雌激素和孕酮对犬子宫内膜基质细胞增殖和孕酮受体表达的影响

本试验应用不同消化分离途径获取犬子宫内膜基质细胞,调节培养液中雌激素(E₂)和孕酮(P₄)的浓度,采用MTT法测定E₂和P₄浓度水平对犬子宫内膜基质细胞体外增殖的影响,利用细胞免疫组织化学法鉴定细胞并测定细胞孕酮受体(PR)表达与激素浓度水平的相关性。结果表明,E₂浓度变化(15、30、100 pg/mL)对犬子宫内膜基质细胞的增殖和PR的表达均没有显著的调节作用(P>0.05);P₄(15、30 ng/mL)对犬子宫内膜基质细胞的增殖有显著促进作用(P<0.05),P₄(3、15、30 ng/mL)对犬子宫内膜基质细胞PR的表达具有显著的抑制作用(P<0.05),其影响程度与浓度和作用时间关系密切。     

Effects of coumestrol administration to maternal mice during pregnancy and lactation on immunoblogulin A‐secreting cells in mammary glands

Mortality and morbidity of neonates continue to be major problems in humans and animals, and immunoblogulin A (IgA) provides protection against microbial antigens at mucosal surfaces. The present study was conducted to clarify the effects of coumestrol administration to maternal mice during pregnancy and lactation on IgA antibody‐secreting cells (ASC) in mammary glands in lactating mice. From 6.5 to 16.5 days post coitus and 1 to 13 days post partum (dpp), maternal mice were administered coumestrol at 200 μg/kg body weight/day. Coumestrol administration increased the number of IgA ASC and the messenger RNA expression of IgA C‐region and vascular cell adhesion molecule‐1 in mammary glands of maternal mice at 14 dpp, but coumestrol administration had no effect on the number of IgA ASC in the ileum. Coumestrol administration increased serum IgA concentration in maternal mice at 14 dpp, but IgA concentrations in serum, stomach contents, intestine and feces of neonatal mice were not affected by treatment. These results imply that coumestrol administration to maternal mice during pregnancy and lactation is effective in increasing the numbers of IgA ASC in mammary glands during lactation owing to the activated messenger RNA expressions of IgA C‐region and vascular cell adhesion molecule‐1 in mammary gland.     

Preliminary experiments on mechanical stretch-induced activation of skeletal muscle satellite cells in vivo

We have shown in vitro that mechanical stretch triggers activation of quiescent satellite cells of skeletal muscle to enter the cell cycle through an intracellular cascade of events including nitric oxide (NO) synthesis that results in the release of hepatocyte growth factor (HGF) from its extracellular association and its subsequent presentation to signaling receptors. In order to explore the activation mechanism in vivo, stretch experiments were conducted in the living animal using our suspension model developed. This system used the weight of the hind portion of rats to stretch the inside muscles of the left hind limb suspended for a period of 0.5–2.0 h. At the end of the stretch period, the rats received an intraperitoneal injection of bromodeoxyuridine followed by immunocytochemistry for its incorporation as an index of satellite cell activation in vivo. Depending on the period of stretch, bromodeoxyuridine labeling was increased significantly over the contralateral unstretched leg or control muscle from untreated rats. A stretched muscle extract prepared from the 2 h stretched tissue by incubating it in PBS, showed the active form of HGF as revealed by immunoblotting and it could stimulate the activation of unstretched satellite cells. Also, administering NO synthase inhibitor L‐NAME prior to muscle stretch abolished the stretch activation of satellite cells. Therefore, the results from these experiments demonstrate that stretching muscle triggers NO synthesis and HGF release, which could activate satellite cells in vivo.     

应用兔肾原代细胞培养 兔粘液瘤病毒的研究

试用兔肾原代细胞传代；增殖兔粘液瘤病毒，呈现典型ＣＰＥ。粘液瘤病毒ＳＧ３３株在兔肾细胞上连续传３代之后，就不产生ＣＰＥ，在鸡胚上传３代再回归兔肾原代细胞，又呈现ＣＰＥ。ＳＧ＿（３３）在鸡胚上不能产生明显痘斑。维持液ｐＨ值影响ＳＧ＿（３３）在兔肾原代细胞上形成ＣＰＥ。     

Long‐term ingestion of low amylose/amylopectin ratio diet affects aspects of meat quality by changing muscle fibre characteristics in growing‐finishing pigs

Amylose plays important role in body health. It is controversial whether changing dietary amylose/amylopectin ratio (DAR) will improve meat quality in growing‐finishing pigs. A total of 48 Duroc × Landrace × Large White castrated male pigs (initial body weight 49.8 ± 2.8 kg) were randomly allotted to two treatments, and fed ad libitum either with a low DAR diet (LR, amylose/amylopectin: 12/88) or a high DAR diet (HR, amylose/amylopectin: 30/70) for 68 days. Feed intake was recorded every day, body weight was weighed at 46th and 68th day to calculate average daily gain (ADG), average daily feed intake (ADFI) and Feed:gain ratio. Blood was collected at ?30 min (fasting 12 hr), 60, 90, 120, 180 min postprandial at 64th day and then serum was obtained by centrifugation of blood at 1,500× g at 4°C. After pigs were slaughtered, samples such as longissimus dorsi, iliopsoas and semitendinosus were collected. Density, diameter and types of muscle fibres were analysed. Results showed that ADG, ADFI, Feed:Gain ratio, cross‐sectional area of longissimus dorsi muscle, backfat thickness, colour scores were not affected by DAR. Ingestion of LR diet increased the fasting glucose (p < 0.05) and insulin (p < 0.05) concentrations in serum. The drip loss and firmness were decreased significantly in LR vs. HR animals (p < 0.05). Densities of muscle fibre in longissimus dorsi, iliopsoas and semitendinosus were greater in LR pigs (p < 0.05). Moreover, ingestion of LR diet significantly increased myosin heavy chain (MyHC) IIa mRNA level and decreased MyHC IIb gene expression in longissimus dorsi muscle (LM) (p < 0.05). Therefore, intake of diet low in amylose/amylopectin ratio induces a better meat quality (lower drip loss and lower firmness), which could attribute to smaller myofibres, a shift to slower and/or more oxidative fibres.     

Effect of lncRNA FEZF1-AS1 on viability and apoptosis of LPS-induced vascular endothelial cells by regulating miR-363-3p expression

AIM To investigate the mechanism of long noncoding RNA （lncRNA） FEZF1-AS1 regulating microRNA-363-3p （miR-363-3p） on the viability and apoptosis of lipopolysaocharide （LPS）-induced vascular endothelial cells. METHODS Human umbilical vein endothelial cells （HUVECs） were cultured in vitro. pcDNA-NC, pcDNA-FEZF1-AS1, anti-miR-NC, anti-miR-363-3p, miR-NC and miR-363-3p mimics were transfected into the HUVECs and LPS stimulation was applied for 24 h. RT-qPCR was used to detect the expression of FEZF1-AS1 and miR-363-3p. The cell viability was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The dual-luciferase reporter experiment was used to verify the targeted regulation of FEZF1-AS1 and miR-363-3p. Western blot was used to determined the expression of cyclin D1, Ki67 and cleaved caspase-3. RESULTS Compared with control group, the expression level of FEZF1-AS1 in LPS group was significantly reduced （P<0.05）, and the expression level of miR-363-3p was significantly increased （P<0.05）. Compared with pcDNA-NC+LPS group, the cell viability in pcDNA-FEZF1-AS1+LPS group was significantly increased （P<0.05）, the apoptotic rate was significantly reduced （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）, and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Compared with anti-miR-NC+LPS group, the cell viability in anti-miR-363-3p+LPS group was significantly increased （P<0.05）, the apoptotic rate was significantly reduced （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）, and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Dual-luciferase reporter experiment confirmed that FEZF1-AS1 targeted miR-363-3p. Compared with miR-NC+pcDNA-FEZF1-AS1+LPS group, the cell viability in miR-363-3p+pcDNA-FEZF1-AS1+LPS group was significantly reduced （P<0.05）, the apoptotic rate was significantly increased （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly reduced （P<0.05）, and the protein level of cleaved caspase-3 was significantly increased （P<0.05）. CONCLUSION Over-expression of FEZF1-AS1 promotes the viability and inhibits apoptosis of LPS induced vascular endothelial cells by inhibiting the expression of miR-363-3p.     

