Abstract:Objective To observe the Pin1 expression pattern in skin and to establish an inducible skin specific Pin1 overexpression mouse model.Methods The mouse Pin1 gene was cloned into modified vector pTRE2 with C-terminal Myc tag. The linearized pTRE2-Pin1 DNA was micro-injected into one-cell embryos followed by implantation into foster mice to produce TRE-Pin1 transgenic mice.Results TRE-Pin1 transgenic founder mice were successfully created. These mice were crossed with transgenic tool mice K14-rtTAto create epithelial specific double transgenic progenies. Pin1 gene was induced by incorporating doxycycline into drinking water of the mice. Pin1 protein overexpression in the skin was confirmed by Western blot and immunohistochemistry. The endogenous Pin1 protein was predominantly expressed in epidermal cells in the skin. Conclusions The inducible skin specific Pin1 overexpression mouse model is successfully established which may serve as a useful model for further study of Pin1 functions in the skin.

Abstract:Objective To knockout Rag2 and IL2rg genes and construct severe combined immunodeficiency mice based on CRISPR/Cas9 technology. Method Design and synthesis of 25 bp sgRNA were made according to the Rag2 and IL2rg sequences in Genbank. After annealing, sgRNA was cloned into pX330 vector. Recombination plasmid Rag2-sgRNA, IL2rg-sgRN and Cas9 were then transcribed into RNA, these RNA were microinjected into zygotes and the zygotes were transplanted into recipient ICR mice. F0 founders were born and mutated F0 founders mated with wild type mice to obtain F1 generation heterozygous mice. Mutated F1 mice were crossed and got F2 generation homozygous mice. Genotype and phenotype of the knockout mice were identified by sequencing, flow cytometry and xenograft model.Results Rag2-sgRNA and IL2rg-sgRNA recombination plasmids were constructed and transcribed into RNA. After microinjection and mating, F0 founders were born and F2 homozygous mice were obtained. The results of sequencing showed that there were two types of genotype in IL2rg gene, 10 bp or 11 bp deletion; however, there was only one genotype in Rag2 gene, which was 8 bp deletion. Compared with wild-type BALB/c mice, the number of CD3⁺, B220⁺ and NKp46⁺ cells in peripheral blood of the knockout mice was reduced significantly. After inoculation of human breast cancer cell line SKBR-2HL cells, tumor size in the xenograft mouse model was increased gradually along with time extension. Conclusion CRISPR/Cas9 is an efficient way to mutate Rag2 and IL2rg gene in mice in vivo, leading to aberrant T cells, B cells and NK cells.

Abstract:Objective To investigate the silencing effect of RNA interference on MSTN gene (myostatin, MSTN) expression, and detect the effects on the downstream genes in Schizopygopsis pylzovi.Methods To construct the recombinant adenovirus vector 1P3 (DSP MSTN 273+250+1737) and 1P2 (DSP MSTN 195+1670) for RNA interference of the MSTN gene in Schizopygopsis pylzovi,and to conduct the RNA interference in vivo experiment by injecting the vector into the muscle tissue of Schizopygopsis pylzovi. Real-time PCR and Western blotting were used to evaluate the silencing effects on MSTN gene expression, and to detect the regulatory function of M-CK at gene transcription level after RNA interference of the MSTN gene.Results The result of real-time PCR showed that compared with the HK team (Virus general negative control group) and N team (blank control group), the 1P3 had significant interference effect on the MSTN gene transcription in Schizopygopsis pylzovi (P<0.05), with an inhibition rate of 53.5%, but the 1P2 had no significant interference effect on the MSTN gene transcription. The result of Western blotting was consistent with the results of real-time PCR. At the same time, after the 1P3 interference, the level of MSTN gene transcription was declined, and the level of M-CK gene expression was significantly increased. Conclusions Our results demonstrate that the expression of MSTN gene can be effectively suppressed, and the expression of M-CK gene can be up-regulated through the RNA interference. Therefore, it proves that MSTN gene can inhibit the transcription of M-CK gene in Schizopygopsis pylzovi,and reveals the regulatory role of MSTN gene in the muscle growth and development in the plateau fish Schizopygopsis pylzovi.

