Multiple skleroz kuprizon fare modelinde gen ekspresyonlarındaki değişikliklerin gerçek zamanlı (qRT PCR) yöntemi ile tayin edilmesi
Determination of gene expresssion changes in multiple sclerosis cuprizone mouse model with quantitative real time (qRT) PCR
Yazar: GÖKHAN TERZİOĞLU
Danışman: DOÇ. DR. EDA TAHİR TURANLI
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / İleri Teknolojiler Ana Bilim Dalı / Moleküler Biyoloji-Genetik ve Biyoteknoloji Bilim Dalı
Derece: Yüksek Lisans, 2012
Konu: Biyoloji = Biology ; Genetik = Genetics ; Nöroloji = Neurology

Özet:
Multiple Skleroz, genelde genç erişkinleri etkileyen ve T hücrelerinin, merkezi sinir sistemindeki sinir hücreleri aksonlarının çevresini saran ve hızlı atlamalı (saltatory) iletimi sağlayan myeline karşı otoreaksiyon geliştirdiği otoimmün bir hastalık olarak kabul edilmektedir. Myelin kaybı, sinirsel iletimde yavaşlamaya ve zaman içinde ölüme sebep olmaktadır. İnsan merkezi sinir sisteminden örnek almak zor olduğu için, MS in işleyişini çalışmak üzere toksin temelli, virüs temelli ya da EAE gibi modeller geliştirilmiştir.Mevcut çalışmada toksin temelli bir model olan, kuprizon modeli kullanılmıştır. Kuprizon, bir bakır şelatörüdür, dolayısı ile sistemde bulunan bakır iyonlarının çökmesine sebep olur.. Kuprizonun diğer bir önemli etkisi ise, besinle karıştırılarak verildiğinde MSS de spesifik olarak corpus callosumda demyelinasyona sebep olmasıdır. Hedef gen olarak seçilen NRG1, CNTF ve CXCR4, merkezi sinir sisteminde myelini oluşturan oligodendrosit hücrelerinin sağkalımında, farklılaşmasında ve oligodendrositleri oluşturan oligodendrosit progenitör (öncül) hücrelerin hasarlı bölgeye göçünde etkili faktörlerdir. Bu faktörlerin ekspresyonundaki değişimin belirlenmesi ile, remyelinasyon ya da myelin onarımındaki rolleri ve uzun süreli kuprizon uygulamasının niçin kronik demyelinasyona sebep olduğu anlaşılmaya çalışılmaktadır. Böylece, yeni tedavi yöntemlerinin geliştirilmesi ya da hastalarda görülen remyelinasyonun arttırılması için önemli bilgiler sağlanmış olacaktır.C57BL 6/J farelerden denek ve kontrol grubu olmak üzere 2 grup oluşturularak, denek grubu %0,2 kuprizon karıştırılmış yem ile kontrol grubu ise normal yem ile beslenmiştir. Kuprizon ile beslemeye 12 hafta boyunca devam edilmiş sonraki 4 haftada ise nırmal besin verilmiştir, 2 haftada bir olmak üzere fareler sakrifiye edilerek beyin örnekleri alınmış ve beyinlerinden RNA izolasyonu gerçekleştirilmiş, RNA nın bir kısmı -80°C de RNA olarak, bir kısmı ise cDNA e çevrilerek -20°C de saklanmıştır. cDNA örnekleri, NRG1, CNTF ve CXCR4 hedef genleri için, 2 referans gen kullanılarak (Gapdh, Pgk1) LightCycler 480 cihazı ve UPL Probe ile qRT-PCR analizine tabi tutulmuştur.Kontrol grubu ile karşılaştırılan denek grubu ekspresyon analizi sonuçlarına göre, NRG1 ekspresyonu ilk 4 haftalık kuprizon uygulaması ile belirgin ölçüde azalırken (p<0,001), 4-12 haftalar arasında aynı düzeyde seyretmektedir (4-10 hafta için p<0,001), 12.haftadan sonra başlangıç düzeyine dönmüştür. CNTF ekspresyonu 0-4. haftalar arasında azaldıktan (p<0,05) sonra 8-12 hafta arasında artarak başlangıç düzeyinin üzerine çıkmış (6-8.hafta p<0,05, 8-10.hafta p<0,001), normal beslenmeye dönüldükten sonra azalarak başlangıç düzeyine inmiştir. CXCR4 ekspresyonu ilk 4 hafta içerisinde düşüş gösterdikten sonra (p<0,05), 6.hafta da maksimum düzeyine ulaşmış (p<0,05), 6-12.haftalar arası dönemde minimum düzeye inmiş (6-10. hafta p<0,005), normal beslenmeye dönüldüğü dönem olan 12-16.hafta döneminde ise başlangıç düzeylerine dönmüştür (p<0,001). 12. hafta sonunda kuprizon yemden çekildikten sonra, NRG ve CXCR4 ekspresyonu artarken, CNTF düşüş göstermiştir. Ekspresyon düzeyindeki değişime bakıldığında, kronik demyelinasyon seyri ile paralellik gösteren hedef genin CXCR4 olduğu düşünülmektedir.CXCR4 ekspresyonundaki değişim, kuprizonun oluşturduğu demyelinasyonun seyri ile paralellik göstermektedir. IFN-? nın CXCR4 ekspresyonunu baskıladığı ve demyelinize bölgede IFN-? salgılandığı bilinmektedir. Dolayısı ile, süreç içerisinde IFN-? miktarının arttığı ve CXCR4 ekspresyonunun bu sebeple baskılandığı düşünülebilir.

