Ultrastructural impairment of islet microvascular endothelial cells in STZ-induced type 1 diabetic mice

AIM: To investigate the ultrastructural changes of islet microvascular endothelial cells in STZ-induced type 1 diabetic mice. METHODS: BALB/c mice were randomly divided into diabetic group and control group. The expression of insulin and platelet-endothelial cell adhesion molecule-1 (CD31) in islet microvessels was detected by immunohistochemical staining. The ultrastructural changes of islet β cells and islet microvessels were observed under transmission electron microscope. RESULTS: Compared with control group, the number of islet β cells, ratio of β cells/α cells, average number of secretory granules in β cells and insulin expression area per islet in diabetic group were significantly decreased (P<0.01). Besides, diabetic group had fewer microvessels with lower expression of CD31 (P<0.01). Mitochondria in islet microvascular endothelial cells and pericytes in diabetic group were swelling. The basement membrane of islet microvessels became thicker in diabetic group (P<0.01). CONCLUSION: Islet microvascular endothelial cells were impaired in type 1 diabetic mice.     

Vitamin C enhanced myocardial differentiation of dedifferentiated fat cells

AIM: In order to observe the myocardial differentiation capacity of the dedifferentiated fat (DFAT) cells treated with vitamin C in vitro. METHODS: DFAT cells were dedifferentiated from the mature rat adipocytes with ceiling adherent culture. The DFAT cells of passage 3 were used in the study. Vitamin C and/or neonatal rat heart tissue lysate were added into the culture medium to induce myocardial differentiation for 3 weeks. The cell morphology was observed under microscope. The myocardial-specific markers, such as cTnT, GATA-4 and NKx2.5, were examined by the methods of immunofluorescence, PCR and Western blot. RESULTS: Mature rat adipocytes dedifferentiated into fibroblast-like DFAT cells after ceiling adherent culture. The DFAT cells spontaneously differentiated into cardiomyocyte-like cells under normal culture condition with a low incidence. After treated with neonatal rat heart cell lysate, the DFAT cells became cardiomyocyte-like cells that had bigger size, longer shape and myotubule-structure. The expression of cTnT, GATA-4 and NKx2.5 was remarkably increased at both mRNA and protein levels as compared with the normal cultured DFAT cells. The expression of cTnT, GATA-4 and NKx2.5 was further increased in DFAT cells after treating with vitamin C. No spontaneous beating cell was observed. CONCLUSION: Vitamin C enhances the differentiation of DFAT cells into cardiomyocyte-like cells.     

Periplaneta americana peptide decreases apoptosis of pig-ovary granulosa cells induced by H2O2 through FoxO1

Oxidative stress can induce apoptosis of granulosa cells and lead to follicular atresia, thereby reducing the number of pigs giving birth. The aim of this study was to investigate the protective effect of Periplaneta americana peptide (PAP) on the apoptosis of the granulosa cells of pig ovaries (PGCs) induced by hydrogen peroxide (H₂O₂) via FoxO1. PGCs were treated with H₂O₂ to establish a cell apoptosis model. Cell viability was measured using the cell counting kit-8 (CCK-8) assay, and cell apoptosis was detected using flow cytometry. The malondialdehyde (MDA) level and nitric oxide (NO) content were detected to reflect the oxidative stress. Western blotting, qRT-PCR and overexpression were undertaken to determine the expression of FoxO1 and caspase-3, and immunofluorescence was used to detect FoxO1 in the nucleus and cytoplasm. PGCs were treated with 100 μM H₂O₂ for 6 hr, which resulted in oxidative damage and apoptosis and an apoptosis rate for PGCs of 32.95%. Next, PGCs were treated with 400 μg/ml PAP for 24 hr to repair the apoptosis induced by H₂O₂. PAP improved cell viability in H₂O₂-stimulated PGCs, the increased MDA level and NO content caused by H₂O₂ stimulation were reversed and the apoptotic rate of PGCs was reduced. The qRT-PCR and Western blotting results indicated that PAP decreased the H₂O₂-induced apoptosis and the expression of FoxO1 and caspase-3 in PGCs. The effect of PAP was the same following FoxO1 overexpression. FoxO1 was expressed in the nucleus when stimulated by H₂O₂ or overexpression; however, it migrated to the cytoplasm following PAP treatment. PAP decreased the apoptosis of PGCs induced by H₂O₂ by regulating FoxO1 expression and nuclear translocation.     

