Expression Of Kdr Mrna In Epithelial Ovarian Serous Tumors And Its Clinical Significance

ObjectivePersistent angiogenesis is needed in the growth, metabolism and metastasis of solid tumor. Vascular endothelial growth factor ( VEGF) is one of the most important factors that could promote the angiogenesis of tumors. Recent reports have showed that VEGF could produce effect by combining to kinase insert domain-containing receptor (KDR) , a specific receptor of VEGF. KDR is the receptor of protein of tyrosine kinase of the cell surface, and is expressed in endothelial cells mainly. But the expression of KDR could be also detected in other cells such as macrophages, blood cells and tumor cells. We detected the expression of KDR mRNA in 80 different cases of ovarian tissues with semi-quantified RT-PCR technique and analyzed the relationship between the expression and clinical pathologic characters to investigate the significance of KDR mRNA expression in origination and development of ovarian serous carcinoma ( OSC ) , thus provide evidences for preventing angiogenesis, metastasis and relapse of the tumor in molecular level.Materials and Method1. Materials70 samples of ovarian serous tumors tissues were taken in operations of Department of Gynecology of the Second Affiliated Hospital of China Medical University during March of 2000 to March of 2002, including 30 ovarian serous carcinoma, 10 ovarian borderline serous tumors and 30 benign serous tumors. Choose 10 normal ovarian tissuesfrom hysterectomy and ovarian resection as controls. The age of all patients is from 20 to 70 with a mean age of 43. 1. After the samples were collected, put them into liquid nitrogen, then preserve in refrigerator of - 10 . Take pathological examination by 2 specialists of pathology and make pathological diagnosis. Staging of OSC according to FIGO (1986) : 8 of I andII ; 22 of I and IV; histological grade; 8 of high differentiation, 22 of middle and low differentiation. In them, 24 were positive in abdominal cellular examination, 14 with lymph node metastasis and 20 with pelvic adhesion.2. Main reagentsTRIzol for extracting total RNA ( production of USA Promega Co. ) ; RT-PCR Kit (provided by Japanese TaRaKa Co. ) ; The primer was synthesized by Beijing Oke biologic Ltd. Co. Agarose for elec-trophoresis was provided by Shenyang lianxing Biologic Co.3. Method(1) Extract total RNA: Take one step extracting technique with TRIzol.(2) Reverse transcription to synthesize cDNA; Sample RNA 2uJ, Buer( x2)10uJ, MgS04(2.5mmol/L)4pa, dNTPs(lOmmol/ L) l,jj, RNase-Inhibitor (400/^J) 0. SjjJ, AMV IjjJ, Oligo dT ( 50/xmol) 1 jxl and ddH20 0. SJJL! were added in 20fjJ reactive system. Then incubate for 3 minutes at 90 ^C and for 30 minutes at 42^ to synthesize cDNA.(3) PCR amplification and production analysis-.See the part of normal text of the article about the production of KDR and (3-actin gene and the length of amplified fragment.PCR reactive system of KDR: 3jjJ cDNA production, 0.05 l of each primer, Buffer( 10)2. 5 l, dNTPs ( 10mmol/L)2 l, 0. 2 lTaq enzyme and 17.2jxl ddH20. Predegeneration for 90s at 951, degeneration for 45s at 94 Tl, annealing for 45s at 591, renaturation for 90s at 12 . After 35 cycles, extend for 10 minutes at 72 . -actin was amplified in another reactive system at the same time.Take 10 l PCR amplification production and perform 2% agarose gel with 0. 5 (jig/ml EB electrophoresis for 1 hour with voltage of lOOv. Observe under extra-violet lamp and examine the content of each amplification band with the help of Kodak electrophoresis gel imaging and automatic analysis instrument ( X174-Hic II DNA Marker was regarded as the standard of molecular weight). During every experiment, KDR mRNA extract of colon cancer was made as positive control, distilled water was as blank control, and (3-actin was internal control.(4) Judgment for resultsThe band that showed 555bp and 690bp as internal control compared with Marker would be judged for positive appearance. The band that showed only 690 bp as internal control was judged for negative appearan...