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.     

凝集素亲和层析研究人血清肝型碱性磷酸酶的糖链结构

用麦胚凝集素(WGA)将人血清肝型和骨型碱性磷酸酶(ALP)分离,再将肝型ALP通过蔓陀萝凝集素(DSA)亲和层析柱作醋酸梯度洗脱,发现正常人、急性肝炎、慢性肝炎、肝硬化和原发性肝癌各组各自的混合血清ALP的层析行为有明显差异。正常人只有不结合组分,而各种良性肝病出现数目不同(2—3个)的弱结合组分,肝癌则还出现3个強结合组分,各组分ALP在不同肝病血清中的出峰时间基本恒定。这种ALP洗脱峰的不均一性在单个病人血清中依然存在,是ALP糖链结构不同而引起的。由此推测肝病时ALP上N糖链的天线数增加,肝病越发展成慢性或恶性,血清ALP和DSA的亲和力也越強。这些结果有可能在临床上鉴别各类不同的肝病。     

凝集素芯片技术检测糖蛋白方法的建立及初步应用

建立了凝集素芯片技术检测糖蛋白的方法,对实验条件进行了优化,应用凝集素芯片初步检测分析了Chang?蒺s liver正常肝细胞总蛋白中的糖蛋白糖链构成．将凝集素ConA、GNA固定于环氧化修饰的玻片表面,用Cy3标记标准糖蛋白RNaseB,利用凝集素识别特异糖链的原理建立凝集素芯片检测糖蛋白的方法．摸索出最佳封闭剂是含1% BSA的磷酸缓冲液,最佳孵育时间及温度为3 h和室温,最佳孵育缓冲液为含1% BSA和0.05% Tween-20的磷酸缓冲液,并用甘露糖抑制实验验证了凝集素芯片结合的特异性．用包含10种凝集素的芯片,成功解析了标准糖蛋白RNaseB、Fetuin的糖链构成,证实了凝集素芯片检测糖蛋白糖链的可行性．最后用凝集素芯片初步检测分析了Chang?蒺s liver正常肝细胞总蛋白中的糖蛋白糖链构成,发现 Chang's liver正常肝细胞总蛋白中的糖蛋白可能有多价 Sia或GlcNAc、terminalα-1,3 mannose、GalNAc、Galβ-1,4GlcNAc这些糖链结构的存在．蛋白质糖基化是一种重要的翻译后修饰,它在微生物感染、细胞分化、肿瘤转移、细胞癌变等生命活动中起着重要作用,因此近年来蛋白质的糖基化研究受到广泛的重视,但由于缺乏一种简便、快速、高通量的检测手段,蛋白质糖基化修饰的研究发展缓慢．凝集素芯片技术的出现实现了对糖蛋白的快速、准确、高通量的检测 分析．     

肿瘤与正常细胞表面糖链结构的流式细胞术分析

为了比较正常与肿瘤细胞表面的糖链结构差异,凝集素用荧光素标记后作为研究细胞膜糖链结构的探针,采用流式细胞技术在分子水平上分析。结果显示正常与肿瘤肝细胞与同一浓度的凝集素探针结合量有明显的不同。与凝集素ConA-FITC结合后,正常肝细胞的荧光峰较肿瘤肝细胞的荧光峰明显右移;与凝集素WGA-FITC、PHA-FITC结合后,正常肝细胞的荧光峰较肿瘤肝细胞的荧光峰明显左移。由于凝集素可识别特定糖链结构,该方法说明肿瘤肝细胞的糖链结构与正常肝细胞相比具有特征变化,即平分型糖链和唾液酸含量丰富,可能出现偏二天线以及天线数的增加。     

