N-糖基化修饰在髓性白血病耐药中的作用

目的研究N-糖基化修饰、糖基因表达调控在髓性白血病耐药中的作用,明确N-糖基化修饰、糖基因与白血病耐药的相关性,从而为预测和诊断髓性白血病耐药性,寻求逆转药物提供新策略和靶点。方法通过修饰白血病耐药细胞株的N-糖基化（衣霉素Tunicamycin和PNGase F处理）,Western Blot检测Pgp、CD147糖蛋白的表达水平;MTT法检测N-糖基化修饰前后髓性白血病耐药细胞株的生长情况及对化疗药物的敏感性,观察上述细胞膜型N-糖基化修饰后对化疗药物耐药性的影响;进一步通过RNA干扰技术干预差异表达的糖基因,MTT法检测干扰前后白血病耐药细胞株的生长情况及对化疗药物的敏感性,观测糖基因的表达调控对髓性白血病耐药的影响。结果 NB4/ADR细胞经N-糖基化修饰后,P-gp、CD147糖蛋白的表达水平发生改变,同时该细胞的药物敏感性也增强（P〈0.05）;当通过RNA干扰技术特异性使NB4/ADR细胞中B3GNT8和ST8SIA4表达下调时,该细胞的药物敏感性增强（P〈0.05）。结论髓性白血病细胞株中N-糖基化修饰、糖基因的改变均与白血病多药耐药具有相关性,为预测和诊断髓性白血病耐药性,寻求逆转药物提供新策略和靶点。     

唾液酸糖基转移酶ST8SIA4对NB4/ADR细胞耐药性的影响

目的通过研究在人急性髓性白血病(AML)中NB4和耐药细胞株NB4/ADR的唾液酸糖基转移酶ST8SIA4的表达差异,探讨ST8SIA4基因的差异表达在AML细胞体内外药敏性的影响,为AML多药耐药的检测提供新的标志物,同时为逆转其耐药提供新策略和靶点。方法采用Real-time PCR、Western blot技术检测人AML细胞株ST8SIA4的表达情况。通过对ST8SIA4特异性调控,检测NB4/ADR细胞在体内和体外干扰前后对化疗药物的敏感性变化、信号通路PI3K/Akt的激活情况和P-gp的表达情况。结果在人AML和耐药细胞株中ST8SIA4的表达具有明显差异;特异性下调ST8SIA4在NB4/ADR细胞中的表达,药敏性在该细胞中增强;PI3K/Akt信号通路主要分子p110α、p-Akt308、p-Akt473在NB4/ADR-ST8SIA4sh RNA细胞中表达较少,同时P-gp的表达量减少。结论 ST8SIA4在人AML和耐药细胞株中表达具有明显的差异,AML多药耐药与ST8SIA4的特征性改变具有相关性;AML的多药耐药性可能是通过介导ST8SIA4的信号通路PI3K/Akt来调控P-gp表达改变的。     

糖基因ST6GAL家族在肝癌转移中的作用

目的通过研究糖基因ST6GAL家族在人肝癌高转移细胞株MHCC97-H和人肝癌低转移细胞株MHCC97-L中的差异表达,明确糖基因ST6GAL家族与肝癌转移的相关性,从而确证肝癌转移诊断及抗肿瘤治疗新靶点。方法采用Real-time PCR、Western Blot分析糖基因ST6GAL家族在人肝癌高、低转移细胞株的差异表达;通过RNA干扰技术干预差异表达的糖基因,检测干扰前后MHCC97-H细胞的体外侵袭能力及体内成瘤性。结果糖基因ST6GAL1在人肝癌高、低转移细胞株中表达差异具有统计学意义,而ST6GAL2的表达差异具无统计学意义;当通过RNA干扰技术特异性使MHCC97-H细胞中ST6GAL1表达下调时,该细胞在体外的侵袭能力下降及体内成瘤性受到抑制(P﹤0.05)。结论人肝癌细胞中糖基因ST6GAL1的差异表达与肿瘤细胞的侵袭、成瘤性密切相关,为肿瘤的化学治疗提供新靶点。     

