Breeding of APPSWE transgenic mice and gene identification of filial generation

AIM: To explore the ideal breeding way of APPSWE transgenic mouse (Tg2576) and identify filial generation mice with APPSWE gene for laying a foundation of AD research. METHODS: (1) Three different copulation ways were adopted to observe the survival rate and positive rate with APPSWE gene of filial generation mice. (2) The genome DNA was extracted from the tails of filial generation mice and PCR method was employed to amplify the APPSWE gene fragment. The gene type was observed by electrophoresis.(3) PCR product was inserted in pGEM-T vector to detect its gene sequence. RESULTS: The male Tg2576 mice mated with female Tg2576, and four litters were reproduced, all of them died during two days. The male Tg2576 mice mated with female C57BL, and the survival rate of their filial generation was 81% and positive rate with APPSWE gene was 43.3%. The male C57BL mice mated with the female Tg2576,and the survival rate and positive rate with APPSWE gene of filial generation mice were 82.4% and 21.9%, respectively. By Wilcoxon's Rank Sum Test, there were no significant different between the survival rates of filial generation mice by two breeding way (P＞0.05), but there were significant different between the positive rates with APPSWE gene (P＜0.05). Electrophoresis result showed the molecular weight of PCR product was 428 bp and was in accordance with that of APPSWE gene fragment. By gene sequencing, gene sequence of PCR product was verified as same as APPSWE gene double mutant. CONCLUSION: It is ideal way of breeding filial generation mice with APPSWE gene that male Tg2576 mouse mate with female C57BL. PCR technique can identify the filial generation with APPSWE gene precisely, and offer an ideal animal model for further research on AD.     

Expression of α-synuclein with aging in APP transgenic model of Alzheimers disease

AIM: To investigate the expression of α-synuclein in the brain of AD-like animal model at different age and to explore the pathology mechanism of α-synuclein in neural degeneration.METHODS: APP V717I transgenic (Tg) mouse model of Alzheimers disease was observed at age of 4,10 and 16 months.The Tg mice were randomly divided into 3 model groups (4,10 and 16 months of age).Control adopted the same age and background C57BL/6J mice.The mRNA expression of α-synuclein was measured by genechips and RT-PCR method.The protein of α-synuclein was detected by immuno-histochemistry and Western blotting.RESULTS: The expression of α-synuclein mRNA increased significantly in hippocampus and cortex in Tg mice at age of 4 months,10 months and 16 months compared with age matched controls.The production of α-synuclein protein also increased significantly in hippocampus and cortex in Tg mice at 3 groups.With aging,α-synuclein expression was increased and aggregated in Tg mice.CONCLUSION: Overexpression and aggregation of α-synuclein in different age of APP transgenic mice may play a key role in learning-memory deficit and the pathology of Alzheimers disease,aging,and neural degeneration.     

Expression and identification of ANO1 in mouse cardiomyocytes

AIM: To explore the expression of anoctamin 1 (ANO1), one of calcium-activated chloride channels (CaCCs), in mouse cardiomyocytes and its functional properties. METHODS: The cardiomyocytes from the myocardial tissues of C57BL/6 mice were isolated with enzyme and purified by the differential adherent method. The cells were stained with monoclonal anti-sarcomeric actin and Cy3 to evaluate the purity of the myocardial cells. RT-PCR was used to detect the mRNA expression of ANO1 in the mouse cardiomyocytes. The protein expression of ANO1 in the mouse cardiomyocytes was determined by Western blotting analysis. The fluorescence quenching kinetics experiment was used to identify the ion transport properties of ANO1 in the mouse cardiomyocytes. RESULTS: The results of RT-PCR confirmed that ANO1 was expressed in freshly isolated myocardial cells. The results of Western blotting clearly demonstrated the protein expression of ANO1 in primarily cultured myocardial cells. Fluorescence quenching kinetics experiment on freshly isolated single myocardial cell revealed a pronounced outward rectifying property of the ANO1. The functional properties were similar to the classic CaCCs. CONCLUSION: ANO1 expression was identified in the mouse myocardial cells. The function of CaCCs was generated by ANO1, suggesting that ANO1 is the molecular basis of CaCCs.     

