Cold damage from wax deposition in a shallow, low-temperature, and high-wax reservoir in Changchunling Oilfield.

Wax deposition is an important factor that influences oil production for high-wax crude oilfield. There are few studies on the formation damage by wax deposition, especially cold damage to the shallow low-temperature reservoir. With laboratory tests conducted on reservoir oil and cores of Changchunling Oilfield, this study aims to experimentally investigate the influence of temperature variations on characteristics of oil-water percolation and cold damage mechanisms, as well as the relative permeability of high-wax reservoirs. Experimental results show that seepage flow of high-wax crude is significantly sensitive to temperature-wax deposition evidently increases, whereas the cold damage such as the pore-throat radius and relative permeability sharply decrease with the decline in formation temperature. The research results can be applied to enhance oil recovery of high-viscosity or high-wax oilfields.

[Occurrence regularity and integrated control of leaf miner in safflower].

Leaf miner is one of the major pests on safflower, which causes yield loss and poor quality seriously. "Weihonghua", "nine safflower varieties" and "three chemical insecticides" as materials that used to evaluate variety and regularity of leaf miner, safflower resistant level, and different proportions insecticides in field efficiency test. The results showed that Liriomyza sativae and L. huidobrensis accounted for 80%, the peak period of two pests was all in July; but Phytomyza horticola is relative less, its peak period occured in June. Three were great difference of resistance to leaf miner among safflower varieties, FQ12 and YJ65 expressed higher resistibility to leaf miner by ratio method. With abamectin 2% emulsifiable concentrate diluted for 2 000 times, or the mixture three insecticides(bifenthrin 20% water emulsions, thiamethoxam 25% water dispersible granule, abamectin 2% emulsifiable concentrate=1∶1∶1) diluted for 3 000 times, which were sprayed on leaves at squaring stage and lethal rate was 96% after 48 h in the study. Through comparative study on the variety and regularity of leaf miner, screen for resistant varieties to leaf miner and for high efficiency pesticide. The study provides theoretical basis and reference for integrated pest management of leaf miner.

[Research on regional distribution and industrial status analysis of genuine medicinal resources of flowers in Henan province].

Flower medicinal materials usually refer to Chinese medicinal materials with a complete flower,inflorescence,or part of a flower as the different medicinal parts,they have an important share in the Chinese herbal medicine market and appeared frequently in Chinese medicine prescriptions. Firstly,the species and regional distribution of the flower medicinal materials resources in China were briefly summarized. Secondly,the characteristics,yield,producing area and origin distribution of the main flower medicinal materials in Henan province were discussed. Finally,the present situation and the main problems of the flower medicinal materials industry in Henan province were comprehensively analyzed,and the corresponding industrial development countermeasures were put forward.This research was intended to provide decision-making demonstration and scientific basis for the rational exploitation and utilization of resources,breeding of new varieties,planting division,production layout and the healthy and sustainable development of the flower medicinal materials industry in Henan province.

Description of Chishuiella changwenlii gen. nov., sp. nov., isolated from freshwater, and transfer of Wautersiella falsenii to the genus Empedobacter as Empedobacter falsenii comb. nov.

A Gram-reaction-negative, strictly aerobic, non-pigmented, non-gliding, rod-shaped bacterium, designated strain BY4(T), was isolated from freshwater. Cells were catalase- and oxidase-positive and indole was produced. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain BY4(T) belonged to the family Flavobacteriaceae and showed 91.6-95.9% sequence similarities to the most closely related strains. The major respiratory quinone was MK-6 and the major polar lipid was phosphatidylethanolamine. The major polyamine was homospermidine and the major fatty acids were iso-C(15 : 0), iso-C(17 : 0) 3-OH and summed feature 3 (C(16 : 1)ω7c and/or C(16 : 1)ω6c). The DNA G+C content was 30.0 mol%. On the basis of phenotypic, phylogenetic and genotypic features, strain BY4(T) is suggested to represent a novel species in a new genus within the family Flavobacteriaceae, for which the name Chishuiella changwenlii gen. nov., sp. nov. is proposed. The type strain of this type species is BY4(T) ( = CGMCC 1.12707(T) = JCM 19633(T)). On the basis of data collected from previous and present studies, it is proposed to reclassify Wautersiella falsenii to the genus Empedobacter as the new combination Empedobacter falsenii comb. nov. (type strain NF 993(T) = CCUG 51536(T) = CIP 108861(T)).

