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Objective To assess the efficacy and safety of morinidazole combined with appendectomy in treating purulent or gangrenous appendicitis.Methods Double-blind randomized controlled multicenter clinical trial was designed and conducted.Totally 437 patients were included,219 in the control group and 218 in the experimental group.Cases of purulent or gangrenous appendicitis were enrolled and assigned to each of the two groups.The control group received ornidazole injection for 5 to 7 days while the experimental group received morinidazole injection.Both groups underwent appendectomy.Clinical response,micrombiological outcomes,overall response were evaluated.Adverse events and side effects were recorded.Results No significant difference was observed between the two groups regarding the clinical healing rate at 5-10 days after medicine withdrawal,anaerobia clearance and overall healing rates.Adverse events occurred in 140 patients (32.1％).Incidence of adverse events in the control group and the experimental group was 34.7％ and 29.4％,respectively (P ＞ 0.05).The overall incidence of side effects was 15.1％ (66 cases).Side effects were less seen in the experimental group compared with that in the control group (11.5％ vs.18.7％,P ＜ 0.05).The most frequent side effects were aminotransferase rising,thrombocytosis,nausea,vomiting and electrocardiographic abnormality.Conclusions The effect of morinidazole plus operation was comparable with ornidazole in treating purulent or gangrenous appendicitis.The safety of morinidazole is better than ornidazole.
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This study investigated the "homing" phenomenon in hepatocellular carcinoma (HCC). The "homing" specificity of endothelial progenitor cells (EPC) by establishing an orthotopic implantation model in nude mice. EPCs harvested from the marrow cells were separated by density gradient centrifugation. Fluorescence microscope, flow cytometry (FCM) and double fluorescence staining with FITC-UEA-I and DiI-ac-LDL, were employed to identify the cells. 4',6-diamidino-2-phenylindole (DAPI) labelling and real-time PCR were used for detecting the expression of CD133 and chemokines to trace and observe the distribution of EPCs. Our results showed that the distribution rate of EPCs was obviously higher than that in other important organs and the negative control group. Detection of CD133 and chemokines yielded similar results in difference tissues. Our experiment confirmed that the chemotaxis of EPCs does exist in HCC. Moreover, HIF-1α, SDF-1 and VEGF might play important roles in the "homing" of EPCs in HCC. EPCs might be a potential candidate for targeting vector of HCC for gene therapy.
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This study investigated the "homing" phenomenon in hepatocellular carcinoma (HCC). The "homing" specificity of endothelial progenitor cells (EPC) by establishing an orthotopic implantation model in nude mice. EPCs harvested from the marrow cells were separated by density gradient centrifugation. Fluorescence microscope, flow cytometry (FCM) and double fluorescence staining with FITC-UEA-I and DiI-ac-LDL, were employed to identify the cells. 4',6-diamidino-2-phenylindole (DAPI) labelling and real-time PCR were used for detecting the expression of CD133 and chemokines to trace and observe the distribution of EPCs. Our results showed that the distribution rate of EPCs was obviously higher than that in other important organs and the negative control group. Detection of CD133 and chemokines yielded similar results in difference tissues. Our experiment confirmed that the chemotaxis of EPCs does exist in HCC. Moreover, HIF-1α, SDF-1 and VEGF might play important roles in the "homing" of EPCs in HCC. EPCs might be a potential candidate for targeting vector of HCC for gene therapy.
Subject(s)
Animals , Mice , Endothelial Cells , Pathology , Liver Neoplasms , Pathology , Mice, Inbred C57BL , Mice, Nude , Stem Cells , PathologyABSTRACT
AIM: To compare the methods of two currently employed isolation methods for endothelial progenitor cells (EPCs): from total peripheral blood mononuclear cells (PBMCs) and from enriched CD133~+ cells, by defining the cell morphology, phenotype, reproductive activities and function in vitro, providing a reference for clinic application. METHODS: PBMCs from the healthy subjects were used for CD133~+ sorting or not. The two groups of isolated cells were suspended in complete medium M199 for 7 d to 14 d. EPCs phenotype were characterized by FACS. The proliferation of differentiated EPCs was studied by MTT assay, and VEGF concentration was measured using an ELISA kit. Matrigel experiment and migration assay were imitated vascularization in vivo. RESULTS: PBMCs produced more colony-forming units (CFU) than CD133~+ cells from the same volume of blood (P<0.01). From 7 d to 14 d, the two groups show decreased expression of hematopoietic stem cell markers and increased level of endothelial markers, but CD144~+ cells in CD133~+ group were lower than those in PBMCs groups (P<0.01). Cells in PBMCs group secreted more VEGF than that in CD133~+ group on 7 d (P<0.01). Compared to CD133~+ group, PBMCs group showed more potential of proliferation and vascularization in vitro. CONCLUSION: CD133~+ sorted cells show a lower capacity of differentiation, secretion, proliferation and vascularization in vitro, which is unable to differentiate to mature endothelial cells, indicating that it's not a preferential way to obtain EPCs for clinic therapy.
