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Objective To construct a mammalian vector encoding angiostatin kringle 5 (K5) under the control of ?-fetoprotein (AFP) enhancer and albumin promoter, and to observe the expression of angiostatin by introducting angiostatin gene into hepatocellular carcinoma cells through gene transfection. Methods Angiostatin cDNA was amplified from normal human eukaryotic cells by using RT-PCR. Meanwhile, AFP enhancer and albumin promoter sequences were directed cloned and were inserted into vector pcDNA3.1. The recombinant vector of pcDNA3.1-AFAB-angiostatin K5-His was constructed, which contained the angiostatin K5 cDNA sequence that was under the control of the AFP enhancer and promoter. Angiostatin K5 cDNA was introduced into human AFP positive hepatocellular carcinoma cell lines with the transfected cultured cells that were mediated with Lipofectamine 2000. The expression of angiostatin K5 was analyzed by Western blot and the protein was dectected with anti-His antibody. Results The 500-base pair of angiostatin K5 was in accordance with the expected sequence and the recombinant vector of pcDNA3.1-AFAB-angiostatin K5-His was also confirmed as the anticipated sequence. The expression of angiostatin K5 in AFP positive hepatocellular carcinoma cells was detected both by SDS-PAGE and Western blot. Conclusion Efficient construction and expression of angiostatin K5 to AFP positive cells make it possible for antiangiogenesis therapy of human hepatocellular carcinomas, which may provide a promising approach.
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@#ObjectiveTo study the protective effects of tumor necrosis factor-α(TNF-α) antibody on pancreatic encephalopathy in rats. MethodsSixty SD rats were randomly divided into the normal control group, acute necrotizing pancreatitis induction group and TNF-α antibody treated group. Acute hemorrhage necrotizing pancreatitis model in rats were induced by retrograde injection of 5% sodium taurocholate into the pancreatobiliary duct. Serum TNF-α was detected and animals were killed 12 h after drug administration. The changes of brain water contents, leucocyte accumulation and adhesion were measured, and pathological studies of pancreas and brain were performed. ResultsIn group of TNF-α antibody treated, serum TNF-α level decreased, brain water contents and leucocytes accumulation and adhension decreased significantly than that of acute necrotizing pancreatitis induction group (P<0.05). The histopathological change of pancrea was alleviative. ConclusionTNF-α antibody can alleviate the brain damage in acute hemorrhage necrotizing pancreatitis.
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Objective To investigate the expression of tumor associated glycoprotein-72 (TAG-72) in human breast cancer cells. Methods Two breast cancer cell lines MCF-7 and Bcap-37 were cultured and prepared. TAG-72 expressions in MCF-7 and Bcap-37 were detected immunochemically with anti-TAG-72 single chain variable fragment (scFv). Results TAG-72 was positively expressed in MCF-7 cells but negatively expressed in Bcap-37 cells. Conclusion Tumor associated antigen TAG-72 is expressed in certain human breast cancer cells, which indicate TAG-72 may be used as a tumor maker and anti-TAG-72 scFv may play a role in the diagnosis and treatment of breast cancer.
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Objective To study the role of down regulation of bcl 2 expression of human cholangiocarcinoma cells with bcl 2 mRNA cleaving ribozyme (RZ). Methods Synthetic gene of bcl 2 mRNA cleaving RZ was recombined with the expression vector of eukaryotic cells. The recombined vector was used to transfect human cholangiocarcinoma cells. The expression of bcl 2 in human cholangiocarcinoma cells was observed by immunocytochemistry and flow cytometry. Results The expression of bcl 2 in human cholangiocarcinoma cells transfected with RZ gene was lower than that of control group. Spontaneous apoptosis was observed in a few of the cells. Conclusion bcl 2 mRNA cleaving RZ can down regulate the expression of bcl 2 of cholangiocarcinoma cells notably and can also induce spontaneous apoptosis.
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Objective To construct the inducible vector carrying green fluorescence protein(GFP). Methods We constructed an inducible vector pMD-GFP, including GFP cDNA, which allowed controlled expression of protein upon addition of 100 ?mol/L Zinc as an external inducer. The whole length of GFP cDNA were transfected into human hepatocellular cancer cells HCC-9204 by the method of lipofectin transfection. The expression of GFP was observed under fluorescence microscopy. Results The inducible vector carrying green fluorescence protein was successfully constructed. The observation under fluorescence microscopy showed that green fluorescence was spread in entire HCC-9204 cells transfected with GFP gene. Conclusion This new kind of the inducible vector could serve as a new tool and method for observing the growth and metastasis of neoplasm.
