守宫硫酸多糖对肝癌SMMC-7721细胞分化和增殖的影响

目的:研究守宫硫酸多糖(Gepsin)对肝癌SMMC-7721细胞分化和增殖的影响,并进一步探讨其可能的机制。方法:将不同浓度的Gepsin加入对数生长的SMMC-7721细胞进行培养,在不同的时间利用台盼兰染色观察细胞的增殖情况。同时,收集细胞培养上清液,利用联合放免法,以溴钾酚绿法分别观察Gepsin对甲胎蛋白(AFP)、白蛋白(ALB)分泌的影响;利用酶联免疫吸附试验法观察Gepsin对转化生长因子(TGF)-β1、血管内皮生长因子(VEGF)分泌的影响;利用光镜观察Gepsin对SMMC-7721细胞形态的影响。结果:随着处理组Gepsin的浓度升高,SMMC-7721细胞的增殖明显受到抑制;而对细胞活率无明显影响。Gepsin可增加培养上清中的ALB分泌量,降低AFP分泌量。光学显微镜下可见加入Gepsin后细胞的形态由圆形变为纺锤形。Gepsin对培养上清分泌的VEGF无影响,但可使TGF-β1增加。结论:Gepsin可明显抑制肝癌细胞SMMC-7721的增殖,诱导SMMC-7721细胞分化。其机制可能是通过上调TGF-β1来诱导肝癌细胞分化实现的。

Effects of Ghrelin Antisense Inhibition on VEGF and Its Receptor Flt-1 mRNA Expression

[ Objective] This study was to investigate the effect of VEGF and its receptor Fit-1 mRNA expression in Mongolia sheep umbilical vein endothelial cells by ghrelin antisense inhibition. [ Method] Experiments were divided into 4 groups： group Ⅰ （blank control group） ; group Ⅱ （liposome group） ; group Ⅲ （SCON group： 20 μmol/L sense oligonucleotide） ; group Ⅳ （ASCON： 20 μmol/L antisense oligonucleotide）. VEGF and its receptor Fit-1 mRNA expression changes were detected by using real-time fluorescence quantitative detection after 24, 36 and 48 h. [ Result] The expression of VEGF mRNA in group Ⅰ, group Ⅱ were insignificantly different at higher expression levels, and did not change significantly with the time; the expression of VEGF mRNA in group Ⅲ assumed a slight decrease, but there were no significant differences between group I and group Ⅱ （P 〉0.05）, the expression of VEGF mRNA in group Ⅳ（antisense oligonucleotide group ） decreased significantly （P 〈 0.05） ; the expression of VEGF receptor FLT-1 mRNA was similar to that of VEGF. [ Conclusion] Antisense inhibition ghrelin has a downward effect to the expression of VEGF and its receptor Fit-1 the mRNA.

Feeder-free maintenance of hESCs in mesenchymal stem cell-conditioned media： distinct requirements for TGF-β and IGF-Ⅱ

A paracrine regulation was recently proposed in human embryonic stem cells （hESCs） grown in mouse embryonic fibroblast （MEF）-conditioned media （MEF-CM）, where hESCs spontaneously differentiate into autologous fibroblastlike cells to maintain culture homeostasis by producing TGF-β and insulin-like growth factor-lI （IGF-Ⅱ） in response to basic fibroblast growth factor （bFGF）. Although the importance of TGF-β family members in the maintenance of pluripotency of hESCs is widely established, very little is known about the role of IGF-Ⅱ. In order to ease hESC cul- ture conditions and to reduce xenogenic components, we sought （i） to determine whether hESCs can be maintained stable and pluripotent using CM from human foreskin fibroblasts （HFFs） and human mesenchymal stem cells （hM- SCs） rather than MEF-CM, and （ii） to analyze whether the cooperation of bFGF with TGF-β and IGF-Ⅱ to maintain hESCs in MEF-CM may be extrapolated to hESCs maintained in allogeneic mesenchymal stem cell （MSC）-CM and HFF-CM. We found that MSCs and HFFs express all FGF receptors （FGFR1-4） and specifically produce TGF-β in response to bFGF. However, HFFs but not MSCs secrete IGF-Ⅱ. Despite the absence of IGF-Ⅱ in MSC-CM, hESC pluripotency and culture homeostasis were successfully maintained in MSC-CM for over 37 passages. Human ESCs derived on MSCs and hESCs maintained in MSC-CM retained hESC morphology, euploidy, expression of surface markers and transcription factors linked to pluripotency and displayed in vitro and in vivo multilineage developmental potential, suggesting that IGF-Ⅱ may be dispensable for hESC pluripotency. In fact, IGF-Ⅱ blocking had no effect on the homeostasis of hESC cultures maintained either on HFF-CM or on MSC-CM. These data indicate that hESCs are successfully maintained feeder-free with IGF-Ⅱ-lacking MSC-CM, and that the previously proposed paracrine mechanism by which bFGF cooperates with TGF-β and IGF-Ⅱ in the maintenance of hESCs in MEF-CM may not be fully extrapolated to hESCs maintained in CM from human MSCs.

