Objective To study the clinical significance of DNA content and expression of nm23-H1, C-erb B-2 and p53 oncoproteins in hepatocellular carcinoma. Methods DNA content was measured after DNA feulgen’s dyeing, the expression of nm23-H1, C-erb B-2 and p53 oncoproteins was detected immunohistochemically. Results The incidence of aneuploid DNA content was 50.0% (12/24) in ≤5 cm group, and was 82.1% (23/28) in ＞5cm group; aneuploid DNA content was related with liver metastasis and cancerous thrombosis. The expression of nm23-H1 in HCC with liver metastasis was higher than that without metastasis. The positive rate of p53 in HCC with canerous thrombosis was higher than that without cancerous thrombosis. The positive rates of nm23-H1 and p53 in HCC with aneuploid DNA content were higher than those with diploid DNA content. The positive rate of C-erb B-2 did not have significantly difference between groups. Postoperative survival rates were possibly related with DNA content as well as expression of nm23-H1, and p53 oncoproteins. Conclusion Aneuploid DNA conten, expression of nm23-H1 and p53 oncoproteins are closely related with the invasiveness of HCC, and have remarkably clinical significance.
Objective To use an improved technique to construct the recombinant adeno-associated virus 2 (rAAV2) mediated gene which can transfer human tissue inhibitor of metalloproteinase-1 (TIMP1). Methods Human TIMP1 gene was amplified from pDNR-LIB plasmid by PCR and cloned into the rAAV2 vector pSNAV to recombinant pSNAV-TIMP1, then was transferred into BHK-21 cells by means of lipofectamine. Using G418 selection, a mixed cell named BHK-21/rAAV2-TIMP1 was isolated, which was capable to express TIMP1. The cell was subsequently infected with recombinant herpes simplex virus 1 (rHSV1-rc/△UL2) that was able to package the rAAV2-TIMP1. After purification, rAAV2-TIMP1 was obtained. Results The rAAV2 carrying human TIMP1 gene was constructed successfully. The viral titer of the rAAV2-TIMP1 was 1×1012 v.g./ml. Conclusion rAAV2-TIMP1 was constructed successfully, which would provide experimental basis for carrying the TIMP1 into hepatocellular carcinoma effectively and inhibiting the invasiveness and migratory capacity of hepatocellular carcinoma in vitro and in vivo models.
Objective To explore the effects of overexpression of human tissue inhibitors of metalloproteinase-1 (hTIMP-1) on proliferation of human liver cancer cell line HepG2 in vitro. Methods A recombinant adenoviral vector containing full-length cDNA of hTIMP-1 was generated and transfected into HepG2. The viral titer was checked by measuring GFP, and the expression of hTIMP-1 in vitro was detected by the techniques of Western blot and semi-quantitative RT-PCR. The ultrastructure was observed by transmission electron microscope and the effects of overexpression of hTIMP-1 on proliferation of HepG2 in vitro was analyzed by MTT assay and growth curve. Results The resultant AdhTIMP-1 was successfully constructed and the expression of hTIMP-1 was detected by Western blot and RT-PCR. The growth and proliferation of HepG2, which had been transfected with AdhTIMP-1, was significantly inhibited. Conclusion The proliferation of HepG2 was markedly inhibited by recombinant adenovirus-mediated overexpression of hTIMP-1, which may pave the way for further application in liver gene therapy.
ObjectiveTo investigate the expression of heat shock proteins 90α(HSP90α) in human hepatocellular carcinoma and the relationship between its expression and biologic behavior of tumor and prognosis. MethodsUsing the immunohistochemical SP method, HSP90α expression was detected in liver tissue from 10 normal individuals, 40 patients with hepatocellular carcinoma(HCC) and adjacent noncancerous liver tissues. ResultsThe positive expression rate of HSP90α was 10.0%,52.5%,72.5% in normal liver tissues,adjacent noncancerous liver tissues,hepatocellular carcinous tissues respectively. A significantly higher distribution of HSP90α positive expression in HCC tissues compared with adjacent noncancerous liver tissues and normal liver tissues was obtained （P＜0.05）. The positive expression of HSP90α in HCC was correlated with clinical stage, tumor differentiation, serosal condition and lymph node metastasis （P＜0.05）, but not to tumor number （P＞0.05）. It was also correlated with prognosis of HCC. The mean tumorfree survival of patients with HSP90α negative expression was 38.6 months while that of HSP90α positive expression was 25.5 months （P＜0.05）. ConclusionHSP90α is overexpressed in human hepatocellular carcinoma. HSP90α could be used as an indicator to judge the clinical stage, tumor differentiation, serosal condition, lymph node metastasis and prognosis of HCC.
