The development characteristics of tertiary public hospitals in China presented differently under the macro environment in different periods. Based on the theory of supply and demand chain of medical services, the authors discussed the key factors affecting the high-quality development of tertiary public hospitals under the new situation from the four elements of production, distribution, circulation and consumption, and put forward countermeasures and suggestions as follows: optimizing the supply of medical talents and system construction in the production process; speeding up the layout of high-quality resources and the construction of an integrated medical service system in the distribution process; strengthening imformation construction and the scientific and technological innovation in the circulation process; expanding healthcare/prevention integration and refining supporting policies in the consumption process, so as to provide reference for accelerating the high-quality development of public hospitals in China.

In the post-genomic era, gene function analysis has attracted much attention. Transformation is often needed to investigate gene function. In this study, an easy, rapid, reliable, and cost-effective colony polymerase chain reaction (PCR) method for screening mushroom transformants was developed: picking up a suitable amount of transformant's tissue (1–10 μg) to 20 μl 0.25% Lywallzyme solution, and vortexing for 10 s followed by incubation at 34 °C for 15 min. Finally, 2 μl of the suspension was used as templates to perform PCR and single target bands were successfully amplified from respective transformants of Tremella fuciformis, Pleurotus ostreatus, and Pleurotus tuber-regium. This procedure could be widely employed for screening transformants in mushroom transformation experiments.

Agrobacterium tumefaciens-mediated transformation (ATMT), as a simple and versatile method, achieves successful transformation in the yeast-like cells (YLCs) of Tremella fuciformis with lower efficiency. Establishment of a more efficient transformation system of YLCs is important for functional genomics research and biotechnological application. In this study, an enzymolysis-assisted ATMT method was developed. The degradation degree of YLCs depends on the concentration and digestion time of Lywallzyme. Lower concentration (≤0.1%) of Lywallzyme was capable of formation of limited wounds on the surface of YLCs and has less influence on their growth. In addition, there is no significant difference of YLCs growth among groups treated with 0.1% Lywallzyme for different time. The binary vector pGEH under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter was utilized to transform the enzymolytic wounded YLCs with different concentrations and digestion time. The results of PCR, Southern blot, quantitative real-time PCR (qRT-PCR) and fluorescence microscopy revealed that the T-DNA was integrated into the YLCs genome, suggesting an efficient enzymolysis-assisted ATMT method of YLCs was established. The highest transformation frequency reached 1200 transformants per 106 YLCs by 0.05% (w/v) Lywallzyme digestion for 15 min, and the transformants were genetically stable. Compared with the mechanical wounding methods, enzymolytic wounding is thought to be a tender, safer and more effective method.
Agrobacterium ; Blotting, Southern ; Digestion ; Genome ; Genomics ; Methods ; Microscopy, Fluorescence ; Oxidoreductases ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Wounds and Injuries

Objective: To explore the correlation between the severity of gastroesophageal reflux cough and degree of gastroesophageal reflux. Methods: A cross-sectional investigation was carried out. Data of 174 cases of chronic cough were collected in Children's Hospital of Fuzhou from March 2009 to December 2016. The esophageal 24 hours pH value dynamic monitoring was used to detect gastric acid reflux index. Cases with abnomal results were divided into mild, moderate and severe groups according to severity of reflux and that of day and night cough symptoms, respectively. They were also divided into infant (1-3 years old), preschool (4-6 years old), and school age (>7 years old) groups according to age. Comparative analysis between groups by chi-square test and rank sum test were performed. Correlation analysis was used to analyze the correlation between cough severity and gastroesophageal reflux index. Results: A total of 174 patients with chronic cough, including 115 males and 59 females, aged from 1 to 15 years with an average age of (8.5±2.3) years, and (1.6±0.8) years of disease duration were enrolled. Among them, 129 cases (74.1%) were positive for esophageal reflux test and 45 cases (25.9%) with no obvious pathological gastroesophageal reflux. Patients with positive esophageal reflux test were divided into severe (n=37, 28.7%), moderate (n=23, 17.8%), and mild (n=69, 53.5%). There was no significant difference in the distribution of gastroesophageal reflux in each age group. (The proportions of mild, moderate and severe reflux in infants were 45.0% (9/20), 25.0% (5/20), and 30.0% (6/20), respectively. The proportions of mild, moderate and severe reflux in preschool children were 53.3% (32/60), 16.7% (10/60), 30.0% (18/60), respectively. The proportions of mild, moderate and severe reflux in school age children were 57.1% (28/49), 16.3% (8/49), 26.5% (13/49), respectively χ²=1.204, P=0.877). There was no correlation between age group and gastroesophageal reflux (r=-0.065, P=0.489).The severity of nighttime cough was positively correlated with percentages of distal esophagus pH≤4 in time, recumbent pH≤4 in time, and DeMeester score<14.72 (r=0.689, 0.621, and 0.707 respectively, all P<0.05). There was no statistically significant correlation between the severity of nighttime cough symptoms and percentage of standing pH≤4 in time (r=0.113, P>0.05). There were no statistically significant correlation between the severity of daytime cough and all gastroesophageal reflux markers (all P>0.05). Conclusion The severity of nocturnal symptoms of gastroesophageal reflux cough is related to the degree of gastroesophageal reflux, to which clinical pediatricians should pay attention.

