血清饥饿可诱导人血管平滑肌细胞再分化

体外培养的分化型血管平滑肌细胞 (vascularsmoothmusclecells ,VSMC)以特异性标志基因表达、长梭形外观及对兴奋剂刺激产生收缩反应为其表型特征 .以血清饥饿法培养处于超汇合 (overconfluence)状态的人VSMC ,观察其分化型标志基因表达活性及其与细胞形态特征和收缩反应性之间的关系 ,探讨细胞生存环境对VSMC基因表达及表型的影响 .研究显示 ,生长至超汇合的VSMC由含血清培养转为血清饥饿后 ,收缩蛋白如SMα肌动蛋白 (SMα actin)、SM2 2α、h1 calponin、肌球蛋白重链 (MHC)SM1和SM2亚型的表达活性明显上调 ,证实血清饥饿诱导的收缩蛋白基因表达和血清应答因子 (serumresponsefactor ,SRF)与CArG顺式元件结合活性的增强有关 .同时 ,血清饥饿还可激活参与VSMC分化调节的转录调控因子SmLIM、Gax和分化相关蛋白HRG 1基因的转录 .随着血清饥饿培养时间的延长 ,VSMC逐渐形成多层、束状、成极性排列的形式 ,对兴奋剂刺激产生的收缩反应明显增强 .结果表明 ,超汇合状态的去分化型VSMC脱离血清刺激后 ,可以再分化成熟并重新获得收缩能力     

Comparison of Cell Growth and Alkaloid Production of Catharanthus roseus Cells Cultured in Shake Flask and in Bioreactor

The cell growth and alkaloid production of Catharanthus roseus (L.) G. Don cells cultured in the shake flasks with different volumes and in the stirred tank bioreactor (10 L) were compared. Cell dry weight and alkaloid production showed no significant difference in the small volume scale-up shake flasks. When more broths were added to a certain volume in the shake flask, both cell weight and alkaloid production were decreased. The maximum cell dry weight was similar between the cell cultures in the shake flask and the bioreactor, but the alkaloid production of cells was much less in the bioreactor. Gas regime and shear stress were recognized to be the main factors contributing the important effect on alkaloid production during the scale-up processes.     

Metabolic Pools of ATP in Cultured Bovine Adrenal Medullary Chromaffin Cells

Cultured bovine adrenal chromaffin cells contain a pool of ATP sequestered within the chromaffin vesicles and an extravesicular pool of ATP. In a previous study it was shown that the turnover of ATP in the extravesicular pool was biphasic. One phase occurred with a t1/2 of 3.5-4.5 h whereas the second phase occurred with a t1/2 of several days. The studies described here were undertaken to characterize further the vesicular and extravesicular pools of ATP by examining the effects of metabolic inhibitors, adenosine, and digitonin on ATP utilization and subcellular localization immediately after and 48 h after labeling with [3H]adenosine and 32Pi. Immediately after labeling a combination of cyanide, 2-deoxy-D-glucose, the beta-glucono-1,5-lactone resulted in a 90-95% depletion of the labeled ATP but only a 25% depletion of the endogenous ATP within 30 min. Forty-eight hours after labeling, addition of the inhibitors resulted in a 70% depletion of the [3H]ATP but only a 25% depletion of the [32P]ATP and endogenous ATP. Addition of 10 microM adenosine to the media resulted in a similar loss of [3H]ATP in cells examined immediately after or 48 h after labeling. Adenosine increased the amounts of [32P]ATP when added immediately after labeling but had no effect on the [32P]ATP content when added 48 h after labeling.(ABSTRACT TRUNCATED AT 250 WORDS)     

骨骼肌卫星细胞的特性及其增殖与分化的调控

成体骨骼肌细胞的数量基本保持恒定,骨骼肌的再生主要依赖肌卫星细胞的增殖与分化。骨骼肌卫星细胞是能够被激活、进而分化为肌细胞的一类成肌细胞。现对肌卫星细胞的发生、体外培养以及增殖与分化的调控进行综述,并对能否通过激活肌卫星细胞的增殖来实现肌肉组织生长的调控进行探讨。     

