首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
Summary 86Rb uptake into LLC-PK1 cells (an established renal epithelial cell line) was found to be comprised of an active ouabain-sensitive component, a loop diuretic-sensitive component which was passive and strictly dependent upon the presence of extracellular Na+ and Cl for activity, and a leak component. The diuretic-sensitive component of influx was investigated further in apical membrane vesicles derived from these cells. A large fraction of86Rb,22Na and36Cl flux into these vesicles was sensitive to inhibition by furosemide and dependent upon the presence of the other two co-ions, in keeping with the presence of a loop diuretic-sensitive Na+K+Cl cotransport system. The kinetic parameters for Na+ and K+ interaction have been analyzed under initial linear zerotrans conditions. The following values were obtained:K mNa+=0.42±0.05 mmol/liter,V max=303±24 pmol/mg/6 sec;K mK+=11.9±1.0 mmol/liter,V maxK+=307±27 pmol/mg/6 sec. For Cl interaction evidence for two cooperative binding sites with different affinities and different specificities were obtained. Thus, a stoichiometry of 1Na+1K+2Cl can be calculated. It is concluded that the apical membrane of LLC-PK1 cells contains a Na+K+2Cl cotransport system with properties similar to those described for the thick ascending limb of the loop of Henle.  相似文献   

2.
Polyclonal activation of human peripheral blood lymphocytes (PBLs)in vitro by preparations ofStreptococcus pyogenes Su strain (OK-432) and other heat-killed strains was investigated. The streptococcal preparations tested induce a proliferative response of PBLs via interleukin-2 (IL-2)-independent pathways. The proliferative response is accompanied by the generation of lymphoblastic cells (LBCs), which consist of heterologous lymphocyte populations: CD4+ helper type of T cells, and CD4CD8 double-negative (DN) lymphocytes, including both CD3+ TcR + T cells and CD2+CD3 immature type of T or non-T cell type of lymphocytes. Almost all the LBCs express Leu19, TfR (transferrin receptor), LFA-1 and CD38 (OKT10) antigens, which are expressed on activated T cells, NK cells and some other lymphocytes. The proliferative response of human PBLs is also accompanied by the generation of potent cytotoxic activity against NK-sensitive and -resistant targets. C-dependent cytolysis and cell sorting experiments of OK-432-activated LBCs revealed that both CD3+ and CD3 types of CD4CD8 DN lymphocytes, but not CD4+ helper T cells, may be major populations responsible for the cytotoxicity induced. On the other hand, CD4CD8 T cells may be required for the proliferation of PBLs and generation of cytotoxic effector cells. These results suggest that the OK-432 and other streptococcal preparations stimulate the human PBLsin vitro to induce the proliferation/activation of CD4+ T cells, mediating the following generation of DN cytotoxic effector lymphocytes.  相似文献   

3.
Summary Homogenates and plasma membranes were isolated from the livers of male Fischer 344 rats ranging in age from 19 hr to 92 days postnatal. These plasma membranes exhibited comparable levels of purity: protein yields were 2–2.5%; relative specific activities of 5-nucleotidase and ouabain-sensitive Na+/K+-ATPase were from 8–11 and from 12–19, respectively. 5-nucleotidase and ouabain-sensitive Na+-K+-ATPase displayed distinct and different developmental patterns. The activity of -glutamyltranspeptidase was found to be at exceptionally high levels in isolated plasma membranes immediately after birth and to decline precipitously thereafter achieving and maintaining low levels from days 3–21 postnatal. Liver plasma membrane -glutamyltranspeptidase activity was observed to increase 9.2 fold from this low point, first rising on day 21, peaking on day 40 and returning to low levels by day 56. From day 56 day to 92 postnatal, -glutamyltranspeptidase activity was expressed at a uniformly low level but a level 2 fold higher than that preceeding the rise at day 40. The hormone determinants of these developmental changes in -glutamyltranspeptidase activity are discussed.  相似文献   

