【作者】 龚祖元；

【导师】 廖彩仙；

【作者基本信息】 南方医科大学 ， 外科学， 2011， 硕士

【摘要】 唾液酸(Sialic acid, SA)是带负电荷的9碳糖,是真核细胞膜上糖蛋白、糖脂的重要成分。唾液酸并不是一种单一物质,而是一个大的家族,其的化学本质是神经氨酸(NeuA)或酮基脱氧壬酮糖酸(KDN)的N-或O-酰基衍生物。它在蛋白质或脂质的糖基化过程中最后添加到寡糖链末端。唾液酸与糖结合的方式有3种：①通过α2,3键位与半乳糖(Gal)结合,②通过α2,6键位与半乳糖(Gal)、N-乙酰胺半乳糖(N-GalNAc)或乙酰胺葡萄糖(GlcNAc)结合,③通过α2,8键位与其他唾液酸连接而形成多聚唾液酸。目前已知,细胞表面的分子中普遍存在唾液酸化修饰现象。由于唾液酸位于细胞表面糖链的末端位置并携带负电荷,因而糖链的唾液酸修饰被认为是调控细胞功能的重要因素。对于肿瘤细胞,它在识别、粘附、转移、免疫逃逸等方面的变化,都可能与其膜表面糖链唾液酸化修饰的变化有关。目前认为肝癌细胞表面糖链的唾液酸化修饰异常还只是推测,只是观察到肝细胞癌病人血清总唾液酸比正常人血清总唾液酸高而得出的推论,至今未见直接测定肝癌细胞表面唾液酸修饰状态的报道。肝细胞癌(hepatocellular carcinoma, HCC)细胞的多种生物学特性可能与细胞膜表面分子末端唾液酸化有关,如癌细胞转移潜能的大小、癌细胞分化程度的高低等。2003年Kongtawelert等的发现,肝细胞癌病人血清总唾液酸含量比正常人血清总唾液酸含量高,据此认为肝癌细胞产生的唾液酸增多和／或肝癌细胞表面唾液酸化修饰异常致脱落或分泌入血的唾液酸增多,而肝细胞癌变后细胞膜表面唾液酸化修饰有无异常并未直接测定,并且至今无报道。肝癌细胞表面唾液酸化修饰变化与癌细胞分化程度的关系同样是既不清楚又无报道。本实验的目的是直接测定肝细胞和HCC细胞的膜表面糖链末端a2,3-SA和a2,6-SA的含量差异,直接测定不同分化程度的HCC细胞膜表面糖链末端a2,3-SA和a2,6-SA的含量差异,据此评价肝细胞发生癌变后膜表面糖链唾液酸化修饰变化、评价癌细胞表面糖链唾液酸化修饰变化与癌细胞分化程度的相关关系。本实验根据FITC荧光标记的植物凝集素MAL和SNA可以分别与a2,3-SA、a2,6-SA特异性结合[3]的原理,将肝细胞癌(HCC)细胞和肝硬化组织肝细胞分别放入含荧光标记的朝鲜槐凝集素(FITC-MAL)或黑接榾凝集素(FITC-SNA)的缓冲液中孵育1 h,使其充分与细胞膜表面糖链末端的a2,3-唾液酸(α2,3-SA)或a2,6-唾液酸(α2,6-SA)结合。最后用流式细胞仪检测细胞表面的荧光强度,并据此评定细胞膜表面a2,3或a2,6-唾液酸的含量。结果发现肝硬化组织肝细胞发生癌变后,膜表面糖链α2,3-SA和a2,6-SA的含量显著增加。HCC细胞的分化程度越低,细胞膜表面α2,3-SA和a2,6-SA的含量就越多。[材料和方法]1、标本获取肝癌组织和肝硬化肝组织从南方医院肝胆外科手术病人的切除标本中获取。纳入标准为：(1)乙型病毒性肝炎后肝硬化；(2)病理检查证实为HCC,肝硬化肝组织取自远离癌灶的无癌肝组织。根据HCC细胞病理检查结果,将HCC细胞分为三组：高分化组、中分化组、低分化组。本次实验纳入统计的标本共28例。其中高分化8例、中分化13例、低分化7例。2、实验方法2.1多点穿刺两步灌流法分离肝细胞主要步骤包括：(1)将10 ml注射器抽取38℃前灌流液,多点穿刺肝组织,灌注前灌流液,使肝硬化组织块由暗红色变为灰白色且流出的灌流液变清亮,此过程10～15 min。(2)用10 ml注射器吸取38℃预温的0.05%Ⅳ型胶原酶溶液多点穿刺灌注,直至组织块变疏松、失去弹性,表面呈龟背状,此过程需15～20min。(3)用无菌眼科剪剪去残存的包膜和纤维结缔组织,再粗剪几下肝组织后将其放入无菌瓶中,加入0.05%Ⅳ型胶原酶液10 ml,37.5℃恒温摇动消化10min。(4)用粗口吸管将消化物吹打为细胞悬液,加入10 ml 4℃DMEM培养基,100目不锈钢滤网过滤,收集肝细胞悬液,移至离心管中。(5)4℃,50 g离心5 min,小心吸去上清及大部分上层红细胞,底层沉淀加入红细胞裂解液室温静置3 min后,继续50 g×2 min,离心1次。(6)收集底层沉淀,用含0.005%DNaseⅠ的细胞洗涤缓冲液重悬细胞,再用200目不锈钢网过滤。(7)4℃下,50 g离心1 min,去上清,无血清DMEM培养基重悬细胞,再次50 g离心1 min,弃上清,沉淀的细胞用3 ml 4℃Hanks液重悬后置冰浴中备用。(8)相差显微镜计数细胞密度、活力和纯度。2.2剪切消化法分离HCC细胞主要步骤包括：(1)将癌组织粗剪几下后用DMEM培养基漂洗两次,吸去多余液体,将组织小块剪成约1mm3大小(此过程大约20 min)后加入DMEM培养基漂洗并用100目过滤网过滤,去除那些过于细碎的组织块及细胞团块,留在过滤网上的大小较均一的肝癌组织块收集于15 m1无菌瓶内。(2)在瓶内加入0.05%Ⅳ型胶原酶10 ml后将瓶置于37.5℃恒温摇动消化30 min。(3)用粗口吸管吹打消化后的细胞悬液,加入4℃DMEM培养基10ml,在冰浴条件下100目不锈钢网过滤,收集癌细胞悬液,移至离心管中。(4)4℃,50 g离心5 min,弃上清,底层沉淀加入红细胞裂解液室温静置3 min后,继续50 g离心2 min,1次。(5)收集底层沉淀,用含0.005%DNase I的细胞洗涤缓冲液重悬细胞,再用200目不锈钢滤网过滤。(6)4℃下,50 g离心1 min,弃上清,再用含0.005%DNase I的细胞洗涤缓冲液重悬细胞,再次50 g离心1 min,弃上清,沉淀细胞用3 ml 4℃Hanks液重悬后冰浴中备用。(7)相差显微镜计数细胞密度、活力和纯度。2.3细胞膜表面α2,3-SA、α2,6-SA含量测定本方法的原理：FITC荧光标记的植物凝集素MAL和SNA分别与a2,3-SA、a2,6-SA特异性结合。操作过程：取6个2 ml微量离心管,分成3组：FITC-MAL组、FITC-SNA组、空白组,每组各两管,分别加入106个肝细胞和HCC细胞,离心后去上清。FITC-MAL组每管加入50μl含4% BSA,0.1% NaN3,4μgFITC-MAL的Hanks液的染色缓冲液, FITC-SNA组每管中加入50μl含4% BSA,0.1% NaN3, 1μg FITC-SNA的Hanks液的染色缓冲液,空白组每管中加入50μl含4% BSA,0.1%NaN3的Hanks缓冲液。上述3组细胞4℃避光孵育1 h,用Hanks液洗涤、离心一次后加入1%多聚甲醛固定细胞,使FITC-SNA、FITC-MAL不容易从细胞上脱落。然后行流式细胞检测细胞表面荧光强度,最后在荧光显微镜下观察上述细胞。