| Part ? The relativity between EGFL7 level and clinic-pathological features and metastasis of HCCObjective:To investigate the relativity between EGFL7 level and clinic-pathological features,metastasis and survival prognosis of HCC patients.Materials and Methods:The serum specimen of 110 patients with primary liver carcinoma was collected and enzyme linked immunosorbent assay(ELISA)was used to test the level of EGFL7 protein.The clinic-pathological features,including gender,ages,HBV history,liver function(Child-Pugh score),number and diameter of lesions,venous invasion,extrahepatic metastasis were also detected to analysis their relation with serum EGFL7 protein level.The tissue specimens of 90 patients with primary liver carcinoma were collected and immunohistochemistry was used to detect the protein expression of EGFL7.The time to metastasis and over survival time of patients were inbestigated to analysis their relation with the expression of EGFL7 in tissue.Results:The serum EGFL7 protein(pg/ml):males(21.7±13.7),females(23.6±12.8)with no significant difference(P>0.05);age less than 60 years(23.5±14.2),age more than 60 years(20.5±12.9)with no significant difference(P>0.05);Child-Pugh score Level A(20.8±13.9),Level B(27.3±9.9),Level C(27.9±13.9)with significant difference(P<0.05);Numbers of lesion,less than three(22.0±13.7),more than three(22.4± 13.4),with no significant difference(P>0.05);diameter of lesion,smaller than 5 cm(21.8±12.9),larger than 5 cm and smaller than 10 cm(21.6±14.5),lager than 10cm(22.6±13.8),with nosignificant difference(P>0.05);venous invasion,yes(32.0±11.8),no(18.8±12.5),with significant difference(P<0.05);extrahepatic metastasis,yes(31.7±12.4),no(18.4±12.2),with significant difference(P<0.05).The expression of EGFL7 in tumor tissue:high in 70 patients,low in 20 patients.The median overall survival of high expression groups and low expression groups were 27 months and 69 months respectively,with significant difference(P<0.05).The median tumor free survival time of high expression groups and low expression groups were 33 months and 79 months respectively,with significant difference(P<0.05).Conclusions:The serum EGFL7 levelis closely correlated with the venous invasion,extrahepatic metastases and liver function.The high level of serum EGFL7 protein is detected in the patients with venous invasion or extrahepatic metastasis or with high Child-Pugh score.Patients with high expression of EGFL7 in tumor are prone to metastasis and poor prognosis.EGFL7 is an oncogene associated with invasion,metastasis and poor prognosis in HCC.Part ? The clinical research of effect of hypoxia on serumEGFL7 protein levelObjective:To investigate the effect of hypoxia onserum EGFL7 protein level in patients.Materials and Methods:The tissue specimens of 50 patients with HCC were stained by immunohistochemistry with HIF-la.The patients were divided into three groups according to the percent of positive cells in slices:HIF-1? negative group(the percent of positive cells<10%),HIF-la positive group(the percentage of positive cells10%?50%),HIF-la strong positive group(the percentage of positive cells>50%).Furthermore,the serum EGFL7 protein of these patients was measured by ELISA.The difference of serum EGFL7 protein in the 3 groups was analyzed by Mann-Whitey test.Results:Among the 50 patients,15 patients were included in HIF-la negative group,24 in HIF-la positive group,and 11 in HIF-1? strong positive.The average serum EGFL7 protein level was 15.1pg/ml in HIF-1? negative group,31.7 pg/ml in HIF-1? positive group,and 31.6pg/ml in HIF-1? strong positive group.The difference between strong positive group and negative group as well as positive group and negative group is statistical significant(P<0.05),and the difference between positive group and strong positive group is not statistical significant(P>0.05).Conclusions:The serum EGFL7 protein in the HCC patients with tumor hypoxia is higher than that in patients without tumor hypoxia.Hypoxia is one of the factorsthat stimulate EGFL7 to overexpression.Part III The cytological study ofeffect of hypoxia on EGFL7 expression in HCC cellObjective:To investigate the effect of hypoxia on EGFL7 expression in HCC cell at cellular level.Materials and Methods:The expression of EGFL7 in Hep3b,SMMC-7721,SMMC-7701,97L,97H cells were detected by Western blot and real-time PCR,thus to select the highest EGFL7 expression cell line and lowest EGFL7 expression cell line for the following research.Two selected HCC cell lines were cultured in oxygen-deficient environment simulated by a tri-gas incubator with 1%oxygen concentration.Western blot and real-time PCR were used to detected EGFL7 protein and EGFL7mRNA after 0,1,2,4,8 hours in oxygen-deficient environment respectively.The difference between different time points was analyzed and compared by one-way ANOVA.Results:Among Hep3b,SMMC-7721,SMMC-7701,97L,97H cell lines,the highest EGFL7 expression is detected in SMMC-7721,and lowest in 97H.In SMMC-7721 cell,the average gray scale of EGFL7 protein tested by Western blot at different time points were 0.140±0.01,0.335±0.02,0.429±0.04,0.656±0.05,0.701±0.04,with significant difference(P<0.001).The relative quantity of EGFL7mRNA detected by real-timePCR in SMMC-7721 cell were 1.019±0.248,0.677±0.090,0.719±0.264,0.870+0.119,0.724±0.058,without significant difference(P>0.05).Similar results were obtained in 97H cell line.The Western blotresult was 0.231±0.01,0.324±0.02,0.549±0.04,0.668±0.05,0.90310.04,with significant difference(P<0.001).Thereal-time PCR result was 1.010±0.176,1.074±0.092,0.988±0.134,0.885±0.018,1.022±0.105,without significant difference(P>0.05).Conclusions:The EGFL7 protein in HCC cell is elevated in hypoxia environment with a time dependent manner.The longer the time cultured in oxygen-deficient environment,the higher EGFL7 proteinis detected.However,there is no change of EGFL7 mRNA in hypoxia environment compared with atmospheric environment.Hypoxia is one of the signalfactorsthat up-regulated EGFL7 proteins.However,this up-regulation effect does not occur at transcriptional level. |
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