柑桔钙离子结合蛋白基因克隆及植物表达载体构建

以伏令夏橙叶片中分离的总RNA为模板,经RT-PCR扩增到一条约600bp、含钙离子结合蛋白基因(CsCaBP)的片段,将此片段克隆到pMD-19T中,经测序分析该片段与甜橙基因组中的对应序列完全吻合。设计2对带有限制性内切酶位点的特异性引物,以cDNA为模板扩增到2个CsCaBP片段,并连接到TA克隆载体pMD-19T上;经双酶切消化后,分别以正反2个方向插入到植物表达载体pFGC5941的查耳酮合成酶(CHSA)内含子两侧,构建成功CsCaBP的RNA干扰载体,但未获得转基因植株。将CsCaBP的正向片段定向克隆到具有CaMV35S启动子的pF-GC5941表达质粒上,构建成功CsCaBP过量表达载体。将构建好的表达载体导入根癌农杆菌LBA4404菌株,转化酸橙下胚轴,经PCR检测,获得9株过量表达转基因植株,荧光定量PCR验证发现目的基因在转基因植株中有不同程度的表达。     

PPARγ gene and protein expression in aortic plaque of different week-old apoE-knock out mice and the relationship with the composition of aortic plaque

AIM: To investigate the relationship of PPARγ gene expression with the composition of aortic plaque in apoE-knock out mice. METHODS: PPARγ gene and protein in aortic area of 20-week-old and 40-week-old apoE-knock out mice were investigated using RT-PCR and immunoblotting. The same aged wild type mice (C57BL/6J) were served as control (n=10). The composition of aortic plaques was analyzed by Movat method and oil red O staining. The expression of antigens such as PPARγ, SM-actin and MOMA-2 in aortic plaque were compared using immunohistochemistry. The relationship of PPARγ with macrophage, smooth muscle cells (SMC), lipid, elastic fiber, collagen and proteoglycan in aortic plaque were analyzed using immunofluorescence. RESULTS: PPARγ gene and protein in aortic wall and plaque of apoE-knock out mice were more significant than that in the same aged C57BL/6J mice (P<0.05). PPARγ expression at 40-week-old apoE-knock out mice was most significant and very low in C57BL/6J mice. More PPARγ expression of gene and protein at 20-week-old C57BL/6J mice than 40-week-old C57BL/6J mice were observed. Compared with 20-week-old apoE^-/- mice, the lipid pool in aortic plaque at 40-week-old apoE^-/- mice were increased remarkably, while elastic fiber, collagen and proteoglycan in plaque were decreased and aortic remodeling was very significant. Even, upregulation of MOMA-2 and downregulation of SM-actin were also detected in latter (P<0.05). In addition to SMC of aortic tunica media, PPARγ also expressed in SMC and macrophages in the aortic plaque of apoE^-/- mice. PPARγ was very enriched in lipid pool of the plaque. CONCLUSION: PPARγ expression level decreases with aging in C57BL/6J mice, while increases with plaque progression in apoE-knock out mice. There is positive correlation between PPARγ expression and lipid composition in plaque. The observed upregulation of PPARγ gene expression in aortic plaque may be a compensatory behavior and protective mechanism in apoE-knock out mice.     

Up-regulation of CD36 induced by casein-induced inflammation promotes renal injury in mice

