Objective To establish a rapid method of real-time fluorescence RT-PCR to quantify Soul virus.Methods The pro-fessional software was adopted to design the primer and the TaqMan-BHQ probe.With artificially synthesized L gene segment as the template of Soul virus,the real-time RT-PCR for detecting Soul virus was researched.Results The Ct value of templates had a good linear relationship with the log value of the template diluted concentration.The standard curve was Y =-3.607X +41.84, r2 =0.998,the PCR amplification efficiency was 108.1%,its lowest detection limit was 53.2 copies/μL.Conclusion Applying the real-time fluorescence RT-PCR by the TaqMan-BHQ probe for detecting nucleic acid of Seoul virus has the characteristics of short time-consuming and high sensitivity.

AIM: To construct a recombinant adenovirus vector carrying AFP promoter to specifically express a targeting gene in hepatocellular carcinoma HepG2 cells. METHODS: Based on the Adeno-X~TM Expression system, the CMV promoter was replaced by a 300 bp a-fetoprotein promoter. The EGFP(enhanced green fluorescent protein) gene as a report gene was inserted to the multiple-cloning site(MCS). The normal liver LO2 cells, hepatocellular carcinoma HepG2 cells and HeLa cells were infected by the recombinant adenovirus, respectively. Northern blotting and fluorescence microscope were used to detect the transcription level of EGFP gene and its protein expression, respectively. RESULTS: Northern blotting showed that the target gene was markedly transcribed in HepG2 cells, but slightly in LO2 and HeLa cells. Under the fluorescence microscope, strong EGFP expression was seen in HepG2 cells but very weakly in HeLa and LO2 cells. CONCLUSION: Under the control of the 300 bp human AFP promoter, the target gene carried by the recombinant adenovirus was expressed in the AFP-producing HepG2 cells at a very high level, but not or very weakly in AFP negative cells. This adenovirus system can be used as a new, potent and specific approach for the gene-targeting therapy for the AFP producing primary hepatoma. [

In order to prove that ectopic over-expression of Pim-2 could induce malignant transformation of human liver cell line L02, three groups of cells were set up including human liver cell line L02 (L02), L02 cells transfected with Pim-2 gene (L02/Pim-2) and L02 cells transfected with empty-vector (L02/Vector). Pim-2 expression levels were detected. The morphology, proliferation level, apoptosis rate and migration ability of the cells were detected respectively. Then the cells were subcutaneously inoculated into athymic mice and the microstructures of the neoplasm were observed. Compared with the controls, Pim-2 expression levels were significantly higher in L02/Pim-2 cells (P<0.05), and their morphology had obvious malignant changes. They also showed a significantly increased proliferation rate (P<0.05) and migration capacity (P<0.05), as well as a significantly decreased apoptosis rate (P<0.05). Only the athymic mice inoculated with L02/Pim-2 cells could generate neoplasm, and the morphology of the neoplasm coincided with that of the hepatoma. The results manifest that ectopic Pim-2 gene could be stably expressed in L02/Pim-2 cells. Both the morphological and biological changes of L02/Pim-2 cells demonstrate the trend of malignant transformation. L02/Pim-2 cells could generate hepatoma in athymic mice. In conclusion, Pim-2 could induce malignant transformation of human liver cell line L02.
Animals ; Apoptosis ; Cell Line ; Cell Movement ; Cell Proliferation ; *Cell Transformation, Neoplastic ; Humans ; Liver/pathology/physiology ; *Liver Neoplasms/genetics/pathology ; Mice ; Mice, Nude ; Neoplasm Transplantation ; *Oncogenes ; Protein-Serine-Threonine Kinases/genetics/*metabolism ; Proto-Oncogene Proteins/genetics/*metabolism

In order to enhance the anticoagulant properties of decellularized biological materials as scaffolds for tissue engineering research via heparinized process, the decellularized porcine liver scaffolds were respectively immobilized with heparin through layer-by-layer self-assembly technique (LBL), multi-point attachment (MPA) or end-point attachment (EPA). The effects of heparinization and anticoagulant ability were tested. The results showed that the three different scaffolds had different contents of heparin. All the three kinds of heparinized scaffolds gained better performance of anticoagulant than that of the control scaffold. The thrombin time (TT), prothrombin time (PT) and activated partial thromboplastin time (APTT) of EPA scaffold group were longest in all the groups, and all the three times exceeded the measurement limit of the instrument. In addition, EPA scaffolds group showed the shortest prepared time, the slowest speed for heparin release and the longest recalcification time among all the groups. The decellularized biological materials for tissue engineering acquire the best effect of anticoagulant ability in vitro via EPA heparinized technique.
Animals ; Anticoagulants ; chemistry ; Biocompatible Materials ; chemistry ; Heparin ; chemistry ; Liver ; Swine ; Tissue Engineering ; Tissue Scaffolds