ObjectiveTo investigate the predicting effect of PIK3CA mutations for the efficacy and prognosis of hepatocellular carcinoma (HCC) patients received surgical resection. MethodsPCR and DNA sequencing were used to detect the PIK3CA mutation status of 79 HCC tissues, its impact on the short and long term effects of the patients were analyzed. ResultsIn this group of patients, mutation rate of PIK3CA gene exon 9 was 39.24% (31/79), PIK3CA mutation rate correlated with lymph node status and tumor differentiation (P < 0.05). The therapeutic effect of patients with PIK3CA mutation was significantly poor than that of the non-mutated group (P < 0.05). The three-year cumulative survival of patients with PIK3CA mutation (33.33%) was significantly lower than non-mutated group's (60.00%) by Kaplan-Meier (P < 0.05). ConclusionPIK3CA gene mutation in exon 9 could impact the efficiency of surgical resection in patients with HCC and could predict a poor survival prognosis.
【Abstract】Objective To construct a recombinant adenoviral vector carrying antisense matrix metalloproteinase2 (MMP2) for use in the gene therapy to inhibit the invasiveness and migratory capacity of hepatocellular carcinoma (HCC) cell line HepG2 in vitro and in vivo models. Methods Total RNA was extracted from HCC, and then a 500 bp fragment at the 5′ end of human MMP2 cDNA was synthesized by polymerase chain reaction (PCR) and was reversely inserted into the multiclone site (MCS) of the shuttle plasmid pAdTrack-CMV,with the resultant plasmid and the backbone plasmid pAdEasy-1,the homologous recombination took place in the E.coli BJ5183 and the recombinant adenoviral plasmid carrying the antisense MMP2 gene was constructed and generated. The adenoviruses(Ad-MMP2AS) were packaged and amplified in the HEK 293 cells.Then the viral titer was checked by GFP. Results The recombinant adenovirus vector carrying antisense MMP2 was constructed successfully, the b green fluorescence was observed in HEK 293 cells under a fluorescence microscopy. The viral titer was 1×108/ml. Conclusion The recombinant adenovirus Ad-MMP2AS constructed by us could introduce the antisense MMP2 into HepG2 effectively,which would provide experimental basis for reversing the overexpression of MMP2 in HCC and for inhibiting the invasiveness and migratory capacity of HepG2 in vitro and in vivo models.
ObjectiveTo investigate the expressions and clinical significance of human telomerase reverse transcriptase (hTERT) mRNA and γglutamyl transpeptidase mRNA-H (GGT mRNA-H) in the peripheral blood of small hepatocellular carcinoma (HCC) patients. MethodsThe expressions of hTERT mRNA and GGT mRNA-H were detected in the peripheral blood of thirty patients with small HCC by RT-PCR, eighteen patients with benign liver diseases, and twelve normal volunteers. ResultsThe positive rate of hTERT mRNA and GGT mRNA-H expression in patients with small HCC were 80.0% (24/30) and 46.7%(14/30), respectively. In patients with hepatitic cirrhosis the positive rate of hTERT mRNA expression was 33.3% (6/18), while the expression of GGT mRNA was not detected. Both the expressions of hTERT mRNA and GGT mRNA-H were negative in all normal volunteers. The combination analysis of hTERT mRNA and GGT mRNA-H expression achieved positive rate of 86.7% in the diagnosis of small HCC, which was significantly higher than the positive rate of AFP (26.7%)， Plt;0.05. ConclusionThe hTERT mRNA and GGT mRNA-H are significantly expressed in small HCC patients, the combination analysis of hTERT mRNA and GGT mRNA-H seems to be useful in the early diagnosis of small HCC.