4.
The parental strain (A+T+) of Saccharomyces cerevisiae and mutants, deficient in catalase T (A+T), catalase A (AT+) or both catalases (AT), grew on ethanol and oleic acid with comparable doubling times. Specific activities of catalase were low in glucose- and ethanol-grown cells. In the two oleic acid-grown A+-strains (A+T+ and A+T) high catalase activities were found; catalase activity invariably remained low in the AT+ strain and was never detected in the AT strain. The levels of -oxidation enzymes in oleic acid-grown cells of the parental and all mutant strains were not significantly different. However, cytochrome C peroxidase activity had increased 8-fold in oleic acid grown A strains (AT+ and AT) compared to parental strain cells. The degree of peroxisomal proliferation was comparable among the different strains. Catalase A was shown to be located in peroxisomes. Catalase T is most probably cytosolic in nature and/or present in the periplasmic space.  相似文献   

5.
Microdissected -cell-rich pancreatic islets fromob/ob-mice were used in studies of transmembrane36Cl efflux. The mean rate coefficient for36Cl efflux was stable at 0.158 min–1 during the initial 10 min. Depolarization of the -cell plasma membrane by acute increases in extracellular K+ (5–130mM) stimulated the36Cl efflux in a concentration-dependent manner. Glucose-induced (20mM) and K+-induced increases in36Cl efflux were largely overlapping, but even at 135.9 mM K+, glucose slightly further enhanced the36Cl efflux rate. The data suggest (1) that pancreatic -cells are equipped with a voltage-dependent Cl permeability, (2) that glucose-induced increase in Cl permeability may, at least partly, be mediated by primary membrane depolarization, and (3) that glucose in addition may activate other mechanisms for -cell Cl transport.  相似文献   

6.
Diurnal variation in hydrological variables and dissolved inorganic nutrients such as PO inf4 sup3– -P, N O inf2 sup– -N, NO inf3 sup– -N and NH inf4 sup+ -N were studied in three interconnected biotopes including freshwater, marine and mangrove brackish water of the Kakinada coastal zone, Andhra Pradesh. Samples were collected at intervals of 3 hours, for a period of 24 hours. In the marine environment salinity varied from 26 to 32 whereas in the mangrove waters it fluctuated from 12 to 20 and in both biotopes salinity showed bimodal type of oscillation. Dissolved oxygen content was high in the mangrove waters during day time but decreased rapidly during the night hours. In the marine environment POf4 p3–-P concentration varied from 0.345 to 1.195 g at l–1, NO inf3 sup– -N from 1.03 to 6.62 g at l–1 and NO inf2 sup– -N from 0.086 to 0.506 g at l–1. The highest and the lowest concentrations of PO inf4 sup3– -P, NO inf3 sup– -N, NO inf2 sup– -N recorded in the mangrove waters were 0.790 and 0.325 g at l–1, 7.10 and 1.60 g at l–1 and 0.278 and 0.060 g at l–1, respectively. The concentration of PO inf4 sup3– -P, NO inf3 sup– -N and NO inf2 sup– -N were high in the freshwater canal, the maximum and minimum values being 1.110 and 0.730 g at l–1, 26.40 and 9.98 g at l–1 and 0.520 and 0.252 g at l–1 respectively. The concentration of ammonia was relatively high in the mangrove water. Gross and net primary production in the mangrove water was 4 times higher than in the marine biotope. There was no export of dissolved nutrients from the mangrove environment to the adjacent marine waters.  相似文献   

7.
Guo  S.  Brück  H.  Sattelmacher  B. 《Plant and Soil》2002,239(2):267-275
In order to investigate the effect of N form on dry matter (DM) formation and water uptake rate, French bean (Phaseolus vulgaris L. `Sotaxa') plants were grown with a split-root system. Three treatments were compared: sole nitrate (NO 3) supply (NN), sole ammonium (NH+ 4) supply (AA) and spatially separated supply of NO 3 and NH+ 4 (NA). The pH of the nutrient solutions was kept constant at 6.3 using a pH-stat system. 9 days after onset of the treatments, NN plants had higher root (36%) and shoot dry matter (11%) than AA plants. N form drastically influenced partitioning of assimilates: in the NA treatment, the root half exposed to NO 3 revealed a 170% higher DM than the root half exposed to NH+ 4. N form affected stable carbon-isotope discrimination () of leaf tissue. In leaves of plants which were supplied with NH+ 4 (AA; NA) was significantly more negative (–29.4, –29.6) than in NN treatment (–28.2). We explain this effect by differences in stomatal conductance. We suppose that the significantly less negative of root tissue under NH+ 4 supply is most probably related to higher PEP-case activity. The water uptake rate was higher in NN than in AA grown plants. This effect was found in both, short- and long-term experiments. In case of NA plants, the water uptake in the root part being exposed to NO 3 was 104% higher than in those receiving NH+ 4. At least in the case of the NA treatment we can exclude shoot growth effects as being responsible for differences in water uptake. We therefore assume that differences in root hydraulic conductivity are responsible for the observed effects.  相似文献   