猪lncRNA TCONS_00791383对骨骼肌卫星细胞增殖分化的影响

为了探究lncRNA TCONS_00791383对猪骨骼肌卫星细胞增殖和分化的影响。本研究利用qRT-PCR技术检测出生7 d内大白仔猪6种组织（心、脾、肺、肾、背肌和腿肌）以及猪骨骼肌卫星细胞增殖分化前后TCONS_00791383的表达水平；通过设计反义核苷酸（antisense oligonucleotides，ASO）片段在猪骨骼肌卫星细胞中对TCONS_00791383进行敲低，检测敲低TCONS_00791383之后增殖分化标志基因的表达量变化；通过trans （co-expression）对TCONS_00791383进行靶基因预测，使用DAVID对其进行GO富集和KEGG通路分析。结果显示，TCONS_00791383在猪心脏中表达量最高，在脾和肾组织中不表达。在骨骼肌卫星细胞从增殖到分化的过程中，TCONS_00791383的表达量逐渐上升，且在分化后30 h表达量达到最高。在使用ASO片段敲低TCONS_00791383之后，与对照组相比，在分化24 h，增殖标志基因Pax3、Pax7表达量显著或极显著降低（P<0.05，P<0.01），分化标志基因MyoG表达量极显著降低（P<0.01），在分化48 h，增殖标志基因Pax3表达量极显著降低（P<0.01），Pax7表达量显著降低（P<0.05），分化标志基因MyHC表达量显著降低（P<0.05）。预测得到的相关靶基因富集到AMPK、ATP等多个与骨骼肌卫星细胞增殖和分化过程相关的重要信号通路。本研究表明，lncRNA TCONS_00791383可能促进猪骨骼肌卫星细胞的增殖和分化。     

消化酶及胎牛血清对奶牛乳腺上皮细胞培养的影响

试验研究了不同浓度及组合消化酶对奶牛乳腺组织分离的影响以及不同FBS浓度培养基对奶牛乳腺上皮细胞纯化和生长的影响。试验结果表明,0.25%胰酶＋0.20%Ⅱ胶原酶对组织块分离效果最好,乳腺上皮细胞生长最多,5%与10%浓度FBS对奶牛乳腺上皮细胞纯化效果最好,5%与10%浓度FBS对细胞生长没有明显的差异（P〉0.05...     

不同日龄滩羊皮肤组织化学特征比较

为比较2、35日龄滩羊皮肤毛囊的发育特点与血管内皮生长因子（vascular endothelial growth factor，VEGF）和血管内皮生长因子受体2（vascular endothelial growth factor receptor 2，VEGFR2）的分布特征，探究出生后滩羊被毛生长发育的变化特点，试验应用常规HE染色及改良Masson胶原纤维染色、Gomori银氨法染色、磷钨酸染色等特殊染色观察2与35日龄滩羊皮肤组织结构特点；应用免疫组织化学法结合免疫荧光染色法观察VEGF及VEGFR2在2与35日龄滩羊皮肤组织中的分布定位，并用IPP图像分析软件进行定量分析。结果显示：与2日龄滩羊皮肤组织比较，35日龄滩羊表皮与真皮间界限更加清晰，毛囊结构发育完整；毛囊密度显著降低（P<0.05）；胶原纤维与弹性纤维含量增加，形成网格状分布。免疫组化及免疫荧光结果显示，VEGF及VEGFR2在滩羊皮肤表皮及毛囊外根鞘、皮脂腺上均有表达。统计表明，VEGF及VEGFR2在2日龄滩羊皮肤组织中的表达量均显著高于35日龄（P<0.05）。综合上述结果，滩羊毛囊发育过程中，胶原纤维和弹性纤维增加明显；VEGF与VEGFR2通路在毛囊角质形成中起直接调节作用。     