DIAGNOSTIC VALUE OF SERUM INSULIN- LIKE GROWTH FACTOR BINDING PROTEIN- 3 IN CHILDREN WITH OR WITHOUT GROWTH HORMONE DEFICIENCY

OBJECTIVE: To study the value of serum insulin-like growth factor binding protein-3 (IGFBP-3) levels in differential diagnosis of growth hormone deficiency (GHD). METHODS: To measure serum IGFBP-3 levels by RIA in normal children and adolescents, GHD children and short-stature children without GHD. RESULTS: Serum level of IGFBP-3 in 129 children with untreated GHD and with no pubertal development was 1.6 +/- 0.9 mg/L, which was less than that in normal group of the same age, but overlapped with the normal children in Tanner stage I. After six-month treatment with recombinant human growth hormone (rhGH), serum level of IGFBP-3 in 59 GHD significantly increased from 1.3 +/- 0.7 mg/L to 2.7 +/- 0.9 mg/L, accompanied by an increase of body heights, growth velocities and serum level of IGF-1. Serum level of IGFBP-3 in 55 short-stature children without GHD was 3.3 +/- 2.2 mg/L, which was not significantly different from that in normal group. CONCLUSION: Serum IGFBP-3 level can reflect the status of GH secretion in children with GHD and is a useful marker for differential diagnosis of GHD.

Reactivation of the insulin-like growth factor-Ⅱsignaling pathway in human hepatocellular carcinoma

Constitutive activation of the insulin-like growth factor (IGF)-signaling axis is frequently observed in human hepatocellular carcinoma(HCC).Especially the over- expression of the fetal growth factor IGF-Ⅱ,IGF-Ⅰ receptor(IGF-IR),and cytoplasmic downstream effectors such as insulin-receptor substrates(IRS)contribute to proliferation,anti-apoptosis,and invasive behavior. This review focuses on the relevant alterations in this signaling pathway and independent in vivo models that support the central role IGF-Ⅱsignaling during HCC development and progression.Since this pathway has become the center of interest as a target for potential anti-cancer therapy in many types of malignancies,various experimental strategies have been developed,including neutralizing antibodies and selective receptor kinase inhibitors,with respect to the specific and efficient reduction of oncogenic IGF-Ⅱ/IGF-IR-signaling.

ERK1/2 contributes negative regulation to STAT3 activity in HSS-transfected HepG2 cells

Signal transducer and activator of transcription 3 (STAT3) is a recently characterized transcription factor which is essential to liver regeneration. We have previously reported that hepatic stimulator substance (HSS), a novel growthpromoting substance, phosphorylated the epidermal growth factor (EGF) receptors and activated downstream RasMAP kinase (extracellular signal-regulated kinases, ERK1/2) cascade. However, whether HSS signal is related to STAT3pathway remains unclear. The present study is aiming to explore the regulatory effect of activation of ERK1/2 evoked by HSS on STAT3 phosphorylation and STAT3 signaling. Human hepatoma cell line HepG2 was stably transfected with HSS cDNA and HSS expression was measured by Northern blot. The results showed that the transfection of HSS into HepG2 resulted in remarkable increase in cellular proliferation as compared with the non-transfected cells, and it was further proved that the cellular proliferation in the HSS-transfected cells was related to ERK1/2 activation. Treatment of the cells with 50 μM of PD98059, an ERK1/2 specific upstream inhibitor, resulted in ERK1/2 inactivation completely.Inhibition of ERK1/2 allowed the tyrosine of STAT3 to be phosphorylated in a dose-dependent manner to PD98059.Furthermore, transient transfection of STAT3 mutant (STAT3S727A) into HSS-bearing cells could remarkably reverse the inhibitory effect of ERK1/2 on STAT3 phosphorylation. Based upon these results, it is concluded that ERK1/2negatively modulates STAT3 phosphorylation and this function is dependent on residual serine-727 (S727) of STAT3.