人主动脉壁硫酸乙酰肝素蛋白聚糖对培养的人主动脉平滑机细胞合成蛋白聚糖的影响

从人正常胸主动脉分离硫酸乙酰肝素蛋白聚糖（ＨＳＰＧ）,观察其对体外培养的人主动脉平滑肌细胞（ＨＡＳＭＣ）合成ＰＧ的影响。ＨＡＳＭＣ在不加（对照）或加ＨＳＰＧ（１９μｇ醛酸／ｍｌ）的￣（３５）Ｓ－硫酸钠培养液中培养,以标记ＰＧ。继之,培养液及细胞层的４ｍｏｌ／Ｌ盐酸胍提取液中的ＰＧｓ经离子交换及凝胶过滤柱层析分离,发现加ＨＳＰＧ后,培养液中的ＨＳＰＧ,硫酸软骨素ＰＧ（ＣＳＰＧ）及硫酸皮肤素－硫酸软骨素ＰＧ（ＤＳＣＳＰＧ）均明显增高,而细胞层中仅ＨＳＰＧ和ＣＳＰＧ增高,且加ＨＳＰＧ后细胞层的ＤＳＣＳＰＧ分子大小有所不同,进一步分析ＤＳＣＳＰＧ中ＤＳ及ＣＳ含量发现加ＨＳＰＧ组ＨＡＳＭＣ细胞层中的ＤＳ％含量略低于对照组。结果提示ＨＳＰＧ可刺激ＨＡＳＭＣ的ＰＧ合成,其可能与血管壁修复及动脉壁脂质沉积有关。     

Amyloid β-Protein Induces Its Own Production in Cultured Degenerating Cerebrovascular Smooth Muscle Cells

Abstract: The progression of Alzheimer's disease and related disorders involves amyloid β-protein (Aβ) deposition and pathologic changes in the parenchyma as well as cerebral blood vessels. The cerebrovascular Aβ deposits in these disorders are associated with degenerating smooth muscle cells in the vessel wall, which have been shown to express the Aβ precursor (AβPP) and Aβ. Here, we show that Aβ_1–42, an abundant cerebrovascular form of Aβ, causes cellular degeneration in cultured human cerebrovascular smooth muscle cells. This stress response is accompanied by a striking increase in the levels of cellular AβPP and soluble Aβ peptide produced in these degenerating cells. These data provide the first experimental evidence that Aβ can potentially contribute to the onset and progression of the cerebrovascular pathology. The present findings suggest that this mechanism may involve a molecular cascade with a novel product-precursor relationship that results in the adverse production and subsequent accumulation of Aβ.     

Rapid Communication Muscle Mitochondrial ATP Production in Progressive Supranuclear Palsy

Abstract: Six patients with progressive supranuclear palsy (PSP) and 12 age-matched disease-free subjects participated in this study designed to compare rates of ATP production by intact mitochondria from biopsied skeletal muscle. When pyruvate and malate were used as metabolic substrates, rates of ATP production were 0.184 ± 0.025 μmol/min/U of citrate synthase (CS) activity (a mitochondrial marker) in control subjects and 0.131 ± 0.051 μmol/min/U of CS in PSP patients. In the presence of succinate, rates of ATP formation were 0.137 + 0.02 μmol/min/U of CS in controls and 0.109 ± 0.04 /4mUmol/min/U of CS in patients. With N,N,N',N' -tetramethyl- p -phenylenediamine (TMPD) and ascorbate, rates were 0.034 ± 0.008 μm Umol/min/U of CS in controls and 0.022 ± 0.01 μmol/min/U of CS in PSP subjects. Differences between the control and PSP populations reached statistical significance with pyruvate/malate and TMPD/ascorbate. No differences in either muscle histopathology or histochemistry were found between patient and control subjects. Results of this study suggest that oxidative phosphorylation defects occur in muscle mitochondria from patients with PSP.     

