Abstract:Objective To compare two separation medium of isolation of adult rat cardiomyocytes, and to observe the characteristics of excitation-contraction coupling of cardiomyocytes. Methods The isolated adult rat heart was hanged on to the Langendorff apparatus for aortic counter-current perfusion and collagenase digestion using two different separation medium. The single cardiomyocytes were cultured and infected with adenovirus. The morphological features of cardiomyocytes were observed with microscope and fluorescent microscope. The shortening-re-lengthening features of sarcomere and the intake-discharge features of calcium were simultaneously recorded by IonOptix equipment. Results 70% rod-shaped with clear-striation adult rat cardiomyocytes could be obtained with the stated two separation medium and cultured in serum-free medium for more than 7 days. GFP could express more than 7 days when the cardiomyocytes were infected with adenovirus. Cardiomyocytes obtained by the first separation medium could not contract with the electrical stimulation, while cardiomyoctyes obtained by the second separation medium could be used for the detection of excitation-contraction coupling. The shortening fraction of sarcomere was 11.61%±2.15% and the relaxing time was (0.177±0.031) s. The amplitude of calcium transient was 30.79%±9.74 % and the decaying time of calcium transient was (0.300±0.074) s. Conclusion With the stated two separation medium, adult rat cardiomyocytes can be well isolated, cultured and infected with adenovirus. The second separation medium can be used for the detection of excitation-contraction coupling characteristics.

Abstract:Objective To observe the expression of insulin-like growth factor 1 (IGF-1),insulin-like growth factor binding protein 3 (IGFBP-3) in rats' serum with non-alcoholic fatty liver disease(NAFLD) and the impact of Polyene phosphatidyl choline. Methods NAFLD model was induced by feeding the SD rats with a high-fat diet, with Polyene phosphatidyl choline to intervene.Observe the pathological changes of the rats' liver tissue dynamically after HE staining.Detect the contents of IGF-1,IGFBP-3 in serum dynamically through the immuno radio metric assay(IRMA). Results There were no obvious exception of the liver tissue pathology in the normal group at each time.With the extension of a high-fat diet,the liver tissue of the model group increased on fatty change,the degree of inflammation,the balloon sample change,the NAFLD activity score at 4,8,12weeks.IGF-1,IGFBP-3 in serum were decreased significantly,and the model set was significantly lower than the normal group at the same phase.After the application of polyene phosphatidyl choline,the degree of rat liver tissue inflammation and the NAFLD activity score were reduced significantly when compared with model group,while the level of IGF-1,IGFBP-3were significantly higher. Conclusion The levels of IGF-1,IGFBP-3 in the rats' serum reduce in the development of non-alcoholic fatty liver disease.

Abstract:Objective To study the isolation,culture, adipogenic and osteogenic induction Tupaia bone marrow mesenchymal stem cells(BM-MSCs). Method The BM-MSCs from tupaia were isolated and expended by combination of gradient centrifugation and adherence culture, then subcultured and observed for morphology under inverted phase contrast microscope. BM-MSCs were induced to adipocytes.and osteoblasts in vitro Result Cells were spindle or triangle-shaped,and clone proliferation.Cells were successfully induced into adipocytes.and osteoblasts Conclusions The method of isolation BM-MSCs from tupaia by combination of gradient centrifugation and adherence culture is simple and feasible, BM-MSCs have differentiation potential into adipocytes and osteoblasts.

