茯苓健脾作用活性部位的研究

通过建立饮食失节、过食肥甘所致的小鼠脾虚模型,以体重、饮食量、外部表现特征及脾指数为指标,从茯苓总三萜类、茯苓总多糖及茯苓水煎剂中确定具有健脾作用的有效活性部位。结果表明:茯苓总三萜类对脾虚小鼠有明显的恢复作用,茯苓水煎剂也有一定作用,而茯苓总多糖作用不明显。说明茯苓总三萜类是茯苓治疗过食肥甘所致小鼠脾虚证的活性部位。     

A novel growth factor in rat spleen which promotes proliferation of hepatocytes in primary culture

We have identified a factor from adult rat spleen which stimulates the proliferation of rat hepatocytes. The activity was found in the spleen soluble matrix fraction (1,300xg supernatant of inter-cellular fraction). No activity was found in the spleen cell homogenate, in the spleen insoluble matrix fraction or rat serum. After 4 days of incubation with the spleen factor, the cell number increased 4-fold higher than that at inoculation. The growth stimulation were observed in both fetal bovine serum supplemented medium and hormonally defined medium which contains insulin, epidermal growth factor, glucagon, growth hormone and prolactin. The level of activity in the spleen soluble matrix was not affected by partial hepatectomy or trypsinization. These data indicate that the spleen factor is different from previously characterized effectors of hepatocyte proliferation. The novel factor has been named spleen derived growth factor (SDGF).     

Effect of cortisol on the release and retention of labelled DNA in the thymus and spleen of mice.

The lymphocytolytic effect of different doses of cortisol was studied in the thymus and spleen of mice previously injected with 3H-thymidine. The results indicate that in thymus the fraction of labelled cells was more resistant to cortisol than the unlabelled cell population. The release of DNA into the fraction of DNA soluble in 0.14 M NaC1 was delayed suggesting that cortisol controls indirectly the lymphocytolytic process. Up to 24 hours after administration of cortisol the loss of labelled spleen cells significantly exceeded the loss of unlabelled cells. The time course of the release of labelled DNA into the fraction of DNA soluble in 0.14 M NaC1 indicates that a fraction of labelled DNA was rapidly removed from the spleen after injection of cortisol.     

Suppressor cells in the spleens of tumor-bearing mice: enrichment by centrifugation on hypaque-ficoll and characterization of the suppressor population.

Spleen cells from mice bearing methylcholanthrene-induced sarcomas or a mammary adenocarcinoma suppressed the mitogen responses of normal spleen and lymph node cells. Lymph node cells from tumor bearers had no suppressive effects. Centrifugation of spleen cells layered on Hypaque-Ficoll (specific gravity of 1.08) produced a dense fraction which pelleted and a light fraction which was retained at the Hypaque-Ficoll-medium interface. Suppressive activity was not found in either fraction of normal spleen cells. In tumor-bearer spleen cells suppressor activity was greatly enriched in the light fraction. Treatment of the suppressor fraction with anti-theta or anti-Ig serum and complement did not remove suppressor activity. However, the suppressor cells were removed by passage through nylon wool or by carbonyl iron treatment. Also, the population which adhered to plastic Petri dishes contained the suppressor cell activity.     

Erythroid colony formation by fetal rat liver and spleen cells in vitro: inhibition by a low relative molecular mass component of fetal spleen.

Liver and spleen hematopoietic cell suspensions from 20-day-old-fetal rats were fractionated on Percoll gradients. A granulocyte-rich splenic fraction inhibited CFUe production by cultures of a CFUe-enriched liver fraction, and by cultures of unfractionated liver and spleen hematopoietic cells. Conditioned medium from the spleen cell fraction contained an inhibitor of relative molecular mass, Mr, 25-35 x 10(3). The sensitivity of spleen cells to the inhibitor varied with the age of the fetus from which they were derived (20-day-old less than 18-and 19-day-old). No such age-dependence was found for liver cells. The inhibitor affects cycling CFUe, blocks the lethal effect of AraC, does not appear to be lineage-specific and its influence can be reversed by washing.     

Further studies on generation of macrophages in in vitro cultures of mouse spleen cells and its inhibition by the capsular polysaccharide of Klebsiella pneumoniae.

Although the number of macrophages detected in cultures of mouse spleen cells at the start of the culture was very small, it markedly increased during further incubation. Macrophages were generated not only from the glass-adherent cell fraction of spleen cells, but also from the nonadherent cell fraction obtained after removal of adherent cells either by incubating in glass petri dishes or by passing through a glass bead column. The generation of macrophages from the nonadherent cell fraction occurred even when it was separated as late as 48 hr after the start of the culture. The phagocytic activity of macrophages newly generated from the nonadherent cell fraction was relatively weak, but it was activated during further incubation. Based on these results, the maturation process of macrophages can be divided into at least the following four stages; glass-nonadherent nonphagocytic precursor cells, glass-adherent nonphagocytic precursor cells, immature macrophages with low phagocytic activity, and mature macrophages with full phagocytic activity. The addition of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) to cultures of spleen cells markedly suppressed the generation of macrophages. The suppressive effect of CPS-K depended on its dosage, and the minimum concentration of CPS-K showing a definite effect was 0.05 μg/ml. CPS-K inhibited further generation of macrophages in either the nonadherent or adherent cell fraction at any time after the start of the culture. The suppressive effect of CPS-K on the generation of macrophages could not be reversed by simple washing of spleen cells which had been kept in contact with CPS-K for 3 hr. There was no evidence which showed that CPS-K exhibited direct cytotoxic effects on spleen cells in the culture.     

Reaginic antibody formation in the mouse. VII. Induction of suppressor T cells for IgE and IgG antibody responses.

Intravenous injections of urea-denatured ovalbumin (UD-OA) into OA-primed high responder mice suppressed the antibody response not only to the priming antigen but also to subsequent immunization with dinitrophenyl derivatives of OA (DNP-OA). The transfer of normal spleen cells or OA-primed spleen cells into UD-OA-treated animals did not restore the capacity of responding to DNP-OA to form anti-DNP IgE and IgG antibodies. The transfer of splenic T cell fraction from the UD-OA-treated animals into normal syngeneic mice diminished both IgE and IgG antibody responses of the recipients to DNP-OA. The B cell-rich fraction from the same donors failed to affect the anti-hapten antibody response and enhanced anti-cancer (OA) IgG antibody response of the recipients. It was also found that the transfer of T cell-rich fraction of OA-primed spleen cells failed to suppress antibody response of the recipients to DNP-OA. The results indicated that spleen cells of UD-OA-treated mice contained suppressor T cells which are distinct from helper cells. Suppressive activity of T cells in the UD-OA treated animals was specific for OA. The transfer of the T cell-rich fraction failed to suppress anti-DNP antibody response of the recipients to DNP-KLH.     

Intracellular localization of anti-tumor immune RNA.

Syngeneic spleen cells from normal, non-immune Fischer 344/N rats and allogeneic spleen cells from normal Wistar-Furth rats became cytotoxic, in vitro, to chemically induced Fischer rat sarcoma (MC3-R) target cells following incubation with xenogeneic Immune RNA (I-RNA) extracted from spleens of guinea pigs immunized with MC3-R tumor cells. I-RNA extracted from intact spleen cells or from the cytoplasmic fraction of spleen cells were equally active. RNA extracted from isolated spleen cell nuclei was inactive, as were all RNA fractions from spleen cells of nonspecifically immunized guinea pigs. Syngeneic I-RNA extracted from intact spleen cells or the cytoplasmic fraction of cells from spleens of Fischer rats bearing growing MC3-R transplants mediated cytotoxic reactions against MC3-R target cells when incubated with normal Fischer rat spleen cells. RNA from the nuclei of spleen cells of rats bearing MC3-R tumors was considerably less active. All RNA fractions from spleen cells of normal non-immune Fischer rats were inactive. The immunologically active component of xenogeneic and Syngeneic I-RNA, therefore, were found to be localized in the cytoplasm of specifically sensitized lymphoid cells.     

Cooperation between humoral factor(s) and Lyt-2+ T cells in effective clearance of Sendai virus from infected mouse lungs

The mechanism of cooperation between the L3T4+ and Lyt-2+ T cell subsets in effective clearance of Sendai virus from infected mouse lungs was studied by adoptive cell transfer using nude mice. Simultaneous transfer of a long-term-cultured Sendai virus-specific L3T4+ T cell line with L3T4+ cell-depleted immune spleen cell (L3T4-) fraction to infected nude mice could result in viral clearance, although single injection with either of these cells was not effective. Instead of the L3T4+ T cells, culture supernatants of the L3T4- T cell line or concanavalin A-stimulated mouse spleen cells and mouse serum immunized with the virus were also active in the cooperative viral clearance with L3T4- fraction. The role of the Sendai virus-sensitized L3T4- cell fraction in cooperative viral clearance with humoral factors could be replaced by neither T cell-deprived immune spleen cell fraction nor normal spleen cells. The 1,500 units of recombinant mouse interleukin 2 (IL-2), which was more than 12 times the IL-2 activity present in the supernatants of the T cell line or concanavalin A-stimulated spleen cells, failed to clear the virus in combination with the L3T4- fraction. Monoclonal antibodies to Sendai or mouse hepatitis viruses were also effective in the cooperative antiviral activity. IL-2 activity was not detected in these monoclonal antibodies and the mouse immune serum. Single injection of any humoral factors failed to clear the virus. These results indicate that Sendai virus-sensitized Lyt-2+ subset of T cells acts cooperatively with humoral factor(s) other than IL-2 or Sendai virus-specific antibody present in supernatants of the T cell line, of concanavalin A-stimulated spleen cells or hybridomas, and in mouse serum immunized with the virus.     

Detection of neuropeptide Y-like immunoreactivity and messenger RNA in rat platelets: the effects of vinblastine, reserpine, and dexamethasone on NPY expression in blood cells.

Rat plasma contains high basal levels (220 pmol/liter) of neuropeptide Y (NPY)-like immunoreactivity (LI) compared to pig (30 pmol/liter) and man (25 pmol/liter). The platelet-enriched fraction (PEF), obtained from rat blood contained 10,061 pmol/g NPY-LI. However, in human and pig blood, the PEF contained very low levels of NPY-LI. Gradient centrifugation of rat blood showed the highest concentration of NPY-LI (10.8 +/- 0.4 pmol/g) in the platelet fraction. The mononuclear cell fraction contained 1.64 +/- 0.16 pmol/g, whereas only 0.56 +/- 0.06 pmol/g of NPY-LI was found in the red blood cell/polymorphonuclear cell fraction. Characterization of NPY-LI in rat plasma and platelets by high-pressure liquid chromatography showed one predominating peak which coeluted with synthetic NPY (1-36) as well as three minor peaks, one of which coeluted with oxidized NPY. Analysis of NPY messenger RNA (mRNA) in bone marrow of the rat revealed a 0.79-kb-long NPY mRNA. This size is intermediate to the 0.82-kb NPY mRNA in brain and the 0.76-kb NPY mRNA in spleen. The highest level of NPY mRNA in rat blood was found in the mononuclear cell fraction but NPY mRNA was also detected in the platelet fraction. No NPY mRNA was detected in bone marrow or blood from pig and rabbit or from human blood or bone marrow. Forty-eight hours after treatment of rats with vinblastine the content of NPY mRNA and NPY-LI in rat blood was decreased, while the level of NPY-LI in bone marrow was markedly enhanced. Reserpine treatment caused an increase in NPY mRNA content in bone marrow and spleen. After administration of dexamethasone the level of NPY mRNA increased in both spleen and peripheral blood cells with increased NPY-LI content in the spleen. It is concluded that in addition to megakaryocytes in spleen and bone marrow, platelets and possibly also lymphocytes/monocytes in peripheral blood of the rat contain NPY mRNA and peptide. The expression of NPY mRNA in bone marrow, spleen, and blood is influenced by vinblastine, reserpine, and dexamethasone.     

Role of alkaline endonucleases in the release of soluble chromatin from thymus, spleen and liver nuclei of normal and irradiated mice.

Thymus, spleen and liver nuclei released a large fraction of soluble chromatin in vitro when incubation was carried out in sucrose media containing low concentrations of CaCl2 and/or MgCl2. A significant fraction of deoxyribopolynucleotides (DPN) was also extracted from nuclei. After 30 min of incubation at 37 degrees C, the maximum release of soluble chromatin was observed near a pH of 8, which corresponds to the optimum pH of the alkaline endonuclease activity from thymus, spleen and liver. The soluble chromatin and DPN were precipitated by increasing the bivalent ion concentration of the medium. The protein/DNA ratio and the molecular weight of DNA suggest that the soluble chromatin and DPN represent nucleosome-like particles. The release of soluble chromatin in the first 4 hours of incubation was significantly increased if the nuclear fraction was isolated from the thymus and spleen of whole-body irradiated mice (1000 rad). This effect was absent in the liver nuclei.     

