic group depleted of CD4+CD25+Treg (group C), mesenchymal stem cel s group (group D), and asthmatic group depleted of CD4+CD25+Treg and administrated with mesenchymal stem cel s (group E). Except group A, mice in the other groups were sensitized and chal enged by ovalbumin to establish asthma models. In group C and group E, each mouse was treated with anti-CD25+monoclonal antibody to deplete CD4+CD25+Treg. In group D and group E, mice were intravenously administered with 0.2 mL mesenchymal stem cel s 1×109/L) at 10 days after sensitization. At 24 hours after the final activation, the number of CD4+CD25+Treg in the peripheral blood was detected by flow cytometry. The total cel number of inflammatory cel s in the bronchoalveolar lavage fluid, and the number of eosinophils, lymphocytes and neutrophils were counted to analyze the degree of inflammation of the airway together with pathological observation. RESULTS AND CONCLUSION:(1) The proportion of CD4+CD25+Treg in peripheral lymphocytes was ranged as fol ows:group B

Objective To investigate intraspecific and interspecific variation within Malassezia iso-lates from patients with pityriasis versicolor by random amplified polymorphic DNA (RAPD) analysis, to learn the difference between RAPD analysis and physiological and biochemical methods in the typing of Malassezia species, and to explore the relationship between RAPD patterns and Malassezia species. Methods A total of 47 Malassezia isolates were obtained from 34 patients with pityriasis versicolor, and they were classified into 5 species by morphological, physiological and biochemical features, I.e., M. Fin'fur, M. Obtusa, M. Globosa, M. Restricta and M. Sympodialis. Genomic DNA was extracted from the 47 clinical isolates and 10 reference strains (including 7 species) of Malassezia. PCR was performed using 4 random primers including S22, S24, S25 and S33. RAPD patterns were analyzed by NTSYS software and dendrogram was autogenerated. Results Genomic DNA of most strains was successfully amplified with four primers, espe-cially with primers S22 and S24 that resulted in rather stable and clear DNA bands. A total of 82 fragments were amplified from all tested strains. These strains showed both interspecifie and intraspecific variation. Multiple swains were isolated from different body sites of 4 patients and identified into different species by biochemical and morphological typing; those swains from same hosts occupied contiguous positions in the dendrogram and exhibited a high genetic convergence. Conclusion The phenomenon that different strains from a co-host show a high genetic convergence indicates that species specificity and evolution of Malassezia are closely related to its hosts.

ObjectiveTo assess the efficacy and safety of Xuebijing injection for the treatment of severe acute pancreatitis (SAP).Methods An extensive search of related literatures from the Cochrane Library, EMBASE, China Biology Medicine (CBM), CNKI, VIP and Wanfang data up to March 2014 was performed. Randomized controlled trials (RCTs) regarding Xuebijing injection for the treatment of SAP were collected regardless of languages. Jadad scale was taken for quality evaluation of the included studies by two researchers. The patients in control group were given conventional treatment, and those of the Xuebijing group were given Xuebijing injection on the top of conventional treatment. The Cochrane Collaboration RevMan 5.2 software was used for data analysis regarding the effect of Xuebijing injection on the mortality, incidence of complication, effective rate, the length of stay in hospital, and the safety of the drug in patients with SAP.Results A total of 15 published reports meeting the inclusion criteria were enrolled. The methodological quality of the trials was low. Meta analysis showed that the mortality in Xuebijing group was significantly lower [odds ratio (OR) = 0.37, 95% confidence interval (95%CI) =0.17 - 0.77,P = 0.008], and the incidence of complication was also significantly decreased (OR = 0.26, 95%CI =0.14 - 0.45,P< 0.000 01) as compared with those of control group. The effective rate in Xuebijing group was significantly higher than that of the control group [relative risk (RR) = 0.85, 95%CI = 0.80-0.91,P< 0.000 01]. The length of stay in hospital in Xuebijing group was significantly shorter than that of the control group [mean difference (MD) = -5.28, 95%CI = -6.69 to -3.86,P< 0.000 01]. Adverse reactions of Xuebijing injection were reported in 2 studies. The adverse reaction in one study was headache and nausea, which were relieved by adjusting the speed of intravenous infusion, and mild rash was reported in another case, and it disappeared after the withdrawal of Xuebijing. Conclusions The currently available evidence shows that Xuebijing injection may have some therapeutic effect on SAP. Because of the low methodological quality of the included trials, multi-center and high-quality RCTs with large sample sizes are needed to provide stronger evidence.