3、统计学处理所有数据用X±S表示。应用SPSS13.0统计分析软件进行统计学处理,假设检验水准a=0.05。比较两种细胞活力和纯度时采用独立样本t检验,比较HCC细胞与肝细胞膜表面唾液酸含量差别时采用配对样本t检验,比较不同病理分级的HCC细胞膜表面唾液酸含量差别时采用方差分析和LSD检验。[结果]1.多点穿刺两步灌流法分离肝硬化肝组织得到的肝细胞活力为(83.96±2.3)%,纯度为(95.4±1.9)%；剪切消化法分离肝癌组织得到的HCC细胞活力为(88.04±3.3)%,纯度为(94.7±1.4)%。两种细胞活力和纯度均没有显著差异。2.不论是FITC-MAL组还是FITC-SNA组,肝细胞表面的荧光强度和HCC细胞表面的荧光强度都有显著差异,并且都是HCC细胞表面的荧光强度显著高于肝细胞表面的荧光强度。即HCC细胞表面的a2,3-SA和a2,6-SA都显著高于肝细胞表面的含量。3.不论是CaMAL组,还是CaSNA组,HCC细胞表面荧光强度在不同分化程度组别间比较都有显著差异,并且是分化程度越低,细胞表面的荧光强度越强。表明HCC细胞的分化程度越低,其细胞表面的α2,3-SA和a2,6-SA含量越高。[结论]1、多点穿刺两步灌流法适用于从肝硬化肝组织中分离肝细胞,剪切消化法适用于从肝癌组织中分离肝癌细胞。2、不论是α2,3-SA还是α2,6-SA,在HCC细胞膜表面的含量均显著高于肝细胞膜表面的含量,表明乙型肝炎后肝硬化患者的肝细胞发生癌变后,细胞表面的糖链结构发生了变化,即糖链末端的α2,3-SA和α2,6-SA增多。3、HCC细胞的分化程度越低,细胞膜表面的α2,3-SA和α2,6-SA含量越多。表明肝癌细胞表面糖链唾液酸化修饰变化与肝癌细胞的分化程度有关。更多还原

【Abstract】 Sialic acid (SA) is a negatively charged nine carbon sugar,which is the important ingredients of glycoproteins and glycolipids on eukaryotic cells membrane. The chemical nature of SA is the N-or O-acyl derivatives of neuraminidase (NeuA) or 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid(KDN).SA is the last ingredient that added to the end of the oligosaccharide chains in the glycosylation process.A determinate number of principal sialyl-linkages has been described so far in mammalian sialoglycoconjugates:sialic acid may be linked either through an a2,3- or an a2,6-bond to galactose (Gal);or through an a2,6-bond to N-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc); or through an a2,8-bond to another sialic acid, forming polysialic acid. Study shows that SA widely exists in the cells membrane surface molecules.Because SA is located in the end of the sugar chain and it carry a negative charge,so it considered to play an important role of cell’s functions.So as for the tumor cells, the sialylation modification of sugar chains on the membrane surface may affect the tumor cell recognition,adhesion and tumor metastasis,and other processes.Now that the liver cell surface sialic acid modification of sugar chain anomaly is only speculation, but observed that serum total sialic acid in hepatocellular carcinoma patients is more than normal people.Now there is no report about the direct determination the amount of sialic acid on liver cancer cells membrane surface.The sialylation of molecules’s end on the HCC cells surface may connect with a variety of biological characteristics,such as the metastasis ability of HCC cells,the differentiation of HCC cells. The present study showed that after hepatocytes carcinomatous change, cell membrane increased total sialic acid synthesis, and shedding or secreted into the blood, leading to abnormal serum SA content increased. 2003 Kongtawelert, P and other research shows that hepatocellular carcinoma patients with higher serum total sialic acid than normal men.Thus it believed that the total sialic acid on HCC cells surface was increased or/and the total sialic acid which secreted from HCC was increased. But there is no reported that whether both ofα2,3 andα2,6- sialic acids on cell’s membrane surface have significantly increased after hepatocytes carcinomatous change. Meanwhile we do not know wether there is some kinds of relationship between the amount of the two membrane surface sialic acids on the HCC cells and it’s pathological differentiation.The purpose of this study:to directly determine the amount ofα2,3-SA,α2,6-SA on cell’s membrane surface terminal sugar chain on liver cells and HCC cells and then to compare the amount ofα2,3-SA,α2,6-SA on the two kinds of cells. Also we discussed the relationship between the amount of the two membrane surface sialic acids and the pathological differentiation of the HCC cells.This study according to the principle that FITC labeled MAL and SNA lectins specific binding with theα2,3-SA,α2,6-SA respectively. Cells were suspended in staining buffer containing either FITC-MAA or FITC-SNA and incubated for 1 hour. Then flow cytometric analysis was carried out to measure the cells surface mean fluorescence intensity (MFI). In the end we found that both ofα2,3 andα2,6- sialic acids on cell’s membrane surface have significantly increased after hepatocytes carcinomatous change. We also found that the lower the pathological differentiation of HCC cells,the moreα2,3-SA andα2,6-SA were expressed on HCC cells surface.[MATERIALS AND METHODS]1, Obtain specimensHepatocellular carcinoma tissue and cirrhosis liver tissue were obtained from the hepatic carcinoma patients who have tumor resection in the Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University.Inclusion criteria were:(1) Hepatitis B liver cirrhosis; (2) pathological examination confirmed HCC, cirrhosis tissue was non-cancerous liver tissues,which was taken far away from tumor site. The HCC cells were divided into three groups follow the result of HCC pathological detection:well differentiated, moderately differentiated, poorly differentiated. The experiment included 28 cases of specimens of statistics,8 cases of well-differentiated,13 cases of moderately differentiated and 7 cases of poorly differentiated.2, The experimental method2.1 Multi-point puncture two-step perfusion was adopted to separate liver cellsThe main steps include:(1) Use 10 ml syringe draw 38℃preheating perfusion fluid then multi-point puncture and perfuse to the liver tissues until the liver tissues change color from dark red to gray and the outflow of perfusion fluid get clean and bright.This process about 10-15 minutes. (2) Use 10 ml syringe draw 38℃preheating 0.05%Ⅳcollagenase perfusion solution and multi-point puncture until the liver tissues get loose, loss of elasticity,and the surface appears back of Turtle-like, this process takes 15～20 min. (3) Tear the liver tissues into pieces bluntedly with sterile scissors,remove the residual amicula and fibrous connective tissue, and then