Differentiation of serum-free mouse embryo cells into an astrocytic lineage is associated with the asymmetric production of early neural, neuronal and glial markers

Serum-free mouse embryo (SFME) cells, the astrocyte progenitor cells in the central nervous system (CNS), were exposed to 10 ng/ml leukemia inhibitory factor (LIF) and 10 ng/ml bone morphogenic protein 2 (BMP2) to induce differentiation, and expression of cell-type specific markers. Nestin, a marker of early neural lineage, betaIII-tubulin, a marker of neuronal lineage, oligodendrocyte marker O4 (O4), a marker of oligodendrocytic lineage and glial fibrillary acidic protein (GFAP), a marker of astrocytic lineage, were analyzed. Characteristics of SFME cells, as a CNS progenitor, were identified and a possible mechanism, underlying SFME cell specification into an astrocytic lineage upon differentiation, was investigated. These markers were present, both at the initial proliferative phase and after induction of differentiation. GFAP expression increased strongly upon differentiation, while expression of the other markers changed very little. These results indicate that astrocytic differentiation is associated with the asymmetric production of these markers, rather than through induction of astrocytic markers.     

In vitro action of leukemia inhibitory factor (LIF) on mid-secretory stage endometrial stromal cells collected from hormone-simulated, ovariectomized monkey and maintained in three-dimensional primary culture

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that is known to play an important role in blastocyst implantation. The putative action of LIF in the regulation of uterine function has been examined using mid-secretory stage monkey endometrial stromal cells cultured on rat-tail collagen type I and treated with recombinant human LIF (rhLIF) or immunoneutralized LIF (in LIF) under serum-free condition. Long-term ovariectomized rhesus monkeys (n=8) underwent simulation of their menstrual cycles with steroid hormones and endometrial tissue samples were collected on cycle day 18; stromal cells were isolated and grown in primary culture on three-dimensional collagen matrix. Significant decline in cellular protein synthesis (P < 0.01) and cell proliferation index (P < 0.05) was observed in cells with increasing doses (0-1000 ng/ml) of rhLIF under serum-free in vitro condition. JAK1 expression in cultured cells increased (P < 0.01) in response to rhLIF as revealed from Western blot and confocal laser scanning microscopic examination, STAT1 and STAT2 expressions were unchanged, while pSTAT3 expression increased (P < 0.01) with increased concentration of rhLIF in culture medium. Autophosphorylation of JAK1 in endometrial stromal cells showed no change with increasing concentration (0.01 to 100 ng/ml) of rhLIF in vitro, but significant (P < 0.05) increase was observed with the time of exposure to rhLIF. Immunoneutralization of LIF or no addition of rhLIF to cultured cells led to significant (P < 0.01) increase in stromal cell proliferation index and significant (P < 0.01) decrease in the level of JAK1 and its autophosphorylation as compared to cells exposed to rhLIF alone. From the present set of experiments we conclude that rhLIF affects the physiological behaviour of monkey mid secretory stage endometrial stromal cells in vitro via the JAK-STAT signaling pathway.     

肝癌中骨形成蛋白2对PTEN蛋白水平影响的研究

目的用骨形成蛋白2(BMP2)干预肝癌细胞系HepG2细胞,从而观察肝癌细胞中PTEN蛋白水平的变化,观察BMP2对PTEN蛋白表达的影响,探讨BMP2与PTEN蛋白水平之间的关系,探索肝癌治疗的新途径。方法将受试对象分为3个组:对照组、BMP2100ng/ml组、BMP2300ng/ml组,作用时间为3个水平:6、12、24h。用免疫组织化学方法测定细胞中PTEN蛋白水平。结果免疫组织化学结果显示在不同浓度BMP2组中HepG2细胞中PTEN蛋白表达差异有统计学意义。不同时间组中PTEN蛋白表达差异有统计学意义。PTEN蛋白阳性表达率在300ng/ml组与100ng/ml组、对照组之间差异有统计学意义(P<0.05),在6h组与12h组、24h组之间差异有统计学意义(P<0.05)。在300ng/ml、24h组中PTEN蛋白阳性表达最高。浓度与时间之间无交互作用。结论在肝癌中BMP2可以增加PTEN蛋白水平,且呈浓度时间依赖。     

Bisphenol A induces leptin receptor expression, creating more binding sites for leptin, and activates the JAK/Stat, MAPK/ERK and PI3K/Akt signalling pathways in human ovarian cancer cell