Abstract:Objective Type A lamins are encoded by LMNA and a major component of the nuclear lamina, which have been suggested to play important roles in chromatin organization, transcription, DNA replication, and cell apoptosis. The aim of this study was to analyze the bioinformation of zebrafish lamins. Methods A phylogeny analysis was figured out with protein sequences of different species by Clustal X and MEGA 4.0 software. Then we compared the lamin protein sequences of different species with that of zebrafish by BLAST tool from NCBI. A figure of synteny analysis results was done with lamin sequence information of humans, murine and zebrafish cited from UCSC, Vega and Ensemble.Results The analysis results showed that lmna, lmnb1, and lmnb2 genes of zebrafish are highly conservative and they may be homology of human LMNA, LMNB1 and LMNB2 genes. Conclusions Zebrafish lamins and human lamins have homologous sequence similarity, indicating that these two genes are orthologous genes.

Abstract:Objective To explore whether Helq deletion affect the pluripotency of stem cells. Methods Helq knockout embryonic stem cells were obtained by CRISPR-Cas9 gene editing technique.Results The results of immunofluorescence analysis showed that the expression of Oct4 and Nanog had no obvious difference to that of the control cells. The Helq^-/- embryonic stem cells could produce viable pups by tetraploid complementation, indicating that their pluripotency was not affected. Meanwhile, we found that day 2 epiblast-like cells also were obtained through differentiation of the Helq^-/- embryonic stem cells in vitro. Immunostaining and real-time PCR analysis showed that the gene expression of Helq^-/- epiblast cells were similar to the wild type cells. Conclusions Taken together, it is proved that the genomic instability caused by Helq deletion does not affect the pluripotency of pluripotent stem cells.

Abstract:Objective To develop a reliable method for the primary culture of airway smooth muscle cells (ASMCs) from mice by adherent tissue culture and to identify them by immunofluorescence microscopy. Methods Airway smooth muscle (ASM) tissue was isolated from BALB/c mice under dissecting microscope, and cut into 1 mm³ pieces. These tissue blocks were washed with PBS with 1% penicillin and streptomycin, and adhered on 6 cm culture dish with 5 mL culture media. The dishes were incubated at 37℃ in an incubator with 5% CO₂. The obtained primary ASMCs were identified by immunofluorescence microscopy, and the cell proliferation was measured by MTT assay.Results We observed that obvious fusiform cells grew out from tissue blocks within 3 to 5 days. After 5 to 6 days, “hill-valley” structure was observed. After cell passage and purification, the immunofluorescence microscopy showed that the purity of isolated ASMCs reached up to 99%. The growth curve of ASMCs was constructed by MTT assay. Conclusions Obtained ASMCs from this simple and economical method show a preferable proliferation ability, density and purity, and can satisfy the need for cell biology research.

Abstract:Objective To establish and evaluate the CaV1.1-R528H gene knock-in mouse model of thyrotoxic hypokalemic periodic paralysis. Methods Thirty-six 8-week-old male CaV1.1-R528H gene knock-in mice and thirty-six 8-week-old wild-type male C57BL/6J mice were used in this study. Using three-factor two-level 2×2×2 factorial design (the three factors including mutation, thyroxine and insulin, and two levels were with or without), the mice were divided into 8 groups. The thyroxine groups were intraperitoneally injected with levothyroxine in a dose of 350 μg/kg once per day for 12 consecutive days to produce thyrotoxicosis. The insulin groups were intraperitoneally injected with short-acting insulin in a dose of 0.8 U/kg after the last administration of levothyroxine, and the potassium levels of different groups were measured and recorded before (0 min) and after insulin injection (30 min, 60 min).Results (1) Compared with the control group, the following phenomena including irritability, dull coat, increased diet and water intake, and slow body weight gain, were observed in the thyrotoxic mice. Thyroid function tests showed that the levels of T3 and T4 in the thyrotoxic mice were significantly higher than those in the corresponding control mice (P<0.05), and the TSH level was significantly lower than that of the corresponding control mice (P<0.05 ).(2) After administration of insulin or thyroxine alone, the potassium levels in the mutant and wild-type mice were not significantly different. However, after combined administration of thyroxine and insulin, the potassium levels in the mutant group were significantly lower than those in the wild-type mice at 30 min and 60 min (P<0.05 for both). (3) The main effects and interactions: Mutation factor or thyroxine factor alone did not influence on the potassium level, only insulin showed hypokalemic effect (P<0.05). There were interactions between thyroxine and mutation, and between insulin and mutation (P<0.05), but no interaction between thyroxine and insulin. Conclusions (1) A thyrotoxicosis state in mice is successfully developed in this study. (2) An CaV1.1-R528H gene knock-in mouse model of thyrotoxic hypokalemic periodic paralysis is successfully established.

