大鼠骨骼肌卫星细胞诱导分化为胰岛素生成细胞的研究

目的探讨大鼠骨骼肌卫星细胞（MDSCs）定向诱导分化为胰岛素生成细胞（IPCs）,为1型糖尿病的干细胞治疗提供一种新的研究思路。 方法通过二次酶消化法和差速贴壁培养法分离、培养大鼠MDSCs,利用不同的诱导培养液使MDSCs定向分化为IPCs,并对诱导后细胞进行形态观察,通过双硫腙染色和免疫组化染色对MDSCs-IPCs形态进行鉴定,采用Q-PCR和Western Blot方法检测MDSCs-IPCs中C-peptide和Insulin的表达,通过胰岛素释放实验检测MDSCs-IPCs的生物学功能,β细胞和MDSCs-IPCs两组间比较采用t检验。 结果MDSCs在接种4 h后开始贴壁部分细胞伸出小的突起,48 h后绝大多数细胞贴壁呈梭形、胞浆丰富、折光度高。随着培养时间的延长,细胞的梭形形状更为明显且生长迅速。免疫组化结果显示细胞表达Desmin、α-Sarcomeric Actinin、MyoD1、Myf5和PAX7。成胰诱导后MDSCs形成胰岛样的圆形细胞团,双硫腙染色呈猩红色,Insulin免疫组化染色阳性。Q-PCR结果显示MDSCs-IPCs中C-peptide和Insulin mRNA表达量分别是β细胞的0.73倍（P > 0.05）和0.79倍（P > 0.05）。胰岛素释放实验显示,5.6 mmol/L和16.7 nmlol/L葡萄糖刺激培养2 h后,β细胞和MDSCs-IPCs分泌胰岛素量分别为[（20.3±4.2）mU/L]、[（16.1±3.7）mU/L]、[（60.5±9.3）mU/L]和[（40.9±7.3）mU/L],葡萄糖可调节MDSCs-IPCs胰岛素的分泌。 结论MDSCs易于分离培养、增殖能力强,体外可诱导分化为有功能的IPCs,适合作为再生医学的种子细胞。     

神经上皮干细胞的分离培养及其体外分化特性的观察

目的探讨大鼠胚胎神经管神经上皮干细胞的分离培养条件,并观察其在体外的分化特性.方法采用显微解剖、机械吹打、无血清悬浮培养方法分离培养神经上皮干细胞,采用巢蛋白(nestin)免疫细胞化学染色技术检测神经上皮干细胞,用NSE和GFAP免疫组化染色检测并计数神经细胞和神经胶质细胞.结果大鼠胚胎神经管神经上皮干细胞在无血清培养基中可形成大量呈nestin抗原阳性细胞构成的神经球,经传代有血清培养后分化为NSE阳性和GFAP阳性细胞,其中NSE阳性细胞占细胞总数的47.7%,GFAP阳性细胞占细胞总数的39.8%.结论胎鼠神经管神经上皮干细胞在无血清培养中可增殖和传代,在有血清培养中可分化为神经细胞和神经胶质细胞,两者之比为47.7∶39.8.     

Modulation of Sirt1 by resveratrol and nicotinamide alters proliferation and differentiation of pig preadipocytes

Sirt1, a NAD⁺-dependent histone deacetylase, may regulate senescence, metabolism, and apoptosis. In this study, primary pig preadipocytes were cultured in DMEM/F12 medium containing 10% fetal bovine serum (FBS) with or without reagents affecting Sirt1 activity. The adipocyte differentiation process was visualized by light microscopy after Oil red O staining. Proliferation and differentiation of preadipocytes was measured using methylthiazolyldiphenyl-tetrazolium bromide (MTT) and Oil red O extraction. Expression of Sirt1, FoxO1, and adipocyte specific genes was detected with semi-quantitive RT-PCR. The results showed that Sirt1 mRNA was widely expressed in various pig tissues from different developmental stages. Sirt1 mRNA was expressed throughout the entire differentiation process of pig preadipocytes. Resveratrol significantly increased Sirt1 mRNA expression, but decreased the expression of FoxO1 and adipocyte marker gene PPARγ2. Resveratrol significantly inhibited pig preadipocyte proliferation and differentiation. Nicotinamide decreased the expression of Sirt1 mRNA, but increased the expression of FoxO1 and adipocyte specific genes. Nicotinamide greatly stimulated the proliferation and differentiation of pig preadipocytes. In conclusion, these results indicate that Sirt1 may modulate the proliferation and differentiation of pig preadipocytes. Sirt1 may down-regulate pig preadipocytes proliferation and differentiation through repression of adipocyte genes or FoxO1.     

A HLA-Cw*0701 restricted Melan-A/MART1 epitope presented by melanoma tumor cells to CD8+ tumor infiltrating lymphocytes

Melan-A/MART1 is a melanocytic differentiation antigen recognized on melanoma tumor cells by CD8+ and CD4+ T cells. In this study, we describe a new epitope of this protein recognized in the context of HLA-Cw*0701 molecules by a CD8+ tumor infiltrating lymphocyte (TIL) clone. This CD8+ TIL clone specifically recognized and killed a fraction of melanoma cells lines expressing Melan-A/MART1 and HLA-Cw*0701. We further show that the Melan-A/MART1_51–61 peptide is the optimal peptide recognized by this clone. Together, these data significantly enlarge the fraction of melanoma patients susceptible to benefit from a Melan-A/MART1 vaccine approach.     