Expression of matrix metalloproteinases in bovine luteal cells induced by prostaglandin F2α, interferon γ and tumor necrosis factor α

We recently demonstrated that luteal cells flow out from the ovary via lymphatic vessels during luteolysis. However, the regulatory mechanisms of the outflow of luteal cells are not known. Matrix metalloproteinases (MMPs) can degrade the extracellular matrix and basal membrane, and tissue inhibitors of matrix metalloproteinases (TIMPs) inhibit the activity of MMPs. To test the hypothesis that MMP expression in luteal cells is regulated by luteolytic factors, we investigated the effects of prostaglandin F2α (PGF), interferon γ (IFNG) and tumor necrosis factor α (TNF) on the mRNA expression of MMPs and TIMPs in cultured luteal cells. Luteal cells obtained from the CL at the mid-luteal stage (days 8–12 after ovulation) were cultured with PGF (0.01, 0.1, 1 μM), IFNG (0.05, 0.5, 5 nM) and TNF (0.05, 0.5, 0.5 nM) alone or in combination for 24 h. PGF and IFNG significantly increased the expression of MMP-1 mRNA. In addition, 1 μM PGF in combination with 5 nM IFNG stimulated MMP-1 and MMP-9 mRNA expression significantly more than either treatment alone. In contrast, IFNG significantly decreased the level of MMP-14 mRNA. The mRNA expression of TIMP-1, which preferentially inhibits MMP-1, was suppressed by 5 nM INFG. One μM PGF and 5 nM IFNG suppressed TIMP-2 mRNA expression. These results suggest a new role of MMPs: luteal MMPs stimulated by PGF and IFNG break down the extracellular matrix surrounding luteal cells, which accelerates detachment from the CL during luteolysis, providing an essential prerequisite for outflow of luteal cells from the CL to lymphatic vessels.     

Role of ghrelin in cell protection by up-regulating HSP70 through ERK1/2 signaling pathway in PC12 cells

AIM:To study the role of ghrelin in cell protection by up-regulating heat shock protein 70 (HSP70) and inhibiting apoptosis induced by oxidative stress through extracellular regulated protein kinases 1/2 (ERK1/2) signaling pathway in the PC12 cells. METHODS:Sodium nitoprusside (SNP) was used to induce oxidative stress injury in the PC12 cells. The cultured PC12 cells were divided into SNP-injured group (incubated with SNP at 0.5 mmol/L for 6, 12, 18 and 24 h), ghrelin pretreatment group (ghrelin at 100 nmol/L was given 30 min before adding SNP); HSP70 inhibitor group (quercetin at 10 μmol/L was added 60 min before ghrelin treatment), ERK inhibitor group (ERK 1/2 inhibitor PD98059 was added 60 min before ghrelin treatment) and control group (added same amount of culture medium only). The apoptotic rate was detected by flow cytometry. The protein expression was determined by Western blot and immunocytochemistry. RESULTS:Compared with control group, the apoptotic rate of PC12 cells in SNP-injured group was significantly increased (P<0.05). Compared with SNP-injured group, ghrelin (100 nmol/L) pretreatment significantly inhibited SNP-induced apoptosis of PC12 cells (P<0.05), and significantly up-regulated the protein expression of HSP70 (P<0.05). Time-effect analysis showed that ghrelin had the most significant effect at 18 h after SNP injury. Quercetin, an inhibitor of HSP 70, significantly reduced the anti-apoptotic effect of ghrelin (P<0.05). Ghrelin pretreatment promoted the phosphorylation of ERK1/2. ERK1/2 inhibitor PD98059 significantly inhibited the effects of ghrelin on up-regulation of HSP70 expression (P<0.05). CONCLUSION:Ghrelin upregulates the expression of HSP70 and inhibits the apoptosis in the PC12 cells induced by oxidative stress by promoting the phosphorylation of ERK1/2.     

The transdifferentiation of Sca-1+ cells from murine fetal liver

AIM:To explore transdifferentiation potential of Sca-1⁺ cells from murine fetal liver. METHODS:2×10³ of Sca-1⁺ cells from male murine fetal liver were transfused into female mouse irradiated lethally with γ ray from ⁶⁰ Co source (10 Gy) via tail vein. Two months later, FISH and immunohistochemistry were used to detect the situation for transdifferentiating of the donor cells (male cells) in tissues of female recipient mouse. RESULTS:The renal tubular epitheliocyte-like and neurocyte-like cells with Y chromosome were found on the sections of renal and brain tissues from female recipient mice. These cells have phenotype characteristics of RCA⁺/CD₄₅^-F_4/80^- and NueN⁺/CD₄₅^-F_4/80^-, respectively. CONCLUSION:The evidence is provided for Sca-1⁺ cells from murine fetal liver to transdifferentiate into both renal and brain tissue cells.