Objective: To investigate the effect of ginsenoside-rg1 (G-Rg1) on rat’s cardiomyocytes H9c2 with its mechanism of signal pathwayin vitro.
Methods: H9c2 cells were cultured and treated in different conditions by following groups:①Blank control group,②Hypoxia alone group, the cells were treated for (2, 6, 12, 24, 48) hr respectively,③G-Rg1 group, the cells were treated by G-Rg1 at (5, 10, 50) μmol/L respectively,④YC-1 group, which is the speciifc inhibitor of hypoxia inducible factor-1α (HIF-1α),⑤YC-1 + G-Rg1 group,⑥Wortmannin group, which is the speciifc inhibitor for protein kinase B (Akt) phosphorylation and⑦Wortmannin + G-Rg1 group. Each experiment was conducted with 5 replicates. The effects of G-Rg1, hypoxia and YC-1 on cell activity and injury were studied; intracellular mRNA expressions of HIF-1α, glucose transporter-1 (GLUT-1) and heme oxygenase-1 (HO-1) were examined by RT-PCR; protein expressions of HIF-1α, GLUT-1, HO-1, activating transcription factor-6 (ATF-6), CCAAT/enhancer binding protein homologous protein (CHOP) and Akt with its signal pathway factors were measured by Western blot analysis.
Results: The time of hypoxia was negatively related to cell activity (r=-0.8580,P<0.05) and positively related to LDH overlfow rate (r=0.9201,P<0.05). G-Rg1 (10μmol/L) group showed increased cell activity than Hypoxia alone (24 hr) group (87.8% vs 62.6 %,P<0.05), while decreased LDH overlfow (25.0% vs 74.8%,P<0.05), and up-regulated mRNA expressions of HIF-1α, GLUT-1 and HO-1, P<0.05. YC-1+ G-Rg1 group had decreased cell activity than G-Rg1 group (68.0% vs 87.8%,P<0.05), while increased LDH overlfow (56.4% vs 25.0%,P<0.05). Meanwhile, YC-1 clashed the effect of G-Rg1 on protein expressions of HIF-1α, GLUT-1, HO-1, ATF-6 and CHOP,P<0.05; wortmannin clashed the effect of G-Rg1 on protein expressions of HIF-1α, CHOP,P<0.05 and suppressed the two phosphorylation sites for Akt activation,P<0.05.
Conclusion: G-Rg1 may protect rat’s H9c2 cellsin vitro by activating expressions of HIF-1α with its downstream factors and inhibiting endoplasmic reticulum stress, which might be related to the effect of G-Rg1 on Akt activation.

OBJECTIVEThe Ministry of Public Health released the National Surveillance project on Shigellosis in August, 2005. This study was to reveal the antimicrobial resistance status of Shigella isolates through the National Shigellosis Surveillance System in 2005 in China, so as to provide evidence for the development of surveillance, prevention and cure of Shigellosis.

METHODSAll the lab assistants received training from Chinese Center for Disease Control and Prevention. The project prescribed the uniform experimentation, quality control method, reagent, etc. Disc diffusion test(K-B) was carried out, following the CLSI methods. Data were analyzed by WHONET 5.4 software.

RESULTS(1) 3 serotypes were identified and S. flexneri was common that accounted for 75.5% of all Shigella isolates followed by 24.4% of S. sonnei, but only 1 strain of S. dysenteriae was separated. (2) The resistant rates to tetracycline and ampicillin in Shigella spp were quite high, as over 90.0%. However, the resistant rate to Cefotaxime was the lowest, only 6.1%. The resistant rates were different between serotypes with the resistant rates of S. flexneri to ampicillin, ampicillin/clavulanate and ciprofloxacin were higher than those of S. sonnei (P < 0.001). (3) The multiple-antibiotic-resistance status in Shigella spp was quite serious and the resistant rate to five and more antimicrobials was 54.9%. The most common resistant patterns were seen on ampicillin, nalidixin, tetracycline and sulfamethoxazole. (4) There were some differences in subtypes and antimicrobial resistance among different provinces.

