【 Abstract 】 Objective To probe into the role of inositol 1, 4, 5-trisphosphate (IP3) and bax gene expression in apoptosis of HepG2 cells induced by genistein (Gen). Methods HepG2 cells were treated with different concentrations including 20, 40, 60 and 80 μ mol/L Gen as HepG2 cells cultured with 0 μmol/L Gen for 72 h was control; HepG2 cells were treated with 60 μmol/L Gen for 6, 12, 24, 48 and 72 h as HepG2 cells treated with 60 μmol/L Gen for 0 h was control. IP3 content, bax mRNA expression and apoptosis rate were assayed by IP3- ［ 3H ］ Birtrak assay, RT-PCR and flow cytometry, respectively. ResultsHepG2 cells incubated with each concentration of Gen for 72 h ， IP3 content was lower than that of control 〔 (17.7 ± 1.3), (11.2 ± 0.9), (4.9 ± 0.5), (4.8 ± 0.3) pmol/106 cells vs (29.4 ± 0.5) pmol/106 cells 〕 , P ＜ 0.01 ； bax mRNA expression (RI which was the gray degree multiply area of bax/the gray degree multiply area of β -actin) was higher than that of control (0.26 ± 0.02, 0.33 ± 0.05, 0.35 ± 0.06, 0.38 ± 0.05 vs 0.09 ± 0.01), P ＜ 0.01 ； The apoptosis rate was higher than that of control 〔 (10.1 ± 0.9)%, (18.7 ± 1.6)%, (28.7 ± 2.5)%, (27.9 ± 2.0)% vs (2.6 ± 0.1)% 〕 , P ＜ 0.01. HepG2 cells were incubated with 60 μ mol/L Gen for 6, 12, 24, 48 and 72 h ， IP3 content was lower than that of control 〔 (22.6 ± 0.9), (12.0 ± 1.4), (7.5 ± 0.8), (5.6 ± 0.5), (4.3 ± 0.6) pmol/106 cells vs (29.2 ± 0.6) pmol/106 cells 〕 , P ＜ 0.01 ； bax mRNA expression was higher than that of control incubated with 60 μ mol/L Gen for above 12 h (0.25 ± 0.06, 0.29 ± 0.02, 0.30 ± 0.02, 0.35 ± 0.04 vs 0.09 ± 0.01), P ＜ 0.01 ； The apoptosis rate in groups incubated with 60 μ mol/L Gen for 24, 48 and 72 h was significantly higher than that in control 〔 (7.4 ± 0.5)%, (20.5 ± 2.0)%, (30.7 ± 1.6)% vs (2.6 ± 0.1)% 〕 , P ＜ 0.01. ConclusionGen induces apoptosis of HepG2 cells by reducing IP3 production and increasing bax gene expression.
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.
Objective To construct the recombinant of hepatocellular carcinoma-targeting adenovirus containing r-Caspase-3 gene and provide the gene therapic strategy for hepatocellular carcinoma. Methods The pAdTrack-EAFP-PALB was constructed and the r-Caspase-3 gene was subcloned into the vector. The linearized shuttle plasmid was homogenously recombined with AdEasy-1 in BJ5183 cells. The candidate clone was analyzed by restriction endonuclease digestion and sequencing, and then pAdEasy-EAFP-PALB/r-Caspase-3 vector was digested with PacⅠand transfected into AD293 cells for packaging and amplifying, recombinant virus was constructed successfully. Infection titer and efficiency of recombinant virus were monitored by green fluorescent protein (GFP) expression. The expression of r-Caspase-3 in infected HepG2 cells was detected by RT-PCR and Western blot. The apoptosis of HepG2 cells was detected by SRB dyeing method. Results Shuttle vector pAdTrack-EAFP-PALB/r-Caspase-3 was correct after identification by restriction endonuclease analysis and sequencing. By PCR and PacⅠ restriction endonuclease analysis， the homologous recombinant of pAdEasy-EAFP-PALB/r-Caspase-3 was successful. The expression of GFP was observed when linearized pAdEasy-EAFP-PALB/r-Caspase-3 was transfected into AD293 cells. AD293 cells could be infected repeatedly by recombinant adenovirus. The expression of r-Caspase-3 gene on HepG2 cells was detected by RT-PCR and Western-blot methods respectively, which confirmed that the Ad-EAFP-PALB/r-Caspase-3 was constructed successfully. The specificity of Ad-EAFP-PALB/r-caspase-3 which targeting induced hepatocellular carcinoma cells was founded by SRB dyeing test. Conclusion The Recombinant of hepatocellular carcinoma-targeting adenovirus containing r-Caspase-3 gene was constructed successfully and which established the foundation of r-Caspase-3 gene therapy in future research to hepatocellular carcinoma.