Is diabetes a causal agent for colorectal cancer? Pathophysiological and molecular mechanisms

The possible relationship between diabetes mellitus (DM) and colorectal cancer (CRC), concerning pathophysiological and molecular mechanisms is highlighted in this review. The most recent and complete articles and developments in this particular field were thoroughly reviewed. Common risk factors, such as obesity, sedentary lifestyle, and Western diet between DM and CRC, led to the theory that DM might be a causal agent for CRC development. Various studies have connected type 2 DM and CRC, either proximal or distal, in both sexes. Additionally, chronic insulin treatment has been linked with increased colorectal tumor risk among type 2 diabetic patients. Interestingly, elevated hemoglobin A1c has been proven to be an independent predictor of aggressive clinical behavior in CRC patients. These mechanisms include the insulin-like growth factor-hyperinsulinemia theory and the participation of oncogenic intracellular signaling pathways. Furthermore, it has been proposed that Cox-2 inhibitors might have a role in decreasing the incidence of CRC. Finally, the use of statins to reduce the risk for colon cancer in patients with diabetes has remained controversial. Diabetic patients over 50 should receive counseling regarding their elevated risk for CRC, and screening colonoscopy should be recommended before initiating insulin therapy. However, there are no current guidelines, and this strategy is not yet applicable to some countries, as the corresponding risk would not allow screening colonoscopy to be adopted. There is strong evidence to indicate that DM is a causal agent for CRC development. This conclusion provides new impetus for re-evaluating CRC screening worldwide.

Liver insulin-like growth factor 2 methylation in hepatitis C virus cirrhosis and further occurrence of hepatocellular carcinoma

AIM: To assess the predictive value of the insulinlike growth factor 2 (Igf2) methylation profile for the occurrence of Hepatocellular Carcinoma (HCC) in hepatitis C (HCV) cirrhosis. METHODS: Patients with: (1) biopsy-proven compensated HCV cirrhosis; (2) available baseline frozen liver sample; (3) absence of detectable HCC; (4) regular screening for HCC; (5) informed consent for genetic analysis were studied. After DNA extraction from liver samples and bisulfite treatment, unbiased PCR and DHPLC analysis were performed for methylation analysis at the Igf2 locus. The predictive value of the Igf2 methylation profile for HCC wasassessed by Kaplan-Meier and Cox methods. RESULTS: Among 94 included patients, 20 developed an HCC during follow-up (6.9 ± 3.2 years). The methylation profile was hypomethylated, intermediate and hypermethylated in 13, 64 and 17 cases, respectively. In univariate analysis, two baseline parameters were associated with the occurrence of HCC: age (P = 0.01) and prothrombin (P = 0.04). The test of linear tendency between the three ordered levels of Igf2 methylation and probability of HCC occurrence was significant (Log Rank, P = 0.043; Breslow, P = 0.037; Tarone-Ware, P = 0.039). CONCLUSION: These results suggest that hypomethylation at the Igf2 locus in the liver could be predictive for HCC occurrence in HCV cirrhosis.