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To elucidate the effect of p27 KIP1 on cell cycle and proliferation of gastric carcinoma cells, we transfected the full e length cDNA of p27 KIP1 into human SGC7910 gastric cancer cells by the method of lipofectin transfection. Expression of p27 KIP1 at protein or mRNA level was analyzed by Western blotting, and RNA dot blotting, respectively. Effect of p27 KIP1 on cell growth was observed by trpan blue exclusion assay and anchorage independent growth in soft agar. Flow cytometry was applied to assess the effect of p27 KIP1 on cell cycle. The results showed that the expression of p27 KIP1 at protein or mRNA level increased evidently in SGC7901 cells transfected with p27 KIP1 . The cell growth was reduced by 42% 48h post induction with zinc as determined by cell viability assay. The rate of anchorage independent growth in soft agar decreased significantly. p27 KIP1 over expression caused cell arrest at G 1 by 36%(from 33 68% to 69 29%, P
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Objective To investigate the relationship between canceration and primary operation mode for congenital choledochal cysts(CCC). Methods The clinical data of 21 patients with CCC treated in the last 30 years were analysed retrospectively. Results In this series, the incidence of carcinoma was 14.8%; the canceration rate after internal drainage operation was significantly higher than that after resection of the cyst(P<0.001); the age of canceration after internal drainage was younger than that of resection of the cyst(P<0.01) and patients without operation(P<0.01); the interval time of canceration after internal drainage operation was significantly less than that of resection of the cyst(P<0.01); the age of carcinoma would be 15.4 years younger in internal drainage operation patients than that in patients without operation. Conclusions Internal drainage, which could accelerate the occurrence of canceration of CCC, should be abandoned; resection of the cyst is recommended as the therapy of the first choice.
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0.05). ConclusionsAdriamycin can induce apoptosis of cancer cells, and this is an important mechanism for its anticancer effect. This effect may be related to the down regulation of Bel-2 (expression).
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Objective To investigate the effect of pancreatic transplantion(PTA) using diverse operative (methods) in rats. Method Inbred SD rats were used as donor and recipient, and were randomly assigned (into) 4 groups:Normal control group( Group NC), consisted of 10 rats;diabetes group(Group DC) consisted of 10 rats;PTA with enteric drainage group(Group E-D), consisted of 22 diabetic rats;PTA using bladder reconstruction group(group B-D), consisted of 22 rats.Arterial supply of transplanted pancreas was (performed) by using end-to-side anastomosis of the donors' abdominal aorta(with splenic artery) and (recipients)′ abdominal aorta; and venous drainage was performed by using end-to-end anastomosis of the (donors)' portal vein segment(with splenic vein) and (recipients)′ left renal vein(cuff method). The fasting blood glucose, body weight, food intake, water intake and urine volume of the recipient were monitored before transplantation and on the 1st, 3rd, 7th, 14th, 30th day after operation, and also the reasons of failures were analyzed. Results The operative time of recipient in E-D group was significantly longer than that in B-D group(P
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Objective In this study,we determine whether Survivin antisense oligonucleotide (ASODN) down-regulates the expression of Survivin mRNA,induces apoptosis,and enhances the sensitivity of pancreatic cancer cells to a chemotherapy agent Gemcitabine. Methods Lipofectin was used to encapsulate ASODN in transfection. Viability of pancreatic cell line BxPC-3 cells treated with ASODN was assessed by MTT method,the expressions of Survivin mRNA were detected by RT-PCR;Flow cytometry and electron microscopy were used to demonstrate apoptotic changes in ASODN treated cells. Result Cell viability was inhibited in dose-dependent and time-dependent manner with an IC 50 of 400 nM at the time of 24 h,Survivin mRNA was down-regulated by 3.38 fold compared to control group. The cell apoptotic ratio was 24.93%?2.97%,early apoptotic morphology was demonstrated by electron microscopy. In combination with Gemcitabine,at the time of 48 h and 72 h,cell viability decreased by 2.76 and 4.58 fold compared to when Gemcitabine was used alone. Conclusion Survivin ASODN down-regulates Survivin mRNA ,induces apoptosis,inhibits proliferation and enhances the sensitivity of pancreatic cancer cells to Gemcitabine.
