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Objective @#To construct myeloid specific Spi1 gene knockout mice and analyze their genotypes , so as to provide animal model basis for the study of pathological mechanism of diseases and drug targets .@*Methods @#According to the principle of CRISPR/Cas9 technology and C re/LoxP system , sgRNA and Donor vectors were de signed and constructed . The transcript of Exon 2 ( Exon 2) was used as the knockout region , and Loxp elements were placed on both sides of Exon 2 . Cas9 protein , sgRNA and Donor vector were mixed and microinj ected into the fertilized eggs of C57BL/6J mice , the fertilized eggs were transplanted into the uterus of C57BL/6J pregnant female mice , and F0 generation was obtained after 19 ~ 20 days . Positive F0 mice were mated with C57BL/6J mice to ob tain stable F1 Spi1 flox/ + mice . Spi1 flox/ + mice of F1 generation were selfed to obtain Spi1 flox/flox mice . Spi1 flox/flox mated with Lyz2-Cre + mice to obtain Spi1 flox/ + /Lyz2-Cre + mice , and then mated with Spi1 flox/flox , the Spi1 flox/flox/Lyz2-Cre + mice were myeloid specific Spi1 gene knockout ( KO) mice . Spi1 flox/flox/Lyz2-cre - mice were used as wild type (WT) mice . DNA of WT and KO mice was extracted , and the genotypes were identified by agarose gel electro phoresis after PCR amplification . Western blot was used to detect the expression of spleen focus forming virus proviral integration oncogene , Spi - 1 /purine rich box - 1(PU . 1) in immune cells of WT and KO mice .@*Results@#The results of PCR identification showed that the genotype of mice with only 220 bp amplified by flox primer was Spi1 flox/flox homozygote , and the genotype of mice with 700 bp amplified by Lyz2-Cre primer was Lyz2-Cre + . Western blot showed that compared with WT group , the protein PU . 1 was not expressed in bone marrow derived macropha ges (BMDMs ) and peritoneal macrophages (PM) in KO group (P < 0.01) . There was no significant difference of statistics in the expression level of PU . 1 in T cells between KO mice and WT mice . The results of PCR and West ern blot showed that myeloid specific Spi1 KO mice were successfully constructed . @*Conclusion @#The myeloid spe cific Spi1 gene KO mice are successfully constructed and identified , which provides animal model basis for further revealing the potential mechanism of PU . 1 inimmune regulation .
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Objective @#To breed and identify the T lymphocyte-conditional Spi1 knockout mice for the further in- vestgation of the specific role of Spi1-encoded protein PU. 1 . @*Methods @#The Lck-Cre mice were mated with Spi1 flox/flox mice to obtain Lck-Cre ×Spi1 flox/flox mice (T lymphocyte-specific Spi1 knockout mice) , and the genotype was determined by polymerase chain reaction (PCR) and agarose gel electrophoresis . Magnetic beads were used to sort out the splenic T lymphocytes , and the knockdown efficiency of PU. 1 in T cells was detected by Western blot , quantitative real-time PCR ( qPCR) and flow cytometry. @*Results @#The Lck-Cre ×Spi1 flox/flox mouse genotype was stably inherited . Compared with Spi1 flox/flox mice , the expression level of PU. 1 was significantly reduced in splenic T cells of Lck-Cre ×Spi1 flox/flox mice . @*Conclusion @#In this study , the T lymphocyte-specific Spi1 knockout mice was successfully constructed by applying Cre/LoxP system and CRISPR/Cas9 technology , which provided a reliable an- imal model for the subsequent experiments of the specific role of PU. 1 in T cell-related diseases .
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Objective@#To investigate awareness of digestive system injury caused by corona virus disease 2019 (COVID-19) in gastroenterologists.@*Methods@#From February 21 to 23 in 2020, the electronic questionnaire was sent out to explore the condition of the basic knowledge of COVID-19 and knowledge of digestive system injury caused by COVID-19 grasped by gastroenterologists. Chi-square test was used for statistical analysis.@*Results@#A total of 2 216 gastroenterologists from 31 provinces, autonomous regions and municipalities nationwide completed the survey. 99.7% (2 209/2 216) of gastroenterologists stated that they had read the COVID-19 diagnosis and treatment guidelines. The percentage of physicians who well knew the diagnostic criteria of suspected and confirmed cases of COVID-19 was 34.9% (774/2 216) and 39.4% (874/2 216), respectively. The percentage of physician who gave the right answer of COVID-19 detectable methods and lung imaging was 68.4% (1 516/2 216) and 71.6% (1 586/2 216), respectively. The percentage of correct answer of digestive system injury caused by COVID-19 in residents, attending physicians, associate chief physicians and chief physicians was 30.9% (134/433), 33.9% (234/691), 32.4% (213/657) and 34.9% (152/435), respectively, however there were no statistically significant differences among physicians of different level (χ2=6.60, P> 0.05). 95.6% (2 119/2 216) of gastroenterologists believed that probiotics could effectively improve bowel function, and 94.0% (2 082/2 216) of gastroenterologists considered that enteral nutrition support could improve patients’ prognosis.@*Conclusions@#The knowledge and dynamic progress of the digestive system injury caused of COVID-19 are still insufficiently grasped by gastroenterologists in China. So it is necessary to carry out systematic and pertinent training for them.
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Objective:To investigate the awareness of digestive system injury caused by coronavirus disease 2019 (COVID-19) in gastroenterologists.Methods:From February 21 to 23 in 2020, the electronic questionnaire was used to learn about the condition of the basic knowledge of COVID-19 and awareness of digestive system injury caused by COVID-19 among the gastroenterologists across the country. Chi-square test was used for statistical analysis.Results:A total of 2 216 gastroenterologists from 31 provinces, autonomous regions and municipalities nationwide completed the survey. 99.7% (2 209/2 216) of gastroenterologists stated that they had read the COVID-19 diagnosis and treatment guidelines. The percentages of physicians who knew clearly about the diagnostic criteria of suspected and confirmed cases of COVID-19 was 34.9% (774/2 216) and 39.4% (874/2 216), respectively. The percentages of physician who gave the right answers of COVID-19 detectable methods and pulmonary imaging was 68.4% (1 516/2 216) and 71.6% (1 586/2 216), respectively. The percentages of correct answers of digestive system injury caused by COVID-19 were physicians and chief physicians was 30.9% (134/433) in residents, 33.9% (234/691) attending physicians, 32.4% (213/657) associated chief physicians and 34.9% (152/435) chief physicians, respectively, however there were no statistically significant differences among physicians with different professional titles ( χ2=6.60, P> 0.05). 95.6% (2 119/2 216) of gastroenterologists believed that probiotics could effectively improve bowel function, and 94.0% (2 082/2 216) of gastroenterologists considered that enteral nutrition support could improve patients’ prognosis. Conclusions:The knowledge and dynamic progress of the digestive system injury caused by COVID-19 are still insufficiently grasped by gastroenterologists in China. So it is necessary to carry out systematic and pertinent training programmes for them.
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Objective To explore the clinical effect of free postnatal delivery in the second stage of labor, and to provide evidence for clinical midwifery. Methods From March 2016 to November 2017, 120 maternity patients from the First People′s Hospital of Hefei were selected and divided into research group and control group, using simple random grouping and the method of random number table, with 60 cases in each group. At the second stage of delivery, the research group used free body position at different stages, while the control group took the position of supine bladder lithotomy position. The delivery outcome, duration of the second stage, postpartum vaginal bleeding within two hours, newborn asphyxia rate, degree of perineal laceration, perineum incision rate, delivery comfort and satisfaction and midwives job satisfaction were compared. Results The vaginal delivery rate of the research group was 96.7％(58/60), which was higher than that of the control group 86.7％ (52/60). The difference was statistically significant (χ2=3.93, P<0.05). The duration of the second stage in the research group was (31.97 ± 9.85) minutes, and that of the control group was (39.50 ± 12.19) minutes. There was a statistically significant difference between the two groups (t=-3.357, P<0.05). There was no significant difference in neonatal asphyxia rate and the amount of postpartum vaginal bleeding within 2 hours between the two groups (P>0.05). The perineum integrity rate in the research group was 10.4％(6/58), higher than that in the control group, which was 0. Perineal side cut rate in the research group was 0, lower than that in the control group, which was 21.1％(11/52). The difference was statistically significant (χ2=11.31, 13.63, P<0.05). Deliverycomfort, delivery satisfaction and midwives job satisfaction in the research group were 91.7％ (55/60), 100.0％ (60/60) and 100.0％ (60/60) respectively, while delivery comfort, delivery satisfaction and midwives job satisfaction in the control group were 38.3％(23/60), 83.3％(50/60) and 76.7％(46/60). The differences were statistically significant (χ2=37.51, 10.91, 15.85, P<0.05). Conclusion The second labor stage free position is effective safe childbirth way of delivery, which can improve the natural births, shorten the labor, lighten the perineal injury, improve the puerpera childbirth comfort and satisfaction, it also raised the midwives job satisfaction, and had no adverse effect on maternal and infant outcomes, so the method has clinical application and dissemination value.
