Reduction of tumorigenicity of SMMC-7721 hepatoma cells by vascular endothelial growth factor antisense gene therapy

AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with control cells. CONCLUSION: Expression of antisense VEGF RNA in SMMC-7721 cells could decrease the tumorigenicity, and antisense-VEGF gene therapy may be an adjuvant treatment for hepatoma.

Expression of vascular endothelial growth factor and its role in oncogenesis of human gastric carcinoma

AIM: To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesis of human gastric carcinoma more directly. METHODS: The expression of VEGF and its receptor kinase-domain insert containing receptor (KDR) in human gastric cancer tissue were observed by immunohistochemical staining. VEGF levels were manipulated in human gastric cancer cell using eukaryotic expression constructs designed to express the complete VEGF(165) complimentary DNA in either the sense or antisense orientation. The biological changes of the cells were observed in which VEGF was up-regulated or down-regulated. RESULTS: VEGF-positive rate was 50%, and VEGF was mainly localized in the cytoplasm and membrane of the tumor cells, while KDR was mainly located in the membrane of vascular endothelial cells in gastric cancer tissues and peri-cancerous tissue. In 2 cases of 50 specimens, the gastric cancer cells expressed KDR, localized in both the cytoplasm and membrane. Introduction of VEGF(165) antisense into human gastric cancer cells (SGC-7901, immunofluorescence intensity, 31.6%)) resulted in a significant reduction in VEGF-specific messenger RNA and total and cell surface VEGF protein (immunofluorescence intensity, 8.9%) (P【0.05). Conversely, stable integration of VEGF(165) in the sense orientation resulted in an increase in cellular and cell surface VEGF (immunofluorescence intensity, 75.4%) (P【0.05). Lowered VEGF levels were associated with a marked decrease in the growth of nude mouse xenografted tumor (at 33 days postimplantation, tumor volume: 345.40 +/- 136.31 mm3)(P【0.05 vs control SGC-7901 group: 1534.40 +/- 362.88 mm3), whereas up-regulation of VEGF resulted in increased xenografted tumor size (at 33 days postimplantation, tumor volume: 2350.50 +/- 637.70 mm3) (P【0.05 vs control SGC-7901 group). CONCLUSION: This study provides direct evidence that VEGF plays an important role in the oncogenesis of human gastric cancer.

Epidermal growth factor receptor-targeted immune magnetic liposomes capture circulating colorectal tumor cells efficiently

AIM To compare the capacity of newly developed epidermal growth factor receptor(EGFR)-targeted immune magnetic liposomes(EILs) vs epithelial cell adhesion molecule(Ep CAM) immunomagnetic beads to capture colorectal circulating tumor cells(CTCs).METHODS EILs were prepared using a two-step method, and the magnetic and surface characteristics were confirmed. The efficiency of capturing colorectal CTCs as well as the specificity were compared between EILs and Ep CAM magnetic beads. RESULTS The obtained EILs had a lipid nanoparticle structure similar to cell membrane. Improved binding with cancer cells was seen in EILs compared with the method of coupling nano/microspheres with antibody. The binding increased as the contact time extended. Compared with Ep CAM immunomagnetic beads, EILs captured more CTCs in peripheral blood from colorectal cancer patients. The captured cells showed consistency with clinical diagnosis and pathology. Mutation analysis showed same results between captured CTCs and cancer tissues. CONCLUSION EGFR antibody-coated magnetic liposomes show high efficiency and specificity in capturing colorectal CTCs.

C-reactive protein,procalcitonin,interleukin-6,vascular endothelial growth factor and oxidative metabolites in diagnosis of infection and staging in patients with gastric cancer