Abstract:Objective This study was aimed to characterize the swine leukocyte antigen(SLA) class I genes of GGTA1^-/- Wuzhishan minipigs and compare their similarity to human leukocyte antigen(HLA). It has important implications for understanding the cellular rejection in xenotransplantation. Methods Specimens of ear tissue from six founding GGTA1^-/- Wuzhishan minipigs were collected, and the SLA class I genes (SLA-1, SLA-3, SLA-2) were amplified by RT-PCR. Purified products were cloned into pEASY-T1 vectors and sequenced, followed by BLAST alignment and using bioinformatc analysis to characterize the SLA class I genes and compare with the similarity to HLA.Results A total of six alleles were detected, among them alleles were previously reported (SLA-1*0703,SLA-2*1102, SLA-3*0401, SLA-3*0403), and the other were novel (SLA-1*0401wz01, SLA-2*11wz01). The homology between alleles of SLA class I genes in Wuzhishan minipigs and HLA was from 70.5% to 72.1%. The homology analysis of critical amino acid residues on HLA binding with human CD8⁺ molecules showed that SLA-1*0401wz01, SLA-1*0703, SLA-2*11wz01, SLA-2*1102 and SLA-3*0401 occurred mutant at amino acid positions 225 and 228 (T→S,T→M), whereas the other loci were highly conserved. There was a high homology at amino acid level between SLA-2*11wz01, SLA-2*1102 and HLA class I genes which are NK cell KIRs binding sites. Conclusions The amino acid sequences of SLA class I genes of GGTA1^-/- Wuzhishan minipigs have a high homology to HLA. From the point of view of cell-mediated xenograft rejection, the amino acid sequences of SLA class I genes of GGTA1^-/- Wuzhishan minipigs have a high homology to HLA, therefore, Wzhishan minipigs may become a good potential donor for pig-human xenotransplantation.

Abstract:Objective To observe the effects of high fat diet and 6-week voluntary wheel running on the structure of hypothalamus, pituitary and testis and sex hormone levels in pubertal male rats.Methods Forty 3-week-old male Sprague-Dawley rats were randomly divided into following groups: the control group fed with normal diet (C), training group fed with normal diet (CE), control group fed with high fat diet (D), and training group fed with high fat diet (DE), 10 rats in each group. The groups C and D were bred for 8 weeks freely, and the groups CE and DE were assigned to have voluntary wheel running twice/d, 1 h/time, 5 d/w, for 6 weeks after a 2-week adaptive feeding. Eight weeks later, blood sample was collected to detect the serum T, E₂, FSH and LH, and the E₂ and T of testis were also detected. The histology of hypothalamic, pituitary, and testis tissues was observed by light microscopy.Results (l) Compared with the group C, the group D had significantly decreased levels of serum T and E₂ and testicular T (P<0.05), and significantly increased serum E₂, FSH and LH levels (P<0.05). In the group D, vacuolar lipid droplets were increased in the hypothalamus, eosinophils and basophils were reduced in the pituitary, and the area of seminiferous tubules, percentage of sperm cells, and quantity of Leygid cells were significantly decreased in the testis. (2) Compared with the group D, the serum T and testicular T concentrations were increased, but the serum E₂, FSH and LH were decreased significantly (P<0.05) in the group DE, and vacuolar lipid drops were increased in the hypothalamus, eosinophil cells were increased in the pituitary gland, and the area of seminiferous tubules was increased but not significantly in the testis. Conclusions 6-week voluntary wheel exercise can improve the high fat diet-induced abnormal secretion of sex hormones, but not effectively improve the histological changes in hypothalamus, pituitary gland and testis.

