A review of 50 years investigation on burn pathology in China and its prospect / 中华烧伤杂志

A great achievement has been made on burn pathology research in China since 1958. These advances include: pathological changes in burn wound, the healing process of burn wound and its mechanism modulated by growth factors especially bFGF, intermingled transplantation of allo-skin or xeno-skin with auto-skin for coverage of extensive third degree burns, characteristic postburn inflammatory reaction, pathological changes and evolution in various internal organs, multiple organ dysfunction syndrome (MODS), pathological changes in phosphorus burn, pathological changes in endotoxemia in burn, the role of vascular endothelial cell in pathogenesis of postburn visceral organ dysfunction as well as steam and smoke inhalation injury.

VEGF-induced tubulogenesis of endothelial cells from human brain malignant glioma in the three dimentional model / 中华病理学杂志

<p><b>OBJECTIVE</b>To compare the tubulogenesis capability of malignant glioma-derived microvessel endothelial cells (GDMEC) from human brain with that of ECV304 cells in a three dimentional model and to explore the significance of GDMEC in the study on angiogenesis.</p><p><b>METHODS</b>The GDMEC were isolated from malignant gliomas of human brain and purified by selective binding to the monoclonal antibody against CD105 bound to the magnetic MACS MicroBeads. GDMEC and endothelial-like cell line ECV304 were compared with their capabilities of formatting tubule-like structure (TLS) in the three dimentional collagen matrix, with or without inducement by various concentration of vascular endothelial growth factor (VEGF).</p><p><b>RESULTS</b>The obtained GDMEC had a high purification (98%) and could be successfully cultured in vitro. GDMECs formed more TLS than ECV304 cells of the same number and at the same time points. VEGF could induce rapid formation of TLS in a dose-dependent manner, however, ECV304 cells were less response to VEGF stimulation.</p><p><b>CONCLUSIONS</b>GDMEC could maintain their endothelial characteristics and potential capability of angiogenesis. They were more response to VEGF than ECV304, therefore, more suitable for in vitro studies on tumor angiogenesis.</p>

Effects of hypoxia on ET and NO levels in cultured umbilical venous endothelial cells of native Tibetan / 中国应用生理学杂志

<p><b>AIM</b>To assay ET and NO in venous blood of native Tibetan and to investigate the effects of hypoxia on ET and NO levels in cultured umbilical venous endothelial cells of native Tibetan.</p><p><b>METHODS</b>ET and NO in venous blood of native Tibetan, immigrant Han and lowland Han were assayed. Umbilical venous endothelial cells (UVECs) from native Tibetan and immigrant Han newborns were cultured and divided into 4 groups: (1) Native Tibetan control group (TC), (2) Native Tibetan hypoxic group (TH), (3) Immigrant Han control group (HC), (4) Immigrant Han hypoxic group (HH). Supernatant was collected and ET and NO were detected.</p><p><b>RESULTS</b>Venous blood NO was significantly higher in native Tibetan than in immigrant Han, while ET lower in native Tibetan than in immigrant Han. ET excretion from UVECs was elevated while NO decreased in both Tibetan and Han groups after exposed to hypoxia. On time-points 12 h and 24 h, ET was significantly lower in TH than in HH, while concentration of NO showed no difference in TH and HH.</p><p><b>CONCLUSION</b>ET released by UVECs was higher in Han than in Tibetan after 12 h and 24 h hypoxic exposure, which may be in favor of lower vascular resistance and better fetal blood supply in Tibetan, and thus plays a role in the mechanisms of less intrauterine growth restriction (IUGR) throughout pregnancy and heavier birth weight of Tibetan newborns.</p>

Effects of ectopic glial fibrillary acidic protein/green fluorescent protein gene expression on cellular differentiation and proliferation of human glioma cell line / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the biological effects of ectopic overexpression of glial fibrillary acidic protein (GFAP) in human malignant glioma cell line, and to explore new method of differentiation induction gene therapy for gliomas.</p><p><b>METHODS</b>A eukaryotic expression vector containing 1.1 kb GFAP cDNA fused with green fluorescent protein (GFP) gene, pIRGFP-GFAP, was transfected into human SHG-44 glioma cell line by lipofectamine. The expression of GFAP/GFP gene and their proteins were detected by fluorescent real-time monitoring, in situ hybridization, Western blot and immunocytochemistry. Flow cytometry, soft agar colony formation and other methods were used to measure the effects of exogenous GFAP expression on cell cycle progression, morphology and growth features of the transfected glioma cells.</p><p><b>RESULTS</b>The expressions of GFAP mRNA and its protein were markedly increased in SHG-44 cells upon stable transfection with pIRGFP/GFAP vector. Profound morphological changes in these cells were also observed, including the formation of abundant, stellate and thin cytoplasmic processes and a reduction of atypia. Cell proliferation rate and its tumorigenecity on soft agar were markedly reduced. In addition, cell cycle analysis revealed a percentage decrease of cell populations at G0/G1 and G2/M phases.</p><p><b>CONCLUSIONS</b>Ectopic overexpression of GFAP gene could significantly suppress the growth of SHG-44 malignant glioma cells along with an induction of differentiation. These results imply that forced over-expression of GFAP gene may provide a new strategy for glioma therapy.</p>

Roles of cyclin-dependent kinase 4 (CDK4) in nordihydroguaiaretic acid-induced inhibitory effect on proliferation of human malignant glioma cells / 第三军医大学学报

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.

Significance of bcl-2 and c-myc gene expression in nordihydroguaiaretic acid-induced apoptosis of a human malignant glioma cell line / 第三军医大学学报

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.

