In order to investigate the influence of cytokine combinations on proliferation and differentiation of human umbilical cord blood CD34(+) cells into megakaryocytes/platelets in vitro, the CD34(+) cells from human umbilical cord blood were amplified in serum-free medium StemSpan(SFEM) supplemented with several cytokine combinations by three-phase culture system. The effects of the cytokine combinations were compared. The results showed that at day 14 of the first culture phase, the CD34(+) cells cultured with cytokine combinations SCF + TPO + FL + IL-3 were amplified (11 000 ± 1 000) times, which were significantly higher than that of cells cultured with SCF + TPO + FL, but were not significantly different from that of cells cultured with SCF + TPO + IL-3 or SCF + TPO + FL + IL-3+ hydroxyl-corticosteroids. At day 7 of the second culture phase, the CD34(+) cells cultured with cytokine combination SCF + TPO + FL + IL-11 were amplified by (204666.7 ± 11718.9) times, which were significantly higher than that of cells cultured with SCF + TPO + FL + IL-3, but were not significantly different from that of cells cultured with SCF + TPO + FL + IL-11 + BMP4 + VEGF. At day 3 and day 6, the CD34(+) platelet-like cells accounted for about (39.8 ± 1.9)%, (39.7 ± 2.6)% and (25.5 ± 1.4)%, (23.1 ± 3.5)% cultured with SCF + TPO + FL + IL-11 and SCF + TPO + FL + IL-11 + BMP4 + VEGF, and significantly higher than that of the cells cultured with SCF + TPO + FL + IL-3. It is concluded that the cytokine combination of SCF + TPO + FL + IL-3 is most suitable cytokines combination for the amplification of CD34(+) hematopoietic progenitor cells. The cytokine combination of SCF + TPO + FL + IL-11 is preferred for the proliferation and differentiation of megakaryocytes, this study lays an experimental basis for investigating the proliferation and differentiation of CD34(+) into megakaryocytes/platelets in vitro.
Antigens, CD34 ; immunology ; Blood Platelets ; cytology ; Cell Differentiation ; Fetal Blood ; cytology ; immunology ; Humans ; Interleukin-11 ; pharmacology ; Interleukin-3 ; pharmacology ; Megakaryocytes ; cytology ; Stem Cell Factor ; pharmacology ; Thrombopoietin ; pharmacology

OBJECTIVE: To investigate the effect of hematopoietic stimulating factors on the expansion of mature megakaryocytes. METHODS: (2, 4, 6, 8, 10) x 10(5)/mL bone marrow single nucleus cells (BMNC) were added in the culture system of colony forming unit-megkaryocyte (CFU-Meg) to find out the relationship of the cultured BMNC with the output of CFU-Meg. rmSCF + rmTPO + rmIL-3 (3HSFs) and rmSCF + rmTPO + rmIL-3 + rmIL-6 (4HSFs) or F-CM were added in the liquid culture system of megkaryocytes respectively. The number of mature megakaryocytes were counted every other day. RESULTS: The number of CFU-Meg increased with the increase of the cultured BMNC. The CFU-Meg productivity of 1 x 10(6) BMNC/mL culture system was more than that of 2 x 10(5) BMNC/mL culture system. 3HSFs and 4HSFs or F-CM significantly promoted the expansion of mature megakaryocytes in the liquid culture system, but the effect was different. The peak time of the number of mature megakaryocytes in 3HSFs and 4HSFs or F-CM were 7 d, 7 d and 5 d respectively. CONCLUSION 3HSFs and 4 HSFs or F-CM had positive effect on the expansion of mature megakaryocytes. 4HSFs was better than 3HSFs and F-CM. 3HSFs was better than F-CM. The peak time of the number of mature megakaryocytes in different culture systems was different.
Animals ; Cells, Cultured ; Colony-Forming Units Assay ; Female ; Hematopoietic Cell Growth Factors ; pharmacology ; Interleukin-3 ; pharmacology ; Interleukin-6 ; pharmacology ; Macrophage Colony-Stimulating Factor ; pharmacology ; Male ; Megakaryocytes ; cytology ; Mice

BACKGROUNDHematopoietic growth factor (HGF) is indispensable to hematopoiesis in the body. The proliferation and differentiation of hematopoietic cells must rely on the existence and stimulation of HGF. This study investigated the effect of catechin, an active component extracted from Spatholobus suberectus Dunn (SSD), on bioactivity of granulocyte-macrophage colony-stimulating activity (GM-CSA), burst-promoting activity (BPA) and megakaryocyte colony-stimulating activity (MK-CSA) in spleen condition medium (SPCM) of mice to clarify the hematopoietic mechanism of catechin and SSD.

