Publication: Fare trombositlerinde GAS 6 (growth arrest spesific-6) varlığının araştırılması
Abstract
Gas6 proteini (Growth arrest specific-6), K vitaminine bağımlı protein ailesinin bir üyesidir ve reseptör tirozin kinazlardan Tyro3 (Sky), Axl ve Mer adlı reseptörlerin (TAM) ligandıdır. Fare trombositlerinde Gas6 proteininin var olup olmadığı konusunda birbiriyle çelişkili yayınlar bulunmaktadır. Biz bu tezde, fare trombositlerinde Gas6 proteini ve reseptörlerinin varlığını, mRNA ve protein seviyesinde araştırmayı amaçladık. Deney hayvanı olarak kullandığımız 25 adet spesifik patojen içermeyen 8-10 haftalık, 25-30 gr ağırlığındaki Balb-C ırkı dişi/ erkek fareleri hafif eter anestezisi altında bayıltıp, kalpten kan örnekleri aldık. Trombosit izolasyonu yaptık. Trombositlerde Gas6 ve TAM reseptörlerine ait mRNA varlığını araştırmak için önce trombositlerden total RNA izolasyonu yaptık. Daha sonra, RT-PZR ile Gas6 ve reseptörlerine ait primerler varlığında mRNA ölçümü yaptık: GAS6 genine ait 400 bç’lik bölge ile Mer genine ait 238 bç’lik bölgenin amplifikasyonunu yaptık. Bulgular bize fare trombositlerinde GAS6 ve Mer mRNA’sının var olduğunu göstermektedir. Buna karşın farklı döngü ve sıcaklıklarda yaptığımız RT-PZR reaksiyonlarında, Sky ve Axl ’a ait mRNA varlığını saptayamadık. Özetle, bu tezde fare trombositlerinde Gas6 ile TAM reseptörlerinden sadece Mer proteinini kodlayan mRNA’ların var olduğunu gösterdik. ELISA’yla Gas6, Axl ve Sky protein seviyelerini ölçülebilir sınırların altında bulduk. Mer için henüz ticari kit olmadığından seviyesi ölçülemedi. Tromboz, hemostaz, restenoz gibi kardiyovasküler fonksiyonları çok yakından ilgilendiren çeşitli hastalıklarda Gas6 ve reseptörlerinin trombositlerdeki etki mekanizmasının moleküler seviyede aydınlığa kavuşturulması için ileri araştırmalara ihtiyaç vardır. Bu sayede Gas6’ nın tromboz ve ateroskleroz tedavisinde farmakolojik yeni bir hedef olup olamayacağı konusunda güvenilir bilgiler edinilecektir. Gas6, K Vitamini, TAM Reseptörleri, Trombosit
The Investigation of The Presence of Gas6 (Growth Arrest Spesific-6) Protein in Mouse Platelets Gas6 is a member of the vitamin K-dependent protein family and is a ligand for three receptors belonging to the receptor tyrosine kinase subfamily: Tyro3 (also called Sky) Axl and Mer. There are contradictory publications about the presence of Gas6 in mouse platelets. The purpose of this study is to investigate the presence of Gas6 and its receptors in mouse platelets as mRNA and protein levels. Used as 25 specific pathogen-free, 8-10 week old, 25-30 g in weight, race of Balb-C male/ female mice were anesthetized under light ether anesthesia and were taken blood samples from their hearts. Platelets were isolated. Total RNA were isolatad from platelets for investigating the presence of mRNA belonging to Gas6 and TAM receptors By RT-PCR experiments. After then, we measured the primers of Gas6 and receptors by RT-PCR. We aplificated both the 400 bp region of GAS6 gene and 238 bp region of Mer gene. These findings show us the presence of mRNAs both of GAS6 and Mer gene in Mouse platelets. However, we couldn’t detect the presence of Sky and Axl of mRNA although we have used different loop and temperatures for RT-PCR reactions. Summary, in this thesis we showed only the presence of mRNA which encoding Gas6 protein and Mer receptor in mouse platelets. We found Gas6, Axl and Sky levels under the measurable limits by ELISA. Due to there isn’t any commercial kit for Mer, we couldn’t measure its level. We need advanced investigations to clarify of molecular effects of Gas6 and its receptors in platelets which are very closely related to cardiovascular diseases such as thrombosis, hemostasis and restenosis. Thanks to this, reliable informations will be gotten for whether Gas6 is a pharmacological new target in trombosis and atherosclerosis treatments. Key Words: Gas6, Vitamin K, TAM Receptors, Platelet
The Investigation of The Presence of Gas6 (Growth Arrest Spesific-6) Protein in Mouse Platelets Gas6 is a member of the vitamin K-dependent protein family and is a ligand for three receptors belonging to the receptor tyrosine kinase subfamily: Tyro3 (also called Sky) Axl and Mer. There are contradictory publications about the presence of Gas6 in mouse platelets. The purpose of this study is to investigate the presence of Gas6 and its receptors in mouse platelets as mRNA and protein levels. Used as 25 specific pathogen-free, 8-10 week old, 25-30 g in weight, race of Balb-C male/ female mice were anesthetized under light ether anesthesia and were taken blood samples from their hearts. Platelets were isolated. Total RNA were isolatad from platelets for investigating the presence of mRNA belonging to Gas6 and TAM receptors By RT-PCR experiments. After then, we measured the primers of Gas6 and receptors by RT-PCR. We aplificated both the 400 bp region of GAS6 gene and 238 bp region of Mer gene. These findings show us the presence of mRNAs both of GAS6 and Mer gene in Mouse platelets. However, we couldn’t detect the presence of Sky and Axl of mRNA although we have used different loop and temperatures for RT-PCR reactions. Summary, in this thesis we showed only the presence of mRNA which encoding Gas6 protein and Mer receptor in mouse platelets. We found Gas6, Axl and Sky levels under the measurable limits by ELISA. Due to there isn’t any commercial kit for Mer, we couldn’t measure its level. We need advanced investigations to clarify of molecular effects of Gas6 and its receptors in platelets which are very closely related to cardiovascular diseases such as thrombosis, hemostasis and restenosis. Thanks to this, reliable informations will be gotten for whether Gas6 is a pharmacological new target in trombosis and atherosclerosis treatments. Key Words: Gas6, Vitamin K, TAM Receptors, Platelet