The Study On Transferrin And?1-acidglycoprotein And Their Highly Branched Glycosylation In HBV Related Diseases

Primary liver cancer(PLC)is the fourth most common malignancy and the third leading cause of cancer death in China.PLC includes two major histological types:hepatocellular carcinoma(HCC)and intrahepatic cholangiocarcinoma(ICC).However,HCC accounts for 83.9%~92.3%of the total number of liver cancer in China.The association between hepatitis B virus(HBV)infection and HCC has long been clear.The proportion of liver cancer caused by HBV infection is as high as 70%~80%,and most liver cancer patients in China have background of HBV infection and cirrhosis.Part?:Changes of transferrin in serum of HBV-associated liver diseases and its clinical significanceTransferrin(TRF,TF)regulates the absorption,transport,utilization and storage of iron and maintains iron balance in the body.The effect of liver diseases on TRF level and its clinical significance are of great research value since TRF is mainly secreted by liver cells.The aim of this study was assessing TRF level and its clinical significance in HCC,ICC,liver cirrhosis(LC),chronic hepatitis B(CHB)and healthy control(HC).Serum TRF level,disease characteristics and relevant clinical indicators were analyzed at the same time.The levels of serum TRF(g/L)of HCC,ICC,CHB,LC and HC,detected by Siemens BN?system,were 1.82±0.50,1.83±0.58,2.32±0.70,2.57±0.82,1.40±0.59(mean±SD,g/L)respectively.The levels of serum TRF in the CHB and LC group was significantly higher than the HCC group(p<0.001).The levels of serum TRF in the HC group was significantly lower than the HCC group(p<0.001).The difference between the HCC and ICC group was not statistically significant.Serum TRF level was negatively correlated with the laboratory indexes such as TAB(R=-0.135),DBIL(R=-0.085);and was positive correlated with the laboratory indicators such as ALB(R=0.262),TP(R=0.218),PLT(R=0.240),RBC(R=0.228),HGB(R=0.238).There was no statistically significant correlation between serum TRF level with some laboratory indexes such as A/G,TBIL,ALT,AST,AFP,CA19-9.There was no statistically significant difference of serum TRF level between the grade of HCC and MVI.To sum up,the serum TRF level of HCC group was located between healthy control and benign diseases,and has no significant difference with ICC.Serum TRF level was probably not suitable to separate HCC from healthy control and benign diseases alone.Part?:Structure analysis of serum transferrin glycan based on lectin microarrayGlycosylation is the most common post-translational modification.Approximately 50%~70%of serum proteins are glycosylated.Aberrant glycosylation is associated with cancer including its oncogenesis,progression and metastases.The application of lectin microarray makes it possible to detect the variety and abundance of various glycan structures simultaneously.We used LecChip ~TMM lectin microarray which including 45 lectins to analyze TRF purified from serum of HCC,ICC,LC and HC group.The result suggested that there were 19 lectins has significant differences between four groups(p<0.05).Lectins fluorescence signals of SNA and SSA were significantly higher in cancer group than non-cancer group.However,signals of LTL,AOL,MAL,PHAL,ACG,ECA,RCA120,NPA,ConA,GNA,TJA?,EEL,UDA,Jacalin,HPA and PTL?were significantly higher in non-cancer group than cancer group.Since lectin can recognize specific glycan structure,according to our research,some glycan structures such as Fuc?1-6GlcNAc,sialidase,Gal?1-4GlcNAc,tri/tetra-antennary complex-type N-glycan have significant differences between four groups.Besides,the sialidase was significantly higher in cancer group than non-cancer group,others were significantly higher in non-cancer group.This study suggested that the types of TRF glycan structure were significantly different in different liver diseases and healthy people.Part?:The establishment of lectin enzyme-linked immunosorbent assay for detection of DSA-TRF and its preliminary application in the diagnosis of primary liver cancerTRF is a high-abundant glycoprotein which is synthesized by liver.There are two N-linked glycosylation sites which usually contain two biantennary glycan structures.However,the increase of branches of sugar chain was observed in HCC.This study created a lectin-enzyme linked immunosorbent assay(Lectin-ELISA)to