AIM:The current study was to determine the serum/pLasma levels of VEGF,IL-6,malondialdehyde (MDA),nitric oxide (NO),PCT and CRP in gastric carcinoma and correlation with the stages of the disease and accompanying infection. METHODS:We examined the levels of serum VEGF,IL-6, PCT,CRP and plasma MDA,NO in 42 preoperative gastric cancer patients and 23 healthy subjects.There were infection anamneses that had no definite origin in 19 cancer patients. RESULTS:The VEGF levels (mean±SD; pg/mL) were 478.05±178.29 and 473.85±131.24 in gastric cancer patients with and without infection,respectively,and these values were not significantly different (P>0.05).The levels of VEGF, CRP,PCT,It-6,MDA and NO in cancer patients were significantly higher than those in healthy controls and the levels of CRP,PCT,It-6,MDA and NO were statistically increased in infection group when compared with non- infection group (P<0.001). CONCLUSION:Although serum VEGF concentrations were increased in gastric cancer,this increase might not be related to infection.CRP,PCT,IL-6,MDA and NO have obvious drawbacks in the diagnosis of infections in cancer patients. These markers may not help to identify infections in the primary evaluation of cancer patients and hence to avoid unnecessary antibiotic treatments as well as hospitalization. According to the results of this study,IL-6,MDA,NO and especially VEGF can be used as useful parameters to diagnose and grade gastric cancer.

Insulin-like growth factor 2 targets IGF1R signaling transduction to facilitate metastasis and imatinib resistance in gastrointestinal stromal tumors

BACKGROUND Gastrointestinal stromal tumors(GISTs)are typical gastrointestinal tract neoplasms.Imatinib is the first-line therapy for GIST patients.Drug resistance limits the long-term effectiveness of imatinib.The regulatory effect of insulin-like growth factor 2(IGF2)has been confirmed in various cancers and is related to resistance to chemotherapy and a worse prognosis.AIM To further investigate the mechanism of IGF2 specific to GISTs.METHODS IGF2 was screened and analyzed using Gene Expression Omnibus(GEO:GSE225819)data.After IGF2 knockdown or overexpression by transfection,the phenotypes(proliferation,migration,invasion,apoptosis)of GIST cells were characterized by cell counting kit 8,Transwell,and flow cytometry assays.We used western blotting to evaluate pathway-associated and epithelial-mesenchymal transition(EMT)-associated proteins.We injected transfected cells into nude mice to establish a tumor xenograft model and observed the occurrence and metastasis of GIST.RESULTS Data from the GEO indicated that IGF2 expression is high in GISTs,associated with liver metastasis,and closely related to drug resistance.GIST cells with high expression of IGF2 had increased proliferation and migration,invasiveness and EMT.Knockdown of IGF2 significantly inhibited those activities.In addition,OEIGF2 promoted GIST metastasis in vivo in nude mice.IGF2 activated IGF1R signaling in GIST cells,and IGF2/IGF1R-mediated glycolysis was required for GIST with liver metastasis.GIST cells with IGF2 knockdown were sensitive to imatinib treatment when IGF2 overexpression significantly raised imatinib resistance.Moreover,2-deoxy-D-glucose(a glycolysis inhibitor)treatment reversed IGF2 overexpressionmediated imatinib resistance in GISTs.CONCLUSION IGF2 targeting of IGF1R signaling inhibited metastasis and decreased imatinib resistance by driving glycolysis in GISTs.

Elevated levels of interleukin-1β, interleukin-6, tumor necrosis factor-α and vascular endothelial growth factor in patients with knee articular cartilage injury

BACKGROUND Inflammatory cytokines play a vital role in the occurrence of osteoarticular injury and inflammation. Whether inflammation-associated factors interleukin-1β(IL- 1β), IL-6, tumor necrosis factor-α(TNF-α) and vascular endothelial growth factor (VEGF) are involved in the pathogenesis of keen articular cartilage injury remains poorly understood. AIM To measure the levels of inflammatory factors [IL-1β, IL-6, TNF-α and VEGF] in patients with knee articular cartilage injury. METHODS Fifty-five patients with knee articular cartilage injury were selected as patient groups, who were divided into three grades [mild (n = 20), moderate (n = 19) and severe (n = 16)] according to disease severity and X-ray examinations. Meanwhile, 30 healthy individuals who underwent physical examination were selected as the control group. The levels of IL-1β, IL-6, TNF-α and VEGF were measured by ELISA and immunohistochemical staining. RESULTS Compared with the control group, patient groups displayed significantly higher levels of IL-1β, IL-6, TNF-α and VEGF, and the extent of increase was directly proportional to the severity of injury (P < 0.05). In addition, the number of cells with positive staining of IL-1β, IL-6, TNF-α and VEGF in the synovial membrane were significantly increased, along with increased disease severity (P < 0.05). After treatment, the scores of visual analogue scale and the Western Ontario and McMaster University of Orthopaedic Index in patient groups were 2.26 ± 1.13 and 15.56 ± 7.12 points, respectively, which were significantly lower than those before treatment (6.98 ± 1.32 and 49.48 ± 8.96). Correlation analysis suggested that IL-1β and TNF-α were positively correlated with VEGF. CONCLUSION IL-1β, IL-6, TNF-α and VEGF levels are increased in patients with knee articular cartilage injury, and are associated with the disease severity, indicating they might play an important role in the occurrence and development of knee articular cartilage injury. Furthermore, therapeutically targeting them might be a novel approach for the treatment of keen articular cartilage injury.

