ObjectiveTo explore the effect of serum containing Zhenwutang on myocardial mast cell apoptosis induced by angiotensin Ⅱ （AngⅡ） and the mechanism of the correlation between apoptosis and mitochondrial autophagy. MethodsIn this experiment， AngⅡ and serum containing Zhenwutang with different concentrations were used to interfere with H9C2 cardiomyocytes for 24 h， and the survival rate of H9C2 cardiomyocytes was detected by cell counting kit-8 （CCK-8） to screen the optimal concentration for the experiment. Enzyme-linked immunosorbent assay （ELISA） was used to detect the content of B-type natriuretic peptide （BNP） in cell culture supernatant， and immunofluorescence was used to detect the cell surface area to verify the construction of the myocardial mast cell model. Subsequently， the experiment was divided into a blank group （20% blank serum）， a model group （20% blank serum + 5×10^-5 mol·L^-1 AngⅡ）， low-， medium-， and high-dose （5%， 10% and 20%） serum containing Zhenwutang groups， an autophagy inhibitor group （1×10^-4 mol·L^-1 3-MA）， and autophagy inducer group （1×10^-7 mol·L^-1 rapamycin）. The apoptosis level of H9C2 cells and the changes of mitochondrial membrane potential were detected by flow cytometry. The lysosomal probe （Lyso Tracker） and mitochondrial probe （Mito Tracker） co-localization was employed to detect autophagy. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect Caspase-3， Caspase-9， B-cell lymphoma 2 （Bcl-2）， Bcl-2-related X protein （Bax）， and cytochrome C （Cyt C） in apoptosis-related pathways and the relative mRNA expression of ubiquitin ligase （Parkin）， phosphatase and tensin homolog （PTEN）-induced kinase 1 （PINK1）， and p62 protein in mitochondrial autophagy-related pathways. Western blot was used to detect cleaved Caspase-3， cleaved Caspase-9， Bax， Bcl-2， and Cyt C in apoptosis-related pathways， phosphorylated ubiquitin ligase （p-Parkin）， phosphorylated PTEN-induced kinase 1 （p-PINK1）， p62， and Bcl-2 homology domain protein Beclin1 in mitochondrial autophagy-related pathways， and the change of microtubule-associated protein 1 light chain 3 （LC3） Ⅱ/Ⅰ ratio. ResultsCCK-8 showed that when the concentration of AngⅡ was 5×10^-5 mol·L^-1， the cell activity was the lowest， and there was no cytotoxicity. At this concentration， the surface area of cardiomyocytes was significantly increased （P<0.01）， and the content of BNP in the supernatant of culture medium was significantly increased （P<0.05）. Therefore， AngⅡ with a concentration of 5×10^-5 mol·L^-1 was selected for the subsequent modeling of myocardial mast cells. Compared with the blank group， the model group and the autophagy inhibitor 3-MA group had a significantly increased apoptosis rate （P<0.01） and significantly decreased mitochondrial membrane potential （P<0.01）. The results of immunofluorescence co-localization showed that compared with the blank group， the model group had a significantly decreased number of red and green fluorescence spots. The results of Real-time PCR showed that compared with that in the blank group， the relative mRNA expression of Bax， Caspase-3， Caspase-9， Cyt C， and p62 in the model group was significantly up-regulated （P<0.01）， while the relative mRNA expression of Bcl-2， Parkin， and PINK1 was significantly down-regulated （P<0.01）. In addition， the relative protein expression of Bax， cleaved Caspase-3， cleaved Caspase-9， Cyt C， and p62 was significantly up-regulated （P<0.01）. The LC3Ⅱ/Ⅰ was significantly decreased， and the relative protein expression of Bcl-2， p-Parkin， p-PINK1， and Beclin1 was significantly down-regulated （P<0.01）. Compared with the model group， the serum containing Zhenwutang groups and the autophagy inducer group had significantly decreased apoptosis rate （P<0.01）， and the decrease ratio of mitochondrial membrane potential is significantly lowered （P<0.01） in a dose-dependent manner. Additionally， both red and green fluorescence spots became more in these groups. In the 3-MA group， the number of red and green fluorescence spots decreased significantly. The relative mRNA expression of Bax， Caspase-3， Caspase-9， Cyt C， and p62 was significantly down-regulated （P<0.05， P<0.01）， while that of Bcl-2， Parkin， and PINK1 was significantly up-regulated （P<0.01）. In the serum containing Zhenwutang groups， the relative protein expression levels of Bax， cleaved Caspase-3， cleaved Caspase-9， Cyt C， and p62 were significantly down-regulated （P<0.05，P<0.01）. The LC3Ⅱ/Ⅰ was significantly increased， and the relative protein expression levels of Bcl-2， p-Parkin， p-PINK1， and Beclin1 were significantly up-regulated （P<0.01）. ConclusionThe serum containing Zhenwutang can reduce the apoptosis of myocardial mast cells and increase mitochondrial autophagy. This is related to the inhibition of intracellular Bax/Bcl-2/Caspase-3 apoptosis pathway and regulation of Parkin/PINK1 mitochondrial autophagy pathway.

;Objective To establish an integrated feeder-free platform for in vitro expansion and gene editing to tack-le the major challenges in clinical applications of cryopreserved primary human natural killer(NK)cells in terms of low expansion efficiency,technical difficulty in genetic modification and safety concerns.Methods A non-viral CRISPR-Cas9 ribonucleoprotein(RNP)-based multiplex gene editing system was developed through systematic op-timization of culture medium and nucleofection conditions.Cell phenotype(CD56+CD3-),viability,editing effi-ciency,and tumor-killing activity were evaluated via flow cytometry and cytotoxicity assays.Results The number of NK cells achieved 5 000-fold expansion over 25 days while maintaining high purity(CD56+CD3-＞95％)and viability(＞90％).Post-thawing viability(＞80％)and tumor-killing capacity were preserved.Cas9 RNP delivery enabled efficient dual knockout of NKG2A and CISH immune checkpoint genes(＞80％),significantly enhanced cytotoxicity against K562 tumor cells(P＜0.05).Conclusions Compared to viral vectors,the non-viral strategy eliminates genomic integration risks and reduces off-target effects.This result may provide a safe and efficient tech-nical platform for clinical application of NK cell immunotherapy and potentially encourage application of multiplex gene editing in cancer therapy.

Objective:To explore the application of a scenario-based onsite first-aid skills station in objective structured clinical examination (OSCE).Methods:Based on common scenarios and cases in medical practice, an evaluation framework of the OSCE onsite first-aid skills station—containing assessment indicators, exam room setting, examiner training, and assessment process—was designed to evaluate the onsite first-aid competencies of medical graduates of the five-year program for three consecutive years. SPSS 24.0 was used to perform the Kruskal-Wallis test and Pearson correlation analysis to calculate the correlation between course examination scores and OSCE onsite first-aid skills station assessment scores. Excel was used to calculate the difficulty index and discrimination index of test items.Results:The graduates' OSCE onsite first-aid skills station assessment scores were improved year by year, with a mean score of about 80 points. The station assessment items showed a moderate difficulty level (0.7-0.8), a good discrimination level (>0.4), and good internal consistency (Cronbach's α>0.7). The examiners and examinees had a high recognition of the design and effectiveness of this station assessment method. There was a positive correlation between the OSCE scores and corresponding course scores (2016, r=0.245, P=0.001; 2017, r=0.108, P=0.026; 2018, r=0.198, P=0.006). Conclusions:Through scientific scoring and strict examination management, the OSCE scenario-based onsite first-aid skills station can effectively evaluate examinees' injury treatment competencies in different situations, which can provide a reference for course teaching.