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Objective To study the protective effects of ulinastatin and tumor necrosis factor-?(TNF-?) antibody on ischemia and reperfusion injury of liver in rats. Methods One hundred and twenty male SD rats were randomly divided into four groups: the normal control group, ischemia and reperfusion group, TNF-? antibody group and ulinastatin plus TNF-? antibody group. And the animals were killed after 60 minutes ischemia of liver followed by reperfusion for 1,3,6 and 12 hours. Serum alanine aminotransferase(ALT) and malondialdehyde(MDA) were detected, and liver histopathologic lesions were observed. Results After ischemia and reperfusion, the serum level of ALT and MDA remarkedly increased, and the hepatic congestion was prominent. Treatment of ulinastatin and TNF-? antibody could decrease the serum level of ALT and MDA significantly, and relieve hepatic congestion. Conclusions Ulinastatin and TNF-? antibody can suppress the inflammatory reaction induced by hepatic ischemia and reperfusion, and has protective effects on rat hepatic ischemia and reperfusion injury.
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Objective To investigate the expression of MUC1 in primary liver carcinoma (PLC) and in cirrhotic liver tissue and its clinical significance in the diagnosis and immuotherapy of PLC. Methods The expression of MUC1 was examined by immunohistochemical analysis in 43 samples of primary liver carcinoma (PLC) , incluing 34 samples of hepatocellular carcinoma (HCC), 9 samples of cholangiocarcinoma (CC) ;and 12 samples of cirrhotic liver tissue and 6 samples of normal liver tissues. Results Immunohistochemical analysis showed over- expression of MUC1 in PLC , which aberrantly localized in the cancer cell membrane; while expression of MUC1 was detected only in 2 cirrhotic liver samples,and no expression in normal liver tissue. The expressional level of MUC1 in PLC was significantly higher than that in cirrhotic and normal liver tissue ( P 0.05),but the expressional levels between with portal cancer emboli group and without portal cancer embloli group was significant difference( P
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ObjectiveTo investigate the expression of MUC1 in thyroid cancer and benign thyroid diseases (nodular goiter and adenoma) and its clinical significance. Methods The expression level of MUC1 was examined by immunohistochemical analysis with MUC1 monoclonal antibody in 68 samples of thyroid carcinoma and in 18 samples of benign diseases and 10 normal thyroid tissues. Results Immunohistochemical analysis showed positive expression of MUC1 in 51 out of 68 cases of thyroid cancer, in 4 out of 18 cases of benign thyroid diseases and 1 out of 10 normal control thyroid tissue, with a difference statistically significant between the malignancy and benign and normal control (? 2=17.20,P0.05).Conclusion The over-expression of MUC1 in thyroid cancer may act as diagnostic markers of thyroid carcinoma.
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ObjectiveTo investigate the effect of p27 KIP1 transfection on cell cycle and apoptosis of hepatocellular carcinoma cells(HCC). MethodsWe used an inducible expression system pMD neo, which allowed controlled expression of protein upon addition of zinc as an external inducer. p27 KIP1 cDNA was transfected into human HCC 9204 cell line. Expression of p27 KIP1 was analyzed and cell growth was observed. ResultsExpression of p27 KIP1 in protein and mRNA increased significantly in HCC 9204 cell line transfected with p27 KIP1 . The cell growth reduced by 35%, p27 KIP1 over expression caused cell growth arrest at G 1 by 35% ( P =0 000). Apoptotic cell index significantly increased ( P =0 000).Conclusionp27 KIP1 may cause cell cycle arrest in G 1 phase and subsequently lead to apoptosis.
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Objective To explore the clinical manifestations, pathogenic mechanisms and treatment of Mirizzi syndrome.Method 65 patients with Mirizzi syndrome were analysed retrospectively.Results According to Csendes typing, patients with Mirizzi syndrome included type Ⅰ in 18 patients(27 6%), type Ⅱ in 25 patients (38 4%), type Ⅲ in 13 patients(20 0%), type Ⅳin 9 patients(13 8%), all patients were healed by corresponding operation and perioperative management.Conclusions Good outcome of Mirizzi syndrome patients can be achieved by accurate diagnosis,exact operation and active perioperative management.