We previously demonstrated that bisphenol A (BPA) promotes proliferation in OVCAR-3 human ovarian cancer cells. This study was designed to investigate the effects of BPA on leptin expression and activity in ovarian cancer. Real-time PCR, Western blot analysis and ELISA assays were used to quantify leptin receptor expression and leptin gene and protein expression after treatment with BPA at doses of 0.2, 2, 8 and 20ng/ml. Our data reveal leptin receptor expression but an absence of leptin gene and protein expression in OVCAR-3 cells. At doses of 8 and 20ng/ml, BPA had stimulatory effects on leptin receptor gene and protein expression. Leptin and BPA alone stimulated cell proliferation but BPA did not potentiate leptin activity. Similarly to leptin, but with different kinetics and duration, BPA induced phosphorylation of Stat3, ERK1/2 and Akt. In co-treatment experiments, the timing of protein phosphorylation represented an additive effect of BPA and leptin treatment. In conclusion, taking into consideration limitation of in vitro study, whether BPA by creating more binding sites for leptin and extending the time of leptin-induced Stat3, ERK1/2 and Akt phosphorylation, can potentiated leptin action in cancer cells, require confirmation by in vivo study.     

Bisphenol A concentration-dependently increases human granulosa-lutein cell matrix metalloproteinase-9 (MMP-9) enzyme output

Bisphenol A (BPA) is an estrogenic contaminant that has been quantified at higher levels in the follicular fluid of women with polycystic ovarian syndrome (PCOS) compared to healthy fertile controls. However, the effect of BPA on granulosa cell function is unknown. Therefore, the objective of the present study was to quantify the effect of BPA on granulosa cell progesterone (P4) output and matrix metalloproteinase (MMP)-2, and -9 output and activity. Granulosa-lutein cells (GLCs) were collected from women undergoing oocyte retrieval in an academic in vitro fertilization (IVF) program. Granulosa-lutein cells were treated with increasing log concentrations of BPA (1-10,000 ng/ml) or 17beta-estradiol (E2, 272 pg/ml or 1.0 nM) and treatment effects on MMP-2 and -9 activity and output, cell viability and cell proliferation were measured by commercial gelatin zymography, MMP-ELISA, MTS and BrdU incorporation assays, respectively. Granulosa-lutein cells in culture secrete MMP-2 and MMP-9. Bisphenol A treatment concentration-dependently increased MMP-9 output by GLCs with a maximal effect observed at 1000 ng/ml. Cell viability/proliferation was unaffected by BPA treatment at concentrations相似文献   

白细胞介素-1β和黄芪对气管上皮细胞转化生长因子-β_1表达的影响

目的检测经白细胞介素(IL)-1β、IL-1β和黄芪共同干预后兔气道上皮细胞转化生长因子-β1(TGF-β1)蛋白的表达水平,探讨IL-1β对气管上皮细胞TGF-β1表达的影响以及黄芪在哮喘气管重塑中的防治作用。方法体外培养兔气管上皮细胞,①实验组加入终浓度为1ng/ml的IL-1β,于不同时间点收集培养上清液及贴有细胞的盖玻片;②实验组分别加入不同浓度的IL-1β;③各组均加入终浓度为10ng/ml的IL-1β,同时实验组分别加入不同浓度的黄芪和地塞米松。②与③均于24h后收集培养上清液及贴有细胞的盖玻片。采用免疫细胞化学染色和双抗体夹心酶联免疫吸附试验(ELISA)测定TGF-β1蛋白的表达。结果①经IL-1β1ng/ml处理后TGF-β1的表达在24h点[上皮细胞吸光度值(0.613±0.022),上清液含量(701±32)pg/ml]明显高于其余各时间点(P分别<0.05和0.01)。②与对照组[(0.138±0.009),(216±28)pg/ml]比较,IL-1β 0.1ng组[(0.156±0.003),(267±12)pg/ml]、1ng组[(0.614±0.020),(710±32)pg/ml]、10ng组[(0.917±0.050),(940±34)pg/ml]TGF-β1表达均增高,差异有统计学意义(P分别<0.05和0.01);经直线相关分析培养上清液中TGF-β1的含量与所加IL-1β的浓度呈正相关(r=0.906,P<0.01)。③与IL-1β组[(0.904±0.047),(935±32)pg/ml]比较,黄芪50mg组[(0.397±0.020),(398±52)pg/ml]、黄芪200mg组[(0.144±0.005),(258±45)pg/ml]、黄芪500mg组[(0.401±0.005),(414±22)pg/ml]和地塞米松组[(0.155±0.003),(247±44)pg/ml]TGF-β1表达水平均降低,差异有统计学意义(P分别<0.05和0.01)。结论IL-1β可促进气管上皮细胞TGF-β1的蛋白表达,黄芪对这一过程有抑制作用,早期应用黄芪可能延缓气管重塑的形成与发展。     