Objective:To study the clinical effect of Mammotome minimally invasive breast circumcision in the treatment of breast cancer.Methods:A prospective study was carried out in 800 breast benign tumor patients admitted to the People's Hospital of Anji County from January 2017 to December 2018.The patients were divided into control group(400 cases) and observation group(400 cases) according to the random digital table method.The control group received open breast tumor resection, and the observation group received minimally invasive mastectomy.The postoperative complications, recovery time, aesthetic effect of breast appearance, serum inflammatory factors and cellular immune function were compared between the two groups.Results:The operation time and bleeding volume in the observation group were (17.59±8.23)min, (23.57±7.64)mL, respectively, which were lower than those in the control group [(29.83±11.91)min, (54.28±17.59)mL]( t=16.910, 30.027, all P<0.05). The incidence of postoperative complications in the observation group was 1.50%(6/400), which was lower than 5.75%(23/400) in the control group(χ 2=10.340, P<0.05). The time of incision healing and hospitalization in the observation group were (4.07±1.39)d, (5.58±2.31)d, respectively, which were shorter than those in the control group [(5.56±1.75)d, (7.92±2.78)d]( t=13.334, 12.948, all P<0.05). The excellent and good rate of breast appearance aesthetics in the observation group was 97.25%(389/400), which was higher than 89.25%(357/400) in the control group(χ 2=20.336, P<0.05). After operation, the serum levels of inflammatory factors in the observation group were lower than those in the control group( t=17.360, 14.717, 14.109, all P<0.05). CD 3+ and CD 4+ /CD 8+ in the two groups were lower than before operation(control group: t=20.788, 16.121; observation group: t=11.616, 8.348, all P<0.05), and CD 3+ and CD 4+ /CD 8+ in the observation group were higher than those in the control group( t=10.655, 7.328, all P<0.05). Conclusion:Mammotome minimally invasive mastectomy can effectively reduce the surgical trauma of breast cancer patients, reduce the postoperative complications, reduce the impact on their cellular immune function, and help to speed up the recovery and improve the appearance of breast.

Objective To investigate application effect of strengthened night nursing to the prevent of vascular crisis in patients after finger replantation. Methods A total of 155 patients with finger replantation in Hand surgery from February 2014 to February 2016 were selected as research object, 90 cases from March 2015 to February 2016 with strengthened night nursing were as the observation group,65 cases from February 2014 to February 2015 with routine nursing were as the control group. The two groups were compared of limb replantation success rate, vascular crisis rate and nursing satisfaction. Results The replantation success rate in the observation group was 90.00%(81/90)higher than 76.92%(50/65)in the control group,the occurrence of vascular crisis rate was 11.11%(10/90)lower than 27.69%(18/65)in the control group,the success rate was 7/10 higher than 10/18 in the control group, and the difference was statistically significant (χ2=4.932, 7.013, 4.216, all P<0.05). The nursing satisfaction in the observation group was (94.32 ± 3.57) points, 93.33%(84/90) higher than (91.22 ± 4.48) points, 83.08%(54/65) in the control group, and the difference was statistically significant (t=4.789, χ2=4.071, P<0.05). Conclusions Strengthened night nursing can effectively prevent vascular crisis in patients after finger replantation,improve nursing satisfaction of patients.

Picrotoxin is an anatagninst of gamma-aminobutyric acid, which is an internal inhibition-transmitter in the central nervous system, Picrotoxin exerts a biphasic action on the blood pressure and heart rate in rats and cats in vivo. That is to say, in the initial stage, picrotoxin can lower the blood pressure and heart rate, and then an elevation of these two even above the original level can be observed, up to the present, from the authors limited literature, there has been no report dealing with the problem whether picrotoxin can act on an isolated heart directly.In this study, the heart of a frog was isolated and routine intubation of the heart was done for its perfusion. Physiological polyconduction instrument was inserted through a mechanical transducer to record the heart rale and myocardial contractility. A suspending glass microelectrode coupling with a microamplifier is used to record the action potential of the ventricular myocardium. Real time analysis of all the data was accomplished with a microcomputer. The dosages of picrotoxin used were 1,5, 3.0, 6.0, and 12.0 mg per kilogram of body weight.It was found that picrotcxin can directly act on the isolated frog heart. The results were as follows.1 ) Picrotoxin exerts inhibition on the special conduction system of the heart,and the A-V node and venous sinus are very sensitive. Complete or partial transmission block can be induced.2 ) It can elicit clearly a fall of the heart rate but no biphasic action can berevealed. 3) It can reduce the myocardial contractility, suggesting that the calciuminflow during the functioning period of the action potential is effected. 4 ) It can reduce the amplitude of the action potential but no effect on themaximal depolarization speed is observed, suggesting that picrotoxin islikely to affect the level of resting potential but not the action potentialin the depolarized period.

