Study Of Angiogenesis In The Genesis And Development Of Hepatocellular Carcinoma

Part One Study of angiogenesis in the genesis and development of hepatocellular carcinomaHepatocellular carcinoma(HCC)is one of the most common cancers worldwide and the third leading cause of cancer-related death.It is particularly prevalent in Asia and sub-Saharan Africa,yet there has been a rising incidence and a progressive increase in HCC-related mortality in the United States and Western Europe.HCC tends to show early invasion into blood vessels as well as intrahepatic metastasis.Despite advances in surgical and nonsurgical therapies,the outcome of HCC is poor.So it is crucial to understand the molecular mechanism of this highly aggressive cancer and to develop novel therapies for it. HCC is a typical hypervaseular cancer and angiogenesis is crucial in tumor growth and development.As a major part of tumor vessles,endothelial cells have attacted much interest in anti-angiogenesis study.As HCC is associated with multiple risk factors and accompanied with complex genetic abnormalities,we used the cDNA microarray to identify the differentially expressed genes in the development and progression of HCC.The selected genes were investigated in the HCC tissues and normal controls.Furthermore,the endothelial cells were isolated from HCC tissues and verified for the angiogenesis related study in HCC.Methods1.Two pairs of samples,including HCC tumor tissues and normal liver tissues were obtained for the oligonucleaotide cDNA microarray study.The pathological types of tissues were diagnosed by HE staining and total RNA was isolated by one-step Trizol reagents.RNA was run on 1%denaturing formaldehyde agarose gel to check the quality.The mRNA synthesis,reverse transcription,fluorescence labeling and purification were then performed. Cy3-(test)or Cy5-(reference)labeled cRNA probes were hybridized to oligonucleotide microarray.Overnight incubation was followed by stringent washing and figures were obtained by LuxScan 10KA.The differentially expressed genes were obtained as Cy3/Cy5 Ratio value≥2 or≤0.5.2.Tumor and non-cancerous tissues were freshly collected from 42 primary HCC patients who received resections at Shandong Provincial hospital during April 2005 to June 2007. Samples were from 34 males and 8 females with an average age of 54.5 years(32～74 years). The mRNA expression of PRL-3,MMP-2,MMP-9,E-cadherin was evaluated by real-time PCR.Western blot and immunohistochemistry were used to detect the expression of these factors at protein level.The microvessle density(MVD)in HCC was detected with immunohistochemistry by CD31 staining.3.Endothelial cells were isolated from freshly collected liver tissues of 30 HCC cases with anti-CD31 conjugated magnetic microbeads.The PRL-3,MMP-2 and MMP-9 expression in endothelial cells was evaluated by real-time PCR and Western blot.Results1.Typical pathological changes were observed on HCC,para-cacerous tissues and normal control liver tissues by HE staining.Total RNAs of matched tissues were of high quality and none of which degraded confirmed by 1%denaturing for maldehyde agarose gel.The screen results on the oligonucleaotide cDNA microarray shows 855 differentially expressed genes including 457 up-regulated genes and 398 down-regulated one.Cluster analysis showed that the development and progression of HCC is very complex.Numbers of kinds of genes involved in the procedure,including oncogenes and anti-oncogenes,genes encoding cellular cyclemodulator,genes associated with cellular stress,genes associated with cellular skeleton and movement,genes related to cellular apoptosis,genes encoding proteins regulating synthesis,repair,and rearangemen to DNA,genes encoding transcription factors and proteins binding with DNA,cytokine receptor genes,immunological protein genes,genes associated with cellular metabolism,genes encoding signal transductional factor,genes encoding factors that regulated protein translation,genes encoding proteins that were consistent with body growth and development and others.There are still some genes that cannot be clustered.2.We found PRL-3 was significantly up regulated in the HCC tumor tissues(0.664±0.053 vs.0.024±0.003,P＜0.001)compared with corresponding noncancerous liver tissues.The mRNA level of PRL-3 in tissues was correlated with the serum alpha-fetoprotein level, vascular invasion and metastasis(P＜0.001).The PRL-3 expression was closely correlated to MVD.Furthermore,we found a significant correlation between PRL-3 mRNA expression and MMP-2,MMP-9 and E-cadherin.3.The isolated endothelial cells presented typical "cobblestone" appearance.The immunostaining of von Willebrand factor was positive in more than 95%of isolated cells. The endothelial cells were confirmed by RT-PCR analysis CD31,CD146,AFP and CD45. PRL-3 was significantly up regulated in the HCC endothelial cells(PRL-3/β-actin 11.5±2.95 vs.1.76±1.04,P＜0.001).Conclusion1.The development and progress of HCC are very complex and involve expression changes of multiple genes.One gene or signal pathway cannot explain the profile of molecular mechanism of HCC.Through comprehensively analysis the differential genes expression profiling of HCC,our research provide a serial of differentially expressed genes, which is of great value for the study of HCC.2.Our results showed that PRL-3 is up-regulated in HCC.It is strongly suggested that PRL-3 play a key role in the angiogenesis and invasion of HCC.MMP-2,MMP-9 and E-cadherin might be involved in the PRL-3 functions in HCC.3.Tumor endothelial cells can be isolated from HCC tissues by Immunomagnetic methods and these cells were verified for angiogenesis research in HCC.PRL-3 was significantly up regulated in the HCC endothelial cells.These data strongly suggest that endothelial cell expression of PRL-3 and MMP-2,MMP-9 might be crucial in the angiogenesis of HCC and could be a useful marker for the anti-angiogenesis therapy.Part Two The study of CD4⁺CD25⁺ regulatory T cells in the pathogenesis of polycythaemia veraObjectiveThere is growing evidence that regulatory T cells(Treg)play an important role in the hematopoietic activity.This study aims to investigate the frequency and significance of Treg cells in the pathogenesis polycythaemia vera(PV).MethodsThe peripheral blood of 21 PV patients and 25 healthy donors were collected.Flow cytometry analysis was used to identify Treg cells stained as CD4⁺CD25⁺FOXP3⁺. CD4⁺CD25⁺ and CD4⁺CD25^- T cells were isolated by magnetic activated cell sorting (MACS).FOXP3 expression in CD4⁺CD25⁺Treg cells was detected by real-time quantatative PCR and western blot.The suppressive activity of CD4⁺CD25⁺ Treg cells was assessed by the proliferation and cytokine secretion of the co-cultured CD4⁺CD25^- fractions in different ratios. ResultsThe results showed that the percentage of Treg cells in the peripheral blood of PV patients was significantly increased compared to healthy controls:10.93±4.02%VS.5.86±1.99%, P＜0.05.Moreover,the mRNA and protein expression of FOXP3 was higher in CD4⁺CD25⁺ Treg cells.Coordinately,when co-cultured with the activated CD4⁺CD25^- cells,the CD4⁺CD25⁺ T_regcells showed enhanced suppressive function in PV.Yet,the underlying mechanism for the increased frequency and function of CD4⁺CD25⁺ Treg cells is still to be defined.ConclusionThe results indicate that CD4⁺CD25⁺ Treg cells expansion might account for the abnormal T cell immunity in PV patients and thus contribute to the pathogenesis of PV.