collect the liver tissues into sterile bottle, and then continue to digest with shake in 0.05%Ⅳcollagenase solution for 10 minutes at 37.5℃. (4) Make the digest into cell suspension with thick-mouth straw,and then add 10 ml 4℃DMEM medium and filter with 100-mesh stainless steel screen,and collect the liver cell suspension with a centrifuge tube on the ice bath. (5) Centrifuge 5min at 4℃with a speed of 50g,remove the supernatant,add some red cell disruption to the sediments,allow it to stand for 3 minutes at room temperature,and then centrifuge at the same speed for 2 minutes. (6) Collect the bottom sediment,re-suspend the cells with liver cell washing buffer solution containing 0.005% DNase I,and then filter with 200-mesh stainless steel screen. (7) Centrifuge lminute with 50g at 4℃,remove the supernatant,re-suspend the cells with serum-free DMEM medium,centrifuge again,discard the supernatant,and re-suspend the sediment cells use 3ml 4℃Hanks solution and put it on the ice bath for use.(8) Ues phase contrast microscope counting cell density, viability and purity.2.2 Cut and digestion was adopted to separate HCC cellsThe main steps include:(1) cancer tissues were rough cut and then rinsed with DMEM twice, and then the cancer tissues were cut into small granules,about 1mm3 size (this process took approximately 20 min).Then the small granules were rinsed using DMEM and filtered with 100-mesh stainless steel screen to remove the tissue that are too small and cell fragment and the cells mass.The granules that left on the screen were collected in 15 ml sterile bottle. (2) Then continue to digest with shake in 0.05%Ⅳcollagenase solution for 30 minutes at 37.5℃. (3) Make the digest into cell suspension with thick-mouth straw,and then add 10 ml 4℃DMEM medium and filter with 100-mesh stainless steel screen,and collect the liver cell suspension with a centrifuge tube on the ice bath. (4) Centrifuge 5min at 4℃with a speed of 50g,remove the supernatant,add some red cell disruption to the sediments,allow it to stand for 3 minutes at room temperature,and then centrifuge at the same speed for 2 minutes. (5) Collect the bottom sediment,re-suspend the cells with liver cell washing buffer solution containing 0.005% DNase I,and then filter with 200-mesh stainless steel screen. (6) Centrifuge 1minute with 50g at 4℃,remove the supernatant,re-suspend the cells with serum-free DMEM medium,centrifuge again,discard the supernatant,and re-suspend the sediment cells use 3ml 4℃Hanks solution and put it on the ice bath for use.