Effect of nordihydroguaiaretic acid on expressions of VEGF and its receptor KDR in vitro / 第三军医大学学报

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.

Inhibitory effects of angiogenesis inhibitor TNP-470 on growth of a human malignant glioma cell line SHG-44 and its xenografted tumor / 第三军医大学学报

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.

Effect of nordihydroguaiaretic acid on the morphological changes of endothelial cells in vitro / 第三军医大学学报

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.

Quantitative study on morphologic features and proliferative activity during DEN-induced hepatocarcinogenesis in rats / 第三军医大学学报

Objective　To explore the relationship between morphologic evolution and proliferative activity during hepatocarcinogenesis in rats. Methods　Imaging analysis technique was used to detect the morphologic parameters of cells in hepatic lesions in both Solt-Farber model and diethylnitrosamine (DEN)-induced liver cancer model. Immunohistochemistry was employed to detect proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU). Results　The oval cells were identified as irregular small proliferating cells in size of one-eighth of and with a nucleus/cytoplasm ratio of 6 times of the normal hepatocyte by image analysis. The morphometric parameters of basophil hepatocyte in precancerous foci and nodule were similar to those of the liver cancer cell. PCNA and BrdU positive cells were mainly localized within the proliferative foci and hepatocellular carcinoma (HCC) tissues. There was a better consistency between the development of hepatic lesions and cellular proliferative activity. Conclusion　The morphologic evolution is closely related to proliferative activity during hepatocarcinogenesis in rats.

Expression of some oncogenes and point mutation of c-Ha-ras1 during hepatocarcinogenesis in rats / 第三军医大学学报

Objective　To study the expressions of oncogenes c-Ha-ras, c-ki-ras, pan-ras and c-myc and point mutation of c-Ha-ras1 during hepatocarcinogenesis in rats. Methods　Immunohistochemistry, in situ hybridization and microdissection of tissue (MDT)-PCR-SSCP were used to detect the oncogene expressions and point mutation of c-Ha-ras1 in both Solt-Farber model and DEN-induced liver cancer model. Results　The overexpression of c-Ha-ras was closely associated with the formation and proliferation of the precancerous basophilic hepatocyte foci, while that of c-myc with the growth of the oval cell foci. The abnormalities of IGF-Ⅱ played an important role in the evolution of precancerous foci/nodules towards hepatocellular carcinoma (HCC). The overexpression of fms was only associated with HCC of some rats. Conclusion　Hepatocarcinogenesis in rats was related with the overexpression of c-Ha-ras, c-myc, IGF-Ⅱand fms and the point mutation of c-Ha-ras1, and overexpression of these oncogenes was associated with morphological evolution.

Roles of cyclin-dependent kinase 4 (CDK4) in nordihydroguaiaretic acid-induced inhibitory effect on proliferation of human malignant glioma cells / 第三军医大学学报

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.

Significance of bcl-2 and c-myc gene expression in nordihydroguaiaretic acid-induced apoptosis of a human malignant glioma cell line / 第三军医大学学报

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.

Effect of nordihydroguaiaretic acid on expressions of VEGF and its receptor KDR in vitro / 第三军医大学学报

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.

Inhibitory effects of angiogenesis inhibitor TNP-470 on growth of a human malignant glioma cell line SHG-44 and its xenografted tumor / 第三军医大学学报

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.

Effect of nordihydroguaiaretic acid on the morphological changes of endothelial cells in vitro / 第三军医大学学报

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.

Quantitative study on morphologic features and proliferative activity during DEN-induced hepatocarcinogenesis in rats / 第三军医大学学报

Objective　To explore the relationship between morphologic evolution and proliferative activity during hepatocarcinogenesis in rats. Methods　Imaging analysis technique was used to detect the morphologic parameters of cells in hepatic lesions in both Solt-Farber model and diethylnitrosamine (DEN)-induced liver cancer model. Immunohistochemistry was employed to detect proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU). Results　The oval cells were identified as irregular small proliferating cells in size of one-eighth of and with a nucleus/cytoplasm ratio of 6 times of the normal hepatocyte by image analysis. The morphometric parameters of basophil hepatocyte in precancerous foci and nodule were similar to those of the liver cancer cell. PCNA and BrdU positive cells were mainly localized within the proliferative foci and hepatocellular carcinoma (HCC) tissues. There was a better consistency between the development of hepatic lesions and cellular proliferative activity. Conclusion　The morphologic evolution is closely related to proliferative activity during hepatocarcinogenesis in rats.

Expression of some oncogenes and point mutation of c-Ha-ras1 during hepatocarcinogenesis in rats / 第三军医大学学报

Objective　To study the expressions of oncogenes c-Ha-ras, c-ki-ras, pan-ras and c-myc and point mutation of c-Ha-ras1 during hepatocarcinogenesis in rats. Methods　Immunohistochemistry, in situ hybridization and microdissection of tissue (MDT)-PCR-SSCP were used to detect the oncogene expressions and point mutation of c-Ha-ras1 in both Solt-Farber model and DEN-induced liver cancer model. Results　The overexpression of c-Ha-ras was closely associated with the formation and proliferation of the precancerous basophilic hepatocyte foci, while that of c-myc with the growth of the oval cell foci. The abnormalities of IGF-Ⅱ played an important role in the evolution of precancerous foci/nodules towards hepatocellular carcinoma (HCC). The overexpression of fms was only associated with HCC of some rats. Conclusion　Hepatocarcinogenesis in rats was related with the overexpression of c-Ha-ras, c-myc, IGF-Ⅱand fms and the point mutation of c-Ha-ras1, and overexpression of these oncogenes was associated with morphological evolution.