Abstract:Objective To investigate a antitumor effects of mouse original monoclonal antibody against hMIC-1 as intravenous administration with human pancreatic tumor in vivo and providing experimental data.Methods The fourty-eight mice were randomized into eight groups for loaded with two pancreatic tumor cell lines panc-1 or sw1990 respectively, and individual tumor growth was observed, antitumor efficacy was evaluated after using mouse original monoclonal antibody against hMIC-1 by intravenous administration.The pathological change with formalin fixed, paraffin embedded tissues section was viewed.Results There was a significant difference in tumor volume and weigt in intravenous injection of mouse original monoclonal antibody against hMIC-1 on load pancreatic tumor with nude mice group compared with that in the control group after four week treatment, and the mouse original monoclonal antibody against hMIC-1 demonstrated a close association between inhibition of tumor volume growth and dose-effective in the two xenograft models examined. Under examined microscope, the pancreatic tumor tissue was destroyed evidently in mouse original monoclonal antibody against hMIC-1 group.Conclusions The antitumor effect of intravenous injection for mouse original monoclonal antibody against hMIC-1 is better than that of systemic using gemcitabine.

Abstract:Objective To measure the range of routine blood counts and blood biochemical parameters,to analyze hemorheology of the rats with congenital cataract. Methods Blood samples were taken from 90 rats with congenital cataract weight about 185~211 g.Routine blood analysis was performed and blood biochemical and hemorheology paramenters were determind using an automatic blood biochemical and hemorheology analyzer. Results There were no significant difference (P > 0.05) between the cataract rats and the normal rats in Blood test results; but there were significant difference between the microphthalmos cataract rats and the normal same-sex rats (P< 0.01 or P< 0.05). The biochemical results is the cataract rats and the normal rats were different significantly in ALB group (P< 0.01 or P< 0.05), and the female microphthalmos cataract rats compared with the control rats had significant difference in Ure (P< 0.01), the female cataract rats ompared with the normal rats were very significant difference in Cr group(P< 0.05 or P< 0.01).The erythrocyte counts of the male cataract rats and male microphthalmos cataract rats were significantly lower than that in the female ones, respectively(P< 0.05, P< 0.01).The platelet counts of the male cataract rats and the male microphthalmos cataract rats were significantly higher than that in the female ones, respectively(P< 0.01), and the creatinine of the male cataract rats and the male microphthalmos cataract rats were significantly lower then that in the female ones, respectively(P< 0.01). There were no significant difference in every group on hemorheology.Conclusions There were significant differences in some blood indexes between the congenital cataract rats and the normal rats. These data may become useful reference for biomedical researcher in this field.

Abstract:Objective To investigate the protective effect and the mechanism of isoflurane preconditioning on rat brain tissue suffering from rat liver ischemia-reperfusion injury. Methods 75 SD rats were randomly divided into five groups, sham group (S group); ischemia-reperfusion group (I/R group): liver ischemia for 60 min, reperfusion for 120 min; isoflurane preconditioning group (ISO group): 60 min before liver I/R,ISO pretreatment for 30 min; CsA+ISO group; CsA (MPTP specific blocker) 50 mg/kg intravenous injection, the same as ISO group after 30 min; CsA group: CsA 50 mg/kg intravenous injection. 30 min before I/R.The animals were killed 24 h after ischemia and their brain were excised for measurement of mitochondrial permeability transition pore (MPTP) and calcium content in mitochondria. The measurement of content of S-100β protein in serum before ischemia and 120min after reperfusion through the application of Elisa kit. Results The mitochondrial Ca²⁺ concentration of I/R group(287.32±26.17)was higher than that in S group(198.54±21.02)and the ISO group(209.74±29.49)(P< 0.05), and Ca²⁺ concentration of CsA+ISO group(267.31±37.52) was higher than the ISO group (P< 0.05); there was not statistical significance between I/R and CsA group(288.63±23.15)(P< 0.05).MPTP were more opened in I/R group(△S:1.73±0.24)than that in S group(△S:2.36±0.35)and ISO group(△S:2.11±0.32)(P< 0.05),but MPTP were more opened in CsA+ISO group than that in ISO group (P< 0.05). The content of S-100β protein in serum was significantly higher in I/R group than that in sham and ISO groups (P< 0.05),and the content of S-100β protein in serum was significantly higher in CsA+ISO group than in the ISO groups (P< 0.05). Conclusion The liver ischemia-reperfusion may injury the brain of the rat and isoflurane preconditioning can protect the brain on the rat. The reduce of mitochondria calcium overload and inhibition of MPTP opening are involved in the mechanism.

