[Cloning of differentially expressed genes of eosinophils from asthmatic patients by suppression subtractive hybridization].
OBJECTIVE
To explore the molecular mechanism of eosinophils for its role in bronchial asthma.


METHODS
The total RNA extracted from the eosinophils of patients during the onset of asthma was used as the tester and the total RNA obtained after treatment served as the driver. cDNA suppression subtractive hybridization (SSH) was performed using the protocols described in the Clontech SMART PCR cDNA Syn thesis Kit and PCR-Select cDNA Subtraction Kit. The subtracted cDNA was then inserted into T vector to generate subtracted cDNA library. Clones of the subtracted cDNA library were screened by hybridization and the insert sequence of the positive clones was compared with the sequence in the GenBank.


RESULTS
A total of 400 clones selected from the subtracted cDNA library were amplified by PCR and about 85% of these clones contained inserts. Six differential cDNA fragments were acquired after two differential screening. These genes were involved in the regulation of proinflammatory response, signal transduction, energy metabolism and cell apoptosis.


CONCLUSIONS
Differentially expressed genes of the eosinophils during the onset and the remission stage of bronchial asthma can be effectively cloned by SSH, which provides a solid foundation for clarifying the molecular mechanism of eosinophils in asthma and a theoretical base for clinical treatment and prevention of asthma.
Xu
Jin-song
JS
Department of Respiratory Diseases, Nanfang Hospital, First Military Medical University, Guangzhou 510515, China.
Cai
Shao-xi
SX
Zou
Fei
F
Tong
Wan-cheng
WC
Zhao
Hai-jin
HJ
D001249:Asthma / Q000097:blood*; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004804:Eosinophils / Q000378:metabolism*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction