[A review of 50 years investigation on burn pathology in China and its prospect].

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.

Unique proteomic features induced by a potential antiglioma agent, Nordy (dl-nordihydroguaiaretic acid), in glioma cells.

Nordy is a chirally synthesized compound of a natural lipoxygenase inhibitor nordihydroguaiaretic acid. In this study, we found that Nordy inhibited the growth of human glioma cell lines in vitro and their tumorigenicity in mice. In addition, Nordy promoted differentiation of highly malignant human glioma cells. Investigation into the mechanistic basis of Nordy activities revealed that it altered the pattern of protein expression profiles in tumor cells. By using 2-DE, we found that in human glioma cell lines, at least six proteins were down-regulated after Nordy treatment, while four proteins were elevated in the same cells. Among the six down-regulated proteins, microsequencing with MALDI TOF MS confirmed the identity of five: proliferation-associated gene A (PAG-A), alternative splicing factor-3 (ASF-3), beta-galactoside binding lectin, eukaryotic translation initiation factor 5A (eIF-5A), and coffilin-1 (nonmuscle). Four up-regulated proteins were GST-pi, glyceraldehyde-3-phosphate dehydrogenase, alpha-enolase, and cyclophilin. All these proteins have been reported to participate in key cellular functions including proliferation, metabolism, differentiation, apoptosis, and gene transcription. Our results suggest that Nordy may constitute a promising drug lead for the development of novel antitumor agents targeting proteins that control tumor cell function at multiple levels.

Preferential expression of chemokine receptor CXCR4 by highly malignant human gliomas and its association with poor patient survival.

OBJECTIVE: CXCR4 is implicated in the growth, metastasis, and angiogenesis of malignant tumors. We investigated the potential role of CXCR4 in human gliomas. METHODS: The expression of CXCR4 messenger ribonucleic acid and protein by human glioma cell lines was examined by reverse-transcriptase polymerase chain reaction and immunocytochemistry analysis. Tumor cell chemotaxis and production of vascular endothelial growth factor induced by the CXCR4 ligand SDF-1beta were measured. Xenograft models were used for evaluation of glioma cell tumorigenesis. CXCR4 expression by xenografted tumors and primary human glioma specimens were evaluated for CXCR4 protein expression. The relationship between CXCR4 expression and patient survival was analyzed. A synthetic lipoxygenase inhibitor, Nordy, was tested for its effects on glioma cell expression and function of CXCR4, as well as on glioma cell tumorigenicity. RESULTS: CXCR4 expression correlated directly with the degree of malignancy of the human glioma cell lines and primary tumors. Activation of CXCR4 induced tumor cell chemotaxis and increased production of vascular endothelial growth factor. Glioma cells expressing higher levels of CXCR4 formed more rapidly growing and lethal tumors in nude mice. Primary human glioma specimens expressing CXCR4 contained high-density microvessels. Patients with CXCR4-positive gliomas had poorer prognosis after surgery. The lipoxygenase inhibitor Nordy diminished CXCR4 expression by glioma cell lines in vitro and reduced their tumorigenicity in nude mice. CONCLUSION: The level of CXCR4 expression seems to correlate with the degree of malignancy of human gliomas and may contribute to their rapid growth.

Increased angiogenic capabilities of endothelial cells from microvessels of malignant human gliomas.

Vascular endothelial cells (ECs) that initiate tumor angiogenesis may acquire distinct properties after conditioning in tumor microenvironment as compared to ECs in non-malignant tissues. Thus far, most in vitro studies of angiogenesis used ECs isolated from normal tissues, which may not fully represent the nature of ECs in tumor vasculature. In this study, glioma-derived microvascular ECs (GDMEC) were purified from human glioma tissues by incubating with magnetic beads coated with anti-CD105 antibody and highly pure (98%) preparations of GDMEC were obtained. These cells exhibited typical EC phenotype, and proliferated rapidly in culture. Interestingly, GDMEC expressed higher levels of VEGF receptors, flt-1 and flk-1, as compared to an established human EC cell line ECV304 and primary human umbilical vascular EC (HUVEC). Functionally, GDMEC were capable of forming intercellular junctions and tubule-like structures (TLS) of various sizes. Stimulation by VEGF further promoted TLS formation with diverse tubular walls by GDMEC. In contrast, TLS formed by ECV304 and HUVEC showed significantly different features. We further observed that Nordy, a synthetic lipoxygenase inhibitor, potently inhibited TLS formation by GDMEC. The results suggest that isolation of highly pure ECs derived from tumor tissues is more appropriate for studies of tumor angiogenesis and for test of potential anti-cancer therapeutic targets.

Activation of chemokine receptor CXCR4 in malignant glioma cells promotes the production of vascular endothelial growth factor.