Effects of epidermal growth factor on the growth of human gastric cancer cell and the implanted tumor of nude mice

AIM: Epidermal growth factor (EGF) plays an important role in the regulation of gastrointestinal tissue growth and development, and it can stimulate epithelial proliferation, cell differentiation and growth. It has been established that the EGF can promote gastric cytoprotection and ulcer healing. But the potential ability of EGF to regulate the gastric cancer growth is unknown. This study is to investigate the influence of EGF on human gastric cancer cell and the implanted tumor growth of nude mice. METHODS: The cell growth rates of human gastric adenocarcinoma cell lines MKN-28, MKN-45, SGC-7901 and normal human gastric epithelial cells 3T3 were assessed when incubated with recombinant human EGF (rhEGF, 0.05, 0.1, 0.5, 1.0, 10, 50, 100 mg.L(-1)) using MTT method. The cells of MKN-28, MKN-45, SGC-7901 (gastric cancer tissue 1.5mm(3)) were implanted in the BALB/cA nude mice for 10 days.The EGF was given intraperitoneally (15, 30, 60 microg.kg(-1)) for 3 weeks. The body weights of the tumor-bearing animals and their tumor mass were measured afterwards to assess the mitogenic effect of rhEGF in the nude mice. RESULTS: Within the concentration range of 0.05-100mg.L(-1), rhEGF could increase the cell growth of normal 3T3 cells (cell growth rate 100% vs 102.8%, P【0.05), but partially restrain the gastric cancer cell growth. The latter effect was related to cell differentiation. In 15-60 microg/kg rhEGF groups, the mean implanted tumor mass of MKN-28 cell were 1.75 g, 1.91 g, 2.08 g/NS group 1.97 g (P】0.05), the mean tumor mass of SGC-7901 cell were 1.53 g, 1.07 g, 1.20 g/NS group 1.07 g (P】0.05), and for MKN-45 cell, the tumor mass were respectively 1.92 g, 1.29 g, 1.77 /NS group 1.82 g (P】0.05). So rhEGF had no obvious effect on implanted MKN-28, SGC-7901 and MKN-45 tumor growth. CONCLUSION: EGF has no stimulating effect on the human gastric cancer cell growth neither in vitro nor in vivo.

Transcription factor EGR-1 inhibits growth of hepatocellular carcinoma and esophageal carcinoma cell lines