8.
DNA polymorphism patterns linked to the A-globin gene were analyzed in healthy Japanese using four different restriction endonucleases. The chromosomes with the A-globin gene were mapped through an evaluation of the presence of seven different restriction sites (HincII 5 to ; HindIII in G and A; HincII in, and 3 to, 1; AvaII in ; Bam-HI 3 to ). Among 36 chromosomes analyzed, 20 chromosomes had a haplotype of [+–––––+]. Among 55 individuals examined, 7 possessed a homozygous haplotye of [+–––––+]. All Japanese with the AT-globin gene had a subhaplotype of [–++–+] 5 to the -globin gene. Their major haplotypes were [–++–+–+] and [–++–++–]. It was expected that the presence of the AT-globin gene in Japanese may be deduced from subhaplotypes 5 to the -globin gene.  相似文献   

9.
Elementary Na+ currents were recorded in inside-out patches from neonatal rat heart cardiocytes to analyze the influence of a site-directed polyclonal anti-serum against the linker region between the domains III and IV (amino acids 1489–1507 of the cardiac Na+ channel protein) on Na+ channel gating and to test whether this part of the -subunit may be considered as a target for modifying agents such as the (–)-enantiomer of DPI 201-106.Anti-SLP 1 serum (directed against amino acids 1490–1507) evoked, usually within 10–15 min after cytosolic administration, modified Na+ channel activity. Antiserum-modified Na+ channels retain a single open state but leave, at –60 mV for example, their conducting configuration consistently with an about threefold lower rate than normal Na+ channels. Another outstanding property of noninactivating Na+ channels, enhanced burst activity, may be quite individually pronounced, a surprising result which is difficult to interpret in terms of structure function relations. Removal of inactivation led to an increase of reconstructed peak I Na (indicating a rise in NP o) and changed I Na decay to obey second-order kinetics, i.e., open probability declined slowly but progressively during membrane depolarization. The underlying deactivation process is voltage dependent and responds to a positive voltage shift with a deceleration but may operate even at the same membrane potential with different rates. Iodatemodified Na+ channels exhibit very similar properties including a conserved conductance. They are likewise controlled by an efficient, voltage-dependent deactivation process. Modification by (–)-DPI 201-106 fundamentally contrasts to the influence of anti-SLP 1 serum and the protein reagent iodate since (–)-DPI-modified Na+ channels maintain their open probability for at least 120 msec, i.e., a deactivation process seems lacking. This functional difference suggests that the linker region between the domains III and IV of the -subunit may not be the only target for (–)-DPI 201-106 and related compounds, if at all.This work was supported by a grant of the Deutsche Forschungs-gemeinschaft (Ko 778/2–4), Bonn.  相似文献   

10.
Following acute liver injury, hepatocytes divide to facilitate regeneration. However, during chronic injury, hepatocyte proliferation is typically blocked and repair is mediated through liver progenitor (oval) cells. Signalling of the p55 tumour necrosis factor (TNF) receptor is central to these processes. Two ligands for p55 are known: TNF and lymphotoxin-alpha (LT). However, one study suggests that another exists that mediates liver injury following viral challenge. We have therefore investigated whether ligands other than TNF and LT are required for liver regeneration following either acute or chronic injury. Wild-type and double TNF/LT knockout (TNF–/–LT–/–) mice were subjected to either partial hepatectomy (PHx) or a choline-deficient ethionine-supplemented (CDE) diet. Proliferating hepatocytes, oval cells and inflammatory cells were identified and quantified in liver sections by immunohistochemistry. Liver inflammatory cells were characterised by cell surface antigen expression. Liver damage and mortality were monitored. Both hepatocyte and oval cell proliferation was reduced in TNF–/–LT–/– mice. Lymphocyte clusters were evident in all TNF–/–LT–/– livers and were heterogeneous, comprising B and T lymphocytes. PHx evoked liver inflammation in TNF–/–LT–/– but not wild-type mice, whereas no difference was apparent between genotypes in CDE experiments. Thus, TNF/LT signalling mediates liver regeneration involving both hepatocytes and progenitor cells. The hyper-inflammatory response following PHx in TNF–/–LT–/– animals, which is absent following CDE-induced injury, demonstrates that the two forms of liver injury evoke discrete inflammatory responses and provides a model in which such differences can be examined further.  相似文献   

