Study Of Variations Of The Amout Of Sialic Acids On Hepatocellular Carcinoma Cell's Membrane Surface

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.