The Expressions And Significance Of EphB4 Receptor And Its Ligand EphrinB2 MRNA In Endometrial Carcinoma

ObjectiveEndometrial carcinoma is one of three malicious tumors of female genital tract. In recent years, its morbidity has a trend to increased. To know the mechanism of the genesis and development of endometrial carcinoma and look for an effective method for diagnosis and treatment is our task.Persistent angiogenesis is needed in the course of tumors'occurrence and development. Many growth factors contribute to this course. The Eph (Erythropoietin — producing epatocellular) family of receptor tyro-sine kinases and their ligands, the eprins, act as the new growth factor to promote angiogenesis. It has been proved that they are highly expressed in many kinds of tumors. Few has studied their expressions in endometrial carcinoma. The expressions of EphB4 mRNA and EphrinB2 mRNA in endometrial carcinoma and nomal endometrial tissues were detected by RT — PCR technique,so as to explore the relationship between these two factors and the genesis and development of endometrial carcinoma ,and provide evidences for the gene therapy of endometrial carcinoma in future.Materials and Method1. Materials33 samples of endometrial carcinoma tissues were taken in operations of the second Clinical College of China Medical University during Octoberof 2003 to January of 2005, with 10 samples nomal endometrial tissues as controls. Tumors were staged according to the criteria proposed by International Federation of Gynecology, including stage I 18 cases, stage II 9 cases, stage DI — IV 6 cases;G1 10 cases, G2~G3 23 cases. The age of all the patients is from 31 to 71, with a mean age 59. After the samples were collected, put them into liquid nitrogen quickly,and then preserved them in refrigerator of — 70°C. All the samples were diagnosed by pathological examination.2. Main reagents:Primers for EphB4^EphrinB2 and internal control;RT - PCR kit;TRIzol.3. Experimental apparatus:PCR amplification apparatus;automatic analysis system of electro-phoresis gel imaging;electrophoresis apparatus,etc.4. Method:(1) Extract total RNA:Take one step extracting technique with TRIzol.(2) Reverse transcription to synthesize cDNA:The operation was taken according to the directions of test kit.(3) PCR amplification and production analysis:Primers for EphB4NEphrinB2 and [3—action were designed by primer 5. 0 in normal text.PCR reactive system: cDNA 3^1;Buffer (10 X ) 2. 5^1;dNTPs (10mmol/L)2fil;Taq enzyme(5U//ul)0. 2p\-,0. 1^1 of each primer;dd ? H2 O 16. 7p\. Predegeneration for 3 min at 90°C , degeneration for 45S at 94°C ,annealing for 60S at 54°C , renaturation for 60S at 72°C. After 35 cycles, extend for 7 min at 72°C.Take 10/^1 production and perform 2% agarose gel electrophoresis, and observe under extra — violet lamp and take photos, then detect the content of each amplification band with the automatic analysis system of electrophoresis gel imaging. Use marker as the standard of molecular weight,and (3—action as internal control,and skin tissue sample as nega-tive control instead of experimental sample.5. Judgement for results:The bands which showed both 509bp and 690 bp as internal control were positive results;and the bands which showed both 503bp and 690 bp as internal control were positive results too;and the bands which showed 690bp only were negative ones.6. Statistical analysis:Take Statistical analysis with software of SPSS 10. 0, t — test or a-nalysis of variance were used to compare relative contents. X2 — test or Fisher's exact probability were used to compare rates.Experimental results1. The expressions of EphB4.. EphrinB2 mRNA in the endometrial carcinoma and normal endometrial tissues.The expression rates and relative content of EphB4 mRNA in endometrial carcinoma were higher than that in normal endometrial tissues significantly. (p0. 05). The relative content of EphB4 mRNA in stage ffi~]V were higher than that in stage I > II significantly (p0. 05). The relative content of EphrinB2 mRNA in stage HI~ IV was much higher than that in stage I significantly (p0. 05).(2) The expression rate and relative content of EphB4 mRNA in G2-—G3 were much higher than that in Gl significantly (p0. 05).(4) In different depth of myometrium invasion, there was no obvious difference in expression rate and relative content of EphB4 mRNA and EphrinB2 mRNA(p>0. 05).(5) There was no statistical significance between endometrial carcinoma with lymphatic metastasis and without lymphatic metastasis in expression rate and relative content of EphB4 mRNA and EphrinB2 mRNA (p>0. 05).Conclusion1. EphB4>EphrinB2 have relationship with the occurrence of endometrial carcinoma, and it maybe another index reflecting the benignancy or malignancy of endometrial disease.2. There was dense relationship between the mRNA expressions of EphB4 and EphrinB2 and the biological behavior of endometrial carcinoma. They play an important role in the development of endometrial carcinoma, and it even maybe do as one of markers that evaluate the patient's condition .3. The mRNA expressions of EphB4 and EphrinB2 has no relationship with the depth of myometrium invasion , histological type and lymphatic metastasis of endometrial carcinoma.4. The mRNA expressions of EphB4 and EphrinB2 in endometrial carcinoma were coincident. This means that the binding of EphrinB2 lig-and with EphB4 receptor maybe the principal form of EphB4 receptor activation. They may interact with each other to exert their biological function.