Taibaiella chishuiensis sp. nov., isolated from freshwater.

A Gram-reaction-negative, strictly aerobic, non-motile and rod-shaped bacterial strain, AY17T, was isolated from the Chishui River in Guizhou Province, South-west China. Strain AY17T grew optimally at pH 7.0 and 20 °C. Flexirubin-type pigments were produced. 16S rRNA gene sequence analysis indicated that strain AY17T belonged to the family Chitinophagaceae within the phylum Bacteroidetes; the closest phylogenetic relative was Taibaiella smilacinae PTJT-5T (95.3% gene sequence similarity). The DNA G+C content was 49.5 mol%. Strain AY17T contained MK-7 as the predominant respiratory quinone and phosphatidylethanolamine as the major polar lipid. The major fatty acids were iso-C15:0, iso-C15:1G and iso-C17:0 3-OH. On the basis of phylogenetic, phenotypic and genetic data, strain AY17T was classified as representing a novel species of the genus Taibaiella, for which the name Taibaiella chishuiensis sp. nov. is proposed. The type strain is AY17T (=CGMCC 1.12700T=JCM 19637T).

Mechanisms and influence of octreotide-induced regulation of somatostatin receptor 2 on hepatocellular carcinoma.

BACKGROUND: Somatostatin receptors (SSTRs) belong to the family of G protein-coupled receptors. Exposure of G protein-coupled receptors to their agonists induces a rapid decrease in their initial response. The goal of this study is to investigate alteration in SSTR2 by the treatment of SSTR agonist octreotide (OCT) in hepatocellular carcinoma (HCC) and the resulting consequence. METHODS: Morphology, proliferation and cell cycle of the human HCC cell line (Bel7402) were evaluated. Effect of OCT on HCC growth and development was assessed in vivo. SSTR2 expression was measured by RT-PCR and detected by immunohistochemistry. RESULTS: Short-term OCT treatment on Bel7402 cells barely changed cell proliferation and morphology, and no apoptosis was induced. The SSTR2 protein level was markedly decreased on Bel7402 cells after exposure to OCT. However, the weight of the HCC xenograft was significantly lower in the OCT treatment group as compared with the control group. In the rat hepatocarcinogenesis model, the mortality and incidence of HCC in the OCT treatment group were remarkably less than those in the control group. Long-term OCT treatment led to increased levels of both SSTR2 mRNA and protein in hepatocytes and HCC cells. CONCLUSION: Short-term OCT treatment could lead to SSTR2 desensitization, resulting in a reduced inhibitory effect on HCC by OCT. However, long-term OCT treatment effectively inhibited the development and growth of HCC probably via resensitization and upregulation of SSTR2.

Expression of protease-activated receptor-2 (PAR-2) in patients with nasopharyngeal carcinoma: correlation with clinicopathological features and prognosis.

We aimed at determining whether the expression of protease-activated receptor 2 (PAR-2) is involved in the progression of nasopharyngeal carcinoma (NPC) and correlated with latent membrane protein 1 (LMP-1), matrix metalloproteinases-9 (MMP9), and angiogenesis of tumor. PAR-2, LMP-1, and MMP9 expressions were detected in 57 biopsies of primary NPC by immunohistochemistry. The presence of Epstein-Barr virus (EBV) was determined using EBER in situ hybridization, and intratumoral microvessels were highlighted by staining endothelial cells for anti-CD34. The correlations with immunostainings and clinicopathological factors, as well as the follow-up data of patients, were analyzed statistically. Strong expression of PAR-2 in 61.4% (35/57) of the biopsies was correlated with extensive lymph node metastasis and advanced stage of NPC. The patients with PAR-2/LMP-1 or PAR-2/MMP9 dual high-expression tumors had a significant worse prognosis than those with single protein high expression and dual low or negative expression tumors (P=0.013 and 0.004, respectively). Angiogenesis in the tumor is related to overall survival of NPC patients (P=0.001), and exhibits strong PAR-2 expression or LMP-1 expression in tumors associated with increased intratumoral microvessel density (P=0.026 and 0.006, respectively). PAR-2 is a possible mediator cooperating with LMP-1 and MMP9 to influence the progression of NPC by inducing angiogenesis and promoting lymph node metastasis.