Anticancer activity of a low immunogenic protein toxin (BMP1) from Indian toad (Bufo melanostictus, Schneider) skin extract

Earlier, a protein (BMP1, MW-79kDa) had been isolated from Indian toad (Bufo melanostictus) skin aqueous extract possessed anticancer activity against EAC bearing mice (Bhattacharjee et al., 2011). In the present study, the anti-proliferative and apoptogenic activities of BMP1 have been evaluated in leukemic (U937 and K562) and hepatoma (HepG2) cells. BMP1 dose dependently inhibited U937 and K562 cell growth having IC₅₀ values of 49 μg/ml and 30 μg/ml respectively. The anti-proliferative activity of BMP1 was observed in MTT assay, proliferating cell nuclear antigen (PCNA) expression and cell cycle arrest study. Flow-cytometric data revealed that BMP1 arrested cell cycle in U937 and K562 cells at Sub-G1 and G1 phases. The BMP1-induced dose dependent expressions of CDKIs (p21^cip1 and p27^kip1) and inhibition of CDK2 and PCNA expression in HepG2 cells support the inhibition of cell proliferation due to G1 arrest. BMP1-induced apoptosis analyzed by annexin-V binding study and the DNA fragmentation by comet assay were correlated with the sub-G1 arrest. The parallel induction of bax and p53 expression in HepG2 cells and the up-regulation of caspase 3 and caspase 9 due to BMP1 treatment indicated the involvement of p53-dependent intrinsic pathway of apoptosis. BMP1 was found to be low immunogenic in nature.     

Alteration of multiple cell membrane functions in L-6 myoblasts by T-2 toxin: an important mechanism of action

Recent studies suggest that T-2 toxin interacts with cell membranes and alters membrane function. This study was done to assess the effect of T-2 toxin on a broad range of cell membrane functions in L-6 myoblasts. The following parameters were assessed after exposure to T-2 toxin for 10 min: (1) the uptake of calcium, rubidium, and glucose; (2) the uptake of leucine and tyrosine and incorporation into protein; (3) the uptake of thymidine and incorporation into DNA; and (4) residual cellular lactate dehydrogenase (LDH) as a measure of cell membrane integrity. The effects of T-2 toxin on these parameters were: (1) The minimal effective concentration (MEC) of T-2 toxin that caused a reduction in the uptake of calcium and glucose was 4 pg/ml. The uptake of rubidium was increased at 0.4 pg/ml and then reduced at 4 pg/ml and higher concentrations. (2) The MEC for reduction of the uptake of leucine and tyrosine and their incorporation into protein was 4 pg/ml. (3) Thymidine uptake and incorporation into DNA showed a biphasic response with an increase at 0.4 pg/ml and a reduction at 4 pg for uptake and 40 pg/ml for incorporation. (4) Intracellular LDH was reduced at 4 ng/ml. (5) Calcium efflux was reduced after 1-, 5-, and 15-min exposures to T-2 toxin in a concentration of 40 ng/ml. All of the changes noted, including protein synthesis inhibition, were present to a significant degree within 10 min of exposure to T-2 toxin. This time interval is too short to attribute all of these effects directly to protein synthesis inhibition since most short-lived proteins have half-lives measured in hours. In conclusion, T-2 toxin appears to have multiple effects on cell membrane function at very low concentrations (0.4 pg/ml to 4 ng/ml), which are independent of protein synthesis inhibition. These likely include effects either direct or indirect on amino acid, nucleotide, and glucose transporters, as well as calcium and potassium (rubidium) channel activities.     

