To investigate the effect of recombinant human growth hormone (rhGH) on JAK2-STAT3 pathway and the growth of gastric cancer cell lines at different GHR expression status, the eukaryotic expression vector targeting human GHR (pGPU6/GFP/Neo-shGHR and pGPU6/GFP/Neo-scramble) was constructed and transfected into MGC803 cells by Lipofectamine 2000. Stable expressive cell lines were obtained by G418 screening. The expression of GHR was analyzed by Western blotting. After being stimulated with rhGH, cell growth was detected by MTT assay. Cell cycle and apoptosis were examined by flow cytometry. The components of JAK2/STAT3 signaling pathway were detected by Western blotting. There is no significant difference of GHR expression between MGC803 and pGPU6/GFP/Neo-scramble-transfected cells (named as MGC803-NC) (P > 0.05). Compared with MGC803, the GHR expression in pGPU6/GFP/Neo-shGHR-transfected cells (named as MGC803-shGHR) decreased significantly (protein decreased 50%). The cells were treated with rhGH at 0, 150 and 300 ng x mL(-1), the growth rate of MGC803 and MGC803-NC increased significantly, PI and the number of G2/M phase cells all increased significantly, and apoptosis decreased significantly. Western blotting revealed that the expression of pJAK2 and pSTAT3 was up-regulated after being treated with rhGH in MGC803 and MGC803-NC cells. In contrast, similar change was not observed in MGC803-shGHR cells. Knockdown of GHR gene may decrease the sensitivity of gastric cancer cells to rhGH, and down-regulating of components of the expression of JAK2/STAT3 signaling pathway may be the potential mechanisms.

Adult ; Brucellosis ; Humans ; Male ; Middle Aged ; Occupational Diseases

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.

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

Objective To observe the effectiveness of introducing Diazepam into combined aesthesia of Sumianxin and ketamine hydrochloride .Method A total of 80 rabbits of both genders for operation were randomly divided into A , B and C groups .The A group was injected with Sumianxin intramuscularly ( 0.3 mL/kg by weight ) .The B group was injected with Sumianxin and ketamine hydrochloride intramuscularly ( 0.3 mL/kg by weight ) .The C group was injected with Diazepam intravenously ( 1.5 mL/kg by weight ) combined with Sumianxin and ketamine hydrochloride injected intramuscularly (0.3 mL/kg by weight).The aesthetic effects, induction time, anesthesia maintaining time, total anaesthetic dose and operation time were observed , recorded and compared .Result The induction time of the C group was significantly shorter than A and B groups (P<0.01).The initial anesthesia maintaining time of the C group was the longest among the three (P<0.01) with least total anaesthetic dose (P<0.01).The operation time of the C group was the least with best aesthetic effects (P<0.01).Conclusion Introducing Diazepam into combined aesthesia of Sumianxin and ketamine hydrochloride can improve the aesthetic effects .Therefore , this is an optional aesthetic method for time-consuming animal operation or sensitive surgical sites of rabbits .

Purpose To investigate the expression level of MED27 in lung cancer tissue samples and lung cancer cell lines and to further study the biological function of MED27 in lung cancer cells.Methods Immunohistochemistry and Western blot were used to detect MED27 expression in 70 lung cancer tissues and 5 different lung cancer cell lines,and the correlation between MED27 expression and gender,age as well as PTNM was also analyzed.The silence sequence of MED27 was designed by the siRNA technique.Western blot was used to detect the silence efficiency of MED27.The proliferation,migration and invasion ability of cells were assessed by CCK-8 assay,Scratch assay and Transwell assay after the MED27 was knocked down.Western blot was used to detect the expression of protein involved in the cell proliferation,migration and invasion.Results The results of immunohistochemistry and Western blot showed that MED27 expression was higher in lung cancer tissues and cells (P ＜ 0.05).The expression of MED27 was positively correlated with lymph node metastasis (x2 =9.438,P =0.002,P ＜ 0.05).However,it was not related with gender,age,tumor size and distant metastasis (P ＞ 0.05).The knockdown of MED27 by MED27 specific siRNA could inhibit the proliferation,migration and invasion of H460 cells (P ＜ 0.05).The expression of MMP-2 and MMP-9 involved in the cell migration that were significantly inhibited in H460 cells transfected by MED27 siRNA,and the expression of E-cadherin,related with cell invasion was also decreased,while E-cadherin negative regulatory protein Snail was increased.Conclusion MED27 is highly expressed in lung cancer tissues and cells and high expression of MED27 predicts poor prognosis in lung cancer patients.The knockdown of MED27 inhibits the proliferation,migration and invasion ability of lung cancer cells.All of the above results suggest that MED27 is expected to be a candidate target of lung cancer gene therapy.

To investigate the effect of recombinant human growth hormone (rhGH) on JAK2-STAT3 pathway and the growth of gastric cancer cell lines at different GHR expression status, the eukaryotic expression vector targeting human GHR (pGPU6/GFP/Neo-shGHR and pGPU6/GFP/Neo-scramble) was constructed and transfected into MGC803 cells by Lipofectamine 2000. Stable expressive cell lines were obtained by G418 screening. The expression of GHR was analyzed by Western blotting. After being stimulated with rhGH, cell growth was detected by MTT assay. Cell cycle and apoptosis were examined by flow cytometry. The components of JAK2/STAT3 signaling pathway were detected by Western blotting. There is no significant difference of GHR expression between MGC803 and pGPU6/GFP/Neo-scramble-transfected cells (named as MGC803-NC) (P > 0.05). Compared with MGC803, the GHR expression in pGPU6/GFP/Neo-shGHR-transfected cells (named as MGC803-shGHR) decreased significantly (protein decreased 50%). The cells were treated with rhGH at 0, 150 and 300 ng x mL(-1), the growth rate of MGC803 and MGC803-NC increased significantly, PI and the number of G2/M phase cells all increased significantly, and apoptosis decreased significantly. Western blotting revealed that the expression of pJAK2 and pSTAT3 was up-regulated after being treated with rhGH in MGC803 and MGC803-NC cells. In contrast, similar change was not observed in MGC803-shGHR cells. Knockdown of GHR gene may decrease the sensitivity of gastric cancer cells to rhGH, and down-regulating of components of the expression of JAK2/STAT3 signaling pathway may be the potential mechanisms.
Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; Human Growth Hormone ; genetics ; pharmacology ; Humans ; Janus Kinase 2 ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Receptors, Somatotropin ; genetics ; metabolism ; Recombinant Proteins ; genetics ; pharmacology ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; Stomach Neoplasms ; metabolism ; pathology ; Transfection

OBJECTIVETo compare the sensitivity of Brown Norway rats (BN) with Guinea pigs (GP) as allergen assessment animal models.

METHODBN rats and GP were randomly assigned to 1 control group, 2 Bovine serum albumin group (BSA), respectively. Animals in BSA groups of BN rats and GPs were sensitized by intraperitoneal injection of 0.6% BSA 1 ml on day 1, 3, 5, respectively, and irritated by intravenous injection of 2.4% BSA 1 ml on day 7 and day 14 after the last sensitization, while the same volume of normal saline was given to control group on each time point mentioned above. The allergic reactions were scored within 1 h after each irritation treatment, and the sera of both BN rats and GPs were collected to detect IgE concentration by using ELISA. The sera were also applied for passive cutaneous anaphylaxis test (PCA test) in SD rats.

RESULTNo obvious allergic reactions were observed in BSA group of GPs after each irritation treat, however, the score of allergic response in BSA group of BN rats was evidently higher than that in control group after first irritation. PCA test by using sera from BSA group of BN rats after both irritations showed the strong positive result characterized as large amount of subcutaneous effusions of Evans blue in SD rats, however, the sera from BSA group of GP were negative in PCA test. Serum IgE concentration did not increase after each irritation in BSA group of both BN rats and GP.

CONCLUSIONBN rats were more sensitive than GPs on initiative systemic anaphylaxis test and passive cutaneous anaphylaxis test. Meanwhile, BN rats has an advantage in experimental treatment compared with Guinea pigs.

