Trombositten Zengin Plazma (Tzp)’Nın Kondrosit Proliferasyonu Üzerine Etkisi Var Mı?

Geleneksel uygulanan trombositten zengin plazmanın elde edilmesinde kullanılan ticari kitlerin birçoğunda kolajen, trombin ve CaCl2 ile aktive edilmeden hazırlanan trombositler, herhangi bir ilaç taşıma sistemi olmaksızın doğrudan hastalara uygulanmaktadır. Yarı ömürleri çok kısa olan büyüme faktörlerinin, canlı dışına alındığında dakikalar içerisinde biyoetkinliği yok olarak ölmektedir. Dahası hangi lökosit konsantrasyonuna sahip trombositten zengin plazmanın kıkırdak hasarlarında daha efektif olacağına yönelik kanıt değeri yüksek çalışma yok denecek kadar azdır. Bu yüzden bu araştırmada biyopolimerik hidrojel içerisine emdirilerek kullanılacak olan farklı lökosit konsantrasyonuna sahip trombositten zengin plazmanın; kondrosit proliferasyonu ve prekondrosit diferansiyasyon proteini olan stage pesific embryonic antigen-1 (SSEA-1) üzerine, geleneksel klinik uygulanan PRP’den daha efektif olabileceğinin moleküler düzeyde gösterilebilmesi amaçlandı. Olguların osteokondral dokularından izolasyonla, standart primer insan kondrosit kültürleri hazırlandı. Osteokondral dokusu kullanılan her bir hastaya ait otolog kan örnekleri alınarak, lökosit içeriği farklı konsantrasyonlarda olan aktive edilmiş trombositten zengin plazmalar elde edildi. Kültürize kondrositleri içeren örnekler beş ana gruba ayrıldı. Moleküler düzeyde, sadece MTT-cell viability, toksisite, proliferasyon ve stage pesific embryonic antigen-1 protein ifadeleri analizlenmedi aynı zamanda, enviromenting scanning electron ve inverted light mikroskobileri de değerlendirildi. Grupların istatistiksel olarak karşılaştırılması sonucunda, hem sağlıklı proliferasyona müsaade eden hem de SSEA-1 proteinin daha fazla ifade edildiği grubun, hidrojel içerisine düşük konsantrasyonda lökosit içeren TZP emdirilen örneklerin olduğu gözlemlendi (p=0.000). SSEA-1 seviyesi ile hücre proliferasyonu arasında korelasyonun olduğu ve bunun istatistiksel olarak anlamlı olduğu raporlandı (rho=0.745; p=0.000). Kıkırdak hasarlarının tedavisinde geleneksel uygulanan TZP yerine, düşük konsantrasyonda lökosit içeren TZP emdirilen ilaç taşıyıcı matrikslerin kullanılmasının acilen in vivo deneysel metotlarla test edilmesinin önemi vurgulandı

İnsan Yumurtalık Kanseri Hücre Soylarında CIP2A Onkogeni Mutasyonlarının Taranması

