AIM:Studies on totipotential stem cells using primordial germ cells (PGCs) have the same application prospect as embryonic stem cells, especially for the animals difficult to isolate embryonic stem cells from blastula. In vitro culture condition of embryo germ cells is the key to control this. This study designs a method to effectively amplify PGCs of mice in vitro and establishes an effective culture method of PGCs. METHODS: Experiments were carried out in the Department of Histology and Embryology of Chongqing Medical University and Key Laboratory of Biochemistry and Molecular Pharmacology from October 2006 to August 2007. ①Clean Kunming pregnant mice (embryos 0.5 dpc) were provided by Animal Experimental Center of Chongqing Medical University. Experimental procedures met the animal ethical standards. ②Allantois and surrounding tissues of 8.5 day post coitum (dpc) embryos, the hindhut and surrounding tissues of 9.5 dpc and 10.5 dpc embryos, the gonadal ridges and surrounding tissues of 11.5 dpc and 12.5 dpc embryos were collected and digested with 0.25%pancreatin-0.02%EDTA, then the cells were cultured in the plastic petridishes which are pretreated with 0.1% gelatine. The formation ratio of primary and the first passaged Embryonic germ (EG) clones were compared among those different time points. The SSEA-1 positive ratio of isolated cells was compared between the two days by immunohistochemical method. The assays of clone numbers counting and MTT were used to compare the different proliferation ability of PGCs from the 11.5 dpc and 12.5 dpc embryos. The effects of expanding PGCs from 11.5 dpc and 12.5 dpc embryos by those two ways above were compared. The EG clones were analyzed by the expression of alkaline phosphatase and the immunologic marker SSEA-1,the undifferentiated marker Nanog and the differentiation ability in vitro were also tested. RESULTS:①The formation ratio of primary and the first passaged EG clone from 11.5 and 12.5 dpc embryos was higher than that from 8.5 to 10.5 dpc embryos and efficiency of expansion was increased (P 0.05). On the third day, the number of the primary EG clones from 11.5 dpc embryos was higher than that from 12.5 dpc embryos (P 0.05), but the two groups had different characteristics. ④The EG clones have representative morphology with high level of alkaline phosphatase activity and SSEA-1, Nanog expression, which can differentiate into the cystic embryoid body. CONCLUSION: 11.5 dpc and 12.5 dpc mouse embryos, especially 11.5 dpc embryos are the optional time points to expand PGCs efficiently. Co-culturing with the tissue and isolating the gonadal ridges both of these two ways are practicable.

Objective To investigate the isolation, purification and culture methods of human embryonic germ cells (EGCs) on feeder layer cells of human embryonic fibroblasts. Methods Primordial germ cells(PGCs) from the genital ridges of 6-11 weeks(post fertilization) human embryos were cultrued on feeder layer cells of human embryonic fibroblasts(HEFs) which were isolated from the 3-4 month embryos and routinely cultured for over 25 passages. The medium composed of growth factors and differentiation inhibitory factors. The cultures were analyzed with the expression of alkaline phosphatase, immunologic markers (SSEA-1,SSEA-4) and the transcription factor Oct-4 that have been used to routinely to characterize EGCS. Results A large-scale EG cells were obtained and meintained on feeder layers for over 8 passages. The cell surface marker showed that the EGCs possess high levels of AP activity. EGCs colonies stained strongly for SSEA-4,SSEA-1 and they expressed the transcription factor Oct-4.Conclusion EGCs have potential to maintain long term proliferation and undifferentiation state on human embryonic fibroblasts in vitro.;

Objective To study the temporal and spatial expression of Nanog in developing primordial germ cells(PGCs) of mouse.Methods Immunofluorescence technique was taken to qualitatively analyze the expression of Nanog in PGCs of 7.5days(post-coitus days)-15.5days mouse embryo.A further quantitative analysis of Nanog expression change in PGCs of 11.5days-15.5days mouse embryo was done by using SSEA-1 and Nanog double labelling immunofluorescence staining with confocal microscopy.Results The PGCs in mouse allantois,hindgut,dorsal mesentery and genital ridge were Nanog positive.Both of the highest positive ratio and the highest fluorescence intensity appeared in PGCs of 11.5days mouse embryo.For the female mice,Nanog expression drops dramatically after 12.5days,and for the male mice,a noticeable decline of Nanog expression was occurred after 13.5 postcoitus days.Conclusion\ Nanog expresses stably in the proliferating PGCs.The down-regulation of its expression may be related with the differentiation of PGCs.