AIM: To investigate the role of CD36 in casein-induced mouse renal injury.METHODS: Eight-week-old male C57BL/6J mice and CD36 knockout (CD36KO) mice were randomly divided into C57BL/6J saline injection group, C57BL/6J casein injection group and CD36KO casein injection group (n=8 in each group). After 14 weeks of treatment with high-fat diet, the mouse serum, 24 h urine and kidney tissue samples were collected for analysis. The serum content of tumor necrosis factor-α (TNF-α) was measured by ELISA. The renal function markers in the serum and urine were determined by an automatic biochemical analyzer. The pathological changes of the kidney were observed by HE staining and Masson staining. The expression of CD36 and cytokines/chemokines (TNF-α, IL-6 and MCP-1) at mRNA and protein levels in the renal tissues were determined by real-time PCR and Western blot. The content of tissue hydrogen peroxide (H₂O₂) was measured by a commercial kit. The protein levels of Nrf2 and TGF-β1 in the renal tissues were measured by immunohistochemical staining.RESULTS: Compared with saline injection group, casein injection increased the level of TNF-α in the serum and in the kidney tissues of C57BL/6J mice (P<0.05), suggesting that casein injection successfully induced chronic inflammation in C57BL/6J mice. Casein injection also promoted the protein expression of CD36 and TGF-β1 in the renal tissues of the C57BL/6J mice, accompanied with glomerular sclerosis, proteinuria, increased serum creatinine content, increased H₂O₂ content, and decreased Nrf2 protein level and the ability of antioxidant in the kidneys (P<0.05). Furthermore, CD36 deficiency protected the mice from casein-induced renal injury, as evidenced by improved kidney pathological changes and decreased proteinuria. The content of H₂O₂ in the kidneys of casein-treated CD36 knockout mice was also lower than that in casein-treated C57BL/6J mice.CONCLUSION: Inflammatory responses promote the oxidative stress and renal injury in a CD36-dependent manner.     

Molecular cloning of ING4 gene and ING4-induced apoptosis in HeLa cells

AIM: To isolate a gene encoding mouse ING4, construct pcDNA3.0-ING4 recombinant eukaryotic expression plasmid and investigate its effects on HeLa cells in vitro. METHODS: The mouse ING4cDNA was amplified by RT-PCR from mouse liver. The eukaryotic expression vector pcDNA3.0-ING4 was constructed by DNA recombination technique. The recombinant plasmid pcDNA3.0-ING4 was identified by PCR, restriction enzyme digestion and DNA sequence analysis, then was transfected into HeLa cells by lipofectamine. The expression was determined by RT-PCR. Apoptosis was detected by fluorescence microscope with Hoechst33258 staining and laser scanning confocal microscope. Cell cycle distribution was measured with flow cytometry. RESULTS: RT-PCR product was about 750 bp specific fragment. Analysis by restricting enzyme digestion and PCR of pcDNA3.0-ING4 recombiant plasmid showed that results were about 750 bp, DNA sequencing revealed that ING4 cloning were successful. With Hoechst fluorescence staining, we found that the percentage of apoptotic rate in HeLa cells transfected with pcDNA3.0- ING4 (21.25%) was higher than that in HeLa cells transfected with pcDNA3.0 (8.91%,P<0.01). Apoptosis was also detected by laser scanning confocal microscope. Cell cycle analysis reavealed the cell number in S phase of HeLa cells transfected with pcDNA3.0- ING4 increased. CONCLUSION: The gene encoding mouse ING4 and construction of pcDNA3.0- ING4 eukaryotic expression vector were successfully obtained, ING4 could enhance apoptosis in HeLa cells.     

苹果MdFT基因对番茄的遗传转化

 通过RT2PCR扩增, 从苹果叶片cDNA中克隆了^FT基因的同源基因^MdFT, 构建了花椰菜病毒35S启动子驱动的MdFT植物表达载体35S: : MdFT, 并利用根癌农杆菌介导法将其导入番茄栽培品种‘中蔬四号’; 同时转化拟南芥A tFT基因作为阳性对照。从添加卡那霉素的筛选培养基上再生了抗性植株,PCR扩增证明, 外源基因MdFT和AtFT已经整合到转基因番茄的基因组, 半定量RT-PCR则证明它们已经在转基因番茄中得到异位过量表达。形态鉴定发现, 转基因番茄植株比非转基因对照植株开花早, 表明成功地从苹果中克隆了成花素基因MdFT, 该基因具有通过转基因缩短苹果树童期的潜在价值。     

Establishment of a transgenic heterozygous mouse model of ApcMin/+ precancerosis of colorectal cancer with p110δ mutation

AIM：To establish a transgenic heterozygous mouse model of precancerous lesions of colorectal cancer with p110δ mutation in the C57BL/6J background for serving the studies on colorectal cancer research mediated by p110δ. METHODS：The transgenic heterozygous mice were generated by crossing in p110δD910A/D910A mouse and ApcMin/+ mouse, and the genotype was detected by PCR. Compared with ApcMin/+ mice, transgenic heterozygous mice (ApcMin/+; p110δD910A/D910A)were counted, and the number and size of intestine polyps were analyzed after methylene blue staining. The intestinal tissue structure was assessed by HE staining. RESULTS：The transgenic heterozygous mouse model of precancerous lesions of colorectal cancer with p110δ mutation was established. The number and size of polyps in the transgenic heterozygous mice were declined. CONCLUSION：A transgenic heterozygous mouse model of precancerous lesions of colorectal cancer with p110δ mutation was successfully established. The initial phenotype of intestinal tumors in transgenic mice was observed. This model will greatly contribute to the related research of colorectal cancer in mice.     