This paper described the practice of medical cost control in Hubei's provincial state-owned hospitals. Their measures taken include combinations of overall planning and classification, total volume control and structural adjustment, external governance and internal management, as well as systematical management and difficulties breakthroughs. This paper proposed to further consolidate the outcomes of the abovementioned efforts by improving the fiscal compensation mechanism, furthering medical insurance payment reform, and continuing to monitor and publicize the medical expenses.

Objective To study the biological behavior of human liver cancer stem cells. Methods Human liver cancer MHCC97H cells in logarithmic growth phase were inoculated in the serum free medium for suspension cultivation and human liver cancer MHCC97H stem cells with stable propagation were acquired. Human liver cancer MHCC97H stem cell microspheres and human liver cancer MHCC97H cells were collected. Messenger ribonucleic acid (mRNA) of cell markers including cluster of differentiation (CD) 133, CD90, epithelial cell adhesion molecule (EpCAM) was detected by reverse transcription polymerase chain reaction (RT-PCR). Colony formation assay, tumor cell invasion assay (Transwell assay) and nude mouse tumorigenicity assay were performed respectively to observe the colony formation, cell invasion and tumorigenicity of two kinds of cells. The experimental data of two kinds of cells were compared by t test or t′test. Results The mean contents of CD133, CD90, EpCAM mRNA in human liver cancer MHCC97H stem cells (32.70±0.20, 66.30±0.32, 115.40±4.00) were significantly higher than those in human liver cancer MHCC97H cells (1.27±0.29, 13.60±1.36, 1.34±0.40) (t′=117.8, 53.97, 83.37;P<0.05). The colony forming efifciency was (213±10)%in human liver cancer MHCC97H stem cells and was (54±11)%in human liver cancer MHCC97H cells, where signiifcant difference was observed (t=13.11, P<0.05). Through the Transwell assay, the membrane permeating cell count was (587±120)/visual ifelds in human liver cancer MHCC97H stem cells, and was (97±13)/visual ifelds in human liver cancer MHCC97H cells, where signiifcant difference was observed (t=6.38, P<0.05). In the nude mouse tumorigenicity assay, subcutaneous transplanted tumors were observed in nude mouse 4 weeks after inoculating 2×103 human liver cancer MHCC97H stem cells and the tumor formation rate was 1/6. Subcutaneous transplanted tumors were observed in all nude mice by inoculating 2×105 human liver cancer MHCC97H stem cells, and the tumor formation rate was 6/6. Subcutaneous transplanted tumors were observed in nude mice by inoculating at least 2×105 human liver cancer MHCC97H cells, and the tumor formation rate was 2/6. Conclusion Compared with the human liver cancer MHCC97H cells, human liver cancer MHCC97H stem cells possess stronger invasion and higher tumor formation rate.

