大鼠白介素10真核表达质粒在大鼠体内外肝细胞中的表达及影响

目的探讨体外重组的大鼠白介素10（rIL-10）真核表达质粒能否在大鼠体内外肝细胞中表达及表达产物对肝细胞的影响。方法通过受体介导的脂介体转染法及尾静脉大容量注射法将rIL-10真核表达质粒分别转入大鼠BRL细胞及体内大鼠肝细胞中,采用RT—PCR法、ELISA法和免疫组织化学法检测体内外肝细胞rIL-10的表达情况,MTT法及流式细胞术检测rIL-10真核表达质粒转染对BRL细胞增殖与凋亡的影响。结果转染rIL-10真核表达质粒的BRL细胞及大鼠肝组织高表达rlL-10基因,BRL细胞培养上清与大鼠血清中rIL-10浓度分别为（12．78±O．94）ng／ml,（61．68±3．60）．g／ml。MTT法及流式细胞术显示rIL-10的表达对肝细胞有-定的保护作用。结论rIL-10真核表达质粒可在大鼠体内外肝细胞中表达并对肝细胞有-定的保护作用。     

SPARC Is a Key Regulator of Proliferation, Apoptosis and Invasion in Human Ovarian Cancer

Background

Secreted protein acidic and rich in cysteine (SPARC), a calcium-binding matricellular glycoprotein, is implicated in the progression of many cancers. In this study, we investigated the expression and function of SPARC in ovarian cancer.

Methods

cDNA microarray analysis was performed to compare gene expression profiles of the highly invasive and the low invasive subclones derived from the SKOV3 human ovarian cancer cell line. Immunohistochemistry (IHC) staining was performed to investigate SPARC expression in a total of 140 ovarian tissue specimens. In functional assays, effects of SPARC knockdown on the biological behavior of ovarian cancer cells were investigated. The mechanisms of SPARC in ovarian cancer proliferation, apoptosis and invasion were also researched.

Results

SPARC was overexpressed in the highly invasive subclone compared with the low invasive subclone. High SPARC expression was associated with high stage, low differentiation, lymph node metastasis and poor prognosis of ovarian cancer. Knockdown of SPARC expression significantly suppressed ovarian cancer cell proliferation, induced cell apoptosis and inhibited cell invasion and metastasis.

Conclusion

SPARC is overexpressed in highly invasive subclone and ovarian cancer tissues and plays an important role in ovarian cancer growth, apoptosis and metastasis.     

惊厥后大鼠海马神经再生与凋亡的动态变化

探讨惊厥持续状态(status convulsion,SC)后大鼠海马神经再生与凋亡的动态变化。建立成年Wistar鼠30minSC模型,在SC后1天至56天的6个时间点上处死动物,处死前1天均腹腔注射5-溴2-脱氧尿嘧啶核苷(5-bromo-2-deoxyuridine,BrdU);采用免疫组织化学方法动态检测BrdU、nestin的表达,确定神经干细胞增殖水平;双重荧光染色标记nestin/TUNEL,确定新生神经干细胞存活时间。与对照组相比,BrdU阳性细胞数目于SC后第7天在CA1区达增殖高峰,28天降至正常水平;于SC后第28天在齿状回达增殖高峰,56天降至正常水平;在SC后第7天,CA3区有大量的BrdU阳性细胞;BrdU和nestin阳性细胞数目无统计学差异。在SC后的前3天,CA1区新增殖的神经细胞呈TUNEL阳性;齿状回新增殖细胞始终表现TUNEL阴性。上述结果提示:SC后能激活自体神经干细胞原位增殖,并且部分新生细胞向损伤区域迁移。     

Cellular Proliferation of Intestinal Epithelia in the Rat Two Months after Partial Resection of the Ileum

Sprague-Dawley rats subjected 2 months previously to partial resection (10 per cent) of the small intestine and their controls were injected with tritiated thymidine and sacrificed at 2 and 23 hours. Segments of the duodenum, jejunum, and ileum were autoradiographed, and the migration of the labelled cells during the period between 2 and 23 hours was measured with an eyepiece micrometer. The cells had migrated 35, 42, and 34 per cent of the total distance from the crypts to the tips of the villi in the control segments of duodenum, ileum, and jejunum respectively, and 43, 90, and 82 per cent, respectively, in similar segments from resected animals. The rate of migration in the portion of the intestine remaining after resection was approximately three times the normal rate in the ileum, twice the normal rate in the jejunum, and showed an increase of one-third in the duodenum. These results demonstrate that the rate of cell renewal is considerably greater in the remaining portion of the intestine of resected animals than in normal intestine. The increased rate of migration after resection, together with the increase in the height of the villi, resulted in an increase in the rate of cell renewal amounting to 141 per cent in the ileum, 114 per cent in the jejunum, and 23 per cent in the duodenum when compared with control segments.     