Prediction of HLA-A 2.1-restricted CTL epitopes from IGFBP7 antigen of lung carcinoma

Objective： With the development of peptide-based cancer specific immunotherapy, the prediction of CTL epitopes from insulin-like growth factor-binding protein 7 （IGFBP7） is very important for some research about tumor metastasis. Because HLA-A2.1-expressing individuals cover 〉50% in the population of China, we aimed at identifying IGFBPT-encoded peptide presented by HLA-A2.1. Methods： In our study, a HLA-A2.1 restricted CTL epitope was identified by using the following two-step procedure： （a） computer-based epitope prediction from the amino acid sequence of IGFBP7 antigen; （b） Validation with epitope molecular modeling. Results： We obtained four epitopes with high immunogenicity scores by all of the three algorithms, i.e., BIMAS, SYFPEITH1 and IMTECH. Each of the four candidates satisfied the criteria of the HLA-A2.1- restricted CTL epitopes in molecular modeling analysis. Conclusion： The combination of BIMAS, SYFPEITHI and IMTECH method can improve the prediction efficiency and accuracy. Due to this research herein, this four epitopes have potential value for further studied, also have potential application in peptide-mediated immunotherapy. These epitopes may be useful in the design of therapeutic peptide vaccine for lung carcinoma and as immunotherapeutic strategies against lung carcinoma after identified by immunology experiment.

hVEGF165 Expression in Escherichia coli Conserves Its Biological Function

The paper describes the expression of human protein VEGF165 in Escherichia coli and its purification. This growth factor isoform contains exon 7, which is essential for binding to extracellular domain of VEGF receptor 2, located on endothelial cells lining the surface of blood vessels. This binding stimulates the cascade of downstream signalling events leading to process known as angiogenesis, hVEGF165 overexpressed with His-tag in BL21 E. coli cells forms inclusion bodies （insoluble protein）, so the research found the procedure for its solubilization and purification on a Nickel based affinity chromatography. Although this eukaryotic signal protein needs posttranslational processing for its full function as a homodimer, author verified the biological activity of our hVEGF165 protein, obtained as monomer, by wound healing test.

Correlation between the expression of vascular endothelial growth factor c and C-erbB-2 in human breast cancer

Objective: We aimed to study the transcription level of VEGF-C in human breast cancer tissue, and explore the correlations with the expression of C-erbB-2. Methods: The expression of VEGF-C mRNA in 51 cases of human breast cancer was assessed by hybridization in situ. The expressions of C-erbB-2 was assessed by immunohistochemistry. Results: The positive rate of VEGF-C mRNA was 54.9% in 51 cases of breast cancer. The transcription level had correlation with tumor size and status of lymph nodes(P < 0.05). The expression of VEGF-C mRNA had a positive correlation with the expression of C-erbB-2(P < 0.05). Conclusion: The up-expression of VEGF-C has a significant correlation with the malignancy level and clinical stage of breast cancer. The combined detection of VEGF-C, C-erbB-2 may help to estimate the prognosis of patients with breast cancer and study on thetherapeutic implications.

Inhibitory Effect of IGF1R siRNA on the growth of human liver cancer SMMC7721 cell xenograft in nude mice

Objective： To investigate the effect of insulin-like growth factor 1 receptor （IGF1R） small interfering RNA （siRNA） on the growth of human liver cancer SMMC7721 cell xenograft in nude mice. Methods： siRNA targeting IGF1R was designed, and plasmid SMMC7721-1GF1R-siRNA was constructed and transfected into SMMC7721 cells （SMMC7721-1GF1R-siRNA cells）; the cells transfected with SMMC7721-1GF1 R-mutation （SMMC7721-1GF1 R-mutation cells） were used as negative con- trol, and untransfected cells as empty control. Stable cell clones were screened by G418, and transplanted into nude mice to establish cancer xenograft. Tumor growth was monitored. Tumor morphology was observed with HE staining. The expression of IGF1R protein in tumor tissues was detected by Western blot. Microvessel density （MVD） in tumor tissues was detected by SP immunohistochemistry. Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling （TUNEL） assay. Results： The tumor volume was significantly smaller in SMMC7721-1GF1R-siRNA group than in SMMC7721-1GF1R-mutation and SMMC7721 groups （P 〈 0.05）. Necrosis and cell apoptosis were found in SMMC7721- IGF1R-siRNA group. The expression of IGF1R protein was significantly lower in SMMC7721-1GF1R-siRNA group than in SMMC7721-1GF1R-mutation and SMMC7721 groups （P 〈 0.05）. MVD was significantly lower in SMMC7721-1GF1R-siRNA group than in SMMC7721-1GF1R-mutation and SMMC7721 groups （11.3 ± 4.4 vs. 36.7 ± 7.6 and 28.4 ±6.5, P 〈 0.05）. The apoptosis rate of tumor cells was significantly higher in SMMC7721-1GF1R-siRNA group than in SMMC7721-1GF1R-mutation and SMMC7721 groups [（50.2 ± 6.4）% vs. （5.4 ± 1.0）% or （6.0 ±2.1）%, P〈0.05]. Conclusion： IGF1R siRNA can inhibit the growth of SMMC7721 cell xenograft in nude mice.