Background Posterior capsular opacification (PCO) is a primary complication after extracapsular cataract extraction.The mechanism of PCO is associated with proliferation,migration and epithelialmesenchymal transition (EMT) of human lens epithelial cells (LECs).To explore the target treatment of PCO is very important.Objective This study was to investigate the biological effects of second mitochondria-derived activator of caspases (Smac) on the proliferation and apoptosis of LECs.Methods Human LECs line (HLE-B3) and Smac-overexpressed LECs line were cultured,and the cells were transfected using small interfering RNA (siRNA)-Smac3 plasmid with green fluorescent protein (GFP) for 24 hours.Different concentration of transforming growth factor-β2 (TGF-β2) (5,10,20 and 50 μg/ml) or 200 μmol/L H2O2 were added respectively into the culture medium to establish PCO model and oxidative stress model.Cell counting kit-8 (CCK-8) assay was used to compare the cell proliferative activity among PBS group,TGF-β2 group and Smac-hyperexpression +TGF-β2 group.Flow cytometry was used to evaluate the apoptotic rate of the PBS group,H2 O2 group and siRNA-Smac+H2 O2 group.The expressions of Smac,caspase-3 and proliferating cell nuclear antigen (PCNA) mRNA and their proteins in the cells were detected by real-time quantitative PCR (RT-PCR) and Western blot.Results The GFP+ cells were≥ 80％ 12 hours after siRNA-Smac3 transfection,with the optimal plasmid of siRNA-Smac3.GFP+ cell rate was (72.32 ± 2.31)％ in the siRNA-Smac3 transfection group,which was significantly higher than that in the blank plasmid group ([4.91 ±0.24] ％) (t=116.342,P＜0.001).The relevant expression levels of Smac was 35.21 ±4.11 in the Smachyperexpression group,and that in the blank plasmid group was 15.24±2.48,with a significant difference between them (t =215.47,P＜0.05).The cell viability of 20 ng/ml TGF-β2 affected PBS group,TGF-β2 group and Smachyperepression+TGF-β2 group was (98.4 ± 1.7) ％,(98.9 ± 0.1) ％ and (64.2 ± 3.1) ％,and the cell viability of Smac-hyperepression+TGF-β2 group was significantly lower in the Smac-hyperepression+TGF-β2 group than that in the TGF-β2 group (P＜0.05).The apoptotic rate in the PBS group,H2 O2 group and siRNA-Smac+H2 O2 group were (2.9 ± 1.2) ％,(45.1 ±4.5) ％ and (27.5 ± 1.8) ％,and the apoptotic rate was evidently lower in the siRNA-Smac +H2O2 group than that in the H2O2 group (P＜0.05).RT-PCR results showed that the expression levels of caspase-3 mRNA in PBS group,H2 O2 group and siRNA-Smac + H2 O2 group were 0.321 ± 0.103,0.715 ± 0.112 and 0.479 ±0.209,respectively.Compared with the H2 O2 group,the relative expression level of caspase-3 mRNA in siRNA-Smac+ H2O2 group was significantly decreased,the difference was statistically significant (P＜ 0.05).The PCNA mRNA expression levels in PBS group,TGF-β2 group and Smac-hyperepression+TGF-β2 group were 0.299±0.013,0.645± 0.102 and 0.490±0.209,respectively.Western blot results showed that the relative expression of caspase-3 protein in siRNA-Smac+H2O2 group and H2O2 group was 0.712±0.012 and 0.973±0.051,with significant difference between the two groups (t =132.52,P＜0.05).The relative expression of PCNA protein in Smac-hyperepression+TGF-β2 group was 0.782±0.212,which was lower than 1.126±0.251 in the TGF-β2 group (P＜0.05).Conclusions Smac may prevent and treat PCO by inhibiting the proliferation and promoting apoptosis of human LECs.

Objective To evaluate the performance of fast Treponema pallidum(TP) detection in voluntary blood donors and optimize the strategy for pre-donation screening.Methods Before blood donation,the gold standard TP test strip was used to make a fast detection.After blood donation,the TP-ELISA was used to test the blood.Then,analyze the donors′ anti-TP positive rate,times and intervals of donating,false positive and negative of TP fast detection.Results From 2014 to 2015,among 73 990 donors who were tested by using fast TP detection,0.71% of them(529 donors) were positive.Among the positive donors,89.2% of them(472 donors) were first-time blood donors.35 donors′ donating intervals were more than 3 years,who accounted for 61.4% of the donors who had donated for more than once.The numbers of the false positive obtained from fast TP detection were 5 and the false negative was 15.By applying the fast TP detection before blood donation,the rate of anti-TP positive had been declined from 0.71% to 0.17%.Conclusion The rejection rate of TP positive can be significantly reduced by using fast TP detection before blood donation.The fast TP detection can be used to optimize the pre-donation screening and promote blood donation service efficiency and level,while donating times and intervals of the blood donors were also considered.