【Abstract】ObjectiveTo discuss the possibility and clinical significance of SSX-1 mRNA used as specific marker for examining HCC cells in peripheral blood of HCC patients. MethodsUsing the RT-PCR method, the SSX1 mRNA of the peripheral blood was examined in 25 cases of HCC patients and 20 non-HCC patients. The same method was used to detect the expression of SSX-1 mRNA in the tumor tissues, para-tumor tissues, cirrhosis tissues and normal hepatic tissues. A randomized sample was extracted for DNA sequencing from positive electrophoresis expression samples of SSX-1 to examine the reliability of results. ResultsThe expression rates of SSX-1 mRNA were 60%(15/25) and 40%（10/25） respectively in tumor tissues of HCC and the corresponding peripheral blood. SSX-1 mRNA were not expressed in para-tumor tissues,cirrhosis tissues and normal hepatic tissues. The DNA sequence -confirmed that the RTPCR products were true target cDNA. No relationships were found between the expression of SSX-1 gene and clinical characteristics, such as age, sex, tumor size, TNM stage， extent of differentiation and serum AFP level (Pgt;0.05). However, in 33%（3/9） patients with normal serum AFP (lt;20 μg/L), specific expression of SSX1 mRNA was observed. ConclusionHigh specific expression of SSX-1 mRNA is observed in the peripheral blood of patients with HCC, it suggests that applying it as a tumor marker to detect HCC cells in peripheral blood is an adjuvant diagnostic tool. The expression of SSX-1 mRNA in the peripheral blood is observed in the group HCC patients whose serum AFP (lt;20 μg/L) are normal, which suggests that applying both SSX-1 mRNA and AFP as tumor markers together might be useful to improve the diagnostic accuracy for HCC.
【Abstract】ObjectiveTo study the mechanism of spontaneous rupture of hepatocellular carcinoma (HCC). MethodsArticles have been reviewed to find out the theory of spontaneous rupture of HCC. ResultsResearchful results suggested that the injury of small arteries was usually followed in patients of spontaneous rupture of HCC. In this review, the immune complex, which composed of hepatitis B virus e antigen, complement C1q and immunoglobulins, was found deposited in the elastic membrane of arteries. Likely as a result of immune complex deposition, vascular injury occurs mainly in the small arteries where the deposition of immune complex was present. The small arteries in which immune complex deposited are readily injuried and cause hemorrhage and rupture of HCC during vascular load increase. ConclusionWe would conclude that immune complex deposition in vessel wall led to the small arteries injury may be the factor involved in the pathogenesis of spontaneous ruptured HCC.
ObjectiveTo evaluate the prognosis value of oncoprotein metadherin (MTDH) in alpha fetoprotein (AFP)negative hepatocellular carcinoma (HCC) patients following curative hepatectomy.
MethodsThe expression of MTDH protein in 152 AFP negative HCC patients underwent curative hepatectomy from 2007 to 2010 in this hospital was detected by immunohistochemical stain. Clinicopathologic data for these patients were analyzed. Survival analysis was evaluated with Kaplan-Meier method and log-rank test to compare survival difference. Cox proportional hazard model analysis was used to assess prognostic significance of MTDH in AFP negative HCC patients.
ResultsThe rate of MTDH high expression in the AFP negative HCC tissue was 60.53% (92/152). MTDH high expression was associated with tumor diameter (P=0.029), Edmondson grade (P=0.032), microvascular invasion (P=0.024), or tumor recurrence (P=0.014). Univariate and Cox proportional hazard model analysis showed that high expression of MTDH was correlated with the poor survival in AFP negative HCC patients (P=0.002, P=0.017).
ConclusionMTDH is an independent predictor for survival in AFP negative HCC patients after curative hepatectomy.