糖基工程酵母表面呈现抗体库的构建及抗真菌β甘露糖型抗体的筛选

目的:通过构建糖基工程酵母表面呈现免疫抗体库,筛选与毕赤酵母表达的禽流感血凝素HA7结合的特异性抗体。方法:利用糖基工程毕赤酵母细胞膜锚定蛋白Sed将人Ig G的Fc段锚定在酵母表面,然后用毕赤酵母表达的HA7免疫小鼠,从小鼠脾脏细胞中扩增抗体轻重链可变区基因构建抗体转化锚定了Fc的糖基工程酵母,构建表面展示免疫抗体库,通过免疫磁珠法筛选与毕赤酵母表达的HA7特异性结合的抗体表达酵母,分析筛选该抗体的特异性结合靶点。结果:构建了多态性良好的抗体表达质粒库,转化糖基工程酵母后筛选得到特异性结合HA7的抗体呈现酵母,Western印迹分析发现该抗体只与糖基化的HA7结合而不与切除糖链后的HA7结合,说明抗体结合区域在HA7的糖链上,进一步实验发现该抗体不与α甘露糖型和哺乳动物复杂型糖型结合,通过β甘露糖苷酶切研究发现抗体结合靶点为酵母表达HA7糖链上的β甘露糖。结论:利用糖基工程酵母构建抗体库,筛选得获得了抗真菌β甘露糖的抗体,动物实验发现抗体具有中和活性。     

慢性髓细胞性白血病病人骨髓单个核细胞中差异表达基因的筛选

目的:筛选慢性髓细胞性白血病(CML)病人骨髓单个核细胞与正常人的差异表达基因,探讨CML的发病机制.方法:提取正常人和CML病人单个核细胞的RNA,逆转录成cDNA并用地高辛标记,应用全基因组表达谱基因芯片对差异表达基因进行研究,采用Jubilant病理/疾病分类法对CML相关差异表达基因进行分析.结果:共筛选出CM...     

应用凝集素芯片检测肝癌细胞膜表面糖链变化

利用凝集素糖链特异亲和原理构建对细胞膜表面糖链进行即时检测的凝集素芯片体系,检测肝癌发生过程中细胞膜糖链的变化.从H22细胞系、正常小鼠和肝癌模型鼠肝组织中提取细胞进行荧光标记,激光扫描仪检测凝集素位点捕获的细胞,根据凝集素特异亲和性确定细胞膜表面糖表达谱,显微镜下观察捕获细胞的形态.对凝集素芯片捕获细胞的最佳条件进行探讨,用甘露糖抑制试验、流式细胞仪和不同血型红细胞验证了凝集素捕获细胞的特异性.结果显示:正常和肝癌小鼠肝细胞膜表面糖链存在较大差异,正常组只有PSA、DSL、STL、NPL凝集素位点捕获到细胞,实验组只有LTL和DBA位点没有捕获到细胞,提示小鼠肝癌组织细胞膜表面糖链显著增加,细胞膜上唾液酸、乙酰葡萄糖、乙酰半乳糖、甘露糖和半乳糖糖链表达增加,这些糖链及其相关糖蛋白可能在肝癌的发生和发展中起一定作用.该凝集素芯片有较好的稳定性和特异性,可以对细胞膜表面糖链进行动态、即时、通量的检测,为研究细胞膜表面聚糖在细胞发育和癌变等过程中的变化提供了一个技术平台.     

植物凝集素与医学应用

植物凝集素是一类能选择性地结合细胞表面糖链的糖结合蛋白,由于植物凝集素所具有的糖结合专一性及细胞毒性使其在治疗方面具有很大的潜能,目前临床医学将它主要用于构成免疫毒素和作为有效的免疫佐剂以及靶向性运载工具等。     

亚砷酸诱导分化的急性早幼粒细胞白血病细胞浸润人脑膜组织的体外模拟及其分子机制研究

目的:探讨急性早幼粒细胞白血病(Acute promyelocytic leukemia,APL)合并中枢神经系统白血病的发病机制。方法:采用流式细胞术检测亚砷酸(Arsenious acid, ATO)诱导分化前后的APL细胞及人APL细胞株NB4细胞表面CD56、CXCR4的表达;用荧光染料-羧基荧光素二醋酸盐琥珀酰亚胺酯标记ATO分化的APL(APL/ATO)、NB4细胞(NB4/ATO);用微重力旋转培养法体外模拟APL细胞浸润人脑膜组织,观察组织学及超微结构。结果:ATO诱导后,APL/ATO细胞表面CXCR4的表达明显高于诱导前(35.2±9.5%vs. 18.6±4.9%);NB4/ATO细胞表面CXCR4的表达明显高于诱导前(39.6±2.6%vs. 21.0±7.3%);APL/ATO细胞表面CD56的表达明显高于诱导前(36.6±8.9%vs. 25.8±5.15%);NB4/ATO细胞表面CD56的表达明显高于诱导前(44.6±8.4%vs. 25.6±2.4%)。组织学实验结果显示对照组脑膜组织未见NB4、APL细胞浸润,实验组可见APL/ATO、NB4/ATO细胞浸润到人脑膜组织中;荧光显微镜下可见被标记的APL/ATO、NB4/ATO细胞浸润到人脑膜组织中,扫描电镜见APL/ATO、NB4/ATO细胞浸润到脑膜组织中。结论:本研究采用微重力旋转培养系统体外模拟了ATO诱导分化的异常早幼粒细胞浸润人脑膜组织,APL细胞和NB4细胞CXCR4、CD56的表达升高可能是ATO诱导治疗APL所致的中枢神经系统浸润的分子机制之一。     