Abstract:Objective To establish the background information of physiological parameters for germ-free (GF) Taihu piglets. Methods In this study we selected 25 days old GF Taihu piglets and 4 conventional (CV) littermates, the male and female ratio was 1∶3, to measure the normal clinical values of hematology and serum biochemistry, immunoglobulin concentration and main organ coefficients. The analysis of relative growths of main organ weight to body weight was conducted in the Taihu GF and CV pigs by allometric scaling model.Results (1) Twelve hematological parameters and 8 blood biochemical parameters in the GF piglets were significantly lower than those in CV pigs (P<0.05). (2) The average body weight, IgM concentration of GF pigs and CV pigs had significant difference (P<0.05), and no mesenteric lymph nodes were found in the GF pigs. (3) The gut weight had the largest allometric association with body weight in the GF pigs, while spleen weight has the largest allometric association with body weight in the CV pigs. Both the weight of heart and stomach in CV and GF pigs had a negative allometric association with body weight (allometric coefficient b<1), respectively. Conclusions Different microbe control grades affect the body weight, hematology and serum biochemistry, expression of immunoglobulin and development of main organs in laboratory pigs.

Abstract:Objective To establish a stable rat model of middle cerebral artery occlusion (MCAO) assisted by laser Doppler flowmetry. Methods Sixteen rats were randomly divided into two groups: (1) experimental group, n=8; (2) control group, n=8. In the experimental group, a sudden decrease of regional cerebral blood flow from baseline (before ischemia) to approximately 30% of baseline confirmed the correct placement of nylon thread. In the control group, 1.8 cm nylon thread insertion was performed without laser Doppler flowmetry. The modified neurological severity scores (mNSS) were tested before and 24 h after MCAO. All rats were killed at 24 h after MCAO. The brains were removed and dissected into 6 pieces of 2 mm coronal sections. The fresh brain slices were stained with 2% 2,3,5-triphenyl tetrazolium chloride and the relative infarct volume was measured.Results In the experimental group, 8 rats showed typical symptom of hemiplegy with stable mNSS (between 10 and 13) and infarct volume (37.5±3.9%). However, only 5 rats in the control group showed stable mNSS and infarct volume. The success rate of the model establishment was 100% in the experimental group compared with 62.5% in the control group (P<0.05). Conclusion The application of laser Doppler flowmetry significantly increases the establishment success rate of stable MCAO models in rats.

Abstract:Objective To compare the timeliness, success and mortality rates between the modified carotid artery puncture method (MCAPM) and standard suture method (SSM) in the establishment of rat model of a middle cerebral artery occlusion (MCAO). Methods Thirty-two male rats were randomly and equally assigned into MCAPM group and SSM group. The MCAO models were established by inserting a thread into the common carotid artery (CCA). 24 h after modeling, the rats of the two groups were evaluated with Bederson neurological scores, and the modeling success rate and mortality rate were analyzed.Results The suture insertion times, success rates and mortality rates of the MCAPM vs. SSM groups were (82.3±17.4)s versus (164.6±22.0)s (P<0.01), 87.5% versus 68.75% (P>0.05), and 6.25% versus 18.75% (P>0.05). Conclusions MCAPM can be used to establish the rat model of MCAO due to its simplicity, mild wound and feasibility.

Abstract:Objective To evaluate the inhibitory effect of highly selective M4 receptor antagonist MT3 on the form deprivation myopia in guinea pigs and its potential mechanism. Methods Thirty-two healthy male guinea pigs were randomly divided into three groups: control group, form deprivation group, and form deprivation+MT3 group, 8 animals in each group. Refraction was measured by retinoscopy after cycloplegia before and after the experiment. The ocular biological dimensions were measured by A-scan ultrasound. RT-PCR was used to detect the relative expression of TGF-β2 mRNA in the retina and choroid.Results Compared with the right eyes of control group, the right eyes of form deprivation+MT3 group developed relative myopia of -1.44±0.50 D (right-left eye) (P=0.001). The vitreous chamber depth and axial length of the right eyes were significantly prolonged by 0.10±0.02 mm and 0.14±0.07 mm (P<0.001, P<0.001), respectively, but the increases of myopia and axial length were significantly smaller than that of the form deprivation group (P<0.001, P<0.001, P<0.001). Down-regulation of relative mRNA expression of TGF-β2 in retina and choroid was found in the form deprivation group (P<0.001, P=0.014) compared with the right eyes of the control group, while up-regulation of relative mRNA expression of TGF-β2 in retina and choroid was found in the form deprivation+MT3 group (P<0.001, P<0.001). Conclusions MT3 can inhibit the development of form deprivation myopia in guinea pigs, which may play an important role by the regulation of TGF-β2 mRNA level in the retina and choroid.