Objective To explore the management strategy and indications for revisionary internal fixation after percutaneous kyphoplasty/percutaneous vertebroplasty (PKP/PVP) in cancellous vertebral fractures.Methods A retrospective analysis was made of the 676 cases of single-segment PKP/PVP at Department of Orthopaedics,The Affiliated Hospital to Qingdao University from January 2008 to January 2019.They were subjected to 4 different managements after their primary PKP/PVP:rehabilitation without any treatment in 637 cases,conservative treatment in 19 cases (including 3 ones who refused any revision),KP/VP revision in 12 cases and internal fixation revision in 8 cases.The rate of volume reduction after bone cement dispersion (Vx) was calculated using software Mimics 17.0 on the basis of primary CT data of all the patients.The correlation regression analysis was made between the revision rate and the approximate quantization value of Vx.The Glasgow Coma Score (GCS) of conscious state was used to evaluate the 39 patients after failure of their primary surgery before the surgical strategy for revision was worked out.The cobb angle,pelvic incidence angle (PI),pelvic inclination angle (PT),sacral inclination angle (SS),sagittal deviation (SVA),pain visual analogue scale (VAS) were measured and recorded before operation and at the last follow-up for the KP/VP revision group and internal fixation revision group,indicated as △cobb,△PI,△PT,△SS,△SVA and △VAS,respectively.The indexes were compared between the 2 groups.Results The incidence of osteoporotic vertebral fractures treated with internal fixation revision was 1.18％ (8/676).The correlation between Vx and revision rate was y =0.53 + 0.04x (P ＜ 0.05).The regression analysis showed that Vx was positively correlated with the revision rate (r2 =0.860,P =0.001) and the fitting curve was correlated (r2 =0.916,P =0.001).The GSC scores revealed 31 normal,6 mild disturbance and 2 moderate disturbance cases.There were no significant differences in gender,age or VAS scores between the KP/VP revision group and the internal fixation revision group (P ＞ 0.05).There was a significant difference in △cobb between the 2 revision groups (6.3° ± 7.5° versus 19.2° ± 14.8°) (P ＜0.05),but there were no significant differences between the 2 groups in △PI (4.1°±5.2° versus 3.3°±6.7°),△PT (0.7°±4.6° versus 0.4° ± 3.2°),△SS (3.7° ± 6.2° versus 3.1° ± 5.3°) or △SVA (-3.2 ± 11.9 mm versus-7.9 ± 9.5 mm) (P ＞ 0.05).Conclusions The outcomes of primary PKP/PVP have a great impact on the decision-making of internal fixation revision.The mode and extent of diffusion after initial vertebral cement perfusion are particularly related to the revision rate.The revision plan should depend on clinical symptoms.The internal fixation revision should be individualized to ensure the quality of life of the patients in line with the principles of "resolving symptoms" and "moderate correction".

Objective? To explore the effects of strengthening out-of-hospital health beliefs on perceived well-being and behavioral change in elderly patients with colon cancer. Methods? Totally 112 elderly patients with colon cancer admitted in Nanyang City Center Hospital from August 2015 to August 2017 were selected by convenient sampling and divided into two groups (n=56) based on the time of admission. Patients in the control group received conventional nursing care, while patients in the observation group received out-of-hospital health beliefs. Both interventions lasted for 6 months. The knowledge needs scale for cancer patients and the Health Promoting Lifestyle Profile-Ⅱ (HPLPⅡ) were used to evaluate the perceived well-being and healthy behaviors in the two groups before and after intervention. Results? The scores of psychological needs, technical needs, medication needs and rehabilitation needs and the total score of the knowledge needs scale for cancer patients in the observation group were higher than those before intervention and those of the control group after intervention. The scores of self-fulfillments, exercise, health responsibility, nutrition, pressure handling and interpersonal support and the total score of HPLP Ⅱ of the observation group were higher than those before intervention and those of the control group after intervention (P＜ 0.05). Conclusions? Strengthening out-of-hospital health beliefs can effectively improve the perceived well-being of elderly patients with colon cancer and enable them to develop healthy behaviors.

Objective To observe the clinical efficacy of extracorporeal shock wave (ESW) plus electroacupuncture in treating periarthritis of shoulder.Method Ninety patients with periarthritis of shoulder were randomized into an electroacupuncture (EA) group, an ESW group, and an ESW plus EA group, 30 cases in each group. The EA group was intervened by EA, the ESW group was treated with ESW, and the ESW plus EA group by ESW and EA. The three groups were treated once every 2 d, with successive 10 sessions as a treatment course. The Visual Analogue Scale (VAS) score and shoulder range of motion (ROM) score were evaluated before and after the treatment in the three groups. Result The VAS scores dropped significantly in the three groups after the intervention (P<0.05); there was no significant difference in comparing the VAS score between the EA group and ESW group after the intervention (P>0.05); the VAS score in the ESW plus EA group was significantly different from that in the EA group and ESW group after the treatment (P<0.05). The ROM scores were significantly improved in the three groups after the intervention (P<0.05); there was no significant difference in comparing the ROM score between the EA group and the ESW group after the intervention (P>0.05); the ROM score in the ESW plus EA group was significant different from that in the other two groups (P<0.05).Conclusion ESW plus EA can more significantly ease the pain and improve the shoulder ROM in treating periarthritis of shoulder compared with the two methods used separately.

