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Study of genetic variation of the structural region of hepatitis C virus

Journal of Third Military Medical University.2001;23(3):296-299.

Objective　To study the role of the structural gene variation of hepatitis C virus(HCV) in the chronicity of HCV infection. Methods　Serum samples from four HCV infection acquired during six-month follow up and four patients with chronic hepatitis C type were obtained. A 1-kb fragment containing the 3' half of core ，completed E1 and 5' half of E2/NS1 was amplified by nested reverse transcription polymerase chain reaction (RT-PCR). For each，30 cloned cDNAs were examined by a method that combined heteroduplex analysis (HAD) and a single-stranded conformational polymorphisim (SSCP)assay to optimize selection of clones with unique gel shift patterns (clonotypes )as a measure of the quasispecies complexity. Results　There was obvious difference between the SSCP bands of cloned cDNAs representing core-E1 and E2 region，but more distinct difference between heteroduplex and the homoduplex of clones spanning E2 region which accurately reflected the degree of heterogeneity. Quasispecies complexity was higher in those with persistent viremia than those with acute viremia. Conclusion　HCV persistence is associated with complex quasispecies in viral structural region.

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Effects of glucagon-like peptide 2 on recovery of small intestinal epithelia from radiation injury in mice

Jundong ZHU ; Yongping SU ; Tianmin CHENG

Journal of Third Military Medical University.2001;23(3):293-295.

Objective　To investigate the effects of glucagon-like peptide 2 (GLP-2) on recovery of small intestinal epithelia from radiation injury in mice. Methods　Mice received a single 8 Gy dose of total body irradiation from 60Co gamma ray followed by treatment with GLP-2 or vehicle. DNA and protein content in small intestinal mucosa were measured, and small intestine was processed for histological examination with light microscope and scanning electron microscope. Results　Small intestinal mucosal DNA and protein content, villus height, and villus number significantly decreased in irradiated mice, partial villus tips were ulcerated. GLP-2 administration caused increase in DNA and protein content, villus height, and villus number as compared with irradiated control group. Meanwhile, the villus tips were lack of ulceration. Conclusion　GLP-2 can promote recovery of small intestinal epithelia from radiation injury in mice.

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Effects of irradiation and W11-a12 on anion-selective channel of mouse peritoneal macrophage

Chongxiang SHU ; Benlan YE ; Tianmin CHENG ; Jiasi XIAO

Journal of Third Military Medical University.2001;23(3):290-292.

Objective　To study the effects of irradiation and W11-a12，a kind of repair-promoting drug，on anion-selective channel in membranes of mouse peritoneal macrophage. Methods　The activity of anion-selective channel was recorded from cell-attached patches with patch clamp techniques． Results　The effects of irradiation on anion-selective channel in membranes of peritoneal macrophage included：①decreasing the mean number of activated channels by the presence of zymosan; ②prolonging the mean time from stimulus to the opening of channels; ③depressing the opening of channels by decreasing open-state probability，shortening open-time and prolonging close-time． The effects of irradiation could partly be depressed by W11-a12. Conclusion　Irradiation will depress the anion-selective channel of peritoneal macrophage, which may be an important way to depress the function of macrophage．

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Effects of systemic irradiation and W11-a12 on neutrophils in wounds

Xiaohong CHEN ; Tianmin CHENG ; Guoping AI

Journal of Third Military Medical University.2001;23(3):287-289.

Objective　To study the effects of systemic irradiation and conglutinant drug W11-a12 on the number and some functions of wound nentrophils (Neu). Methods　Wound Neu was collected from sponges which were implanted in rat's dorsum incision. The number of Neu, as well as the phagocytic function and motility of wound Neu were measured. Results　After 4,6,8 Gy systemic irradiation, the number of white blood cells and Neu in wound, as well as the phagocytic function and chemotactic motility of wound Neu, were significantly decreased at 24 h, 48 h after wounding. W11-a12 markedly increased the number of wound Neu, improved the phagocytic function and chemotactic motility of wound Neu at 24 h, 48 h after wounding despite the rats were radiated or not. Conclusion　The results indicated that the decreased number and function of wound Neu in the early stage of wound healing contributed to the impairment of repair after systemic irradiation. W11-a12 accelerated normal and irradiation-impaired wound healing partly by increasing the number of wound Neu and improving the Neu function.