METHODSSpleen cells of mice were separated and spleen condition medium (SPCM) was prepared from spleen cell culture. Bone marrow cells of mice were separated and cultured in a culture system including 10% (v/v) SPCM (induced by catechin in vivo or ex vivo) for 6 days. Granulocyte-macrophage colony forming units (CFU-GM), erythrocyte burst-colony-forming units (BFU-E) and megakaryocyte colony-forming units (CFU-Meg) formation were employed to assay the effects of different treatment on the bioactivity of GM-CSA, BPA and MK-CSA in SPCM.

RESULTSSPCM induced by 100 mg/L catechin ex vivo could promote the growth of CFU-GM, BFU-E and CFU-Meg, which indicated that catechin could stimulate the production of GM-CSA, BPA and MK-CSA in SPCM. SPCM prepared at the fourth day of spleen cell culture showed the best stimulating activity. The bioactivity of GM-CSA, BPA and MK-CSA in the SPCM prepared after intraperitoneally injecting catechin into mice was also increased. The number of CFU-GM, BFU-E and CFU-Meg gradually increased as the dose of catechin increased and the time of administration prolonged. CFU-GM, BFU-E and CFU-Meg of the high-dose catechin group were significantly higher than those of the control group (P < 0.01) and reached the maximum at the seventh day after administration.

CONCLUSIONSThis study suggests that catechin extracted from the active acetic ether part of Spatholobus suberectus Dunn can regulate hematopoiesis by inducing bioactivity of GM-CSA, BPA and MK-CSA in SPCM of mice. This may be one of the mechanisms for the hematopoietic-supportive effect of catechin and Spatholobus suberectus Dunn.

Animals ; Catechin ; pharmacology ; Granulocyte-Macrophage Colony-Stimulating Factor ; physiology ; Hematopoiesis ; drug effects ; Interleukin-3 ; physiology ; Mice ; Thrombopoietin ; physiology

Prolonged thrombocytopenia is a puzzling problem following umbilical cord blood (CB) transplantation. It might be the result of inadequate megakaryocyte progenitors and the arrest in the megakaryocyte maturation. It is an important method to solve the problem by transfusing ex-vivo expanded CB megakaryocyte progenitor cells into the patients to shorten the duration of platelet recovery. However, the most optimal condition of expansion has not been established so far. In the study, cord blood mononuclear cells (MNC) were cultured in serum-free medium with TPO, IL-3, SCF and IL-6. The numbers of MNC, CFU-MK and CD41(+) cells were measured at 0, 6, 10 and 14 days, in order to find the best cytokine combination and optimal harvest time point for clinical use. The results showed that the megakaryocyte progenitors most efficiently expanded with the cytokine combination including TPO, IL-3, SCF and IL-6. The time point of maximal CFU-MK growth is day 10. At 10 days, the numbers of CFU-MK and CD41(+) cells expanded by 6.8- and 8.8-fold respectively. In conclusion, in vitro, the cytokine cocktail including TPO, IL-3, SCF and IL-6 was the most optimal cytokine combination which stimulates the ex vivo expansion of megakaryocyte progenitors in CB MNC and serum-free medium. The maximal CFU-MK colonies were harvested at 10 days, that may be an optimal harvest time for clinical transfusion.
Antigens, CD34 ; analysis ; Cell Division ; Culture Media, Serum-Free ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Interleukin-3 ; pharmacology ; Interleukin-6 ; pharmacology ; Megakaryocytes ; cytology ; Platelet Membrane Glycoprotein IIb ; analysis ; Stem Cell Factor ; pharmacology ; Thrombopoietin ; pharmacology