地衣霉素对细胞膜表面运铁蛋白受体功能的影响

应用抑制糖蛋白Ｎ－糖链合成的地衣霉素处理ＳＭＭＣ－７７２１人肝癌细胞,３Ｈ甘露糖掺入实验显示细胞膜表面糖蛋白Ｎ－糖链的合成受到显著抑制,但细胞膜表面运铁蛋白受体内吞再循环的过程无显明变化,进一步的研究表明受体与运铁蛋白的亲和力亦无改变,但细胞膜表面运铁蛋白受体数减少。结果提示用地衣霉素处理细胞后,在内质网合成的无Ｎ－糖链的运铁蛋白受体影响其运输到细胞膜表面表达。     

NPM1突变基因通过MMPs参与调控白血病细胞体外侵袭

核仁磷蛋白基因(nucleophosmin,NPM1)突变是目前急性髓系白血病(AML)中突变率最高的基因改变,在白血病的发生发展过程中发挥重要的调控作用。为探讨NPM1突变参与调控白血病髓外浸润的分子机制,将表达质粒pEGFPC1-NPM1-mA转染THP-1细胞系,筛选稳定表达NPM1突变蛋白的白血病细胞株(THP-1-mA)。利用RT-PCR及Western blot分析了THP-1-mA细胞与亲代细胞间MMP-2、MMP-9、TIMP-1、TIMP-2表达水平的差异。结果显示,具有体外高侵袭能力的THP-1-mA组细胞MMP-2的mRNA水平和蛋白水平均明显高于两对照组,而MMP-9 mRNA表达水平虽有所增高,但蛋白表达水平却明显降低。同时,与空载体转染组和未处理组细胞相比,THP-1-mA组细胞TIMP-2的mRNA水平和蛋白水平表达显著降低,差异具有统计学意义;TIMP-1表达水平无明显改变。提示MMP-2及其抑制剂TIMP-2在NPM1突变参与调控的白血病细胞髓外浸润中可能发挥重要作用。     

Glycomic alterations are associated with multidrug resistance in human leukemia

Correlations of disease phenotypes with glycosylation changes have been analyzed intensively in tumor biology field. In this study we describe glycomic alterations of multidrug resistance in human leukemia cell lines. Using multiple glycan profiling tools: real-time PCR for quantification of glycogenes, FITC-lectin binding for glycan profiling, and mass spectrometry for glycan composition, we compared the glycomics of drug-resistant K562/ADR cells with parental K562 line. The results showed that the expression of glycogenes, glycan profiling and N-glycan composition were different in K562/ADR cells, as compared with those in K562 cells, whereas O-glycans of the two cell lines showed no different mass spectra. Further analysis of the N-glycan regulation by way of tunicamycin application or PNGase F treatment in K562/ADR cells showed partial inhibition of biosynthesis and increased sensitivity to chemotherapeutic drugs in vitro. We targeted glycogene B3GNT8 and ST8SIA4, which were over-expressed in K562/ADR cells, and silenced the expression levels of two glycogenes after using RNA interference approach. The results showed that the silencing of B3GNT8 or ST8SIA4 in K562/ADR cells resulted in increased chemosensitivity to anti-tumor drugs. In conclusion, glycomic alterations are responsible for the overcoming multidrug resistance in human leukemia therapy and the N-linked oligosaccharides are associated with the drug resistance of cancer cells.     