ObjectiveTo study the mechanism, diagnosis and treatment of spontaneous rupture of hepatocellular carcinoma (HCC) for the purpose of screening out the effective methods of diagnosis and treatment. MethodsArticles have been reviewed to find out the difference among many kinds of diagnosis and treatment. ResultsThe mechanism of spontaneous rupture was not clear. Possible factors included tumor necrosis, coagulopathy, cirrhosis and portal hypertension. Diagnosis of spontaneous rupture of HCC can be made by abdominal paracentesis, ultrasonography and CT. Judging from the reported results the first choice of emergency treatment for hemostasis was transcatheter arterial embolization (TAE). The rational treatment of patients with ruptured HCC was TAE followed by hepatectomy if the lesion was resectable. ConclusionThe prognosis of patients with ruptured HCC could be improved if the correct diagnosis and treatment can be adopted.
Objective To study the prokaryotic expression of the anti-tumor associated glycoprotein-72 (TAG-72) singlechain fragment variable (scFv) antibody and its specific affinity to hepatocellular carcinoma cell lines and tissues. Methods The cDNA of anti-TAG-72 scFv antibody was inserted into pCANTAB5E to obtain phage vector anti-TAG-72 -scFv-pCANTAB5E. Isopropyl-β-D-thiogalactoside (IPTG) was used to induce the expression of anti-TAG-72 scFv antibody. SDS-PAGE and Western blot were used to identify the anti-TAG-72 scFv antibody. Human hepatocellular carcinoma cells were cultured, and TAG-72 was determined with the obtained scFv by immunohistochemistry in the cells and paraffin-embedded hepatocellular carcinoma tissues. Results SDS-PAGE and Western blot showed that the anti-TAG-72-scFv antibody was successfully expressed. Anti-TAG-72 scFv could bind to hepatocellular carcinoma cell lines SMMC7721, HepG2, and HHCC, but not to BEL7402, suggesting that SMMC7721, HepG2, and HHCC cells expressed TAG-72. For the 40 cases of hepatocellular carcinoma tissues, the positive rate of TAG-72 in stage Ⅰ and Ⅱ-Ⅲ was 23.08% (3/13) and 62.96%(17/27), respectively. While no TAG-72 expression was found in the 10 normal cases. TAG-72 expression was significantly different between hepatocellular carcinomas and normal tissues (Plt;0.05). Conclusions The prokaryotic expression of anti-TAG-72 scFv antibody is successfully achieved, and can be used to identify TAG-72 antigen. TAG-72 is highly expressed in hepatocellular carcinoma, but not in normal liver tissue, which may be suggested as cancer marker of hepatocellular carcinoma.
Objective To investigate the expressions of CD90, IGF1R, and hTERT protein in hepatocellular carcinoma, and the correlations of each other in the development of carcinoma. Methods The expressions of CD90, IGF1R, and hTERT protein in hepatocellular carcinoma were detected by S-P immunohistochemical staining, 20 cases of normal liver tissues were collected as contrast, and to compare the relations between expression and prognosis or survival rate. Results The positive rate of CD90, IGF1R, and hTERT protein in hepatocellular carcinoma group were obviously higher than that in contrast group（P＜0.05）, which was 63.9% vs. 0, 52.8% vs.5.0%, and 47.2% vs.0, respectively. The positive rate of CD90, IGF1R, and hTERT protein were higher in UICC Ⅲ-Ⅳ stage group than that in UICC stage Ⅰ-Ⅱ group（P＜0.05）, which was 79.2% vs.33.3%, 70.8% vs.16.7%, and 62.5% vs.16.7%, respectively. There was a statistically significant positive correlation observed between the expressions of CD90 and IGF1R protein （Kendall’s tau-b=0.563 1, P＜0.05）, so it was with CD90 and hTERT protein （Kendall’s tau-b=0.363 6, P＜0.05）. The survival rates of positive expressions of CD90, IGF1R, and hTERT protein were lower than negative expressions of CD90, IGF1R, and hTERT（P＜0.05）, which was 21.7% vs.50.0%, 17.6% vs.43.8%, and 20.0% vs.38.9%, respectively. Conclusions The expressions of CD90, IGF1R, and hTERT may have correlations with the progress of HCC, and may serve as a marker for HCC prognosis potentially.