Allergens ; administration & dosage ; toxicity ; Anaphylaxis ; chemically induced ; Animals ; Guinea Pigs ; Hypersensitivity ; etiology ; Male ; Models, Animal ; Rats ; Rats, Inbred BN ; Rats, Sprague-Dawley

OBJECTIVETo identify factors associated with YMDD mutation in patients with chronic hepatitis B before and after lamivudine treatment in Zunyi region.

METHODS53 patients with chronic hepatitis B were enrolled in this study, HBV DNA,HBV markers, ALT, AST, TBil, albumin in the serum were examined at 0, 3, 6, 12, 18 and 24 months after lamivudine treatment. HBV genotype and YMDD mutation were determined by sequencing before lamivudine treatment. YMDD mutation was checked again if serum HBV DNA rebound to more than 1 x 10(4) copies/ml after the initial decrease.

RESULTSHBV genotype in Zunyi region is constitute of B, C and B+C genotype. YMDD mutation occurred in 18 cases after lamivudine treatment, the rate of YMDD mutation was 15.1%, and 34.0% after 1 year and 2 years treatment. There are four types of mutation: rtL180M/M204V, rtL180M/M204I, rtM204I, rtL180M. rtM204V mutation in C gene was always accompanied by rtL180M mutation (100%). The rate of rtL180M/M204V mutation in genotype C group was significantly higher than that in genotype B group (77.8% to 25.0%), the same was true for the rtL180M/ M204I mutation (22.2% to 12.5%). There was no point mutation in genotype C group. The point mutation of rtM204I and rtL180M appeared only in genotype B group. Gender, nation, family history of hepatitis B and HBeAg were not associated with YMDD mutation (P more than 0.05), while the mutation rate was associated with the disease course and severity of disease. YMDD mutation did not occur in patients with low HBV DNA level (less than 10(5) copies/ml).

CONCLUSIONYMDD mutation after lamivudine therapy is associated with HBV genotype and P gene mutation type, and prolonged treatment increases the the mutation rate. In order to reduce the incidence of YMDD mutation, patients with shorter disease course, lower HBV DNA level, more serious liver damage should be treated with lamivudine.

Adult ; Alanine Transaminase ; blood ; Antiviral Agents ; pharmacology ; therapeutic use ; Aspartate Aminotransferases ; blood ; China ; epidemiology ; DNA Mutational Analysis ; DNA Primers ; DNA, Viral ; blood ; genetics ; Drug Resistance, Viral ; Female ; Genotype ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; blood ; drug therapy ; virology ; Humans ; Lamivudine ; pharmacology ; therapeutic use ; Male ; Mutation ; Polymerase Chain Reaction

Objective To investigate the assessment and influencing factors of the cerebrovascular function in 40-60 years old women in Guiyang city,and to provide information for the prevention of stroke in menopausal women.Methods Self-designed questionnaire was used among randomly selected healthy women aged 40-60 years who received health checkup from April 2016 to April 2017 in the Medical Examination Center of Affiliated Hospital of Guizhou Medical University.Levels of height,weight,blood pressure,blood lipid,blood glucose,blood uric acid and the cerebral hemodynamics were checked,comparing the influence of different factors for cerebrovascular function integral value differences,and assess the risk of stroke.Results A total of 439 cases were included in the study,the number of cases with abnormal cerebro vascular function (the score of cerebro vascular function score ＜75) accounted for 13.2％ (58/439).Increased age,being ethnic minorities,menopause,obesity,high blood pressure,high blood sugar,high blood uric acid,increased high-density lipoprotein cholesterol (HDL-C),total cholesterol and triglycerides were related to the abnormal scores of brain function,and the difference was statistically significant (P＜0.05).Logistic regression analysis indicated that minority,High HDL-C [OR(95％CI):0.38 (0.25-0.58),P=0.036],obesity [OR(95％CI):11.62 (5.17-26.14),P=0.001] and hypertension [OR(95％CI):14.87(2.46-89.75),P=0.001] are the factors influencing the cerebro vascular function.Conclusion The proportion of abnormal cerebrovascular function was 13.2％.Increased HDL-C is the protective factor for perimenopausal cerebro vascular function.Minority,obesity and high blood pressure are risk factors for cerebro vascular function,of which,the association between hypertension and cerebro vascular function worsens with elevated blood pressure.