OBJECTIVE: The goal of this study was to study the mechanism of substance P (SP)-mediated the neural control of mast cell (MC) degranulation. METHOD: Bone marrow mast cells from mice were cultured with stem cell factor (SCF), IL-3 and IL-4 (group A) and SCF, IL-3 (group B) for four weeks. Then the cells were harvested and reserved for studies. Western Blot hybridization technique was used to detect the expression of FcεR I α and NK-1R on MCs from the two groups. Then such cells were activated with SP (0, 0. 01, 0. 10, 1. 00, 10. 00 µg/ml, respectively) for 30 min. The histamine released into the supernatant and stored in the protoplasm was quantified by enzyme linked immunosorbent assay (ELISA). And the percentage of histamine release was calculated as a percent of total histamine content. RESULT: The expressions of FcεR I α and NK-1R on these mast cells in group A were statistically higher than in group B (P<0. 05). The MCs from two groups can be actived when stimulated by SP, but the level of MC degranulation in group A was higher than group B (P<0. 05). CONCLUSION Neuropeptide may stimulate MC degranulation through immunological and non-immunological pathways. In summary, the current study provides us with better understanding of the mechanism of neuropeptide-controlled MC deranulation, and this should be helpful for the further research involved in the mechanism and treatmemt of airway hyper-reactivity.
Animals ; Bone Marrow Cells ; Cell Degranulation ; Cells, Cultured ; Culture Media ; chemistry ; Histamine ; metabolism ; Interleukin-3 ; pharmacology ; Interleukin-4 ; pharmacology ; Mast Cells ; cytology ; metabolism ; Mice ; Stem Cell Factor ; pharmacology ; Substance P ; pharmacology

This study aims to explore the effect of "Trichosanthis Fructus-Allii Macrostemonis" combination(GX) on the activation of NOD-, LRR-, and pyrin domain-containing protein 3(NLRP3) inflammasome, the release of inflammatory cytokines, and the level of autophagy in RAW264.7 macrophage damaged by lipopolysaccharide(LPS), and the mechanism of GX against inflammatory response in macrophages. To be specific, LPS was used to induce the injury of RAW264.7 cells. Cell Counting Kit-8(CCK-8) assay was employed to measure the survival rate of cells, and Western blot to detect the protein expression of NLRP3, apoptosis-associated speck-like protein(ASC), cysteine-aspartic acid protease(caspase)-1, interleukin(IL)-18, IL-1β, microtubule-associated protein light chain 3(LC3)-Ⅱ, and selective autophagy junction protein p62/sequestosome 1 in RAW264.7 macrophages. ELISA was used to measure the levels of IL-18 and IL-1β in RAW264.7 cells. Transmission electron microscopy was applied to observe the number of autophagosomes in RAW264.7 cells. Immunofulourescence staining was used to detect the expression of LC3-Ⅱ and p62 in RAW264.7 cells. The result showed that GX significantly reduced the protein expression of NLRP3, ASC, and caspase-1 in RAW264.7 cells, significantly increased the protein expression of LC3Ⅱ, decreased the expression of p62, significantly inhibited the secretion of IL-18 and IL-1β, significantly increased the number of autophagosomes, significantly enhanced the immunofluorescence of LC3Ⅱ, and reduced the immunofluorescence of p62. Furthermore, 3-methyladenine(3-MA) could reverse the inhibitory effect of GX on NLRP3, ASC, and caspase-1 and reduce the release of IL-18 and IL-1β. In summary, GX can increase of the autophagy activity of RAW264.7 and inhibit the activation of NLRP3 inflammasome, thereby reducing the release of inflammatory cytokines and suppressing inflammatory response in macrophages.
Inflammasomes/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Interleukin-18/metabolism* ; Lipopolysaccharides/pharmacology* ; Macrophages ; Cytokines/metabolism* ; Caspase 1/metabolism* ; Autophagy ; Interleukin-1beta/metabolism*

