表皮生长因子和碱性成纤维细胞生长因子联合应用于深度褥疮的护理研究

[目的]探讨表皮生长因子和碱性成纤维细胞生长因子联合应用于深度褥疮的护理效果。[方法]选择褥疮45例处,其中Ⅱ期褥疮19例处、Ⅲ期21例处、Ⅳ期5例处,随机分为实验组和对照组,实验组将表皮生长因子和碱性成纤维细胞生长因子联合喷洒于褥疮创面,结合微波治疗;对照组用抗生素药液湿敷,予红外线局部照射,观察创面修复情况及愈合速度。[结果]实验组创面愈合速度为0.1mm/d～0.3mm/d,对照组为0.07mm/d～0.15mm/d,差异有统计学意义(P<0.05);实验组有效率高于对照组。[结论]表皮生长因子和碱性成纤维细胞生长因子联合应用于深度褥疮的治疗,疮面愈合效果好于传统的抗生素湿敷及红外线照射法。

PD98059抑制aFGF诱导的人膀胱癌细胞株EJ细胞增殖

目的探讨细胞外信号调节激酶(ERK)激酶MEK抑制剂PD98059对酸性成纤维细胞生长因子(aFGF)诱导的人膀胱癌细胞株EJ细胞增殖的抑制作用。方法以不同浓度的aFGF和PD98059作用EJ细胞,通过细胞计数法、MTT比色法观察PD98059对细胞增殖的抑制作用;流式细胞仪测定细胞各周期细胞百分数的变化;利用[γ3-2P]ATP掺入外源性底物的方法,液体闪烁测定ERK活性的变化。结果aFGF使EJ细胞株增殖比明显增加,而PD98059使EJ细胞株增殖比明显下降,在一定浓度范围内,其程度均随浓度增高而增强。EJ细胞受到aFGF刺激后,由G0+G1期进入S和G2+M期的细胞明显增多,在PD98059的作用下,由G0+G1期进入S和G2+M期的细胞明显减少,并在一定浓度范围内成剂量依赖性,与对照组比较差异显著(P<0.05);随着aFGF浓度的增加,EJ细胞ERK活性增高,PD98059抑制细胞内ERK活性,与PD98059浓度呈剂量依赖效应。结论PD98059对aFGF引起的EJ细胞增殖具有抑制作用,aFGF可能通过激活Ras-Raf-ERK信号转导途径来调控EJ细胞增殖,PD98059可有效阻滞此传导途径。

人羊膜上皮细胞培养液抑制角膜新生血管的实验研究

背景角膜新生血管（CNV）是一种常见的眼部病变，研究其发病机制及其抑制剂是眼科研究的热点和难点。目的研究人羊膜上皮细胞（AECs）培养液对CNV的抑制作用及机制。方法消化法培养及鉴定人AECs，并收集培养液，酶联免疫吸附试验（ELISA）法检测色素上皮衍生因子（PEDF）和白细胞介素1受体拮抗剂（IL-1Ra）在培养液中的质量浓度。兔角膜上皮细胞分离后分别用无血清DMEM培养基、人AECs培养液、混合培养液（DMEM＋人AECs培养液）培养48h，采用实时定量聚合酶链反应（QRT—PCR）法检测不同培养液培养的角膜上皮细胞中血管内皮生长因子（VEGF）及碱性成纤维细胞生长因子（bFGF）的表达。用含质量分数10％胎牛血清的DMEM培养基培养人脐静脉血管内皮细胞（UVECs），并分别加入无血清DMEM、混合培养液和人AECs培养液，划痕法和细胞计数试剂盒8（CCK8）法检测各组培养液对人UVECs迁移的影响。分别在上述3种培养基中加入终质量浓度为50txg／L的bFGF，CCK8法检测各培养液中人UVECs的增生情况。在原子力显微镜（AFM）下观察人AECs培养液对人UVECs超微结构的影响。结果人羊膜培养和传代细胞角蛋白免疫组织化学染色阳性证实为人AECs。与无血清DMEM组相比，人AECs培养液组的兔角膜上皮细胞VEGFmRNA（1．00＋0．22VS．2．98_＋0．46）及bFGFmRNA（1．00＋0．36VS．2．55-＋0．48）的表达均明显下降，差异均有统计学意义（p_-0．001、0．002）；培养后不同时间人AECs培养液组人UVECs的增生吸光度（A）值明显下降，差异均有统计学意义（P〈0．05），人UVECs的迁移率下降，差异有统计学意义（P〈0．05）。AFM观察见人AECs培养液组的血管内皮细胞膜粗糙、表面颗粒紊乱，细胞间连接及伪足减少。ELISA法检测人AECs培养液中PEDF的质量浓度为（70．41±0．68）Ixg／L，IL-1Ra的质量浓度为（153．56±0．36）-s／L，无血清DMEM组中未检出。结论人AECs培养液可抑制角膜上皮VEGF及bFGF的表达，抑制血管内皮细胞的增生和迁移，细胞结构和功能改变，这可能是其抑制CNV的机制之一。