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Objective:To obtain the volume of thoracolumbar vertebral(T12-L5)in elderly people by CT post processing software, and establish the calculation formula of lumbar vertebral body volume from L1 to L4.Methods: Collect the elderly lumbar CT examination cases, 60 normal elderly people without lumbar lesions were included in this study. The volume of each vertebral body from T12 to L5 was obtained by processing software after a lumbar CT scan. The volume values of vertebral body from L1 to L4 were regarded as dependent variables and the volumes of upper and lower vertebral body was considered as independent variables. All the data were analyzed with multiple linear regression method.Results: There was a correlation between the volume of each lumbar vertebral from L1 to L4 and volumes of adjacent vertebral bodies, and the multivariate linear regression equation between them was obtained successfully.Conclusion: The volume of vertebral body can be accurately measured by CT processing software, and the calculation formula can be used to predict the normal vertebral volume value before fracture. The ratio of bone cement quantity to vertebral volume value can be earned and used to guide the operation of bone cement implantation for the further study.
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Objective:To establish the detection method of double-antibody sandwich ELISA about CYFRA 21-1 in human ser-um.Methods:The paired antibody were screened among four strains mAbs of CYFRA 21-1, which was marked by sodium periodate method.The detecting method of double antibody sandwich ELISA was optimizted , and evaluated by specificity , stability and sensitivi-ty.Results:The results showed that a paired of antibody , which was 2F9 as the coated antibody and 6F11 as the labeled antibody, was selected from four mAbs .It was the optimum condition of double antibody sandwich ELISA that the coating antigen concentration of 2F9 was 0.50 μg/ml, while the labeled antibody of 6F11 was diluted 6 000 times.The linear range of standard curve was 0.7-25 ng/ml with r2 =0.990 8, while the limit of detection was 0.666 8 ng/ml, the recovery rate was 98.14%.The cross-reactions with the oth-er analogues in serum were less than 0.1%.The coefficient of variation in group (n=10) was 6.8%, whereas coefficient of variation among group(n=5) was 11.4%.The correlation compared with other foreign ELISA kit was 91.42%.Conclusion:In brief, we suc-cessfully established the method of double antibody sandwich ELISA detecting CYFRA 21-1 level in human serum , laying the foundation for the production of CYFRA21-1 ELISA kit.
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Objective To construct and identify norepinephrine ( NE) complete antigen for the preparation of high sensitive and specific anti-NE monoclonal antibody .Methods Glutaraldehyde ( GA) and 1-Ethyl-3-(3-Dimethylaminopropyl ) carbodiimide ( EDC) were used to cross-link NE with carrier pro-teins (BSA, OVA) for NE complete antigen preparation under conditions of pH 4.5 or pH9.0.Three assays including UV scanning , SDS-PAGE and FeCl3 color reaction were performed for identification of NE com-plete antigen.Serum antibody titers were evaluated in mice model induced by intraperitoneal immunization with NE complete antigen .Results NE complete antigens were successfully prepared as indicated by the three identification assays .The coupling ratio was significantly increased in a time-depended manner under the condition of pH9.0 in comparison to that in the condition of pH 4.5.Indirect ELISA results showed that , when coating antigens and serum antibodies were prepared with the same cross -linking method , the serum antibody titers were significantly higher than those with different methods .Conclusion Anti-NE antibodies were successfully prepared by immunizing mice with NE complete antigens .
