| Cancer of the cervix is the second leading cause of cancer deaths in women worldwide and a leading cause of mortality among women of reproductive age in developing countries . Although its morbility reduced in recent years , its 5 - year - survive rate is low . and in those women whose age below fifty its incidence increase 10.3% , as well as increase at 3% each year. GIN (cervical intraepithelial neoplasia) also doubled in young women . Hence, It has become the more important for morden gynecologists to find early genetic index as well as valid methods to treat cervical cancer, furthermore reveal its molecular biologic mechanism of producing and developing .It has been developed to genetic level for cervical cancer etiologic research and treatment in recent years. Especially, nowadays European . American and Japanese scientists had made quantity research to find COX genetic structure . function and the relationship with tumor.COX is the rate - limiting enzyme in the conversion of arachidon-ic acid to prostanoids . Its inducible isoenzymes COX ?2 is directly related to tumor development . Investigation revealed that COX - 2 expression is elevated in gastric adenocarcinomas . prostaglandin cancer . bladder tumor . human no small cell lung cancer and adenocarcino-ma. Colorectal adenomas and carcinomas. So it is considered thatCOX - 2 is directly related to tumor development and progression . Little is known , however , about the expression of COX - 2 in cervical cancer and in cervical intraepithelial neoplasia. COX - 2mRNA expression was assessed by reverse transcription - polymerase chain reaction ( RT - PCR) in cervical cancer and CIN to demonstrate the associations between COX - 2 expression and cervical cancer . in order to find early genetic diagnosis target and genetic remedy in cervicalcancer.Materials and Methods(一)SpecimensSeveral tissue specimens were selected from surgical section andpathologic examination in China Medicaal University from March 2000- October 2002. thirty cases of cervical cancer . ten cases of CIN andthirty cases of chronic cervicitis . Samples were stored at - 70 T! untilanalysis.(二)Methods1. Extraction - RNA was extracted using TRIZOL reagent according to the protocol recommended by the manufacture . Briefly 50 ?100 mg of each tissue was homogenized in 1 ml of TRIZOL, subsequently 0.2 ml of chloroform was added and the mix was centrifuged (13000 g 15 min 4℃ ) This separated the solution into an aqueous phase containing RNA , an interphase containing DNA, and an organic phase containing protein . The aqueous layer was carefully aspirated and added to 0.5 ml of isopropanol for RNA precipitation ,Following this ,the solution was centrifuged ( 13000 g 15 min 4℃) and the pellet was washed with 75% ethanol , After that ,RNA was collected into 50ul ofDEPC - treated water according to the size of the pellet. The RNA solution was kept frozen at 70℃ until analysis.2. Reverse transcription The reaction solution was obtained by mixing reagents as follows : 2 x buffer; 10ul; 2 mmol MgSO44ul;10 mmol dNTPs; BcaBest Polymerase 1ul; Rnase Inbitor 0. 5ul Oligo dT1ul;dd H2O 0. 5ul . The reaction was performed in an incubator at 42℃: for 30min and then inactivated by heating at 90℃ for 3 min.3. PCR - The primers for sequence were: COX - 25' -TTCAAATGAGATTGTGGGAAAATTGCT - 3 ' (forwad) and :5' -AGATCATCTCTGCCTGAGTATGTTT - 3 ' , ( reverse ) yielding a 305bp product and for control , 3 - actin 5 ' - TGTATGCCTCTG-GTCGTACCAC - 3 ' ( forward) and 5 ' - ACAGAGTACTTGCGCT-CAGGAG -3' (reverse) yielding 690bp product.ResultsmRNA forB - actin was seen in all samples , mRNA for COX - 2 was delected in 28/30(93. 3% ) samples of cervical cancer ,mRNA for CINM was detected in 4/10(40% ) ,and chronic cervicitis 12/30 (40% ). COX - 2mRNA expression was siginificantly increased in cervical cancer (P <0. 01) ,However ,there is no difference in COX -2 expression between CIN HI and chronic cervicitis ( P>0.05 )Discussion一, COX -2 is a... |
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