Tesis

Pengaruh asidifikasi pH ekstraseluler PBMCs terhadap stres oksidatif dan viabilitas sel serta kaitannya dengan ekspresi mRNA CA9 dan HIF-1α = Effect of extracellular pH acidification of PBMCs on oxidative stress and cell viability: the relationship with expressions of CA9 and HIF-1α mRNA.

Latar Belakang: pH ekstraseluler (pHe) perlu dipertahankan dalam sel normal untuk menjalankan fungsi sel dengan baik. Adanya perubahan di lingkungan ekstraseluler meliputi asidifikasi pHe akan berdampak pada fisiologi sel dan menginduksi patologis pada beberapa penyakit degeneratif sistemik. Namun, studi in vitro tentang respons homeostasis terhadap asidifikasi pHe masih terbatas. Tujuan penelitian ini adalah menganalisis pengaruh asidifikasi pHe peripheral blood mononuclear cells (PBMCs) manusia normal terhadap stres oksidatif dan viabilitas sel serta kaitannya dengan ekspresi mRNA CA9 dan HIF-1α. Model ini digunakan untuk menganalisis respons homeostasis seluler terhadap kondisi sistemik dan menentukan strategi pencegahannya. Metode: PBMCs dikultur dengan berbagai pH medium selama 24, 48, dan 72 jam. pH dari medium kultur diatur menjadi pH 7,4; 7,2; 7,0 dan 6,6 menggunakan 0,01 M asam klorida (HCl) dan diukur menggunakan pH meter. Viabilitas sel dihitung menggunakan metode eksklusi trypan blue. Kadar superoksida dianalisis menggunakan dihydroethidium (DHE) probe dan kadar hidrogen peroksida dianalisis menggunakan 2’-7’-dichlorofluorescein diacetate (DCFH-DA) probe. Ekspresi mRNA HIF-1α, CA9, dan MnSOD dianalisis menggunakan qRT-PCR. Aktivitas spesifik MnSOD dianalisis menggunakan RanSOD Kit dan aktivitas CAT juga dianalisis. Konsentrasi MDA diukur menggunaan metode Wills. Hasil: ∆pHe meningkat secara bertahap setelah asidifikasi ekstraseluler pada 24, 48, dan 72 jam. Setelah 72 jam perlakuan asidifikasi, kadar ROS dan ekspresi mRNA HIF1α, CA9, dan MnSOD meningkat, sementara aktivitas MnSOD menurun dan dilanjutkan dengan aktivitas CAT yang meningkat. Konsentrasi MDA meningkat dan berdampak pada penurunan viabilitas sel. Kesimpulan: PBMCs masih menunjukkan respons homeostasis yang cukup baik pada kondisi asidifikasi pHe, namun berdampak pada peningkatan stres oksidatif dan penurunan viabilitas sel.
Kata kunci: PBMCs, pH ekstraseluler, stres oksidatif, CA9, HIF-1α


Background: The extracellular pH (pHe) needs to be maintained in normal cells to carry out cell functions properly. Changes in the cellular environment including acidification of the extracellular induce pathologies in systemic degenerative diseases. However, in vitro studies about homeostasis responses of pHe acidification are still limited. This study aimed to analyze the effect of pHe acidification of human peripheral blood mononuclear cells (PBMCs) on oxidative stress and cell viability along with its association to the expressions of CA9 and HIF-1α mRNA. This model will be used to analyze cellular homeostasis responses to the systemic condition and determine prevention strategies. Methods: PBMCs were cultured with various pH medium for 24-, 48-, and 72-h. The pH of culture medium was adjusted to pH 7.4, 7.2, 7.0, and 6.6 by using 0.01 M hydrochloric acid (HCl) and measured by pH meter. Cell viability were counted by using the trypan blue exclusion method. Superoxide levels were analyzed using a dihydroethidium (DHE) probe and hydrogen peroxide levels were analyzed using a 2’7’-dichlorofluorescein diacetate (DCFH-DA) probe. HIF-1α, CA9, dan MnSOD mRNA expressions were analyzed using qRT-PCR. MnSOD activity was analyzed using RanSOD Kit and CAT acitivity was analyzed. MDA concentration was measured by Wills method. Results: ∆pHe gradually increased after 24-, 48-, and 72-h extracellular acidification. After 72-h of acidification treatment, the ROS levels and HIF-1α, CA9, MnSOD mRNA expressions was increased, while the MnSOD spesific activity decreased and was followed by increased CAT activity. The concentration of MDA increases and had an impact on decreasing cell viability. Conclusion: PBMCs showed a homeostasis response under extracellular acidification, but impact to increasing oxidative stress and decreased cell viability
Key words: PBMCs, extracellular pH, oxidative stress, CA9, HIF-1α