Translational regulation of ferritin synthesis in rat spleen: effects of iron and inflammation

Translational control of ferritin synthesis was studied in rat spleen, and compared with that for liver, heart and brain, in response to iron and inflammation. Spleen concentrations of total RNA in the ribonucleoprotein (mRNP) fraction was comparable to that for liver, while polyribosomal RNA was less. Both fractions were ten-fold lower in heart and brain. In untreated animals, the mRNP fraction of all tissues had the largest portion of the ferritin mRNA, as determined by slot blot hybridization with 32P-labeled cDNA for the L subunit. Acute treatment with ferric ammonium citrate shifted the spleen ferritin mRNA to the polyribosome fraction. This was also so in liver but not in the heart and brain which took up much less iron. The findings were confirmed by hybridization studies of mRNPs and polyribosomes separated in sucrose gradients. Turpentine-induced inflammation also caused a shift in ferritin mRNA from the mRNP to the polyribosome fraction of spleen and liver, over 12 h. We conclude that as in liver, spleen ferritin synthesis is under translational control by iron, and that both tissues also respond to inflammation by shifting of ferritin mRNA to the polyribosomes.     

Induction of nitrite production in mouse spleen cells by immunization.

Changes of nitrite production in mouse spleen cells of in vitro secondary antibody response were investigated. Mouse spleen cells immunized with gamma globulin fraction of rat serum produced nitrite 3 days after in vitro challenging with the same antigen. Nitrite production of rabbit IgG-challenged spleen cells was found to be about 2.9-times higher than that of spleen cells primed with the gamma globulin fraction of rat serum. Nitrite production in this system was completely suppressed by T cell depletion (99.7% inhibition). Furthermore, nitrite production in these cells significantly decreased by addition of anti-interferon gamma antibody (62.9% inhibition). These data indicate that nitrite production in antigen-immunized spleen cells is affected with the immunogenicity of an antigen and regulated by T cells, especially interferon (IFN) gamma.     

Effect of the U-2 fraction of splenic extract from Horsfield's terrapin on the hematopoiesis of mammals

Intraperitoneal administration of a spleen extract from Testudo horsfieldi and its U-2 fraction increases the number of endogenous splenic haemopoietic colonies. The U-2 fraction administered to irradiated (4 Gy) mice increases the number of bone marrow CFUs. Bone marrow cells of exposed (4 Gy) mice preincubated in vitro with the U-2 fraction also increase the number of exogenous colonies in the recipient's spleen.     

Preferential distribution of group-II-like phospholipase A2 in mononuclear phagocytic cells in rat spleen and liver

The localization of calcium-dependent phospholipase A2, (PLA2) immunochemically closely related to the enzyme of the viperid and crotalid type (group II), in cells isolated from rat spleen and liver was examined using a polyclonal antibody directed against rat spleen group II, PLA2 (PLA2M). In isolated spleen cells, the monocyte/macrophage fraction had the highest PLA2 activity (1.28 +/- 0.35.min-1.10(6) cells-1) which was almost completely inhibited by the anti-PLA2M antibody. An immunoblot analysis confirmed the presence of the enzyme in this fraction. An immunocytochemical study revealed that the PLA2 was present in spleen macrophages. In the isolated liver cells, Kupffer cells (0.92 +/- 0.22 nmol.min-1.10(6) cells-1) contained higher anti-PLA2M-antibody-inhibitable PLA2 activity than parenchymal cells (0.26 +/- 0.06.min-1.10(6) cells-1). The immunocytochemical study showed that cells immunopositive with anti PLA2M antibody were Kupffer cells. These results suggest that the mononuclear phagocytic cells in rat spleen and liver have relatively high activity of group-II-like PLA2. Subcellular distribution patterns of the anti-PLA2M-antibody-inhibitable phospholipase A2 activity in different cell populations from spleen and liver were compared. A mode of the distribution of the enzyme in the spleen macrophages was essentially similar to that in the spleen lymphocytes. The distribution in Kupffer cells was similar to that in parenchymal cells.     

Role of adhesive lymphoid cell subpopulation in areactivity to hepatoma 22a]

The role of adhesive fraction of T lymphocytes in nonreactivity of mice to hepatoma 22a was studied. It was shown that the removal of the adhesive fraction from the spleen suspension enriched with T lymphocytes promotes intensification of cell immunity in tumor-tolerant mice.     

