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1.
p75NGFR在哺乳动物神经细胞中的表达及其在诱导细胞凋亡中的功能 总被引:2,自引:0,他引:2
将从人胎儿基底前脑提取的总RNA用RT-PCR扩增并克隆在pUC12中的人神经生长因子低亲和力受体(p75NGFR)基因分两步克隆到逆转录病毒表达载体pXT-1中,经PA317细胞体外包装后收集假病毒上清感染大鼠小脑神经细胞系R2,在RNA转录和蛋白质合成水平获得了表达。转染了p75NGFR的R2细胞在去血清培养时可以诱导细胞凋亡的发生,此凋亡可被RNA合成抑制剂放线菌素D和蛋白质合成抑制剂环己酰亚胺抑制。表明p75NGFR诱导的细胞凋亡过程中有活跃的基因表达参与。 相似文献
2.
本研究目的在于探讨丝裂素活化蛋白激酶(MAPK)是否在AngⅡ(10-8mol/L)诱导的培养新生大鼠心肌成纤维细胞(FB)的增殖反应中起重要作用。实验以FB数目和DNA合成速率(3H-胸腺嘧啶掺入率)为增殖指标,[γ-32P]ATP掺入法和免疫印迹法分别测定FBMAPK的活性和含量,结果发现(1)AngⅡ处理FB24h后,DNA合成速率和细胞数比对照组分别增加60%和39%;(2)AngⅡ处理FB5min后,MAPK活性比对照组增高203%;(3)培养新生大鼠FB含有两个MAPK同型体-p44mapk和p42mapk,其中p44mapk含量高于p42mapk,分别为总量的58%和42%。AngⅡ处理5min后,MAPK蛋白含量(p44+p42〕增高429%,其中p44mapk的增加明显大于p42mapk的增加,分别比相应对照增高486%和349%。以上结果表明,AngⅡ诱导的MAPK活性和含量的增加,参与了FB的增殖反应,其中p44mapk的作用较为显著 相似文献
3.
可溶性55kD人肿瘤坏死因子受体(shTNFR55)在变铅青链霉菌中的分泌表达 总被引:1,自引:0,他引:1
从 He La 细胞中提取总 R N A,采用反转录 P C R 技术,从该总 R N A 中扩增了约 530 bp 的sh T N F R55 基因的 c D N A,并克隆至质粒 p U C m el中酪蛋白酶 m el Cl 分泌信号肽编码序列的下游,构建成含融合基因 m el/ T N F R 的重组质粒 p U C m el/ T N F R.把融合基因 m el/ T N F R 插入链霉菌表达质粒 p I J459 的多克隆位点,使之位于 erm 强启动子的下游,得到重组表达质粒 p I J459 m el/s T N F R.经 Southern 杂交证明重组质粒 p I J459 m el/s T N F R 插入了 s T N F R55 基因片段.对重组菌株 Streptom yces lividans(p I J459 m el/s T N F R)的发酵液进行 S D S P A G E、受体配基杂交( Ligand blot)分析、对 T N F 敏感的 L929 细胞的细胞毒性中和试验表明,可溶性肿瘤坏死因子受体 s T N F R55 在链霉菌中得到了分泌表达,表达产物具有生物学活性.表达产物的分子量约在 26~28 k D 之间. 相似文献
4.
双歧杆菌活菌制剂对慢性乙肝病人细胞免疫功能影响的观察 总被引:2,自引:1,他引:2
在应用双歧杆菌活菌制剂治疗慢乙肝期间,重点观察了T细胞亚群(CD3,CD4,CD8)、NK细胞(CD(16))、白细胞介素Ⅱ(IL-2)分泌细胞、肿瘤坏死因子(TNF)等细胞免疫指标治疗前后的动态变化,同时观察了病人血内毒素水平的动态变化和乙肝病毒标志物(HBVM)的改变。结果表明:(1)与对照组比较,双歧杆菌活菌制剂可使慢乙肝病人CD3+,CD4+数目明显增多,而对CD8+细胞数目无明显影响;(2)双歧杆菌活菌制剂可使CAH组的CD16+细胞显著增多(p<0.05);使CAH组和CPH组的IL-2分泌细胞均有非常显著和显著增加(分别p<0.01和p<0.05);(3)CAH组病人血中内毒素和TNF水平在双歧杆菌活菌制剂治疗后,匀出现非常显著降低(p<0.01);CPH组TNF水平较对照组无显著变化,但内毒素水平较对照组显著降低(p<0.05);(4)满疗程后(60天)CAH组有6例,CPH组有5例HBeAg阴转(分别为26.06%和25.0%),而对照组仅2例阴转(13.33%),两治疗组与对照组比较有显著性差异(p<0.05)。 相似文献
5.
