白血病抑制因子受体不同亚基细胞内区与HL—60细胞内STAT3激活的关系

目的　观察白血病抑制因子（LIF）受体gp190亚基和gp130亚基胞内区在人白血病细胞系HL-60中与STAT3表达及激活的关系,了解白血病抑制因子（LIF）引发白血病细胞增殖抑制和分化的机制。方法　用基因重组技术将gp130和gp190的细胞内区互换以构成两个嵌合体受体基因（130/190,190/130）并分别在HL-60细胞表达。用免疫组化和免疫印迹杂交方法分析形成受体亚基细胞内区同源性二聚体后的磷酸化STAT3的水平和STAT3的表达水平。结果　转染pED130/190,LIF诱导10min后,HL-60细胞内的STAT3磷酸化增加（P＜0.01）,经LIF诱导的转染pED130/190的HL-60细胞的STAT3磷酸化水平存在时间依赖性,转染pED190/130的HL-60细胞,LIF诱导6h后,STAT3的表达降低。结论　白血病抑制因子受体gp190亚基细胞内区在LIF诱导下参与HL-60细胞中STAT3的激活。     

流式细胞术检测磷酸化STAT3

为建立流式细胞术检测细胞内的信号转导及转录活化因子 3 (STAT3 )的方法 ,用IL 5刺激HL 60细胞株 ,活化细胞内的STAT3 ,然后用抗酪氨酸磷酸化STAT3抗体及相应的二抗标记 ,进行流式分析。结果表明 :( 1)IL 5能活化HL 60细胞内的STAT3 ,且STAT3的酪氨酸磷酸化水平与IL 5的浓度之间存在剂量依赖关系 ;( 2 )可用流式细胞术测得的平均荧光强度反映细胞内酪氨酸磷酸化STAT3的相对浓度。应用流式细胞术能快速、定量地测定细胞内的磷酸化STAT3水平。本法可用于STAT3相关疾病的筛选和临床监测及对其他胞内信号转导分子的研究。     

C3b-induced eosinophil degranulation involves PI3-kinases and is inhibited by protein kinase C activity

Carlson M, Venge P, Lampinen M. C3b‐induced eosinophil degranulation involves PI3‐kinases and is inhibited by protein kinase C activity. APMIS 2010; 119: 119–26. Selective release of individual eosinophil granule proteins has been demonstrated in eosinophilic conditions and in vitro using different stimuli. The aim of this study was to investigate if selective release of eosinophil cationic protein (ECP), eosinophil protein X/eosinophil derived‐neurotoxin (EPX/EDN) and eosinophil peroxidase (EPO) could be due to the involvement of different signal transduction pathways. Peripheral blood granulocytes from healthy donors were incubated with Wortmannin, LY294002, Genistein, Staurosporine, GÖ6976 or PD98059 prior to the induction of degranulation by C3b. The released amounts of ECP, EPO and EPX/EDN were determined by immunoassays, and related to the total cell content of respective protein. Wortmannin caused a significant, dose‐dependent inhibition of all three granule proteins. LY294002 (10^?6 M) also inhibited the release of all proteins. Genistein (10^?6 M) inhibited the release of ECP, whereas the release of EPO was increased. However, there was a tendency towards similar concentration‐dependent patterns of release of all three proteins. Staurosporine (10^?7 M), GÖ6976 (10^?6 M) and PD98059 (10^?5 M) caused an increased release of the three proteins. PI3‐kinases play an important role in the C3b‐induced release of ECP, EPO and EPX/EDN, whereas protein kinase C seems to have inhibitory effects on C3b‐induced degranulation.     

Highly expressed FYN promotes the progression of placenta accreta by activating STAT3, p38, and JNK signaling pathways

Placenta accreta is an abnormality of the placenta caused by the chorionic villi invading the muscular layer, which can cause serious bleeding, infection, shock, bladder invasion, uterine perforation, and even death. However, the etiology of placental accreta is not entirely clear. In the present study, high-throughput sequencing results showed that FYN is highly expressed in the placental accreta position in the placenta accreta group and is a key regulator of cell invasion and migration. Therefore, we aimed to evaluate the role and potential molecular mechanism of FYN in placenta accreta. The results showed that FYN was highly expressed in the placenta tissues of the placenta accreta group. Furthermore, the levels of phosphorylated STAT3, p38, and JNK in the placenta accreta group were remarkably increased compared with those in the control group. In addition, FYN knockdown considerably decreased the migration and invasion rates of trophoblast cells (HTR8/SVneo) and inhibited the levels of phosphorylated STAT3, p38, and JNK. After subsequently blocking these signaling pathways, the invasion and migration abilities of HTR8/SVneo cells were substantially decreased. In conclusion, FYN may promote excessive trophocyte cell invasion by activating STAT3, p38, and JNK pathways and can be a new target for placenta accreta prevention and treatment.     

