Apoptotic Gene Expression Profiling Of Placental Trophoblast Cells In Patients With PIH

§1 Observation on Placental Trophoblast Apoptotic Phenomenon in Patients with PIHObjective To analysis the features of apoptosis in placental trophoblasts of patients with PIH, and study the relationship between the trophoblastic apoptosis and the etiology and pathophysiology of PIH. Methods To obtain the evidences of apoptosis in the placental trophoblasts of patients with PIH, the techniques of the electron microscope scanning, TUNEL and FCM were applied. Results (1) In the ultrastructure of PIH placental trophoblasts under the electron microscope scanning, there also were typic morphologic changes of apoptosis including anomalo-nucleus, pyknosis, abnormal distribution of heterochromatin, deeply-stained mitochondrial matrix in cytoplasm, clouding or disarrangement of mitochondrial crest, vacuoles in placental trophoblast cells, and so on. (2) Under the detection of TUNEL technique, it was found that there were more numbers of apoptotic trophoblasts in the placenta of PIH with fewer apoptotic cells appearing in the placenta of NC. (3) In normal placental tissues, no markedly apoptotic peak (Apo peak) was found in the diagrams of FCM. However, there all were the markedly apoptotic peaks in the FCM diagrams of the PIH placental tissues. Conclusion Trophoblstic apoptosis in the placenta may contribute to the etiology or pathophysiology of PIH. §2 Apoptotic Gene Expression Profiling of Placental Trophoblast Cells in Patients with PIHObjective To explore the function of placental trophoblast cells on the pathogenesis mechanism of PIH, the character and changers of gene expression patterns of trophoblast cells in PIH patient placenta were analysed by the technique of gene microarrays. Methods 20 cases of PIH and 10 cases of normal pregnancy placental tissues were acquired at the termination of the pregnancy. 5 cases were randomly selected in each group and theirs apoptosis gene expression patterns were studied. Target genes (provided by poxing company, Shanghai) come through cDNA colone,PCR amplifying,purification,spotting and then preparation of microarrays for following use. To obtain purfied trophoblast cells, placental tissues for studying of apoptotic gene expression patterns were processed density gradient centrifugation respectively. After mergering trophoblast cells based on experiment and control group (in order to decrease the individual heredity difference), total RNA were extracted by single step method and then preparation of microarrays. The microarray was scanned with a ScanArray 3000 Scanner after washing. The acquired image was analyzed by ImaGene 3.0 sofeware. Differentially expressed gene were defined as follow:①The absolute value of the natural logarithm of the signal ratio of cy5/cy3 was greater than 0.69.②One of the signal values of cy3 and cy5 was greater than 800. Results Ten differentially expressed genes were acquired through apoptotic gene expression experiment (5 percent of total apoptosis gene expression chips). There was a significant decrease in expression in PIH patient placental tissues (e.g. the ratio of cy5/cy3 was lower than 1). Among them, there were genes that possess significant anti-apoptosis functions (including SFRP2,IAP2,DHCY24 and ATPIA1). Conclusion On the basis of the previous experiment, which be found that notable apoptosis exits in trophoblast cells in PIH patient placental tissues, the results suggest that significant decrease in genes, which possess anti-apoptosis functions, could result in the apoptosis of placental trophoblast cells and thus contribute to the pathogenesis of PIH.§3 Verification of Differentially Expressed Genes in Placenta of Patients with PIHObjective To verify the expressive levels of the differentially expressed genes in placenta of PIH and identify the reliability and confidence level of the result in gene expression profiles of PIH placenta. Methods The total RNA were extracted from PIH placenta and normal placenta. RT-PCR and Western Blot techniques were used to detect the expressive levels of the differentially expressed genes in placenta of PIH and normal placenta. Results It was found that the expressive levels of the differentially expressed genes were significantly lower in the placenta of PIH than those in the normal placenta. Conclusion The tendencies in the expressive alteration of all verified differentially expressed genes were in coincidence with the result of gene expression profiles of placenta in the patients with PIH. It was suggested that the experiment of cDNA microarray be of reliability and confidence. To further analysis the relationship between the differentially expressed genes and PIH may provide the etiology and pathophysiology of PIH with new theory gist.