Tumor Angiogenesis Correlated with bFGF and FGFR-1 in Lung Cancer

To study the relationship between angiogenesis and the expression of bFGF andFGFR-1 in lung cancer. Methods: The specimens of 56 patients with lung cancer treated with surgerywere collected. Anti-Von Willebrand factor antibody was used to measure microvascular density (MVD)by means of SABC immunohistochemical technique, and antibody to basic fibroblast growth factor(bFGF) and its receptor (FGFR-1) to detect the expression of these three proteins in the tumortissues. The survival time was compared between low MVD and high MVD groups by the Kaplan-Meiermethod. Results: (1) The expression of MVD showed no significant difference in some clinicalcharacteristics, including sex, age, T stage, M stage and pathologic type, but significantdifference in N stage (P 【 0.01) and clinical stage (P 【 0.05). (2) Survival analysis showed thathigh MVD group was associated with a risk of death (P 【 0.01). (3) The expression of bFGF and FGFR-1were both related to lymphatic metastasis and clinical staging (P 【 0.05). (4) Significantdifference was seen between low MVD and high MVD groups in the bFGF expression in lung cancer (P 【0.01), whereas no correlation in FGFR-1. (5) High co-expression of bFGF and FGFR-1 was consistent intumor cells. Conclusion: (1) MVD is a good prognostic factor for patients of lung cancer, and thesame as bFGF. (2) The angiogenesis may be induced after bFGF binding to FGFR-1.

Involvement of MAPK/ERK kinase-ERK pathway in exogenous bFGF-induced Egr-1 binding activity enhancement in anoxia-reoxygenation injured astrocytes

Objective Intravenous administration of basic fibroblast growth factor （bFGF） is effective to reduce the volume of cerebral infract due to ischemia. This study was designed to investigate the molecular mechanism, especially the signal transduction pathways, involved in this protective role of bFGF. Methods Anoxia-reoxygenation treated atrocytes were used to study the role of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase （MAPK/ERK kinase, MEK）-ERK signaling pathway after exogenous bFGF administration by Western blot. Electrophoretic mobile shift assay was used to detect the binding activity of early growth response factor-1 （Egr-1）, an important transcription factor for endogenous bFGF. Results bFGF could protect some signal transduction proteins from the oxygen-derived free radicals induced degradation. ERK1/2 was activated and involved in Egr-1 binding activity enhancement induced by exogenous bFGF. Conclusion MEK-ERK MAPK cascade may be an important signal transduction pathway contributed to bFGF induced enhancement of Egr-1 binding activity in anoxia-reoxygenation injured astrocytes.

Effects of basic fibroblast growth factor on ischemic gut and liver injuries

AIMS To explore the possible effects of basic fibrob- last growth factor (bFGF) on ischemic gut and liver in- juries after trauma. METHODS Animal model of super mesenteric artery (SMA) occlusion was used in this study. Seventy-two Wistar rats were divided into three groups of 24 rats each. Each animal in group 1 (bFGF treated) was in- jected with 4 μg of bFGF in 0.15 ml of normal saline solution containing 0.1%(w/v) heparin through the jugular vein at the onset of reperfusion. Animals in group 2 (saline treated) received the same vehicle, but without bFGF. Group 3 (sham-operated) ani- mals were treated with the same operations,but without SMA occlusion. Liver function parameters, serum TNFα,bacterial examination and pathological study were used to evaluate the results. RESULTS In group 1,the amounts of ALT and AST and serum TNFα were reduced significantly at 6,24 and 48 hours as compared with group 2. Bacterial ex- amination showed that the bacterial translocation from gut to liver,spleen and MLN in group 1 was much lower than that in group 2. The pathological results support the concept of significant protecting effects of bFGF. CONCLUSIONS Venous administration of bFGF may help reduce gut and liver injuries after ischemia and reperfusion. Its mechanism of action may involve the mitogenic and non-mitogenic effects of bFGF.

Study on Agrobacterium tumefaciens-mediated Transformation of Brassica campestris L. with Fusion Gene Ycoil-bFGF

[ Objective] The study is to generate pharmaceutical protein via plant transgenic technique. [Methed] Using the cotyledons with petiole as transformation receptor, the fusion gene of rapeseed oil-body gene and bFGF was introduced into the rapeseed （ Brassica campestris L. ） by Agrobacterium tumefaciens-mediated transformation; meanwhile regeneration conditions of rapeseed were also optimized, and the regenerated resistant plantlets were detected by PCR and Southern blot. [ Result] This fusion gene had been integrated into rapeseed genome successfully, and the optimized conditions of transformation and regeneration were as follows： explants pre-culture for 2 d, co-culture for 3 d, bacteria solution OD600 for 0.3 and infection time for 5 min. [ Conclusion] The results laid a solid foundation for extraction, isolation and purification of protein in transgenic plant seeds.

Cellular and molecular mechanisms of intestinal fibrosis

Fibrosis is a chronic and progressive process characterized by an excessive accumulation of extracellular matrix (ECM) leading to stiffening and/or scarring of the involved tissue. Intestinal fibrosis may develop in several different enteropathies, including inflammatory bowel disease. It develops through complex cell, extracellular matrix, cytokine and growth factor interactions. Distinct cell types are involved in intestinal fibrosis, such as resident mesenchymal cells (fibroblasts, myofibroblasts and smooth muscle cells) but also ECM-producing cells derived from epithelial and endothelial cells (through a process termed epithelialand endothelial-mesenchymal transition), stellate cells, pericytes, local or bone marrow-derived stem cells. The most important soluble factors that regulate the activation of these cells include cytokines, chemokines, growth factors, components of the renin-angiotensin system, angiogenic factors, peroxisome proliferator-activated receptors, mammalian target of rapamycin, and products of oxidative stress. It soon becomes clear that although inflammation is responsible for triggering the onset of the fibrotic proc-ess, it only plays a minor role in the progression of this condition, as fibrosis may advance in a self-perpetuating fashion. Definition of the cellular and molecular mechanisms involved in intestinal fibrosis may provide the key to developing new therapeutic approaches.

Effect of basic fibroblast growth factor(bFGF) on the treatment of exposure of the orbital implants

Objective: To evaluate the efficacy and the indication of basic fibroblast growth factor (bFGF) in the treatment of exposure of orbital implants. Design: Retrospective and observational case series. Methods: We reviewed 41 patients (41 eyes) suffering exposure of orbital implants from Jan. 2000 to June 2006. The study group patients with mild exposure received com-bined treatment with bFGF and antibiotic drops, and while the control group patients with mild exposure were treated with anti-biotic drops only. The study group patients with moderate and severe exposure received combined treatment with bFGF and antibiotic drops, and after 2 months they were subjected to amniotic membrane transplantation, while the control group patients with moderate and severe exposure underwent amniotic membrane transplantation after using antibiotic drops. Observation of the growth of conjunctival epithelium and comparison of the healing rate of the two groups. Results: The healing rates of the mild, moderate and severe exposure study group were 100% and 92.3%. The healing rates of the mild, moderate and severe exposure control group were 55.6% and 66.7% respectively. The difference of the healing rates of the mild exposure study group and the control group was significant (P=0.033). And the difference of the healing rates of the moderate and severe exposure study group and the control group was not significant (P=0.167). Conclusion: bFGF may promote obviously the healing of orbital implant exposure, particularly it can be the first choice for the treatment of mild degree exposure. For the moderate and severe cases, it can be administered before surgical repair to enhance neovascularization and will tend to increase the success rate of surgical repair.