AIM: The transcription factor EGR-1 (early growth response gene-1) plays an important role in cell growth, differentiation and development. It has identified that EGR-1 has significant transformation suppression activity in some neoplasms, such as fibrosarcoma, breast carcinoma. This experiment was designed to investigate the role of egr-1 in the cancerous process of hepatocellular carcinoma (HCC) and esophageal carcinoma (EC), and then to appraise the effects of EGR-1 on the growth of these tumor cells. METHODS: Firstly, the transcription and expression of egr-1 in HCC and EC, paracancerous tissues and their normal counterpart parts were detected by in situ hybridization and immunohistochemistry, with normal human breast and mouse brain tissues as positive controls. Egr-1 gene was then transfected into HCC (HHCC, SMMC7721) and EC (ECa109) cell lines in which no egr-1 transcription and expression were present. The cell growth speed, FCM cell cycle, plate clone formation and tumorigenicity in nude mice were observed and the controls were the cell lines transfected with vector only. RESULTS: Little or no egr-1 transcription and expression were detected in HCC, EC and normal liver tissues. The expression of egr-1 were found higher in hepatocellular paracancerous tissue (transcription level P=0.000; expression level P=0.143, probably because fewer in number of cases) and dysplastic tissue of esophageal cancer (transcription level P=0.000; expression level P=0.001). The growth rate of egr-1-transfected HHCC (HCC cell line) cells and ECa109 (EC cell line) cells was much slower than that of the controls. The proportion of S phase cell, clone formation and tumorigenicity were significantly lower than these of the controls' (decreased 45.5% in HHCC cells and 34.1% in ECa109 cells; 46.6% and 41.8%; 80.4% and 72.6% respectively). There were no obvious differences between SMMC7721 (HCC) egr-1-transfected cells and the controls with regard to the above items. CONCLUSION: The decreased expression of egr-1 might play a role in the dysregulation of normal growth in the cancerous process of HCC and EC. Egr-1 gene of transfected HHCC and ECa109 cells showed obvious suppression of the cell growth and malignant phenotypes, but no suppression in SMMC7721 (HCC cell line) cells.

骨代谢生化指标临床应用专家共识(2023修订版)

骨代谢生化指标的临床应用,为骨质疏松的诊断、鉴别诊断、预测骨折风险及抗骨质疏松治疗疗效评价提供了分子生物学依据,并在骨质疏松流行病学研究、发病机制、骨质疏松药物开发研究方面具有重要意义。由于骨代谢生化指标检测特异性强、灵敏度高,其应用日趋广泛。该文检索了大量中外文献,编审了《骨代谢生化指标临床应用专家共识》(2023修订版),对骨代谢生化指标的分类、骨代谢生化指标的方法学以及生物学意义、骨代谢指标的检测变异等进行了论述。

Oncogenic Osteomalacia Associated with Phosphaturic Mesenchymal Tumor of the Knee: Case Presentation and Review of the Literature

Oncogenic osteomalacia (OOM) is an uncommon metabolic and bone disease caused by fibroblast growth factor 23 (FGF23), a phosphaturic factor produced by phosphaturic mesenchymal tumors (mixed connective tissue variant, PMTMCTV) characterized by phosphate leakage from kidneys and subsequent hypophosphatemia. In this paper, we present the case of a patient, 42-year-old woman affected by left side limp and pain involving lumbar spine, pelvis and hip joints, referred to the Rheumatology Department of our Hospital for the treatment of a suspected sero-negative spondilo-arthritis. During hospitalization patient began an immuno-suppressive therapy with TNF-alpha inhibitors associated with Pamidornate, Indometacin, Esomeprazole and vitamin D3. Nevertheless pain did not decrease and a new examination found a worst hypophosphatemia (1 mg/dl) with normal Ca and PTH’s plasma values. During the same check-up a painful bulge on the anterior part of the right knee was observed and the Magnetic Resonance Imaging scan revealed an ovular solid lesion in the soft tissue closed to the upper part of the patella. Histological analysis identified the lesion as a PMTMCTV. After surgical removal patient got complete recovery. We will discuss about diagnostic evaluation, differential diagnosis and treatment.

Immunohistochemical demonstration of transforming growth factor－β and bone morphogenetic protein in salivary gland tumors

Transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP)were related to embryonic development and the differentiation of many types of cells. Recent studies showed that they might play an important role in regulating the differentiation o

Effects of endostatin on expression of vascular endothelial growth factor and its receptors and neovascularization in colonic carcinoma implanted in nude mice