Expression of kinesin superfamily genes in striatum and substantia nigra in MPTP-treated mice

AIM: To investigated the effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on the expression of kinesin superfamily (KIF) genes in striatum and substantia nigra in C57BL mice. METHODS: The Parkinson's disease model was established by consecutive administration of MPTP to C57BL mice. The levels of mRNA for five kinesin superfamily genes, KIF1A, KIF2, KIF3A, KIF4, and KIF5A, in striatum and substantia nigra of mice, were estimated by RT-PCR. RESULTS: In substantia nigra, the expression of KIF genes were decreased after MPTP treatment except KIF2 that showed no significant change. However, the expression of KIF1A, KIF3A and KIF4 were increased in striatum after MPTP treatment, while the expression of KIF2 and KIF5A were similar to that in substantia nigra. CONCLUSION: The lose of dopaminergic neurons in nigrostriatal pathway after MPTP treatment may be related to the expression of KIF genes.     

Role of resistin in hepatic insulin resistance and the underlying mechanism

AIM: To investigate the role of resistin in hepatic insulin resistance and its mechanism. METHODS: A mouse model of hyperresistinemia in C57BL/6 mice was established by intravenous administration of the recombinant adenovirus encoding mouse resistin. Using periodic acid-Schiff staining we observed the effects of resistin on hepatic glycogen storage. Western blotting was used to measure AMPK-α protein and phosphrylated AMPK-α (Thr172) protein. mRNA levels of phosphoenolpyruvate carboxykinase gene and glucose-6-phosphatase gene were measured by real-time PCR. RESULTS: On day 5 after Adv injection, the concentration of plasma resistin was much higher in Adv-resistin-EGFP-treated mice than that in saline- or Adv-EGFP-treated mice. Semiquantitation of hepatic glucogen storage by PAS showed that the mice with hyperresistinemia had decreased glycogen particles compared to normal control and Adv-EGFP groups. The ratio of phosphorylated AMPK (Thr172)-α to total AMPK-α was used to evaluate hepatic AMPK activation. Compared with normal control and Adv-EGFP groups, the Adv-resistin-EGFP-treated mice had significantly lower ratio of p-AMPK/AMPK, and higher expression levels of G6Pase and PEPCK mRNA in liver. CONCLUSION: Resistin may decrease AMPK activation with downregulating expression of gluconeogenic enzymes, resulting in increased glucose production and decreased hepatic glycogen storage. Resistin may play an important role in hepatic insulin resistance.     

Effect of bone marrow mesenchymal stem cells on engraftment of hematopoietic stem/progenitor cells in sensitized mice

AIM: To evaluate the effects of bone marrow-derived mesenchymal stem cells (MSCs) on engraftment of hematopoietic stem/progenitor cells in sensitized mice. METHODS: Mouse bone marrow-derived MSCs were cultured by adherent culture method. MSCs combined with or without hematopoietic stem/progenitor cells were implanted into the sensitized mouse model, which was established by allogeneic splenocyte transfusion, and were divided into 6 groups: MSC intervention groups, including sensitized mice with MSCs on day 11, sensitized mice with MSCs on day 0 and sensitized-mice with MSCs both on day 11 and day 0; control groups, including sensitized mice without MSC intervention, non-sensitized mice without MSC intervention and non-sensitized mice without MSCs or transplantation of hematopoietic stem/progenitor cells. The survivors were assessed after transplantation and hematopoietic recovery was monitored weekly including hematological change, immune function reconstruction, bone marrow cell recovery, chimera analysis and graft-versus-host disease development. RESULTS: Compared with different control groups, MSC intervention did not prolong the survival rates of the sensitized model mice after lethal irradiation. CONCLUSION: Under the experimental conditions, MSC combined with C57BL/6 bone marrow hematopoietic stem/progenitor cells fail to promote the growth of engraftment in C57BL/6 allogeneic splenocyte-sensitized BALB/c mice in vivo.     