Increased Activity of the Human Telomerase Catalytic Subunit Promoter by the VEGF Enhancer in Human Cancer Cells

We attempted to improve the activity of hTERT promoter by fusing the vascular endothelial growth factor （VEGF） enhancer. To determine the potential as cancer specific promoters, we measured the reporter gene transfection assay driven by the hTERT promoter and the VEGF enhancer in human cancer cells. We found that the hTERT promoter containing VEGF enhancer conferred strong expression of the reporter gene only in different cancer cell lines but not in normal human cells. Retrovirus vector expressing HSV-TK controlled by the hTERT promoter and the VEGF enhancer was constructed. A549 cells infected with LN-enh-hT-TK was significantly suppressed and induced to apoptosis more than those infected with LN-hT-TK. The apoptosis ratio ofA549 cell infected with two kinds of retrovirus cell with GCV in lower concentration is 20.94% and 50.7%. It suggested that there is significant differentiation between the assay groups. Our results demonstrated the possible application of hTERT promoter and the VEGF enhancer in targeted cancer gene therapy.

Effect of acupuncture on the luteal function of rats with embryo implantation dysfunction

Objective To observe the effects of acupuncture on the luteal function of rats with embryo implantation dysfunction, and to explore its mechanism. Methods The early pregnant rats were randomly divided into normal control group （N）, model group （M）, acupoint group （A）, and non-acupoint group （AC）. The model of embryo implantation dysfunction in Group M, A, and AC was established with Mifepristone. For rats in Group A, bilateral ＂Housanli＂ （后三里 ST 36） and ＂Sanyinjiao＂ （三阴交 SP 6） were needled, while the non-acupoints beside the acupoints were needled in Group AC. In this experiment, the serum levels of luteinizing hormone （LH）, estradiol （E2）, and progesterone （P） were detected with radioimmunoassay, the expression of vascular endothelial growth factor （VEGF） in the rat ovarian tissue was detected by using Western-blot. The mRNA expressions of VEGF and luteinizing hormone receptor （LHR） in the ovarian tissue were detected by using RT-PCR. Results The serum levels of LH and P were significantly higher in group A than in group M and AC （all P〈0.05）, showing no statistical significance when compared with group N. The VEGF content, and expressions of VEGF mRNA and LHR mRNA in the ovarian tissue of group A were significantly elevated than those in group M and group AC （all P〈0.05）, showing no statistical significance when compared with group N. Conclusion Needling at ＂Housanli＂ （后三里 ST 36） and ＂Sanyinjiao＂ （三阴交 SP 6） could elevate the serum levels of LH and P of rats with embryo implantation dysfunction, and up-regulate the expression of LHR mRNA, VEGF and its mRNA in the ovarian tissue. It may enhance the luteal function of rats with embryo implantation dysfunction and improve its embryo implantation environment.

Effects of nerve growth factor on neuronal nitric oxide production after spinal cord injury in rats

Objective: To explore the protective effects of nerve growth factor (NGF) on injured spinal cord. Methods: The spinal cord injury (SCI) model of Wistar rats was established by a 10 g× 2.5 cm impact force on the T 8 spinal cord. NGF (60 μg/20 μl) was given to the rats of the treatment group immediately and at 2, 4, 8, 12, 24 hours after SCI. The level of neuronal constitutive nitric oxide synthase (ncNOS) and the expression of ncNOS mRNA in the spinal cord were detected by the immunohistochemistry assay and in situ hybridization method. Results: Abnormal expression of ncNOS was detected in the spinal ventral horn motorneuron in injured rats. The levels of ncNOS protein in the NGF group were significantly lower than those in the normal saline group (P< 0.05 ). The ncNOS mRNA expression was found in the spinal ventral horn motorneuron in injured rats and the expression in the NGF group was significantly decreased compared with that in the normal saline group (P< 0.01 ). Conclusions: NGF can protect the injured tissue of the spinal cord by prohibiting abnormal expression of nitric oxide synthase and the neurotoxicity of nitric oxide.

