Expression of insulin-like growth factor-1 mRNA and protein level of corpora striata in ischemic side at the early stage of middle cerebral artery ischemia/reperfusion in rhesus monkeys

BACKGROUND： Insulin-like growth factor-I（IGF-1）, as one of the important members of growth factor family, participants in the regulation of many physiological functions and behaviors, having very strong neuroprotective effect. However, the expression of IGF-1 following cerebral ischemia/reperfusion is still disputed. OBJECTIVE： To observe the expression of IGF-1 and protein of corpora striata in ischemic side at the early stage of middle cerebral artery ischemia/reperfusion in rhesus monkey. DESIGN ： A completely randomized grouping design, controlled animal experiment SETTING ： Institute of Cerebrovascular Disease, Affiliated Hospital of Medical College of Qingdao University MATERIALS： ① Totally 17 rhesus monkeys , of either gender, aged 4 to 5 years, were enrolled . Seven rhesus monkeys observed with gene chip were randomly divided into 2 groups： sham operation group （n=3） and ischemia/reperfusion group 〈n=4〉. Ten rhesus monkeys observed with in situ hybridization and immunohistochemistry method were randomly divided into 2 groups： sham operation group 〈n=3 〉and ischemia/reperfusion group （n=7）. Rhesus monkeys observed under microscope were divided into 2 groups： sham operation group （n=6） and ischamia/reperfusion group （n=-11）.②Materials used in the experiment： cresyl violet （Sigma Company, America）; immunohistochemical reagent kit （ Huamei Bio-engineering Company）; In situ hybridization reagent kit （Boshide Bio-engineering Co.Ltd, Wuhan）; 12 800 dots chip （Boxing Company, Shanghai）. METHODS ： This experiment was carried out at the Institute of Cerebrovascular Disease, Affiliated Hospital of Medical College of Qingdao University from January 2001 to December 2003.① The onset area of middle cerebral artery was blocked for 2 hours, middle cerebral artery ischemia/reperfusion models were created.② After ischemia/reperfusion for 24 hours, cerebral tissue sections of rhesus monkeys were prepared and stained with cresyl violet. Image analysis was performed with 5001W image analysis software. Morphological change of corpora striata of operative side was observed in the rhesus monkeys between two groups. Total RNA was extracted from cerebral tissue. ③ Detection of gene chip： Cy3-duTP and Cy5-duTP were used to respectively perform reverse transcription labeling. The sample was reversely transcribed into cDNA, then hybridized with cDNA of cerebral tissue. Genes with the separate absolute value of cy3 and cy5〉800, cY3/cy5 〉 2（high expression） or 〈 0.5 （low expression） were found out. Those were genes with differential expression. ④ The expressions of IGF-1 mRNA and protein level of corpora striata in ischemic side of rhe- sus monkeys were detected between sham operation group and ischemia/reperfusion group at 9 and 24 hours after ischemia/reperfusion with in situ hybridization method and immunohistochemical method. Brown granules were IGF-1 protein positive cells. ⑤ Analysis of variance was used in the difference comparison of measurement data among groups. MAIN OUTCOME MEASURES ： ① Change of morphological structure of corpora striata at ischemic side in rhesus monkeys. ② Change of cerebral gene expression profiles at ischemia/reperfusion in rhesus monkeys between two groups.③ Expression of IGF-1 mRNA and protein level of corpora striata at ischemia/reperfu- sion in rhesus monkeys between two groups. RESULTS ： ① Pathological change ： Obvious pathological change of cerebral infarction appeared in the ischemia and reperfusion group, while there was no such pathological change in the sham operation group.② Change of gene expression profile ： There were 4480 genes with difference expression in the ischemia/reperfusion group and sham-operation group, in which, 260 genes had high expression and their absolute value was over 800, and 63 genes had low expression, cy3/cy5 of IGF-1 was 0.379, being relative low ex- pression. ③ IGF-1 mRNA and protein positive cell counts in corpora striata at cerebral ischemic side[IGF-1 mRNA： 〈9.72±1.18）,（9.11 ±0.76）,（14.77±0.60） counts/field：lGF-1 protein： （15.11 ±1.83）,（15.39±0.78）, （34.62±0.97）counts/field, P 〈 0.05-0.01]. CONCLUSION： IGF-1 mRNA and protein are lowly expressed in middle cerebral artery of rhesus monkeys at ischemia/reperfusion.

Quantitative analysis of hepatic hypoxia-inducible factor-1α and its abnormal gene expression during the formation of hepatocellular carcinoma

BACKGROUND:Hepatic hypoxia-inducible factor-1(HIF-1) is activated in the progression of hepatocellular carcinoma (HCC).This study aimed to investigate the dynamic alterations of HIF-1αand its gene expression so as to explore the relationship between HIF-1αexpression and hepatocarcinogenesis at the early stage of HCC. METHODS:A hepatoma model was made with 2-fluorenyl- acetamide(2-FAA)in male Sprague-Dawley rats.Morphological changes of rat hepatocytes were assessed pathologically (HE staining).The dynamic expression of hepatic and circulating HIF-1αwas quantitatively analyzed by ELISA. The gene fragments of hepatic HIF-1αmRNA were amplified by RT-PCR and confirmed by sequencing.The cellular distribution of hepatic HIF-1αexpression was confirmed by immunohistochemistry. RESULTS:Histological examination confirmed granulelike degeneration to atypical hyperplasia and HCC development in rat hepatocytes and progressive increases in the levels of hepatic and circulating HIF-1αand its gene expression during the course.The levels of HIF-1α expression in the liver and blood of rats with hepatoma were significantly higher than those in normal ratsand those with degeneration.Immunohistochemical analysis confirmed the positive expression and hepatocyte distribution of HIF-1αin the development of rat hepatoma. A positive relationship was found between HIF-1α expression in the liver and blood(P<0.01). CONCLUSIONS:The above observations support the hypothesis that the overexpression of HIF-1αand its gene are closely associated with the malignant transformation of hepatocytes and play an important role at the stage of hepatocarcinogenesis.