[Objective]This study was designed to investigate the genetic evolution of the neuraminidase(NA)gene of seasonal A/H1N1 and 2009 novel A/H1N1 inflilenza virus,and discuss the genetic variation of influenza A virus.[Methods]The virus strains were separately isolated from the clinical samples collected in 2006 and 2009,and then identified as seasonal A/H1N1 and novel A/H1N1.The full length of the NA gene of these strains was amplified by RT-PCR.Then the genetic evolution and mutations of important functional sites were analyzed.[Results]The homology of NA gene between the 2009 novel A/H1N1 isolates and 2006 seasonal A/H1N1 isolates was low(77.9%～78.8%),so was the homology of NA gene between the 2009 novel A/H1N1 isolates and representative strains of different periods and 1979-2001 WHO recommended vaccine strains(78.1%～79.3%).But compared with the WHO recommended vaccine strains of 2009 novel A/H1N1,the homology reached more than 99%.The genetic evolution analysis revealed that NA gene of 2009 novel A/H1N1 had the closest genetic relationship with the swine influenza A virus(A/swine/Belgium/1/1983)from Eurasian Iineage,and some of the antigenic sites and neuraminidase active sites of NA gene of seasonal A/H1N1 were mutated after 2005.[Conclusion]The NA gene of 2009 novel A/H1N1 may originate from Eurasian Iineage of swine influenza virus.The variation of NA gene of seasonal A/H1N1 has occurred in a certain degree.Hence,it is very necessary to continuously monitor the variant of influenza A virus.

Objective To detect dengue virus infection by serological method and to determine the sequences of E gene of dengue virus isolated from Guangzhou in 2006.so as to clarify the possible origin of dengue fever.Methods IgM and IgG antibodies to dengue virus were detected by immunochromatographic test(ICT);NSI antigen and IgM antibody were detected by enzyme-linked immunosorbent assay(ELISA).The virus was cultured and isolated from the serum samples within 2 days using C6/36 cell lines and was identified by immuno-fluorescence assay(IFA)and RT-PCR.The E gene of isolated virus DV1-GZ42/06 was sequenced;homological analysis and phylogenetic tree analysis were performed by comparing with the reference strains and epidemic virus strains.Results The positive rates of IgM and IgG of dengue virus in patients were 89.5%(433/484)and 38.0%(184/484)by ICT,respectively.The positive rates of NS1 antigen were 92.7%(38/41)in day 1 to day 2,83.3%(70/84)in day 3 to day 5,and 10.9%(5/46)in day 6 to day 10;and the IgM detection rates were 2.4%(1/41),51.2%(43/84)and 97.8%(45/46)at the same period by ELISA.Twenty-five strains of dengue virus were isolated from 41 serum samples(6 1.O%)and were identified as type 1 dengue virus by IFA and RT-PCR.The sequencing and phylogenetic analysis of the E gene showed that the homology between the isolated Guangzhou/42/06 strain and standard strain Hawaii/45 was 94.6%.and it had a high homology with the Thailand/NI09V104,Vietnam/06.and Vietnam/07 isolates(99.0%,98.6%and 98.6%,respectively)and belonged to the same cladogram,but had low homology with the isolated strain from Guangdong before 2006.Conclusions The detection of NS1 antigen is important in the early diagnosis of dengue fever.The outbreak of dengue fever in Guangzhou in 2006 was possibly caused by the cases from neighboring countries.

This article summarizes the virtual reality(VR) technology's bases and characters and discusses the application scope of VR in modern medicine thoroughly.For example,the application of VR in digitized virtual human,the simulation of surgical operation,long-distance medical diagnosis,virtual hospital,higher medical education technology and so on.It also covers the existing problems and prospect of VR.

Objective: To observe the expression of hMLH1 protein in esophagus squamous cell carcinoma, atypical hyperplasia tissue and normal esophagus tissue, so as to discuss the relationship between hMLH1 expression and esophagus carcinogenesis. Methods: The specimens of esophagus squamous cell carcinoma, atypical hyperplasia and normal esophagus tissue were obtained from 92 esophagus carcinoma patients. Immunohistochemistry (IHC) staining technique was used to detect expression of hMLH1 protein. The relationship between hMLH1 expression with clinical parameters, such as gender, age, cancer differentiation level, infiltration depth, tumor stage, lymphatic metastasis, was analyzed. Results: The positive rates of hMLH1 protein in esophagus squamous cell carcinoma, and atypical hyperplasia tissue and normal esophagus tissue were 36.96%,56.52%, and 84.78%,respectively,with the former 2 significantly lower than the latter (P