Objective To investigate the inhibition rate of cell proliferation, cell apoptosis rate and their effects on the cell cycle proceeding of the SSMC7721 cell line when 5-FU combined with thermotherapy is induced into the cells, and then provide theoretical bases to the combined therapy of hepatocellular carcinoma. Methods The inhibition rate of cell proliferation was detected by the MTT under different conditions, the cell cycle proceeding and the cell apoptosis rate was detected by flow cytometry and the subcellular structure was detected by the electronmicroscope. Results The cell inhibition rate of the thermotherapy group, 5-FU group and the combinedgroup were 18.4% ,28. 3% and 52. 7% ,respectively. The inhibition rates in the latter two groups were significantly different to the thermotherapy group. The results of flow cytometry showed that the cell numbers increased in G1 stage decreased in S stage,and increased in G2/M stage;the cell apoptosis rate increased. There was significant difference between different groups(P ＜ 0.01 or P ＜0.05). The results of the electronmicroscop showed that the nuclear chromatins agglutinated in the borderline and the mitochondriums became swelled. Conclusions The 5-FU combined with thermotherapy could significantly improve the inhibition rate of cell proliferation, inhibit the cell cycle proceeding from G1 stage to S stage, and induce cells apoptosis and change the subcellular structures in the SSMC7721 cell line.

Abstract: Objective To observe the phenotypic characteristics of 3 wild-type plague phages under different experimental environments, providing scientific evidence for the identification of phage biological characteristics and the study of their interaction with host bacteria in the future. Methods The sensitivity of 3 wild-type plague phages were detected by using liquid culture method, emisolid medium method and micro-liquid culture method based on OmniLog TM microbial identification system. Results The growth result based on LB liquid medium showed that the growth of plague phage 476 for 20-24 hours at both 28 ℃ and 37 ℃was better than that of plague phages 087 and 072204 at 37 ℃, and the growth of plague phages 087 was better than that of plague phages 072204 at 37 ℃. With the attenuated plague bacterium EV76 as the host bacterium, phage 476 was able to form visible plaque on double-layer agar medium for 20-20 hours at both 28 ℃ and 37 ℃, phages 087 and 072204 were only able to form opaque plaque on double-layer agar medium for 20-24 hours at 37 ℃. The growth results based on OmniLogTM system showed that when plague phage was lysed in EV76 strain at 33 ℃, the first row appeared as a straight line with a peak of no more than 100 in the 96-well microplate curve chart. As the phage quantity decreased, the dilution plate appeared with growth curve similar to EV76 strain in turn, and the color of tetrazolium dyes in the experimental wells gradually deepened as the phage number decreased and the host bacteria number increased. Therefore, it indicates that phage 476 was sensitively at both 28 ℃ and 37 ℃, while phage 087 and 072204 were temperature-dependent only at 37 ℃ to attenuated plague bacterium EV76. Conclusions The lysing ability of 3 wild-type plague phages are temperature-dependent, and the growth results are consistent under the three experimental conditions.

Objective To investigate the biological characteristics and epidemiological significance of Yersinia pestis strains in Qilian County,Qinghai Province,in order to provide a scientific basis for plague prevention and control.Method Totally 67 strains were separated from kinds of host in Qilian County,Qinghai Province from 1958 to 2011,to do biochemical test,toxicity test,virulence factors evaluation,plasmid analysis and different region (DFR) genotyping.Results According to biochemical typing,48 of the 50 strains tested were Qing-Tibet Plateau ecotype,15 were Qilian Mountain ecotype,and the remaining 4 were different ecotypes from the plague foci in Qinghai plateau.The strains had 8 genomovars,and were given priority to genomovar8 (42 strains),secondly,genomovar44 (15 strains),genomovar5 (4 strains),genomovar7 (2 strains),genomovar19 (1 strain),genomovar30 (1 strain),genomovar32 (1 strain),and genomovar34 (1 strain).A proportion of 95.52％ (64/67) of the strains contained 3 kinds of plasmid-6 × 106,45 × 106,and 52 × 106;85.07％ (57/67) contained all the four virulence factors,and 96.00％ (48/50) were velogenic strains.Conclusion The strains separated in Qilian County,Qinghai Province have the characteristics of Qinghai-Tibet Plateau plague's pathogen and have strong toxicity,so we should enhance the plague monitoring and give more publicity to plague prevention to prevent animal plague spreading to human.

