Cloning And Primary Identification Of Differentially Expressed Genes In Placentas From Preeclamptic Patients

OBJECTIVESPreeclampsia is a common complication of pregnancy and the major cause of maternal and perinatal morbidity and mortality, The pathogenesis of preeclampsia is not totally clear so far, it is the focus for studying in obstetrics all the time. Its pathophysiologic change which controlled by mult'igene involves of many systems and organs. so we dedicate to look for susceptible genes of preeclampsia and research their expressing situation which may be helpful to define molecular alterations in preeclampsia. mRNA differential display(DD) was a method of isolating and cloning genes . In this study , we dedicate to clone placental genes related to preeclampsia by DD and using other methods to identify them primaryly.MATERIALS AND METHODSWe extracted total RNA from placentas of 3 sever preeclamsia patientes and 3 normal pregnancy women, Equivalent high qualitative RNA were used for fluorescent differential display(FDD)and reamplified by PCR. 12 pairs of primers combined by 3 anchored primers with fluorescence and 4 arbitrary primers were applied for the experiment. After PCR products were seperated by polyacrylamide gelelectrophoresis, the differentially expressed placental cDNA bands were identified through fluorescent scanning and excised from the gel and reamplified, then they were being cloned by linked with T-easy vector and transformated E. coli. After the recombinant was filtrated > purificated and identified ,all cDNA fragments we obtained were sequenced. the cDNA sequence were analysed for homologue in GenBank database by using BLASTN software. Northern blot was performed for determining the expression of cDNA fragments in 2 groups of tissues, mRNA expression of genes was measured by semi-quantitative RT-PCR in placentas of 15 preeclampsia and 15 normal pregnancy woman, protein expression was detected by immuhohistochemistry in placentas of 30 preeclampsia and normal pregnancy women.RESULTS72 differential expressed cDNA bands were cutted from polyacrylamide gel, 20 of them were reamplif ied, cloned and sequenced, we have obtained 17 cDNA fragment. 10 of them are highly expressed in placentas of preeclamptic patients, 5 of them are respectively high homologous to human protein phosphatase 2A catalytic subunit-beta (PP2ACP) ^ ABC transporter G2(ABCG2h delta-aminolevulinate dehydra-tase(6-ALADK cytochrome c oxidase subunit Vic (C0X6c)and vacuolar protein sorting 29(VPS29), 2 of them are homologous to genes which function is unknown so far, 3 of them have low identity with known genes and are submitted to GenBank , which were given new accession numbers of CV99805K CV998052^ CV998053. 7 of them are lowly expressed in...