[General surgery].
Explore the source record for details and available documents.
Biomedical Research↗
Biomedical subjects
Xisheng Leng
Publications and source records attributed to Xisheng Leng.
[Construction, packaging and titration of recombinant adeno-associated virus vectors contaning antitumor genes].
OBJECTIVE: To construct, package, and titrate vectors that express antitumor genes using adeno-associated virus (AAV) containing human alpha-fetoprotein (AFP) promoter. METHODS: Recombinant AAV vectors were constructed by blunted ligation of plasmids rAAV-AFP-GFP and pTR-UF5 and p16 cDNA and p21 cDNA. Packaging cell lines, 293 and C12 cells, were transfected with rAAV vectors, plasmid PspRC72, and adenovirus cosmid pAdc with the help of LipofectAMINE and then infected by wild adenovirus 48 hours later. The total viral particle titre was measured by PCR. Electron microscopy was used to observe the morphology of rAAV. C12 cells were infected with rAAV and wild adenovirus (MOI 10) and the titre of infectious virus was measured. by RCA method. Hepatocellular carcinoma cells were cultured and infected with rAAV containing green fluorescent protein (GFP), the infectious rate was observed 48 hours later. RESULTS: Recombinant vectors, rAAV-AFP-p16, rAAV-AFP-p21, rAAV-CMV-p16, and rAAV-CMV-p21, were constructed and verified by DNA sequencing and enzyme digestion. The total viral titre was 10(13) to 10(14)/ml. The titre of infectious rAAV containing GFP was 5 x 10(10)/ml. Electron microscopy showed that the rAVV was round and plaqued with a diameter between 20 and 30 nm. When the MOI was 100, the infectious rate of hepatocellular carcinoma cells was at least 70%. Four rAAV vectors carrying p16 or p21 genes have been constructed. rAAV packaging and titration methods were developed to produce highly-purified AAV stocks. These approaches help in research of gene therapy in liver cancer.
Cell Line↗
[Interleukin-10 inhibits the activation of cultured rat hepatic stellate cells induced by Kupffer cells].
OBJECTIVE: To investigate the effect of interleukin-10 (IL-10) on the activation of cultured rat hepatic stellate cells (HSC) induced by Kupffer cells (KC) and relevant mechanisms. METHODS: HSC and KC were isolated and purified from rat liver by collagenase IV perfusion and density gradient centrifugation with Nycodenz. After primary culture for 2 days, HSC and KC were divided randomly into three groups: HSC group, KC group and HSC + KC group (coculture group), then were stimulated by IL-10 of concentrations of 2 ng/ml or 20 ng/ml respectively. After 2 days, the proliferation of HSC were determined with (3)H-TdR incorporating test, the expression of alpha-smooth muscle actin (alpha-SMA) in HSC was detected by Western blotting, and tumor necrosis factor-alpha (TNF-alpha) protein concentration in the supernatant was determined by ELISA. RESULTS: IL-10 showed no significant effect on HSC proliferation (P > 0.05) and alpha-SMA expression (P > 0.05) in HSC group. The levels of proliferation and alpha-SMA expression of HSC in coculture group were 2.4 times (P < 0.001) and 6 times (P < 0.001) higher respectively than that in HSC groups. In coculture group, HSC proliferation and alpha-SMA expression were decreased by 23% (P < 0.001) and 35% (P < 0.05) respectively after stimulation of 2 ng/ml IL-10, and were decreased by 33% (P < 0.001) and 49% (P < 0.05) respectively after stimulation of 20 ng/ml IL-10 in a dose dependent way. TNF-alpha was not detected in HSC group. The level of TNF-alpha in HSC + KC coculture group was 74% higher than that in KC group (P < 0.01). 2 ng/ml and 20 ng/ml IL-10 reduced the TNF-alpha level by 27% (P < 0.01) and 36% (P < 0.01) respectively in coculture group, and reduced the TNF-alpha level by 29% (P < 0.05) and 42% (P < 0.01) respectively in KC group in a dose dependent way. CONCLUSION: IL-10 reduces the level of TNF-alpha secreted by KC dose-dependently. Through reducing cytokine production by KC, IL-10 inhibits the activation of cultured rat HSC induced by KC, which may play a protective role against liver fibrosis.
Animals↗
[Expression of mitogen-activated protein kinase and its upstream regulated signal in human hepatocellular carcinoma].
