The Clone Of FasL Gene And The Distribution Of Its MRNA In Lung Cancer Cell

Objective: To adop RT-PCR technique and molecular cloning technique to clone human FasL gene fragment and make cRNA probe, to adop in situ hybridization technique to observe the distribution of FasL-mRNA in lung cancer celi,and study its clinical significance.Method: Healthy human peripheral blood monocty were acquired (its concetration is50Mm)and treated by phytohaemagglutinin-p for 15 minutes to induce expression of FasL gene.total RNA was extracted with QIAamp RNA Blood Mini kit,and its cDNA fragment were amplified by RT-PCR with specific primers. Through sepharose electrophpresis, there was a DNA amplifying strap which matched expertation in molecular weight,the PCR production were retrieved with QIAquick Gel Extration kit,then were amplified and puredWe use Pst I and EcoR I emzymes to make the production and plasmid DNA complement, inserted it into the MCS ofPspt19 withT4 ligase to construct recombinant plasmid,then transfer the plasimd into competence colibacillus JM109, positive recombinant ones were selected by Aminobenzylpenicillin resistance in LB plate,the FasL gene fragement was testified by sequencing. The recombinant plasmid Pspt 19 was linearized by EcoR I to synthesize the Dig-cRNA probe with SP6 polymerse.All lung cancer specimens were collected from the thoracic surgery department in hospital affiliated Qingdao university medical college(3 squamous carcinoma,2 adenocarcinoma, 1 small cell carcimoma), the specimens were put in Eppendorf,freezed quickly by liguid nitrogen and stored in -70 "C refrigerator. The specimens were treated by 4% Polymerisatum and 15% Saccharu, then were ready for cryo-section which was 6um in depth, and then fixed again with 4% Polymerisatum, we used nonion detergent to treat cell membrane We can observe the distribution of FasL mRNA in lung cancer cells by using in situ hybridization technique on ctyostat sections.Result:The sequence of FasL cDNA fragment inserted in plamid is right by sequencing;Dig-FasL cDNA probe is efficient by labeling efficiency detecting;We can see blue-puple hybridizition signals in lung cancerjts means that lung cancer cells express FasL mRNA.Conclusion: The cloning of FasL gene is a tool to study lung cancer cells apoptosis.FasL cRNA probe labeled by digitoxin is a efficient method to futher study the distribution of FasL mRNA in lung cancer cells; As an apoptosis gene, FasL relates closely to the occurrence,development of lung cancer.Peng Chuanliang(thoratic surgery) Directed by Professor ShenYi...