CIP2A geni son dönemde tanımlanmış bir insan proto-onkogenidir. CIP2A proteininin birçok farklı tipte insan kanser dokusunda ve kanser hücre hatlarında ifadesinin arttığı tespit edilmiştir. Ancak bu artışın temelinde yatan moleküler mekanizmalar henüz aydınlatılamamıştır. CIP2A genindeki promotor ve gen içi genomik değişimlerin bu artışın temelindeki rolü araştırılmamıştır. Projemizde, farklı düzeyde CIP2A proteini ifadesine sahip olan, 10 farklı insan yumurtalık kanseri hücre soyunda, CIP2A onkogeni mutasyonlarının DNA dizi analizi yöntemiyle taranması planlanmıştır. Bu amaçla, öncelikle elimizde mevcut olan 10 farklı insan yumurtalık kanseri hücre soyuna ait genomik DNA üzerinden CIP2A geni 12 bölgeye ayrılarak özgün primerlerle çoğaltıldı. PCR ürünleri, agaroz jel elektroforezi ile kontrol edilmelerini takiben, DNA dizileme işlemleri için Namık Kemal Üniversitesi, Bilimsel ve Teknolojik Araştırmalar Uygulama ve Araştırma Merkezi’ne (NABİLTEM) gönderildi. Elde edilen DNA dizileri referans dizilerle karşılaştırılarak CIP2A geni üzerindeki değişimler tespit edildi. CIP2A ifadesinin yüksek olduğu bilinen hücre soylarından Ov-CAR3’da düşük olanlardan farklı olarak rs2278911, intron 4’te +206 (TA)n(TG)n, intron 6’ da +212A>G ve Caov-3 hücre soyunda ise intron 4’te +206 (TA)n(TG)n, intron 6’ da +212A>G değişimleri heterozigot formda bulunmuştur. Bu proje ile ortaya konan genomik değişimlerin insan kanserlerine ait klinik örneklerde tanı, tedavi ve hastalığın seyrinin öngörülmesi açısından araştırılması gerekmektedir.CIP2A is a recently identified human proto-oncogene. CIP2A over expressions have been found in many different types of human cancers and cancer cell lines. However, underlying molecular mechanisms have not been elucidated. Genomic variations within the gene and its promoter region have not been studied yet. In our project, mutation screening of the CIP2A gene by sequencing has been planned in 10 different human ovarian cancer cell lines having different levels of CIP2A expression. For this purpose, we amplified the CIP2A gene with specific primers by dividing into 12 regions in 10 different human ovarian cancer cell lines. PCR products were checked by agarose gel electrophoresis and have been sent to the Research and Application Center for Scientific and Technological investigations of Namik Kemal University (NABILTEM) for DNA sequencing. The DNA sequencing results were compared with the reference sequence of the CIP2A gene. rs2278911, +206 (TA)n (TG)n in intron 4, + 212 A>G in intron 6 and +206 (TA)n (TG)n in intron 4, + 212 A>G in intron 6 have been detected as heterozygous form at the high CIP2A expressor cell lines Ov-car3 and Caov-3, respectively. The genomic changes found by this project should be investigated in human samples for potential clinical relevance at the diagnosis, treatment and prediction of prognosis of the disease

Screening of CIP2A Oncogene Mutations in Ovarian Cancer Cell Lines

Amaç: Kanserle ilişkili genetik değişimlerin belirlenmesi erken tanı, hastalığın takibi ve hedefe yöneliktedavi yaklaşımlarının geliştirilmesi açısından önem taşımaktadır. CIP2A, birçok insan kanserleri ileilişkilendirilmiş yeni tanımlanmış bir onkoproteindir. Birçok kanserde CIP2A gen ekspresyonununartışı gösterilmiştir ancak bu güne kadar AGS, HeLa, HT1080 kanser hücre soylarında CIP2A promotorbölge mutasyonlarının araştırıldığı ve tarafımızdan yapılmış olan bir çalışma dışında herhangibir kanser tipinde CIP2A geninin kodlayan dizilerindeki mutasyonlarını araştıran bir çalışma yapılmamıştır.Bu çalışmamızda CIP2A gen ekspresyonunun kanserdeki artışına ilişkin moleküler mekanizmalarınaydınlatılmasına katkıda bulunmak amacı ile bu genin onkogenik etkisinin oluşmasında rolüolması muhtemel regülatör bölge ve gen içi mutasyonlar araştırılmıştır.Gereç ve Yöntemler: CIP2A geninin kodlayan dizileri, ekzon-intron bağlantı bölgeleri ve promotorbölgesi DNA dizi analizi yöntemiyle SK-OV-3, Ov-CAR3 ve Caov-3 insan yumurtalık kanseri hücresoylarında taranmıştır.Bulgular: Ov-CAR3 hücre soyunda ekson 3, intron 6 ve intron 8’de, Caov-3 hücre soyunda ise sadeceintron 6 ve intron 8’de genomik değişimler saptanmıştır.Sonuç: Bulgularımız, yumurtalık kanseri için CIP2A ekspresyonundaki artışta CIP2A’daki genomikdeğişimlerin etkisini dışlamaktadır. CIP2A onkoproteinini yüksek düzeylerde ekspresyon eden hücrelerdebu duruma neden olabilecek diğer mekanizmaların da araştırıldığı fonksiyonel çalışmalaragereksinim vardır.Objective: Determination of genetic changes associated with cancer is important for early diagnosis, follow-up of the disease and development of targeted therapeutic approaches. CIP2A is a newly identified oncoprotein associated with many human cancers. In many cancers, an increase in the CIP2A gene expression has been shown, but until now there has been no study investigating CIP2A promoter region mutations except our previous study about the mutations in the coding sequence of the CIP2A gene in AGS, HeLa, HT1080 cancer cell lines. The aim of this study was to contribute to the elucidation of the molecular mechanisms of CIP2A over expression in cancer and investigate the possible regulatory regions and intra-genic mutations that may play a role in the oncogenic effect of this gene. Material and Methods: The sequences encoding the CIP2A gene, exon-intron linkage regions, and the promoter region were screened in the SK-OV-3, Ov-CAR3, and Caov-3 human ovarian cancer cell lines by DNA sequence analysis. Results: Genomic changes were detected at exon 3, intron 6 and intron 8 in the Ov-CAR3 cell line and at intron 6 and intron 8 in the Caov-3 cell line. Conclusion: Our findings exclude the effect of genomic alterations in CIP2A in the increase in CIP2A expression for ovarian cancer. For cells expressing CIP2A oncoprotein at high levels, there is a need for functional studies to examine the underlying mechanisms