AIM:To investigate the antiangiogenic effects of endostatin on colonic carcinoma cell line implanted in nude mice and its mechanism. METHODS:Nude mice underwent subcutaneous injection with LS-174t colonic carcinoma cell line to generate carcinoma and were randomly separated into two groups.Mice received injection of vehicle or endostatin every day for two weeks. After the tumor was harvested,the tumor volumes were determined,and the expressions of CD34,VEGF and FIk-1 were examined by immunohistochemical method. RESULTS:Tumor volume was significantly inhibited in the endostatin group(84.17%)and tumor weight was significantly inhibited in the endostatin group(0.197±0.049) compared to the control group(1.198±0.105)(F=22.56, P=0.001),microvessel density(MVD)was significantly decreased in the treated group(31.857±3.515)compared to the control group(100.143±4.290)(F=151.62,P<0.001). Furthermore,the expression of FIk-1 was significantly inhibited in the treated group(34.29%) ompared to the control group(8.57%)(X^2=13.745,P=0.001).However no significant decrease was observed in the expression of vascular endothelial growth factor(VEGF)between these two groups(X^2=0.119,P=0.730). CONCLUSION:Endostatin can inhibit tumor growth and angiogenesis by blocking Vegf/FIk-1 pathway.This experiment provides the theory basis for developing a new anti-carcinoma drug through studying the properties of anti-angiogenesis inhibitors.

Tumor-induced osteomalacia with elevated fibroblast growth factor 23: a case of phosphaturic mesenchymal tumor mixed with connective tissue variants and review of the literature

Tumor-induced osteomalacia (TIO), or oncogenic osteomalacia (OOM), is a rare acquired paraneoplastic disease characterized by renal phosphate wasting and hypophosphatemia. Recent evidence shows that tumor-overexpressed fibroblast growth factor 23 (FGF23) is responsible for the hypophosphatemia and osteomalacia. The tumors associated with TIO are usually phosphaturic mesenchymal tumor mixed connective tissue variants (PMTMCT). Surgical removal of the responsible tumors is clinically essential for the treatment of TIO. However, identifying the responsible tumors is often difficult. Here, we report a case of a TIO patient with elevated serum FGF23 levels suffering from bone pain and hypophosphatemia for more than three years. A tumor was finally located in first metacarpal bone by octreotide scintigraphy and she was cured by surgery. After complete excision of the tumor, serum FGF23 levels rapidly decreased, dropping to 54.7% of the preoperative level one hour after surgery and eventually to a little below normal. The patient's serum phosphate level rapidly improved and returned to normal level in four days. Accordingly, her clinical symptoms were greatly improved within one month after surgery. There was no sign of tumor recurrence during an 18-month period of follow-up. According to pathology, the tumor was originally diagnosed as "glomangioma" based upon a biopsy sample, "proliferative giant cell tumor of tendon sheath" based upon sections of tumor, and finally diagnosed as PMTMCT by consultation one year after surgery. In conclusion, although an extremely rare disease, clinicians and pathologists should be aware of the existence of TIO and PMTMCT, respectively.

Demethylation of tumor necrosis factor-α converting enzyme promoter associated with high hepatitis B e antigen level in chronic hepatitis B

AIM: To evaluate tumor necrosis factor-α converting enzyme(TACE) methylation status in patients with chronic hepatitis B(CHB).METHODS: Eighty patients with hepatitis B e antigen(HBe Ag)-positive CHB, 80 with HBe Ag-negative CHB, and 40 healthy controls(HCs) were randomly enrolled in this study. Genomic DNA was extracted from peripheral blood mononuclear cells and methylation status of TACE promoter was determined by methylation-specific polymerase chain reaction. The clinical and laboratory parameters were collected.RESULTS: One hundred and thirty of 160 patients with CHB(81.25%) and 38 of 40 HCs(95%) displayed TACE promoter methylation. The difference was significant(χ2 = 4.501, P < 0.05). TACE promoter methylation frequency in HBe Ag-positive CHB(58/80, 72.5%) was significantly lower than that in HBe Ag-negative CHB(72/80, 90%; χ2 = 8.041, P < 0.01) and HCs(χ2 = 8.438, P < 0.01). However, no significant difference was observed in the methylation frequency between HBe Agnegative CHB and HCs(χ2 = 0.873, P > 0.05). In the HBe Ag-positive group, TACE methylation frequency was significantly negatively correlated with HBe Ag(r =-0.602, P < 0.01), alanine aminotransferase(r =-0.461, P < 0.01) and aspartate aminotransferase(r =-0.329, P < 0.01). CONCLUSION: Patients with HBe Ag-positive CHB have aberrant demethylation of the TACE promoter, which may potentially serve as a biomarker for HBe Ag seroconversion.