三结构域重组FN多肽表达质粒的构建及其表达产物性质的初步鉴定 总被引:1,自引:0,他引:1
为探讨三结构域重组迁连蛋白(FN)在肿瘤治疗中的作用,构建了两个三结构域重组FN表达质粒pF94-62和pF94-82,它们分别编码两个重组多肽:CH62(FNPro1239-Ser1515经Met、Ala 1690-Val2049相连)和CH82(从CH62中删除了HerpⅡC端和CellⅡ结构域N端的Pro1953-Glu1978)。含表达质粒pF94-82的工程菌经37℃培养,CH82得到表 相似文献
6.
大鼠肝癌发生过程中p53的突变和甲胎蛋白的表达 总被引:2,自引:0,他引:2
采用免疫组织化学ABC和PAP法,对二乙基亚硝胺(DEN)诱发大鼠肝癌发生过程中突变型p53蛋白(mp53)和甲胎蛋白(AFP)在肝细胞中的表达进行了系统观察。结果显示:(1)DEN诱发大鼠肝癌发生率为100%;(2)正常大鼠及诱癌第4周大鼠的肝细胞均不表达mp53,至诱癌第8周,可见少量肝细胞表达mp53,诱癌晚期的癌结节内大部分肝癌细胞呈mp53阳性表达,mp53免疫反应阳性产物为胞核内棕褐色颗粒;(3)正常大鼠肝细胞不表达AFP,诱癌早期(4~8周)的大鼠肝小叶内可见少量AFP阳性肝细胞,多为小肝细胞,呈散在分布,此后AFP阳性肝细胞逐渐增多,晚期的癌结节内大部分癌细胞呈AFP阳性,AFP免疫反应阳性产物为胞浆内棕褐色颗粒。结果提示,mp53和AFP可作为分析肝癌进展的病理学指标 相似文献
7.
血管内皮细胞生长因子受体Flt—1胞外区基因的克隆及其在中国仓?… 总被引:1,自引:0,他引:1
朋原代培养的人脐静脉血管内皮细胞(HUVEC)提取细胞总RNA,采用逆转录PCR(RT-PCR)方法得到VEGF受体Flt-1胞外区前3个IgG样区域cDNA片段(Flt-1n3)。将获得的受体基因克隆到真核表达载体pcD-NA3.1中,得到重组质粒pcDNA3.1/Flt-1n3,通过南体转染方法将其转入中国仓鼠卵巢细胞(CHO),用G418筛选得到稳定表达目的蛋白的细胞砍隆。经固相结合实验筛选 相似文献
8.
研究表明,缺乏神经生长因子(NGF)的营养支持是Alzheimer's等神经元退行性疾病发生发展的重要原因,而NGF和/或NGF受体的过度表达则与一些神经系统肿瘤的发生发展有着十分密切的因果关系。采用(125)Ⅰ-NGF受体特异结合实验作为NGF受体活性物质筛选实验模型从中药牛膝中筛选出了能强烈地抑制(125)Ⅰ-NGF受体结合的活性成分N42-A(ⅠC(50)=6.18±3.43,n=4);细胞培养实验表明,N42-A对NGF诱导大鼠嗜铬神经瘤PCl2细胞的分化也具有很强的剂量依赖性抑制作用(对0.1nmol/L和0.2nmol/LNGF诱导的大鼠嗜铬神经病PC12细胞轴突生长的半数抑制浓度分别为6μg/mL和21μg/mL)。这表明,N42-A是神经元上介导NGF诱导轴突生长的特异受体抑制剂,不仅对NGF及其受体过度表达所致的神经系统肿瘤的防治具有潜在的应用价值,而且对Alzheimer's等神经元退行性疾病防治药物的开发研究具有十分重要的意义。 相似文献
9.
由反转录病毒载体转移的马立克病毒糖蛋白D基因在感染细胞中的表达 总被引:6,自引:0,他引:6
用EcoRI、PstI将已分离和克隆的马立克氏病病毒糖蛋白D基因从重组pMgD18质粒中切出,克隆进反转录病毒质粒载体(RCAS)的连接质粒载全PUCCla112N的相同位点。用ClaI再次gD基因切出,构建于RCAS的ClaI位点。通过原位杂交筛选重组RCAS,并结合酶切分析鉴定出gD插入方向正确的重组RCAS。用磷酸钙沉淀法将gD重组RCAS转染鸡胚成纤维细胞(CEF),转染后第9天收集细胞上 相似文献
10.