氧化修饰HDL剌激培养人主动脉平滑肌细胞增殖

平滑肌细胞（smooth muscle cell, SMC）增殖在动脉粥样硬化（atherosclerosis, AS）形成中起着重要作用.氧化修饰HDL（oxidized HDL, OX-HDL）可刺激 ³H-TdR掺入培养人动脉SMC的DNA,促进SMC增殖.以四甲基偶氮唑盐(MTT)法直接观察OX-HDL对培养人动脉SMC增殖细胞数的影响.结果显示,天然HDL（native HDL N-HDL）对SMC增殖没有影响,而OX-HDL则显著刺激SMC增殖（P＜0.01）,N-HDL显著抑制OX-LDL刺激SMC增殖作用（P＜0.01）,而OX-HDL则显著增加OX-LDL刺激SMC增殖作用（P＜0.01）     

Skeletal Muscle Satellite Cells Are Committed to Myogenesis and Do Not Spontaneously Adopt Nonmyogenic Fates

The developmental potential of skeletal muscle stem cells (satellite cells) remains controversial. The authors investigated satellite cell developmental potential in single fiber and clonal cultures derived from MyoD^iCre/+;R26R^EYFP/+ muscle, in which essentially all satellite cells are permanently labeled. Approximately 60% of the clones derived from cells that co-purified with muscle fibers spontaneously underwent adipogenic differentiation. These adipocytes stained with Oil-Red-O and expressed the terminal differentiation markers, adipsin and fatty acid binding protein 4, but did not express EYFP and were therefore not of satellite cell origin. Satellite cells mutant for either MyoD or Myf-5 also maintained myogenic programming in culture and did not adopt an adipogenic fate. Incorporation of additional wash steps prior to muscle fiber plating virtually eliminated the non-myogenic cells but did not reduce the number of adherent Pax7+ satellite cells. More than half of the adipocytes observed in cultures from Tie2-Cre mice were recombined, further demonstrating a non-satellite cell origin. Under adipogenesis-inducing conditions, satellite cells accumulated cytoplasmic lipid but maintained myogenic protein expression and did not fully execute the adipogenic differentiation program, distinguishing them from adipocytes observed in muscle fiber cultures. The authors conclude that skeletal muscle satellite cells are committed to myogenesis and do not spontaneously adopt an adipogenic fate.     

Effects of Basal [Ca]i on Calcium Handling in Ca-Overloaded Rat Cultured Heart Muscle Cells

To elucidate the relationship between intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and Ca²⁺-signalling by the sarcoplasmic reticulum (SR) in Ca²⁺-overloaded heart muscle cells, the direct effects of “basal” [Ca²⁺]_i on calcium waves were investigated by altering the membrane potential. When basal inter-calcium wave (BCW) [Ca²⁺]_i was maintained at a high level, (i) calcium waves showed more gradual and more rapidly suppressed increase in [Ca²⁺]-profile (P < 0.005), and (ii) calcium waves occurred at a significantly higher frequency and velocity (259% and 137%), than when low BCW [Ca²⁺]_i was maintained. Similar investigations on inhibition of the Na⁺-Ca²⁺ exchanger, however, showed that membrane potential did not elicit direct effects on calcium waves. These results showed that the elevation of BCW [Ca²⁺]_i per se directly influences Ca²⁺-signalling in heart muscle cells through non-equilibrated release-restoration Ca²⁺-handling by the SR.     

Artemisia princeps Extract Promoted Glucose Uptake in Cultured L6 Muscle Cells via Glucose Transporter 4 Translocation

Artemisia princeps is a familiar plant as a food substance and medicinal herb. In this study, we evaluated the effects of an ethanol extract of A. princeps (APE) on glucose uptake in differentiated L6 muscle cells. Treatment with APE elevated deoxyglucose uptake, and translocation of the insulin-responsive glucose transporter (GLUT4) to the plasma membrane in L6 myotubes occurred. The PI3K inhibitor LY294002 attenuated glucose uptake induced by APE. Phosphorylation of the Ser⁴⁷³ residue of Akt was not observed, but phosphorylation of PI3K, Akt (Thr³⁰⁸), and atypical PKC was. In addition, APE stimulated phosphorylation of AMP-activated protein kinase (AMPK) at a level similar to 5′-amino-5-imidazolecarboxamide-riboside (AICAR). These results indicate that APE stimulates glucose uptake by inducing GLUT4 translocation, which is in part mediated by combination of the PI3K-dependent atypical PKC pathway and AMPK pathways.     

Transient Transfection Factors for High-Level Recombinant Protein Production in Suspension Cultured Mammalian Cells

The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells.     

