Empirical Study On The Expression And Significance Of Endostatin, BFGF, CD34 In Gallbladder Carcer

PrefaceGallbladder cancer is the most frequent malignant tumor of the biliary system. Because it is hard to be diagnosed early, clinical patients were mostly in advanced stage, its 5 year survival rate is very low. It is a hot spot to try finding new antitumor drugs in gallbladder cancer comprehend treasment.Tumor's generation, development and diversion rely on neovascularization. Neovascularization makes it easy for tumor to grow rapidly, to enter blood circulation and to transfer to distant place. Basic fibroblast growth factor (bFGF) is a drastic Vascularization promoter. A great quantity of studies have approved that bFGF can promote cell caryocinesis and induce neovascularity and have close relations with tumor differentiation, infiltration, growth, metastasis and neovascularization. Vascularization is a process in which many factors play their parts, and which is precisely controlled by 'Vascularization switch'. Angiogenesis factors hold the balance with anti-angiogenesis factors. Endostatin is an ideal Vascularization inhibitor found to day. It can inhibit tumor neovascularization and metastasis, promote tumor cell apoptosis. Folkman proposed that malignant tumor growth and metastasis rely on neovascularization, that we could inhibit tumor growth by means of inhibiting neovascularization. By now, Folkman's propose has been proved to be true. Endostatin is regarded as a broad-spectrum and low toxical neovascularization inhibitor. Microvessel density (MVD) is a quantitive index for neovascularization, can be used as an objective index for tumor prognosis, prognosis evaluation and demixing treatment. Now, we believe that bFGF has close relation with neovascularization, tumor growth, stage, metastasis, prognosis and curative effect in gallbladder cancer. But reports seem not enough. And no report is found correlating the relations between Endostatin and gallbladder cancer.ObjectiveThis experiment detected Endostatin, bFGF, CD34 expression in paraffin sections of post-operation gallbladder cancer specimens by means of immunohistochemical method. The purpose was to try to find the relations between Endostatin and bFGF expression with clinical and pathological parameters of gallbladder cancer.Materials1. Specimens61 paraffin sections of post-operation gallbladder cancer and 10 normal gallbladder specimens collected from the 1st Affiliated Hospital of China Medical University.2. Chemical and reagentsEndostatin polyclonal antibody (Neomarkers, USA); bFGF polyclonal antibody (Santa, USA); CD34 monoclonal antibody, SP immunohistochemistry kit and positive sections (Maixin, Fuzhou).3. Experimental instrumentsFluorescent light inverted microscope (Olympus ix 51, Japan); Micro image collection system CCD (Olympus DP 71, Japan)Methods1. Immunohistochemistry:Routine SP immunohistochemical method was used.Inves 3μm paraffin section on polylysine smeared glass slide, on fire at 75℃for 1.5 hours. Dimethyl benzene to disparaffin to water. Put in 0.05 (PH 7.2)PBS to wash for 5 minutes. Antigen repair at high temperature in 1 M citrate buffer solution (PH6.0). 0.3% hydrogen dioxide methanol to block endogenous Oxidase activity for 10 minutes, 5% normal nonimmune animal blood serum to block endogenous antigen for 5 minutes, add the first and second antibody, put in SP solution for 20 minutes, DAB to color, hematoxylin to afterstain, dry out and clear, mount with neutral gum. The first antibody was substituted by PBS as negative control while already known gallbladder cancer positive section was used as positive control. Score method: Immunal reactive score (IRS) = Staining intensity (SI)×Positive percentage (PP). IRS 0-3 negative; 3-6 weakly positive; 6-9 positive; 9-12 strong positive. MVD refered to Weidner method.2. Statistical analysisEnumeration data was analyzed by X test, measurement data was analyzed by T-test, ANOVA and Q test, using SPSS 10.0 software.Results1. Endostatin expression:Positive expression rate in normal gallbladder tissue and gallbladder cancer were 20% and 67.21% respectively, showing statistic difference (P<0.05). In 61 cases of gallbladder cancer, expression intensity of Endostatin had correlation with clinical stage and lymph node metastasis (P<0.01, P<0.05), but had nothing to do with sex, age, tumor location, size and histological class.2. bFGF expression:Positive expression rate in normal gallbladder tissue and gallbladder cancer were 40% and 77.05% respectively, showing statistic difference (P<0.05). In 61 cases of gallbladder cancer, expression intensity of Endostatin had correlation with clinical stage and lymph node metastasis (P<0.05, P<0.05), but had nothing to do with sex, age, tumor location, size and histological class.3. CD34 expression:MVD was calculated by means of CD34 expression. The mean value of MVD in gallbladder cancer was 76.66±20.15, much higher than in normal tissue (29.53±5.03)( P<0.01). In 61 cases of gallbladder cancer, the mean MVD value of Nevin stage III-V was 80.53±17.98, much higher than Nevin stage I~II (46.79±5.38)(P <0.01). Mean MVD value of cases with lymph node metastasis was 94.60±7.28, much higher than those without lymph node metastasis (58.12±9.24)(P<0.01). Mean MVD value of cases of histological class G1, G2, G3 were 60.59±14.71, 83.08±15.30, 96.53±6.92 respectively, showing statistic difference among groups (P<0.01) .But had nothing to do with sex, age, tumor location and size.4. Correlations among Endostatin, bFGF and CD34 expression:There was strong correlation between Endostatin and CD34 expression (P<0.01), so was bFGF and CD34 (P<0.01). But no correlation was found between Endostatin and bFGF expression.DiscussionIn normal tissue, angiogenesis promoters hold the balance with angiogenesis inhibitors. Blood vessels are in resting state called quiescence. After tumorigenesis, angiogenesis promoters increase while angiogenesis inhibitors decrease correspondingly, causing Vascular proliferation which makes it easy for tumor to grow and metastasize. We found no reports regarding the relations between expression of angiogenesis promoter bFGF and angiogenesis inhibitor Endostatin in gallbladder cancer and their interaction. This study detected Endostatin and bFGF expression in gallbladder cancer by means of immunohistochemical method, using MVD, which was calculated through CD34 expression, as an objective index. Our experiment proved that there existed high correlation between Endostatin expression and MVD. So did bFGF expression and MVD. Their roles in the course of gallbladder carcinogenesis and development are obvious.Conclusions1. Endostatin expression in gallbladder cancer was significantly higher than in normal tissue, playing certain role in gallbladder carcinogenesis and development. So did bFGF expression and MVD.2. Endostatin expression was greatly enhanced with clinical stage increasing and lympe node metastasis. Enhanced expression showed higher malignant extent and more powerful invasion.3. bFGF expression was greatly enhanced with clinical stage increasing and lympe node metastasis. Enhanced expression showed higher malignant extent and more powerful invasion.4. MVD was greatly increased with clinical stage increasing, lympe node metastasis and histological class increasing. Those with more MVD showed higher malignant extent.5. Endostatin expression had close relations with MVD, so did bFGF expression, showing that angiogenesis promoters and angiogenesis inhibitors play significant roles in Angiogenesis. No relations was found between Endostatin and bFGF expression, showing that Endostatin's inhibiting role might had its independent mechanism, except for fighting for receptors with bFGF.