Objective The aim is to utilize CRISPR/Cas9 gene editing technology to construct Dmd gene mutant mice with a point mutation in exon 23 of the Dmd gene. Subsequently, the phenotypic changes of the mice in muscles and immune systems are analyzed and verified, providing an evaluation model for Duchenne muscular dystrophy and other related diseases.MethodsBased on the sequence characteristics of exon 23 of the Dmd gene, small guide RNA (sgRNA) was designed and synthesized. Cas9 mRNA, sgRNA fragments, and oligo donor DNA were microinjected into fertilized eggs of C57BL/6J mice. After transferring the fertilized eggs to surrogate mice, F0 generation mice were born. After mating with F0 generation mice, offspring mice were obtained, and Dmd gene positive mutant (Dmd^Mu/+) mice were obtained after genotype identification. Male hemizygous Dmd^Mu/+(Dmd^Mu/Y) mice were selected for phenotype validation. The body weight of live 3- and 9-month-old mice were recorded. Muscle tension was evaluated through the grid test. Hearts and semitendinosus muscles were collected, and the histopathological changes were observed using HE staining. Further, the expression of Dmd protein in muscle tissue of 9-month-old mice was analyzed by Western blotting.An acute inflammation model was established in Dmd^Mu/Y mice using lipopolysaccharide induction. Peripheral blood from the submandibular vein was collected, and the changes in the proportion of neutrophils and monocytes were detected by flow cytometry.Results The results of genome sequencing and Western blotting confirmed the successful construction of Dmd gene point mutant mice (Dmd^Mu/+ mice). Dmd protein expression was not detected in skeletal muscle and myocardium of Dmd^Mu/+ mice, and it was significantly reduced compared to wild-type C57BL/6J mice (P<0.05). Compared with wild-type mice of the same background, Dmd^Mu/Y mice at 3 and 9 months of age showed significant weight loss (P<0.01) and decreased muscle tension (P<0.05). 9-month-old Dmd^Mu/Y mice exhibited significant pathological changes in skeletal muscle and myocardium, including widening of intermuscular space. Under normal condition, compared with wild-type mice, the proportion of neutrophils and monocytes in the peripheral blood of 3-month-old Dmd^Mu/Y mice was significantly lower than that of wild-type mice (P<0.01). After lipopolysaccharide stimulation, the proportion of neutrophils in peripheral blood of 3-month-old Dmd^Mu/Y mice remained significantly lower compared to that of wild-type mice (P<0.01). The proportion of neutrophils in peripheral blood of 9-month-old Dmd^Mu/Y mice significantly decreased after lipopolysaccharide induction (P<0.01), with a trend of change observed in monocytes between groups.Conclusion The successful construction of the Dmd gene mutant mouse model has confirmed the vital function of Dmd gene in maintaining normal muscle tissue morphology and muscle tone. It preliminarily indicated that Dmd gene deletion could significantly reduce the proportion of neutrophils in peripheral blood, offering a new perspective for the study of immune system alterations in Duchenne muscular dystrophy patients.

Objective:To investigate the relationship between Helicobacter pylori(Hp)infections in Parkinson's disease(PD)patients at high altitude and peripheral inflammatory markers.Methods:In this prospective study, 120 PD patients in Qinghai Province(altitude: 2260 m)were enrolled and evaluated using PD motor symptom scales and a non-motor symptom scale.The 13C-Urea breath test was used to detect Hp, and patients were divided into an Hp infection group and a non-Hp infection group based on test results.The levels of high-sensitivity C-reactive protein(hs-CRP), white blood cell counts and ratios, serum interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α)were measured. Results:The incidence of Hp infections in PD patients was 56.67%(68/120).The scores of Unified Parkinson's Disease Rating Scale(UPDRS)-Ⅲ, UPDRS-Ⅳ, total UPDRS, Hoehn-Yahr(H-Y)score, constipation scoring system(CSS)and Leeds dyspepsia questionnaire(LDQ)in the Hp infection group were higher than those in the non-Hp infection group, while the mini-mental state examination(MMSE)score was lower in the non-Hp infection group(all P<0.05).The neutrophil count, neutrophil-to-lymphocyte ratio(NLR), monocyte-to-lymphocyte ratio(MLR), serum IL-6 and TNF-α in the Hp-infection group were elevated compared with the non-Hp infection group(all P<0.05).Multivariate Logistic regression analysis showed that IL-6, TNF-α, NLR and H-Y score were independent risk factors for Hp infections in PD patients( OR=1.103, 1.188, 3.320, 4.593, respectively, all P<0.05).Correlation analysis showed that IL-6, TNF-α and NLR had positive correlations with UPDRS-Ⅲ( r=0.676, 0.644, 0.488, respectively), UPDRS-Ⅳ( r=0.679, 0.660, 0.430, respectively), UPDRS-total score( r=0.391, 0.448, 0.319, respectively), H-Y( r=0.610, 0.750, 0.460, respectively), CSS( r=0.529, 0.366, 0.212, respectively)and LDQ( r=0.581, 0.440, 0.263, respectively), but were negatively correlated with MMSE score( r=-0.617, -0.596, -0.321, respectively)(all P<0.05). Conclusions:Peripheral inflammation caused by Hp infections may be involved in the occurrence and development of Parkinson's disease at high altitude and serum IL-6, TNF-α and NLR could serve as indicators to evaluate PD patients with Hp infections.