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Effects of nordihydroguaiaretic acid treatment combined with Methotrexate or Vincristine on a human malignant glioma cell line

Jian CAO ; Fu WU

Journal of Third Military Medical University.2001;23(3):284-286.

Objective　To observe the effect of nordihydroguaiaretic acid treatment combined with Methotrexate or Vincristine on a human malignant glioma cell line. Methods　The effect of drugs on the cells was measured by MTT, and the protein expression of cyclin D1 gene in the cells was measured by immunohistochemistry assay. Results　①The anti-tumor effects of drugs on the cells were increased with the increasing of concentrations, and had a combined function between NDGA and MTX or VCR. ②There was no difference between the effect of treating NDGA with MTX or VCR given at the same time and MTX or VCR given after 24 h of NDGA treatment, but the effects were apparently different when NDGA was administrated after MTX or VCR treatment for 24 h. ③The protein expression of cyclin D1 gene in the cells was apparently reduced after NDGA treatment. Conclusion　There is a cooperative function between NDGA and MTX or VCR on the cells, and the function is probably related to the NDGA-induced down-regulation of protein expression of cyclin D1 gene in the cells.

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Effect of nordihydroguaiaretic acid on the morphological changes of endothelial cells in vitro

Huiqin SUN ; Zhaozheng CHEN ; Yisheng CHEN ; Jingquan SHI ; Xiuwu BIAN ; Rong XIN

Journal of Third Military Medical University.2001;23(3):280-283.

Objective　To investigate morphological changes of endothelial cells after nordihydroguaiaretic acid (NDGA) treatment in vitro. Methods　The morphological changes of human umbilical vein endothelial cells (HUVEC) cell line ECV-304 and the cell apoptosis rate in sub-G0 phase were observed with invert, light and electron microscope and flow cytometry after NDGA treatment at different concentrations or with PBS (0.01 mol/L) as control. Results　①After the treatment of NDGA at 50～200 μmol/L for 1～3 d or up to 8 d at 100 μmol/L, ECV-304 cells tended to elongate into a shuttle-like sparse appearance and those in mitosis were decreased, indicating the suppression of cell proliferation. All these alteration was in a time-and dose-dependent manner. ②NDGA-treated ECV-304 cells displayed morphological features of apoptosis, especially at the 48th h after the treatment. With flow cytometry, the cells in sub-G0 phase were significantly increased, and reached its peak at hour 12 (20.42%) after NDGA treatment. In addition, the degeneration and necrosis of ECV-304 cells were related to the concentrations of NDGA. Conclusion　NDGA can inhibit the proliferation and growth of endothhelial cells, and induce apopotosis, which might also inhibit angiogenesis.

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Inhibitory effects of angiogenesis inhibitor TNP-470 on growth of a human malignant glioma cell line SHG-44 and its xenografted tumor

Linlin DU ; Xiuwu BIAN ; Yisheng CHEN ; Jingquan SHI

Journal of Third Military Medical University.2001;23(3):272-275.

Objective　To investigate the effects of TNP-470 on the growth of a human malignant glioma cell line SHG-44 in vivo and in vitro. Methods　The colorimetric MTT assay, soft agar culture, flow cytometry，light and electron microscopy were used to determine the proliferation, the cloning efficiency, cell cycle and the morphological changes of SHG-44 cells as well as the growth of its xenografted tumor. Results　TNP-470 (20～2 000 ng/ml) significantly inhibited the proliferation of SHG-44 cells in vitro (the 50% inhibitory concentration was 200 ng/ml). Cloning efficiency reduced obviously. The number of cells in G0/G1 phase increased, while that in S, G2/M phases decreased significantly. Weight and volume of xenografted tumors treated with TNP-470 (30 mg/kg, injected subcutaneously every other day) reduced notably. Furthermore, there were necrotic area and apoptosis in the tumor. No severe side effect of TNP-470 was found in this study. Conclusion　The inhibitory effect of TNP-470 on the growth of SHG-44 cells correlates with its functions of regulating cell cycle and inducing apoptosis, which suggests that the angiogenesis inhibitor TNP-470 has strong inhibitory effect on human malignant gliomas.