11.
The binding abilities of silver(I) to mammalian MT 1 have been studied and compared with those of copper(I), recently reported [Bofill et al. (2001) J Biol Inorg Chem 6:408–417], with the aim of analyzing the suitability of Ag(I) as a Cu(I) probe in Cu–MT studies. The Zn/Ag replacement in recombinant mouse Zn7–MT 1 and corresponding Zn4-MT 1 and Zn3-MT 1 fragments, as well as the stepwise incorporation of Ag(I) to the corresponding apo-MTs, have been followed in parallel by various spectroscopic techniques including electronic absorption (UV–vis), circular dichroism (CD) and electrospray mass spectrometry coupled to capillary zone electrophoresis (CZE-ESI-MS). A comparative analysis of the sets of data obtained in the titration of Zn7–MT 1, Zn4–MT 1 and Zn3-MT 1 with AgClO4 at pH 7.5 and 2.5 has led to the reaction pathways followed during the incorporation of silver to these proteins under these specific conditions, disclosing unprecedented stoichiometries and structural features for the species formed. Thus, the Zn/Ag replacement in Zn7–MT 1 at pH 7.5 has revealed the subsequent formation of Ag4Zn5–MT, Ag7Zn3–MT, Ag8Zn3–MT, Ag10Zn2–MT, Ag12Zn1–MT, Agx–MT, x=14–19, whose structure consists of two additive domains only if Zn(II) remains coordinated to the protein. A second structural role for Zn(II) has been deduced from the different folding found for the Agx–MT species of the same stoichiometry formed at pH 7.5 or 2.5. Comparison of the binding features of Cu(I) and Ag(I) to the entire MT at pH 7.5 shows that, among all the xZny–MT (0y<7) species found, only MI4Zn5–MT [(Zn4)(4Zn1)] and MI7Zn3–MT [(3Zn2)(4Zn1)], which form during the first stages of the Zn(II)/M(I) metal replacement, show comparable 3D structures; thus, they are the only species where Ag(I) ions can be predicted to be an adequate probe for Cu(I).Electronic Supplementary Material Supplementary material is available in the online version of this article at .  相似文献   

12.
The sodium contents of -cell-rich pancreatic islets fromob/ob-mice were measured with an integrating flame photometer. After washing to an apparent steady state with different types of ice-cold media, islets incubated in the absence of glucose contained 79–108 mmol sodium kg–1 dry weight. Exposure to glucose resulted in 25 % reduction of the islet content of sodium. This effect became manifest in the presence of 5 mM glucose, there being no additional reduction with a further increase of glucose to 20 mM. Depression of Na+ activity may partially explain why glucose, under certain conditions, can lower cytoplasmic Ca2+ and even inhibit insulin release.  相似文献   

13.
Astroglial cells are known to release taurine in response to stimulation by a variety of stimuli including -adrenergic receptor agonists such as isoproterenol (IPR). The effects of changing osmolarity and extracellular [K+] on IPR-stimulated taurine release were studied with LRM55 cells, a continuous astroglial cell line. IPR-stimulated taurine release decreased almost 8% for each 1% increase in osmolarity, indicating that IPR-stimulated release is highly regulated by the osmolarity of the medium. IPR-stimulated taurine release was greatly enhanced when external [K+] was increased isosmotically by substituting KCl for NaCl but was strongly suppressed when external [K+] was increased hyperosmotically by adding KCl to the medium. Both IPR-stimulated and K+-stimulated taurine release depended on external [Cl]; IPR-stimulated release declined approximately in parallel to K+-stimulated release as [Cl] in the medium was reduced. The high sensitivity of IPR-stimulated release to factors that change cell volume (osmolarity, external [K+], external [Cl]) is consistent with the idea that IPR, elevated [K+], and reduced osmolarity all elicit taurine release via a single tension-controlled mechanism.Special issue dedicated to Dr. Claude Baxter.  相似文献   