detect the highly branched TRF.Lectin-ELISA was similar to“sandwich”ELISA.TRF antibody was coated on the 96-well plate to catch the TRF in the serum sample.Datura stramonium agglutinin(DSA)was used to detect the TRF with tri/tetra-antennary complex-type N-glycan(DSA-TRF).Mixed serum with serial dilution was used to build concentration-gradient curve.The repeatability and anti-interference capability were evaluated.The results showed that the maximum intra-batch variation coefficient(CV%)was 7.72,and the maximum inter-batch variation coefficient(CV%)was 9.34,The interfering substances have no obvious interference to the system.We detected the 685 serum samples of HCC,ICC,CHB,LC and HC with Lectin-ELISA.Results suggested that DSA-TRF of HCC(0.282±0.06)was significantly higher than disease control(ICC,CHB,LC)(0.200±0.07)and health group(0.238±0.100)(p<0.001).DSA-TRF of cancer group was significantly higher than non-cancer group(0.266±0.072 vs 0.208±0.084,p<0.001).DSA-TRF of HCC patients with negative AFP was significantly higher than non-HCC(0.276±0.067 vs 0.208±0.084,p<0.001).Logistic regression was used to construct the diagnostic model LogitTRF1(=55.711×DSA-TRF/TRF-8.057×DSA-TRF+0.017×AFP-0.077×TBIL+0.357×ALB-16.437)for the differential diagnosis between HCC and high-risk group(LC and CHB)and LogitTRF2(=24.125×DSA-TRF-2.279×TRF-24.346×DSA-TRF/TRF-0.059×TBIL+3.315)for the differential diagnosis between HCC with negative AFP and non-HCC.Receiver operating characteristic curve(ROC)was used to assess the diagnostic value of DSA-TRF and LogitTRF1,LogitTRF2.When identifying HCC from high-risk group,the area under the ROC(AUC)of DSA-TRF reached 0.859(95%CI:0.818~0.900),and the AUC of LogitTRF1 reached 0.981(95%CI:0.971~0.992).When identifying HCC with negative AFP from non-HCC,the AUC of DSA-TRF reached 0.775(95%CI:0.726~0.824),and the AUC of LogitTRF2 reached 20.839(95%CI:0.799~0879).This study suggested that DSA-TRF and diagnostic model LogitTRF1,LogitTRF2 might have prospects in the differential diagnosis of HCC and HCC with negative AFP.Part?:Detection of DSA-AGP by lectin enzyme-linked immunosorbent assay and its preliminary application in the diagnosis of primary liver cancer?1-acidglycoprotein(AGP/AAG)is an acute phase protein synthesized and secreted into plasma by liver cells.It is one of the specific proteins with high sugar content in serum and has 5 N-linked glycosylation sites.In HCC,aberrant fucosylated AGP has been reported,but there is a lack of relevant literature about the change of multi-antennary glycan structure of AGP in HCC.We established enzyme-linked immunosorbent assay(Lectin-ELISA)and detected the AGP with tri/tetra-antennary complex-type N-glycan(DSA-AGP)in HCC,ICC,CHB,LC and HC.Meanwhile we detected levels of matched serum AGP.We found that DSA-AGP of HCC(0.471±0.07)was significantly higher than disease control(0.443±0.09)and health control(0.389±0.10)(p<0.001),AGP of HCC was significantly higher than health group(0.63±0.30 vs 0.45±0.15,g/L,p<0.001).DSA-AGP and AGP in cancer group was significantly higher than non-cancer group(0.462±0.08 vs 0.428±0.10;0.67±0.32vs 0.52±0.28,g/L,p<0.001).DSA-AGP and AGP was significantly higher in HCC with negative AFP than in non-cancer group(0.478±0.09 vs 0.428±0.10,p<0.001;0.60±0.28 vs 0.52±0.29,g/L,p<0.05).Logistic regression was used to construct two diagnostic models combining DSA-AGP,AGP and laboratory indexes(AFP,TBIL,ALB):LogitAGP1(=6.578×DSA-AGP+1.445×AGP+0.013×AFP-3.258),for the differential diagnosis of HCC and non-cancer group,and LogitAGP2(=6.912×DSA-AGP-0.126×TBIL-0.193×ALB+6.417),for the differential diagnosis of HCC with negative AFP and non-cancer group.The diagnostic value of DSA-AGP?AGP?LogitAGP1 and LogitAGP2 was tested by ROC.When identifying HCC from non-HCC,the AUC of LogitAGP1 reached 0.836(95%CI:0.797~0.874),which was significantly higher than DSA-AGP(0.651,95%CI:0.594~0.707)?AGP(0.632,95%CI:0.577~0.687)and AFP(0.803,95%CI:0.748~0.836)respectively.When identifying HCC with negative AFP from non-cancer group,the AUC of LogitAGP2was 0.929(95%CI:0.899~0.959),which was significantly higher than DSA-AGP(0.669,95%CI:0.603~0.735)?AGP(0.607,95%CI:0.536~0.678)respectively.This study suggested that DSA-AGP and diagnostic model LogitAGP1,LogitAGP2 might have prospects in the differential diagnosis of HCC and HCC with negative AFP.