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Effect of nordihydroguaiaretic acid on expressions of VEGF and its receptor KDR in vitro

Huiqin SUN ; Yisheng CHEN ; Jingquan SHI ; Xiuwu BIAN ; Zhongmin ZOU ; Deyu GUO ; Rong XIN

Journal of Third Military Medical University.2001;23(3):268-271.

Objective　To explore the effect of nordihydroguaiaretic acid (NDGA) on the expressions of vascular endothelial growth factor (VEGF) and its receptor, kinase-inserted domain containing receptor(KDR) and the possible mechanism. Methods　The expression of VEGF in human malignant glioma cell line SHG-44 and that of KDR in human umbilical vein endothelial cell (HUVEC) line ECV-304 were observed 1～3 d after NDGA treatment with immunohistochemistry, in situ hybridization and image analysis. Results　The expression of VEGF was declined at protein or mRNA levels in SHG-44 cells after treated with 100 μmol/L NDGA for 1 to 3 d. The expression of KDR in endothelial cells with 100 μmol/L NDGA treatment for 1 to 3 d was decreased too, in a more obvious way compared with the decline of VEGF expression in SHG-44 cells. Conclusion　The results suggest that NDGA inhibits the expression of VEGF in glioma cells as well as that of VEGF receptor KDR in endothelial cells, which may be the important molecular mechanism of anti-angiogenesis of NDGA.

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Significance of bcl-2 and c-myc gene expression in nordihydroguaiaretic acid-induced apoptosis of a human malignant glioma cell line

Deyu GUO ; Yisheng CHEN ; Xiuwu BIAN ; Jingquan SHI ; Ziqiang CHEN ; Rong XIN

Journal of Third Military Medical University.2001;23(3):260-263.

Objective　To investigate the changes and their significance of bcl-2 and c-myc in nordihydroguaiaretic acid (NDGA)-induced apoptosis of human malignant glioma cell line SHG-44. Methods　The apoptosis of SHG-44 cells was observed with light and electron microscopy and TUNEL method. The expression of bcl-2 and c-myc gene was measured with immunohistochemistry, in situ hybridization and image analysis. Results ① The SHG-44 cell apoptosis was induced by NDGA at a concentration lower than 200 μmol/L in a time-dependent manner. ② The expressions of bcl-2 and c-myc gene in SHG-44 cells were decreased after the treatment of 100 μmol/L NDGA with the elapse of time, indicating a close association with cell apoptosis. ③ The expressions of bcl-2 and c-myc mRNA in SHG-44 cells were decreased after the treatment with 100μmol/L NDGA, which was apparently consistent with the immunohistochemical results. Conclusion　The NDGA-induced apoptosis in human malignant glioma cells might be related with the down-regulated expressions of bcl-2 and c-myc gene. The exact mechanism needs further research.

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Roles of cyclin-dependent kinase 4 (CDK4) in nordihydroguaiaretic acid-induced inhibitory effect on proliferation of human malignant glioma cells

Deyu GUO ; Yisheng CHEN ; Xiuwu BIAN ; Jingquan SHI ; Ziqiang CHEN ; Rong XIN

Journal of Third Military Medical University.2001;23(3):257-259.

Objective　To investigate roles of cyclin-dependent kinase 4 (CDK4) in nordihydroguaiaretic acid (NDGA)-induced inhibitory effect on proliferation of human malignant glioma cells. Methods　The techniques of cell culture, cell counts, flow cytometry, immunoprecipitation, immunohistochemistry and image analysis were employed in this study. Results　①A concentration-dependent inhibition of proliferation was demonstrated in the SHG-44 cells incubated for 24 hours in the presence of NDGA, and cell proliferation was blocked in the G1→S phase. ②The activity of CDK4 was decreased apparently in the SHG-44 cells treated for 24 hours with 10 to 200 μmol/L NDGA in a concentration-dependent way. ③The expression of CDK4 gene was downregulated in the cells after NDGA treatment. Conclusion　CDK4 plays an important role in NDGA-induced inhibition of glioma cell proliferation.

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