Study On The Mechanisms Of Intercellular Adhesion Molecule-1in Radiation Pulmonary Leision

Radiation pulmonary leision (RPL) is a general term for damage to thelungs which occurs as a result of exposure to ionizing radiation. whichcommonly occurs as a result of radiation therapy administered to treat thoraciccancers. RPL belongs to the non-infectious inflammation and is alwaysdifficult to treat[1], therefore it is one of the dose-limiting factors in thoracicradiotherapy. The pathogenesis of RPL is not very clear at present, so there areno independent predictor and no effective predictation and treatment to RPL.Intercellular adhesion molecule-1(ICAM-1), also known as CD54(Cluster ofdifferentiation54) is one of important cell adhesion molecules andinflammatory mediators. ICAM-1is involved in a series of importantphysiological and pathological processes such as cell signaling and activation,cell stretching, movement, growth, differentiation, inflammation, thrombosis,tumor metastasis and wound healing[2]. With the roles of ICAM-1inleukocyte adhesion and aggregation in the alveolar region, ICAM-1is one ofthe key factors to lead to pnuemonocyte injury and cytokine release sustainlyin RPL. There have been some reports that the anti-ICAM1antibody canreduce the degree of lung injury[3-4], but it remains to be further studied thatthe specific mechanism of ICAM-1in RPL and that ICAM-1can be used as anew target to reduce radiation-induced lung injury. In this parper, the ICAM-1gene silencing cell lines was constructed with RNA interference technologyand the mechanisms of ICAM-1was analysed on cell and gene levels，Which is helpful to provide theoretical basis and new ideas for the prediction andtreatment of RPL.Objective:1. To investigate the expression of ICAM-1and related Cytokines in theprocess of RPL with the mouse RPL model due to60Co γ ray exposition.2. To investigate the changes of biological characteristics and gene profilingexpression by constructing the ICAM-1gene silencing cell lines.3. To explore the effect of ICAM-1gene silencing on lewis lung cell (LLC)radiation sensitivity.Methods:1. The mice were exposed to single fraction irradiation60Co γ ray to establishthe mouse model of RPL. The pulmonary injure was observed afterhematoxylin-eosion (HE) staining and the expression levels of ICAM-1,transforming growth factor-β1(TGFβ1) and tumor necrosis factor (TNF-α)detected with immunohistochemistry (IHC).2.①Three shRNA gene fragments of the mouse ICAM-1synthetisedaccording to its mRNA sequence were used to constructed three specificshRNA expression vectors pRNAT-U6.1/Neo-ICAM1-shRNA. Thecorrectness of the inserted sequence was verified with enzyme digestion andDNA sequencing.②LLC cells were transfected with liposomes and the positive clones weresingled out with G418. RT-PCR and Western blot assay were used to detectthe silencing efficiency of ICAM-1gene.③The inverted microscope was used to observe cell morphology changesand count the cell numbers. The growth curve and the MTT colorimetricassay was used to investigate the cell proliferation activity, Gene microarray was involved to analyse the genome-wide expression spectrum changes ofICAM-1silenced cells in order to select the different genes related to thesignaling pathway of ICAM-1and to find the transduction process of theICAM-1signaling pathway.3. The ICAM-1silenced cells were exposed to60Co γ ray at the accumulateddose of4Gy. MTT colorimetric assay was used to investigate the cellproliferation activity. Clone formation test were used to detect the radiationsensetivity, and flow cytometry was used to check the cell apoptosis andcell cycle progression.Results:1. Compared with the control group, the mice of the irradiated group becamedroopy and sluggish with presence of loss of hair, there slowed lungcongestion.Being nohomogeneous and alveolar wall thickened andinfiltration of inflammation cells. The expression levels of ICAM-1andTGF-1were increased significantly (P<0.01, P<0.05). TNF-α was alsoincreased, but not significant compared with the control group (P>0.05).2.①Three shRNA expression vectors that targetedly inhibit the expression ofICAM-1gene were constructed and were verified by means of enzymedigestion and DNA sequencing.②The stable cell clones that inhibit the expression of ICAM-1werescreened successfully, the inhibition rates on the levels of mRNA andprotein were69.07%and71.12%by RT-PCR and Western blot analysis.③C ompared with the control group, the morphology of the cells thatICAM-1gene was silenced were changed, the cycle of growth and divisionwas extended; MTT results showed that the proliferation activity of silentcells were decreased; gene chip results suggest that there were290genesexpressed differentially, including238up-regulated genes and52down-regulated genes. 3. MTT and colone formation tests showed that the irradiation sensitivity ofthe ICAM-1gene silenced group were decreased, at the same dose ofirradiation conditions, the ICAM-1gene silencing group were damagedslightly, the proliferative activity and capacity were enhanced afterirradiation than the control; flow cytometry apoptosis results showed thatthe apoptosis rate of ICAM-1gene silencing group were smaller than thecontrol after24h,48h irradiated. the percentage of the cell were in the G2/M phase increased significantly after24h irradiated, after irradiated48h thepercentage were decreased but still higher than the non-irradiated group, it’sconsistent with the apoptosis results.Conclusions:1. From the study of the mouse RPL model established by16Gy60Co γ ray, itfound that ICAM-1protein in the lung tissue express abnormally, andconsistent with the expression of TGF-β1, TNF-α. It prompted theexpression of ICAM-1may be related to RPL and had interactions withTGF-β1, TNF-α. It can be used as an important factor to forecast orevaluate the degree of RPL.2.It has laid the foundation for establishing the ICAM-1genesilencing cell lines and the follow-up studies, with the successful constructi-on of recombinant plasmid vector pRNAT-U6.1/Neo-ICAM1-shRNAtargeted ICAM-1gene.3. The ICAM-1gene silencing cell lines have been established successfully.The study of biological characteristics of silencing cell lines suggested thatICAM-1may be involved in the physiological activities of LLC, forexample the activity of proliferation.4. Analyzing the changes of genome-wide expression profile after the ICAM-1gene silencing, it implied that ICAM-1may be involved in the processes ofproliferation, apoptosis and other physiological processes throng the signaling pathway of Bax/Bcl-2, FasL/Fas, MAPK.5. It can be found that interfering with the expression of ICAM-1gene canreduce the radiation sensitivity of the LLC lines significantly by themechanism study of ICAM-1in the irradiated LLC lines. It implied thatICAM-1may be a potential molecule target as a gene therapy in RPL andprovide a new and effective treatment strategy for the clinical.