骨代谢生化指标临床应用专家共识(2020)

骨代谢生化指标包括钙磷代谢调节指标、骨形成标志物、骨吸收标志物、激素与细胞因子。骨代谢生化指标分别来源于骨、软骨、软组织、皮肤、肝、肾、小肠、血液及内分泌腺体等,是由成骨细胞或破骨细胞分泌的酶和激素,以及骨基质的胶原蛋白或非胶原蛋白代谢产物。骨代谢生化指标可及时反映骨转换状态,灵敏度高、特异性强,用于骨质疏松诊断分型、预测骨折风险、抗骨质疏松治疗疗效评价,以及代谢性骨病的诊断与鉴别诊断。并且在骨质疏松流行病学、发病机制、骨质疏松药物的研究方面具有重要的临床意义。本文对《骨代谢生化指标临床应用专家共识(2019)》进行了修订,共有41处修改,保留了经典文献,删减了部分内容,增加了近三年的文献,收集整理了骨代谢指标实验检测参考范围。

Expression of p-STAT3 and vascular endothelial growth factor in MNNG-induced precancerous lesions and gastric tumors in rats

AIM: To investigate the dynamic expression of p-signal transducer and activator of transcription 3 (STAT3) and vascular endothelial growth factor (VEGF) in the formation of gastric tumors induced by drinking water containing N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) in Wistar rats. METHODS: One hundred and twenty Wistar rats were randomly divided into two groups (60 in each group): Control group and Model group. The rats in each group were then randomly divided into three groups (20 in each group): C/M15, C/M25 and C/M40 (15, 25 and 40 represent the number of feeding weeks from termination). Rats in the control group received normal drinking water and rats in the model group received drinking water containing 100 μg/mL MNNG. Stomach tissues were collected at the end of the 15th, 25th and 40th week, respectively, for microscopic measurement using hematoxylin and eosin staining. The expression of p-STAT3 and VEGF in different pathological types of gastric tissue, including normal, inflammation, atrophy, hyperplasia and gastric stromal tumor, was observed by immunohistochemistry and Western blot, and the corelation between p-STAT3 and VEGF was analyzed. RESULTS: (1) The expression of p-STAT3 in tissue with gastritis, atrophy, dysplasia and gastric stromal tumor were significantly increased in the model group compared with the control group (2.5 ± 1.0, 2.75 ± 0.36, 6.2 ± 0.45, 5.67 ± 0.55 vs 0.75 ± 0.36, P = 0.026, 0.035, 0.001, 0.002, respectively); the expression of p-STAT3 in tissue with dysplasia was higher than that in samples with gastritis or atrophy (6.2 ± 0.45 vs 2.5 ± 1.0, P = 0.006; 6.2 ± 0.45 vs 2.75 ± 0.36, P = 0.005, respectively); however, the expression of p-STAT3 in gastritis and atrophy was not significantly different (P > 0.05); (2) the expression of VEGF in tissue with gastritis, atrophy, dysplasia and gastric stromal tumor was significantly increased in the model group compared with normal gastric mucosa; and the expression of VEGF in tissue with dysplasia was higher than that in tissue with inflammation and atrophy (10.8 ± 1.96 vs 7.62 ± 0.25, P = 0.029; 10.8 ± 1.96 vs 6.26 ± 0.76, P = 0.033, respectively); similarly, the expression of VEGF in tissue with gastritis and atrophy was not significantly different (P > 0.05); and (3) the expression of VEGF was positively correlated with p-STAT3. CONCLUSION: p-STAT3 plays an important role in gastric cancer formation by regulating the expression of VEGF to promote the progression of gastric tumor from gastritis.