Influence of ginsenoside Rg1, a panaxatriol saponin from Panax notoginseng, on renal fibrosis in rats with unilateral ureteral obstruction

Total saponins of Panax notoginseng （PNS） have been shown to ameliorate renal interstitial fibrosis. Ginsenoside Rg 1, a panaxatriol saponin, is one of the major active molecules from PNS. The present study was undertaken to investigate the effect of ginsenoside Rgl on renal fibrosis in rats with unilateral ureteral obstruction （UUO）. The rats were randomly divided into 3 groups： sham-operation （n=15）, UUO （n=15） and UUO with ginsenoside Rgl treatment （n=15, 50 mg per kg body weight, intraperitoneally （i.p.） injected）. The rats were sacrificed on Days 7 and 14 after the surgery. Histological examination demonstrated that ginsenoside Rgl significantly inhibited interstitial fibrosis including tubular injury as well as collagen deposition, u-smooth muscle actin （α-SMA） and E-cadherin are two markers of tubular epithelial-myofibroblast transition （TEMT）. Interestingly, ginsenoside Rgl notably decreased α-SMA expression and simultaneously enhanced E-cadherin expression. The messenger RNA （mRNA） of transforming growth factor-β1 （TGF-β1）, a key mediator to regulate TEMT, in the obstructed kidney increased dramatically, but was found to decrease significantly after administration of ginsenoside Rg 1. Further study showed that ginsenoside Rgl considerably decreased the levels of both active TGF-β1 and phosphorylated Smad2 （pSmad2）. Moreover, ginsenoside Rgl substantially suppressed the expression of thrombospondin-1 （TSP-1）, a cytokine which can promote the transcription of TGF-β1 mRNA and the activation of latent TGF-β1. These results suggest that ginsenoside Rgl inhibits renal interstitial fibrosis in rats with UUO. The mechanism might be partly related to the blocking of TEMT via suppressing the expression of TSP-1.

Toxicarioside A inhibits SGC-7901 proliferation,migration and invasion via NF-κB/bFGF signaling

AIM:To investigate the inhibitory role of toxicarioside A on the gastric cancer cell line human gastric cancer cell line(SGC-7901) and determine the underlying molecular mechanism.METHODS:After SGC-7901 cells were treated with toxicarioside A at various concentrations(0.5,1.5,4.5,9.0 μg/mL) for 24 h or 48 h,cell viability was determined by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl2H-tetrazolium bromide assay,and the motility and invasion of tumor cells were assessed by the Transwell chamber assay.Immunofluorescence staining,reverse transcription polymerase chain reaction and Western blotting were performed to detect the expression of basic fibroblast growth factor(bFGF) and fibroblast growth factor receptor-1(FGFR1),and nuclear factorkappa B(NF-κB) activation was examined by electrophoretic mobility shift assay.RESULTS:The results showed that toxicarioside A was capable of reducing cell viability,inhibiting cell growth,and suppressing cell migration and invasion activities in a time-and dose-dependent manner in SGC-7901 cells.Further analysis revealed that not only the expression of bFGF and its high-affinity receptor FGFR1 but also the NF-κB-DNA binding activity were effectively blocked by toxicarioside A in a dose-dependent manner compared with the control group(P < 0.05 or P < 0.01).Interestingly,application of the NF-κB specific inhibitor,pyrrolidinedithiocarbamate(PDTC),to SGC-7901 cells significantly potentized the toxicarioside A-induced down-regulation of bFGF compared with the control group(P < 0.05).CONCLUSION:These findings suggest that toxicarioside A has an anti-gastric cancer activity and this effect may be achieved partly through down-regulation of NF-κB and bFGF/FGFR1 signaling.

Transforming growth factor-β1 induces intestinal myofibroblast differentiation and modulates their migration

AIM： To investigate the effects of transforming growth factor β1 （TGF-β1） on the differentiation of colonic lamina propria fibroblasts （CLPF） into myofibroblasts in vitro.METHODS： Primary CLPF cultures were incubated with TGF-β1 and analyzed for production of m-smooth muscle actin （α-SMA）, fibronectin （FN） and FN isoforms. Migration assays were performed in a modified 48-well Boyden chamber. Levels of total and phosphorylated focal adhesion kinase （FAK） in CLPF were analyzed after induction of migration.did not change α-SMA levels, while TGF-β1 treatment for 6 d significantly increased α-SIVlA production. Short term incubation （6 h） with TGF-β1 enhanced CLPF migration, while long term treatment （6 d） of CLPF with TGF-β1 reduced migration to 15%-37% compared to untreated cells. FN and FN isoform mRNA expression were increased after short term incubation with TGF-β1 （2 d） in contrast to long term incubation with TGF-β1 for 6 d. After induction of migration, TGF-β1-preincubated CLPF showed higher amounts of FN and its isoforms and lower levels of total and phosphorylated FAK than untreated cells.CONCLUSION： Long term incubation of CLPF with TGF-β1 induced differentiation into myofibroblasts with enhanced α-SMA, reduced migratory potential and FAK phosphorylation, and increased FN production. In contrast, short term contact （6 h） of fibroblasts with TGF-β1 induced a dose-dependent increase of cell migration and FAK phosphorylation without induction of α-SMA production.