采用核仁组成区嗜银蛋白(Ag-NOR)技术,对40例胸腹水的患者进行细胞学观察研究。结果发现,良性病变胸腹水内细胞Ag-NOR计数在1.1~2.0之间,银颗粒小,大小比较均一。恶性病变Ag-NOR计数在4.3~6.2之间,银颗粒大,不规则。肉瘤与癌所致的胸腹水内细胞Ag-NOR颗粒数目未发现有明显差别。提示在临床病理工作中,Ag-NOR技术在胸腹水脱落细胞学检查中,对良、恶性肿瘤的鉴别诊断具有一定的实用意义。 相似文献
11.
Bovine adrenal chromaffin granules are a site of synthesis of atrial natriuretic factor 总被引:2,自引:0,他引:2
H Ong C Lazure T T Nguyen N McNicoll N Seidah M Chrétien A De Léan 《Biochemical and biophysical research communications》1987,147(3):957-963
We have previously reported the existence of a peptide factor in the adrenal medulla which inhibits aldosterone secretion in cultured bovine zona glomerulosa cells. The acid extracts of chromaffin granules from bovine adrenal medulla were purified by a four step high performance liquid chromatography procedure. Two active fractions exhibited sequence homology with bovine atrial natriuretic factor ANF (Ser99-Tyr126) and its polypeptide precursor (Asn1-Tyr126). The occurrence of both precursor and mature forms of ANF within chromaffin granules indicates the endogenous character of ANF in the adrenal medulla and suggests the potential usefulness of cultured adrenal chromaffin cells for investigating the synthesis, maturation and secretion of atrial peptides. 相似文献
12.
Atrial natriuretic factor immunoreactivity (ir-ANF) was examined in thyroid tissue sections and cultured thyroid cells using immunohistochemical staining with a monoclonal anti-ANF antibody. Localization of ir-ANF in perinuclear granules in cultured cells and in the basal region of follicular cells in sectioned tissue suggests that ir-ANF is a basally secreted product. Thyroidal ir-ANF was also characterized using reversed phase high performance liquid chromatography (RP-HPLC) of acidic thyroid extracts. An ir-ANF peak coeluting with synthetic rat ANF(99-126) suggests that thyroidal ir-ANF may be identical in form to circulating atrial ANF. However, the detection of ir-ANF in cultured thyroid cells confirms that the immunoreactivity is locally produced. Saturation analysis revealed high affinity ANF receptors (Kd = 0.1 nM; MBC = 17.2 fmol/mg protein) on these cultured cells, and a competition study demonstrated the ability of extracted thyroidal ir-ANF to inhibit 125I rat ANF binding to the membrane receptors. The evidence presented here suggests that ir-ANF in the thyroid may be secreted locally to exert an autocrine effect on neighboring follicular cells. 相似文献
13.
Atrial natriuretic factor in the vena cava and sinus node 总被引:2,自引:0,他引:2
C Sola G Thibault H Haile-Meskel M B Anand-Srivastava R Garcia M Cantin 《The journal of histochemistry and cytochemistry》1990,38(8):1123-1135
We investigated the localization of atrial natriuretic factor (ANF) mRNA and of immunoreactive ANF in the vena cava and sinus node of rat and, for comparative purposes, in atria and ventricles. In situ hybridization with an ANF cRNA probe revealed that the supradiaphragmatic portion of the inferior vena cava contains almost as much mRNA as the atria, whereas the levels were less in the superior vena cava and higher than in ventricles in the sinus node. Immunoreactive ANF (high Mr form) was found to be 22 times less abundant in the supradiaphragmatic vena cava and 148 times less abundant in the superior vena cava than in atrial cardiocytes. The wall of the supradiaphragmatic portion of the vena cava and the valve (eustachian valve) that separates the atrial cavity from that of the vein are made up of atrial-like cardiocytes containing secretory granules. The subendothelial area of the superior vena cava also contains atrial-like cardiocytes with secretory granules, whereas the outer portion of the vein is made up of "transitional cells" without or with only a few secretory granules. Secretory granules in the vena cava and nodal cells, as well as transitional cells, contain immunoreactive ANF. With immunocryoultramicrotomy, virtually all cells, whether atrial-like, transitional, or nodal, and even those without secretory granules, were found to contain immunoreactive ANF in their Golgi complex and in secretory vesicles in the vena cava and in the sinus node. 相似文献
14.
Double immunocytochemical labeling applying the protein A-gold technique 总被引:23,自引:0,他引:23
M Bendayan 《The journal of histochemistry and cytochemistry》1982,30(1):81-85
In the present study we report the modifications and the different steps of the protein A-gold (pAg) technique that allow the simultaneous demonstration of two antigenic sites on the same tissue section. The labeling is carried out in the following manner: face A of the tissue section is incubated with an antiserum followed by a pAg complex prepared with large gold particles; face B of the same tissue section is then incubated with a second antiserum followed by a pAg complex prepared with small gold particles. Each of the pAg complexes reveals a different antigenic site on opposite faces of the tissue section. The transparency of the section in the electron beam allows the visualization of the gold particles present on both faces. The double labeling pAg technique was applied for the simultaneous demonstration of two secretory proteins in the same Golgi, condensing vacuoles, and zymogen granules of the rat pancreatic acinar cells. 相似文献
15.
The secretion of atrial natriuretic factor-(99-126) by cultured cardiac myocytes is regulated by glucocorticoids 总被引:2,自引:0,他引:2
Atrial natriuretic factor (ANF) is stored in mammalian atria primarily as ANF-(1-126), the precursor to the known circulating form of the hormone ANF-(99-126). When primary cultures of atrial myocytes were maintained in a complete serum-free medium, they contained and secreted an ANF-(1-126)-like peptide. The addition of dexamethasone to the culture medium, however, resulted in the secretion of a molecule with chromatographic characteristics identical to ANF-(99-126), although the intracellular storage form of ANF was unchanged. Radiosequencing and amino acid analysis confirmed that the cultures maintained in dexamethasone secreted authentic ANF-(99-126). Chronic exposure of the cells to dexamethasone also resulted in a significant increase in the quantity of immunoreactive ANF both contained and secreted by the cultures. Dexamethasone stimulated ANF processing and secretion by atrial cultures in a dose-dependent manner, with an approximate EC50 of 10 nM. This stimulation could be reversed by removing the glucocorticoid from the culture medium. ANF processing was also stimulated by the specific glucocorticoid receptor agonist RU 28362, and both DEX- and RU 28362-stimulated ANF processing was inhibited by the specific glucocorticoid receptor antagonist RU 38486. Ventricular cells, which possess few granules and release ANF in a constitutive fashion, were also capable of processing ANF in a glucocorticoid-dependent fashion. Medium freshly removed from atrial cultures did not convert ANF-(1-126) to ANF-(99-126) nor was exogenous ANF-(1-126) efficiently processed when added to the medium of actively processing cultures. These results indicate that the post-translational processing of ANF-(1-126) to ANF-(99-126) likely occurs within or in close association with the cardiac myocytes and is not dependent on the presence of large quantities of secretory granules. Furthermore, it is apparent that both the expression and the post-translational processing of ANF by cultured cardiac myocytes is specifically regulated by glucocorticoids. 相似文献
16.
Neonatal atria and ventricles secrete atrial natriuretic factor via tissue-specific secretory pathways 总被引:23,自引:0,他引:23
The cellular mechanisms regulating secretion of the peptide hormone atrial natriuretic factor (ANF) differ in neonatal atrial and ventricular cardiocytes. We demonstrate that although both cell types synthesize and secrete ANF, only atrial cells store peptide in abundant secretory granules. Neonatal ventricular cells secrete ANF rapidly after synthesis and lack secretory granules. We propose that ventricular ANF is released by a constitutive secretory pathway whereas atrial ANF is stored and released by a regulated pathway. Furthermore, ventricular ANF mRNA and hormone concentrations decrease during the first week of life. Developmental variation in the use of ANF secretory pathways may reflect changing requirements for maintenance of intravascular volume and pressure. Tissue-specific modulation of hormone secretory pathways appears to be a novel response to developmentally induced changes in the requirements for a peptide hormone. 相似文献
17.