GC NÜKLEOTİD ORANI YÜKSEK VE UZUN GEN BÖLGELERİNİN ÇOĞALTILMASINI SAĞLAYAN PCR PROTOKOLLERİNİN GELİŞTİRİLMESİ

Birçok genetik hastalığın tanısında kullanılan Polimeraz Zincir Reaksiyonu (PCR) ilgilenilen gen bölgelerinin çoğaltılması, sonraki işlemlerin gerçekleştirilebilmesi için gerekli olan ilk basamaktır. Fakat bazı durumlarda ilgilenilen gen bölgesinin “GC” nükleotidleri içeriği bakımından zengin ve uzun bir bölge olması PCR işlemini başarısızlığa uğratmakta, bu nedenle genetik hastalıkların tanısının zorunlu olduğu durumlarda çok daha fazla zaman, iş gücü ve maliyet gerektiren başka işlemler kullanılmakta veya söz konusu hastalığın laboratuvardaki tanısı gerçekleştirilememektedir. Diğer yandan eğer yürütülen çalışma bir araştırma ise çoğu zaman çalışma başarısızlıkla sonuçlanmaktadır. GC’ ce zengin bölgelerin PCR yöntemi ile çoğaltılmasında karşılaşılan güçlükleri aşmak için de bu güne kadar PCR katkı kimyasallarının ve modifiye nükleotidlerin kullanımı ayrıca ısı döngülerinin optimizasyonu gibi değişik yaklaşımlar ortaya konmuştur. Bilimsel araştırmaların yanı sıra birçok firma da GC nükleotid oranı yüksek ve uzun olan gen bölgelerinin çoğaltılmasını sağlayacak kitlerin üretimine çalışmaktadır. Ancak, birçok hastalıkla ilşikili GC’ce zengin gen bölgelerinin PCR’ la çoğaltılmasındaki güçlükler aşılamamıştır. Bu proje ile tanı amaçlı çalışan moleküler genetik laboratuvarlarında sıklıkla çalışılan Frajil-X sendromu, Huntington Hastalığı, Miyotonik Distrofi başta olmak üzere GC’ ce zengin ve uzun gen bölgeleri içermeleri nedeniyle klasik PCR yöntemi ile tanısı konulamayan hastalıkların tanısının hızlı ve ucuz bir yöntemle konulmasına olanak sağlayacak PCR protokollerinin geliştirilmesi hedeflenmektedir.Polymerase Chain Reaction (PCR) is widely used in diagnosis of many genetic diseases. Amplification of the gene of interest with PCR is the first step required for afterwards process. But in some cases gene of interest is "GC" rich and too long for a sufficient PCR. In this kind of situations diagnosis of genetic diseases require more complicated molecular techniques costing much time and payment. Even the results are often unsuccessful. To overcome these difficulties, usage of chemical additives modified nucleotides and optimization of PCR conditions demonstrated with many research. Also many companies are working on the production of kits which can duplicate GC rich sequences of disease related genes responsible for Fragile X syndrome, Huntington's disease and Myotonic Dystrophy. The aim of this project is to develop a quick and cheap PCR protocol could be used in genetic diagnosis

Effects of idiopathic erythrocytosis on the left ventricular diastolic functions and the spectrum of genetic mutations: A case control study