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Objective To explore the mechanism of melanoma differentiation associated gene-7(mda-7) in combination with adriamycin(ADM) killing the HCC HepG2 cells and reversing their multidrug resistance (MDR). Methods The experiment was conducted in three groups including the combined group, ADM group and mda-7 group. MTT assay and FCM were used to determine the differences among the 3 groups and clarify the reversing effect of combined treatment on multidrug resistance of the tumor cells. Expression levels of MDR-1, STAT-3, BCL-2, BAXmRNA were determined with real-time PCR. Western blotting was performed to observe the changes of proteins gp-l70, stat3,P-stat3, PKB, bcl-2,bax in all 3 groups. Result After transfection with 100VP/cell Ad. mda-7,the growth suppression rate of HepG2 treated by ADM (1.5 mg/L) rose from 17.46% to 79. 5%.According to the changes, killed HepG2 cells were increased by a factor of 4.55. times. MDR-1 mRNA was decreased from (16.49 ± 0. 11) to (5.48±0.05) and STAT-3 mRNA increased from (13.17±0. 08) to (21. 57±0. 11)(P＜0.05). Western blotting also showed that P-170 and PKB was decreased and the phosphorylation-stat-3 increased after the combined treatment. Conclusion Ad.mda-7 can reverse the multidrug resistance HepG2 cells. It inhibits the expression of MDR-1 mRNA,then arrests PKB protein and the signaling pathway of active stat-3 to induce apoptosis of HCC cells.
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Objective To investigate the plasma level of SDF-1a in patients with breast cancer after modified radical mastectomy and chemotherapy, and to evaluate the value of serum SDF-1a half-life for predicting breast cancer recurrence and metastasis. The correlation of SDF-1 a half-life and breast cancer recurrence and metastasis after treatment were retrospectively analyzed. Methods Serum chemokine SDF-1a of 112 cases of breast cancer were detected before and after modified radical surgery, and 1 day beforeeach chemotherapy session. SDF-1a levels and the dynamic changes in the process were observed and calculated. Results In 85 cases with no recurrence and metastasis the plasma level of SDF-1 a decreased rapidly to normal level ,while that in 27 cases with recurrence and metastasis was on high level with the halflife of SDF-1a being longer than that in no recurrence group(P ＜0. 01 ). Taking SDF-1a half-life ≥14 d as cut off point to predict breast cancer recurrence and metastasis after treatment, the sensitivity is 81.5%,specificity is 70. 6 %, and accuracy is 73.2%. Conclusions Serum SDF-1a half-life is a valuable marker in predicting postoperative breast cancer recurrence and metastasis.
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Two isolation methods for sorting of endothelial progenitor cells (EPCs): from peripheral blood mononuclear cells (PBMCs) and CD133(+) enriched cells were compared, by defining the cell morphology, phenotype, reproductive activities and function in vitro, to provide a reference for clinical application of EPCs. PBMCs from healthy subjects were used either directly for cell culture or for CD133(+) sorting. The two groups of cells were cultured in complete medium 199 (M199) for 7 to 14 days and the phenotypes of EPCs were analyzed by FACS. The proliferation of differentiated EPCs was studied by MTT assay, and the VEGF concentration was measured using an ELISA kit. ECM gel experiment and migration assay were performed in vivo. The results showed that PBMCs produced more colony-forming units (CFU) than CD133(+) enriched cells from the same volume of blood (P<0.01). From day 7 to 14, the two groups showed decreased expression of hematopoietic stem cell markers and increased level of endothelial markers, but CD144(+) cells in CD133(+) group were less than in PBMCs group (P<0.01). PBMCs group secreted more VEGF than CD133(+) group on the day 7 (P<0.01). As compared with CD133(+) group, PBMCs group had more potent potential of proliferation and vascularization in vitro. It was concluded that CD133(+) sorted cells showed a lower capacity of differentiation, secretion, proliferation and vascularization in vitro, suggesting that CD133-negative cells may be a preferential way to get EPCs for clinical therapy.