Perinatal exposure to Bisphenol A disturbs the early differentiation of male germ cells

Understanding the effects of Bisphenol A (BPA) on early germ cell differentiation and their consequences in adult life is an area of growing interest in the field of endocrine disruption. Herein, we investigate whether perinatal exposure to BPA affects the differentiation of male germ cells in early life using a transgenic mouse expressing the GFP reporter protein under the Oct4 promoter. In this model, the expression of GFP reflects the expression of the Oct4 gene. This pluripotency gene is required to maintain the spermatogonial stem cells in an undifferentiated stage. Thus, GFP expression was used as a parameter to evaluate the effect of BPA on early germ cell development. Female pregnant transgenic mice were exposed to BPA by oral gavage, from embryonic day 5.5 to postnatal day 7 (PND7). The effects of BPA on male germ cell differentiation were evaluated at PND7, while sperm quality, testicular morphology, and protein expression of androgen receptor and proliferating cell nuclear antigen were studied at PND130.We found that perinatal/lactational exposure to BPA up-regulates the expression of Oct4-driven GFP in testicular cells at PND7. This finding suggests a higher proportion of undifferentiated spermatogonia in BPA-treated animals compared with non-exposed mice. Moreover, in adulthood, the number of spermatozoa per epididymis was reduced in those animals perinatally exposed to BPA.This work shows that developmental exposure to BPA disturbed the normal differentiation of male germ cells early in life, mainly by altering the expression of Oct4 and exerted long-lasting sequelae at the adult stage, affecting sperm count and testis.     

Comparison of ribosome-inactivating proteins in the induction of apoptosis

The aim of this study was to evaluate the ability of verocytotoxin-1 (VT1), VT1 B chain alone, ricin and a hybrid toxin (RASTA2) consisting of ricin A chain linked to VT1 B chain to inhibit protein synthesis and to induce apoptosis. The lethal effects of the toxins were compared using vero cells (originating from green African monkey kidney tissue). As previously described cell death occurred through apoptosis which was quantified using the diphenylamine assay. DNA fragmentation was seen with VT1 at 10 pg/ml but there was no effect with B chain alone. Fragmentation with ricin was seen at 10 ng/ml and with RASTA2 at 1 ng/ml. Protein synthesis inhibition was measured by [³⁵S]methionine incorporation. VT1 had an IC50 of 0.0024 ng/ml, B chain alone was ineffective at inhibiting protein synthesis. Ricin had an IC₅₀ of 0.39 ng/ml and RASTA2 of 1.7 ng/ml. In vero cells the B chain of these toxins does not participate in cell killing.     

Combined effects of BPA and PFOS on fetal cardiac development: In vitro and in vivo experiments

Analyses of the combined effects of different EDCs are both important and difficult. This study attempts to evaluate the individual and combined effects of BPA and PFOS on heart development. Sprague-Dawley rats received individual or combined PFOS and BPA for 19 days during pregnancy. The results show that the combined BPA and PFOS exposure could lead to a morphological change in the fetal rat heart. An increase in the interventricular septal thickness (IVS) of approximately 20 % (391 μm in control vs 464 μm in combined exposure) was observed in the fetal rat hearts after the combined exposure to nearly 2000 μg/L PFOS and 100 μg/L BPA through drinking water. The total collagen and dynamin-related protein 1 (Drp1) mRNA level was increased in the fetal hearts exposed to the combination of 2000 μg/L PFOS and 100 μg/L BPA. However, the cell number in the IVS did not significantly change. Based on the previous literature, we believe that the combined exposure to BPA and PFOS had a synergistic effect on the thickness of the IVS. The combined exposure to 40 μg/L PFOS and 2 μg/L BPA failed to cause significant damage to the embryonic heart.The individual and combined effects and the mechanism of the effects of BPA and PFOS on heart development were further investigated by an in vitro study. Embryonic stem cells were administered individual or combined 10 ng/mL BPA and 100 ng/mL PFOS for 14 days during the cardiac differentiation period. The results show that exposure to the combination of 100 ng/mL PFOS and 10 ng/mL BPA could increase the cardiomyocyte size and collagen content. A selective inhibitor of Drp1, Mdivi-1, could inhibit the cardiomyocyte size enlargement but not the collagen content increase caused by the combined exposure. Thus, we believe that although the combined exposure to PFOS and BPA could affect mitochondrial biogenesis and collagen expression, these two effects seem to be relatively independent. Based on these results, this research concludes that combined exposure to PFOS and BPA could specifically lead to increased collagen and IVS thickening in heart development.     

Lipopolysaccharide inhibits the self-renewal of spermatogonial stem cells in vitro via downregulation of GDNF expression in Sertoli cells

Lipopolysaccharide (LPS) can reduce sperm count and sperm quality. The molecular mechanisms underlying this process are not fully understood. In this report, we investigated the effects of LPS-treated Sertoli cells on self-renewal and differentiation of spermatogoinial stem cells (SSCs). Sertoli cell cultures were established and incubated with LPS (10 μg/ml) for 1, 2 or 3 days, respectively. The culture media were collected and used as conditioned media (CM) to culture SSCs. The expression of glial cell-derived neurotrophic factor (GDNF), stem cell factor (SCF) and bone morphogenetic protein 4 (BMP4) in Sertoli cells treated with LPS was analyzed by RT-PCR and Western blotting. The results showed that the expression of SSC differentiation markers, c-kit and Sohlh2, was increased, while the expression of SSC self-renewal markers, plzf, oct4, and PCNA, was repressed when cultured in CM from LPS-treated Sertoli cells. GDNF levels in Sertoli cells and CM reduced dramatically after LPS treatments, while SCF and BMP4 levels did not show any significant changes. Moreover, correlated with the GDNF levels in CM, GDNF target genes, Bcl6b and Etv5, were reduced markedly in SSCs. Our results suggest that LPS inhibits the expression of GDNF in Sertoli cells, and might prevent the SSC self-renewal via down-regulation of GDNF target genes.     

Functional characterization of bone morphogenetic protein binding sites and Smad1/5 activation in human vascular cells

Mutations in the bone morphogenetic protein (BMP) type II receptor (BMPR2) gene cause familial pulmonary arterial hypertension (FPAH), a disease characterized by excessive smooth muscle and endothelial cell proliferation. However, the specific receptors mediating responses to BMPs in human vascular cells are not known. We show that human pulmonary artery smooth muscle cells (HPASMCs) express high specific (125)I-BMP4 binding, whereas human microvascular endothelial cells (HMEC-1) and human pulmonary artery endothelial cells (HPAECs) exhibit low binding. BMP4 competes for both high- and low-affinity (125)I-BMP4 binding sites on HPASMCs, yet BMP2 competes only at the low-affinity binding sites. In addition, BMP4, but not BMP2, induced Smad1/5 phosphorylation at low concentrations in HPASMCs. Conversely, HMEC-1 cells exhibited a single binding site population with equal affinity for BMP2 and BMP4. In both cell types, growth differentiation factor-5 (GDF5), BMP6, and BMP7 stimulated Smad1/5 phosphorylation and competed for (125)I-BMP4 less efficiently than BMP2 or BMP4. HPAECs exhibited weak Smad responses to BMPs. Expression analysis suggested the low binding in endothelial cells corresponded to lower ALK3 and ALK6 expression. Although transfection of small interfering RNAs (siRNAs) for ALK3 and BMPR-II abrogated Smad1/5 phosphorylation to BMP4, BMP2, and GDF5 in HMEC-1 and HPASMCs, they had little effect on (125)I-BMP4 binding. ALK6 siRNA did not alter binding or Smad1/5 responses, even to GDF5, a reported ALK6 selective ligand. Therefore, ALK3/BMPR-II is the BMP4/BMP2/GDF5-responsive receptor in human vascular cells, but these studies suggest that a BMP4/GDF5 selective binding protein exists in HPASMCs. These cell-specific differences in BMP responses are important for understanding the pathogenesis of FPAH.     

RANTES对肥大细胞Toll样受体4表达的作用

目的探讨趋化因子T细胞激活分泌调节因子(RANTES)对肥大细胞Toll样受体4(TLR4)表达的调节作用。方法将小鼠肥大细胞P815分为:RANTES 0.1、1.0、10、100ng/ml处理组(分别为A1、A2、A3、A4组)、RANTES 100ng/ml+RANTES阻断抗体10、30μg/ml组(分别为B1、B2组)以及空白对照组(C组)。作用2、6、16h后,采用流式细胞术、免疫荧光、RT-PCR检测肥大细胞上TLR4的表达情况。结果与C组相比,RANTES呈剂量依赖性地上调P815细胞中TLR4mRNA和蛋白表达(P<0.05),而B1、B2组能明显抑制该上调过程(P<0.05)。结论 RANTES上调P815细胞中TLR4的表达,从而加重过敏反应。     

Involvement of activating ERK1/2 through G protein coupled receptor 30 and estrogen receptor α/β in low doses of bisphenol A promoting growth of Sertoli TM4 cells