岩藻糖糖链与肝癌细胞的迁移作用

通过凝集素印迹转移电泳和亲和层析技术,对岩藻糖糖基化蛋白在肝癌细胞中的作用进行了研究.在化学诱发的大鼠肝癌过程中, 分子质量在23 ku到40 ku范围内与荆豆凝集素(UEA)及扁豆凝集素(LCA)结合的岩藻糖糖基化蛋白显著减少, 诱癌至17～20周这些条带重新恢复,而分子质量为80 ku的条带却在诱癌过程中逐周增加.比较高、低转移性肝癌细胞的岩藻糖糖基化蛋白, 发现高转移性肝癌细胞具有多种增强的条带.利用橘果粉胞凝集素(AAL)和LCA亲和层析柱分离了这些岩藻糖基化糖蛋白, 并用这些糖蛋白直接作用于肝癌细胞,发现AAL-糖蛋白具有显著抑制肝癌细胞迁移的作用,迁移细胞数从对照的(100±4.9)%下降到(48.1±2.5)% (P＜0.01), LCA-糖蛋白也有类似作用.用胰酶和木瓜蛋白酶水解蛋白质部分后,形成的糖肽抑制肝癌细胞迁移的作用并不改变,甚至增强.此外直接用肝癌转移灶的组织测定了岩藻糖转移酶活性,发现α1,6岩藻糖基转移酶活性显著比正常肝组织高,而α1,3岩藻糖基转移酶活性没有显著的改变.用系列凝集素分析发现这些糖链主要能结合伴刀豆凝集素A, 也能结合E-型及L-型植物凝集素, 显示这种糖蛋白的糖链可能含有较多的高甘露糖型.这些结果提示糖链在诱癌过程中结构有了改变,使之在肝癌细胞的迁移和转移中起重要作用.     

视黄酸对人肝癌细胞表面N糖链类型及天线数的影响

本文采用系列凝集素柱层析法,并配合外切糖苷酶处理研究了在视黄酸(RA)作用1—5天过程中人肝癌细胞株SMMC-7721细胞表面N糖链结构的变化。结果表明,RA促进3~H-甘露糖(Man)参入细胞表面N糖链,使高甘露糖型N糖链的百分比下降,复杂型百分比上升,并促进二天线N糖链的生物合成,使多天线特别是四天线和C_2,C_(21)b三天线N糖链的合成减少。结果提示,N糖链结构的这些变化可能是RA诱导SMMC-7721细胞向正常方向分化的结果。     

铁过载引发人肝细胞HH4 N-糖链表达差异研究

肝脏铁过载是血液系统疾病患者进行骨髓移植后的典型并发症之一,长期铁过载可引发肝脏细胞凋亡和器官损坏,然而铁过载的分子调控机理迄今仍不清楚。以培养的枸橼酸铁铵过载人肝细胞HH4和正常人肝细胞HH4为研究对象,细胞裂解提取总蛋白,分子筛超滤管分离获得总糖肽,PNGase F酶解释放出N-糖链,Sepharose 4B除盐纯化N-糖链,再利用基质辅助激光解析电离飞行时间质谱(MALDI-TOF/TOF-MS)技术比较N-糖链变化情况。同时,采用荧光细胞凝集素免疫组化方法验证糖链分析结果。结果在铁过载人肝细胞和正常细胞中均检测到16种N-糖链,但糖链丰度存在明显差异。与正常人肝细胞相比,铁过载人肝细胞中高甘露糖型糖链的含量降低,而杂合型、复杂型、平分型、岩藻糖化和唾液酸化糖链的含量明显升高。凝集素免疫组化结果显示铁过载后细胞对凝集素Con A的亲和作用减弱,而对PHA-E、AAL、LCA和MAL-II的亲和作用增强,与糖链质谱分析结果相一致。研究为进一步探索人肝细胞铁过载模式下N-糖链表达差异的分子机理提供了技术支持。     

大鼠诱发肝癌过程中N乙酰氨基葡萄糖转移酶V的高效液相层析测定

提纯人血浆运铁蛋白(Tf),经链霉蛋白酶水解,再经肼解法制备Tf中的二天线N糖链,后者经还原末端的氨基吡啶(PA)化进行荧光标记,再切除外链的唾液酸和半乳糖残基,获得Gn_2Man_3Gn_2-PA荧光标记糖链,以此制备的糖链为受体底物,UDP-GlcNAc为供体底物,用反相HPLC分离底物和产物,建立了N乙耽氨基葡萄糖转移酶V(GnT-V)的测定法。用GnT-V作为肿瘤生化标志物,观察到在二乙基亚硝胺诱发大鼠肝癌的过程中,此酶在诱癌第4周轻度上升,第6周恢复,第10周后持续升高,至18周达正常鼠肝的20倍以上,与病理学上的癌变期相一致。     

