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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 (DmdMu/+) mice were obtained after genotype identification. Male hemizygous DmdMu/+(DmdMu/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 DmdMu/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 (DmdMu/+ mice). Dmd protein expression was not detected in skeletal muscle and myocardium of DmdMu/+ 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, DmdMu/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 DmdMu/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 DmdMu/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 DmdMu/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 DmdMu/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.
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ObjectiveOptimize the latex perfusion technique and apply it to the construction of a murine craniofacial venous vascular model.Methods A total of nine 8-week-old male C57BL/6 mice weighing (25.0±1.3) g were randomly divided into three groups: 60% latex physiological saline group, 60% latex heparin group, and 30% latex heparin group. After completion of the perfusion, the specimens were immersed in 4 °C formalin fixative for 24 h, followed by dissection, observation, and measurement of the extracranial blood vessel diameters. Results After 200 μL latex perfusion solution was injected into the external jugular vein, the supraorbital vein, infraorbital vein, temporal vein, retrofacial vein, masseter vein and external jugular vein were perfused in each group.After comparing the perfusion degree of the distal branches of blood vessels, sublingual vein and tip venule, it was found that the 30% latex heparin group had the best perfusion effect, followed by the 60% latex heparin group, and the 60% latex saline group had the worst perfusion effect.ConclusionThe optimized latex perfusion technique can effectively infuse the veins in the head and face of mice, and this technique can provide a good reference for the study of the direction and morphology of facial veins in mice.
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Objective Exploring the relationship between anxiety symptoms and neurometabolism in the ventrome-dial prefrontal cortex(vmPFC)of adolescents with bipolar depression.Methods Thirty-six adolescent patients with bi-polar depression were assessed and grouped by using the 14-item Hamilton anxiety rating scale(HAMA),including 20 pa-tients with anxiety symptoms and 16 patients without anxiety symptoms.The severity of depressive symptoms was assessed using the 24-Hamilton depression scale(HAMD),and 1H-magnetic resonance spectroscopy(1H-MRS)was used.The dif-ference of vmPFC neurometabolism between 2 groups was compared.Results Compared with the group without anxiety symptoms,the HAMD score[24.50(24.00,26.75)vs.23.00(22.00,24.00)]and the proportion of family history(40.0%vs.0)were significantly higher in the group with anxiety symptoms than in the group without anxiety symptoms(P<0.05).The level of mI/Cr was higher in the group with anxiety symptoms than that in the group without anxiety symptoms(0.58±0.12 vs.0.47±0.11),and the difference was significant(P<0.05).Cho/Cr and HAMD scores in patients with anxiety symptoms were positively correlated(r=0.589,P=0.006),and mI/Cr was negatively correlated with disease duration(r=-0.481,P= 0.032).Conclusions Anxiety symptoms in adolescent bipolar depression patients may be related to elevated levels of mI,a neurometabolite in the brain region of vmPFC.
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AIM: To investigate the effect of streptozotocin-induced diabetes on the expression of NADPH oxidase, and to determine the effect of exercise on the expression of NADPH oxidase.METHODS: One weeks after successful modeling, the effect of diabetes mellitus on the expression of NADPH oxidase subunits in the cardiac tissue was determined.The effect of exercise for 8 weeks on the protein expression of NADPH oxidase subunits and the effect of diabetes on the protein expression of collagen in the heart were observed, and whether 8 weeks of exercise affected the expression of collagen were also measured.RESULTS: Diabetes mellitus resulted in the increased expression of NADPH oxidase subunits p47phox and gp91phox in the cardiac ventricle, and exercise for 8 weeks inhibited the increase in the expression of NADPH oxidase subunits p47phox and gp91phox.Diabetes mellitus significantly increased the expression of p47phox in the atrial muscle, while exercise inhibited this increase.Diabetes mellitus significantly increased the levels of cardiac collagen III, while exercise reduced the increase in collagen protein.CONCLUSION: Reduction of diabetic rat heart p47phox and gp91phox expression by effective exercise therapy may be an important mechanism of improving the cardiac matrix by inhibiting myocardial oxidative stress and decreasing collagen expression, thus preventing diabetic cardiomyopathy.
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Targeting of tumor cells is crucial for gene therapy of malignant diseases.This can be achieved by tumor targeted gene transfer or tumor specific gene expression, as well as secretion of tumor targeted therapeutic molecules by autologous normal cells.Tumor targeted gene transfer is mediated by the recognition of a class of tumor specific antigens or receptors by corresponding vector fused antibodies or ligands, while therapeutic genes can be selectively expressed in tumor cells under the control of tumor or tissue specific promoters or enhancers, as well as the induction of certain physical, chemical or physiological factors.
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Aim To clone apoptosis-related genes from human MCF-7 breast cancer cells and to analyze the character of the method used in the process. Methods A poptotic cell model of MCF-7 cells was established with the apoptotic tumor cells induced by the all-trans-retinoic acid. The apoptotic gene was cloned from the model by improved PCR-based subtractive hybridization. Results 5 clones were identified to be related to apoptosis by reverse dot blot, 4 of them were known genes, and 3 were related to apoptosis. A novel gene， named apmcf-1, coded for 47 amino acid was identified. This gene was accepted by Genbank, the accession number was AF141882. Conclusion This improved PCR-based subtractive hybridization may be an efficient way in cloning differential expression gene.
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A hammerhead RZ DNA was designed and synthesized, which can specifically cleave the bcl-2 mRNA. After demonstration of right sequences by sequencing and cleavage activity of RZ by in vitro cleaving experiment, The RZ DNA was recombinated into the pDOR - neo vector to form the recombinant pDOR - RZ. Using lipofectin - mediated DNA transfectionpDOR-RZ was successfully introduced into HL - 60 cells. The RZ expression was observed by Southern, RNA dot blot hybridization and flow cytometry (FCM) . The results demonstrated that (a) the RZ was expressed in 72 hours after transfection; (b) the synthesis of Bel - 2 protein was inhibited by the expression of RZ; (c) apoptotic peak appeared in FCM.