Further purification of bovine spleen inhibitors of lymphocyte DNA synthesis (chalones).

Partially purified lymphocytic factors were obtained from boving spleen; these factors are non-cytotoxic and biologically active in vitro and in vivo: [3H]Thymidine incorporation into DNA of mitogen-stimulated mouse spleen cells in culture is inhibited; similar results are obtained with phytohaemagglutinin-stimulated peripheral human lymphocytes, where blast cells transformation is blocked. [3H]Thymidine incorporation into DNA of mitogen stimulated lymphocytes withdrawn from in vivo treated mice, is also reduced. The two factors in vitro and in vivo seem to act preferentially on mouse spleen cells compared to their action on liver, kidney and testicle cells in cluture, as far as thymidine incorporation into DNA is concerned. The following techniques were applied for their purification: 1. Homogenization of the fresh tissue in water, centrifugation, dialysis of the supernatant, centrifugation, fractionation of the supernatant by alcoholic precipitation and finally concentration in vacuo and lyophilization of the material soluble at 75% of alcohol yielded fraction F. 2. Preparation of an acetone powder from the spleen, extraction of the dry powder with water, then high speed centrifugation, followed by lyophilization of the supernatant produced fraction F'. Both fractions F and F' were further fractionated by chromatography on a Sephadex G75 column: 7 peaks were obtained (F1--F7). Biological activity was found in fraction F1, corresponding to high molecular weight material, and in fraction F6, corresponding to low molecular weight substances. By rechromatography on Sephadex G 75, it is easy to dissociate from F1 a small molecular weight fraction which might be similar to F6 as far as elution volume and biological properties are concerned.     

Suppression of natural killer (NK) cell activity of spleen cells by thymocytes

The in vitro influence of thymus cells on natural killer cell activity of spleen cells against prelabeled target cells (YAC-I and RL♂I) has been studied in syngeneic as well as in allogeneic murine models. In mixing experiments to demonstrate suppression, total thymocytes have been found to have no effect on NK activity of syngeneic or allogeneic spleen cells. Among several thymocyte fractions separated by velocity sedimentation, a relatively faster sedimenting fraction showed remarkable suppression of NK activity by spleen cells against two target cells. The suppressive effect of this particular fraction on NK activity was demonstrated to be proportional to the cell dose. The suppressive function was resistant to irradiation at 1000 or 2000 rad administered in vitro and was not restricted by the major histocompatibility complex. Moreover, the thymocyte fraction which induced suppression was not sensitive to NK-mediated cytolysi? by syngeneic spleen cells. The suppression of NK cytolysis in vitro by certain subpopulations of thymocytes as observed in the present studies may be consistent with a role for the thymus in regulating NK activity in vivo.     

Possible endogenous substrate proteins for cytosolic protein-tyrosine kinase from porcine spleen.

1. Endogenous phosphate acceptor proteins by cytosolic protein-tyrosine kinase from porcine spleen (CPTK-40) were studied using subcellular fractions of porcine spleen and supernatant fraction of rat various tissues. 2. At least 13 phosphate acceptor proteins ranging from 94 to 26 kDa were observed in all but mitochondrial subcellular fractions. 3. Among the supernatant fraction of rat tissues, brain, testis and spleen contained many phosphate acceptor proteins. 4. The most heavily phosphorylated band of around 55 kDa which was commonly recognized among various tissues was confirmed as tubulin by the immunoreactivity with anti-tubulin antibody. 5. The results obtained in this paper indicate that CPTK-40 has the potential to catalyze the phosphorylation of numerous endogenous proteins including tubulin.     

Transfer Agent of Immunity

Using erythrocytes as antigen particles, number of antibody-forming cells was enumerated by immunocytoadehesion technique, in which formation of rosette was shown to be inhibited by anti-mouse immunoglobulin sera. This number increased in vitro after treatment of spleen cells of mice for 60 min with RNA fraction extracted from spleen of mice immunized with erythrocytes used in the enumeration, and incubation of cells for 12 hr at 37 C. Response of cells treated with immune RNA fraction was immunologically specific and was inhibited by puromycin or cycloheximide. The activity of immune RNA capable of converting nonimmune cells to antibody-forming cells was shown to be sensitive to ribonucleases but resistant to deoxyribonuclease and proteolytic enzyme.