Identification Of Differently Expressed Genes In Eosinophils Of Asthma Suing Suppression Subtractive Hybridization

Asthma is an airway inflammation disease, which is characterized by airway hyperactivity and airway inflammation. Now, it is commonly believed that eosinophils, especially low-density eosinophils are key cells in the late asthmatic response and airway hyperactivity. The asthma was once named chronic desquamating eosinophlia bronchitis because the main pathologic character of allergic inflammation was believed to be featured by eosinophils infiltration. And now, asthma is believed to be caused by multi-genes and multi-agents. Therefore, there may exist one or more genes that trigger asthma; meanwhile, some other genes may influence human's susceptibility to asthma risk factor. Those genes may be inherited, or obtained from gene mutation through interaction with environmental factors. Understanding the function and expression pattern of those genes has been becoming increasingly important, especially after human genome project completed. So, it's scientific significance is very important, to study the differently expressed genes in eosinophils of patient with asthma.Object: To study the differently expressed genes in eosinophils of patient with asthma, a PCR based different expression technology with highly discriminating ability, the Suppression Subtractive Hybridization (SSH), wasintroduced to find out differently expressed genes and to further reveal the possible gene-marker during the process of onsetting asthma.Methods: The eosinophils were respectively isolated from peripheral blood of pre-and post-treatment patient with asthma, by using uncontinuous density gradient method. Then, the total RNAs were isolation. The cDNAs were synthesized by the technology of SMART?based on RT-PCR and digested with Rsa I. After ligating the digested cDNA with adaptors, two steps of hybridization and two rounds of PCR were followed to normalize and enrich differently expressed genes and construct the subtractive cDNA library. A PCR-select differential screening procedure was employed to reduce the background, clones containing the differently expressed genes was selected for DNA sequencing and analysised with BLAST program in GenBank wired NCBI website. Some sense genes were identified by Virtual Northern blot.Results: The highly purity eosinophils was respectively isolated from peripheral blood of pre-and post-treatment patient with asthma. Total RNAs were exacted and purified in good quality and quanity. Double strand cDANs were reversing transcripted integrally, and then cute by Rsa I into even length short segments. Ligation was effective. After two stage of subtractive hybridization, the differently expressed cDNA subtractive library of eosinophils in pre- and post-treatment patients with asthma was successfully constructed. Nest -PCR was selectively enlarged differently expressed genes. Products of PCR were ligated with T vector and then transformed into Escherichia DH5 a , which dispersed differently expressed genes mixture into single ones. 96 target gene segments were isolated primarily from it. After differential screening, 15 clones containing differently expressed genes were identified. 12 differently expressed genes were obtained. Sequences was submitted to GeneBank for homogenous contrast to makd claear their property.Conclusions: The cDNAs were synthesized by the technology of SMART? It solved the contradiction of how to synthesize enough cDNAusing trace total RNA. SSH PCR had been applied successfully in our study to construct eosinophil differently expressed genes from patient with asthma.12 differently expressed genes were found, which encode proteins including cGMP gated channel, TAKl-like protein, ATP synthase FO subunit 6, CoxII/D-loop DNA fusion protein, cytochrome c oxidase subunit I and II, DAZ associated protein 2, NADH dehydrogenase, PTH-responsive osteosarcoma D1 protein, similar to Sarcoplasmic reticulum histidine-rich calcium-binding protein. It had indirectly confirmed the genes of eosinophils that regulation of proinflammatory response, signal transduction, energy metabolism and ce...