Transplantation of microencapsulated umbilical-cord-blood-derived hepatic-like cells for treatment of hepatic failure

AIM: To investigate intraperitoneal transplantation of microencapsulated hepatic-like cells from human umbilical cord blood for treatment of hepatic failure in rats. METHODS: CD34+ cells in umbilical cord blood cells were isolated by magnetic cell sorting. In the in vitro experiment, sorted CD34+ cells were amplified and induced into hepatic-like cells by culturing with a combination of fibroblast growth factor 4 and hepatocyte growth factor. Cultures without growth factor addition served as controls. mRNA and protein levels for hepatic-like cells were analyzed by reverse transcription-polymerase chain reaction, immunohistochemistry and immunofluorescence. In the in vivo experiment, the hepatic-like cells were encapsulated and transplanted into the abdominal cavity of acute hepatic failure (AHF) rats at 48 h after D-galactosamine induction of acute hepatic failure. Transplantation with PBS and unencapsulated hepatic-like cells served as controls. The mortality rate, hepatic pathological changes and serum biochemical indexes were determined. The morphology and structure of microcapsules in the greater omentum were observed. RESULTS: Human albumin, alpha-fetoprotein and GATA-4 mRNA and albumin protein positive cells were found among cultured cells after 16 d. Albumin level in culture medium was significantly increased after culturing with growth factors in comparison with culturing without growth factor addition (P < 0.01). Compared with the unencapsulated group, the mortality rate of the encapsulated hepatic-like cell-transplanted group was significantly lower (P < 0.05). Serum biochemical parameters, alanine aminotransferase, aspartate aminotransferase and total bilirubin in the encapsulated group were significantly improvement compared with the PBS control group (P < 0.01). Pathological staining further supported these findings. At 1-2 wk post-transplantation, free microcapsules with a round clear structure and a smooth surface were observed in peritoneal lavage fluid, surviving cells inside microcapsules were found by trypan blue staining, but some fibrous tissue around microcapsules was also detected in the greater omentum of encapsulated group by hematoxylin and eosin staining. CONCLUSION: Transplantation of microencapsulated hepatic-like cells derived from umbilical cord blood cells could preliminarily alleviate the symptoms of AHF rats.

Significant roles of anti-aging protein klotho and fibroblast growth factor23 in cardiovascular disease

The klotho gene has been identified as an aging suppressor that encodes a protein involved in cardiovascular disease （CVD）. The inac- tivation of the klotho gene causes serious systemic disorders resembling human aging, such as atherosderosis, diffuse vascular calcification and shortened life span. Klotho has been demonstrated to ameliorate vascular endothelial dysfunction and delay vascular calcification. Fur- thermore, klotho gene polymorphisms in the human are associated with various cardiovascular events. Recent experiments show that klotho may reduce transient receptor potential canonical6 （TRPC6） channels, resulting in protecting the heart from hypertrophy and systolic dys- function. Fibroblast growth factor23 （FGF23） is a bone-derived hormone that plays an important role in the regulation of phosphate and vi- tamin D metabolism. FGF23 accelerates urinary phosphate excretion and suppresses 1,25-dihydroxy vitaminD3 （1,25（OH）2D3）synthesis in the presence ofFGF receptorl （FGFR1） and its co-receptor ldotho, principally in the kidney. The hormonal affects of circulating klotho pro- tein and FGF23 on vascular and heart have contributed to an understanding of their roles in the pathophysiology of arterial stiffness and left ventricular hypertrophy. Klotho and FGF23 appear to play a critical role in the pathogenesis of vascular disease, and may represent a novel potential therapeutic strategy for clinical intervention.