CONCLUSIONCefotaxime seemed the best in curing Shigellosis at the clinic level. Programs regarding monitoring subtypes and antimicrobial resistance of Shigella should be in a continuous manner so as to understand the pathogens timely and to control the disease pertinently.

Anti-Bacterial Agents ; pharmacology ; China ; Drug Resistance, Microbial ; Dysentery, Bacillary ; drug therapy ; Humans ; Population Surveillance ; Serotyping ; Shigella ; drug effects

Objective To investigate the changing antibiotic resistance profile of Haemophilus influenzae and Moraxella catarrhalis strains collected from children in Sichuan province from 2013 to 2016,provide some reference for rational utilization of clinical antimicrobial agents.Methods Collected the infection data of Haemophilus influenzae and Moraxella catarrhalis strains isolated from children which reported in Sichuan Province Drug Resistance Monitoring Report from 2013 to 2016.The experimental results were analyzed by WHONET5.6 software.Results The prevalence of H.influenzae increased with time from 8.95％ in 2013 to 16.6％ in 2016.The prevalence of M.catarrrhalis increased with time from 4.16％ in 2013 to 6.34％ in 2016.Among the 15 896 clinical strains of H.influenzae,the highest resistance rate was to ampicillin,which was 71.6％ in 2016.The resistance rate to cefaclor also increased from 26.1％ in 2013 to 59.5％ in 2016 for increase of 33.4％.The insensitivity rate to azithromycin increased from 8.3％ in 2013 to 25％ in 2016.However,the insensitivity rate to ceftriaxone and moxifloxacin decreased in recent years and the susceptibility rate to ceftriaxone,cefotaxime,levofloxacin and moxifloxacin were higher than 90％ in each year.The resistance rate of H.influenzae strains from children were higher than the stains from all patients.The insensitivity rate to azithromycin in strains from children and all patients increased from 8.3％,10.2％ in 2013 to 25％,22.1 ％ in 2016,respectively.The 5 625 clinical strains of M.catarrrhalis re mained highly susceptible to the amoxicillin-clavulanic acid,ceftriaxone,cefotaxime,levofloxacin,ciprofloxacin (greater than 90％).The resistance rate to cotrimoxazole increased from 15.1 ％ in 2013 to 59.1 ％ in 2016.Conclusion H.influenzae are still susceptible to the third generation cephalosporins (greater than 90 ％),which can be used as the first choice in clinical practice.Nearly 70 ％ of these strains were resistant to ampicillin and cotrimoxazole,which is inappropriate for clinical therapy.The resistance rate to cotrimoxazole in the M.catarrrhalis strains from children increased from 15.1％ in 2013 to 59.1 ％ in 2016,and the resistance rate to the other test drug in M.catarrrhalis did not change much in the-year period.

Aim To investigate the effect of the novel benzoxazolone derivative 4-( 5′-dimethylamino )-naph-thalenesulfonyl-2 ( 3H )-benzoxazolone ( W3D ) on TLR4-MyD88-NF-κB signaling pathway in LPS-in-duced RAW264.7 cells. Methods The cell viability was detected by MTT assay, and the contents of TNF-α, IL-6, IL-1β and COX-2 in the cell supernatant were analyzed using ELISA methods. The protein ex-pression of IL-6, TLR4, MyD88, p-IRAK4 and NF-κB were investigated by western blot analysis, and the mRNA expressions of TLR4, MyD88 and IL-6 were an-alyzed by RT-PCR. Results W3D could obviously in-hibit the production of TNF-α, IL-6 and IL-1β in LPS- induced RAW264.7 cell supernatant, but it had no effect on the release of COX-2. Compared with the model group, the expressions of TLR4, MyD88 and IL-6 were decreased significantly in a dose dependent manner. Meanwhile, the expressions of p-IRAK4 and nucleus of NF-κB were decrease in W3D treated group compared with the model group. Conclusion The no-vel compound W3D could inhibit the release of the in-flammatory mediators through the regulation of TLR4-MyD88-NF-κB signaling pathway.