[Changes in TGF-beta1/Smads signaling pathway in rats with chemical hepatocarcinogenesis].

OBJECTIVE: To investigate the changes in transforming growth factor beta 1 (TGF-beta1)/Smads signaling pathway in rats with chemical hepatocarcinogenesis. METHODS: Fresh diethylnitrosamine (DENA) solution was administered in SD rats to induce hepatocellular carcinoma (HCC). The protein expressions of TGF-beta1, phosphorylated Smad2, Smad4 and Smad7 were detected in these rats with immunohistochemistry, and the mRNA expression of Smad4 was evaluated with RT-PCR. RESULTS: Cirrhotic nodules occurred in the rats 8 weeks after DENA treatment, and HCC nodules were found 16 weeks after the treatment. In the normal liver tissue, very low levels of TGF-beta1 and Smad4 expressions, low Smad7 expression and high phosphorylated Smad2 expression were detected. The development of liver cirrhosis was accompanied by increased expressions of TGF-beta1, Smad4 and Smad7 but at 8 weeks after DENA treatment, the expression of phosphorylated Smad2 was significantly decreased, followed then by gradual increment till nearly the normal level. Twenty-two weeks after DENA treatment, Smad4 expression in liver tissue decreased markedly as compared with the levels at 8 and 16 weeks. The expressions of Smad4 and phosphorylated Smad2 in the HCC tissue was significantly lower than those in normal liver tissue. CONCLUSION: Hepatocarcinogenesis involves very complex mechanisms, can can be related partially to the decreased Smad4 and phosphorylated Smad2 expression and TGFbeta1 and Smad7 overexpression in advanced stage of liver cirrhosis.

[Expression of estrogen receptor, progesterone receptor, and human epithelial growth factor receptor 2 in breast cancer and the significance thereof: analysis of 910 cases].

OBJECTIVE: To investigate the expression of estrogen receptor (ER), progesterone receptor (PR), and human epithelial growth factor receptor 2 (HER2) in breast cancer, and to explore their correlation with the age of patient, and size, clinic stage, and lymph node metastasis of the tumor. METHODS: The data of 910 breast cancer, 89.4% of invasive ductal carcinoma, 1.7% of invasive lobular carcinoma, 44 cases 5% of ductal carcinoma in situ, and 4.9% of other pathologic types, 29.9% being less than 2 cm, 45.6% being 2-5 cm, and 24.5% bigger than 5 cm in size, 54.2% without metastasis in lymph node, 25.5% with metastasis in 1-3 lymph nodes, and 20.3% with metastasis in more than 3 lymph nodes respectively, were analyzed retrospectively. Immunohistochemistry was used to detect the expression of ER, PR, and HER2. RESULTS: The ER negative expression rate was 33.0%, and PR negative expression rate was 27.4%, and HER2 overexpression rate was 20.3%. The possibility of lymph node metastasis decreased along with the increase of age (P < 0.001). Tumor size was negatively correlated with the expression of ER and PR (both P < 0.001), and positively correlated with the expression of HER2 (P < 0.001). There was no significant difference between the situation of lymph node metastasis and the expression of ER, PR and HER2 in primary tumors. CONCLUSION: As good prognostic markers of breast invasive ductal cancer, ER and PR are negatively correlated with the HER2 expression, as a worse prognostic marker. ER/PR positive or HER2 negative tumors are morel likely to be diagnosed at earlier stages.

[Suppression of osteosarcoma in vitro by coexpression of antisense VEGF165 cDNA and thymidine kinase gene].

OBJECTIVE: To investigate the effect of VEGF expression in osteosarcoma cell line and the target killing effect of HSV1-TK/GCV system on transfected osteosarcoma cells under hypoxia conditions. METHODS: Eukaryotic expression plasmid with HRE promoter was constructed to express the antisense VEGF165 cDNA and Hygromycin phospho-transferase-thymidine kinase (HyTK) fusion gene. The recombinant vectors were then transfected into osteosarcoma cell line MG63 with lipofectin mediated gene transfer methods. PCR and RT-PCR were used to confirm the presence and expression of TK gene. The sensitivity of transfected cells to GCV and "bystander effect (BSE)" of HSV1-TK/GCV system under normoxia or hypoxia conditions were measured by MTT assay and mixed co-culture experiment. The expression of VEGF protein was detected by ELISA under hypoxia condition. Cell cycle phase distribution was determined by flow cytometry. In addition, electromicroscopy was used to document ultrastructural alterations. RESULTS: The eukaryotic expression vector pBI-HRE-AsVEGF165 -HyTK was constructed successfully. The transfected cell line MG63TV was established and confirmed by PCR and RT-PCR of the presence of transgene and its mRNA expression. GCV was toxic to transfected cells in a concentration-dependent manner. The sensitivity to GCV toxicity was 100 times higher under hypoxia condition than that under normoxic condition. The mixed culture experiments showed that the "bystander effect" was enhanced significantly under hypoxia condition. VEGF expression of transgene cells under hypoxia condition decreased 50% compared to that of normal condition. Under hypoxia and GCV, DNA synthesis of MG63TV cells was inhibited along with an increase of cells at G0 approximately G1 phase, apoptosis and necrosis. CONCLUSIONS: Antisense VEGF expression driven by HRE promoter in combination with hypoxia can provide a target inhibition of VEGF expression in human osteosarcoma cells, with an enhanced selective killing effect and BSE of the HSV-TK/GCV system. The double-gene co-expression system in study provides experimental basis for therapy against osteosarcoma by a synchronous antiangiogenic and suicide gene approach.

[Study on the regular pattern of the distribution of skin epidermal stem cells in the different parts of a healthy human body].

OBJECTIVE: To investigate the regular pattern of the distribution of skin epidermal stem cells (ESCs) in the different parts of a healthy human body, and to evaluate the feasibility of the identification of ESCs by P63 and CD29 with single and double labeling. METHODS: Full-thickness skin samples from 21 parts (including scalp, dorsum of foot, sole of foot, pubic region, and scrotum) of 5 healthy persons were harvested for the study. Immunohistochemistry method with biotin-streptavidin-horseradish peroxidase (SP) was employed with P63 and CD29 as the first antibody to carry out single and double labeling. The staining results were subjected to image analysis. The distribution of the ESCs in the skin from the above parts was observed and expressed as positive unit (PU) value. RESULTS: It was found by P63 single labeling and P63 and CD29 double labeling that the PU value in the dorsum of foot was the lowest while that in the scalp was the highest among all the parts of a healthy body. It was also found by CD29 single labeling that the PU value in the dorsum of foot was the lowest [(11.9 +/- 1.5)%] while highest in the scalp [(29.1 +/- 5.0)%]. The PU value in the hairy region of a human body was evidently higher than that in the non-hairy region (P < 0.01), when examined by P63 and CD29 single and double labeling. But there was no difference in the PU values between the trunk and limbs by means of P63 and CD29 single and double labeling (P > 0.05). CONCLUSION: There are more ESCs in the skin from the scalp, mons pubis and scrotum than other parts of the body. Single P63 or CD29 labeling exhibits higher sensitivity but lower specificity in the identification of ESCs. While the double labeling method exhibits higher specificity but lower sensitivity. Above all, it seems that the double labeling may be a simple and effective method for the identification of ESCs.