Sertoli cells play a pivotal role in supporting proliferation of germ cells and differentiation during spermatogenesis in mammals. Nanomolar concentrations of Bisphenol A (BPA) can significantly stimulate the proliferation of mouse immature Sertoli (TM4) cells. However, mechanisms by which BPA caused these effects were still unclear. In the present study, an inverse U-shaped curve was observed when treating TM4 cells with increasing doses of BPA: 1 to 10 nM BPA significantly stimulated the proliferation of TM4 cells and increased the proportion of cells in S phase; >1 μM BPA caused lesser proliferation of cells. Exposure of TM4 cells to G15 or ICI 182,780, which are specific antagonists of GPR30 and estrogen receptor α/β (ERα/β), respectively, abolished BPA-induced proliferation of cells, which suggests that both GPR30 and ERα/β were involved in the observed effects of BPA. Furthermore, exposure to BPA caused rapid (5 min) activation of ERK1/2 via both GPR30 and ERα/β. Blocking the GPR30/EGFR signal transduction pathway by antagonists suppressed both phosphorylation of ERK and BPA-induced cell proliferation. BPA up-regulated mRNA and protein expression of GPR30 in a concentration-dependent manner. In summary, the results reported here indicated that activating ERK1/2 through GPR30 and ERα/β is involved in low doses of BPA that promoted growth of Sertoli TM4 cells. The GPR30/EGFR/ERK signal is the downstream transduction pathway in BPA-induced proliferation of TM4 Sertoli cells.     

Bisphenol A Increases the Migration and Invasion of Triple‐Negative Breast Cancer Cells via Oestrogen‐related Receptor Gamma

Triple‐negative breast cancer (TNBC) is characterized by great metastasis and invasion capability. Our study revealed that nanomolar bisphenol A (BPA), one of the most ubiquitous endocrine disruptors, can increase wound closure and invasion of both MDA‐MB‐231 and BT‐549 cells. BPA treatment can increase protein and mRNA expression of matrix metalloproteinase‐2 (MMP‐2) and MMP‐9, while had no effect on the expression of vimentin (Vim) and fibronectin (FN) in TNBC cells. The expression of G‐protein‐coupled receptor (GPER), which has been suggested to mediate rapid oestrogenic signals, was not varied in BPA‐treated MDA‐MB‐231 and BT‐549 cells. Its inhibitor G15 also had no effect on BPA‐induced MMPs expression and cell invasion. Interestingly, BPA treatment can significantly increase the mRNA and protein expressions of oestrogen‐related receptor γ (ERRγ), but not ERRα or ERRβ, in both MDA‐MB‐231 and BT‐549 cells. The knock‐down of ERRγ can markedly attenuate BPA‐induced expression of MMP‐2 and MMP‐9 in TNBC cells. BPA treatment can activate both ERK1/2 and Akt in TNBC cells. Both inhibitors of ERK1/2 (PD98059) and Akt (LY294002) can attenuate BPA‐induced ERRγ expression and cell invasion of MDA‐MB‐231 cells. Collectively, our data revealed that BPA can increase the expression of MMPs and in vitro motility of TNBC cells via ERRγ. Both activation of ERK1/2 and Akt participated in this process. Our study suggests that more attention should be paid to the roles of xenoestrogens such as BPA in the development and progression of TNBC.     

Shp-2和LIF在影响HL-60细胞增殖分化中的相关性研究

目的探讨酪氨酸蛋白磷酸酶Shp-2和白血病抑制因子(LIF)影响HL-60细胞增殖与分化过程中的相互关系。方法采用Shp-2(c>s)作为对照,将Shp-2和Shp-2(c>s)分别转染白血病细胞HL-60,给予LIF刺激,用免疫组化、RT-PCR、免疫印迹和流式细胞术等方法检测其增殖细胞核抗原(PCNA)和CD15的表达水平。结果在胞质内Shp-2加强表达的情况下,PCNA表达明显下降;在LIF的刺激后,PCNA表达下降更加明显;然而,Shp-2、Shp-2(c>s)转染细胞之间无明显差别。同时,Shp-2和Shp-2(c>s)转染细胞都有CD15表达升高的现象;在LIF刺激后各组CD15表达均有增加,但以Shp-2转染细胞增幅为最高。结论Shp-2和LIF两者在HL-60细胞增殖抑制作用方面无关联,而Shp-2的大量表达可以协同LIF的促白血病细胞分化作用,其中Shp-2的N-SH2结构域参与了LIF激活白血病细胞分化的信号传递。