Objective To study the biological characteristics and epidemiological significance of Yersinia pestis in Chengduo County of Qinghai Province,in order to provide scientific basis for plague prevention and control in this area.Methods Thirty one strains of Yersinia pestis isolated from Chengduo County of Qinghai Province from 1980 to 2011 were selected as study subjects.Biochemical test,virulence factors evaluation [Fra1 (F1),pesticin Ⅱ (Pst Ⅱ),virulence antigen (VW),pigmentation (Pgm)] and different region (DFR) genotyping were carried out.Nineteen of the 31 strains Yersinia pestis were selected according to different time,different areas and different hosts to determine their toxicity in mice,MLD ≤ 10 000 was strong toxic strain,10 000 ＜ MLD ＜ 100 000 was moderate toxic strain.Results Among thirty one strains of Yersinia pestis,23 strains were isolated from human,the Himalaya marmot and its fleas and lice,and their biological type was classical,biochemical type was Qinghai-Tibet plateau;21 strains genotype was type 5,1 was type 16,1 was type 32,and they contained all four kinds of virulence factors (F1,Pgm,Pst Ⅱ,VW),and toxicity test showed all strains (14) were strong toxic strains.The rest 8 strains of Yersinia pestis isolated from the Microtus fuscus and its fleas,and their biological type was Microtus,biochemical type was Chuanqing plateau;they could produce F1 and Pgm,of which 87.5％ (7/8) strains could produce Pst Ⅱ,but could not produce VW antigen factor,the genotype was 14,and the toxicity results showed that they were strong (3)and moderate (2) toxic strains.Conclusion The strains separated in Chengduo County of Qinghai Province from 1980 to 2011 have the pathogen characteristics of Qinghai-Tibet plateau plague,they are mainly strong toxic strains;the work on prevention and control of plague should not be neglected.

Objective To analyze the biological characteristics of Yersinia pestis strains in Haixi Prefecture,Qinghai Province,in order to provide a scientific basis for plague prevention and control in future.Methods Totally 181 strains were separated from variety kinds of host in Haixi Prefecture,Qinghai Province from 1957 to 2011,and these strains were conducted biochemical test,virulence factor evaluation,plasmid analysis,different region (DFR) genotyping,drug and disinfectant resistant genes detection;79 of the 181 strains were examined by toxicity test and classified according to the criteria (minimum lethal dose:MLD≤ 10 000 was velogenic strain,10 000 ＜ MLD ＜ 100 000 was moderate virulence strain,MLD ≥ 100 000 was hypovirulent strain).Results According to six biochemical typing about gelatin candy,rhamnose,maltose,melibiose,glycerin and denitrification,the 181 strains of Yersinia pestis were antique biovar and Qing-Tibet Plateau ecotype.Aproportion of 81.22％ (147/181) of Yersiniapestis strains contained all the four virulence factors (F1,Pst Ⅰ,VW,Pgm).Totally 63.54％ (115/181) of the strains contained 3 kinds of plasmid-6 × 106,45 × 106,and 52 × 106;31.49％ (57/181) of the strains contained 3 kinds of plasmid-6 × 106,45 × 106,and 65 × 106.The strains had 8 genomovars,and were given priority to genomovar 8 (109 strains),secondly,genomovar 32 (33 strains),genomovar 5 (20 strains),genomovar 1b(i4 strains),genomovar 44 (2 strains),genomovar 7 (1 strain),genomovar 37 (1 strain),and genomovar 49 (1 strain).Among the 181 Yersinia pestis strains,strains with genes related to streptomycin resistance,sulfanilamide resistance,beta lactam resistance and disinfectant resistance were not found;and 75 of 79 strains were velogenic strains by toxicity test (MLD ≤ 10 000),accounted for 94.94％ (75/79).Conclusion The strains separated in Haixi Prefecture,Qinghai Province have the characteristics of Qinghai-Tibet Plateau plague's pathogen and have strong toxicity;all strains don't have the characteristics of drug and disinfectant resistance genes.

