WT1联合多参数流式在急性髓系白血病预后的临床观察

目的 研究WT1基因定量联合多参数流式（FCM）在急性髓系白血病（AML）预后的临床观察。方法62例AML患者分为低危组、中危组、高危组;治疗上参照《成人急性髓系白血病（非急性早幼粒细胞白血病）中国诊疗指南2017年版》,用RT-qPCR方法测定患者WT1基因表达水平;同时用多参数流式细胞分析技术（FCM）分析患者低水平微小残留（MRD）水平;比较不同预后患者WT1表达及WT1表达与预后的关系。观察WT1基因定量联合FCM对AML患者预后评估的临床意义。对患者进行随访不低于2年。结果 AML患者WT1高表达比例为77.42%（48/62）,不同预后分型的AML患者WT1高表达有统计学差异（P <0.05）。在中、高危组的患者WT1高表达明显高于低危组。初发的WT1高表达与WT1低表达患者在诱导缓解率无统计学意义（P >0.05）,但WT1高表达患者的2年无病生存率、2年总生存率均WT1低表达患者,差异有统计学意义（35.41% vs 71.43%,47.92% vs 85.71% P < 0.05）。WT1基因联合FCM预测AML早期复发的敏感性、特异性均高于单独WT1基因和单独的FCM（P < 0.05）。结论 WT1在AML患者中高表达,与患者预后相关,联合FCM可有效的预测患者早期复发,可作为临床治疗及预后判断的靶点。     

骨髓增生异常综合征的中西医治疗

骨髓增生异常综合征(MDS)以难治性全血细胞减少和易转化为急性白血病为特征。患者的预后、合并症和年龄是决定治疗策略时必须考虑的因素。治疗策略经常以支持治疗为主,包括造血细胞生成因子、输注红细胞和血小板以及抗感染治疗。另外诱导分化、免疫抑制治疗、刺激造血、靶向治疗、化疗、异基因骨髓移植等根据具体病情选择应用。我们明确提出“气阴两虚,血瘀内阻”的中医复合证候为MDS临床主证,以“益气养阴活血”为治疗大法,制成“益髓颗粒剂”,进行了大量实验及临床研究,获得了确切疗效。     

Scanning for sisnatures of geosraphically restricted selection based on population genomics analysis

Natural selection, as the driving force of human evolution, has direct impact on population differentiation. However, it is still unclear to what extent the genetic differentiation has been caused by natural selection. To explore this question, we performed a genome-wide scan with single nucleotide polymorphism （SNP） data from the International HapMap Project. Single locus FsTanalysis was applied to assess the frequency difference among populations in autosomes. Based on the empirical distribution of FsT, we identified 12669 SNPs correlating to population differentiation and 1853 candidate genes subjected to geographic restricted natural selection. Further interpretation of gene ontogeny revealed 121 categories of biological process with the enrichments of candidate genes. Our results suggest that natural selection may play an important role in human population differentiation. In addition, our analysis provides new clues as well as research methods for our understanding of population differentiation and natural selection.     

应用傅立叶变换红外光谱研究白血病骨髓血红细胞

研究了急性粒细胞白血病(AML)和慢性粒细胞白血病(CML)患者的骨髓血红细胞的红外光谱差异性：与CML红细胞相比,AML红细胞酰胺Ⅰ带较宽;1540cm-1与1 084cm-1吸收峰面积之间的比值较低,范围较窄。该结果提示AML和CML骨髓血红细胞系中有核红细胞的数量分布不同,同时提示骨髓红细胞的成熟可能与白血病细胞的增殖有关。骨髓血细胞的红外光谱的差异性对白血病的分析和评价具有重要的意义。     