大鼠卵巢的生后发育

目的研究大鼠卵巢中卵泡生长发育的增龄性变化,为生殖生物学研究寻找动物模型.方法 用组织学方法,观察了0天～15月大鼠卵巢的形态结构.结果 0天见卵母细胞聚集呈团索状,未见卵泡;4天见大量原始卵泡;1周出现大量初级卵泡;2周见次级卵泡;3周次级卵泡数量增加,卵泡腔扩大;1月可见不同发育阶段的卵泡和闭锁卵泡;45天出现黄体;2月～5月,有大量黄体和生长卵泡;6月～8月,卵巢体积缩小,卵泡数量减少;12月萎缩,未见正常卵泡和黄体;15月,纤维化,可见囊泡.结论 大鼠从6月开始,生殖功能随月龄增长而进行性衰减,机体逐渐衰老.大鼠可用作研究女性生殖与衰老关系的动物模型.     

Deficiency in Endothelin Receptor B Reduces Proliferation of Neuronal Progenitors and Increases Apoptosis in Postnatal Rat Cerebellum

Endothelins regulate cellular functions in the mammalian brain through the endothelin receptors A and B (EDNRA and EDNRB). In this study, we investigated the role of EDNRB on cell proliferation in the cerebellum by using the spotting lethal (sl) rat, which carries a naturally occurring deletion in the EDNRB gene. Proliferating cells in the three genotypes, wild-type (+/+), heterozygous (+/sl) and homozygous mutant (sl/sl) rats were labelled by intraperitoneal injection of 5-bromo-2′-deoxyuridine (BrdU) at postnatal day 2. The density of BrdU-positive cells (per mm²) in the external germinal layer of sl/sl rats (Mean ± SEM, 977 ± 388) was significantly reduced compared to +/+ (4915 ± 631) and +/sl (2304 ± 557) rats. Subsequently, we examined the effects of EDNRB mutation on neural apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labelling assay. This showed that the density of apoptotic cells in the cerebella of sl/sl rats (9.3 ± 0.5/mm²) was significantly more increased than +/+ rats (4 ± 0.7). The expression of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) were measured with standard ELISA, but were unchanged in all genotypes. These results suggest that ENDRB mediates neural proliferation and have anti-apoptotic effects in the cerebellum of the postnatal rat, and that these effects are independent of changes in the expression of BDNF and GDNF. Our findings will lead to better understanding of the morphological changes in the cerebellum of Hirschsprung’s disease patients with congenital EDNRB mutation.     

CKIP-1: 一种新型的凋亡促进因子

酪蛋白激酶2相互作用蛋白1(caseinkinase2interactionprotein1,CKIP-1)是近年来发现的一种重要分子,它通过与其他分子的相互作用在许多细胞行为中都发挥着重要的作用。最新研究发现,CKIP-1还具有促进细胞凋亡的作用。     

The TATA-Binding Protein as a Regulator of Cellular Transformation

No abstract available.     

Ghrelin and Nicotine Stimulate Equally the Dopamine Release in the Rat Amygdala

The orexigenic peptide ghrelin plays a prominent role in the regulation of energy balance and in the mediation of reward processes and reinforcement for addictive drugs, such as nicotine. Nicotine is the principal psychoactive component in tobacco, which is responsible for addiction and relapse of smokers. Ghrelin and nicotine activates the mesolimbicocortical dopaminergic pathways via growth hormone secretagogue receptors (GHS-R1A) and nicotinic acetylcholine receptors (nAchR), respectively, resulting in the release of dopamine in the nucleus accumbens, the amygdala and the prefrontal cortex. In the present study an in vitro superfusion of rat amygdalar slices was performed in order to investigate the direct action of ghrelin and nicotine on the amygdalar dopamine release. Ghrelin increased significantly the dopamine release from the rat amygdala following electrical stimulation. This effect was inhibited by both the selective GHS-R1A antagonist GHRP-6 and the selective nAchR antagonist mecamylamine. Under the same conditions, nicotine also increased significantly the dopamine release from the rat amygdala. This effect was antagonized by mecamylamine, but not by GHRP-6. Co-administration of ghrelin and nicotine induced a similar increase of amygdalar dopamine release. This stimulatory effect was partially reversed by both GHRP-6 and mecamylamine. The present results demonstrate that both ghrelin and nicotine stimulates directly the dopamine release in the amygdala, an important dopaminergic target area of the mesolimbicocortical pathway.     