Objective:To investigate the predictive value of platelet reactivity for early neurological deterioration (END) in patients with acute ischemic stroke.Methods:Patients with acute ischemic stroke within 48 h of onset admitted to the Department of Neurology, the Affiliated Haikou Hospital of Xiangya School of Medicine, Central South University from January 2017 to March 2019 were enrolled prospectively. Aspirin was taken on the day of admission, and the platelet aggregation rate was detected using a PL-11 Platelet Function Analyzer 7 d after taking it. END was defined as the National Institutes of Health Stroke Scale (NHISS) score at any time point within 7 d after admission increased by ≥2 or the motor function item score increased by ≥1 from baseline. The demographics, baseline data, imaging examination and laboratory findings of patients in the END and non-END groups were compared. Multivariate logistic regression analysis was used to determine the independent risk factors for END. Receiver operating characteristic (ROC) curve was used to analyze the predictive value of platelet aggregation rate for END. Results:A total of 230 patients were included in the study. They aged 63.24±9.75 years, 126 were females (51.4%). The median baseline NIHSS score was 6 (interquartile range, 4-10). The median time from onset to admission was 15 h (interquartile range, 9-28 h). There were 54 patients (23.5%) in the END group and 176 (76.5%) in the non-END group. There were significant differences in arachidonic acid-induced maximum platelet aggregation ratio (MAR-AA), epinephrine-induced maximum platelet aggregation ratio (MAR-EPI) and collagen-induced maximum platelet aggregation ratio (MAR-COL) between the END group and the non-END group (all P<0.05). Multivariate logistic regression analysis showed that MAR-AA (odd ratio [ OR] 1.165, 95% confidence interval [ CI] 1.091-1.243; P<0.001) and MAR-EPI ( OR 1.035, 95% CI 1.006-1.067; P=0.023) were the independent risk factors for END in patients with acute ischemic stroke. ROC curve analysis showed that MAR-AA had good predictive value for END, and the area under the curve was 0.775 (95% CI 0.707-0.843; P<0.001). The optimal cut-off value was 21.80%. The sensitivity and specificity of MAR-AA for predicting END were 72.2% and 77.3%, respectively. Conclusions:The platelet function measured by PL-11 is closely related to the risk of END in patients with acute ischemic stroke. It has a better predictive value for END.

Objective : To investigate the mental health status of the pupils from Grade Four and Five in Jiading District of Shanghai, and to provide evidence for mental health intervention. Methods : All the students from Grade Four and Five in Jiading District, Shanghai were selected as survey objects. The MHT was used to evaluate their mental health status, and the influencing factors of mental health problems were analyzed by multivariate logistic regression model. Results : A total of 3 788 questionnaires were issued, of which 3 100 ones were valid and the effective rate was 81.84%. The detection rate of mental health problems was 17.06%. The detection rates of impulse tendency, terror tendency, physical symptoms, sensitivity tendency, self-blame tendency, loneliness tendency, anxiety towards people and learning anxiety were 11.77%, 15.55%, 12.97%, 16.19%, 10.10%, 4.81%, 13.16% and 14.00%, respectively. The results of multivariate logistic regression analysis showed that female students ( OR=1.617,95%CI: 1.325-1.974 ) , poor self-rated appearance ( OR=3.683, 95%CI: 2.428-5.586 ) ,only one or two friends ( OR=1.806, 95%CI: 1.342-2.428 ) , poor academic performance ( OR=1.626, 95%CI: 1.167-2.267 ) , primary school education or below of mother ( OR=1.539, 95%CI: 1.107-2.139 ) general or poor relationship with mother ( OR=1.785, 95%CI: 1.303-2.444 ) or father ( OR=1.745, 95%CI: 1.336-2.280 ) were risk factors for mental health problems. Conclusion The fourth-to fifth-grade primary school students in Jiading District of Shanghai have high detection rate of psychological problems. Gender, the number of friends, academic performance, education level of mother and the relation with parents are associated.