Abstract:Objective To study the effect of TGF-β1 gene therapy on the rat model of postpartum stress urinary incontinence and explore a novel non-operative treatment of this disease. Methods Two hundred and forty 6-month old SD female rats were used to prepare the model of postpartum stress urinary incontinence by vaginal dilation with a water sac. 148 rats from the 185 successfully prepared model rats were selected, and randomly divided into 5 groups: the TGF-β1 gene therapy, clentuterol treatment, electric stimulation therapy, injection of empty vector plasmid, and non-treated groups. In addition, 20 normal rats were selected as blank control group. Sneeze test and urodynamic test were conducted, the pelvic floor pubococcygeus muscle contractile force/muscle weight ratio was calculated, serum TGF-1 was detected by ELISA, and TGF-1 protein was detected by immunohistochemistry at 1, 21, 42 and 63 days after the treatment.Results At 21 days after treatment, all the maximum bladder capacity, leak point pressure, and urine or contractile force / muscle weight ratio of the TGF-β1 gene therapy group showed even better effects than those of the electrical stimulation group, but the differences were statistically not significant (P>0.05). Conclusions TGF-β1 gene therapy shows good therapeutic effect on the rat models of postpartum stress urinary incontinence, suggesting that TGF-β1 gene therapy may become a new type of non-surgical treatment for this disease.

Abstract:Objective To observe the emotional response to inflammatory pain model induced by complete Freund’s adjuvant (CFA), and to explore the relationship between phosphate kinase C (PKC)zeta in the anterior cingulate cortex (ACC) and emotional response to inflammatory pain. Methods Twenty-four healthy 2-month old female Sprague-Dawley rats were randomly divided into control group and model group. The inflammatory pain model was established by injecting subcutaneously with 0.1 ml CFA into the right hind paw of rats. To observe the dynamical changes of body weight and pain threshold before modeling, and at 3, 7, 14, 21, and 28 days after modeling. Detecting the total distance, distance in open arm, the number of times entering into the open arm, percentage of time spent in open arm of elevated O maze were measured at 14, 21, 28 days after modeling. The total distance, distance in the central quadrat, the number of times entering the central quadrat, and time spent in the central quadrat of open field were determined at 14 and 29 days after modeling. The protein expression of PKCzeta in the ACC was detected by Western blot at 14 and 29 days after CFA-injection.Results There were no significant differences of body weight between the control and model groups (P>0.05). Compared with the control group, the pain threshold of rats in the model group was significantly decreased at 1, 3, 7, 14, 21, and 28 days after CFA-injection (P<0.05), the distance in open arm and percentage of time spent in open arm of the model group were markedly reduced at 28 days after modeling (P<0.05), distance in the central quadrat and the number of times entering into the central quadrat of model group were significantly decreased at 29 days after modeling (P<0.05), and the protein expression of PKCzeta in ipsilateral ACC was increased at 29 days after CFA-injection (P<0.05). There was a negative correlation between distance in open arm and protein expression of PKCzeta in the ipsilateral ACC, percentage of time spent in open arm and protein expression of PKCzeta in the ipsilateral ACC, distance in the central quadrat and protein expression of PKCzeta in ipsilateral ACC, the number of times entering into the central quadrant and protein expression of PKCzeta in the ipsilateral ACC, respectively. Conclusions Abnormal emotional behavior may occur in rats with chronic inflammation pain induced by CFA-injection. This pain emotion behavior may be associated with high protein expression of PKCzeta in the ipsilateral anterior cingulate cortex.