Effect of keratinocyte growth factor-2 on proliferation of human adult keratino cytes

To investigate the proliferative ef fect of keratinocyte growth factor (KGF 2) on human adult keratinocytes. Methods: The standard medium was keratinocyte growth medium wit hout bovine pituitary extract (BPE), hydrocortisone or epidermal growth factor ( EGF). Keratinocytes from a 48 year old subject were cultured and seeded on dis hes with standard medium of EGF in cell density of 2×10 4/32 mm 2. After 24 hours, the medium was replaced by the standard medium with 0, 4, 16, 125 and 50 0 ng/ml KGF 2, respectively. The standard medium with EGF was used as the posit ive control and the standard medium without EGF or KGF 2 was used as the negati ve controls. The growth of keratinocytes was monitored by 3 (4,5 dimethythiazo l 2 yl) 2,5 dipheyl tetrazolium bromide (MTT) assay and by photographs on day s 3, 5 and 7, respectively. Results: KGF 2 in concentrations of 4 500 ng/ml showed a sign ificant proliferative effect on days 5 and 7 as compared with that of the negati ve controls (P< 0.01 ). On day 3 the cells were prolifer ated to 1.5 2.5 fold, on day 5 to 3 5 fold and on day 7 to 3 12 fo ld in KGF 2 medium as that of the negative controls. The optimal response occur red when the concentration of KGF 2 was 125 ng/ml on day 7. Cell proliferation was also consistently higher in all KGF 2 concentrations as compared with that of the positive controls. Conclusions: KGF 2 has significant effects on the proliferatio n of adult keratinocytes, which are more effective than that of EGF. This study supports KGF 2 can improve the healing of chronic wounds in adults in clinic.

HER-2 overexpression and survival in colorectal cancer: a meta-analysis

Objective： Numerous studies examining the relationship between human epidermal growth factor receptor 2 （HER-2） overexpression and survival in patients with colorectal cancer （CRC） have yielded controversial results. We therefore performed a meta-analysis more precisely to estimate its prognostic value. Methods： Published studies investigating the effect of HER-2 overexpression on CRC survival were identified; the hazard ratios （HRs） and their corresponding 95% confidence intervals （95% Cls） were pooled in terms of disease-specific or overall survival. Results Eleven studies were included in the meta-analysis. The pooled data showed that HER-2 overexpression was negatively related to CRC survival （HR=1.10, 95% CI： 0.77-1.44）. Subgroup analyses regarding test method and study quality also demonstrated little association between HER-2 overexpression and CRC survival （HR=0.89, 95% CI： 0.50-1.29; HR=0.90, 95% Ch 0.43-1.37, respectively）, Conclusions： Regardless of several limitations, our study suggested that HER-2 overexpression probably had little impact on CRC survival.

A novel strategy to derive iPS cells from porcine fibroblasts

Induced pluripotent stem (iPS) cell technology demonstrates that somatic cells can be reprogrammed to a pluripotent state by over-expressing four reprogramming factors.This technology has created an interest in deriving iPS cells from domesticated animals such as pigs,sheep and cattle.Moloney murine leukemia retrovirus vectors have been widely used to generate and study mouse iPS cells.However,this retrovirus system infects only mouse and rat cells,which limits its use in establishing iPS cells from other mammals.In our study,we demonstrate a novel retrovirus strategy to efficiently generate porcine iPS cells from embryonic fibroblasts.We transfected four human reprogramming factors (Oct4,Sox2,Klf4 and Myc) into fibroblasts in one step by using a VSV-G envelope-coated pantropic retrovirus that was easily packaged by GP2-293 cells.We established six embryonic stem (ES)-like cell lines in human ES cell medium supplemented with bFGF.Colonies showed a similar morphology to human ES cells with a high nuclei-cytoplasm ratio and phase-bright flat colonies.Porcine iPS cells could form embryoid bodies in vitro and differentiate into the three germ layers in vivo by forming teratomas in immunodeficient mice.