Objective In order to acquaint with the prevalence of Tibetan sheep plague in this area, we conducted a serum epidemiological investigation of Tibetan sheep plague in Qinghai Province. Methods Indirect hemagglutination assay (IHA) and colloidal gold immunochromatography (GICA) were applied to test serum samples of Tibetan sheep and whole blood samples from jugular vein of Tibetan sheep were collected in 8 Prefectures of Qinghai Province from 2013 to 2016. Results A total of 86 positive Tibetan sheep serum samples with plague F1 antibody were detected by both methods, and the positive rate was 0.68％ (86/12710), the samples collected in Xinghai County Hainan Prefecture had the highest positive rate, which was 5.20％ (27/519). The Haixi Prefecture and Yushu Prefecture were historical epidemic areas, the positive rates were 0.65％(15/2313) and 0.26％(6/2293), respectively. Hainan Prefecture, Guoluo Prefacture and Huangnan Prefecture were newly confirmed epidemic areas, the positive rates were 1.61％ (28/1741), 1.01％ (15/1481), and 1.44％(19/1316), respectively. The antibody titers were 1:20 to 1:5120, the samples collected in Maqin County Guoluo Prefecture had the highest titer, namely 1 :5120. Conclusions In Qinghai Province, Tibetan sheep plague is endemic, and there are outbreaks in some regions. So we have to enhance the Tibetan sheep plague monitoring especially in Marmot plague epidemic area.

Objective:To analyze the pathogenic characteristics of Yersinia pestis in a plague natural foci in Qinghai-Tibet Plateau. Methods:In this study, 1 378 strains of Yersinia pestis isolated from different regions, hosts and vectors in Qinghai-Tibet Plateau from 1954 to 2016 were taken as the research objects. Phenotypic characteristics, plasmid spectrum and genotype of the strains were studied by using conventional techniques and molecular biological techniques. The etiology and geographical distribution of the plague were studied. Results:There were 6 biochemical types of Yersinia pestis in Qinghai-Tibet Plateau, namely Qinghai-Tibet Plateau, Qilian Mountain, Gangdis Mountain, Kunlun Mountain A, Kunlun Mountain B and Chuanqing Plateau. This study found that the Qinghai-Tibet Plateau type strain was not only distributed in north Tibet Plateau, but also distributed in south Tibet, and the distribution of Gangdis Mountain type strain extended to south Tibet. Four virulence factors (capsule antigen, yersinin, virulence antigen and pigmentation factor) were found in 79.97% (1 102/1 378) Yersinia pestis. The results also showed that there were 12 kinds of plasmids carried by Yersinia pestis strains in Qinghai-Tibet Plateau, which constituted 17 kinds of plasmid spectrum. There were 3 kinds of the largest plasmids with taxonomic properties, forming their respective relatively independent distribution areas. The study of different regions (DFR) type showed that 5, 8, 14, 19, 32 and 44 of 1 378 strains were the main genotypes, and the main genome types had obvious geographical distribution. Conclusions:All the tested strains have the characteristics of plague pathogen in Qinghai-Tibet Plateau. The polymorphism of the main hosts, vectors and the ecological landscape of plague geography in the plague foci in Qinghai-Tibet Plateau may lead to the diversity of biochemical characters, plasmid spectrum and geno types of Yersinia pestis.

Objective:To observe the toxic effects of different doses of baicalin on liver and kidney of rats after different time administration, and provide experimental reference for the safety of clinical medication.Methods:In April 2019, 42 Wistar male rats were randomly divided into a control group (0.9% sodium chloride solution) and baicalin administration groups (100, 200 mg/kg) , 14 rats in each group, and one was given by oral gavage. 7 times/d, 6 times/week, 7 rats in each group were sacrificed 28 and 56 days after the administration. The wet weights of liver and kidney were weighed and the organ coefficients were calculated. The hematoxylin-eosin (HE) staining was used to detect the histomorphological changes. And the levels of serum alanine aminotransferase (ALT) , aspartate aminotransferase (AST) , alkaline phosphatase (ALP) , blood urea nitrogen (BUN) and creatinine (CRE) were detected.Results:After 56 days of administration in baicalin 200 mg/kg rats, the body weight and kidney coefficient were lower than those of the control group. Histopathology showed that glomerular atrophy became smaller, renal tubules were significantly atrophied, and epithelial cell necrosis occurred. No obvious abnormalities in liver was observed. After 56 days of administration in baicalin 200 mg/kg rats, the levels of BUN and CRE in the serum were higher than those of the control group, and the differences were statistically significant ( P<0.05) . There were no obvious abnormalities in the baicalin 100 mg/kg group and the 28 d of administration in baicalin 200 mg/kg group. Conclusion:Under the conditions of this test, baicalin has certain renal toxicity in male rats.