Abstract:Objective To study the changes of total SOD activity and expression in major organs and observe the effects of antioxidant N-acetylcysteine (NAC) on antioxidant status in diabetic rats. Methods Normal rats (group C, n=8) or streptozotocin (STZ)-induced diabetic rats (group D, n=8) were treated with NAC 1.5 g/kg/d (Group D+N, n=8) or equal volume of normal saline once daily by gastric gavage for 4 weeks. Blood samples were collected and the 15-F2t-isoprostane, total antioxidant concentration and SOD activity were assayed. The heart, lung, liver and kidney tissues were collected for assay of the total SOD activity using a kit and the protein expression of SOD isoforms, including Cu/Zn-SOD and Mn-SOD, was determined by western blot.Results The levels of 15-F2t-isoprostane and total antioxidant concentration in plasma, total SOD activity in the heart, protein expression of Cu/Zn-SOD in the heart and lung, and Mn-SOD expression in the liver were significantly increased (PPConclusions The levels of total SOD activity and protein expression of Cu/Zn-SOD and Mn-SOD in diabetic rats are tissue specific, and antioxidant NAC can attenuate the target organ damages to a certain degree by restoring the antioxidant levels in diabetic rats.

Abstract:Objective To investigate the antioxidant effects of polysaccharide extraction from Eupolyphaga sinensis Walker (ESW) in vitro and in vivo. Methods The methods of spectrophotometry, pyrogallol autoxidation and Fe²⁺ phenanthroline were used to detect the free radicals scavenging (DPPH·,O₂^-·and·OH), erythrocyte oxidative hemolysis and liver mitochondrial swelling of polysaccharide extraction from ESW in vitro. The mice were administered with different doses of polysaccharide extraction from ESW (0, 40, 80, 160 mg/kg) for 20 days in vivo. The contents of MDA in different tissues of the mice were determined by TBA method, and spectrophotometry was used to assay the activity of antioxidant enzymes (SOD, CAT, GSH-Px).Results Compared with the control group, free radical (DPPH·, O₂^-·and·OH) scavenging rate was significantly improved with the increasing concentration of polysaccharide extraction. There was a significant decrease in the oxidation hemolysis of erythrocytes and mitochondrial swelling in vitro. MDA contents were decreased in the polysaccharide group, and the activities of SOD, CAT and GSH-Px were significantly higher than those in the control group. Conclusions Polysaccharide extraction from ESW can eliminate free radicals, reduce cell oxidative damage caused by free radicals, inhibit the formation of peroxide in tissues, and increase the activity of antioxidant enzymes, indicating a good antioxidant property.

Abstract:Objective To establish a zebrafish model of thrombosis induced by three kinds of inducers and observe the anti-thrombotic effect of a Chinese traditional medicine, Guanxinning tablet (GXN). Methods The zebrafish models of thrombosis was induced by using 1.5 μmol/L phenyl hydrazine, 80 μmol/L arachidonic acid and 5 mg/L ponatinib, respectively, and were treated with various concentration of GXN, clopidogrel or asprin. The thrombus in the tail vein was observed under microscope, Erythrocytes in the zebrafish heart were stained with o-dianisidine and the erythrocyte staining intensity was assessed with a NIS-Elements DTM image analyzer, and the anti-thrombolic effect of GXN was calculated.Results Venous thrombus was significantly increased and the staining intensities of erythrocytes in the heart were significantly decreased after induction by phenyl hydrazine, arachidonic acid or Ponatinib (P<0.001), respectively. At the same time, GXN showed an incresing anti-thrombolic effect in the zebrafish models (P<0.001) in a dose-effect manner, with a IC₅₀ of GXN of 44.32 mg/L,138.5 mg/L and 459.5 mg/L, respectively. Conclusions The zebrafish models of thrombosis are successfully established by phenyl hydrazine, arachidonic acid or Ponatinib, respectively, by different formation mechanisms. GXN has been shown to have an anti-thrombosis effect, probably, by multiple target effects.

Abstract:In recent years, stem cells and translational medicine become more and more popular.Due to the reason that stem cells have multiple differentiation potential and a high degree of self-renewal capacity,stem cells can be used as functional stock cells to repair functional cells in living organisms.It is attracting more and more attention worldwide in regenerative medicine and tissue engineering fields.The clinical application of stem cells brings hope to the treatment of many kinds of diseases, and it overcomes the limitations of conventional clinical treatment. For the present, clinical application of stem cells is already stepping into a rapid development stage. The Chinese government also attaches great importance to stem cell research, increases investment in research, strengthens the standardized management and developing relevant policies related to clinical application of stem cells.If the stem cell therapy takes use of translational medicine mode, links the clinical and application research more closely, and pays attention to the research and application of stem cell therapy using large laboratory animal model such as pig, dog and monkey models of human diseases,it will considerably promote the development of stem cells translational medicine and clinical application.