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"Latar Belakang: pH ekstraseluler (pHe) perlu dipertahankan dalam sel normal untuk menjalankan fungsi sel dengan baik. Adanya perubahan di lingkungan seluler meliputi asidifikasi akan berdampak pada fisiologi sel dan menginduksi kematian sel. Namun, studi terntang pengaruh asidifikasi pHe terhadap stres oksidatif dan regulasinya masih terbatas. Tujuan penelitian ini adalah menganalisis pengaruh asidifikasi pHe PBMCs terhadap stres oksidatif dan viabilitas serta kaitannya dengan ekspresi mRNA CA9 dan HIF-1alfa.Metode: PBMC dikultur dengan berbagai pH medium selama 24, 48, dan 72 jam. pH medium kultur diatur menjadi pH 7,4, 7,2, 7,0, dan 6,6 menggunakan 0,01 HCl. Viabilitas sel dihitung menggunakan Trypan blue. Kadar ROS diukur menggunakan DHE dan DCFH-DA probes. Ekspresi mRNA HIF-1alfa, 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 pada waktu inkubasi 24, 48, dan 72 jam. Kadar ROS dan ekspresi mRNA HIF-1alfa, CA9, dan MnSOD meningkat, sementara aktivitas MnSOD menurun dan CAT meningkat. Konsentrasi MDA meningkat dan berdampak pada penurunan viabilitas sel.Kesimpulan: Asidifikasi pHe PBMCs berdampak pada peningkatan stres oksidatif dan penurunan viabilitas sel. Selain itu, respons pada mRNA CA9 dan HIF-1alfa masih cukup baik.

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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 affect to cell physiology and induce cell death. However, studies about effect of pHe acidification on oxidative stress and its regulation are still limited. This study aimed to analyze the effect of pHe acidification of PBMCs on oxidative stress and cell viability with expressions of CA9 and HIF-1alpha mRNA.

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 HCl. Cell viability was calculated using trypan blue. ROS levels was measured using DHE and DCFH-DA probes. HIF-1alpha, CA9, dan MnSOD mRNA expressions were analyzed using qRT-PCR. MnSOD spesific activity was analyzed using RanSOD Kit and CAT acitivity was analyzed. MDA concentration was measured by Wilss method.

Results: pHe increased gradually at 24-, 48-, and 72-h incubation. ROS levels and HIF-1alpha, CA9, MnSOD mRNA expressions were increased, while the MnSOD spesific activity decreased and CAT activity increased. MDA concentration incease and had an impact on decreasing cell viability.

Conclusions: pHe acidification increased oxidative stress levels and decreased cell viability. In additon, PBMCs had a response to of CA9 and HIF-1alpha mRNA."

"Latar belakang: Prematuritas merupakan salah satu kelainan yang masih menjadi masalah global. Kejadian prematuritas tidak hanya terjadi di negara berkembang tetapi juga di negara maju. Beberapa kondisi ibu hamil dapat memicu keadaan hipoksia dalam rahim sehingga menyebabkan kelahiran prematur. Keadaan plasenta menggambarkan kesejahteraan janin intra uteri. Kondisi hipoksia seluler memicu ekspresi HIF-1α yang menjadi faktor transkripsi bagi CA9 sebagai penanda hipoksia. Penelitian ini bertujuan menganalisis pengaruh hipoksia terhadap plasenta prematur. Metode: Sampel menggunakan plasenta prematur yang hipoksia (H) dan nonhipoksia (N) sebagai kontrol. Parameter yang dinilai adalah struktur histologis plasenta (Hematoksilin-Eosin), regulator hipoksia HIF-1α (imunohistokimia), dan penanda hipoksia CA9 (ELISA). Hasil: Penilaian struktur histologis menunjukkan adanya perbedaan jumlah pembuluh darah fetus antara kedua kelompok secara bermakna, dimana pada kelompok hipoksia jumlah pembuluh darah fetus lebih banyak dibandingkan kelompok non-hipoksia. Distribusi intensitas ekspresi HIF-1α kedua kelompok juga berbeda bermakna. Rerata kadar CA9 kedua kelompok tidak berbeda bermakna, namun terdapat kecenderungan rerata kadar CA9 kelompok hipoksia lebih tinggi 28% dibandingkan yang non-hipoksia. Kesimpulan: Pengaruh hipoksia terhadap plasenta prematur pada tingkat molekuler berupa stabilitas protein HIF-1α yang menyebabkan peningkatan jumlah pembuluh darah fetus dan terjadi kecenderungan peningkatan sintesis protein CA9.

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Background: Prematurity is a disorder that is still a global problem. Incidence of prematurity is a problem in developing and also in developed countries. Certain condition accompanying pregnancies may trigger uterine hypoxia, causing premature birth. The placental condition is related with the intra-uterine fetal condition. Cellular hypoxic condition caused by systemic chronic hypoxia, lead to stabilization of HIF-1α protein, a transcription factor of CA9. This study aimed to analyze the effect of hypoxia on the premature placenta.

Methods: Samples from hypoxic premature placenta (H) and non-hypoxic premature placenta (N) were collected. Parameters assessed were histological structure of the placenta (Hematoxylin-Eosin), expression of HIF-1α (immunohistochemistry) and the level of CA9 (ELISA).

Results: Assessment of histological structure showed the number of fetal blood vessels were differed significantly between the two group, wherein the hypoxia group was more than the non-hypoxia. The distributions of HIF-1α expression between the two groups were also differed significantly. The average level of CA9 between two groups were not significant, but there is a tendency of higher level of CA9 in the hypoxia group (28% higher compared to the non-hypoxia group).

Conclusion: It is concluded that the effect of the hypoxia on premature placenta in this study occured at molecular level and lead to HIF-1α protein stability that causes an increase of the number of fetal blood vessel and synthesis of CA9 protein."