A J de Bold 《Canadian journal of physiology and pharmacology》1987,65(10):2007-2012
Investigations culminating at the beginning of this century clearly established that the cardiac muscle cell (cardiocytes) is differentiated for excitation, conduction, and contraction. All of the physiology and pathophysiology of the heart was developed subsequently based on this concept. However, morphological investigations in the mid 1950s suggested a secretory function for mammalian atrial cardiocytes. These cells contain storage granules, the specific atrial granules, which resemble granules found in polypeptide hormone-producing cells. The development of techniques for the study of these granules using a combined biochemical-morphological approach during the 1970s defined their general chemical nature and their behaviour under different experimental conditions. Because the number of atrial granules change dramatically following upsets of water and electrolyte balance, atrial muscle extracts were tested for effects on kidney function. In 1981, it was reported that atrial extracts contain a natriuretic factor (ANF) capable of inducing massive diuresis, increases in hematocrit, and lowering of blood pressure. It was demonstrated soon thereafter that ANF is stored within specific atrial granules. More recent work has defined ANF as a polypeptide hormone that appears to modulate or antagonize the renin-angiotensin-aldosterone system. Current work attempts to define the physiological and pathophysiological role for ANF as well as possible therapeutic uses. 相似文献
18.
Terje H. Larsen Henrik S. Huitfeldt Ole Myking Thorvald Sætersdal 《Cell and tissue research》1993,272(2):201-210
A close spatial relationship between specific granules containing atrial natriuretic factor (ANF) and microtubules was demonstrated in primary cultures of neonatal rat cardiac myocytes. For the detection of specific granules and microtubules, the myocytes were double immunolabelled with antibodies against -ANF and -tubulin and examined by conventional fluorescence or laser scanning confocal microscopy. In addition, the ultrastructural distribution of specific granules was demonstrated by electron microscopy. In the atrial myocytes, ANF was stored in numerous specific granules that were mainly localized in the perinuclear sarcoplasm. In the ventricular myocytes, however, a minority of the cells (10%) exhibited limited ANF immunoreactivity after 4 days in culture. Microtubules were present throughout the sarcoplasm of the myocytes. They were most densely packed in the perinuclear regions. Depolymerization of the microtubules with nocodazole was followed by dispersal of ANF immunostaining both in the atrial myocytes and in the ventricular myocytes exhibiting ANF immunoreactivity. When the microtubules were allowed to recover, the perinuclear distribution of specific granules, as seen in non-treated myocytes, reappeared. Measurements of secreted immunoreactive ANF by radioimmunoassay revealed that the secretion of ANF from atrial myocytes into the medium was significantly reduced following nocodazole treatment, whereas a similar decrease in secretion from ventricular myocytes was not observed. These findings indicate that ANF-containing specific granules are closely associated with microtubules within the myocytes. It is suggested that secretion of ANF from the atrial myocytes, in contrast to the ventricular myocytes, is microtubule-dependent. 相似文献
19.
Stimulatory effects of atrial natriuretic factor on phosphoinositide hydrolysis in cultured bovine aortic smooth muscle cells 总被引:11,自引:0,他引:11
The effects of atrial natriuretic factor (ANF) on phosphoinositide hydrolysis were examined in preparations of cultured bovine aortic smooth muscle cells. In homogenates or particulate fractions from cultured bovine aortic smooth muscle cells, ANF and atriopeptin I increased the formation of inositol phosphates and GTPase activity. The effects on inositol phosphates were markedly enhanced with guanosine 5'[gamma-thio]triphosphate. Both atrial peptides also stimulated the formation of diacylglycerol in intact cultured cells. In these experiments, atriopeptin I was about 10-fold more potent than ANF. These studies indicate that atrial peptides have stimulatory effects on phosphoinositide hydrolysis which are mediated through a guanine nucleotide regulatory protein. The greater potency of atriopeptin I on GTPase activity and the accumulation of inositol phosphates suggests that the nonguanylate cyclase-coupled receptor for ANF (ANF-R2) mediates the stimulatory effects of ANF on phosphoinositide hydrolysis through a guanine nucleotide regulatory protein. 相似文献
20.
心房钠尿因子对豚鼠窦房结自律性的影响 总被引:2,自引:0,他引:2
用微电极技术研究表明,分别灌流心房钠尿因子(ANF)0.025和0.05μmol/L 7min 后,豚鼠窦房结细胞的自发节律无明显变化,而用0.1μmol/L 灌流时,其自发节律明显降低7%(P<0.01)。但当上述三种浓度 ANF 和异丙肾上腺素混合液灌流时,自发节律分别降低4,12和22%。这些结果表明,ANF 能抑制异丙肾上腺素的阳性变时效应。其机理可能与 ANF 阻滞窦房结细胞的 T 型钙通道电流有关。 相似文献