Ghrelin is present in teeth

Ghrelin belongs to the family of a gut-brain hormone that promotes food intake and controls energy balance. Recently, it has also been shown to regulate bone formation directly. Dental tissue shares several functional, developmental and anatomical similarities with bone, and in the present study we have investigated the presence of ghrelin in 44 human teeth using immunocytochemistry and radioimmunoassay. Both methods showed that the hormone is present in canines and molars, mainly in the odontoblasts but also in the pulp. Ghrelin could potentially play interesting physiological roles in teeth.     

Phosphatidylinositol 3-Kinase Is a Negative Regulator of Cellular Differentiation

Phosphatidylinositol 3-kinase (PI3K) has been shown to be an important mediator of intracellular signal transduction in mammalian cells. We show here, for the first time, that the blockade of PI3K activity in human fetal undifferentiated cells induced morphological and functional endocrine differentiation. This was associated with an increase in mRNA levels of insulin, glucagon, and somatostatin, as well as an increase in the insulin protein content and secretion in response to secretagogues. Blockade of PI3K also increased the proportion of pluripotent precursor cells coexpressing multiple hormones and the total number of terminally differentiated cells originating from these precursor cells. We examined whether any of the recently described modulators of endocrine differentiation could participate in regulating PI3K activity in fetal islet cells. The activity of PI3K was inversely correlated with the hepatocyte growth factor/scatter factor–induced downregulation or nicotinamideinduced upregulation of islet-specific gene expression, giving support to the role of PI3K, as a negative regulator of endocrine differentiation. In conclusion, our results provide a mechanism for the regulation of hormone-specific gene expression during human fetal neogenesis. They also suggest a novel function for PI3K, as a negative regulator of cellular differentiation.     

Processes of Proliferation and Apoptosis in the Brain of the Amur Sturgeon

Using techniques of immunoperoxidase staining of proliferating cell nuclear antigen (PCNA) and TUNEL labeling of fragmented DNA, we studied sites of proliferation and apoptosis in the myelencephalon, cerebellum, tectum opticum, thalamus, and hypothalamus of the Amur sturgeon (Acipenser schrenckii). We found that the processes of proliferation and apoptosis are maintained in the brain of 3-year-old sturgeon individuals; the ratio of these processes in different cerebral regions varied significantly. The maximum intensity of proliferative activity was found in the periventricular zone of the myelencephalon (proliferation index, on average, 21.0 ± 1.3%). This fact allows us to consider this cerebral region a most important zone were adult neurogenesis occurs in the sturgeon. In the medial reticular formation, dorsal thalamic nuclei, inner fibrous layer of the tectum, and lateral hypothalamus, the maximum numbers of apoptotic elements were found. Therefore, these zones in the brain of the sturgeon correspond, apparently, to the regions where postmitotic neuroblasts are localized. In sensory centers (tectum and nuclei of the V, VII, and X nerves), significantly varying ratios of intensities of proliferation and apoptosis were found; this is indicative of dissimilar rates of growth and differentiation in visual and chemosensory centers of the sturgeon brain. The high proliferative activity in sensory and motor cerebral centers of the sturgeon allows us to hypothesize that a neotenic pattern is preserved in these CNS regions of adult sturgeons over a long period after the embryogenesis has been completed.     

Bile Acid-induced Epidermal Growth Factor Receptor Activation in Quiescent Rat Hepatic Stellate Cells Can Trigger Both Proliferation and Apoptosis