[Genetic effects on seed traits in soybean].

The genetic effects of seed traits in soybean, including 100-seed weight, seed length, seed width, seed thickness, length/width, length/thickness and width/thickness, were analyzed using an incomplete diallel cross of eight varieties with its F1 and F2 populations. The results showed that the above seven traits were controlled by direct genetic effects of seed and affected to different extent of maternal and cytoplasmic effects simultaneously. Among the traits, the inheritance of 100-seed weight, seed length, length/width, length/thickness, and width/thickness were mainly controlled by cytoplasmic effects, while those of seed width and thickness were mainly by maternal effects. Both the seed direct heritabilities and the cytoplsmic heritabilities of 100-seed weight, seed length, length/width and width/thickness were medium-sized. The individual selection and seed selection of above four traits at late generation may create good results. The maternal heritabilities of seed width and thickness were pretty high. To increase these two traits, an individual maternal selection should be done at early generation. Our results showed that varieties P2 and P7 could be used as ideal parents for improvements of 100-seed weight, seed length/width, length/thickness and width/thickness, while varieties P1, P4 and P6 are the ideal parents for increasing seed length, width and thickness respectively.

[Influence of aerosols on the expression of cyclin B1, cyclin C and proliferating cell nuclear antigen in wound tissue healing of burned rat models].

OBJECTIVE: To investigate the influence of aerosols on the expression of cyclin B(1), cyclin C and proliferating cell nuclear antigen (PCNA) in wound tissue healing of burned rat models. METHODS: Sprague Dawley (SD) rats were inflicted as the deep partial thickness burn models. Rats were randomly divided into experimental group and control group. The experimental group were treated with aerosols. Samples were collected in 1 approximately 10 postburn days. Immunohistochemistry and image analysis methods were conducted to examine the expression of cyclin B(1), cyclin C and PCNA in both experimental and control groups. RESULTS: The expression of cyclin C in experimental group was detected in nucleus of skin basal cell on the second postburn day, increased evidently at the fifth days and sustained at high expression level up to the tenth days after treatment. The expression of cyclin C in experimental group was significantly higher than control group (P < 0.05). The expression of PCNA was first observed in skin basal cell nucleus and hair follicle cell nucleus in both experimental and control group on the third postburn day. The expression of PCNA increased evidently at the fifth days in experimental after treatment and that increased evidently at the seventh days in control group, which showed there were lots of active proliferation cell. And the difference of the expression of PCNA between experimental and control group was significant (P < 0.01). The expression of cyclin B(1) was detected in nucleus and cytoplasm of skin basal cell in both groups on the third postburn day, and no difference between the experimental and control group (P > 0.05). CONCLUSIONS: Aerosols can up-regulate the expression of cyclin C and PCNA in skin basal cell nucleus. Therefore the aerosols can accelerate wound tissue healing.

[The role of CD44 in the proliferation, adhesiveness and invasiveness of osteosarcoma cell lines].

OBJECTIVE: To study the influence of CD44 a cell-matrix adhesion molecule on the proliferation, adhesiveness and invasiveness of osteosarcoma cell lines, in order to investigate the growth and invasion mechanism of osteosarcoma. METHODS: Three osteosarcoma cell lines MG-63, HOS and U2-OS were routinely cultured. Flow cytometry and Western blot analysis were used for detecting the positive rates and relative amount of CD44 protein in the three cell lines. RT-PCR method was also used to compare the differences in the expression of CD44 mRNA among the 3 cell lines. Then, MTT method, adhesion detection, and Microcon-migration assay were used to detect the changes of the cells' proliferation rate, adhesive and invasive abilities after blocking the role of CD44 by using a special neutralizing antibody. RESULTS: The results of flow cytometry showed that the percentage of CD44 positive cells were both over 99% in HOS and U2-OS, while that in MG-63 was only (2.10 +/- 0.46)%. The average fluorescence density of CD44 in HOS was significantly higher than in U2-OS. Western blot also showed that the relative content of CD44 protein in HOS was notably higher than that in U2-OS, while CD44 was negatively expressed in MG-63. The expression of CD44 mRNA was significantly lower in MG-63 than in both HOS and U2-OS, which were consistent with the expression of CD44 protein. The proliferation rates and adhesive abilities of MG-63 and HOS have no significant difference, but both were significantly higher than that of U2-OS. The invasive abilities of HOS was dramatically higher than MG-63 and U2-OS. After the role of CD44 was blocked by anti-CD44 neutralizing antibody, the proliferation rates of the 3 cell lines did not change significantly. While the HOS and MG-63 adhesion indices decreased dramatically (P < 0.05), the invasive abilities of HOS and U2-OS also decreased notably (P < 0.01). CONCLUSIONS: CD44 could promote the adhesiveness and invasiveness of osteosarcoma cell line HOS. CD44 may take part in promoting the process of U2-OS invasion and the adhesion of MG-63. On the other hand, CD44 could not affect the osteosarcoma cell proliferation rates.