Effects of recombinant adenovirus-mediated chronic hyperresistinemia on glucose metabolism in mice

AIM: To establish the animal model of hyperresistinemia and to observe the effects of resistin on glucose metabolism and insulin sensitivity in vivo. METHODS: We established a mouse model of hyperresistinemia in C57BL/6 mice by intravenous administration of the recombinant adenovirus encoding mouse resistin. Then we observed the fasting blood glucose and insulin levels. We also investigated glucose tolerance by IPGTT, and insulin sensitivity by IPITT. RESULTS: On 5 d after the injection, the concentration of plasma resistin was more than 15-fold higher in Adv-resistin-EGFP-treated mice than that in saline- or Adv-EGFP-treated mice. In the fasting state, no difference in glucose levels was observed among three groups. However, mice injected with Adv-resistin showed higher insulin levels, impaired glucose tolerance and insulin sensitivity. CONCLUSION: Hyperresistinemia affects glucose metabolism in mice and it may play an important role in the pathogenesis of insulin resistance and type 2 diabetes.     

Improvement of insulin sensitivity by osteocalcin inhibits inflammation in the adipose tissue of obese mice

AIM: To explore the improving effect of osteocalcin on obesity-related insulin resistance and inflammation in the adipose tissue of obese mice.METHODS: The C57BL/6 mice were fed with high-fat diet for 12 weeks to obtain obese mice. Osteocalcin (30 ng/kg or 3 ng/kg) and saline solution (control) were intraperitoneally injected for other 4 weeks. The fat mass, body weight, serum triglycerides and serum free fatty acid were analyzed. Intraperitoneal glucose tolerance test and insulin tolerance test were carried out. Macrophage infiltration degree in the adipose tissue was observed by immunohistochemical staining. The mRNA expression of monocyte chemotactic protein-1 (MCP-1) and CD68 was detected by real-time fluorescence quantitative PCR.RESULTS: Osteocalcin (30 ng/kg or 3 ng/kg) treatment for 4 weeks significantly reduced the body weight, fat mass and insulin level, and improved abnormal glucose tolerance and insulin resistance in the obese mice. Moreover, the macrophage infiltration decreased, and the mRNA expression of MCP-1 and CD68 was down-regulated in the adipose tissue of obese mice treated with osteocalcin at 30 ng/kg.CONCLUSION: Osteocalcin at 30 ng/kg significantly reduces body weight and fat mass, and attenuates the severity of insulin resistance through down-regulating the mRNA expression of MCP-1 and CD68 and inbihiting macrophage infiltration in the adipose tissue of obese mice induced by high-fat diet.     

Establishment of an acute GVHD animal model in EL9611 erythroleukemia mice

AIM: To establish an acute graft-versus-host disease (GVHD) model in EL9611 erythroleukemia mice. METHODS: Using C57BL/6 (H-2^b) mice as the donor and BALB/c (H-2^d) mice as the recipient in allogeneic bone marrow transplantation (allo-BMT), the acute GVHD model was established. The mice were divided into leukemia group (n=10), radiation control group (leukemic mice given radiation without allo-BMT, n=4), GVHD group (leukemic mice given radiation+allo-BMT, n=10) and normal control group (n=4). In leukemia group, 2×10⁶/mouse EL9611 erythroleukemic cells were transfused via tail vein into BALB/c mice to build the erythroleukemia model. In GVHD group, 7 days after leukemic cell transfusion, the mice received total dose of 8.0 Gy γ of total body irradiation(TBI), and within 5 h, 2×10⁶ C57BL/6 bone marrow cells and 1×10⁷ C57BL/6 spleen cells per mouse were transfused via tail vein to build the acute GVHD model in EL9611 erythroleukemia mice. The clinical manifestations of posture, fur, stool and so on were observed. Pathological examination was conducted to examine the changes of liver, spleen, skin, small intestine and peripheral blood. The survival rate was also calculated. RESULTS: (1) In leukemia group, the mean survival time (MST) was (14.5±2.1) days,or (7.5±0.7) days when irradiation day was as day 0(P<0.01 compared with GVHD group). The death rate was 100% with no spontaneous remission. The dead mice showed splenohepatomegalia and high WBC count . Pathological examination showed disorganization of normal tissues and leukemic cell infiltration. (2) In radiation control group, MST was (9.0±0.7) d, with significant difference as compared with GVHD group and normal group (P<0.01). The death rate was 100%. Pathological examination showed hematopoiesis exhaustion. (3) In GVHD group, MST was (32.0±3.2) d (P<0.01 compared with other groups). The death rate was 100%, the symptoms were observed on day 10-13 after allo-BMT. Clinical manifestations and pathological examination corresponded to those of I degree to II degree of GVHD. CONCLUSION: Intravenous infusion of 2×10⁶/mouse EL9611 leukemic cell successfully establishes the EL9611 erythroleukemia animal model. Seven days after EL9611 leukemic cell transfusion, lethal dose of TBI and allo-BMT can successfully build the acute GVHD model of EL9611 leukemic mice.     

Increase in proliferation of adventitial fibroblasts at early stage of atherosclerosis in apoE(-/-) mice

AIM: To study the relationship between the proliferation of adventitial fibroblasts and the early formation of atherosclerotic lesion. METHODS: ApoE(-/-) mice and wild-type C57BL/6 black mice (6-week old) were used in this experiment. 5-Bromo-2-deoxyuridine (BrdU) was administered 24 h before the animals were sacrificed at time points of 0, 2, 4 or 10 weeks after hyperlipidic diet, respectively. The ascending aorta was removed for serial sectioning. The morphological changes of the aortic tissues were observed under microscope with HE staining. Some sections of the aorta were used to observe the BrdU labeling in the adventitia and intima at different time points by the method of immunohistochemistry. In addition the arterial adventitial fibroblasts derived from apoE(-/-) mice and C57BL/6 mice after hyperlipidic diet for 2 weeks were cultured. The proliferation of the cells was examined by BrdU incorporation. The cell cycle was examined by flow cytometry. RESULTS: In vivo the BrdU-labeled cells were first found in the adventitia of apoE(-/-) mice after feeding with hyperlipidic diet for 2 weeks, the time point that no visible intimal lesion formation was found, and then subsequently emerged in the intimal lesion. No BrdU labeling was observed in C57BL/6 mice at any time point. The number of BrdU labeled cells in the adventitial fibroblasts of apoE(-/-) mice was significantly higher than that in C57BL/6 mice in vitro (P<0.01). The adventitial fibroblasts of apoE(-/-) mice exhibited higher percentages of S and G₂/M phases than those in C57BL/6 mice (P<0.05). CONCLUSION: The proliferation of arterial adventitial fibroblasts might participate in the early formation of atherosclerotic lesions.     

Study on thrombomodulin and von Willebrand factor in microvasculature in mice lungs with pulmonary fibrosis induced by bleomycin

AIM:To explore the distribution of thrombomodulin (TM) and von Willebrand factor (vWf) on endothelial cells of lung microvasculature as well as the phenotype change of this cell in the process of pulmonary fibrosis in C57BL/6 mice. METHODS:It was carried out with dual immunofluorescent stain and quantitative analysis of fluorescent intensity of TM and vWf in normal and pulmonary fibrosis model induced by bleomycin (BLM) administration. RESULTS:① The normal lungs showed multiple continuous linear positive staining of TM and seldom positive of vWf on the surface of endothelium of alveolar capillaries. Meanwhile, blood vessels exhibited considerable positive of vWf in endothelial cell in normal C57BL/6 mice. ② The fibrotic lungs revealed a statistically significant diminution of TM expression, and at the same time, an increase of vWf expression when comparing with normal lung sections.CONCLUSION:These results suggest that TM dominant phenotype endothelial cells, rather than vWf dominant phenotype, are the major ones of alveolar capillaries in normal C57BL/6 mice lungs. TM dominant phenotype endothelial cells changed into vWf dominant ones as pulmonary fibrosis develops. Both TM and vWf antigen might be considered as markers of endothelium injury of lung microvasculature.