Production of cyclic AMP from extracellular ATP by intact LM cells

Intact LM cells, a line of cultured mouse fibroblasts, exhibited and adenylate cyclase (APT pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity in the presence exogenous [α-³²]ATP which was 20–30% of that observed with comparable preparations of lysed cells. The extent of NaF and prostaglandin E₁ stimulation was comparable in intact cells and lysed cells. 96% of the added ATP and 92% of the cyclic AMP produced by intact cells could be isolated extracellularly in the incubation medium. Cellular integrity under assay conditions was monitored by trypan blue exclusion. These data suggest that LM cells contain an endenylate cyclase activity whic is accessible to extracellular ATP.     

黄芩苷通过降低活性氧生成抑制血管平滑肌细胞伪足形成和迁移

已知黄芩苷（baicalin）通过削弱肌动蛋白相关蛋白（actin-related protein, Arp）2/3复合物的活性抑制血管平滑肌细胞（vascular smooth muscle cell, VSMC）伪足形成和迁移,然而,其抑制该信号途径的机制尚不明确。本研究证明,黄芩苷通过抑制VSMC活性氧（reactive oxygen species,ROS）生成降低Arp2/3活性,发挥阻止细胞伪足形成和迁移的功能。分别利用TRITC 鬼笔环肽和ROS荧光探针标记VSMCs,结果显示,黄芩苷能显著抑制血小板源性生长因子（platelet derived growth factor, PDGF）-BB诱导的VSMC伪足形成和迁移,伴有ROS生成减少。用超氧物歧化酶（superoxide dismutase, SOD）清除胞内过氧化物后,PDGF-BB引发的VSMC伪足形成被逆转,且该过程与降低皮层肌动蛋白微丝（F-actin）成核蛋白Arp2/3活性有关。免疫沉淀分析结果进一步表明,黄芩苷降低p47phox磷酸化水平,与ROS生成减少相一致。体内的实验也表明,黄芩苷（70 mg/kg/d）能有效抑制球囊损伤诱导的大鼠颈总动脉ROS生成。以上结果表明,黄芩苷通过抑制NADPH氧化酶介导的ROS生成,降低细胞皮质区F-actin成核活性,阻止细胞伪足形成、迁移,进而发挥血管保护作用。     

Multiple Molecular Forms of Acetylcholine Receptors in Cultured Skeletal Muscle Cells: Subcellular Localization and Characterization

Abstract: Skeletal muscle cells of newborn rats, cultured in the absence of neuronal influence, were found to contain two types of cell surface acetylcholine receptors as demonstrated by isoelectric focusing. The isoelectric points of the two types of receptors were indistinguishable from those of junctional and extrajunctional types of receptors in mature animals. The cultured cells had two classes of intracellular α-bungarotoxin (αBT) binding components; one had the same sedimentation coefficient as that of surface receptors (9S), and the other had much smaller apparent molecular weights. Only a single major component was detected by isoelectric focusing analysis of the 9s intracellular aBT binding component, with a PI value close to that of the extra junctional receptor. These results suggest that the junctional and extrajunctional types of receptors may be synthesized through a common precursor.     

胡桐悬浮培养细胞中苯丙氨酸解氨酶活性与红厚壳素产量的关系

胡桐CR2细胞悬浮培养过程中,苯丙氨酸解氨酶(PAL)活性上升后红厚壳素产量即增加.加入真菌诱导子后,PAL活性与红厚壳素产量呈一定的正相关.以PAL抑制物降低PAL活性后,红厚壳素产量也降低;诱导物与抑制物同时加入,PAL活性与红厚壳素产量均界于诱导物处理与未处理之间.     

Endothelin- and Sarafotoxin-Induced Phosphoinositide Hydrolysis in Cultured Canine Tracheal Smooth Muscle Cells