PCR检测白血病患者外周血中EB病毒的DNA

目的：了解白血病患者ＥＢ病毒感染情况。方法：收集２１例急性淋巴细胞白血病、１例慢性淋巴细胞白血病、１５例急性粒细胞白血病、８例慢性粒细胞白血病患者及３２例正常对照组的外周血,分离单个核细胞,提取ＤＮＡ,应用ＰＣＲ方法检测ＥＢ病毒ＤＮＡ。结果：在１例初诊慢性粒细胞白血病病人样本中发现ＥＢ病毒阳性,余均为阴性。结论：白血病患者存在ＥＢ病毒感染情况,但并不普遍。     

全反式维甲酸对脐血红系祖细胞增殖过程中HOXB6基因表达的影响

探讨脐血造血干祖细胞向红系增殖过程中HOXB6 mRNA表达及全反式维甲酸对HOXB6 mRNA表达的影响.采用体外培养技术,以全反式维甲酸持续干扰造血干祖细胞,观察集落生成情况,采用实时荧光定量PCR技术检测造血祖细胞增殖分化过程中HOXB6基因的表达水平,用DNA相对拷贝数和RNA表达相对量表示HOXB6基因相对表达量.结果表明,人类造血干祖细胞向红系祖细胞增殖分化过程中,各祖细胞HOXB6基因均表达,与正常对照组比较,全反式维甲酸可上调HOXB6基因的表达,说明HOXB6可能是造血干祖细胞向红系祖细胞正常增殖分化过程中的调控基因之一.     

Effect of Spatholobus suberectus Dunn on proliferation of progenitor cells in mice with bone marrow depression

AIM： To study the effect of Spatholobus suberectus Dunn on the prolilferation and hematonic mechanism of Spatholobus suberectus Dunn. Methods： The techniques of culture of hematopoietic cell and hematopoietic growth factor （HGF） assay were used. The method of semi-solid culture with methylcellulose of CFU-GM, CFU-E, BFU-E, CFU-Meg was adopted in bone marrow depressed mice which treated with Spatholobus suberectus Dunn for a long time. Results： Spatholobus suberectus Dunn could obviously promote the proliferation of bone morrow cells and spleen lymphocytes in healthy and anaemic mice. The cuhure medium of spleen cell, macrophage, lung and skeletal muscle treated with Spatholobus suberectus Dunn had much stronger stimulating effects on hematopoietic cells. The numbers of CFU-GM, CFU-E, BFU-E, CFU-Meg in bone marrow depressed mice were raised distinctly under the control of Spatholobus suberectus Dunn as compared with those of contrast group. Conclusions： Spatholobus suberectus Dunn may enhance hematopoiesis by stimulating directly and/or indirectly stroma cell in hematopoietic inductive microenvironment and muscle tissue to secrete some HGF （Epo, GM-CSF, IL, and MK-CSF）. It can promote the proliferation and differentiation of hematopoietic cells in anaemic mice. This is one of the biological mechanisms for hematonic effect of Spatholobus suberectus Dunn.     

As2O3对脐血红系祖细胞HOXB6基因表达的调控作用

目的：探讨脐血造血干祖细胞向红系祖细胞增殖过程中HOXB6mRNA表达及As2O3对HOXB6mRNA表达的影响．方法：①采用外培养技术,以As2O3干扰造血干祖细胞,观察HSPC经促红素诱导后,CFU-E集落生成情况．②采用实时荧光定量HR技术检测造血祖细胞增殖分化过程中HOXB6基因的表达．结果：①造血于祖细胞红系祖细胞增殖过程中,各组细胞HOXB6基因均表达,②与正常对照组比较,As2O3可下调HOXB6基因的表达．结论：①HOXB6可能是造血干祖细胞向红系祖细胞增殖分化中的调控基因之一．②As2O3能下调HOXB6基因的表达．     