Advantages of the doxorubicin-resistant, acute myelogenous leukemia cell line, AML-2/DX100, for the screening of multidrug resistance protein inhibitors and pro-oxidants

The doxorubicin-resistant, acute myelogenous leukemia cell line, AML-2/DX100, characterized by the over-expression of multidrug resistance protein (MRP) and the down-regulation of catalase, has advantages for the screening of MRP inhibitors as well as for cytotoxic substances producing potential reactive oxygen species. The screening power of AML-2/DX100 cells for an MRP inhibitor, probenecid, was approximately 4-fold stronger than that of another resistant cell line, HL-60/Adr, over-expressing MRP. AML-2/DX100 was approximately 2- to 5-fold more sensitive to pro-oxidants such as Paraquat, H₂O₂ and t-butyl hydroperoxide, when compared with its parental cells.     

胃癌多药耐药相关microRNA的筛选和鉴定

目的:研究胃癌多药耐药相关microRNA并对其进行鉴定、靶基因预测和预测靶基因的生物信息学分析。方法:运用microRNA芯片对胃癌多药耐药细胞SGC7901/ADR和其亲本细胞SGC7901进行microRNA表达谱分析;采用实时定量PCR的方法对差异表达的miRNA进行验证;再运用生物信息学方法对差异表达的miRNA进行靶基因预测;再对预测的靶基因进行GO和KEGG通路分析。结果:与SGC7901相比SGC7901/ADR表达上调超过2倍的miRNA有6个,表达下调超过2倍的有11个。实时定量PCR对共同差异表达的microRNA进行验证显示与芯片结果的一致性。对这17个差异表达的miRNA进行靶基因预测,再对预测得到的靶基因进行GO和KEGG通路分析显示预测的靶基因参与了肿瘤相关通路、MAPK通路、Focal Adhesion通路等。结论:我们初步筛选得到了胃癌多药耐药相关miRNA并对其进行了生物信息学分析,为进一步地探索miRNA在胃癌多药耐药中的作用及其分子机制奠定了基础。     

Mad2beta, an alternative variant of Mad2 reducing mitotic arrest and apoptosis induced by adriamycin in gastric cancer cells

Mad2beta is an alternative splicing variant of spindle checkpoint gene mad2, which was previously found by us and was related to the drug resistance in gastric cancer cells. In this paper, we explored the molecular mechanisms that Mad2beta variant promoted the formation of multidrug resistance in gastric cancer cells. We found that Mad2beta variant was detected only in the two human drug resistant gastric cancer cell sublines SGC7901/VCR and SGC7901/ADR, and it did not appear in its parental cell line SGC7901 and other detected gastric cancer cell lines. Expressions of Mad2 mRNA and protein in SGC7901 cells transfected with Mad2beta, SGC7901/VCR and SGC7901/ADR were significantly lower than that in SGC7901 cells. Moreover, SGC7901 cells overexpressing Mad2beta variant became more resistant to adriamycin, vincristine and mitomycin by abrogating mitotic arrest and apoptosis. This suggests that expression of Mad2beta variant decreases the relative expression of efficient MAD2, which may help gastric cancer cells to develop the phenotype of multidrug resistance.     

Increased sensitivity of an adriamycin-resistant human small cell lung carcinoma cell line to mitochondrial inhibitors.

The energy metabolism of an atypical multidrug resistant human small cell lung carcinoma cell line (GLC4/ADR) was studied. The glycolytic rate was 30% reduced and the glucose-6-phosphate dehydrogenase activity 2-fold increased in GLC4/ADR compared to the parental sensitive line (GLC4). Although mitochondrial respiration activities were similar in both cell lines, GLC4/ADR was more sensitive to the antimitochondrial drugs doxycycline and oligomycin, while cross-resistance was observed for the glycolytic inhibitor 2-deoxyglucose and for the antimitochondrial drug rhodamine-123. Continuous incubation with doxycycline induced a dramatic reduction of mitochondrial mRNAs in both cell lines, whereas a strong reduction of the nuclear-coded mRNA for subunit IV of cytochrome c oxidase was induced in GLC4/ADR only. Incubation with doxycycline had an additive effect on the cytotoxicity of adriamycin in both cell lines. Thus, a form of collateral sensitivity to antimitochondrial drugs may exist in atypical multidrug resistant cell lines.     

