2015

Hücre İçi Serbest Ca2+ Konsantrasyon Dinamiğinin Floresans Yöntemler ile İncelenmesi

Hücre membranına gelen uyarıların bir çoğu, sitoplazmik serbest Ca+2 derişiminde ([Ca+2]S) artışa neden olur. Ca+2'un sitozoldeki konsantrasyonunun oldukça düşük (≈10-7 M), hücre dışı sıvıdaki ve endoplazmik retikulumdaki (ER) (≈10-3 M) konsantrasyonunun ise yüksek olmasından dolayı, pek çok hücrede Ca+2 hücre içi sinyal molekülü ve ikinci haberci olarak kullanılır. Ca+2’u, plazma ve ER membranlarından sitozole doğru iten böyle büyük bir elektro-kimyasal gradiyent nedeni ile plazma ya da ER membranlarında bulunan Ca+2 kanallarının geçici olarak açılmasını sağlayacak bir sinyal, Ca+2 un hızla sitozole akmasına ve [Ca+2]S’nun 10-20 kat artmasına neden olur. Böylece hücre içinde bulunan Ca+2’a duyarlı proteinler aktive olarak bir çok hücresel fonksiyonun yerine getirilmesi sağlanır. Örneğin gen ekspresyonu, hücre çoğalması, bölünmesi, apoptoz ve ayrıca kas hücrelerinde kasılma, salgı hücrelerinde degranülasyon ve sinirsel iletim gibi olayların tetiklenmesinde Ca+2 kilit rol oynar. Bu nedenle [Ca+2]S’nun dinamik olarak ölçülebilmesi, hücrede gerçekleşen pek çok sinyalizasyon mekanizmasının anlaşılmasında önemli bir yer tutar. Floresans görüntüleme teknikleri, Ca+2’un hücre içinde uzay/zaman değişim desenlerini takip edilebilir duruma getirmiştir ve bu sayede bu tekniklerin kullanıldığı yöntemler de son yıllarda gelişerek artmıştır. Özellikle floresans boyaların kullanım kolaylıkları ve hücre hemostazına müdahalenin diğer metotlara göre minimum oranda kalması, bu metotların kullanımının önünü açmıştır.

Anahtar Kelimeler:

Floresans, Ca+2 görüntüleme, IP3 sinyal yolağı

Investigation of Intracellular Free Ca2+ Concentration Dynamics with Fluorescence Methods

Most of the extracellular stimulus arrive to the cell membrane result with the increase in cytoplasmic free Ca+2 concentration [Ca+2]i. Because of the huge Ca+2 concentration differences between the cytoplasm (≈10-7 M) and extracellular fluid and endoplasmic reticulum (ER - which is the major Ca+2 storage organelle in especially non electrically excitable cells) (≈10-3 M), a large electro-chemical gradient repel Ca+2 to the plasma or ER. Therefore a signal which temporarily opens Ca+2 channels, induce a fast influx of Ca+2 through the cytosol and increase its concentration about 10-20 fold. At this organization free Ca+2 functions as an intracellular signalling molecule and a second messenger. In this way many intracellular signalling proteins activated and cellular functions like gene expression, cell proliferation and division, apoptosis, and also myocyte contraction, endocrine cell degranulation and neuronal transmission are regulated. Thus, the key role of Ca+2 in many intracellular process, makes the dynamic measurements necessary for an understanding of the signalling mechanisms. Fluorescence imaging techniques make possible of monitoring the spatiotemporal Ca+2 response patterns in cytoplasm. In the last decades, especially their ease of loading, and minimum manipulations to the cell homeostasis, make these techniques unique with respect to the other methods.

Keywords:

Fluorescence, Ca+2 imaging, IP3 signaling pathway,

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