Hypophyseal corticosteroids stimulate somatotrope differentiation in the embryonic chicken pituitary gland

Although it is known that glucocorticoids induce differentiation of growth hormone (GH)-producing cells in rodents and birds, the effect of mineralocorticoids on GH mRNA expression and the origin of corticosteroids affecting somatotrope differentiation have not been elucidated. In this study, we therefore carried out experiments to determine the effect of mineralocorticoids on GH mRNA expression in the chicken anterior pituitary gland in vitro and to determine whether corticosteroids are synthesized in the chicken embryonic pituitary gland. In a pituitary culture experiment with E11 embryos, both corticosterone and aldosterone stimulated GH mRNA expression and increased the number of GH cells in both lobes of the pituitary gland in a dose-dependent manner. These effects of the corticosteroids were significantly reversed by pretreatment with mifepristone, a glucocorticoid receptor (GR) antagonist, or spironolactone, a mineralocorticoid receptor (MR) antagonist. Interestingly, an in vitro serum-free culture experiment with an E11 pituitary gland showed that the GH mRNA level spontaneously increased during cultivation for 2 days without any extra stimulation, and this increase in GH mRNA level was completely suppressed by metyrapone, a corticosterone-producing enzyme P450C11 inhibitor. Moreover, progesterone, the corticosterone precursor, also stimulated GH mRNA expression in the cultured chicken pituitary gland, and this effect was blocked by pretreatment with metyrapone. We also detected mRNA expression of enzymes of cytochrome P450 cholesterol side chain cleavage (P450scc) and 3β-hydroxysteroid dehydrogenase1 (3β-HSD1) in the developmental chicken pituitary gland from E14 and E18, respectively. These results suggest that mineralocorticoids as well as glucocorticoids can stimulate GH mRNA expression and that corticosteroids generated in the embryonic pituitary gland by intrinsic steroidogenic enzymes stimulate somatotrope differentiation.     

Fast co-evolution of sevenless and bride of sevenless in endopterygote insects

Activation of Ras signaling by the receptor tyrosine kinase Sevenless plays important roles during retinal patterning and male germline development in Drosophila. Sevenless is orthologous to the vertebrate receptor tyrosine kinase c-ros. Remarkably, vertebrate ligands of c-Ros as well as non-Drosophila orthologs of the Sevenless ligand Bride of sevenless have remained elusive. Using newly available insect genome sequence information, we investigated the evolutionary conservation of the seven transmembrane domain protein gene bride of sevenless. Single orthologs were identified in the genomes of mosquito, flour beetle, and honeybee due to strong sequence conservation in the seven transmembrane domain. The extracellular region, however, is only detectably conserved within but not outside Diptera. Analysis of domain-specific substitution rates demonstrates correlated fast rates of evolutionary change in the extracellular domains of both bride of sevenless and sevenless. The rapid pace of sequence change explains why Sevenless ligands are difficult to detect by sequence similarity in distantly related phyla. Second, the conservation of bride of sevenless in flour beetle and honeybee raises the possibility of conserved Sevenless signaling controlled patterning processes in endopterygote insects. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of Echinacea augustifolia extract on cell viability and differentiation in mammary epithelial cells

Echinacea spp. are popularly used as an herbal medicine or food supplement for enhancing the immune system and activating biological property in different tissues. In this study we show the biological effect of Echinacea augustifolia extract on cell viability and cell differentiation in mammary epithelial cell lines. These effects have been observed in two different cell line derived from mouse (HC11) and bovine (BME-UV). Echinacea extract enhanced cell liability from 100 to 1000 ng/ml in association with growth factors, epidermal growth factor (EGF) or insulin, but also without EGF (p<0.05) up to 37% vs. control. This effect may be modulated by MAPK and Akt activation that Echinacea extract treatment increased and/or by a reduction of caspase 3 activity, showed a dose–response decrease after Echinacea treatment. Finally Echinacea extract was able to increase (p<0.05) at 100 ng/ml β-casein expression in association with PRL (5 μg/ml). These data demonstrate that Echinacea angustifolia extract can stimulate mammary epithelial cell physiology and may be considered a candidate to support mammary gland activity during a mammogenetic and lactogenetic state.     

Physiological levels of insulin and IGF-1 synergistically enhance the differentiation of mesenteric adipocytes

Visceral adipose tissue, particularly mesenteric adipose tissue, is important in the pathogenesis of metabolic syndrome. Here, we present a physiologically relevant differentiation system of rat mesenteric-stromal vascular cells (mSVC) to mesenteric-visceral adipocytes (mVAC). We optimized the insulin concentration at levels comparable to those in vivo ( approximately 0.85 ng/ml) by including physiological concentrations of IGF-1. We found that the insulin-like growth factor (IGF-1) and insulin worked synergistically, since IGF-1 alone could induce CCAAT/enhancer binding protein alpha (C/EBPalpha) and adipocyte lipid binding protein (aP2) mRNA expression but not lipid droplet accumulation associated with maturation. Using real-time PCR analyses on 180 adipocyte-related genes, we identified a dramatic effect by IGF-1 plus insulin. We also demonstrated the state of insulin resistance at pathologically high insulin concentrations. This culture system will contribute to understanding the physiological differentiation process and the patho/physiology of mVAC.     

Realgar-induced differentiation is associated with MAPK pathways in HL-60 cells

The clinical efficacy and safety of realgar (arsenic sulfide, As(4)S(4)) in the treatment of acute promyelocytic leukemia in China have given rise to an upsurge in research on the underlying mechanism. We prepared realgar nanoparticles (RNPs) to examine their effect on the differentiation of HL-60 cells. Treatment with RNPs at 6 microM for 72 h induced cell differentiation that was assessed by morphological change, NBT reductive ability, and elevation of CD11b expression at both mRNA and protein levels. The RNP-induced differentiation was synergized, enhanced and suppressed by the inhibition of p38 MAPK, JNK and ERK pathways, respectively. Our findings demonstrate that MAPK signaling pathways are closely related to the RNP-induced differentiation in HL-60 cells.