Numerous studies have showed that chemokine receptors, such as CXCR4, contribute to the growth and metastasis of a variety of malignant tumors. In this study, we investigated the role of CXCR4 in the production of angiogenic factor, vascular endothelial growth factor (VEGF), in various human glioma cells from astrocytic origin. The expression of CXCR4 mRNA and protein in three glioma cell lines, U87-MG, SHG-44, and CHG-5, was determined by RT-PCR and immunocytochemistry, respectively. The malignancies of three gliomas were evaluated by expression of glial fibrillary acidic protein and vimentin, the differentiation markers of astrocytic cells. The role of functional CXCR4 in tumor cell migration was studied with chemotaxis assay. Ca2+ mobilization and VEGF production were measured in the cells after stimulation with CXCR4 ligand, SDF1beta. The results showed that the levels of functional CXCR4 expression at both mRNA and protein levels by several human glioma cell lines were correlated with the degree of differentiation of the tumor cells. Activation of CXCR4 induced glioma cell chemotaxis and could trigger the increase of intracellular [Ca2+]i. Such an activation could result in the increased production of VEGF by the stimulated tumor cells. Our results suggest that CXCR4 may contribute to the high level of VEGF produced by malignant glioma cells and thus constitute a therapeutic target for antiangiogenesis strategy.

[VEGF-induced tubulogenesis of endothelial cells from human brain malignant glioma in the three dimentional model].

OBJECTIVE: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

[Effects of hypoxia on ET and NO levels in cultured umbilical venous endothelial cells of native Tibetan].

AIM: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Effects of ectopic glial fibrillary acidic protein/green fluorescent protein gene expression on cellular differentiation and proliferation of human glioma cell line].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Genomic instability of murine hepatocellular carcinomas with low and high metastatic capacities.

AIM: To investigate the frequency of genomic instability in murine hepatocellular carcinoma (HCC) cell lines Hca/A2-P(P) and Hca/163-F(F) with low and high metastatic capacity, and to explore its association with the occurrence and metastasis of hepatocellular carcinomas. METHODS: Forty microsatellite markers were randomly selected to examine P and F cells for genomic instability using PCR-simple sequence length polymorphism (PCR-SSLP) analysis. RESULTS: Allelic genes on the chromosomes of P cell line with thirty informative microsatellite loci were paralleled to those of inbred strain C(3)H mouse, while those of F cell line with 28 loci were paralleled to those of inbred strain C(3)H mice. The frequency of microsatellite alterations was 37.5% and 42.5% in P cell line and F cell line, respectively. There were different alterations of allelic band 9 at loci between P and F cells, among which, the frequency of microsatellite alterations was most commonly seen on chromosomes 3, 7, 11 and 16. CONCLUSION: Genomic instability in mouse chromosomes 3, 7, 11 and 16 may play a more important role in the development and progression of HCC in mice. It is suggested that these two sub-clones derived from a same hepatic tumor in homozygous mouse present different genetic features.

The effect of lipopolysaccharides on the expression of CD14 and TLR4 in rat Kupffer cells.

OBJECTIVE: To assess the effect of lipopolysaccharides (LPS) on the expression of CD14 and TLR4 in rat Kupffer cells (KCs). METHODS: In rat KCs induced by LPS, the changes of CD14 and TLR4 expression were measured by RT-PCR and immunohistochemistry, and the expressions of TNF-alphamRNA, IL-6mRNA or the concentrations of TNF-alpha, IL-6 were estimated by in situ hybridization, radioimmunoassay, and others. RESULTS: The expressions of CD14 and TLR4 in KCs induced by LPS were markedly increased in a dose-dependent manner (10 mg/L-1 microg/L) or in a time-dependent manner (0.5 h-24 h), with the peaked expression of CD14 at 3-6 hours. The expressions of CD14 and TLR4 in KCs stimulated by the active mediators from KCs which had been exposed to LPS for 1 hour were obviously increased. CONCLUSIONS: There is a close relationship between LPS or the active mediators from KCs induced by LPS and the expressions of CD14, TLR4. It is implied that the increase of TLR4, CD14 expression may be induced by LPS within 1-3 hours, and further increase of TLR4, CD14 expression may be correlated with the cytokines produced by KCs.

Preliminary study on the production of transgenic mice harboring hepatitis B virus X gene.

AIM:To establish transgenic mice lineage harboring hepatitis B virus X gene and to provide an efficient animal model for studying the exact role of the HBx gene in the process of hepatocarcinogenesis.METHODS:The HBx transgenic mice were produced by microinjecting the construct with X gene of HBV (subtype adr) DNA fragment into fertilzed eggs derived from inbred C57BL/6 strain; transgenic mice were identified by using Nested PCR; expression and phenotype of HBx gene were analyzed in liver from transgenic mic at the age of 8 weeks by RT-PCR, pathologic examination and periodic acid-schiff staining (PAS), respectively.RESULTS:Five hundred and fourteen fertilized eggs of C57 BL/6 mice were microinjected with recombinant retroviral DNA fragment, and 368 survival eggs injected were transferred to the oviducts of 18 pseudopregnant recipient mice, 8 of them became pregnant and gave birth to 20 F1 offspring. Of 20 offsprings, four males and two females carried the hybrid gene (HBx gene). Four male mice were determined as founder, named X 1, X 5, X 9 and X 15. These founders were back crossed to set up F1 generations with other ibred C57BL/6 mice or transgenic littermates, respectively.Transmission of HBx gene in F1 offspring of X 1, X 5 and X 9 except in X 15 followed Mendelian rules. The expression of HBx mRNA was detected in liver of F1 offspring from the founder mice (X 1 and X 9), which showed vacuolation lesion and glycogen positive foci.CONCLUSION:Transgenic mice harboring HBx gene were preliminarily established.