Reversal Effect of ST6GAL 1 on Multidrug Resistance in Human Leukemia by Regulating the PI3K/Akt Pathway and the Expression of P-gp and MRP1

β-galactoside α2, 6-sialyltransferse gene (ST6GAL) family has two members, which encode corresponding enzymes ST6Gal I and ST6Gal II. The present atudy was to investigate whether and how ST6GAL family involved in multidrug resistance (MDR) in human leukemia cell lines and bone marrow mononuclear cells (BMMC) of leukemia patients. Real-time PCR showed a high expression level of ST6GAL1 gene in both MDR cells and BMMCs (*P<0.05). Alternation of ST6GAL1 levels had a significant impact on drug-resistant phenotype changing of K562 and K562/ADR cells both in vitro and in vivo. However, no significant changes were observed of ST6GAL2 gene. Further data revealed that manipulation of ST6GAL1 modulated the activity of phosphoinositide 3 kinase (PI3K)/Akt signaling and consequently regulated the expression of P-glycoprotein (P-gp, *P<0.05) and multidrug resistance related protein 1 (MRP1, *P<0.05), which are both known to be associated with MDR. Therefore we postulate that ST6GAL1 is responsible for the development of MDR in human leukemia cells probably through medicating the activity of PI3K/Akt signaling and the expression of P-gp and MRP1.     

Upregulation of miR-142-3p Improves Drug Sensitivity of Acute Myelogenous Leukemia through Reducing P-Glycoprotein and Repressing Autophagy by Targeting HMGB1

miR-142-3p was reported to be downregulated in acute myelogenous leukemia (AML) and acted as a novel diagnostic marker. However, the regulatory effect of miR-142-3p on drug resistance of AML cells and its underlying mechanism have not been elucidated. Here, we found that miR-142-3p was significantly downregulated and high mobility group box 1 (HMGB1) was dramatically upregulated in AML samples and cells, as well as drug-resistant AML cells. P-gp level and autophagy were markedly enhanced in HL-60/ADR and HL-60/ATRA cells. miR-142-3p overexpression improved drug sensitivity of AML cells by inhibiting cell viability and promoting apoptosis, and inhibited P-gp level and autophagy in drug-resistant AML cells, whereas HMGB1 overexpression obviously reversed these effect. HMGB1 was demonstrated to be a target of miR-142-3p, and miR-142-3p negatively regulated HMGB1 expression. In conclusion, our study elucidated that upregulation of miR-142-3p improves drug sensitivity of AML through reducing P-glycoprotein and repressing autophagy by targeting HMGB1, contributing to better understanding the molecular mechanism of drug resistance in AML.     

Loss of multidrug resistance protein 1 expression and folate efflux activity results in a highly concentrative folate transport in human leukemia cells

We studied the molecular basis of the up to 46-fold increased accumulation of folates and methotrexate (MTX) in human leukemia CEM-7A cells established by gradual deprivation of leucovorin (LCV). CEM-7A cells consequently exhibited 10- and 68-fold decreased LCV and folic acid growth requirements and 23-25-fold hypersensitivity to MTX and edatrexate. Although CEM-7A cells displayed a 74-86-fold increase in the reduced folate carrier (RFC)-mediated influx of LCV and MTX, RFC overexpression per se cannot induce a prominently increased folate/MTX accumulation because RFC functions as a nonconcentrative anion exchanger. We therefore explored the possibility that folate efflux activity mediated by members of the multidrug resistance protein (MRP) family was impaired in CEM-7A cells. Parental CEM cells expressed substantial levels of MRP1, MRP4, poor MRP5 levels, whereas MRP2, MRP3 and breast cancer resistance protein were undetectable. In contrast, CEM-7A cells lost 95% of MRP1 levels while retaining parental expression of MRP4 and MRP5. Consequently, CEM-7A cells displayed a 5-fold decrease in the [(3)H]folic acid efflux rate constant, which was identical to that obtained with parental CEM cells, when their folic acid efflux was blocked (78%) with probenecid. Furthermore, when compared with parental CEM, CEM-7A cells accumulated 2-fold more calcein fluorescence. Treatment of parental cells with the MRP1 efflux inhibitors MK571 and probenecid resulted in a 60-100% increase in calcein fluorescence. In contrast, these inhibitors failed to alter the calcein fluorescence in CEM-7A cells, which markedly lost MRP1 expression. Replenishment of LCV in the growth medium of CEM-7A cells resulted in resumption of normal MRP1 expression. These results establish for the first time that MRP1 is the primary folate efflux route in CEM leukemia cells and that the loss of folate efflux activity is an efficient means of markedly augmenting cellular folate pools. These findings suggest a functional role for MRP1 in the maintenance of cellular folate homeostasis.