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Objective To investigate the application valve of bFGF to improve the viability of trans-plantation flap in Wistar rat. Methods Forty-eight Wistar rats were randomly divided into two groups based on the age: group A,B(1.5 month old, every group was 12 rats) and group C,D (3 month old, every group was 12 rats). After an ischemic model completed, recombinant bovine basic fibroblast growth factor (rb-bFGF) was given to groups A and C in ischemie zone by vascularization flap injection and the equality of normal saline to groups B and D. 14 days postoperatively, the muscular tissue was sent for histology, the blood vessel density was calculated by image analysis, and positive VEGF was detected by immunohistochem-istry. Results The member of capillaries and positive VEGF were more in group A than that in B, and also were morein group C than D(P < 0.05), but there were not statistic difference between group A and C (P > 0.05). Conclusion Recombinant bovine bFGF can stimulate angiogenesis and improve the ischemie vascu-larization flap of rat, which is not associated with their age.
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Objective To discuss the clinical therapeutic efficacy of the clinical study of thumb tip defect reconstruction with free distal second toe in emergency. Methods Fifteen cases of thumb tip defect were reconstructed with free distal second toe in emergency.The blood supply was restored by anastomosis of digital and toe'S artery and nerves,the dorsal surface and other side vein of fingers all can be anastomosed with toe's vein.but the first was main. Results All of fifteen fingers survived.Followed-up from 3 months to 3 years,all reconstruction thumbs has a satisfying outward appearance,normal nail and fingerprint,well pinch and hold functions, none effect to the foot. Conclusion Repair of thumb tip defects using free distal second toe in emergency enables favorable functional recovery and satisfying outward appearance,the method is simple and rats recover quickly.
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Objective To investigate the effects of TNF-?,IL-12,IL-15 and underglycosylation IgA_1 in mesangial deposition in the clinicopathological study of IgA nephropathy(IgAN).Methods Seventy-four patients with IgAN were enrolled in the study,with 10 patients randomly selected after remission,10 MCD and 10 healthy volunteers as control groups.The levels of TNF-? in serum and urine were examined by radioimmunoassay,and the levels of IL-12 and IL-15 were examined by ELISA.Immunohistochemistry was used to detect the expression of TNF-? in renal tissue,and the directed immunofluorescence was used to detect the underglycosylation of IgA_1(UGIgA_1)in mesangial deposition.Results The levels of TNF-?,IL-12 and IL-15 in serum of patients with IgAN were higher than those of MCD group,healthy control group and remission group.Compared with the negative group,33 patients with UGIgA_1 positive in renal tissues had lower levels of TNF-? in serum,longer course and less class of renal tissue injury.The positive areas of TNF-? in renal tissue and levels of TNF-? in serum were positively correlated with urinary protein(r=0.249,0.702,P=0.000).There was negative correlation between the levels of TNF-? in serum and Ccr(r=-0.231,P=0.048).The levels of IL-15 was positively correlated with the course of disease(r=0.266,P=0.022) and negatively with Scr(r=-0.320,P=0.005).Conclusions The TNF-?,IL-12 and IL-15 are all involved in the onset of proteinuria in IgAN patients,and IL-15 acts as a factor against renal injury.The patients with UGIgA_1 positive in mesangial have longer course and less pathological changes damaged than those of the UGIgA_1 negative patients.The TNF-? may serve as an important factor in the aggravation of IgAN.
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Electrical potential difference (PD) was measured across the wall of reverted small intestinal saes of mice. The effect of glycyrrhizic acid on transmural potential related to Na+ and glucose transport were investigated. It was found that both zhigancao (100g/L ) and glycyrrhizic acid (2. 5mmol/L ) decreased the PD (P＜0.01).According to the theory of active transport of glucose, we inferred that zhigancao and glycyrrhizic acid possibly produce an inhibitiory effect on Na+. K+-ATP ase,resulted in a blocking effect on sodium channel.