喂二乙基亚硝胺大鼠肝染色质体外转录的研究

在二乙基亚硝胺(DENA)诱发大鼠肝癌过程中,无论在E.Coli RNA聚合酶或大鼠肝RNA聚合酶Ⅱ作用下,肝染色质的体外转录活性都比正常大鼠的高,并有随肝脏恶化程度而增高的趋势。进一步研究证明在RNA聚合酶Ⅱ作用下,喂DENA大鼠肝染色质的转录起始点数量比正常肝的多;而在E.Coli RNA聚合酶作用下,两种染色质的转录起始点数量未见明显差异,但喂DENA大鼠的肝染色质转录的RNA链较长。癌变过程中,肝染色质组蛋白及非组蛋白含量都无明显改变,而RNA含量则较正常肝略有增高。     

基于凝集素芯片的不同转移潜能肝癌细胞膜蛋白糖谱比较

评估采用凝集素芯片技术寻找肝癌细胞表面侵袭和转移相关特征性糖谱的适用性．首先选取一对模式细胞株(中华仓鼠卵巢细胞CHO和其N-乙酰葡萄糖胺转移酶Ⅰ缺陷株Lec1)验证凝集素芯片系统的可靠性．然后通过凝集素芯片比较正常肝细胞L02、非转移肝癌细胞Hep3B、高转移肝癌细胞HCCLM3的细胞表面糖谱,同时采用细胞凝集素组织化学的方法验证芯片结果．细胞Hep3B和L02相比,对凝集素PHA-L、ConA、AAL、MPL的亲和作用增强而对凝集素WGA的亲和作用减弱,提示在肝癌细胞表面可能出现了增多的复杂寡糖分支、高甘露糖、末端岩藻糖、黏蛋白T抗原和减少的N-乙酰葡萄糖胺和/或多价唾液酸结构．细胞HCCLM3和Hep3B相比,对凝集素LCA、MAL-Ⅰ、MAL-Ⅱ、WGA、PHA-E的亲和作用增强而对凝集素RCA-I的亲和作用减弱,提示在高转移肝癌细胞HCCLM3的表面可能出现了增多的核心岩藻糖、唾液酸(主要是α2-3链接方式)、N-乙酰葡萄糖胺、平分型GlcNAc结构以及减少的末端β1-4链接半乳糖结构．细胞凝集素组织化学的结果支持芯片结果．研究证明,凝集素芯片技术是解析生物学进程中糖谱改变的适用工具．     

Comparative study of the sugar chains of gamma-glutamyltranspeptidases purified from human hepatocellular carcinoma and from human liver

The sugar chains of gamma-glutamyltranspeptidases, purified from human hepatoma and from normal human liver, were released quantitatively as oligosaccharides by hydrazinolysis. Comparative study of their structures revealed that the following structural alterations are induced by hepatocyte carcinogenesis: 1) high mannose type sugar chains are detected in the hepatoma enzyme but not in the normal liver enzyme; 2) abnormal biantennary sugar chains containing C-2,4 outer chain branches newly appeared; 3) the total amounts of tri- and tetraantennary sugar chains containing C-2,6 outer chain branches increased up to three times.     

A possible mechanism of induction and translocation into blood stream of rat alkaline phosphatase activity by bile duct ligation