Objective To study the impacts of lipopolysaccharide on expressions of Cx43 and TLR4 proteins in bladder cancer cell lines and on cell proliferation and apoptosis. Methods 5637 cells were cultured in vitro. After stimulation with LPS,expressions of Cx43 and TLR4 proteins were detected by Western blot in bladder cancer 5637 cell. The proliferation and apoptosis in the 5637?control group,5637?LPS group,and 5637?LPS +cisplatin group were detected by CCK?8(cell counting kit)and flow cytometry. Results The gene expression of Cx43 in the 5637?LPS group was significantly higher than that in the 5637?control group(t=3.892,P=0.012). The expressions of TLR4 and Cx43 in 5637?LPS group were significantly higher than those in the 5637?control group(t=7.029,P=0.019;and t=18.17,P=0.003). The proliferation was significantly decreased in the 5637?control group as compared with the 5637?LPS group (t = 8.756,P = 0.018). The apoptotic rate was (8.3 ± 1.58)% in the 5637?control group and (7.8 ± 2.03)% in the 5637?LPS group,with no significant statistical difference(t = 2.935,P = 0.099). However,the rate of the 5637?cisplatin group(60 ± 4.35)%was higher than that in 5637?cisplatin + LPS group(52 ± 6.25)%,the difference was statistically significant(t = 6.992,P =0.019). Conclusions Under the stimulation of LPS,bladder tumor cells may induce tumor cells to escape immune surveillance by increasing the expressions of TLR4 and Cx43.

Anthrax is a highly lethal infectious disease caused by the spore-forming bacterium Bacillus anthracis. The major virulence factor of B. anthracis consists of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA binds with LF to form lethal toxin (LT), and PA binds with EF to form edema toxin (ET). Antibiotics is hard to work in advanced anthrax infections, because injuries and deaths of the infected are mainly caused by lethal toxin (LT). Thus, the therapeutic neutralizing antibody is the most effective treatment of anthrax. Currently most of the anthrax toxin antibodies are monoclonal antibodies (MAbs) for PA and US FDA has approved ABTHRAX humanized PA monoclonal antibody for the treatment of inhalational anthrax. Once B. anthracis was artificially reconstructed or PA had mutations within recognized neutralization epitopes, anti-PA MAbs would no longer be effective. Therefore, anti-LF MAbs is an important supplement for anthrax treatment. Most of the anti-LF antibodies are murine or chimeric antibodies. By contrast, fully human MAbs can avoid the high immunogenicity of murine antibodies. First, we used LF to immunize the transgenic mice and used fluorescent cell sorting to get antigen-specific memory B cells from transgenic mice spleen lymphocytes. By single cell PCR method, we quickly found two strains of anti-LF MAbs with binding activity, 1D7 and 2B9. Transiently transfected Expi 293F cells to obtain MAbs protein after purification. Both 1D7 and 2B9 efficiently neutralized LT in vitro, and had good synergistic effect when mixed with anti-PA MAbs. In summary, combining the advantages of transgenic mice, fluorescent cell sorting and single-cell PCR methods, this study shows new ideas and methods for the rapid screening of fully human monoclonal antibodies.

The present paper was aimed to study the security of burning during the continuous monitoring of transesophageal arterial oxygen saturation. 20 rabbits were randomly divided into two groups: control group and high-frequency electrotome group. All rabbits were implanted with the oxygen saturation detection probes into the lower esophageal after anesthesia. The probes were made up with two light-emitting diode (LED) and then connected with oxygen saturation sensor to monitoring for 2h and 6h, respectively. We then intercepted two luminous points as esophageal specimens and dist the esophageal tissue detection device headend 1cm gastric side esophageal specimens (self-contr01) after monitoring, and carried out pathological examination. The pathological results of the LED-1, LED-2 esophageal biopsy and self-control section were mucous integrity, submucosal mildedema, hyperemia, a small number of lymphocytes and mononuclear cell infiltration. All the score of the esophageal tissue histologic was 2. The results showed that montioring the SteO2 not more than 6h was safe with montiored using a matching of oxygen saturation sensor and monitoring esophageal arterial oxygen saturation.
Animals ; Esophagus ; pathology ; Female ; Male ; Mucous Membrane ; pathology ; Oximetry ; adverse effects ; methods ; Rabbits ; Safety

Objective:To discuss the clinical significance of overlapping infection of HGV in blood donorsand viral hepatitis. Methods :HGV-RNA was detected by reverse transcription-polymerase chain reaction.Results :The infectious rate of HGV in blood donors was 4% ,that in the patients with HBV and HCV was13. 9% and 15.8% respectively. Conclusion:Our results indicate that the HGV infection was widespread.Further study of immune response and status of viral replication in the liver tissue in overlapping infectionwith HBV and HCV,was needed.