Abstract:Objective To observe the effect of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), catalase (CAT) and endothelin (ET-1) and tumor necrosis factor alpha (TNF-α) on rat renal tissue under acute hypoxia. Methods 24 male Wistar rats, weight 180～220 g, were randomly divided into control group and acute hypobaric hypoxia group. Acute hypoxia group was divided into 2 groups hypoxia 1 and hypoxia 2, 8 rats for each group. After acute hypobaric hypoxia 10min and 24h, rats were sacrificed.The left removed kidneys were analyzed for biochemical indexes, and the right parts were observed by immunohistochemistry to evaluate the expression level of renal endothelin (ET-1) and tumor necrosis factor alpha (TNF-α). Results After acute hypobaric hypoxia, the activity of SOD of the rats kidney was greatly decreased (P< 0.01), CAT activity of hypoxia group 1 was significantly decreased (P< 0.01), GSH activity of hypoxia group 2 was significantly decreased (P< 0.05), but the MDA content had no obvious change (P> 0.05). The immunohistochemical staining showed that, the expression level of ET-1 and TNF-αwas increased remarkably, but it was reduced after 24 h. Conclusion The obviously decreased activity of SOD, CAT, GSH and significantly increased expression of ET-1 and TNF-α, may be involved in the pathogenesis of renal hypoxic injury.

Abstract:Objective To establish the sperm specific Sleeping Beauty (SB) transposase-expression transgenic mouse for the study of the genetic modification mediated by transposon system in mouse. Methods Prm1 promoter was cloned from mouse genomic DNA to drive the expression of SB transposase. The transgenic mice were generated by microinjection. The gene type of transgenic line was identified by PCR. The expressing level in testis was determined by western blot and immunohistochemistry (IHC) staining. Results Five lines of transposase transgenic mice were obtained by microinjection and three can be germline. One mouse line with higher expression level of transposase in the testis was obtained. Conclusion One transgenic mouse model with Sleeping Beauty transposase-expression was successfully established. This model will greatly contribute to the research of genetic modification mediated by transposon in mouse

Abstract:Objective To study the influence of IL-33 on the Hematopoietic stem cells and progenitor cells. Methods Cells from the peripheral blood, spleen, thymus and bone marrow were stained with indicated antibodies and analyzed by flow cytometry. The LT-HSCs were sorted and culture using in vitro clonogenic assay. Results The percentage of B cells and T cells was decreased and the percentage of M cells was increased in the peripheral blood from IL-33 transgenic mice. Compared with the wildtype mice, the number of HSCs, MPPs and CLP was decreased; meanwhile the number of CMP and GMP was increased in the bone marrow from IL-33 transgenic mice. An in vitro clonogenic assay showed that LT-HSCs increased the ability to self-renew from IL-33 transgenic mice. And the percentage of S-G2-M stage hematopoietic stem cell was increased from IL-33 transgenic mice. Conclusion IL-33 increase the myeloid differentiation in hematopoietic stem cells.

Abstract:Objective To obtain more physiological data of Leptin knockout SD rats available for the user, the long-term observation of fasting blood glucose and pathological phenotypes were performed. Methods The protein expression levels in liver tissues were determined by western blot. Body weight of Leptin knockout rats (Leptin-/-) and littermate lean rats (Leptin+/+) were weighed up at 1,3,6,8 months of age. Fasting blood glucose of Leptin+/+ rats and Leptin-/- rats at 1,3,6,8 months of age were measured using One Touch brand blood glucose monitoring systems. Pathological changes of pancreas and livers of Leptin-/- rats were observed by the method of HE staining and Immunohistochemistry (IHC). Results Short null Lepin proteins were expressed in liver tissues from Leptin-/- rats. Leptin-/- rats become heavier than Leptin+/+ rats since they were one month old. The body weight of Leptin-/- rats at 8 months of age was twice as heavy as Leptin+/+ rats, female Leptin-/- rats weighing 884g, and male Leptin-/- rats weighing 1200g. Overt hyperglycemia was observed during the first month after birth. Compared with Leptin+/+ female rats,the fasting blood glucose of Leptin-/- female rats was increased by 40%-26% from 1 to 6 months old. After that, blood glucose values decreased and eventually become nearly normal at 8 months of age. Pathological examination indicated that Leptin-/- rats at 8 months of age had a fatty liver, more pancreas islets with lager volume and more beta cells with increased insulin secretion. Conclusion Leptin-/- rat were characterized by obesity, fatty liver, islet cell hyperplasia and early hyperglycemia.