14.
Effects of human natural interferon (nIFN) alone, human natural tumor necrosis factor (nTNF) alone and their combination (OH-1) were tested on three human mesothelioma lines implanted in nude mice. Tumors were transplanted subcutaneously by trocar on treatment day –12. nIFN was given intraperitoneally (i.p.) at a dose of 2 × 107 or 2 × 108 IU kg–1 day–1, 5 days a week for 3 weeks. nTNF was given i.p. at a dose of 2 × 107 or 2 × 108 U kg–1 day–1 in the same schedule as that of nIFN. Tumor diameters were serially measured and tumor volumes were calculated. Antitumor effects were assessed by two methods: comparison of final tumor volumes in treated and control groups (T/C), and changes in median average total tumor volume. The treatment produced no clinically discernible toxicities. nIFN had strong inhibitory activity against all three human mesothelioma lines. nTNF alone had modest activity only at the high dose used. The combination of the two produced activity essentially similar to that produced by nIFN alone. High-dose nIFN may have a role as an active agent in the treatment of patients with mesothelioma.  相似文献   

15.
The maximum productivity of -glucosidase by Saccharomyces cerevisiaerecombinants under the control of GALI promoter was 100 IU l–1 h–1. The highest productivity of -glucosidase by a S. cerevisiae recombinant was 16-fold more than that supported by Cellulomonas biazotea. The recombinants also co-produced ethanol from cellobiose: maximum product yield and productivity were 0.5 and 1.1 g ethanol g–1 cellobiose and g ethanol l–1 h–1, respectively.  相似文献   

16.
The effect of -alany-L-histidinato zinc (AHZ) on bone cell function was investigated in osteoblastic MC3T3-E1 cells. Cells were cultured for 3 days at 37°C in a CO2 incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus AHZ (10–7–10–5 M) or other reagents, and the cells were cultured further for appropriate periods of time. The presence of AHZ (10–7–10–5 M) produced a remarkable increase of alkaline phosphatase activity and protein concentration in osteoblastic cells. Thus increases were seen with the prolonged cultivation (12–21 days). With the culture of 1, 3 and 12 days, the effect of AHZ (10–6 M) to increase alkaline phosphatase activity and protein concentration was more intensive than the effect of zinc sulfate, (10–6 M). The AHZ effects were completely abolished by the presence of cycloheximide (10–6 M), indicating that AHZ stimulates protein synthesis in the cells. The present study suggests that AHZ has a stimulatory effect on cell differentiation, and that this effect is partly involved on protein synthesis in osteoblastic cells.  相似文献   

17.
Fed-batch cultures of Bacillus licheniformis produced poly--glutamic acid (PGA), a water-soluble biodegradable polymer. PGA reached 35 g l–1 with a productivity of 1 g l–1 h–1 by pulsed-feeding of citric acid (1.44 g h–1) and l-glutamic acid (2.4 g h–1) when citric acid was depleted from the culture medium.  相似文献   