(7) Ues phase contrast microscope counting cell density, viability and purity.2.3 To determine the amount of a2,3-SA, a2,6-SA on cells surfaceThe principle of the method:FITC labeled MAL and SNA lectins specific binding with theα2,3-SA,α2,6-SA respectively The process of the operation：6 microcentrifuge tubes(2 ml) were divided into 3 groups:FITC-MAL group, FITC-SNA group, the control group, each group had two microcentrifuge tubes.In each group,one tube were filled with 106 liver cells and another one were filled with 106 HCC cells,and then all tubes were centrifuged and supernatant were removed.In FITC-MAL group,50μl Hanks solution staining buffer which containing 4% BSA, 0.1% NaN3,4μg FITC-MAL were added to each tube.In FITC-SNA group,50μl Hanks solution staining buffer which containing 4% BSA,0.1% NaN3,1μg FITC-SNA were added to each tube.In control group,50μl Hanks solution which containing 4% BSA,0.1% NaN3 were added to each tube. All tubes were incubated for 1 hour at 4℃in the dark,then cells were washed with Hanks solution and centrifuged once,and then add 1% paraformaldehyde to fix cells,so the FITC-SNA, FITC-MAL is not easy to fall off from the cells.Then flow cytometric analysis was carried out to measure the cells surface mean fluorescence intensity (MFI), and finally the cells were observed under fluorescence microscope.3. Statistical analysisAll data show in X±S. Application of SPSS 13.0 statistical analysis software for statistical analysis with hypothesis testing level a= 0.05. Significance between paired simples was determined by Paired t-test. Significance between multiple groups was determined by One-ANOVA, LSD test and in two comparisons was evaluated by the Independend-Samples T Test.[Results]1.The liver cells that are separated by multi-point puncture two-step perfusion have a viability (83.96±2.3)%, purity (95.4±1.9)%; the HCC cells that are separate by cut and digestion have a viability (88.04±3.3)%, purity (94.7±1.4)%. Both cells’viability and purity are not significant difference.2.1n both FITC-MAL group and FITC-SNA group, the two kinds of cells surface mean fluorescence intensity are significant difference, the HCC cells surface mean fluorescence intensity is higher than that of the liver cells. That is the amount of both a2,3-SA and a2,6-SA on HCC cells surface are significantly more than those on liver cell.3.HCC Cells surface mean fluorescence intensity are significantly different among different pathological differentiation in both CaMAL group and CaSNA group. That is the lower the HCC cell’s pathological differentiation is, the stronger the fluorescence intensity is on cell surface.[Conclusion]1. Multi-point puncture two-step perfusion is suitable for separating liver cells from cirrhosis liver tissues, cut and digestion is suitable for separating HCC cells from hepatocellular carcinoma tissues.2. The amount of both a2,3-SA and a2,6-SA on HCC cells surface are significantly more than those on liver cell.This indicating that the cells surface sugar chains changes after the cirrhosis liver cells changes to HCC cells. That isα2,3-SA and a2,6-SA increased in the end of the sugar chain.3. The lower the HCC cell’s pathological differentiation is, the more a2,3-SA and a2,6-SA will be expressed on HCC cells’surface.更多还原