We investigated the effects of bile duct ligation on alkaline phosphatase (ALP) activities in liver, calvarium, duodenum, and ileum in rats and its possible mechanism of action. ALP isozyme activities in the ligated rats were significantly elevated in the liver and duodenum, while those in the ileum and calvarium were markedly decreased. The ALP isozyme activity elevated by the ligation was obviously suppressed by prior administration of indomethacin, an inhibitor of prostaglandin synthesis. Moreover, phorbol ester also elevated the ALP activity as well as the phosphatase level in the ligated rat. However, other drugs, such as an inhibitor of protein kinase C and calmodulin, showed different effects: calmodulin stimulated an 11.0-, 1.3-, or 1.5-fold increase in ALP activity in the ileum, duodenum, or calvarium, respectively; whereas the hepatic enzyme activity was not affected. The induction by calmodulin was markedly different from that by the ligation. Moreover, imipramine, an inhibitor of protein kinase C, had little effect. These results suggest that prostaglandin is a possible ALP inducer in ligated rats, probably working by elevating the cAMP level. On the other hand, the ligation induced simultaneously de novo synthesis of the membranous and soluble ALP isozymes; and the release rate of the soluble enzyme was greater than that of the membranous isozymes, indicating that the soluble enzyme might be a main source of the induced serum ALP. Lectin affinity chromatography indicated that the soluble enzyme or induced serum enzyme may contain more fucose than that of the membranous one, suggesting that the sugar moiety in the ALP molecule may relate to the clearance of ALP from or its release into the circulation.     

Localization of the type III isozyme of hexokinase at the nuclear periphery.

The distribution of the type III isozyme of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) in rat kidney, liver, spleen, lung, and brain was determined immunohistochemically, using a monoclonal antibody generated against the enzyme purified from rat Novikoff hepatoma.In all tissues, specific cell types exhibited intense staining at the nuclear periphery, as confirmed by analysis using confocal microscopy. Isolated nuclei from kidney or liver were devoid of detectable type III hexokinase, although the enzyme was found in the "soluble" fraction from kidney or liver homogenates; these results suggest that the type III isozyme is associated in a labile manner with the external surface of the nucleus, with this association being disrupted by conventional homogenization and nuclear isolation procedures. The nuclear localization of the type III isozyme contrasts with previously demonstrated association of the type I and II isozymes with mitochondria. The physiological significance of a nuclear localization for the type III isozyme remains unclear. However, it was noted that many of the cells exhibiting prominent nuclear staining for type III hexokinase are endothelial or epithelial cells, suggesting a possible relationship between nuclear type III hexokinase and transport functions which are prominent in such cells.     

Beta-glycosidases and diabetic microangiopathy. II. An insulin-dependent isozyme of beta-N-acetylglucosaminidase.

Previously we reported that beta-glycosidase activities were markedly decreased in the kidney but increased in the serum of diabetic rats. To examine these changes, the isozymes of beta-N-acetylglucosaminidase [EC 3.2.1.30] of rats were examined by DEAE-cellulose column chromatography. At least 3 major isozymes were found in both the kidney and liver. The main isozyme was type II isozyme in normal rat kidney and type III in normal rat liver. The activity of the type II isozyme in the kidney was markedly lowered when the total activity was decreased in diabetes and its normal activity was restored on insulin treatment, in parallel with increase in the total activity in diabetes. No significant change was found in the chromatographic pattern of isozymes in the liver in diabetes. In diabetic rat serum, the increase of total activity was found to be due to increase of type I and II isozymes.     

Activities of some enzymes concerned with citrate and glucose metabolism in transplanted rat hepatomas

1. Certain enzymes concerned with citrate and glucose metabolism have been measured in two transplanted rat hepatomas, one induced with ethionine (minimal deviation type) and one induced with dimethylaminoazobenzene. In these hepatomas both citrate-cleavage enzyme and NADP-linked isocitrate dehydrogenase in the soluble fraction of the cell were approximately one-third of the values for normal rat liver. These changes have been discussed in relation to the increased citric acid content of tumours and depressed rate of fatty acid synthesis. 2. The glucose-ATP-phosphotransferase activity was below normal liver values in the ethionine-induced tumour but greater than normal in the dimethylaminoazobenzene-induced hepatoma. The apparent K(m) values for the glucose-ATP phosphotransferases of these hepatomas were approx. 8x10(-5)m; no evidence was found for an enzyme with a high K(m) for glucose equivalent to liver glucokinase. 3. Of the enzymes of the pentose phosphate pathway, glucose 6-phosphate-dehydrogenase activity was three to five times as great whereas 6-phosphogluconate-dehydrogenase activity was the same or lower than normal liver in the ethionine-and dimethylaminoazobenzene-induced tumours respectively.     