Background: We have aimed at exposing left ventricular diastolic functions and the presence of known genetic mutations for familial erythrocytosis, in patients who exhibit idiopathic erythrocytosis. Methods: Sixty-four patients with idiopathic erythrocytosis (mean age, 46.4 +/- 2.7 years) and 30 age-matched healthy subjects were prospectively evaluated. The regions of interest of the erythropoietin receptor, hemoglobin beta-globin, von Hippel-Lindau, hypoxia-inducible factor 2 alpha, and Egl-9 family hypoxia-inducible factor genes were amplified by PCR. Left ventricular (LV) mass was measured by M-mode and 2-dimensional echocardiography. LV diastolic functions were assessed by conventional echocardiography and tissue Doppler imaging. Results: As a result of genetic analyses, genetic mutations for familial erythrocytosis were detected in 5 patients. It has been observed in our study that the risk of cardiovascular disorders is higher in patients. Interventricular septum thickness, left atrial diameter, and some diastolic function parameters such as deceleration time and isovolumetric relaxation time have been found to be significantly higher in idiopathic erythrocytosis group than in the controls. Conclusion: This study has shown that LV diastolic functions were impaired in patients with idiopathic erythrocytosis. In this patient group with increased risk of cardiovascular disorders, the frequent genetic mutations have been detected in 5 patients only. Therefore, further clinical investigations are needed as novel genetic mutations may be discovered in patients with idiopathic erythrocytosis because of cardiovascular risk.scientific and technological research council of Turkey [Tubitak-215S524]This research was funded by scientific and technological research council of Turkey (Tubitak-215S524)

The effects of rivaroxaban, an oral anticoagulant, on human IVD primary cultures

Introduction: The present study aimed to investigate the potential effects of rivaroxaban, an oral anticoagulant that inhibits the effects of factor Xa, on intact intervertebral disc tissue cells and the extracellular matrix (ECM). Material and methods: Rivaroxaban was applied to primary human cell cultures prepared from tissues of the intervertebral disc. Comparative molecular analyses were performed on non-drug-treated control group samples. Descriptive statistics were presented as the mean +/- standard deviation. An analysis of variance test was performed to determine whether there were significant differences in the mean across the groups. When differences across groups were observed, Tukey's honestly significant difference post-hoc test was used for multiple pairwise comparisons. The significance of the obtained data was determined statistically. The alpha significance value was < 0.05. Results: The cells in the control group and in the rivaroxaban-treated group were viable, healthy, and proliferated (p < 0.05). However, the expression levels of the chondroadherin gene (CHAD), cartilage oligo matrix protein (COMP), matrix metalloproteinase (MMP)-13, and MMP-19 genes were changed (p < 0.05). Conclusions: Although rivaroxaban does not suppress cell proliferation due to morphological, biological, and biochemical changes in the intervertebral disc tissue, it may change the expression of genes that are related to ECM maintenance

The association between different molecular weights of hyaluronic acid and CHAD, HIF-1 alpha, COL2A1 expression in chondrocyte cultures

The aim of the present study was to investigate the effects of three different formulations of hyaluronic acid (HA): Low molecular weight (MW) Sinovial One((R)), medium MW Viscoplus((R)) and high MW Durolane((R)), on chondrocyte proliferation and collagen type II (COL2A1), hypoxia-inducible factor 1 alpha (HIF-1 alpha) and chondroadherin (CHAD) expression in primary chondrocyte cultures. Standard primary chondrocyte cultures were established from osteochondral tissues surgically obtained from 6 patients with gonarthrosis. Cell morphology was evaluated using an inverted light microscope; cell proliferation was determined with a MTT assay and confirmed with acridine orange/propidium iodide staining. Levels of CHAD, COL2A1 and HIF-1 alpha expression were assessed using specific TaqMan gene expression assays. The results demonstrated the positive effect of HA treatment on cell proliferation, which was independent from the MW. COL2A1 expression increased in the medium and high MW HA treated groups. It was observed that HIF-1 alpha expression increased in the high MW treated group alone. CHAD expression increased only in the medium MW HA treated group. Evaluation of gene expression revealed that levels of expression increased as the duration of HA application increased, in the medium and high MW HA treated groups. In terms of increased viability and proliferation, a longer duration of HA application was more effective. Taken together, it may be concluded that the administration of medium and high MW HA may be a successful way of treating diseases affecting chondrocytes in a clinical setting

