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 purify native F1 antigen from E pestis EV76 strain and determine its ef-ficacy against Y. pestis. Methods A new purification method was developed by the substitution of physical disruption ( glass beads) for organic solvent ( acetone and toluene) one, followed by a combination of ammo-nium sulfate fractionation and SephacrylS-200HR column filtration chromatography. Groups of mice were im-munized with F1 antigen adsorbed to 25% aluminum hydroxide in PBS by intramuscular route. The immu-nized animals were challenged subeutaneously(s, c. ) with 104 CFU of Y. pestis strain 141 at 18 weeks after the primary immunization. Results There was no IgG titre difference between two groups of mice with one-dose immunization, whereas in the two-dose immunization groups, the group F1-40 μg induced a statistically higher antibody titre than the group F1-20 μg. Complete protection was observed for animals immunized with purified F1 antigen by s.c. route. In contrast, the control mice immunized with aluminum hydroxide suc-cumbed to a same dose of Y. pestis 141 challenge. Conclusion This purification strategy is a simple and ef-fective, and can be operated in a large scale. Native F1 antigen extracted from Y. pestis EV76 is highly im-munagenic, and can be used as a key antigen component to develop sub-unit vaccine of plague.

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 investigate the short-term outcomes of Da Vinci robotic versus laparoscopic and open surgery for locally advanced Siewert type Ⅱ and Ⅲ adenocarcinoma of esophagogastric junction (AEG).Methods:The propensity score matching and retrospective cohort study was conducted. The clinicopathological data of 404 patients with locally advanced Siewert type Ⅱ and Ⅲ AEG who underwent radical gastrectomy in the First Hospital Affiliated to Army Medical University from January 2009 to April 2019 were collected. There were 331 males and 73 females, aged from 34 to 90 years, with a median age of 62 years. Of the 404 patients, 104 undergoing Da Vinci robotic radical gastrectomy were allocated into robotic group, 205 undergoing laparoscopic radical gastrectomy were allocated into laparoscopic group, and 95 undergoing open radical gastrectomy were allocated into open group. Observation indicators: (1) the propensity score matching conditions and comparison of general data among the three groups after propensity score matching; (2) surgical situations; (3) intraoperative lymph node dissection; (4) postoperative situations; (5) postoperative complications; (6) follow-up. Patients were followed up at postoperative 1 month by outpatient examination and telephone interview to detect survival and severe complications up to June 2019. The propensity score matching was used to perform 1∶2∶1 nearest neighbor matching by SPSS 23.0 and R software 3.6.1 Matchit among the robotic group, laparoscopic group and open group. Measurement data with normal distribution were represented as Mean± SD, and comparison among groups was done using one-way ANOVA analysis. Measurement data with skewed distribution were represented as M (range), and comparison was done using the Kruskal-Wallis H test. Comparison of ordinal data was analyzed using the Mann-Whitney U test. Count data were represented as absolute numbers or percentages, and comparison among groups was done using the chi-square test. Results:(1) The propensity score matching conditions and comparison of general data among the three groups after propensity score matching: 312 of 404 patients had successful matching, including 78 in the robotic group, 156 in the laparoscopic group, and 78 in the open group. The age, cases in G1, G2, G3 (histopathological classification) and cases with proximal gastrectomy or total gastrectomy (surgical resection range) before matching were (62.2±1.0)years, 0, 37, 67, 13, 91 in the robotic group, (60.9±8.1)years, 0, 98, 107, 31, 174 in the laparoscopic group, and (58.5±9.8)years, 1, 32, 62, 27, 68 in the open group, showing significant differences among the three groups ( F=4.269, 6.356, χ2=10.416, P<0.05). The above indicators after matching were (61.2±10.8)years, 0, 28, 50, 12, 66 in the robotic group, (60.7±8.0)years, 0, 56, 100, 25, 131 in the laparoscopic group, and (60.7±8.4)years, 0, 25, 53, 18, 60 in the open group, showing no significant difference among the three groups ( F=0.074, 0.379, χ2=2.141, P>0.05). (2) Surgical situations: the surgical time, volume of intraoperative blood loss, length of surgical incision, length of proximal margin after matching were 300.0 minutes(range, 188.0-420.0 minutes), 137.5 mL(range, 50.0-400.0 mL), 6.0 cm(range, 3.0-12.0 cm), 2.5 cm(range, 1.5-5.5 cm) in the robotic group, 276.0 minutes(range, 180.0-400.0 minutes), 150.0 mL(range, 40.0-800.0 mL), 6.0 cm(range, 3.0-12.0 cm), 3.0 cm(range, 1.0-5.0 cm) in the laparoscopic group, and 244.5 minutes(range, 125.0-461.0 minutes), 200.0 mL(range, 55.0-800.0 mL), 20.0 cm(range, 18.0-25.0 cm), 2.0 cm(range, 1.0-5.5 cm) in the open group, showing significant differences among the three groups ( χ2=27.619, 30.069, 179.367, 11.560, P<0.05). (3) Intraoperative lymph node dissection: the number of lymph node dissected, the number of lymph node dissected in the first station, the number of diaphragmatic and periesophageal lymph node dissected were 30.5(range, 10.0-70.0), 18.0(range, 6.0-42.0), 4.0(range, 0-13.0) in the robotic group, 29.0(range, 12.0-79.0), 19.0(range, 6.0-47.0), 5.0(range, 0-15.0) in the laparoscopic group, and 29.0(range, 18.0-58.0), 18.0(range, 12.0-38.0), 5.0(range, 0-8.0) in the open group, showing no significant difference among the three groups ( χ2=3.676, 1.014, 0.827, P>0.05). The number of lymph node dissected in the second station, the number of lymph node dissected in the superior pancreatic region, the number of No.110 lymph node dissected, the number of No.111 lymph node dissected after matching were 9.0(range, 2.0-30.0), 9.0(range, 2.0-30.0), 1.0(range, 0-4.0), 0(range, 0-3.0) in the robotic group, 6.5(range, 0-25.0), 7.0(range, 0-25.0), 0(range, 0-3.0), 0(range, 0-4.0) in the laparoscopic group, and 6.5(range, 0-19.0), 6.5(range, 0-19.0), 0(range, 0-1.0), 0(range, 0-1.0) in the open group, showing significant differences among the three groups ( χ2=19.027, 24.368, 19.236, 11.147, P<0.05). (4) Postoperative situations: the time to first flatus, time to initial out-of-bed activities, duration of postoperative hospital stay, treatment expenses after matching were 3 days(range, 2-5 days), 2 days(range, 1-4 days), 9 days(range, 5-20 days), 10.6×10 4 yuan [range, (5.4-18.0)×10 4 yuan] in the robotic group, 3 days(range, 2-8 days), 2 days(range, 1-7 days), 9 days(range, 6-56 days), 8.6×10 4 yuan[range, (5.7-40.8)×10 4 yuan] in the laparoscopic group, and 4 days(range, 2-10 days), 4 days(range, 2-10 days), 11 days(range, 8-41 days), 8.4×10 4 yuan[range, (5.8-15.2)×10 4 yuan] in the open group, showing significant differences among the three groups ( χ2=28.487, 95.069, 39.443, 83.899, P<0.05). (5) Postoperative complications: the incidence of overall complications, incidence of severe complications (Clavien-Dindo classification ≥grade 3), incidence of gastrointestinal complications, incidence of incisional complications, incidence of respiratory complications, incidence of infection were 21.8%(17/78), 5.1%(4/78), 10.3%(8/78), 1.3%(1/78), 7.7%(6/78), 2.6%(2/78) in the robotic group, 21.8%(34/156), 7.1%(11/156), 5.1%(8/156), 1.3%(2/156), 11.5%(18/156), 3.8%(6/156) in the laparoscopic group, and 29.5%(23/78), 6.4%(5/78), 9.0%(7/78), 2.6%(2/78), 14.1%(11/78), 2.6%(2/78) in the open group, showing no significant difference among the three groups ( χ2=1.913, 0.321, 2.394, 0.866, 1.641, 0.335, P>0.05). (6) Follow-up: 312 patients after propensity score matching were follow up at postoperative 1 month. During the follow-up, 2 cases with severe complications died after discharge. No severe complication such as obstruction of input or output loop, dumping syndrome was found in the other 310 patients. Conclusions:The Da Vinci robotic radical gastrectomy is safe and feasible for locally advanced Siewert type Ⅱ and Ⅲ AEG. Compared with laparoscopic and open radical gastrectomy, Da Vinci robotic radical gastrectomy has more advantages in the number of lymph node dissected in the second station (especially in the superior pancreatic region).