Transactivation of the TIEG1 confers growth inhibition of transforming growth factor-β-susceptible hepatocellular carcinoma cells

AIM:To investigate the role of transforming growth factor(TGF)-β-inducible early gene 1(TIEG1) in TGF-β-induced growth inhibition in hepatocellular carcinoma(HCC) cells.METHODS:Human hepatocyte and HCC cell lines with varied susceptibilities to TGF-β1 were tested by methylthiazoletetrazolium(MTT) assay.The expression changes of Smad2,Smad3,Smad4,Smad7,TIEG1 and TIEG2 gene following treatment with TGF-β1 in a TGF-β-sensitive hepatocyte cell line(MIHA),a TGF-β-sensitive hepatoma cell line(Hep3B) and two TGF-β-insensitive hepatoma cell lines(HepG2 and Bel7404) were examined.SiRNA targeting TIEG1 was transfected into Hep3B cells and the sensitivity of cells to TGF-β1 was examined.Overexpression of TIEG1 was induced by lentiviral-mediated transduction in TGF-β1-resistant hepatoma cell lines(Bel7404 and HepG2).MTT assay and 4',6-Diamidino-2-phenylindole staining were used to identify cell viability and apoptosis,respectively.The expression level of stathmin was measured by reverse transcriptase polymerase chain reaction and Western-blotting analysis,and stathmin promoter activity by TIEG1 was monitored by a luciferase reporter gene system.RESULTS:TIEG1 was significantly upregulated by TGF-β1 in the TGF-β1-sensitive HCC cell line,Hep3B,but not in the resistant cell lines.The suppression of TIEG1 by siRNAs decreased the sensitivity of Hep3B cells to TGF-β1,whereas the overexpression of TIEG1 mediated growth inhibition and apoptosis in TGF-β1-resistant HCC cell lines,which resembled those of TGF-β1-sensitive HCC cells treated with TGF-β1.Our data further suggested that stathmin was a direct target of TIEG1,as stathmin was signif icantly downregulated by TIEG1 overexpression,and stathmin promoter activity was inhibited by TIEG1 in a dose-dependent manner.CONCLUSION:Our data suggest that transactivation of TIEG1 conferred growth inhibition of TGF-β-susceptible human HCC cells.

前囊膜划伤诱导小鼠晶状体上皮细胞早期生长反应因子-1的表达

目的观察前囊膜划伤对小鼠晶状体上皮细胞早期生长反应因子-1(early growth response factor-1,Egr-1)表达的影响。方法36只成年雄性昆明小鼠,随机分为6组,制作小鼠晶状体前囊膜划伤模型。采用Westernblot和逆转录聚合酶链反应(RT-PCR)观察前囊膜划伤后不同时间(0,0.5,0.75,1,2,5h)晶状体上皮细胞Egr-1蛋白和mRNA的表达变化。结果在正常未划伤的晶状体上皮细胞,Egr-1mRNA低表达,划伤后0.5h表达即增强,并在2h时达到峰值,随后逐渐减弱。正常未划伤的晶状体上皮细胞Egr-1蛋白表达微弱,划伤后0.5h,Egr-1蛋白表达即显著增强,并在2h时达到峰值,随后开始减弱。结论前囊膜划伤可诱导小鼠晶状体上皮细胞Egr-1mRNA和蛋白的表达。

低氧预适应对自体肝移植大鼠肝细胞EGR-1表达的影响

目的探讨低氧预适应对大鼠肝移植术后早期生长反应因子1（early growth response factor-1，EGR．1）的影响。方法采用门静脉灌注的大鼠自体肝移植模型：A组：低氧预适应大鼠自体肝移植组；B组：大鼠自体肝移植组；C组：正常大鼠对照组。比较大鼠自体肝移植术后肝脏组织病理学改变、肝组织中低氧诱导因子（hypoxic induce factor1，HIF-1）、肿瘤坏死因子（tumor necrosis factor，TNF）的mRNA表达变化和早期生长反应因子1（EGR-1）的WB结果。结果A组大鼠因术前低氧诱导致HIF-1RNA的表达比B组和C组更明显，术后6h明显高于B组（t=9．601，df=10，2．tailedSig=0．000，P〈0．05）；A组和B组的Egr-1蛋白表达量术后都增多，但A组明显低于B组。而B组肿瘤坏死因子RNA的RT．PCR表达比A组和c组更明显，其中术后6h明显高于A组（t=-12．067，df=10。2-tailedSig=0．000，P〈0．05），与Egr-1蛋白表达呈正相关。肝细胞病理结果示A组肝小叶结构损伤较B组轻，肝细胞轻微肿胀，肝组织无明显改变。结论低氧预适应后大鼠移植肝中EGR．1生成适度增加，与大鼠自体肝移植组相比明显减少，降低了TNF等炎性因子的产生从而减少肝脏的病理损害。