Tesis

Peran Enzim Asam Amino Levulinat Dehidratase terhadap Ekspresi mRNA Sitoglobin: Kaitannya dengan Proliferasi dan Viabilitas Fibroblas Keloid = The Role of Amino Levulinic Acid Dehydrates on Cytoglobin mRNA Expression: Its Correlation with the Proliferation and Viability of Keloid Fibroblasts.

Keloid dapat ditandai dengan peningkatan aktivasi dan proliferasi fibroblas. Peningkatan aktivasi dan proliferasi keloid tentu diikuti dengan peningkatan kebutuhan energi. Pembentukan energi terbesar terjadi di mitokondria dan memerlukan suplai oksigen yang cukup. Hem diketahui berperan penting dalam mendukung fungsi mitokondria tersebut. Penggunaan suksinilaseton (SA) sebagai inhibitor enzim ALAD dapat menghambat proses sintesis hem, dan memengaruhi ekspresi hemoprotein tertentu serta fungsi mitokondria yang berimbas kepada proliferasi maupun viabilitas sel. Hingga saat ini, belum diketahui bagaimana pengaruh biosintesis hem terhadap proliferasi sel serta ekspresi CYGB, yang berperan dalam suplai oksigen, pada fibroblas keloid. Penelitian ini bertujuan untuk menganalisis pengaruh sintesis hem terhadap ekspresi CYGB dan proliferasi fibroblas keloid. Penelitian dilakukan pada fibroblas yang diisolasi dari kultur primer jaringan keloid. Kemudian dilakukan analisis populasi fibroblas dengan penanda CD73+; CD90+; dan CD105+. Inhibisi sintesis hem pada fibroblas keloid dilakukan dengan menggunakan SA 1 mM; 2,5 mM; dan 5 mM. Penentuan aktivitas enzim ALAD, diukur dengan metode kolorimetri. Ekspresi mRNA CYGB dianalisis dengan qRT-PCR. Analisis viabilitas fibroblas dilakukan dengan uji MTT, sedangkan analisis proliferasi dilakukan dengan uji BrdU. Hasil menunjukkan bahwa penggunaan SA menyebabkan penurunan aktivitas enzim ALAD, ekspresi mRNA CYGB, viabilitas, serta proliferasi fibroblas keloid yang sebanding dengan peningkatan kadar SA. Disimpulkan bahwa sintesis hem dapat memengaruhi ekspresi CYGB, viabilitas, serta proliferasi fibroblas keloid.
Kata kunci: fibroblas keloid; ALAD; suksinilaseton; CYGB; proliferasi.


Keloids can be characterized by increased activation and proliferation of fibroblasts. This increase impacts the energy requirements. Most of the energy occurs in the mitochondria and requires an adequate oxygen supply. The hem is known to play an essential role in supporting the function of these mitochondria. The use of succinylacetone (SA) as an inhibitor of the ALAD enzyme can inhibit the process of heme synthesis and affect the expression of certain hemoproteins and mitochondrial functions that affect cell proliferation and viability. Until now, it is not known how the effect of haem biosynthesis on cell proliferation and expression of CYGB, which plays a role in oxygen supply, in keloid fibroblasts. This study aimed to analyze the effect of heme synthesis on CYGB expression and keloid fibroblast proliferation. First, the study was conducted on fibroblasts isolated from the primary culture of keloid tissue. Then, the fibroblast population analysis was performed with the CD73+ marker; CD90+; and CD105+. Heme synthesis inhibition in keloid fibroblasts was performed using 1 mM SA; 2.5 mM; and 5 mM. Determination of ALAD enzyme activity, measured by colourimetric method. CYGB mRNA expression was analyzed by qRT-PCR. The analysis of fibroblast viability was carried out by the MTT test, while the BrdU test carried out the analysis of fibroblast proliferation. The results showed that the use of SA caused a decrease in ALAD enzyme activity, CYGB mRNA expression, viability, and proliferation of keloid fibroblasts that were proportional to the increase in SA levels. It was concluded that heme synthesis could affect CYGB expression, viability, and proliferation of keloid fibroblasts.
Keywords: keloid fibroblast; ALAD; succinylacetone; CYGB; proliferation.