Objective To analyze the distribution and antimicrobial resistance of pathogens isolated from department of hematology during the past three years.Methods Pathogenic strains isolated from patients hospitalized in a hematology de-partment between January 2011 and December 2013 were collected,antimicrobial susceptibility testing was performed by Kirby-Bauer disk diffusion method or automatic system,antimicrobial susceptibility testing results were judged according to American Clinical and Laboratory Standards Institute 2011, data were analyzed by WHONET 5.6 software. Results A total of 462 clinical isolates were collected in 2011—2013,including 161 gram-positive cocci isolates,279 gram-negative bacilli,and 22 fungi.Of Staphylococcus spp ,detection rate of methicillin-resistant coagulase negative Staphylo-coccus(MRCNS)and methicillin-resistant Staphylococcus aureus (MRSA)was 81.37% and 62.50%respectively.The re-sistant rate of Staphylococcus spp .and Enterococcus spp .to linezolid was 1.69% and 3.57% respectively,resistant rate of Staphylococcus spp .to teicoplanin was 3.39%,vancomycin-resistant gram-positive coccus was not found.Enterobacte-riaceae strains Escherichia coli and Klebsiella pneumoniae were highly susceptible to carbapenems,the sensitivity rates were 97.56%—98.88%;while nonfermentative gram-negative bacilli Pseudomonas aeruginosa and Acinetobacter bauman-nii strains were obviously resistant to carbapenems,the resistance rates were 38.71%—64.00%.Conclusion Antimicrobial resistance of major pathogenic strains from hematology department is high,antimicrobial agents should be used according to pathogenic distribution characteristics and antimicrobial susceptibility testing results,healthcare-associated infection control should be strengthened to reduce antimicrobial resistant rate.

Objective To investigate the current situation of healthcare-associated infection(HAI)management in secondary and above medical institutions in Shaanxi Province,analyze development trend,and put forward sugges-tions for improvement.Methods In May-June,2016,170 secondary and above hospitals in 10 cities were selected for surveying through stratified random sampling method.Survey content included basic situation of hospitals,HAI management,HAI monitoring,and so on.Results Available questionnaires were obtained from 165 hospitals (43 tertiary hospitals,and 122 secondary hospitals).Of 165 hospitals,more than 90% have established HAI manage-ment organizations and regulations,but hospital risk management should be paid more attention,only 63.03% of hospitals perfected the risk management system and 66.06% conducted risk assessment.99.09% of hospitals im-plement training on HAI to all staff regularly and 88.41% conducted effective feedback.In the aspect of staff alloca-tion,88.48% of the hospitals assigned enough professionals for HAI management,but only 34.55% have specific training programme for these personnel.Only 33.94% of hospitals have special funds for HAI control;in the aspect of monitoring on HAI,21.21% of hospital installed and used HAI monitoring software;In the aspect of implemen-tation of monitoring programme,about 90% of hospitals developed monitoring on HAI cases and environmental hy-giene,but only 34.55% and 23.64% of hospitals conducted targeted monitoring on intensive care unit and neonatal intensive care unit respectively.Conclusion Organizational structure of HAI management in Shaanxi Province is perfect,relevant rules and regulations are basically established,basic monitoring projects are universal,but the awareness of risk management needs to be strengthened,professional allocation and professional quality develop-ment are both imbalance,informational monitoring is inadequate.

Objective To understand the cognition about healthcare-associated infection(HAI) management among directors in secondary and above hospitals in Shaanxi Province.Methods Questionnaire survey was adopted to investigate hospital directors who participated in The third session of Shaanxi Provincial HAI management training course for hospital directors.Results A total of 181 questionnaires were distributed, 173 (95.58％) were qualified.74.57％ of surveyed hospitals were secondary hospitals, 61.85％ were comprehensive hospitals, 67.05％ of respondents received HAI training in recent 3 years, 81.50％ and 55.49％ of hospital directors thought the main factors influencing the HAI management were health care workers'' awareness on HAI and leaders'' attention respectively.58.96％, 60.12％, and 46.82％ of hospital directors thought the director of HAI management department should have intermediate and above professional title, bachelor degree or above education, and preventive medicine professional requirements respectively.The awareness rate of HAI management-related knowledge was 86.71％, difference in awareness rate of HAI management-related knowledge among respondents of different job, gender, and HAI training in recent 3 years were all significantly different(all P<0.05).Conclusion Hospital directors'' cognition on HAI management affect the development of HAI work, strengthen the training on HAI knowledge among administrators can improve hospital administrators'' awareness on HAI prevention and control.