Bile acids have been reported to induce epidermal growth factor receptor (EGFR) activation and subsequent proliferation of activated hepatic stellate cells (HSC), but the underlying mechanisms and whether quiescent HSC are also a target for bile acid-induced proliferation or apoptosis remained unclear. Therefore, primary rat HSC were cultured for up to 48 h and analyzed for their proliferative/apoptotic responses toward bile acids. Hydrophobic bile acids, i.e. taurolithocholate 3-sulfate, taurochenodeoxycholate, and glycochenodeoxycholate, but not taurocholate or tauroursodeoxycholate, induced Yes-dependent EGFR phosphorylation. Simultaneously, hydrophobic bile acids induced phosphorylation of the NADPH oxidase subunit p47^phox and formation of reactive oxygen species (ROS). ROS production was sensitive to inhibition of acidic sphingomyelinase, protein kinase Cζ, and NADPH oxidases. All maneuvers which prevented bile acid-induced ROS formation also prevented Yes and subsequent EGFR phosphorylation. Taurolithocholate 3-sulfate-induced EGFR activation was followed by extracellular signal-regulated kinase 1/2, but not c-Jun N-terminal kinase (JNK) activation, and stimulated HSC proliferation. When, however, a JNK signal was induced by coadministration of cycloheximide or hydrogen peroxide (H₂O₂), activated EGFR associated with CD95 and triggered EGFR-mediated CD95-tyrosine phosphorylation and subsequent formation of the death-inducing signaling complex. In conclusion, hydrophobic bile acids lead to a NADPH oxidase-driven ROS generation followed by a Yes-mediated EGFR activation in quiescent primary rat HSC. This proliferative signal shifts to an apoptotic signal when a JNK signal simultaneously comes into play.Hydrophobic bile acids play a major role in the pathogenesis of cholestatic liver disease and are potent inducers of hepatocyte apoptosis by triggering a ligand-independent activation of the CD95² death receptor (1–5). The underlying molecular mechanisms are complex and involve a Yes-dependent, but ligand-independent activation of the epidermal growth factor receptor (EGFR), which catalyzes CD95-tyrosine phosphorylation as a prerequisite for CD95 oligomerization, formation of the death-inducing signaling complex (DISC), and apoptosis induction (6, 7). Bile acids also activate EGFR in cholangiocytes (8) and activated hepatic stellate cells (HSC) (9), however, the mechanisms underlying bile acid-induced EGFR activation in HSC remained unclear (9). Surprisingly, bile acid-induced EGFR activation in HSC does not trigger apoptosis but results in a stimulation of cell proliferation (9). The behavior of quiescent HSC toward CD95 ligand (CD95L) is also unusual. CD95L, which is a potent inducer of hepatocyte apoptosis (10–12), triggers activation of the EGFR in quiescent HSC, stimulates HSC proliferation, and simultaneously inhibits CD95-dependent death signaling through CD95-tyrosine nitration (13). Similar observations were made with other death receptor ligands, i.e. tumor necrosis factor-α (TNF-α) and TNF-related apoptosis-inducing ligand (TRAIL) (13). The mitogenic action of CD95L in quiescent, 1–2-day cultured HSC is because of a c-Src-dependent shedding of EGF and subsequent auto/paracrine activation of the EGFR (13). This unusual behavior of quiescent HSC toward death receptor ligands may relate to the recent findings that quiescent HSC might represent a stem/progenitor cell compartment in the liver with a capacity to differentiate not only into myofibroblasts but also toward hepatocyte- and endothelial-like cells (14). Thus, stimulation of HSC proliferation and resistance toward apoptosis in the hostile cytokine milieu accompanying liver injury may help HSC to play their role in liver regeneration. During cholestatic liver injury quiescent HSC are exposed to increased concentrations of circulating bile acids, but it is not known whether this may lead to HSC proliferation (as shown for activated HSC) (9), HSC apoptosis (as shown for hepatocytes) (1–7), or both of them. Therefore, the aim of the current study was (a) to identify the molecular mechanisms underlying bile acid-induced EGFR activation and (b) to elucidate whether bile acid-induced signaling can couple to both cell proliferation and cell death in quiescent HSC.The present study shows that cholestatic bile acids trigger a rapid NADPH oxidase activation in quiescent HSC, which leads to a Yes-mediated EGFR phosphorylation and HSC proliferation. In contrast to hepatocytes, hydrophobic bile acids do not induce a JNK signal in HSC. However, when JNK activation is induced by coadministration of either cycloheximide (CHX) or hydrogen peroxide (H₂O₂), the bile acid-induced mitogenic signal is shifted to an apoptotic one.     

Maduramicin Inhibits Proliferation and Induces Apoptosis in Myoblast Cells

Maduramicin, a polyether ionophore antibiotic derived from the bacterium Actinomadura yumaensis, is currently used as a feed additive against coccidiosis in poultry worldwide. It has been clinically observed that maduramicin can cause skeletal muscle and heart cell damage, resulting in skeletal muscle degeneration, heart failure, and even death in animals and humans, if improperly used. However, the mechanism of its toxic action in myoblasts is not well understood. Using mouse myoblasts (C2C12) and human rhabdomyosarcoma (RD and Rh30) cells as an experimental model for myoblasts, here we found that maduramicin inhibited cell proliferation and induced cell death in a concentration-dependent manner. Further studies revealed that maduramicin induced accumulation of the cells at G₀/G₁ phase of the cell cycle, and induced apoptosis in the cells. Concurrently, maduramicin downregulated protein expression of cyclin D1, cyclin-dependent kinases (CDK4 and CDK6), and CDC25A, and upregulated expression of the CDK inhibitors (p21^Cip1 and p27^Kip1), resulting in decreased phosphorylation of Rb. Maduramicin also induced expression of BAK, BAD, DR4, TRADD and TRAIL, leading to activation of caspases 8, 9 and 3 as well as cleavage of poly ADP ribose polymerase (PARP). Taken together, our results suggest that maduramicin executes its toxicity in myoblasts at least by inhibiting cell proliferation and inducing apoptotic cell death.