Objective:To observe the toxic effects of different doses of baicalin on liver and kidney of rats after different time administration, and provide experimental reference for the safety of clinical medication.Methods:In April 2019, 42 Wistar male rats were randomly divided into a control group (0.9% sodium chloride solution) and baicalin administration groups (100, 200 mg/kg) , 14 rats in each group, and one was given by oral gavage. 7 times/d, 6 times/week, 7 rats in each group were sacrificed 28 and 56 days after the administration. The wet weights of liver and kidney were weighed and the organ coefficients were calculated. The hematoxylin-eosin (HE) staining was used to detect the histomorphological changes. And the levels of serum alanine aminotransferase (ALT) , aspartate aminotransferase (AST) , alkaline phosphatase (ALP) , blood urea nitrogen (BUN) and creatinine (CRE) were detected.Results:After 56 days of administration in baicalin 200 mg/kg rats, the body weight and kidney coefficient were lower than those of the control group. Histopathology showed that glomerular atrophy became smaller, renal tubules were significantly atrophied, and epithelial cell necrosis occurred. No obvious abnormalities in liver was observed. After 56 days of administration in baicalin 200 mg/kg rats, the levels of BUN and CRE in the serum were higher than those of the control group, and the differences were statistically significant ( P<0.05) . There were no obvious abnormalities in the baicalin 100 mg/kg group and the 28 d of administration in baicalin 200 mg/kg group. Conclusion:Under the conditions of this test, baicalin has certain renal toxicity in male rats.

OBJECTIVE: To explore the effect of reactive oxygen species(ROS) and cell cycle in bone marrow cells in benzene-induced aplastic anemia(AA) mouse model. METHODS: Specific pathogens free male CD1 mice were randomly divided into control group and exposure group(n=10, each group). The mice in exposure group were subcutaneously injected with benzene at a dose of 2 mL/kg body weigh diluted 1 ∶1 in corn oil, while the mice in control group were treated with equal volume of corn oil, 3 times a week for a total of 25 times. After exposure, the blood routine and reticulocyte percentage of peripheral blood of mice were examined. The femur histopathology was performed. The levels of benzene and its metabolites hydroquinone, and phenol in blood, liver and bone marrow were tested by solid-phase extraction gas chromatography mass spectrometry. The level of ROS and the changes of cell cycle in bone marrow mononuclear cells(BMMNCs) were determined by flow cytometry. The protein expression of Cyclin D1 and cyclin-dependent kinase 4(CDK4) in BMMNCs was detected by Western blot. RESULTS: Since the 10 th benzene exposure, the body mass of mice in the exposure group was lower than that in the control group at the same time point(P<0.05). After the benzene exposure, all the counts of white blood cell, red blood cell, platelet, and hemoglobin level and reticulocyte percentage in peripheral blood of mice in the exposure group were decreased when compared with the control group(P<0.05). Bone marrow histopathological examination showed that bone marrow hematopoietic cells were decreased and non-hematopoietic cells were increased in the exposure group. In this study, a mouse model of AA induced by benzene was successfully established. The levels of benzene, hydroquinone and phenol in exposure group increased in blood, liver, and bone marrow compared to control group(P<0.05). Furthermore, the level of benzene from high to low were blood, liver and bone marrow, while the levels of hydroquinone and phenol were mainly stored in the blood and bone marrow in exposure group. Compared with the control group, the level of ROS, S phase fraction, and the relative protein expression of Cyclin D1 and CDK4 in BMMNCs increased, while the G1 phase fraction decreased in exposure group(P<0.01). CONCLUSION: The results suggest that benzene and its metabolites induce an increase of ROS level and S phase cell arrest, that play an important role in the pathogenesis and development of benzene-induced AA.