Objective To investigate the effects of matrine on proliferation and apoptosis of human Tenon capsule fibroblasts (HTFs) in.vitro.Methods After treated with 0,0.3,0.6 and 0.9 g/L matrine in vitro,cell counting kit-8 (CCK-8) method was used to assay the proliferation of HTFs at 24,48 and 72 hours,Western blot and PCR were performed to evaluate the expression of apoptosis-associated factor caspase-3 on both protein and RNA levels.Results The activity of human Tenon capsule fibroblast at 48 hours and 72 hours after treated with 0.3,0.6,0.9 g/L matrine was significantly inhibited when compared with the 0 g/L matrine group,and the inhibitory effect was dose-dependent and time-dependent (F ion =1 019.51,P =0.00;Ftime =5 848.66,P =0.00;Fi ion =147.45,P=0.00).After treated with 0,0.3,0.6 and 0.9 g/L matrine,the early apoptosis rate of HTFs was (2.68±0.30)％,(5.08±0.47)％,(6.97±0.69)％ and (10.30±1.20)％,the grey value ofcaspase-3 protein was 1.00±0.13,1.90±0.19,2.50±0.30 and 2.67±0.30,the relative expression of caspase-3 mRNA was 0.98 ±0.12,2.01 ±0.34,6.15 ± 0.60 and 11.40 ± 1.12,respectively,with significant differences among them (F =55.74,66.01,154.50;all at P＜0.01),the early apoptosis rate of HTFs,the grey value of caspase-3 protein and the relative expression of caspase-3 mRNA were all increased significantly as the concentration of matrine increased,with significant differences between any two groups (all at P＜0.05).Conclusions Matrine can inhibit the proliferation of HTFs and induce the apoptosis of HTFs in a time-and dose-depended manner.

Objective To investigate clinical efficacy of lateral tibia bone removal micro vessel regeneration combined with hyperbaric oxygen (HBO) in the treatment of diabetic foot ulcer.Methods A retrospective analysis was made on the clinical practice of lateral tibia bone removal micro vessel regeneration combined with HBO in 21 patients (the treatment group)with diabetic foot ulcer who received treatment in the hospital from November 2015 to March 2017.The patients in the treatment group were compared with the 21 patients (the control group) with diabetic foot ulcer who were treated with HBO at the same period.For the patients in the treatment group,a bone window with an area of about 2 cm × 10 cm was performed at medial surface of the affected lateral tibia,and a special external fixation lateral traction frame was placed and debridement was performed on the affected foot at the same time.On the first day after surgery,the patients received HBO therapy with a pressure of 0.20 MPa,once a day,10 days a treatment course,with a total of 3 courses.On the fifty day after surgery,the bone window was slowly removed laterally (1 mm/day).For the patients in the control group,debridement of the affected foot was performed,and on the first day after surgery,HBO therapy was also implemented in the control group with the same treatment scheme as the treatment group.Before surgery and 3 months after surgery,corresponding dada of healing time,ankle brachial index (ABI) and Michigan neuropathy sensation instruments (MNSI) were closely observed in the patients of the 2 groups.Results The patients of the two groups all had medical followed-ups for an average of 9.3 months.In the control group,the wound of one patient with Wagner Ⅴ failed to heal and in the end had amputation,but the wound of the remaining patients all healed,with an average healing time of (39.52 ± 8.43) days.The wound of the treatment group all healed,with an average healing time of (31.74 ± 6.12) days.Three months after surgery,the ABI level of the treatment group increased from pre-surgical level 0.56 ± 0.18 to post-surgical level 0.94 ± O.16,and the MNSI level decreased from pre-surgical level 5.92 ± 1.13 to post-surgical level 4.11 ± 1.25.Three months after surgery,the ABI level of the control group increased from pre-surgical level 0.54 ±0.21 to post-surgical level 0.81 ±0.20,and the MNSI level decreased from pre-surgical level 5.74 ± 1.26 to post-surgical level 4.98 ± 1.34.Statistical significance could be noted in wound healing time when comparisons were made between the two groups.Three months after surgery,statistical significance could also be found in ABI and MNSI levels as compared with those detected before surgery.The treatment group was superior to the control group,also with statistical significance.Conclusions Lateral tibia bone removal micro vessel regeneration could be used to reconstruct vasoganglion below lower leg and promote surrounding vessel and nerve function recovery.Combined with HBO therapy,it could promote wound surface healing,with the advantages of simple surgery,accurate curative effect,high success rate of limb-salvage.For this reason,it was one of the ideal methods for the treatment of diabetic foot ulcer.