Inhibition of rheumatoid arthritis by blocking connective tissue growth factor

The pathogenesis of rheumatoid arthritis(RA) remains to be completely elucidated so far; however, it is known that proinflammatory cytokines play a pivotal role in the induction of RA. Tumor necrosis factor(TNF-α), in particular, is considered to play a central role in bone destruction by mediating the abnormal activation of osteoclasts or the production of proteolytic enzymes through direct or indirect mechanisms. The use of TNF-α blocking agents has a significant impact on RA therapy. Anti-TNF-α blocking agents such as infliximab are very effective for treatment of RA, especially for the prevention of articular destruction. We have previously shown that several proteins exhibited extensive changes in their expression after amelioration of RA with infliximab treatment. Among the proteins, connective tissue growth factor(CTGF) has a significantrole for the development of RA. Herein, we review the function of CTGF in the pathogenesis of RA and discuss the possibility of a novel treatment for RA. We propose that CTGF is a potentially novel effector molecule in the pathogenesis of RA. Blocking the CTGF pathways by biological agents may have great beneficial effect in patients with RA.

The role of endotoxin,TNF-α,and IL-6 in inducing the state of growth hormone insensitivity

AIM: Critical illnesses such as sepsis, trauma, and burns cause a growth hormone insensitivity, which leads to an increased negative nitrogen balance. Endotoxin is generously released into blood under these conditions and stimulates the production of proinflammatory cytokines such as TNF-alpha, IL-6, and IL-1, which may play a very important role in inducing the growth hormone insensitivity. The objective of this current study was to investigate the role of endotoxin, TNF-alpha and IL-6 in inducing the growth hormone insensitivity at the receptor and post-receptor levels. METHODS: Spague-Dawley rats were injected with endotoxin, TNF-alpha, and IL-6, respectively and part of rats injected with endotoxin was treated with exogenous somatotropin simultaneously. All rats were killed at different time points. The expression of IGF-I, GHR, SOCS-3 and beta-actin mRNA in the liver was detected by RT-PCR and the GH levels were measured by radioimmunoassay, the levels of TNF-alpha and IL-6 were detected by ELISA. RESULTS: There was no significant difference in serous GH levels between experimental group and control rats after endotoxin injection, however, liver IGF-I mRNA expression had been obviously down-regulated in endotoxemic rats. Liver GHR mRNA expression also had a predominant down-regulation after endotoxin injection. The lowest regulation of liver IGF-I mRNA expression occurred at 12h after LPS injection, being decreased by 53% compared with control rats. For GHR mRNA expression, the lowest expression occurred at 8h and had a 81% decrease. Although SOCS-3 mRNA was weakly expressed in control rats, it was strongly up-regulated after LPS injection and had a 7.84 times increase compared with control rats. Exogenous GH could enhance IGF-I mRNA expression in control rats, but it did fail to prevent the decline in IGF-I mRNA expression in endotoxemic rats. Endotoxin stimulated the production of TNF-alpha and IL-6, and the elevated IL-6 levels was shown a positive correlation with increased SOCS-3 mRNA expression. The liver GHR mRNA expression was obviously down-regulated after TNF-alpha iv injection and had a 40% decrease at 8h, but the liver SOCS-3 mRNA expression was the 4.94 times up-regulation occurred at 40 min after IL-6 injection. CONCLUSION: The growth hormone insensitivity could be induced by LPS injection, which was associated with down-regulated GHR mRNA expression at receptor level and with up-regulated SOCS-3 mRNA expression at post-receptor level. The in vivo biological activities of LPS were mediated by TNF-alpha and IL-6 indirectly, and TNF-alpha and IL-6 may exert their effects on the receptor and post-receptor levels respectively.

Experimental and clinic-opathologic study on the relationship between transcription factor Egr-1 and esophageal carcinoma