18.
HER-2/neu is an immunogenic protein eliciting both humoral and cellular immune responses in patients with HER-2/neu-positive (+) tumors. Preexisting cytotoxic T lymphocyte (CTL) immunity to HER-2/neu has so far been mainly evaluated in terms of detection of CTL precursor (CTLp) frequencies to the immunogenic HLA-A2–binding nona-peptide 369-377 (HER-2(9369)). In the present study, we examined patients with HER-2/neu+ breast, ovarian, lung, colorectal, and prostate cancers for preexisting CTL immunity to four recently described HER-2/neu–derived and HLA-A2–restricted "cytotoxic" peptides and to a novel one spanning amino acids 777–785 also with HLA-A2–binding motif. We utilized enzyme-linked immunosorbent spot (ELISpot) assay, which allows a quantitative and functional assessment of T cells directed against specific peptides after only brief in vitro incubation. CTL reactivity was determined with an interferon (IFN-) ELISpot assay detecting T cells at the single cell level secreting IFN-. CTLp were defined as peptide-specific precursors per 106 peripheral blood mononuclear cells (PBMCs). Patients' PBMCs with increased CTLp were also tested against autologous tumor targets and peptide-pulsed dendritic cells (DCs) in cytotoxicity assays. We also studied patients with HER-2/neu-negative (-) tumors and healthy individuals. Of the HER-2/neu+ patients examined, 31% had increased CTLp to HER-2(9952), 19% to HER-2(9665), 16% to HER-2(9689), and 12.5% HER-2(9435), whereas only 2 of 32 patients (6%) responded to HER-2(9777). The CTLp recognizing HER-2(9952) were extremely high in two patients with breast cancer, one with lung cancer, and one with prostate cancer. None of the HER-2/neu- patients or healthy donors exhibited increased CTLp to any of these peptides. Besides IFN- production, preexisting CTL immunity to all five HER-2/neu peptides was also shown in cytotoxicity assays where patients' PBMCs with increased CTLp specifically lysed autologous tumor targets and autologous peptide-pulsed DCs. Our results demonstrate for the first time that (1) preexisting immunity to peptides HER-2(9435), HER-2(9952), HER-2(9689), HER-2(9665), and HER-2(9777) is present in patients with HER-2/neu+ tumors of distinct histology, (2) HER-2(9777) is a naturally processed peptide expressed on the surface of HER-2/neu+ tumors, as are the other four peptides, and (3) HER-2/neu+ prostate tumor cells can be recognized and lysed by autologous HER-2 peptide-specific CTL. Our findings broaden the potential application of HER-2/neu-based immunotherapy.  相似文献   

19.
We investigated the effect of adenosine 5-triphosphate (ATP), -tocopherol and H202 on the micropropagation of cucumber (Cucumis sativus L.) from nodal explants. The effect of the size of the liquid culture vessel (250-ml flask vs. 2.5-l airlift bioreactor) was also evaluated. The addition of ATP alone caused a significant increase in the number of branches (internodes) and in internodal length, but also a reduction of leaf number, compared to control cultures. It also increased significantly the accumulation of NO3 and K+ . The application of ATP + -tocopherol was associated with a significant increase in bud, internodal, leaf and petiole number, internodal, petiole and root length, as well as plant fresh weight, but reduced PO43– and Ca2+ accumulation. The combined application of ATP + -tocopherol + H2O2 was associated with maximum petiole length and increased plant fresh weight but reduced Ca2+ accumulation. Compared to all other treatments, application of ATP + -tocopherol in bioreactor-incubated cultures produced significantly larger plants, with an increased bud number, internode lenght and soluble carbohydrate concentration, but also with a reduced fresh weight, root length and reduced NO3 and PO43– and Ca2+ concentrations. These effects were associated with changes in the redox status of the regenerants, as well as dehydrogenase and peroxidase activities. The perspective for an application of ATP and antioxidants as novel, cost-efficient growth regulators in the micropropagation of this commercially important vegetable species is discussed.  相似文献   

20.
The effect of -alanyl-L-histidinato zinc (AHZ) on protein components in osteoblastic MC3T3-E1 cells was investigated. Cells were cultured for 3 days at 37°C in CO2 incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus various concentrations of AHZ or other reagents, and the cells were cultured further 3 or 6 days. The homgenate of cells was analyzed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The presence of AHZ (10–7 to 10–5 M) caused an appreciable increase of many protein components in cells. Especially, the 67 killo-dalton (kDa) and 44 kDa proteins which are the major components from control cells were clearly increased by the presence of AHZ. Furthermore, the concentrations of osteocalcin, insulin-like growth factor-I and transforming growth factor- in the culture medium secreted from osteoblastic cells were markedly increased by the presence of AHZ (10–6 and 10–5 M). The effect of AHZ was a greater than that of zinc sulfate (10–6 and 10–5 M). The present findings suggest that AHZ can increase many proteins which are involved in the stimulation of bone formation and cell proliferation in osteoblastic cells.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号