Abstract:Objective To investigate the influence of Siraitia Grosvenori and Rehmannia Glutinosa on the Hematopoietic stem cells proliferation and function. Methods Cells from the peripheral blood, spleen and bone marrow of mice were stained with indicated antibodies, and analyzed by flow cytometry. Mice were divided 3 groups: control group, Siraitia Grosvenori treatment group and Rehmannia Glutinosa treatment group. After 4.5Gy IR treatment, mice divided 4 groups: control group, 4.5Gy IR treatment and feed with normal food, 4.5Gy IR treatment and feed with Siraitia Grosvenori and 4.5Gy IR treatment and feed with Rehmannia Glutinosa for 1 month. Results Mice fed with Siraitia Grosvenori and Rehmannia Glutinosa decreased the percentage of B cells and increased the percentage of M cell. For HSCs, the number of HSCs was increased, especially the number of LT-HSCs. After 4.5Gy IR treatment, mice fed with Siraitia Grosvenori and Rehmannia Glutinosa increase the number of HSCs, and increased the percentage of M cells. Conclusion Siraitia Grosvenori and Rehmannia Glutinosa promote the hematopoietic stem cells and progenitor cells proliferation and function and recover the damage that caused by IR treatment.

Abstract:Objective To study the relationship of insulin receptor substrate-1 (Irs1) and metabolic disease, we generated Irs1 gene knockout rat by CRISPR/Cas9 system. Methods Two sgRNA targeting sites were designed for Irs1 targeting. The Cas9 and sgRNAs were transcribed by T7 RNA polymerase in vitro. Cas9 mRNA and sgRNA mixtures were pooled and microinjected into one-cell fertilized eggs of SD rats to generate rats with targeted mutation. Results Five rats with the mutations were detected with the efficiency of 83%. Conclusion The Irs1 gene knockout rats generated in this study can be transmitted by germline.

Abstract:Objective To develop a model that could roundly show the phenotypes of human alzheimer disease (AD), the triple-transgenic rat model harboring APP(Swe), PS1dE9, and TAU transgenes was established in view of the advantage of rat as an important animal model on the research of nerve system. Methods APPswe/PS1dE9/TAU triple transgenic rat AD rats were generated on a SD background by co-injecting rat pronuclei with two human genes driven by the mouse prion promoter:‘Swedish’mutant human APP (APPsw) and exon 9 mutant human presenilin-1 (PS1dE9) and human microtubule-associated protein tau gene under the control of PDGF promoter. Transgene integration was confirmed by genotyping and expression levels were evaluated by western blot (WB) of brain homogenates. The pathological changes were detected by human Abeta, TAU and Phospho-PHF-TAU immunohistochemistry staining (IHC). The behavioral and cognitive changes were evaluated by Morris water maze. Results One transgenic rat lines with high human APP (Swe), PS1dE9, and TAU transgenic expression was selected from three transgenic founders.Compared with the wild type rat, the transgenic rat showed significant learning and memory impairments in the Morris water maze at 6 months of age. The triple transgenic rat manifested hyperphosphorylated tau and obvious aggregation of amyloid-β (Aβ) in the brain cortex and hippocampus. Conclusion APPswe/PS1dE9/TAU triple transgenic rat AD model was established. The triple transgenic AD rat fills a critical need for a next-generation animal model to enable basic and translational AD research.