OBJECTIVETo study the biological and genetic characteristics of 119 strains of Yersinia (Y.) pestis isolated from plague patients in Qinghai province, from 1958-2012.

METHODSBoth regular methods and different region(DFR)molecular typing techniques were used to study the epidemiological characteristics on 119 strains of Y. pesticin Qinghai during 1958-2012. Sources of Y. pestis from two outbreaks, in Nangqian county in 2004 and in Xinghai county in 2009,Qinghai province were also analyzed.

RESULTS105 strains of Y. pestis were identified as Qinghai-Tibet Plateau Ecotype while the other 6 strains as Qilian Mountains Ecotype. 84.03% (100/119) of the tested strains carried 4 virulence factors F1(+), Pst I(+), VW(+) and Pgm(+)). 97.30% (72/74) of the tested strains showed high virulence. Strains that carrying 52×10(6), 65×10(6), 92×10(6) plasmids were distributed in Hainan, Haibei, Haixi,Yushu,Guoluo, Huangnan and Huangyuan counties. Genomovar 5 and 8 were the main gene types that circling around Qinghai Lake. Genomovar 10 was found in strains of Y. pesticin Nangqian county while Genomovar 8 was found in the strains isolated from human plague patient during the epidemics in Xinghai county in Qinghai.

CONCLUSIONData from biological and genetic analyses on the epidemics of human plague in Nangqian county in 2004 and in Xinghai county in 2009 demonstrated that methods as DFR genotyping and virulence factors profiles, as well as plasmids profiles were powerful tools in confirming the human plague epidemics and sources of infection.

China ; epidemiology ; Genotype ; Humans ; Plague ; epidemiology ; microbiology ; Yersinia pestis ; genetics ; isolation & purification