AIM: To observe the growth suppression effect of exogenous introduction of early growth response gene-1 (Egr-1 gene) on esophageal carcinoma tissue as well as on esophageal carcinoma cell line Eca109 and to explore the potential application of Egr-1 gene in gene therapy of tumor. METHODS: Eukaryotic expression vector of PCMV-Egr-1 plasmid was introduced into Eca109 cell line which expressed no Egr-1 protein originally with lipofectamine transfection method. The introduction and expression of PCMV-Egr-1 plasmid into Eca109 cell line was confirmed by G418 selection culture, PCR amplification of neogene contained in the vector, Western blot analysis and immunocytochemical analysis. The cell growth curve, soft agar colony formation rate and tumorigenicity in SCID mice were examined to demonstrate the growth suppression effect of exogenous Egr-1 gene on Eca109 cell line. The Egr-1 mRNA and Egr-1 protein were also detected in 50 surgical specimens of esophageal carcinoma by in situ hybridization and immunohistochemistry. RESULTS: Exogenous Egr-1 gene was introduced successfully into Eca109 cell line and expressed Egr-1 protein stably. The transfected Eca109 cell line grew more slowly than control Eca109 as shown by cell growth curves, the soft agar colony formation rate (4.0% vs 6.9%, P 【 0.01) and the average growth rate of tumor in SCID mice (35.5 +/- 7.6 vs 65.8 +/- 7.6, P 【 0.05). The expression level of Egr-1 mRNA and protein significantly increased in dysplastic epithelia adjacent to cancer rather than in cancer tissues (65.8% vs 20.0% by ISH and 57.9% vs 0.01). CONCLUSION: Exogenous Egr-1 gene shows the strong effect of growth inhibition in Eca109 cell line. Egr-1 in the cancer tissue shows down-regulated expression that supports the inhibited function of Egr-1 in cancer growth and suggests Egr-1 may have an important role in gene therapy of esophageal carcinoma.

Dual neuronal response to tumor necrosis factor-alpha following spinal cord injury

BACKGROUND： Numerous studies have shown that tumor necrosis factor α （TNF-α） is closely correlated with spinal cord injury （SCI）, but the mechanisms of TNF-α and therapeutic treatments for SCI are still poorly understood. OBJECTIVE： To determine the role of TNF-α in the pathogenesis of SCI. DESIGN, TIME AND SETTING： An in vivo experiment based on genetically engineered animals was performed at the Medical University of South Carolina, Charleston, South Carolina, USA, between June 2007 and October 2008. MATERIALS： TNF-α transgenic rats （Xenogen Biosciences in Cranbury, New Jersey, USA） were utilized in this study. METHODS： TNF-α transgenic （tg） and wild-type （WT） rats underwent a complete single-level laminectomy at the 10^th thoracic vertebra （T10）. MAIN OUTCOME MEASURES： Motor function of rat hindlimb was assessed using the Basso, Beattie, and Bresnahan hindlimb locomotor rating scale. Histological evaluation of spinal cord tissue loss was conducted. Immunohistochemistry for astrocytes, microglia/macrophages, and TNF receptors （TNFRs） was performed on spinal cord tissue sections. TNF-α mRNA expression was detected by real-time polymerase chain reaction. The concentrations of nerve growth factor （NGF） and brain-derived neurotrophic factor （BDNF） in the supernatant were determined using an enzyme-linked immunosorbent assay kit for rat NGF or BDNF, respectively. The rats were injected subcutaneously with etanercept to verify that TNF-α was the direct effect of the modulation of behavioral and neurodegenerative outcomes in the TNF-α tg rats. RESULTS： TNF-α tg rats showed higher expression of TNF-α mRNA in the spinal cord prior to SCI. TNF-α tg rats showed worse motor deficits than WT rats in the acute period （〈 3 days） after SCI （P 〈 0.01）, while in the chronic period, TNF-α tg rats exhibited persistent elevated baseline levels of TNF-α mRNA and improved recovery in motor function and tissue healing compared to WT rats （P 〈 0.01 ）. Following SCI, the number of microglia/macrophages in TNF-α tg rat was always greater than in WT rat （P 〈 0.01）. There were no significant differences in NGF and BDNF levels in the supernatant of spinal cord homogenates. TNFR1 expression was significantly greater in the TNF-α tg rats compared to the WT rats （P 〈 0.01）. However, TNFR2 expression did not reveal a significant increase in the TNF-α tg rats compared to the WT rats. Finally, treatment with etanercept reduced injury acutely, but exacerbated the injury chronically. CONCLUSION： Overexpression of TNF-α is deleterious in the acute phase, but beneficial in the chronic phase in the response to SCI. The role of TNF-α post-injury may depend on TNF-α expression in the spinal cord and its differential binding to TNFRI. Our observations may have clinical relevance that antagonists or inhibitors of TNF-α could be administered within the early time window post-injury, and appropriate amounts of TNF-α could be administered during the chronic stage, in order to improve the final neurological recovery in patients with SCI.