Targeted systemic therapies for hepatocellular carcinoma:Clinical perspectives,challenges and implications

Hepatocellular carcinoma （HCC） is a lethal disease in most patients, due to its aggressive course and a lack of effective systemic therapies for advanced disease. Surgical resection and liver transplantation remain the only curative options for a small subset of patients. Few patients with HCC are diagnosed early enough to be eli- gible for curative treatment. Angiogenesis inhibition is a natural therapeutic target for all solid tumors, but par- ticularly for the highly vascularized HCC tumors. With the approval of the targeted agent sorafenib, there are now additional options for patients with HCC. Although sorafenib does produce some improvement in survival in HCC patients, the responses are not durable. In addi- tion, there are significant dermatologic, gastrointestinal, and metabolic toxicities, and, as importantly, there is still limited knowledge of its usefulness in special sub- populations with HCC. Other angiogenesis inhibitors are in development to treat HCC both in the first-line set- ting and for use following sorafenib failure; the furthest in development is brivanib, a dual fibroblast growth factor pathway and vascular endothelial growth factor receptor inhibitor. Additional agents with antiangiogenic properties also in phase IT and Ⅲ development for the treatment of patients with HCC include bevacizumab, ramucirumab, ABT-869, everolimus and ARQ 197.

Overexpression of the receptor tyrosine kinase EphA4 in human gastric cancers

AIM: To clarify the expression and role of Ephrin receptor A4 (EphA4) in gastric cancer in relation to clinicopathological characteristics and the expression of fibroblast growth factor receptor 1 (FGFR1) and ephrin ligands. METHODS: Eleven gastric carcinoma cell lines, 24 paired surgical fresh specimens of gastric adenocarcinoma and adjacent nontumor tissue, 74 conventional formalin-fixed, paraffin-embedded tumor specimens, and 55 specimens spotted on tissue microarray (TMA) were analyzed. Reverse transcription-PCR (RT-PCR), real-time RT-PCR, immunohistochemistry, and cell growth assays were performed. RESULTS: Overexpression of EphA4 mRNA expres-sion was observed in 8 (73%) of 11 gastric cancer cell lines and 10 (42%) of 24 gastric cancer tissues. Over-expression of EphA4, analyzed by immunohistochemistry, was observed in 62 (48%) of 129 gastric cancer tissues. EphA4 overexpression, at the protein level, was significantly associated with depth of invasion and recurrence. EphA4 overexpression was also correlated with FGFR1 overexpression. Patients with EphA4-positive cancer had significantly shorter overall survival periods than did those with EphA4-negative cancer (P = 0.0008). The mRNAs for ephrin ligands were coexpressed in various combinations in gastric cancer cell lines and cancer tissues. Downregulation of EphA4 expression by siRNA in EphA4-overexpressing gastric cancer cell lines resulted in a significant decrease in cell growth. CONCLUSION: Our results suggest that overexpres-sion of EphA4 plays a role in gastric cancer.

Basic fibroblast growth factor alleviates brain injury following global ischemia reperfusion in rabbits

The study was to explore the protective effect of basic fibroblast growth factor (bFGF) on brain injury following global ischemia reperfusion and its mechanisms. Brain injury following global ischemia was induced by four vessels occlusion and systemic hypotension. Twenty-four rabbits were randomized into three groups: group A, only dissection of vessels; group B, intravenous infusion of normal saline after reperfusion for 6 h; group C, 30 μg/kg bFGF injected intravenously at the onset of reperfusion, then infused with 10 μg/(kg&middoth) for 6 h. Serum neuron specific enolase (NSE), S-100B, tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), interleukin-8 (IL-8) were measured before ischemia, 30 min after ischemia, 0.5, 1, 3, 6 h after reperfusion. Brain water content was determined and cerebral histopathological damages were compared. NSE and S-100B were increased 1 h after reperfusion and reached their peaks 6 h after reperfusion, but were much higher in group B than those in group C 3, 6 h after reperfusion. In groups B and C, TNF-a was increased after ischemia and IL-1 and IL-8 were increased significantly 0.5 h after reperfusion, then reached their peaks 6 h, 3 h, 6 h after reperfusion respectively. TNF-a and IL-8 at the time points of 1 h and 3 h and IL-1 at 3 h and 6 h in group C were correspondingly lower than those in group B. These indices in group A were nearly unchanged. There were less severe cerebral histopathological damages in group C compared with group B, but no difference in brain water content. It could be concluded that bFGF alleviates brain injury following global ischemia and reperfusion by down-regulating expression of inflammatory factors and inhibiting their activities.