过表达MyBPC1对牛骨骼肌卫星细胞增殖分化的影响

旨在探究肌球蛋白结合蛋白C1（myosin binding protein C1,MyBPC1）对牛骨骼肌卫星细胞增殖与成肌分化的影响,为进一步研究MyBPC1在细胞分化和肌肉发育过程中的调控作用提供依据。本研究利用西门塔尔胎牛原代牛骨骼肌卫星细胞体外诱导成肌分化模型模拟牛骨骼肌的生长发育过程。采用qRT-PCR和Western blot检测MyBPC1的细胞时序表达谱。试验分为两组。在RNA水平每组4个重复,每个重复20 μL;在蛋白水平每组3个重复,每个重复15 μg。采用qRT-PCR和Western blot检测牛骨骼肌卫星细胞转染MyBPC1的过表达效果,并进一步检测细胞增殖期标志因子Pax7、Ki67以及细胞分化期标志因子MyHC、MyOG的表达变化情况,观察牛骨骼肌卫星细胞肌管形成状态。结果,MyBPC1在牛骨骼肌卫星细胞分化前后表达水平存在极显著差异,牛骨骼肌卫星细胞诱导分化后MyBPC1的mRNA和蛋白表达量均极显著高于增殖期（P<0.01）。过表达MyBPC1后,细胞分化形成的肌管数量明显多于对照组,增殖标志因子Pax7的mRNA水平和蛋白表达水平无显著差异,分化标志因子MyHC的mRNA水平和蛋白表达水平极显著高于对照组（P<0.01）。过表达MyBPC1可以促进牛骨骼肌卫星细胞体外成肌分化,为进一步开展MyBPC1对牛骨骼肌卫星细胞的调控机制奠定基础。     

Fas对镉激活PC12细胞线粒体凋亡通路的影响

旨在探究死亡受体Fas在镉致大鼠肾上腺嗜铬细胞瘤细胞（PC12）凋亡中的作用及其对线粒体通路的调控机制,用10 μmol·L^-1镉处理Fas基因沉默的PC12细胞株12 h,通过Western blot检测BH3相互作用域死亡激动剂（BID）、半胱氨酸蛋白酶-9（caspase-9）、半胱氨酸蛋白酶-3（caspase-3）、多聚二磷酸腺苷核糖聚合酶（PARP）的活化情况,Bcl-2相关X蛋白（Bax）、B细胞淋巴瘤/白血病-2基因（Bcl-2）、凋亡诱导因子（AIF）、核酸内切酶G（Endo G）的表达情况,以及细胞色素C（Cyt C）在细胞内的分布情况,免疫荧光染色检测AIF核转位。结果显示,镉极显著上调tBID/BID比值和Bax/Bcl-2比值,诱导Cyt C从线粒体释放到细胞质,激活caspase-9、caspase-3和PARP,增加AIF和Endo G蛋白表达水平（P<0.01）,并诱导AIF核转位;沉默Fas极显著抑制镉引起的tBID/BID比值和Bax/Bcl-2比值升高,Cyt C从线粒体释放到胞浆,caspase-3、PARP蛋白活化和AIF、Endo G蛋白表达水平极显著升高（P<0.01）,显著抑制镉激活的caspase-9（P<0.05）,并抑制AIF核转位。综上表明,Fas通过调控线粒体通路参与镉致PC12细胞凋亡。     

Mesh-like vascular changes in copper deficiency-induced rat cardiomyopathy

The pathological effects of copper deficiency (COD) are well known. However, the pathogenesis of cardiomyopathy resulting from COD remains unclear. In this study, aimed to elucidate the pathogenesis of COD-induced cardiomyopathy by examining the morphology of the cardiovascular system in copper-deficient rats using histopathology, immunohistochemistry, and scanning and transmission electron microscopy. Changes detected in the myocardium and interstitium were consistent with those reported for COD. Morphological changes included mesh-like changes in the capillary endothelial cells that appear to be a novel finding in COD-induced cardiomyopathy. These changes are hypothesized to result from abnormal vascular remodeling following damage to the basement membrane and due to the mechanical effects of myocardial contractions. Although cardiomyopathy may be associated with microcirculatory disorders arising from these lesions, further investigations are necessary to demonstrate a causal relationship between the pathogenesis of cardiomyopathy and the contribution of these lesions to disease progression.