[Expression of glycoprotein 90K in non-Hodgkin's lymphoma and its clinical significance].

BACKGROUND & OBJECTIVE: 90K, a kind of secretory glycoprotein, is related to prognosis of some tumors. So far, little is reported on its relation to prognosis of non-Hodgkin's lymphoma (NHL). This study was to explore the 90K expression in naive NHL tissues and its clinical significance. METHODS: 90K expression in 110 specimens of NHL was detected by immunohistochemistryû its correlations to short-term treatment efficacy of CHOP regimen and long-term survival of the patients were analyzed retrospectively. RESULTS: Of the 110 NHL specimens, 36 (32.7%) showed negative expression of 90K, 27 (24.5%) showed weak expression, 17 (15.5%) showed fairly strong expression, and 30 (27.3%) showed strong expression; the positive rate of 90K in NHL tissue was 67.3%. 90K expression had no correlation to gender, age, IPI and other clinicopathologic characteristics. All patients received treatment of standard CHOP regimen; the response (complete remission and partial remission) rate was significantly higher in low 90K expression (negative and weak expression) group than in high 90K expression (fairly strong and strong expression) group [82.5% (52/63) vs. 65.2% (30/46), P=0.039], but 90K expression had no correlation to long-term survival (P > 0.05). CONCLUSIONS: 90K expression may be applied to predict short-term efficacy of CHOP regimen on NHL. However, whether 90K could become a prognostic tumor marker of NHL needs further research.

[The study of inhibition effect of octreotide on the growth of hepatocellular carcinoma xenografts in situ in nude mice].

OBJECTIVE: To observe the effect of octreotide (OCT) on inhibiting hepatocellular carcinoma (HCC) and investigate its mechanisms. METHODS: Nude mice bearing xenografts in situ were treated with OCT or saline control for 7 weeks since tumor implantation. The immunohistochemistry for somatostatin receptor 2 (SSTR2), cMet, transforming growth factor beta1 (TGFbeta1), phospho-Smad2, Smad4 and Smad7 was performed. SSTR2 and Smad4 mRNA expression was measured by semi-quantitative RT-PCR. RESULTS: After OCT treatment, the mean tumor weight in mice given OCT (0.17 +/- 0.14 g) was significantly lower than that of the control group (0.53 +/- 0.06 g). The inhibition rate of tumor was 67.9%. mRNA and protein expression of SSTR2, Smad4 in tumor cells of the treatment group were significantly more than that of the control group. cMet expression in OCT group was remarkably lower than that in control group. Between two groups, the expression of TGFbeta1, phospho-Smad2 and Smad7 were not remarkably different. In addition, phospho-Smad2 expression in HCC was significantly less than that of the normal hepatic cell. CONCLUSION: OCT can inhibit the growth of HCC xenografts markedly. The mechanisms of OCT-induced inhibition effect may be related to up-regulating SSTR2 expression, down-regulating cMet, and recovering the function of TGFbeta/Smads-induced antitumor. In addition, the decreased expression of phospho-Smad2 may be an important feature of Bel7402 cells.