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In order to investigate the relationship between the VEGF level and the counts of dendritic cells (DCs) in peripheral blood of patients with hepatocellular carcinoma (HCC) before and after transcatheter arterial chemoembolization (TACE), the peripheral blood was obtained from 37 patients with HCC who treated by TACE. The blood was obtained on the day before TACE, the first day, the 7th day and the 15th day after TACE respectively. The counts of DCs were quantified by flow cytometry. The plasma VEGF level was measured by ELESA kit. It was shown after TACE, the counts of DCs in peripheral blood were decreased significantly (P＜0.05), and the VEGF level in peripheral blood was increased significantly (P＜0.05). The counts of DCs in peripheral blood had an inverse correlation with the plasma VEGF level (r=-0.57, P＜0.05) after TACE. It was concluded that in patients with HCC after TACE, the increased plasma VEGF level appeared to have the effect to suppress the maturation of DCs, which may contribute to reduction of the body's anti-tumor immunity effect, with a consequence of recur and metastasis of tumor.
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In order to investigate the relationship between the VEGF level and the counts of dendritic cells (DCs) in peripheral blood of patients with hepatocellular carcinoma (HCC) before and after transcatheter arterial chemoembolization (TACE), the peripheral blood was obtained from 37 patients with HCC who treated by TACE. The blood was obtained on the day before TACE, the first day, the 7th day and the 15th day after TACE respectively. The counts of DCs were quantified by flow cytometry. The plasma VEGF level was measured by ELESA kit. It was shown after TACE, the counts of DCs in peripheral blood were decreased significantly (P<0.05), and the VEGF level in peripheral blood was increased significantly (P<0.05). The counts of DCs in peripheral blood had an inverse correlation with the plasma VEGF level (r=-0.57, P<0.05) after TACE. It was concluded that in patients with HCC after TACE, the increased plasma VEGF level appeared to have the effect to suppress the maturation of DCs, which may contribute to reduction of the body's anti-tumor immunity effect, with a consequence of recur and metastasis of tumor.
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To investigate the expressions and significance of the tumor suppressor gene phosphatase and tensin homlog deleted on chromosome ten protein (PTEN) and vascular endothelial growth factor (VEGF) in hepatocellular carcinoma (HCC), and to analyze the relationship between their expressions and the tumor's invasion and their pericarcinomatous tissues, the correlation of their expressions with the tumor's clinicopathological characteristics and invasion potential were studied. Our study showed that the expression level of PTEN in HCC was remarkably lower than that in pericarcinomatous liver tissues, while the expressions of both VEGF and MVD were higher than that in pericarcinomatous liver tissues. Correlation analysis revealed that the expression of PTEN was negatively related to the progression of the pathological differentiation and invasion of tumor, whereas the expressions of VEGF and MVD were positively related. Moreover, there was a negative relationship between the expression of PTEN and the expressions of VEGF and MVD, and a positive one between VEGF and MVD. The expressions of PTEN and VEGF may reveal the degree of differentiation and the invasive potential of HCC tissues. The mechanism by which the lack of PTEN expression probably induces abnormal hyperexpression of VEGF may play an important role in the invasion and metastasis of HCC.
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To investigate the expressions and significance of the tumor suppressor gene phosphatase and tensin homlog deleted on chromosome ten protein (PTEN) and vascular endothelial growth factor (VEGF) in hepatocellular carcinoma (HCC), and to analyze the relationship between their expressions and the tumor's invasion and their pericarcinomatous tissues, the correlation of their expressions with the tumor's clinicopathological characteristics and invasion potential were studied. Our study showed that the expression level of PTEN in HCC was remarkably lower than that in pericarcinomatous liver tissues, while the expressions of both VEGF and MVD were higher than that in pericarcinomatous liver tissues. Correlation analysis revealed that the expression of PTEN was negatively related to the progression of the pathological differentiation and invasion of tumor, whereas the expressions of VEGF and MVD were positively related. Moreover, there was a negative relationship between the expression of PTEN and the expressions of VEGF and MVD, and a positive one between VEGF and MVD. The expressions of PTEN and VEGF may reveal the degree of differentiation and the invasive potential of HCC tissues. The mechanism by which the lack of PTEN expression probably induces abnormal hyperexpression of VEGF may play an important role in the invasion and metastasis of HCC.