OBJECTIVE: To detect protein expression of ERK(1), ERK(2), JNK(1), p38 and MEK(1), MEK(2) in human hepatocellular carcinoma and adjacent non-neoplastic liver. METHODS: In 16 surgically resected hepatocellular carcinoma and para-carcinoma tissues, Western blotting was used to detect expression of ERK(1), ERK(2), JNK(1), p38 and MEK(1), MEK(2). RESULTS: In all cases, ERK(1), ERK(2), p38 expression in hepatocellular carcinoma was significantly higher than that in para-carcinoma: integral optic density (IOD) of ERK(1) was 300 +/- 98 in carcinoma and 98 +/- 48 in para-carcinoma tissues (t = 2.519, P < 0.01); IOD of ERK(2) was 587 +/- 83 in carcinoma and 232 +/- 96 in para-carcinoma tissues (t = 2.745, P < 0.01); IOD of p38 was 270 +/- 85 in carcinoma and 107 +/- 88 in para-carcinoma tissues (t = 2.491, P < 0.01). JNK(1) expression in hepatocellular carcinoma was significantly lower than that in para-carcinoma; IOD of JNK(1) was 111 +/- 93 in carcinoma and 292 +/- 109 in para-carcinoma tissues (t = 2.473, P < 0.01). Protein levels of MEK(1) and MEK(2) in carcinoma were significantly higher than in para-carcinoma. IOD of MEK(1) was 1 418 +/- 244 in carcinoma and 806 +/- 90 in para-carcinoma tissues (t = 2.546, P < 0.01). IOD of MEK(2) was 1 041 +/- 122 in carcinoma and 468 +/- 40 in para-carcinoma tissues (t = 2.861, P < 0.01). CONCLUSIONS: ERK(1), ERK(2), MEK(1) and MEK(2) in the signal transduction pathway for cell proliferation are significantly overexpressed and the expression of JNK(1) is lower in hepatocellular carcinoma. Their unbalance is one of the important reasons for the over growth and infinite proliferation of the hepatocellular carcinoma cell. The p38 and JNK(1) may be activated by different pathway.
Adult↗
[Expression and clinical significance of MAGE-4 gene in human hepatocellular carcinoma].
OBJECTIVE: To explore the possibility of MAGE-4 gene encoding protein used as a target for immunotherapy in HCC patients. METHODS: The expression of MAGE-4 gene in tumor tissues and tumor adjacent non-HCC liver tissues was examined by the RT-PCR method. The relationship between positive expression rate of MAGE-4 gene and other clinical and lab data including AFP, AFU, anti-HCV, HBsAg, AFP mRNA, and the diameter of the tumors in HCC patients was also determined. RESULTS: The positive expression rate of MAGE-4 gene was significantly higher in the tumor than in tumor surrounding tissues (38.7% vs 0%, P<0.01), while the positive expression rate of MAGE-4 gene had no relationship with the clinical and lab data (P>0.05). CONCLUSIONS: The high frequency of MAGE-4 gene expression in HCC suggests the possibility of MAGE-4 gene encoding protein as a target for immunotherapy in HCC patients, but the expression has no relationship with the tumor metastasis and the recurrence of HCC.
Antigens, Neoplasm↗
[Long-term results of preoperative regional intraarterial chemotherapy against colorectal cancer].
OBJECTIVES: To summarize the clinical experience of preoperative intraarterial chemotherapy (PRAC) and evaluate the long-term results of multimodality against colorectal cancer. METHODS: Seldinger procedure was used to intubate the tube to the artery branch which supplied blood to the tumor. The tumor was imaged to make sure the diagnosis and irrigate the chemotherapeutic drugs. Ten days after PRAC, the patients received radical operation and 6 chemotherapeutic courses with FCF regimen. Concurrent patients receiving surgical treatment yet no PRAC therapy were chosen as controls. RESULTS: One-year survival rate was 93.05% in the PRAC group and 80.78% in the controls (P = 0.023). COX multivariate analysis was used to analyse the prognostic factors. Dukes'staging and the PRAC prescription or not were found to be independent prognostic factors of colorectal cancer patients. Patients in the PRAC group survived longer than those in the control group. CONCLUSION: PRAC can improve the survival of colorectal cancer patients.
Antineoplastic Agents↗
Induction of cytotoxic T lymphocytes from the peripheral blood of a hepatocellular carcinoma patient using melanoma antigen-1 (MAGE-1) peptide.