Abstract: The endothelins (ETs) and sarafotoxin are two structurally related classes of potently contractile peptides. To understand the mechanism of action of ETs, we have examined the effect of ETs and sarafotoxin on phosphoinositide (PI) hydrolysis in cultured canine tracheal smooth muscle cells (TSMCs). ET-1, ET-2, ET-3, and sarafotoxin caused dose-dependent accumulation of inositol phosphates (IPs) and tracheal smooth muscle contraction. BQ-123, an ET_A receptor antagonist, had a high affinity to block the ET-1-induced IP accumulation and tracheal smooth muscle contraction with pK_B values of 7.3 and 7.4, respectively. Pretreatment of TSMCs with cholera toxin impaired the ability of ET-1 and ET-2 to stimulate IP formation, whereas there was no effect by treatment with pertussis toxin. Stimulation of PI turnover by these peptides required the presence of extracellular Ca²⁺ and was blocked by treatment with EGTA. The addition of Ca²⁺(3–620 nM) to digitonin-permeabilized TSMCs directly stimulated IP accumulation. A further Ca²⁺-dependent increase in IP formation was obtained by inclusion of either GTPrS or ET-1. The combined presence of GTPrS and ET-1 elicited an additive effect on IP formation. Short-term exposure to phorbol 12-myristate 13-acetate (PMA, 1 μM) abolished the stimulation of PI hydrolysis induced by these peptides. The inhibitory effect of PMA on ET-induced response was reversed by staurosporine, a protein kinase C (PKC) inhibitor, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Prolonged incubation of TSMCs with PMA resulted in a recovery of receptor responsiveness that may be due to down regulation of PKC. Inactive phorbol ester, 4α-phorbol 12, 13-didecanoate at 1 μM, did not inhibit this response. The site of this response was further investigated by examining the effect of PMA on AIF₄^?-induced IP accumulation in canine TSMCs. AIF₄^?-induced IP accumulation was inhibited by PMA treatment, suggesting that G protein(s) can be directly activated by AIF₄^?, which was uncoupled to phospholipase C by PMA treatment. These data conclude that ET-stimulated PI hydrolysis and tracheal smooth muscle contraction are mediated by the activation of ET_Areceptors coupling to a G protein and dependent on the external Ca²⁺. The transduction mechanism of ETs is sensitive to feedback regulation by PKC.     

在静态和动态培养条件下杂交瘤细胞的生长和代谢

通过对WuT3杂交瘤细胞在静态和动态培养条件下的比较,发现细胞生长和代谢有很大不同。在静态培养条件下,细胞培养周期较长,细胞密度较高;但在动态培养条件下,细胞代谢更加旺盛,葡萄糖、氨基酸等营养物质的消耗加快,乳酸、氨、丙氨酸等代谢产物的比生成速率较大。     

Effects of Reserpine and Tetrabenazine on Catecholamine and ATP Storage in Cultured Bovine Adrenal Medullary Chromaffin Cells

The in vivo storage relationship between catecholamines and ATP in chromaffin vesicles of cultured bovine adrenal medulla cells was investigated using drugs that block vesicular catecholamine uptake. Three-day treatments with reserpine and tetrabenazine causing 85-90% depletion of catecholamines resulted in 41-46% reductions in cellular ATP content. Subcellular fractionation of reserpine-treated cells indicated that the ATP is lost from the chromaffin vesicle pool. This was confirmed in experiments using metabolic inhibitors to differentiate the vesicular and extravesicular ATP pools. The vesicular ATP loss was not proportional to that of catecholamines, resulting in a reduction by 50% in the chromaffin vesicle mole ratio of catecholamines to ATP after 48 h of treatment. In metabolic labeling studies, it was found that reserpine treatment reduced the incorporation of [3H]adenosine into vesicular ATP selectively, but it reduced the incorporation of 32Pi into both the vesicular and extravesicular pools. The reduction of the [3H]adenosine incorporation was not due to diminished vesicular nucleotide uptake resulting from low catecholamine levels, because when the catecholamines were depleted by tetrabenazine pretreatment followed by removal of the drug before labeling, no reduction in [3H]adenosine incorporation was observed. When present during the labeling, tetrabenazine was found to be a reversible inhibitor of plasma membrane adenosine uptake. The observed loss of adenine nucleotides from catecholamine-depleted chromaffin vesicles in vivo provides evidence that interactions between ATP and catecholamines are important in the vesicular storage of high concentration of these compounds.     

人主动脉壁硫酸乙酰肝素蛋白聚糖对培养的人主动脉平滑肌细胞生长的影响

从动脉粥样硬化（ＡＳ）高（北京）、低（南宁）发区人正常胸主动脉内－中膜分离ＨＳＰＧ,观察其对体外培养的ＨＡＳＭＣ生长的影响,细胞计数、~３Ｈ－ＴｄＲ参入及形态观察均表明ＡＳ高、低发区人主动脉ＨＳＰＧ都能剂量依赖性地抑制ＨＡＳＭＣ增殖,但抑制百分数未见显著差异,结果提示,人动脉壁中ＨＳＰＧ的含量可能与ＡＳ发病有关．