ObjectivesTo compare the survival outcomes between hepatocellular carcinoma and hepatic angiosarcoma, and to develop and validate a nomogram predicting the outcome of hepatic angiosarcoma.MethodsThe Surveillance, Epidemiology and End Results (SEER) database was electronically searched to collect the data of hepatic angiosarcoma patients and hepatocellular carcinoma patients from 2004 to 2016. Propensity score matching (PSM) was used to match the two groups by the ratio of 1:3. Cox regression analysis was used to compare the survival outcomes between hepatic angiosarcoma and HCC. In the angiosarcoma group, population was divided into training set and validation set by 6:4. Nomograms were built for the prediction of half- and one- year survival, and validated by concordance index (C-index) and calibration plots.ResultsA total of 210 histologically confirmed hepatic angiosarcoma patients and 630 hepatocellular carcinoma patients were included. The overall survival of HCC was significantly longer than angiosarcoma (3-year survival: 18.4% vs. 6.7%, median survival: 5 months vs. 1 month, P<0.001), and the nomogram achieved good accuracy with an internal C-index of 0.751 and an external C-index of 0.737.ConclusionsThe overall survival of HCC is significantly longer than angiosarcoma. The proposed nomograms can assist to predict survival probability in patients with hepatic angiosarcoma. Due to limitation of the data of included patients, more high-quality studies are required to verify above conclusions.
ObjectiveTo exclusively compare the short-and long-term outcomes of hepatic resection (HR) patients with multifocal tumors meeting the Milan criteria between locating in same and different sections.
MethodsA total of 219 consecutive HR patients with multifocal tumors meeting the Milan criteria were divided into group SS (n=97, same section) and group DS (n=122, different sections) according to their anatomical location (Couinaud's segmentation).
ResultsThe 1-, 3-, and 5-year overall survival (OS) and recurrence-free survival (RFS) rates were significantly higher in the group SS than those in the group DS (P < 0.05). The subgroup analysis showed that patients with 2 tumors and those undergoing en bloc resection were associated with better OS and RFS (P < 0.05).
ConclusionsFor HCC patients with multifocal tumors meeting the Milan criteria, those with tumors locating in same hepatic section may have better longterm survival and lower HCC recurrence rates than those locating in different sections after HR.
ObjectiveTo identify the risk factors of postoperative recurrence and survival for patients with hepatocellular carcinoma within Milan criteria following liver resection.
MethodsData of 267 patients with hepatocellular carcinoma within Milan criteria who received liver resection between 2007 and 2013 in our hospital were retrospectively analyzed.
ResultsAmong the 267 patients, 123 patients suffered from recurrence and 51 patients died. The mean time to recurrence were (16.9±14.5) months (2.7-75.1 months), whereas the mean time to death were (27.5±16.4) months (6.1-75.4 months). The recurrence-free survival rates in 1-, 3-, and 5-year after operation was 76.8%, 56.3%, and 47.6%, respectively; whereas the overall survival rates in 1-, 3-, and 5-year after operation was 96.6%, 82.5%, and 74.5%, respectively. Multivariate analyses suggested the tumor differentiation, microvascular invasion, and multiple tumors were independent risk factors for postoperative recurrence; whereas the tumor differentiation, positive preoperative HBV-DNA load, and preoperative neutrophil-to-lymphocyte ratio adversely influenced the postoperative survival.
ConclusionsFor patients with hepatocellular carcinoma within Milan criteria after liver resection, the tumor differentiation, microvascular invasion, and multiple tumors contribute to postoperative recurrence; whereas the tumor differentiation, positive preoperative HBV-DNA load, and preoperative neutrophil-to-lymphocyte ratio adversely influence the postoperative survival.
Objective To dynamically study the formation of multidrug resistance(MDR) of human hepatocellular carcinoma cell SMMC-7721 induced by Adriamycin (ADM) and the role of multidrug resistance-associated protein(MRP) in its mechanisms.Methods Hepatocellular carcinoma cell SMMC-7721 was cultured in RPMI-1640 medium containing ADM with progressively increased concentration or directly cultured in medium containing different concentrations of ADM. Resistant index of drug-resistant variants of SMMC-7721 cell was determined by drawing cell dosage-reaction curves.Levels of MRP mRNA expression were detected by reverse transcription-polymerase chain reaction(RTPCR). Intracellular rubidomycin(DNR) concentration was examined by flow cytometry(FCM).Results With progressive increasing of ADM concentration in medium resistant index and levels of MRP mRNA expression were correspondingly increased but intracellular DNR concentration was markly reduced. When parental cells were directly cultured in medium containing different concentrations of ADM, the higher the ADM concentration, the higher the level of MRP mRNA expression, but intracellular DNR concentration was kept at the similar high level and most cells died. Conclusion ADM may progressively induce SMMC-7721 cell resistant to multiple chemotherapeutic drugs with reduced intracellular DNR accumulation associated with the overexpression of MRP gene.