OBJECTIVE: To investigate the possibility of using melanoma antigen-1 (MAGE-1) peptide as a tumor vaccine to treat hepatocellular carcinoma (HCC). METHODS: The expressions of MAGE-1 in 8 HCC cell lines and in liver cancer tissue from a patient were detected using RT-PCR. The type of human leucocyte antigen I(HLA I) of both 8 HCC cell lines and peripheral blood mononuclear cells of the patient was detected using a microcytotoxicity method to screen out target cell lines for the cytotoxicity assay. Peripheral blood mononuclear cells from the HCC patient pulsed with an MAGE-1 peptide (NYKCRFPEI) were used as antigen presenting cells. Autogenous peripheral blood mononuclear cells were stimulated with antigen presenting cells every 7 days for 4 times to elicit cytotoxic T lymphocytes. The phenotype of effector cells was analyzed using flow cytometry. The cytotoxicity of effector cells was detected with a lactate dehydrogenase releasing assay. RESULTS: The expressions of both MAGE-1 and HLA-A24 were detected in BEL7405 cell line which were used as the positive target cell line in the cytotoxicity assay. The expression of MAGE-1 alone was detected in HLE, BEL7402, BEL7404, QGY7703 and SMMC7721 cell lines, and the expression of neither MAGE-1 nor HLA-A24 was shown in QGY 7701 and HpG2 cell lines. The last 7 cell lines could be used as negative target cell lines in the cytotoxicity assay. Peripheral blood mononuclear cells expanded 32 folds during 28-day culture. The ratio of CD3(+) T cells increased by 16% (from 54% to 70%), and the ratio of CD8(+) T cells increased by 20% (from 36% to 56%) during 28-day culture. When the ratio of effector cells to target cells was 10:1, effector cells exhibited 62.5% cytotoxicity against autogenous lymphoblasts pulsed with the peptide (NYKCRFPEI) of MAGE-1 antigen, 40.25% cytotoxicity against BEL7405 cells, compared with 17.88% cytolysis observed against autogenous lymphoblasts, 19.55% against HLE cells, and 1.6% against QGY7701 cells. When the ratio of effector cells to target cells was 3.3:1, the cytotoxicity of effector cells against the peptide pulsed autogenous lymphoblasts was 53.6%, which was much higher against autogenous lymphoblasts, HLE cells and QGY7701 cells at 15.6%, 13% and 1%, respectively. CONCLUSION: The results demonstrate that cytotoxic T lymphocytes with the ability to specifically lyse target cells expressing both MAGE-1 and HLA-A24 could be successfully induced by the MAGE-1 peptide NYKCRFPEI in vitro. This indicates that a good result might be anticipated if this peptide is used as a tumor vaccine to treat HLA-A24 HCC patients.
Adult↗
mRNA of MAGE genes as specific markers in detection of tumor cells in the peripheral blood of patients with hepatocellular carcinoma.
OBJECTIVE: To detect tumor cells in the peripheral blood of patients with hepatocellular carcinoma (HCC) by using the mRNA of the MAGE-1 and MAGE-3 genes as specific tumor markers. METHODS: Peripheral blood was obtained from 25 HCC patients and 20 healthy volunteers. The mRNA of the MAGE-1 and MAGE-3 genes in the peripheral blood mononuclear cells (PBMCs) was detected by nested RT-PCR. The MAGE-1 and MAGE-3 transcripts in the tumor tissues of these HCC patients were also detected by RT-PCR. RESULTS: Of the 25 HCC patients, MAGE-1 and MAGE-3 mRNA were positive in 44% (11/25) and 36% (9/25) of PBMCs respectively, and in 68% (17/25) and 56% (14/25) of HCC tissues respectively. In the PBMCs of the 25 HCC patients, 16 (64%) samples were detected to express at least one type of MAGE mRNA. MAGE mRNA were not detected in the PBMCs from the patients whose tumors did not express the MAGE genes, nor in the PBMCs from the 20 healthy donors. The positive rate of MAGE mRNA in the PBMCs was closely correlated with the TNM stages and the diameter of tumors, but there was no correlation between the positive rate of MAGE mRNA in PBMCs and tumor differentiation degree or serum alpha-FP level. Of 9 HCC patients whose serum alpha-FP was normal or slightly elevated (< 50 ng/ml), 6 were MAGE-1 and/or MAGE-3 mRNA positive in their PBMCs. CONCLUSION: MAGE-1 and MAGE-3 mRNA could be specifically detected with high percentage in the PBMCs of HCC patients by our method. They can be used as specific tumor markers for the detection of the circulating HCC cells, and the detection results may be helpful to evaluate the prognosis of HCC patients.
Antigens, Neoplasm↗