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Summary: In order to investigate the effect of small ubiquitin-like modifier-1 (SUMO-1) on the p53-induced HepG2 cell apoptosis, HepG2 cells were transfected by recombinant plasmids as pwtp53, pMDM2 and pSUMO-1 respectively. Western blot was employed to detect the protein expression of the transfected recombinant plasmids and the rate of apoptosis was measured by flow cytometry. The results showed that in cells transfected with pwtp53 and pwtp53+pSUMO-1, the apoptosis rate was (16.79±1.62) % and (18.15±1.36) % respectively, while transfected with pwtp53+pMDM2, the rate was decreased to (5.17±1.23) %. The apoptosis rate was (14.06±1.84) % in the cells transfected with pwtp53+pMDM2+pSUMO-1, significantly higher than that in the cells Transfected with pwtp53+pMDM2 (P<0.01). The apoptosis rates in the cells were all less than 2 % and had no significant difference among the groups. It was suggested that in the HepG2 cells, SUMO-1 can increase the apoptosis induced by wild-type p53 through binding to p53 protein, post-translational modification and inhibiting the p53 degradation by MDM2.
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Summary: To investigate the difference in expression of hTERT gene between HbsAg-positive human hepatocellular carcinoma (HCC) and HbsAg-negative HCC and to explore the relationship between HBV infection and hTERT gene expression in HCC. The expression of hTERT protein in 30 cases of HbsAg positive HCC and 17 cases of HbsAg negative HCC was detected by immunohistochemistry (SP method), and the expression of hTERT mRNA was analyzed by reverse transcription polymerase chain reaction (RT-PCR). t-test, Chi-squared test and cochran- armitage trend test were used to see whether there was an interrelation between HBsAg and hTERT gene in HCC. The expression of hTERT protein was mostly located in plasm and occasionally in the nucleus of liver cancer cells. The positive rate of hTERT protein and hTERT mRNA in HbsAg positive HCC- 93.33 % (28/30) and 83.33 % (25/30) respectively which were much higher than those in HbsAg negative HCC- 52.94 % (9/17), 47.06 % (8/17) (P<0.01) respectively. HbsAg is related to hTERT gene expression in human hepatocellular carcinoma. The hTERT gene activated by the efficacious ingredient of HBV may play an important role in hepatocellular transformation and carcinogenesis.
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In order to evaluate the effects and mechanisms of celecoxib in inhibiting proliferation and inducing apoptosis on human pancreatic carcinoma cells, the anti-proliferative effect was measured by using methabenzthiazuron (MTT) assay. Cell cycle and apoptosis were analyzed by using flow cytometry (FCM), and the PGE2 levels in the supernatant of cultured pancreatic carcinoma cells were quantitated by enzyme-linked immunoabsordent assay (ELISA). Our results showed that celecoxib suppressed the production of PGE2 and inhibited the growth of JF-305 cells, and the anti-proliferative effect of celecoxib could be abolished by addition of PGE2. FCM revealed that celecoxib could inhibit proliferation and induce apoptosis by G1-S cell cycle arrest. It was concluded that cyclooxygenase-2 specific inhibitor celecoxib could inhibit proliferation and induced apoptosis of human pancreatic carcinoma cells via suppression of PGE2 production in vitro.