补阳还五汤与高血压病气虚血瘀证“方证相关”的蛋白质组学研究

揭示补阳还五汤与高血压病气虚血瘀证"方证相关"的现代生物学基础,制作高血压病气虚血瘀证细胞模型。用补阳还五汤含药血清干预细胞模型,利用双向凝胶电泳(2-DE)及基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS/MS)技术筛选及鉴定差异表达的蛋白点。并对其进行生物学分析。结果高血压病气虚血瘀证组与健康对照组比较,差异蛋白质点有30个。其中,有16个蛋白上调,14个蛋白质下调。补阳还五汤组与高血压病气虚血瘀证组比较,差异蛋白质点有14个。其中,有9个蛋白上调,5个蛋白质下调。MALDI-TOF-MS/MS鉴定出:高血压病气虚血瘀证组与健康对照组差异蛋白点共有8个蛋白被成功鉴定出来。表达上调的蛋白有肽基脯氨酰异构酶A、肽基脯氨酸顺反异构酶A样1亚型、真核翻译起始因子5A-1 B亚型、微管蛋白beta-2C、CRA_b亚型、3-羟酰辅酶A脱氢酶2型异构体2。表达下调的蛋白有丙酮酸激酶同工酶M1亚型、抑制蛋白-1、抑制蛋白-1 1亚型、补阳还五汤组与高血压病气虚血瘀证组差异蛋白点共有3个蛋白被成功鉴定出来。表达上调的蛋白有丙酮酸激酶CRA_c亚型、热休克蛋白27;表达下调的蛋白有膜联蛋白A1,CRA_b亚型。这些蛋白多与促进或抑制细胞凋亡有关。说明高血压病气虚血瘀证存在着细胞凋亡,补阳还五汤可以纠正高血压病气虚血瘀证引起的细胞凋亡。这些差异蛋白可以作为高血压病气虚血瘀证的标志蛋白或补阳还五汤的作用靶点,抑制细胞凋亡可能是补阳还五汤与高血压病气虚血瘀证"方证相关"的现代生物学基础之一。     

Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides

Murine embryonic stem (ES) cells are pluripotent cell lines established directly from the early embryo which can contribute differentiated progeny to all adult tissues, including the germ-cell lineage, after re-incorporation into the normal embryo. They provide both a cellular vector for the generation of transgenic animals and a useful system for the identification of polypeptide factors controlling differentiation processes in early development. In particular, medium conditioned by Buffalo rat liver cells contains a polypeptide factor, ES cell differentiation inhibitory activity (DIA), which specifically suppresses the spontaneous differentiation of ES cells in vitro, thereby permitting their growth as homogeneous stem cell populations in the absence of heterologous feeder cells. ES cell pluripotentiality, including the ability to give rise to functional gametes, is preserved after prolonged culture in Buffalo rat liver media as a source of DIA. Here, we report that purified DIA is related in structure and function to the recently identified hematopoietic regulatory factors human interleukin for DA cells and leukaemia inhibitory factor. DIA and human interleukin DA/leukaemia inhibitory factor have thus been identified as related multifunctional regulatory factors with distinct biological activities in both early embryonic and hematopoietic stem cell systems.     

Effects of STAT3 antisense RNA on IL-6-induced differentiation and growth arrest of Ml leukemia cells

To investigate the biological roles of STAT3 in the regulation of growth and differentiation of leukemia cells, we modified a murine myeloid leukemia cell line Ml with STAT3 antisense RNA. The effects of STAT3 antisense RNA on the growth arrest and terminal differentiation of Ml cells induced by interleukin 6 (IL-6) were determined. It was found that STAT3 antisense RNA blocked the activation of STAT3, and reduced the growth arrest and terminal differentiation of IL-6-induced Ml leukemia cells. These results indicate that STAT3 activation is a necessary process for IL-6-induced growth arrest of Ml cells and for the differentiation of Ml cells into macrophage.     