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.

Fibroblast growth factor-4 and hepatocyte growth factor induce differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocytes

AIM： To investigate the differentiation of human umbilical cord blood （HUCB）-derived mesenchymal stem cells （MSCs） into hepatocytes by induction of fibroblast growth factor-4 （FGF-4） and hepatocyte growth factor （HGF）, and to find a new source of cell types for therapies of hepatic diseases. METHODS： MSCs were isolated by combining gradient density centrifugation with plastic adherence. When HUCB-derived MSCs reached 70% confluence, they were cultured in Iscove modified Dulbecco medium （IMDM） supplemented with 10 mL/L FBS, 20 ng/mL HGF and 10 ng/mL FGF-4. The medium was changed every 4 d and stored for albumin, alpha-fetoprotein （AFP） and urea assay. Expression of CK-18 was detected by immunocytochemistry. Glycogen storage in hepatocytes was determined by PAS staining. RESULTS： By combining gradient density centrifugation with plastic adherence, we could isolate MSCs from 25.6% of human umbilical cord blood. When MSCs were cultured with FGF-4 and HGF, approximately 63.6% of cells became small, round and epithelioid on d 28 by morphology. Compared with the control, the level of AFP increased significantly from d 12 to 18.20：1=1.16 μg/L （t = 2.884, P〈0.05） in MSCs cultured with FGF-4 and HGF, and was higher （54.28±3.11 μg/L） on d 28 （t = 13.493, P〈0.01）. Albumin increased significantly on d 16 （t = 6.68, P〈0.01） to 1.02±0.15 μg/mL, and to 3.63±0.30 μg/mL on d 28 （t = 11.748, P〈0.01）. Urea （4.72±1.03 μmol/L） was detected on d 20 （t = 4.272, P〈0.01）, and continued to increase to 10.28±1.06 μmol/L on d 28 （t = 9.276, P〈0.01）. Cells expressed CK-18 on d 16. Glycogen storage was observed on d 24. CONCLUSION： HUCB-derived MSCs can differentiate into hepatocytes by induction of F-GF-4 and HGF. HUCB derived MSCs are a new source of cell types for cell transplantation therapy of hepatic diseases.

Genomic imbalances in esophageal carcinoma cell lines involve Wnt pathway genes

AIM： To identify molecular markers shared across South African esophageal squamous cell carcinoma （ESCC） cell lines using o/togenetics, fluorescence in situ hybridization （FISH） and single nucleotide polymorphism （SNP） array copy number analysis. METHODS： We used conventional cytogenetics, FISH, and multicolor FISH to characterize the chromosomal rearrangements of five ESCC cell lines established in South Africa. The whole genome copy number profile was established from 250K SNP arrays, and data was analyzed with the CNAT 4.0 and GISTIC software. tions involved the following chromosomal regions and genes： 11q13.3 （CCND1, FGF3, FGF4, FGF19, MYEOV）, 8q24.21（C-MYC, FAM84B）, 11q22.1-q22.3 （B[RC2, BIRC3）, 5p15.2 （CTNND2）, 3qll.2-q12.2 （MINA） and 18p11.32 （TYMS, YES1）. The significant deletions included 1p31.2-p31.1 （CTH, GADD45a, DIRAS3）, 2q22.1 （LRPIB）, 3p12.1-p14.2 （FHIT）, 4q22.1-q32.1 （CASP6, SMAD1）, 8p23.2-q11.1 （BNIP3L） and 18q21.1-q21.2 （SMAD4, DCC）. The 3p11.2 translocation breakpoint was shared across four cell lines, supporting a role for genes involved at this site, in particular, the EPHA3 gene which has previously been reported to be deleted in ESCC.CONCLUSION： The finding that a significant number of genes that were amplified （FGF3, FGF4, FGF19, CCND1 and C-MYC） or deleted （SFRP2 gene） are involved in the Wnt and fibroblast growth factor signaling pathways, suggests that these pathways may be activated in these cell lines.