Abstract:
Multiple Sclerosis is a demyelinative autoimmune disease and generally seem in young adults. In MS, Blood brain barrier (BBB) is breakdown, immune cells infiltrate into CNS and T cells attack the myelin sheath of nerve axons in CNS which ensheath and insulate axons. Myelin is a lipid rich insulation material, Normally, myelin increase the speed of axonal signal transmission and radius of axon, Myelin loss cause neural transmission slowdown and may cause death in time. Plaques are seen in demyelination sites of brain. At early stage of Multiple Sclerosis, demyelinated sitescan be repaired and remyelinated, but after the threshold demyelinated sites can not be remyelinated. For remyelination, myelin debris must be remove with phagosytic activity by macrophages. Oligodendrocytes are glial cells which myelinate the Central Nervous System, and this cells differentiated from Oligodendrocyte progenitor cells which are originated from Subventricular zone and subgranular zone in brain. Oligodencyte cells injection to demyelinated site can not cause remyelination, only oligodendrocte progenitor celll injection cause the remyelination in CNS. Because of this, first step of remyelination is oligodendrocyte progenitor cell migration to demyelination site. After the migration, oligodendrocyte progenitor cells differentiate into mature oligodendrocytes which build myelin sheath.Different models are used for MS studies such ase virus based (TMEV), toxin based models (cuprizone), autoimmune (EAE) models, because sample isolation from the human central nervous system (CNS) is a hard process and dangerous for patient. Theiler Murine Encephalomyelitis Virus is a common virus model, another is Semliki Virus. Most common animal model is the Experimental Autoimmune Encephalomyelitis which is an autoimmune MS model. In EAE, myelin parts or whole myelin inject with adjuvant to mice circulation and this cause autoimmune response to myelin and oligodendrocytes in CNS. Blood brain barrier destroyed and immune cells pass into brain. Another model is the Cuprizone Toxic Model, cuprizone is a copper (Cu) chelator which bind and settle the Cu ions and abnormal mitochondria formation (Megamitochondria). Cuprizone is different from other toxin based models, other models such as Lisolesitin and ethidium bromide only cause local demyelination at injection site, but cuprizone cause global demyelination in brain and cuprizone only effect when mix into chow. Cuprizone cause global demyelination in CNS (particularly Corpus callosum and subcortical region for C57BL/6j mice)), when % 0,2 cuprizone mix into chow of the murines. If cuprizone concentration increase to % 0,5, mortality rate increase. First 6 weeks of cuprizone chow cause acute demyelination which is remyelinated after the cease of cuprizone. But 6-12 cuprizone feeding cause chronic demyelination, remyelination fail. For cuprzione model, Blood brain barrier is intact and cuprizone model is not an immune model, T cell infiltration can?t be seen. Additionaly, cuprizone treat- ment repress the Experimental Autoimmune Encephalomyelitis? effects in brain.At this study, a toxic demyelination model of cuprizone was used. NRG1 (neuregulin 1), CNTF (Ciliary Neurotophic Factor), CXCR4 (C-X-C Chemokine Receptor 4) are target genes of this study and these target genes have important roles in oligodendrocyte survival, maturation and oligodendrocyte progenitor cell migration to the demyelination site. Particularly, CXCR4 is an important receptor for CXCL12 chemokine, and CXCR4-CXCL12 relation is important for oligodendrocyte progenitor cell and other progenitor cells migration in brain. CXCR4 is expressed by stem cells and CXCL12 is expressed by astrocytes and microglias in demyelinated site. Previous studies show that CXCL12 expression levels decrease with cuprizone treatment. Nrg 1 and CNTF are important factors for oligodendrocyte maturation and survival. CNTF is an polypeptid hormone which affect the central nervous system. CNTF is expressed by neurons and microglias in CNS. CNTF injection to developing rat optic nerve, increase cell surival %80. Nrg1 is a protein and have 6 alternative splicing form. NRG 1 is expressed on neurons' cell surface and bind to Erbb receptors on oligodendrocytes. NRG1 promote the oligodendrocyte survival through Erbb receptors on oligodendrocytes. But, Nrg1 is not an essential factor for central nervous system myelination. Nrg 1 knock-out mice central nervous system can be myelinated. Nrg 1 is an essential factor for Schwann cell survival, schwann cells are myelin cells in peripheral nervous system. Beacause of this, NRG 1 is an impotant and essential factor for perfiphera nervous system myelination. If these factors expression changes are determined, roles in remyelination and causes of chronic demyelination are understood from the their expression change in mice brain with longtime cuprizone exposure, thus new therapies