[Expression of hepatocyte growth factor/c-Met system in nasopharyngeal carcinoma and its biological significance].

OBJECTIVE: To investigate the expression of hepatocyte growth factor (HGF), and its receptor c-Met protein in nasopharyngeal carcinoma (NPC) and CNE-2 NPC cell line, to correlate their expression level with clinicopathologic features and to study the effect of HGF/c-Met system on the invasive and metastatic potential of NPC. METHODS: Forty-five biopsies were collected from pre-treatment NPC patients during the period from 1999 to 2003. Immunohistochemical staining was used to detect the expression of HGF-alpha subunit and c-Met protein in NPC tissues. The association between expression of these proteins and clinicopathologic features was statistically analyzed. The expression of HGF and c-Met, as detected by flow cytometry, in CNE-2 NPC cell line (with or without exogenous HGF) was compared. Western blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) were also applied to evaluate the protein and mRNA expression of c-Met in CNE-2 cells. RESULTS: In the 45 cases studied, the expression rate of c-Met was 91.1% (41/45). Only 1 case (2.2%, 1/45) showed positive signal for HGF in neoplastic cells. Instead, HGF was expressed in surrounding lymphocytes. The expression of c-Met positively correlated with lymph node metastasis (P = 0.024). There was also a positive correlation between expression of c-Met by tumor cells and expression of HGF by surrounding lymphocytes (r(s) = 0.450, P = 0.002). Moreover, the expression of c-Met was higher if there was a higher expression of HGF by lymphocytes (P = 0.009). However, there was no association between expression of c-Met and clinicopathologic features, such as age, gender, histopathologic type and clinical stage. After treatment with HGF for 24 hours, the percentage of c-Met-positive cells was significantly increased in CNE-2 cell line, from (46.6 +/- 9.02)% to (85.8 +/- 6.05)% (P = 0.003). The c-Met protein expression and c-Met mRNA level were also enhanced in CNE-2 cells with HGF treatment. However, endogenous HGF was not detected in CNE-2 cells, regardless of HGF treatment. CONCLUSIONS: HGF may play an important role in the development of NPC metastasis by inducing the expression of c-Met in tumor cells via a paracrine, instead of an autocrine, pathway.

[Targeted inhibition of vascular endothelial growth factor (VEGF) expression in human osteosarcoma cell line by antisense VEGF165 cDNA promoted by hypoxia reaction element].

OBJECTIVE: Utilizing the hypoxia inducible factor 1/hypoxia reaction element (HIF-1/ HRE) gene regulation system to construct antisense vascular endothelial growth factor (VEGF165) cDNA eukaryotic expression vector promoted by HRE, and investigate its targeted inhibiting VEGF expression of osteosarcoma cells in hypoxia environment. METHODS: Eukaryotic expression plasmid with HRE promoter was constructed containing luciferase reporter gene and antisense VEGF165 cDNA by using PCR and recombinant DNA techniques. The recombinant vectors were transfected into osteosarcoma cells with lipofectin method. Hypoxia-inducible reporter gene expression was determined by liquid scintillation analyzer and the expression of VEGF protein was detected by ELISA method. RESULTS: The eukaryotic expression plasmid containing antisense VEGF165 and luciferase promoted by HRE was constructed successfully. After being transferred into MG63 cells, luciferase expression was increased 3.5 x 10(2) times and VEGF protein expression decreased 45% under hypoxia condition. CONCLUSION: Antisense VEGF165 cDNA expression, efficiently realized by HRE promoter under hypoxia condition, provides an experimental basis for targeted antiangiogenesis of tumors.

[Effect of survivin antisense mRNA transfection on the growth and chemotherapy sensitivity of lymphoma cells].