This study explores the effect of total flavonoids of Rhododendra simsii(TFR) on middle cerebral artery occlusion(MCAO)-induced cerebral injury in rats and oxygen-glucose deprivation/reoxygenation(OGD/R) injury in PC12 cells and the underlying mechanism. The MCAO method was used to induce focal ischemic cerebral injury in rats. Male SD rats were randomized into sham group, model group, and TFR group. After MCAO, TFR(60 mg·kg~(-1)) was administered for 3 days. The content of tumor necrosis factor-α(TNF-α), interleukin-1(IL-1), and interleukin-6(IL-6) in serum was detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes of brain tissue and cerebral infarction were observed based on hematoxylin and eosin(HE) staining and 2,3,5-triphenyltetrazolium chloride(TTC) staining. RT-qPCR and Western blot were used to detect the mRNA and protein levels of calcium release-activated calcium channel modulator 1(ORAI1), stromal interaction molecule 1(STIM1), stromal intera-ction molecule 2(STIM2), protein kinase B(PKB), and cysteinyl aspartate specific proteinase 3(caspase-3) in brain tissues. The OGD/R method was employed to induce injury in PC12 cells. Cells were randomized into the normal group, model group, gene silencing group, TFR(30 μg·mL~(-1)) group, and TFR(30 μg·mL~(-1))+gene overexpression plasmid group. Intracellular Ca~(2+) concentration and apoptosis rate of PC12 cells were measured by laser scanning confocal microscopy and flow cytometry. The effect of STIM-ORAI-regulated store-operated calcium entry(SOCE) pathway on TFR was explored based on gene silencing and gene overexpression techniques. The results showed that TFR significantly alleviated the histopathological damage of brains in MCAO rats after 3 days of admini-stration, reduced the contents of TNF-α, IL-1, and IL-6 in the serum, down-regulated the expression of ORAI1, STIM1, STIM2, and caspase-3 genes, and up-regulated the expression of PKB gene in brain tissues of MCAO rats. TFR significantly decreased OGD/R induced Ca~(2+) overload and apoptosis in PC12 cells. However, it induced TFR-like effect by ORAI1, STIM1 and STIM2 genes silencing. However, overexpression of these genes significantly blocked the effect of TFR in reducing Ca~(2+) overload and apoptosis in PC12 cells. In summary, in the early stage of focal cerebral ischemia-reperfusion injury and OGD/R-induced injury in PC12 cells TFR attenuates ischemic brain injury by inhibiting the STIM-ORAI-regulated SOCE pathway and reducing Ca~(2+) overload and inflammatory factor expression, and apoptosis.
Animals ; Male ; Rats ; Apoptosis ; Brain Ischemia/metabolism* ; Caspase 3 ; Interleukin-1 ; Interleukin-6 ; Rats, Sprague-Dawley ; Reperfusion Injury/metabolism* ; Tumor Necrosis Factor-alpha/genetics* ; Flavonoids/pharmacology* ; Rhododendron/chemistry*

In order to compare the effects of GM-CSF + IL-4 with the effects of IL-3 + IL-4 on the differentiation and development of dendritic cells (DCs) from mouse bone marrow, we cultured bone marrow hematopoietic progenitor cell in vitro and examined whether DCs induced by IL-3 + IL-4 (IL-3 DCs) differed from the DCs induced by GM-CSF + IL-4 (GM-CSF DCs) in morphology, yields, phenotype and endocytic activity. Scanning electron microscope, laser scanning confocal microscope (LSCM) and flow cytometer (FCM) were used. The results showed that the DCs induced by IL-3 + IL-4 and the DCs induced by GM-CSF + IL-4 had similar morphology, and the DCs induced by IL-3 + IL-4 expressed high level of major histocompability complex (MHC) class II. Besides, IL-3 DCs had the characteristics of higher yields and better consistency. But CD80 and CD86 were expressed at low levels or absent on IL-3-treated DCs. The capability of uptaking antigen of IL-3 DCs was more powerful than that of GM-CSF DCs. IL-3 could substitute for GM-CSF in culturing DCs from mouse bone marrow cells and could obtain the tolerance DCs that lack costimulatory molecules.
Animals ; Antigen-Presenting Cells ; cytology ; Bone Marrow Cells ; cytology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Dendritic Cells ; cytology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Interleukin-3 ; pharmacology ; Interleukin-4 ; pharmacology ; Male ; Mice ; Mice, Inbred BALB C

OBJECTIVETo generate engineering Th17 cells from mice CD4(+)CD25(-) naïve T cells, and to evaluate whether the phenotypes or functions of these engineering cells were similar to natural Th17 cells.