Objective: To investigate the clinicopathological features of indolent T-cell lymphoproliferative disorder of the gastrointestinal tract. Methods: Five cases of indolent T-cell lymphoproliferative disorder of the gastrointestinal tract from the Affiliated Hospital of Qingdao University from 2016 to 2019 were retrospectively reviewed. The clinical and pathological parameters were analyzed by combining clinical data and reviewing the available literature of 35 cases (34 cases abroad and 1 case in China). Results: There were 4 males and 1 female with a median age of 47 years (18-66 years). All patients had abdominal pain and constitutional symptoms including diarrhea, emaciation, intermittent mucous stool or oral and epiglottic ulcers. Endoscopic manifestations included multiple punctate congestion, erosion and ulcer at the terminal ileum and colorectum. Two cases had congestion and erosion of antrum and angle of stomach, and the lesions did not fuse and form tumors. Histologically, the lamina propria was expanded by a dense, medium to small lymphocyte infiltration, which was monomorphic, with slightly irregular nuclei without prominent nucleolus or lymphoepithelial lesions. There were admixed small amount of plasma cells and eosinophils. In 4 cases, immunohistochemistry showed the lesional cells were positive for CD3, CD8, TIA1, and negative for CD4, CD56, granzyme B and Ki-67 index was ≤10%. In situ hybridization showed that EBER was negative and clonal TCR gene rearrangement was detected. One consultation case was CD3⁺, CD5^- and Ki-67 index of 10%, although other indicators were not done. All five patients were treated with symptomatic support. In follow-up observation for 2 to 25 months, all patients were alive with the disease. Conclusions Indolent T-cell lymphoproliferative disorder of the gastrointestinal tract is a newly classified monoclonal T-cell proliferative disease, with low incidence, clinical inertia and long-term survival. It has unique clinicopathological features but pathologically it is easily misdiagnosed as inflammatory bowel disease or T-cell lymphoma. Correct diagnosis is of great important clinical significance.

Objective:To explore the neural mechanism of language dysfunction in patients with subacute stroke using functional near-infrared spectroscopy (fNIRS).Methods:Sixteen patients with non-fluent aphasia after subacute stroke (aphasia group), 16 patients with non-aphasia after stroke (non-aphasia group), and 16 healthy middle-aged and elderly subjects (control group) were enrolled into our study. The 6-min resting-state data of fNIRS were collected. Four language-related regions, Broca area, Wernicke area, dorso lateral prefrontal cortex (DLPFC), and supplementary motor area (SMA), were selected as regions of interest (ROIs), and the whole brain functional connection strength and functional connection strength in ROIs and between each two ROIs were analyzed by NirSpark software.Results:Compared with the control group (0.53±0.15) and non-aphasia group (0.47±0.12), the aphasia group had significantly decreased whole brain functional connection strength (0.29±0.14, P<0.05). Compared with the control group and non-aphasia group, the aphasia group had significantly decreased functional connection strength in the left Wernicke area, right Wernicke area, left Broca area, left SMA area, right SMA area and left DLPFC area ( P<0.05, FDR). Compared with the control group and non-aphasia group, the aphasia group had significantly decreased functional connection strength in the right Wernicke-left Wernicke area, right Wernicke-right Broca area, right Wernicke-left Broca area, right Wernicke-right DLPFC area, right Wernicke-left DLPFC area, right Wernicke-right SMA area, right Wernicke-left SMA area, left Wernicke-right Broca area, left Wernicke-left Broca area, left Wernicke-right DLPFC area, left Wernicke-left DLPFC, left Wernicke-right SMA area, left Wernicke-left SMA area, right Broca-left Broca area, right Broca-left DLPFC area, right Broca-right SMA area, right Broca-left SMA area, left Broca-right DLPFC area, left Broca-left DLPFC area, left Broca-right SMA area, left Broca-left SMA area, right DLPFC-left DLPFC area, right DLPFC-right SMA area, right DLPFC-left SMA area, left DLPFC-right SMA area, left DLPFC-left SMA area, and right SMA-left SMA area ( P<0.05, FDR). Conclusion:Abnormal functional connectivity strength of the whole brain and language-related key brain areas might be the neural mechanism of language dysfunction in patients with non-fluent aphasia after subacute stroke.

Objective To study the epidemiological characters of an influenza A/H1N1 outbreak in a middle school in Huaian city,and to provide scientific measures for prevention and control of influenza A/ H1N1.Method The outbreak of influenza A/H1N1 was investigated through the field epidemiology.The samples were used for virus isolation.Nucleotide sequences of H1N1 HA1 region were determined by RT-nested-PCR and sequencing.H1N1 genotypes were analyzed using bioinformatic programs by MEGA 5.0 software,and phylogenetic tree was constructed.Result 51 cases were reported during 11 days,the attack rate was 2.49％ (51/2051).The sequence analysis showed that the sequence homologous rates of H1N1 virus HA gene nucleotide were 99.6％-99.9％ with H1N1 genotype in 2014,98.5％-98.8％ in 2013,and lower than 98.4％ in 2011.Conclusion The popularity of this outbreak was low.Timely isolation of H1 N1 patients and close contacts played an important role in control the epidemics.Implementation of health propaganda and immunization of influenza A/H1N1 should be strengthened.