Alkaline phosphatase expression in human cell lines derived from primary hepatomas

Isozyme patterns of alkaline phosphatase (ALP) were electrophoretically examined in human cell lines derived from one hepatoblastoma, five hepatocellular carcinomas (HCCs) and two cholangiocellular carcinomas. Most of the cell lines tested had a liver-type ALP isozyme. In addition, an abnormal ALP isozyme, which was similar to variant ALP, was detected in one hepatoblastoma and two HCC cell lines. One HCC cell line of these variant-like ALP-positive cell lines was alpha-fetoprotein (AFP)-negative. These findings suggest that variant-like ALP may be useful for the identification of human hepatoma cell lines, especially in AFP or albumin-negative cell lines.     

Lipid composition of the plasma membrane isolated from hyperplastic nodules of rat liver

Hyperplastic nodules and hepatomas were induced in livers of rats fed a diet containing 0.05% N-2-fluorenylacetamide (2-FAA). The lipid contents, and phospholipid and fatty acid compositions were analyzed in plasma membranes (PM's) isolated from these tissues and normal rat liver, and the following trends were observed. The molar ratio of cholesterol to phospholipid-phosphorus (phospholipid-P) increased in the order: hepatoma less than normal liver less than hyperplastic nodules. The molar percentage of plasmalogen to phospholipid-P decreased in the order: hepatoma = hyperplastic nodules greater than normal liver. The percentages of choline phosphoglycerides (sum of phosphatidylcholine and lysophosphatidylcholine) and ethanolamine phosphoglycerides (sum of phosphatidylethanolamine and lysophosphatidylethanolamine) both decreased in the order: hepatoma greater than hyperplastic nodules greater than normal liver. On the other hand, the percentages of sphingomyelin and phosphatidylserine both increased in the order: hepatoma less than hyperplastic nodules less than normal liver. As regards fatty acid composition, the percentages of both 18:1 and 18:2 decreased in the order: hepatoma greater than hyperplastic nodules greater than normal liver. Those of 18:0 and 20:4 increased in the order: hepatoma less than hyperplastic nodules less than normal liver. These results suggested that the lipid bilayer in PM of hyperplastic nodules has characteristics roughly intermediate between those of hepatoma and liver PM's, although the molar ratio of cholesterol to phospholipid-P in hyperplastic nodules PM was not intermediate.     

Enzymatic,biophysical and ultrastructural changes of plasma membranes in chemical-induced rat hepatoma

Plasma membranes from liver of control rats or from chemical-induced hepatoma were prepared. The basal activity of adenylate cyclase was increased significantly in the rat plasma membranes of DEN-induced hepatoma compared to normal tissue. The glucagon-induced response on the cellular effector systems via guanine nucleotide-binding regulatory proteins (G proteins) was inhibited in hepatoma plasma membranes. These findings suggest that in hepatoma membranes, unlike normal hepatic membranes, the response to hormonal stimuli through regulatory G proteins results in a loss of response to glucagon, as well as to GTP plus glucagon or to GTPγS. However, the activating effects of forskolin, which catalyses the formation of cyclic AMP from ATP acting on the catalytic subunit, were to some extent retained. The methyltransferase-I behaved in the opposite direction to the adenylate cyclase, showing a decreased activity in hepatoma plasma membranes compared to control membranes. In contrast, the activity of the ecto-5′-nucleotidase was significantly increased in hepatoma. These enzymatic changes have been found to influence the membrane fluidity and to be responsible for the ultrastructural modifications of hepatoma plasma membranes which are induced by chemical carcinogens.     

High expression of an N-acetylglucosaminyltransferase III in 3'-methyl DAB-induced hepatoma and ascites hepatoma

Ascites hepatoma AH-66 and 3'-Me-DAB-induced hepatoma of rats contain highly active N-acetylglucosaminyltransferase III (GnT-III) which catalyzes the addition of N-acetylglucosamine through a beta 1-4 linkage (bisecting N-acetylglucosamine) to the beta-linked mannose of the trimannosyl core of asparagine-linked sugar chains, whereas normal rat liver contains very little. The high activity was also detected in fetal rat liver, newborn rat liver, hyperplastic nodules and various transplantable hepatomas.