OBJECTIVE: To study the effect of transfecting survivin antisense mRNA on growth and chemotherapy sensitivity of lymphoma cells. METHODS: Eukaryotic expression plasmid pcDNA3. 1-antisense (As) survivin was constructed and transfected into Jurkat T lymphoblastic lymphoma cell lines with high expression survivin mRNA by use of lipofectmine gene transfer technique. Expression of survivin mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemical and Western blot. The effect of transfecting survivin antisense mRNA on the growth of Jurkat cell lines was monitored by population doubling time (PDT) and Apoptotic indexes (AI). The morphologic features were observed in transfected cells by light and electric microscopes. MTT assay was used to analyze the response of transfected cells to CTX and MTX. RESULTS: Compared with the control cells, the expression of survivin mRNA and protein were reduced after transfected pcDNA3. 1-Assurvivin 48 h, 5 w and 6 w, PDT (52 h) was prolonged. Apoptotic indexes were higher in transfected antisense survivin mRNA cells [20.2% (48 h)], 6.2% (5 w) and 6.8% (6 w) than control ones [2.1%, 1.3% (48 h)] and [1.3% (5 w) and 1.0% (6 w)]. The cells grow slowly and the dead cells increase and some swelling and apoptotic cells were observed in transfected pcDNA3. 1-Assurvivin groups by invert, light and electric microscopes. The Jurkat cell line of transfected pcDNA3. 1-Assurvivin had higher sensitivity to CTX and MTX. The rate of inhibition was higher in transfected group. There is a significant difference between the transfected group and untransfected one, P < 0.05. CONCLUSIONS: The result indicated that survivin gene was very important for growth of Jurkat cells. To inhibit the expression of survivin will be significant in therapy of T lymphoblastic lymphoma. Survivin gene might be a target of therapy.

Association of E-cadherin and beta-catenin with metastasis in nasopharyngeal carcinoma.

BACKGROUND: This study was designed to detect methylation of E-cadherin gene promoter and gene mutation of beta-catenin in exon 3 and their expression of protein and mRNA in primary tumor and lymph node metastatic tumor of nasopharyngeal carcinoma (NPC), and investigate the mechanism of invasion and metastasis of neoplastic cells in NPC. METHODS: Fourty-two fresh biopsy samples were taken from untreated NPC patients at the Affiliated Hospital of Sun Yat-sen Medical College, Sun Yat-sen University, Guangzhou, China during the period of 1999-2002. Among them 21 were taken from primary tumors and the other 21 from lymph node metastatic tumors. The gene promoter methylation of E-cadherin was detected by methylation-specific PCR (MSP). The mutation in exon 3 of beta-catenin was detected by direct sequencing analysis. RT-PCR, Western blot and immunohistochemical staining were used to detect the mRNA and protein expression patterns in both primary and metastatic tumors of NPC. RESULTS: Down-regulated expression of E-cadherin in metastatic tumor was compared with that in primary tumor. Reduced expression of E-cadherin was found to be correlated with lymph node metastatic tumor of NPC (P = 0.004); but there was no obvious correlation between primary and metastatic tumors in the expression of beta-catenin (P = 0.698). The mRNA expression level of E-cadherin in metastatic tumors decreased significantly compared with that in primary tumors. However, little change was observed in the mRNA level of beta-catenin in different tumor tissues. Only 4 samples (19.1%) displayed gene promoter methylation of E-cadherin in primary tumor and 10 samples (47.6%) showed methylated form of E-cadherin. The gene promoter methylation of E-cadherin was more common in metastatic tumor than in primary tumor of NPC (P = 0.024). Only 2 (4.76%) of the 42 samples showed mutations in exon 3 of beta-catenin at 41 (T41A, ACC-->GCC) and codon 47 (S47T, AGT-->ACT). The cytoplasmic and nuclear expression of beta-catenin in tumor was not found in any samples of NPC. CONCLUSIONS: The results suggest that the downregulation of E-cadherin results from the gene promoter aberrant methylation of E-cadherin and that the methylation of E-cadherin plays an important role in invasion and metastasis of tumor cells in NPC. However, beta-catenin mutation is an infrequent event in NPC, and beta-catenin is not a critical factor influencing the invasion and metastasis of tumor cells in NPC.