Objective:To analyze the causes of death of elderly inpatients,so as to provide direction for the precaution of geriatrics.Methods:The first page data of hospitalized medical records of elderly inpatients in medical department in the Second Hospital of Dalian Medical University from 2102 to 2019 were extracted, and the causes of death were analyzed. The statistical analysis was performed by Excel and SPSS 21.0 statistical software.Results:The number of deaths of elderly inpatients in medical department from 2012 to 2019 was 5 249. The proportion of deaths in ICU was 34.43%(1 807/5 249), in oncology department was19.03%(999/5 249), and in cardiovascular department was12.08%(634/5 249). The average age was (78.52 ± 7.82) years. Besides, the age of men was younger than women: (78.18 ± 8.00) years vs. (79.02 ± 7.52) years, and the differences was statistically significant ( P<0.01). Men(59.1%, 3 099/5 249) were more than women (40.96%, 2 150/5 249). The largest number of deaths was in the age of 75-84 years (42.56%, 2 234/5 249). The number of cases with combined above five diseases was 4 552(86.72%, 4 552/5 249). The top three causes of deaths of elderly inpatients in medical department from 2012 to 2019 were cardiocerebrovascular diseases (27.21%, 1 428/5 249), malignant tumor (25.74%, 1351/5 249) and respiratory system diseases (22.10%, 1160/5 249). From 2012 to 2015, the top three causes of deaths were malignant tumor, cardiocerebrovascular diseases and respiratorysystem diseases. From 2016 to 2019,the top three causes of deaths were cardiocerebrovascular diseases, malignant tumor, and respiratory system diseases. The most common cause in cardiocerebrovascular diseases of death was coronary heart disease (51.47%,735/1 428), cerebrovascular disease (43.70%,624/1 428),and hypertension(4.34%, 62/1 428). Among the patients with malignant tumor death, first cause of death waslung malignant tumor (37.53%, 507/1 351), the others in turn were gastric carcinoma (11.10%,150/1 351) and intestinal cancer (11.10%,150/1 351). Among the patients with respiratory system diseases, first cause of death was pulmonary infection (69.66%,808/1 160), the others in turn were chronic obstructive pulmonary disease (15.43%, 179/1 160) and interstitial lung disease (5.09%, 59/1 160). Conclusions:The average age of elderly inpatients in medical department is (78.52 ± 7.82) years. The death age of male is less than that of female, and the number is slightly more than that of female. The top three causes of deaths of elderly inpatients in medical department are cardiocerebrovascular diseases, malignant tumor, respiratorysystem diseases. From 2012 to 2015 the first cause of deaths is malignant tumor. From 2016 to 2019, the cardiocerebrovascular diseases rise to the first.

Objective Stathmin, a microtubule-destabilizing protein , has high expression in esophageal squamous cell carci-noma(ESCC), while taxol is a common chemotherapy microtubule-targeted drug for esophageal cancer .This study aimed to investigate the impact of stathmin expression and its influence on taxol sensitivity in ESCC . Methods We established 2 cell models with ST-MN1 gene overexpression in KYSE 510 and KYSE 170 cell lines, including KYSE 510-Stathmin, KYSE 170-Stathmin, KYSE 510-Control and KYSE 170-Control.MTT assay and colony formation were applied to compare the taxol sensitivity between experimental group and control group .Flow cytometry was used to measure the apoptosis of KYSE 510-Stathmin and KYSE 510-Control after taxol treatment.Western blot was used to test the changes of related factors to apoptosis and autophagy . Results ①Stathmin protein ex-pressions in KYSE 510-Stathmin and KYSE 170-Stathmin cells were higher than those of control cells (P<0.01).② The percentages of inhibition were significantly decreased in KYSE 510-Stathmin and KYSE 170-Stathmin cells 24 h after 50, 100,250 nmol/L taxol treat-ment compared with KYSE 510-Stathmin cells(P <0.01).③The percentages of inhibition were significantly reduced in KYSE 510-Stathmin and KYSE 170-Stathmin cells after 250 nM taxol treatment for 24, 48, 60 h (P<0.01).④After taxol treatment,the number of colony formation in KYSE 510-Stathmin cells was higher com-pared with KYSE 510-Control cells (P<0.01).⑤The percentage of cell apoptosis in KYSE 510-Stathmin was significantly lower than that of KYSE 510-Control cells by flow cytometry (11.90%±0.78%vs 29.63%±3.26%, P<0.05).Western blot showed the ap-optosis of associated proteins such as the activation of Caspase 8 and Caspas9. Conclusion The result indicates that overexpression of stathmin inhibits taxol sensitivity in ESCC cell lines .