Effects of etanercept, a tumor necrosis factor receptor fusion protein, on primary cell cultures prepared from intact human intervertebral disc tissue

The aim of the present study was to investigate the effects of etanercept (ETA), a tumor necrosis factor (TNF) inhibitor, on human cell cultures prepared from intact intervertebral disc tissue. ETA is used as a treatment for cases of rheumatoid arthritis, psoriatic arthritis, axial spondyloarthritis and ankylosing spondylitis accompanied by moderate or severe joint pain. ETA was applied to primary cell cultures [annulus fibrosus and nucleus pulposus (NP) from intact intervertebral disc tissue]. Cell cultures without ETA treatment served as the control group. Morphological and quantitative molecular analyses of the two groups were performed. The number of viable cells and cell proliferation decreased in the ETA-treated cultures as compared with those in the control group. Furthermore, in the treatment group, the chondroadherin gene, an NP-specific marker, was not expressed after 24 h. By contrast, the cartilage oligo matrix protein was expressed 24, 48 and 72 h post-ETA treatment, while its expression was significantly lower than that in the control group. In addition, the expression of interleukin-1 beta, as well as matrix metallopeptidase-7 and -19, was markedly decreased. Overall, the cell proliferation and gene expression in the ETA-treated cells were significantly different from those in the control group (P<0.05). These results suggest that the treatment duration and dosage of TNF inhibitors, which are used to suppress active inflammation, should be considered in the clinical setting. These biological agents may delay the healing of intervertebral disc tissue damage by slowing cell proliferation and altering gene expression via anabolic and catabolic pathways

Evaluation of the effect of apixaban on the primary intact intervertebral disc cell cultures

Aim: Apixaban is a frequently preferred pharmacological agent in clinics to prevent deep vein thrombosis and pulmonary embolism.Such new oral anticoagulants may cause hemorrhage’s in tissues and/or organs or may cause gastrointestinal symptoms withoutbleeding. It is also reported in the literature that it may lead to mental disorders, unwanted disorders in the urinary tract and skeletalmuscle system. However, when the literature is examined, there are no studies, which are of high-evidential value, evaluating theefficacy of apixaban on healthy, intact intervertebral disc tissue, and matrix-like structures. In this pharmaco-molecular study, itwas aimed to investigate the effects of a new oral anticoagulant agent containing the active ingredient apixaban on the intactintervertebral disc tissue cells, extracellular matrix (ECM) structure and to evaluate its positive and / or negative effects on geneexpressions of cartilage oligo matrix protein (COMP), chondroadherin (CHAD), and Matrix Metalloproteinase (MMP)s.Material and Methods: The primary cell cultures were prepared from the intact tissues of the patients with the traumatic intervertebraldisc herniation. Apixaban was administered to the cultures and molecular analyses were performed for 21 days. The data obtainedfrom the apixaban-administered and non-apixaban-administered samples were evaluated statistically and the significance valuewas accepted as P <0.05.Results: The changes were observed in the cell proliferation and the expressions of the mentioned genes in the apixabanadministered group. The suppression of COMP value and the increase in MMP-13 value may be indicative of the development ofmatrix degeneration in the apixaban-administered group, compared to the non-drug-administered control group.Conclusion: The selectivity is one of the most important features of the drugs. However, it should not be forgotten that no drug willonly produce the desired effect

Toxicity of the acetyl-para-aminophenol group of medicines to intact intervertebral disc tissue cells

The present study aimed to investigate the effects of paracetamol, an analgesic and antipyretic that is used in emergency departments and neurosurgery departments for postoperative pain management on intervertebral disc tissue. Paracetamol-treated human primary cell cultures and untreated cell cultures were compared using molecular analyses. Cell proliferation and gene expression were statistically analyzed. Cell proliferation was suppressed on days 10 (P=0.05) and 20 (P<0.05) in the paracetamol-treated groups. Gene expression of chondroadherin, matrix metalloproteinase (MMP)-7, MMP-13 and MMP-19 was higher in the paracetamol-treated samples while gene expression of Cartilage Oligomeric Matrix Protein and interleukin-1 beta was lower (P<0.05). Paracetamol, which appears innocuous compared with many analgesics, may increase the expression of MMPs, which serve a significant role in catabolic reactions and suppress the proliferation of intact intervertebral disc tissue cells.Namik Kemal Universit