METHODSRecombinant lentivirus carrying mouse RORγt (pXZ9-RORγt) and mock control pXZ9 were generated by co-transfected three-plasmids into 293FT packing cells. CD4(+)CD25(-) naïve T cells were purified from mice spleens by magnetic activated cell sorting, and stimulated by anti-CD3ε, anti-CD28 mAb plus IL-2. The stimulated cells were further infected by pXZ9-RORγt or pXZ9 virus with or without polarization by TGF-β plus IL-6 and divided into five groups: pXZ9-RORγt (group A), pXZ9 + TGF-β + IL-6 (group B), pXZ9-RORγt + TGF-β + IL-6 (group C), pXZ9 (group D) and control (group E). Production efficiency of engineering Th17 cells was referred as the percentage of IL-17A producing cells. Cytokine production profiles of these cells were assayed by realtime RT-PCR and cells function was evaluated by susceptibility of mouse experimental autoimmune encephalomyelitis (EAE).

RESULTS(1) High-title lentivirus was prepared and was succeeded to transduce CD4(+)CD25(-) naïve T cells. Forced expression of RORγt (group A) resulted in (40.25 ± 5.46)% CD4(+)CD25(-) naïve T cells converted into engineering Th17 cells and the convert efficiency increased to (60.59 ± 8.15)% in addition of TGF-β and IL-6 (group C), or decreased to (14.36 ± 5.27)% when presence of TGF-β and IL-6 only (group B). (2) IL-17A, IL-17F and IL-21 production of pXZ9-RORγt infected cells combined with TGF-β and IL-6 were most similar to natural Th17 cells while cells over expression of RORγt alone showed deficiency in IL-21 production. (3) Both pXZ9-RORγt infected cells, TGF-β and IL-6 polarized cells and polarized of RORγt transduced cells could promote the susceptibility to mouse EAE in C57BL6 mice models.

CONCLUSIONHigh yield of engineering Th17 cells was prepared from CD4(+)CD25(-) naïve T cells by over expression RORγt plus TGF-β and IL-6 polarization. These engineering Th17 cells were similar to the natural Th17 cells in phenotypes and functional identification.

Animals ; Cells, Cultured ; Genetic Techniques ; Interleukin-17 ; pharmacology ; Interleukin-6 ; pharmacology ; Lentivirus ; genetics ; Mice ; Mice, Inbred C57BL ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; genetics ; Th17 Cells ; cytology ; metabolism ; Transforming Growth Factor beta ; pharmacology

This study was aimed to investigate the effect of some culture system composed of various cytokine combinations (TPO, SCF, FL, IL-1, IL-3, IL-6) on ex vivo expansion of megakaryocytic progenitors induced from CD34+ cells of peripheral blood and to seek a optimal cytokine combination and culture time. Mononuclear cells were isolated from mobilized peripheral blood (MPB) by density gradient centrifugation over Ficoll. CD34+ cells were purified by using an immunomagnetic bead separation system. The selected CD34+ cells were seeded in Iscove's modified Kulbecco's medium (IMDM) supplemented with fetal calf serum (FCS) and various kinds of cytokines. After 15 days of culture, the content of CD41+ cells in culture system were determined by flow cytometry, and the number of megakaryocyte colony-forming unit (CFU-MK) was measured simultaneously. The results showed that after definited days of culture, the cytokine combination TPO/FL/IL-6/IL-3 was the most suitable for MPB to obtain high number of MK, and better than any other three groups (P < 0.05). The increase multiple of CD41+ cells was 93.97 +/- 17.27 on day 5 and 131.23 +/- 18.26 on day 10. On day 15, the proportion and the increase multiple of CD41+ cells decreased obviously. The expansion multiples of CFU-MK were 93.33 +/- 10.02 on day 5 and 120.67 +/- 13.01 on day 10, higher than any other groups. It is concluded that TPO/FL/IL-6/IL-3 combination was the best optimal for expansion ex vivo of megakaryocytic progenitors from MPB, and its suitable duration of culture was 10 days; a culture system for expansion ex vivo of megakaryocytic progenitors have been established in this study.
Antigens, CD34 ; biosynthesis ; Blood Cells ; cytology ; Blood Platelets ; cytology ; Cell Culture Techniques ; methods ; Cell Differentiation ; drug effects ; Hematopoietic Stem Cell Mobilization ; methods ; Humans ; Interleukin-3 ; pharmacology ; Interleukin-6 ; pharmacology ; Megakaryocytes ; cytology ; Stem Cells ; cytology ; Thrombopoietin ; pharmacology