Objective To investigate clinical efficacy of lateral tibia bone removal micro vessel regeneration combined with hyperbaric oxygen (HBO) in the treatment of diabetic foot ulcer.Methods A retrospective analysis was made on the clinical practice of lateral tibia bone removal micro vessel regeneration combined with HBO in 21 patients (the treatment group)with diabetic foot ulcer who received treatment in the hospital from November 2015 to March 2017.The patients in the treatment group were compared with the 21 patients (the control group) with diabetic foot ulcer who were treated with HBO at the same period.For the patients in the treatment group,a bone window with an area of about 2 cm × 10 cm was performed at medial surface of the affected lateral tibia,and a special external fixation lateral traction frame was placed and debridement was performed on the affected foot at the same time.On the first day after surgery,the patients received HBO therapy with a pressure of 0.20 MPa,once a day,10 days a treatment course,with a total of 3 courses.On the fifty day after surgery,the bone window was slowly removed laterally (1 mm/day).For the patients in the control group,debridement of the affected foot was performed,and on the first day after surgery,HBO therapy was also implemented in the control group with the same treatment scheme as the treatment group.Before surgery and 3 months after surgery,corresponding dada of healing time,ankle brachial index (ABI) and Michigan neuropathy sensation instruments (MNSI) were closely observed in the patients of the 2 groups.Results The patients of the two groups all had medical followed-ups for an average of 9.3 months.In the control group,the wound of one patient with Wagner Ⅴ failed to heal and in the end had amputation,but the wound of the remaining patients all healed,with an average healing time of (39.52 ± 8.43) days.The wound of the treatment group all healed,with an average healing time of (31.74 ± 6.12) days.Three months after surgery,the ABI level of the treatment group increased from pre-surgical level 0.56 ± 0.18 to post-surgical level 0.94 ± O.16,and the MNSI level decreased from pre-surgical level 5.92 ± 1.13 to post-surgical level 4.11 ± 1.25.Three months after surgery,the ABI level of the control group increased from pre-surgical level 0.54 ±0.21 to post-surgical level 0.81 ±0.20,and the MNSI level decreased from pre-surgical level 5.74 ± 1.26 to post-surgical level 4.98 ± 1.34.Statistical significance could be noted in wound healing time when comparisons were made between the two groups.Three months after surgery,statistical significance could also be found in ABI and MNSI levels as compared with those detected before surgery.The treatment group was superior to the control group,also with statistical significance.Conclusions Lateral tibia bone removal micro vessel regeneration could be used to reconstruct vasoganglion below lower leg and promote surrounding vessel and nerve function recovery.Combined with HBO therapy,it could promote wound surface healing,with the advantages of simple surgery,accurate curative effect,high success rate of limb-salvage.For this reason,it was one of the ideal methods for the treatment of diabetic foot ulcer.

Objective To explore more accurate method about the time of pregnancy and gestational age of SD rats.Methods 7 to 8-week-old SD rats (18 females,6 males) were selected,the ratios of male to female were 2:1,5:2,1:1 and 4:1.Through vaginal plug observation,vaginal smear method,weight comparison method to verify the exact date of pregnancy in the next morning.Results Vaginal observed at milky vaginal plug sperm could be observed as sickle rat sperm head.Body weight of pregnant rats changed obviously.Three detection methods were used to detect the pregnancy time for 18 females.The positive rate of vaginal plug observation was 5.6％.The positive rate of vaginal smear method was 44.4％.The false negative rate of vaginal smear method was 11.1％.The positive rate of body weight method was 11.1％.The false positive rate was 5.6％.Conclusion The accuracy of vaginal smear is higher than the vaginal plug method and weight comparison method,the vaginal plug method can be used as the premise of the other two methods.Comparison method can be used as supplementary that the other two methods missed.

Aged ; Bone Marrow ; metabolism ; Cytokines ; metabolism ; Female ; Humans ; Karyotype ; Male ; Mesenchymal Stromal Cells ; Middle Aged ; Myelodysplastic Syndromes ; genetics ; metabolism

Objective: To investigate the effect of Tanreqing injections combined with azithromycin in the treatment of children bronchial pneumonia. Methods:Totally 56 cases of pediatric bronchial pneumonia were selected in our hospital from September 2012 to September 2013 and randomly divided into the observation group and the control group with 28 cases in each. The control group was with azithromycin treatment, and the observation group was with Tanreqing injections additionally. The effect and adverse reactions of the two groups were studied and compared. Results:In the observation group, the effective rate was 92. 9%, which was significantly higher than that in the control group(P<0. 05). The hospitalization time and the symptoms disappearance time in the observation group were significantly shorter than those in the control group (P<0. 05). The adverse reactions showed no significant difference be-tween the two groups. Conclusion:Tanreqing injections in the treatment of children bronchial pneumonia is rapid, effective and safe.