Viewpoints on the proinflammation state of leukemia

Proinflammation represents a pathophysiological state on the early stage of a number of diseases, especially the infectious and immunological ones. In recent years, proinflammation has attracted much attention, and the term 損roinflammation factors?appears frequently in the literature. While investigating leukemia and leukemic cells from the angle of 損roinflammation state? we got some intriguing findings, e.g. we detected the significantly elevated expression of proinflammation factor IL-18 in patients with acute myeloid leukemia (AML), which could up-regulate matrix metalloproteinases (MMP) and specific tissue inhibitors (TIMPs). The increased MMP may play a role in the aggressiveness of myeloid leukemic cells, and be associated with a poor prognosis. This phenomenon reflects an ignored aspect of leukemia. Investigations from the angle of 損roinflammation state?have broaden the fields of tumor and leukemia study.     

Anti-TOSO antibody treatment promotes T cell activation-induced cell death （AICD） in vitro and in vivo

T cell activation-induced cell death （AICD）, that involves the induction of Fas-mediated apoptosis, is very important for the maintenance of immune homeosta- sis. TOSO was firstly described as an inhibitor of Fas- mediated apoptosis and overexpressed in chronic lymphocytic leukemia. Recently, TOSO was identified as IgM FcR. In this study, we produced anti-TOSO monoclonal antibody （mAb） that could block the binding of IgM to TOSO and found that T cell apoptosis is negatively cor- related with TOSO expression during T cell activation. Treatment of activated T cells with anti-TOSO blocking mAb promoted T cell AICD in in vitro AICD model, and treatment of xenogeneic-GVHD mice with the antibody also increased the sensitivity of activated T cells to Fas- induced apoptosis, which was accompanied by reduction of c-FLIPL expression and up-regulation of AP-1 complex. In summary, our data indicate the anti-apoptotic effect of TOSO in T cell AICD and open up new therapeutic prospects for the treatment of hematologic malignancies and immune disorders.     

转录因子GATA—1基因在白血病中的表达

转录因子ＧＡＴＡ家族在造血细胞的正常发育中起着重要的作用。利用聚合酶链反应方法分析了１１６例各种白血病中红系统特异转录因子ＧＡＴＡ－１的表达情况。ＡＮＬＬ、ＣＭＬ、Ｃ－ＡＬＬ和ＣＬＬ中的表达率分别为４３．７５％、８８．２４％、１４．２９％和３３．３３％；３例Ｔ－ＡＬＬ均不表达该基因。     

Induction of embryonic stem cells to hematopoietic cellsin vitro

In order to get hematopoietic cells from embryonic stem (ES) cells and to study development mechanisms of hematopoietic cells, the method of inducing embryonic stem cells to hematopoietic cells was explored by differenciating mouse ES cells and human embryonic cells in three stages. The differentiated cells were identified by flow cytometry, immunohistochemistry and Wright’s staining. The results showed that embryoid bodies (EBs) could form when ES cells were cultured in the medium with 2-mercaptoethanol (2-ME). However, cytokines, such as stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3), interleukin-6 (IL-6), erythropoietin (EPO) and granular colony stimulating factor (G-CSF), were not helpful for forming EBs. SCF, TPO and embryonic cell conditional medium were useful for the differentiation of mouse EBs to hematopoietic progenitors. Eighty-six percent of these cells were CD34⁺ after 6-d culture. Hematopoietic progenitors differentiated to B lymphocytes when they were cocultured with primary bone marrow stroma cells in the DMEM medium with SCF and IL-6. 14 d later, most of the cells were CD34^－CD38⁺. Wright’s staining and immunohistochemistry showed that 80% of these cells were plasma-like morphologically and immunoglubolin positive. The study of hematopoietic cells from human embryonic cells showed that human embryonic cell differentiation was very similar to that of mouse ES cells. They could form EBs in the first stage and the CD34 positive cells account for about 48.5% in the second stage.     