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The clinical significance of phosphatase and tensin homolog deleted on chromosome ten (PTEN) protein expression and the correlation between the expression of PTEN and phosphorylation of protein kinase B (PKB/AKT) in human hepatocellular carcinoma (HCC) were investigated.The expression of PTEN and phospho-AKT was detected by SP immunohistochemical technique and Western blotting in 35 cases of HCC, 15 cases of liver cirrhosis and 8 cases of normal tissues. The correlation between the expression of PTEN and PKB/AKT in HCC was analyzed. The results showed that the positive expression of PTEN in HCC (62.9 %, 0. 085±0. 021) was significantly lower than that in liver cirrhosis and normal tissues (P＜0.01). The expression level of PTEN was related to the differentiation degree of HCC and the status of metastasis (P＜0. 05). Western blotting revealed a significant inverse correlation between PTEN and phospho-AKT (r=-0. 818, P＜0.01). These results demonstrated that down-regulation or loss of PTEN, which may not be able to effectively inhibit the hyper-phosphorylation of PKB/AKT, might play an important role in tumorigenesis and progression of HCC.
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Objective To investigate the clinical significance of the expression of fragile histidine triad (FHIT) protein in primary hepatocellular carcinoma (HCC).Methods Immunohistochemical (S-P) method was used to detect the expression of FHIT in the 46 patients with HCC and 10 normal controls.Results In the patients with HCC,the expression rate of FHIT in the tumor tissue was 56.52%,significantly lowerthan that in adjacent non-tumor tissue and normal tissue.The absence of FHIT protein was correlated neither with the size,tumor capsule,serum AFP level nor with chronic hepatitis B virus infection and cirrhosis of liver.There was significant relationship between the expression of FHIT and differentiation and thrombus in the portal vein.Conclusion The loss of FHIT gene protein is a frequent event in HCC.Furthermore,the loss is closoly related to tumor prognosis prognosis and FHIT loss.
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To clone the murine alpha-fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1-6 cells, and then the murine alpha-fetoprotein gene was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8 kb murine alpha-fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.
Subject(s)
Cricetinae , CHO Cells , Cloning, Molecular , DNA, Complementary , Eukaryotic Cells/metabolism , Genetic Vectors , Reverse Transcriptase Polymerase Chain Reaction , Transfection , alpha-Fetoproteins/biosynthesis , alpha-Fetoproteins/geneticsABSTRACT
In order to investigate the changes of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) expression in residual hepatocellular carcinoma (HCC) after transcatheter arterial chemoembolization (TACE), the expression levels of VEGF and bFGF expression in specimens surgically removed from 48 HCC patients were detected by immunohistochemical methods, and staining intensity of VEGF and bFGF was assessed by a computer-assisted image-analyzer. Among the 48 patients, 25 underwent partial hepatectomy alone (single operating group), and 23 were subjected to second stage surgical resection after TACE (TACE group). The results showed that the average absorbance value (A) of VEGF was higher in TACE group than that in single operating group (0.152 +/- 0.021 vs 0.131 +/- 0.012, P < 0.01). The Average A of bF-GF in TACE group was 0.127 +/- 0.023, higher than in single operating group (0.111 +/- 0.016, P < 0.05). These results suggested that TACE of HCC can up-regulate the expression of VEGF and bFGF in HCC tissues possibly due to anoxia and ischemia.
Subject(s)
Aged , Female , Humans , Middle Aged , Antineoplastic Combined Chemotherapy Protocols , Carcinoma, Hepatocellular , Chemistry , Pathology , Therapeutics , Catheterization, Peripheral , Chemoembolization, Therapeutic , Cycloleucine , Fibroblast Growth Factor 2 , Fluorouracil , Liver Neoplasms , Chemistry , Pathology , Therapeutics , Mitomycin , Vascular Endothelial Growth Factor AABSTRACT
To clone the murine alpha-fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1-6 cells, and then the murine alpha-fetoprotein gene was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8 kb murine alpha-fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.
Subject(s)
Animals , Cricetinae , Humans , Mice , CHO Cells , Cloning, Molecular , DNA, Complementary , Eukaryotic Cells , Metabolism , Genetic Vectors , Reverse Transcriptase Polymerase Chain Reaction , Transfection , alpha-Fetoproteins , GeneticsABSTRACT
Objective To investigate the change of vascular endothelial growth factor (VEGF) expression in HepG2 cells under hypoxia. Methods HepG2 cells were cultured under hypoxia(hypoxia group) and normal condition (control group). VEGF expression of HepG2 cells was examined by immunohistochemical staining. The growth of HepG2 cells was examined by MTT colorimetry and cell count. VEGF level in the culture medium was measured by ELISA.Results After 48 h and 72 h of culture, the growth rate of HepG2 cells in hypoxia group was lower than that in control group (P