Abstract:Objective To established cardiac-specific transgenic mice of the cTnC^D145E and cTnC^G159D and compare the HCM and the DCM. Methods The cTnC^D145E and cTnC^G159D were generated by site-directed mutagenesis and the transgenic plasmids were constructed by insertion of the mutant genes under the control of α-MHC, which is a myocardium specific promoter. The transgenic mice were generated by microinjection and were all maintained on a C57BL/6J genetic backgroud. The cardiac structure and function of the transgenic mice were compared and analysized by echocardiographic and pathological observation at different ages. Results The cTnC^D145E and cTnC^G159D transgenic mice were established and developed to HCM and DCM, respectively, with aging. The left ventricular end-systolic volume (ESV) and left ventricular end-diastolic volume (EDV) decreased and ejection fraction (EF) and left ventricular end-systolic posterior wall thickness (ESPWT) increased in the cTnC^D145E transgenic mice, while EDV and ESV increased and EF and ESPWT decreased in the cTnC^G159D transgenic mice at 12 months of age. Conclusions Cardiac-specific human cTnC^D145E transgenic mice showed HCM phenotypes, and cardiac-specific human cTnC^G159D transgenic mice showed DCM phenotypes, which can be used as different models for comparative study of the pathogenesis of cardiomyopathy.

Abstract:Objective To investigate the functional role of 25 microRNAs in breast cancer,and to find new tumor suppressor microRNAs that may serve as specific targets of new gene therapies. Methods Twenty-five microRNAs expression vectors were constructed and stably transfected into mouse mammary tumor cells 4To7 by Lipofectamine2000. Cells were selected with G418 and sorted by Flow cytometry. The cells in logarithmic phase were collected and 2×10⁵ cells/mouse was inoculated into BALB/c mice via tail vein. Lungs were harvested 14 days after tumor cell inoculation, and the number of metastasis foci was counted. Results Mice inoculated with mir-449a-expressing 4To7 cells via tail vein developed reduced lung metastases compared with mice inoculated with negative control cells. Mice inoculated with mir-1935-expressing 4To7 cells via tail vein developed increased lung metastases compared with mice inoculated with negative control cells. Other twenty-three microRNAs neither promoted nor inhibited lung metastases of breast cancer. Conclusions Two of twenty-five microRNA were identified to be associated with breast cancer metastasis. MiR-449a may play a tumour suppressor role in the regulation of migration and metastasis in breast cancer. miR-1935 transgenic over-expression promoted tumor growth and metastasis.

Abstract:Objective To study the effects of NRG2 on cardiac structure and function, we established the cardiac-specific human NRG2 transgenic mice and investigate the effect of NRG2 on cardiac structure and function under pressure overload situation. Methods The transgenic vector was constructed by insertion of the human NRG2 gene under the α-MHC promoter. The transgenic mice were generated by microinjection and were all maintained on a C57BL/6J genetic background. The genotype of transgenic mice was identified by PCR and the expression level of target gene was determined by western blot. Transverse aortic constriction (TAC) was applied to prepare the pressure overload induced cardiomyopathy mice model. The cardiac structure and function of the transgenic mice were compared and analysized by echocardiographic and pathological observation. Results Transgenic mice with high level of NRG2 in heart tissues were established. The left ventricular wall thickness (LVPWD) was increased, and to 15.6% at 3 months old compared with that of the non transgenic (NTG) mice. The hypertrophy of left ventricular wall caused by pressure overload was removed due to the expression of NRG2. Meanwhile, cardiac disarray and fibrosis were increased obviously compared with that of the NTG mice. Conclusion The transgenic expression of NRG2 in heart tissues could shorten the pathological process of hypertrophy, but accelerated the process of heart failure (HF).