Supporting the hypothesis of pregnancy as a tumor: survivin is upregulated in normal pregnant mice and participates in human trophoblast proliferation

PROBLEM: Survivin, a tumor-promoting antiapoptotic molecule, is expressed in the human placenta. Here, we analyzed its expression during normal and pathological murine pregnancy and investigated its participation in human first trimester trophoblast cell survival and proliferation. METHOD OF STUDY: We first analyzed the expression of survivin on the mRNA and protein level at the fetal-maternal interface of normal pregnant (CBA/J x BALB/c) and abortion-prone (CBA/J x DBA/2J) mice at different pregnancy stages by RT-PCR and immunohistochemistry. We also evaluated apoptosis in murine trophoblasts in both mating combinations by TUNEL technique. Functional studies were carried out by knockdown survivin by means of siRNA methodology in two human first trimester trophoblast cell lines [Swan.71 (Sw.71) and HTR8 (H8)]. RESULTS: We observed a peak in mRNA levels on day 5 and a peak of protein levels on day 8 of pregnancy in both combinations. The level of survivin in animals from the abortion-prone group was decreased compared with normal pregnant mice on day 8, which was accompanied by elevated apoptosis rates. In later pregnancy stages (days 10 and 14), survivin levels decreased to levels comparable to those observed right after fecundation in both groups. Transfection of human first trimester cell lines (H8 and Sw.71) with siRNA targeting the survivin gene led to a 76-82% reduction of its expression leading to reduced trophoblast cell viability and proliferation. CONCLUSION: Our findings suggest an important role of survivin to promote trophoblast cell survival and proliferation during placentation, thus maintaining pregnancy. The pregnancy-associated expression of a cancer molecule such as survivin supports the 'pseudo-malignancy' hypothesis of pregnancy. Our data may contribute to the better understanding of trophoblast cell development during implantation and placentation.     

肝细胞生长因子与妊娠期高血压疾病

肝细胞生长因子(hepatocyte growth factor,HGF)是一种具有多效性作用的肝素结合性酸性蛋白.HGF在胎盘中主要由绒毛核心间质细胞表达,旁分泌作用于邻近滋养细胞表面受体c-met.妊娠期高血压疾病时,HGFmRNA及蛋白产物表达下降.HGF在调节滋养细胞浸润能力、抗细胞凋亡和胎盘发生方面与妊娠期高血压疾病关系密切.     

Distribution of immunocompetent cells in decidua of controlled and uncontrolled (choriocarcinoma/hydatidiform mole) trophoblast invasion

Problem:  Pregnancy has been considered as a model of successfully controlled tissue invasion where trophoblast cells infiltrate the maternal decidua without being rejected or without destroying the tissue. In choriocarcinoma (CC) and hydatidiform mole (HM), a dysregulation of invasive (malignant/benign) trophoblast cells is present. Immunocompetent cells (IC) are known to be involved in rejection pathways of malignant cells and can also be identified in early pregnancy decidua. The aim of the present study was to identify the phenotype of IC in decidua of women with normal pregnancy (NP), CC and HM.
Methods:  Immunocompetent cells were detected by immunohistochemistry in decidual tissue from first trimester NP ( n  = 10), CC ( n  = 12) and HM ( n  = 11) using antibodies against CD8⁺, CD3⁺, CD56⁺, CD68⁺ cell surface markers and mast cell tryptase (MCT). A scaled eye piece was used for cell counting to obtain semiquantitative results. Statistical analysis was performed using Wilcoxon rank/Mann–Whitney tests.
Results:  We observed a significantly increased number of lymphocytes positive for CD8, CD3 and MCT positive granulocytes in CC and HM compared with the samples from NP (all P  ≤ 0.001). Lymphocytes positive for natural killer (NK) cell marker CD56 were significantly decreased in CC and HM versus NP ( P  ≤ 0.001). The number of CD68 positive cells (macrophages) were not significantly different among the tissue pools.
Conclusion:  The increase of CD8/CD3 T cells and mast cells in CC and HM and the decrease of CD56 cells, compared with NP, suggests the necessity of a balance between T and NK cells in controlling trophoblast invasion.     

苯那普利对糖尿病大鼠肾皮质JAK/STAT信号蛋白磷酸化的影响

目的: 探讨血管紧张素Ⅱ转换酶抑制剂苯那普利对糖尿病大鼠肾皮质p-JAK2、p-STAT1和p-STAT3信号蛋白表达的影响。方法: 雄性Wistar大鼠随机分为对照组、糖尿病组和苯那普利治疗组。腹腔注射STZ诱发糖尿病大鼠模型，治疗组每日灌胃给予苯那普利10 mg/kg，共2周。采用免疫沉淀和Western blotting检测JAK2磷酸化，Western blotting检测肾皮质p-STAT1、p-STAT3和TGF-β₁蛋白的表达，半定量RT-PCR检测肾皮质细胞TGF-β₁ mRNA的表达。结果: 糖尿病组肾皮质p-JAK2、p-STAT1、p-STAT3及其TGF-β₁表达明显高于对照组，同时TGF-β₁ mRNA表达亦明显强于对照组；苯那普利治疗组p-JAK2、p-STAT1和p-STAT3表达低于糖尿病组，同时TGF-β₁ 蛋白及其mRNA 表达亦低于糖尿病组。结论: JAK/STAT信号途径可能参与糖尿病早期肾脏变化的过程，苯那普利的肾脏保护作用可能部分是通过影响JAK/STAT信号途径的激活而实现。     

SAP increases FynT kinase activity and is required for phosphorylation of SLAM and Ly9

The free Src homology 2 (SH2) domain protein SAP, encoded by the X-linked lymphoproliferative disease gene SH2D1A, controls signal transduction initiated by engagement of the SLAM-related receptors in T and NK cells. Here we demonstrate that SAP is required for phosphorylation of both SLAM and Ly9 in thymocytes and peripheral T cells. Furthermore, in vitro protein interaction studies and yeast two-hybrid analyses indicated that SAP binds directly to FynT and Lck. While SAP bound to both the SH3 domain and to the kinase domain of FynT, SAP bound solely to the kinase domain of Lck. The existence of a strong interaction between SAP and the SH3 domain of FynT prompted us to study the role of SAP in modulating the activity of FynT. In vitro addition of SAP to the autoinhibited form of FynT caused a large increase in FynT catalytic activity. By contrast, the SAP mutant R78E, which is unable to bind to the FynT SH3 domain, did not increase FynT activity and also displayed a reduced adaptor function upon transfection into T cells. Our results demonstrate that SAP is an adaptor that bridges SLAM and Ly9 with Src-like protein tyrosine kinases (PTKs), and has the ability to activate FynT.     

{beta}2-lntegrin dependent aggregate formation between LB T cell lymphoma and spleen cells: assessment of correlation with spleen invasiveness

LB is an aggressive T cell lymphoma which rapidly invades thespleen and lymph nodes of BALB/c mice after s.c. inoculation.We previously reported that mAb directed against the ß₂chain of the leukocyte function-associated antlgen-1 (LFA-1)adhesion molecule (CD18) blocked the invasion of LB cells intothe spleen but not into the lymph nodes. The same antibody alsoblocked in vitro aggregate formation between normal spleen cellsand LB cells. However, aggregate formation between normal lymphnode cells and LB cells was not detected, regardless of ratio.In an attempt to evaluate the association between aggregateformation and tumor invasion of the lymphold organs, we havenow extended the study. Intravenous injection of anti-CD18 mAb,which blocked spleen invasion by LB cells, also blocked theformation of ex vivo aggregates, spontaneously generated inspleen, but not in lymph node, cell suspensions of BALB/c mices.c. Inoculated with LB cells. In contrast, mAbs unable to blockspleen invasion were ineffective inhibitors of both in vitroand ex vivo aggregate formation between spleen and LB cells.Spleens of nude mice that did not provide a supportive environmentfor lymphoma invasion, were also deficient in target cells formingaggregates with LB cells. In line with this observation, enrichedT cells formed more aggregates with LB cells than did enrichednon-T cells, Indicating the lymphoma's preferential bindingto splenic T cells. Aggregate-borne LB cells and LB cells whichwere not included in aggregates, Invaded the spleen and thelymph nodes to the same extent. However, non-aggregated LB cellsacquired the ability to form aggregates after 1 week of in vitrocultivation, suggesting that this capacity may also be acquiredin vivo. Antl-CD44 mAb, in distinct contrast to antl-CD18 mAb,blocked LB cell invasion of the lymph node, but not of the spleen.However, when antl-CD44 mAb was co-Injected with antl-CD18 mAbit antagonized the blocking effect of antl-CD18 mAb on spleeninvasion and formation of ex vivo splenic aggregates. The interpretationof these results in conjunction with the association betweensplenic aggregate formation and spleen invasion by LB cellsis discussed.     

妊娠肝内胆汁淤积症胎盘因素与胎儿不良结局的关系

妊娠期肝内胆汁淤积症（ICP）是发生于妊娠中晚期的妊娠特有并发症,此病可以造成胎儿及新生儿的不良妊娠结局如死胎、死产、早产新生儿窒息死亡等,而这些结果的产生都与胎盘的病理改变有关。本综述就妊娠期肝内胆汁淤积症胎盘因素与胎儿不良妊娠结局的关系进行综述,以便更好的预防不良妊娠结局的发生。