for MS and promotion of remyelination can be developed.C57BL/6j mice are seperated into 2 group which are subject and control. Subject group is fed with %0,2 cuprizone mixed chow for 12 weeks and after 12. weeks, they fed with normal chow for 4 weeks. Control group is fed with normal chow for 16 weeks. Mice are sacrified once at two weeks, and brain is dissected. RNA isolated from brains, a part of RNA keep in -80°C, a part of RNA turn into cDNA with reverse transcrition kit and keep at -20°C. Before the qRT-PCR, cDNAs' are measured with Nanodrop for 260/280nm. cDNAs are analyzed with upl probe qRT-pcr for CNTF, NRG1 and CXCR4 target genes expression levels. Gapdh and Pgk1 are used as reference genes. Gapdh is a common reference gene for qRT PCR studies, and used in some related studies, Pgk 1 is anew reference gene, and its expression is most stabil in development of central nervous system of mice. Gapdh and Pgk 1 expression levels mean is used for relative quantification calculation. Lightcycler 480 and Universal Probe Library probes used for qRT-PCR. Probes and primers are designed Universal Probe Library Design Center and controlled with Primer Blast for specificity. Probes are more specific and confidential method than Cybr Green qRT-PCR, because of this, UPL probes are preferrred. Expression levels calculate with LightCycler 480 Advanced Relative Quantification program. Each measurement repeat for threee times. Statistical P values calculate. p<0,05 accept as a statistical significant.According to the quantitative Real Time Polymerase chain reaction results which are compared with control group, Neuregulin 1 gene expression significantly decrease after 4 weeks of cuprizone treatment (p<0,001), but almost not change in 4-12. weeks period (4-10. week p<0,001). After the 12. week, cuprizone replace from the chow and NRG1 expression increase to the initial level. Ciliary neurotrophic factor expression decrease in first 4 weeks of cuprione treatment (p<0,05), but increase in 8-12 weeks of cuprizone treatment (6-8. weeks p<0,05 8-10 weeks p<0,001) . After the 12. week , CNTF expression level decrease to initial level. Similarly, CXCR4 expression decrease in 0-4. week period (p<0,05), and reach maximum level at 6th week (p<0,05). In 6-12th week period (6-10. weeks p<0,005), CXCR4 expression decrease and reach the minimum at 12th week. At 12th week, cuprizone replace from the chow and CXCR4 expression level increase to the initial level in 4 weeks (p<0,001). Only CNTF gene expression level decrease after the 12th week, NRG1, CXCR4 level increase after the 12th week. CXCR4 expression change pattern correlate with acute demyelination to chronic demyelination transition. CXCR4, CNTF, NRG 1 genes? expressions turn into the initial level after the chease of cuprizone from the chow.According to previous studies, Nrg 1 is not an essential factor for central nervous system myelination, Nrg 1 knock-out mice central nervouse system can be myelinated. But in this study, Nrg 1 expression decrease with cuprizone treatment and not increase in 12 week cuprizone feeding period. Cuprizone maybe affect the alternative pathways which are related with central nervous sytem remyelination. Expression of Silier Neurotrophic factor gene increase in 8-12. week period, and ciliary neurotrophic factor is important for oligodendrocyte survival, but oppositely remyelination wreck at 12. week. Because of this, Central nervous system remyelination maybe decline at early steps, possibly at oligodendrocyte progenitor celll migration.C-X-C chemokine receptor 4 expression pattern is almost similar with Cuprizone Demyelination pattern. Decrease in CXCL12 expression can not answer the quaestions about CXCR4 expression decrease, because CXCL12 expression decrease is little. Possibly, Interferon gamma cause the decrease in CXCR4 expression. Previous studies show that, damaged astrocytes release interferon ? and macrophages are called with IFN? to the demyelination site for phagocytosis of demyelination wrecks and IFN? is detected in corpus callosum for cuprizone model, interferon gamma decrease the number of C-X-C receptor 4 receptors on cell surface and decrease the CXCR4 expression. Thus, interferon gamma expression increase and this can be repressed the expression of CXCR4 in central nervous system. For Multiple Sclerosis, different mechanisms can be suggested, and one of this is Ubiqutin based mechanism. Ubiqutin can be bound the CXCR4 receptor, and ubiqutin have important role in immunsupression. ubiqutin bound molecules goes into the destruction. But central nervous system is almost immunsupressed, in Multiple Sclerosis, blood brain barrier break and immune cellls pass into the central nervous system. Because of this, Ubiqutin level increase for supression of autoimmune response, additionally may affect the CXCR4 level in CNS and may prevent Oligodendrocyte cell migration to demyelination site in brain.