Action mechanisms of a new erythrocyte-derived depressing factor

To investigate the action mechanisms of a new erythrocyte-derived depressing factor (EDDF), the focus is placed on the effect of EDDF on both cytosolic and nuclear free calcium (Ca²⁺) transportation in vascular smooth muscle cell (VSMC), as well as the apoptosis and cell cycle of VSMC of rats. EDDF has been extracted from human erythrocytes. The changes of Ca²⁺ levels in cytoplasm ([Ca²⁺]_i) and nucleus ([Ca²⁺]_n) have been observed using a laser scanning confocal microscope together with fluo-3/AM as a calcium indicator. Flow cytometric technique was used to study the effect of EDDF on cell cycle and apoptosis of VSMC. [Ca²⁺], and [Ca²⁺]_n were significantly decreased through several different pathways: ( i ) it reduced the Ca²⁺ influx by blocking L-type voltage-dependent calcium channel (L-VDC) and R-type voltage-dependent calcium channel (R-VDC); (ii) it inhibited the Ca²⁺ release from inositol 1, 4, 5-trisphosphate (IP₃) sensitive calcium store; and (iii) activated Ca²⁺-ATPase of sarcoplasmic reticulum (SR) and promoted the transportation of Ca²⁺ from cytoplasm to SR. However, EDDF seemed to have little inhibitory effect on the Ca²⁺ release from ryonodine sensitive calcium pool. It was also found that EDDF (10⁻⁴ g/mL) significantly decreased the proportion of S phase of human umbilical vein (HUV) and inhibited the proliferation of VSMC induced by angiotensin II (Angll, 10⁻⁵ mol/L). The apopotosis did not occur when VSMC was cultured under normal condition. While VSMC apoptosis was induced by Angll (10⁻⁵ mol/L) and EDDF (10⁻⁴ g/mL) seemed to have little effect on it. The inhibitory effect of EDDF on the elevation of [Ca²⁺]_i and [Ca²⁺]_n of VSMC might play an essential role in its action mechanisms and the ways it affects the Ca²⁺ handling of VSMC demonstrate that EDDF was different from other endogenous blood pressure regulators and some known antihypertensive drugs. EDDF could inhibit the proliferation of VSMC, which indicated that it might be beneficial to the prevention and treatment of hypertension and arteriosclerosis.     

Knockdown of <Emphasis Type="Italic">MRP4</Emphasis> by lentivirus-mediated siRNA improves sensitivity to adriamycin in adriamycin-resistant acute myeloid leukemia cells

Chemotherapy remains the standard treatment for acute myeloid leukemia;however,the emergence of drug resistance is a major hurdle in the successful treatment of leukemia.The expression of multidrug resistance-associated protein 4(MRP4)induces re- sistance in the adriamycin-resistant acute myeloid leukemia cell line,K562/ADR.The aim of this study was to investigate whether knockdown of MRP4 by lentivirus-mediated siRNA could improve the sensitivity of K562/ADR cells to adriamycin.Five lenti- virus-mediated short hairpin RNAs(lv-shRNAs-MRP4)were designed to trigger the gene silencing RNA interference(RNAi) pathway.The efficiency of lentivirus-mediated siRNA infection into K562/ADR cells was determined using fluorescence mi- croscopy to observe lentivirus-mediated GFP expression.MRP4 expression in infected K562/ADR cells was evaluated by real- time PCR and Western blot analysis.The MTS assay was used to measure cell viability and flow cytometry was used to measure apoptosis.The transfection efficiency of K562/ADR cells was over 80 percent.The gene silencing efficacy of lv-shRNA1-MRP4 was superior to the other constructs.Infection of K562/ADR cells with lv-shRNA1-MRP4 led to strong inhibition of MRP4 mRNA and protein expression.Combined treatment with lv-shRNA1-MRP4 and adriamycin decreased cell growth and increased apoptosis compared to treatment with lv-shRNA1-MRP4 or adriamycin alone.These data indicate that in K562/ADR cells MRP4 is involved in drug resistance mechanisms and that lentivirus-mediated knockdown of MRP4 may enhance sensitivity to adriamycin.