| Pregnancy-induced hypertension (PIH) is one of the main causes of perinatal morbidity and mortality. It is characterized by the spontaneous relieve of its symptoms and signs after the delivery of placenta. Placenta is a temporary organ with the relatively simple structure and sophisticated endocrine functions, consisting of trophoblasts and interstitial tissue and hence sharing the paternal and maternal genetic features. Accumulating data have demonstrated alterations in the placentas from PIH patients using histological, endocrinological and molecular approaches.Current basic researches in this field focus on the disease-associated gene and gene expression profiles with the human genome program finished recently. We have performed the following research works to describe the difference of gene expression between the preeclamptic placenta and its normal counterpart, the relationship between the gene expression and clinical manifestation and the consistency of the gene expression and according proteins.1. We collected clinical data and placental tissues from 42 PIH patients and 22 matched normotensive primipara to construct the PIH database during the period from March, 2001 to Sep, 2002 in Xijing Hospital, Tangdu Hospital, Women and Children's Hospital of Shaanxi, No.4 Hospital of Xi'an. For all cases, pregnancy was terminated by Cesarean section before labor to exclude the alteration of gene expression caused by uterine contraction. Placenta tissue was dissected and frozen in liquid nitrogen overnight and then kept at -80 till use.2. Placenta tissues were embedded in paraffin after fixed in 4% buffered Formaline. One hundred and fifty-spot customized tissue array, with 2 or 3 spots taken from each sample, were manufactured from paraffin-embedded blocks.3. Suppression subtractive hybridization (SSH) was performed to screen thedifferentially expressed genes using two severe PIH cases as 'tester' and 4 normotensive primipara as 'driver'. Of two PIH cases, one delivered male neonate and another one delivered female neonate; for the 4 normotensive primipara, 2 delivered male neonates and the other ones delivered 2 female nconates. Total RNA from 2 PIH placentas were pooled to act as 'Tester RNA' and total RNA from 4 normotensive placentas were pooled to act as 'Driver RNA' after the elcctrophoresis of RNA showed the quality of RNA was satisfactory.PIH subtracted cDNA library was set using the PCR-seclect suppression subtractive hybridization kit. According to the product manual, forward and reverse SSH were performed while the control SSH was done in parallel to evaluate the efficiency of experimental SSH.The final PCR-generated forwarded subtracted cDNA mixture, in which, tester cDNA was from preeclamptic placentas and driver cDNA from normotensive placentas, was cloned into the cloning vector pGEM-T Easy and transformed into Coli strain DH5 . A total number of 384 white clones were picked randomly. Inserts were amplified by nested PCR and the product were then dotted onto membranes for reverse dot hybridization.Forward and reverse probes, respectively from the PCR products of forward subtracted cDNA library and reverse subtracted cDNA library, were labeled by -32P dCTP. Two nylon filters, on which 384 clones were dotted, were hybridized with the labeled probe. Radioactive signals were scanned and quantitated using a computer program. The signal intensity of each dot on forwarded hybridization membrane was compared to those corresponding dots on reverse hybridization filter. The signal intensity ratio was calculated. Clones showing a ratio exceeding 3 on the forward membrane were selected for subsequent sequencing.One hundred and three clones were sequenced by the T7 primer methodology. Ninty clones were sequenced successfully and 13 clones failed to be sequenced because of complicated structure or poor sequence data. BLASTN search in NCBI was used to compare the insert sequence to those in Gcnebank/EMBL databases. Inserts with 95% or more homogeneity were defined putative candidate...
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