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Purpose: To explore the autocrinism of vascular endothelial growth factor ( VEGF) in human hepatocellular carcinoma cell and its significance. Methods: The expression of VEGF receptors was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) in human hepatocellular carcinoma cell lines SMMC7721, HHCC and HepG2 in vitro. The expression of VEGF in human hepatocellular carcinoma cells was inhibited by synthetic antisense oligodeoxynucleotide (ODN). Phenotypic alterations in human hepatocellular carcinoma cells by antisense reduction of VEGF expression were observed. And the alterations for expression of VEGF receptors in human hepatocellular carcinoma cell membranes were analyzed by flow cytometry. Results: Flt-1 was expressed faintly in SMMC-7721 cell whereas KDR was expressed in HHCC cell and HepG2 cell in vitro. Compared to control, antisense ODN against VEGF inhibited HHCC cell proliferation in vitro in a dose-dependent manner and had no effects on SMMC-7721 cell and L929 cell. When the concentration of antisense ODN was 2.5 ?mol/L,5 ?mol/L and 10 ?mol/L, the proliferation of HHCC cell was inhibited by 26%, 41 % , and 50% respectively. Compared with control, flow cytometry analysis showed a decrease in cell number in S phase and a pre-apoptosis peak in HHCC cell treated by antisense ODN. There was no alteration of cell cycle in SMMC-7721 cell. The positive rate of KDR expression in HHCC cell membrane was 19. 2%, 29. 8% and 31. 2% in the antisense ODN, sense ODN and control groups, respectively. The expression of KDR in HHCC cell membrane was markedly inhibited by antisense ODN against VEGF compared with the control group, as measured by flow cytometry. There was no alteration for expression of Flt-1 in SMMC-7721 cell membrane. Conclusions: VEGF produced by human hepatocellular carcinoma cells acts directly upon human hepatocellular carcinoma in an autocrinal manner and may promote the proliferation and differentiation of human hepatocellular carcinoma cells via the activation of the KDR-dependent signaling pathway.
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Aim To investigate the expression of vascular endothelial growth factor(VEGF) and its receptors in human hepatocellular carcinoma cell lines SMMC7721, HHCC and HepG2. Methods Immunohistochemical staining and reverse-transcriptase polymerase chain reaction(RT-PCR) were used to detect the mRNA and expression of VEGF and its receptors: VEGF-R1(Flt-1) and VEGF-R2 (KDR) in three human hepatocellular carcinoma cell lines, SMMC7721, HHCC and HepG2, as compared with ECV304 cells(human umbilical vein endothelial cells) and L929 cells(mouse fibroblast). Results All three human hepatocellular carcinoma cell lines expressed VEGF protein. Flt-1 mRNA and protein could be detected in SMMC7721 cells while KDR in HHCC and HepG2 cells. Conclusion The expressions of Flt-1 and KDR suggests that VEGF may be an autocrine growth factor for human hepatocellular carcinoma, at least for cell lines in vitro.
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Aim To study relationship between expressions of apoptosis-related gene bax and bcl-x in heopatocellular carcinoma (HCC) tissues and its clinical feature. Mothods The expression of Bax and Bcl-x were determined by immunohistochemistry with EnvisionTM system. Results Bcl-x and Bax proteins were detectable in 20 and 19 out of 40 HCCs, respectively. Their expressions in HCC cells were lower than those in cirrhosis(P＜ 0.05), The expression of Bax and Bcl-x in highly or intermediatly differentiated HCC cells were highed than that in low-diffe-rehtiated HCC cells suggesting that there was significant relationship among bcl-x and bax expressions and differential degree， clinical stages of HCC cells， and AFP level. However, the expression of this proteins showed no relationship with patient's sex and age(＞ 60 or ＜ 60 years old) (＞ 0.05). Conclusion Apoptosis-related genes bcl-x and bax participate partially in apoptotic regulation of HCC cells, and show that their expressions have correlation with differential degree, clinical stage of HCC cells and AFP levels.
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Aim To investigate the effect of intrauterine injection of rat fetal hepatocytes on homograft rejection reaction. Methods The skin graft from a male LOU/CN rat was transplanted to a female recipient CHN rat of 7 to 9 weeks after parturition, and then survival time of the graft was observed. Simultaneously, homograft rejection reaction was examined by mixture lymphocyte culture. Results As compared with control group, survival time of transplanted skin graft was obviously prolonged. Mixure lymphocyte culture demonstrated that homograft rejection reaction was inhibited markedly. Conclusion Intrauterine injection of rat fetal hepatocytes wuld obviously inhibite homograft rejection reaction, thus prolonging survival time of the graft.