Tesis

Efek ko-kultur sel stelata hepatik (LX-2) dengan sel punca CD34 + asal darah tali pusat pada morfologi sel, ekspresi TGFβ, tenascin-C dan kolagen tipe 1A1 = Effect of hepatic stellate cells (LX-2) and umbilical cord blood CD34 + stem cells co-culture on cell morphology, TGF-β, tenascin-C and collagen type 1A1 expression.

Latar Belakang : Penggunaan sel punca sebagai anti fibrosis hati cukup menjanjikan. Sel punca CD34+ asal darah tali pusat sudah banyak digunakan dalam studi anti fibrosis hati. Penelitian ini menjelaskan efek ko-kultur antara sel stelata hepatik (HSC) LX-2 dan sel punca CD34+ asal darah tali pusat dalam morfologi sel dan ekspresi TGF-β, tenascin-C dan kolagen tipe 1A1. Metode : Sel CD34+ diisolasi dari sel darah tali pusat manusia yang dikriopreservasi menggunakan separasi magnet. Sel HSC LX-2 dikultur sebagai kontrol monokultur. Sebagian dipanen dan dihitung untuk dilakukan ko-kultur dengan sel CD34+ dalam rasio 1:1. Ko-kultur CD34+ dan LX-2 dilakukan dengan metode kultur konvensional (2D) dan 3D hanging drop. Hasil monokultur dan kokultur dipanen pada hari ke1, 2 dan 3 dan dilakukan pewarnaan imunositokimia tenascin-C ekstraksi RNA untuk analisis kuantitatif dengan real time PCR ekspresi TGF-β dan kolagen tipe 1A1. Hasil : Hasil menunjukkan perbedaan morfologi ko-kultur 2D dan 3D hanging drop dibandingkan kontrol monokultur. Pada ko-kultur 2D terdapat mikromassa, sedangkan pada monokultur 2D tidak ada mikromassa yang terbentuk. Pada kokultur 3D hanging drop, terdapat spheroid yang lebih kecil (hambatan pembentukan spheroid) dibandingkan monokultur 3D hanging drop. Sel CD34 memiliki efek direk terhadap aktivitas sinyal sel stelata hepatik dengan adanya kecenderungan penurunan ekspresi TGF-β. Analisis imunositokimia tenascin-C dalam mikromassa dan spheroid masih perlu dioptimasi. Ko-kultur 2D dan 3D hanging drop method sel punca CD34+ asal darah tali pusat dan sel stelata hepatik memiliki efek terhadap penurunan ekspresi kolagen tipe 1A1. Kesimpulan : Sel punca CD34+ asal darah tali pusat memiliki efek direk terhadap morfologi sel, inhibisi aktivitas sel stelata hepatik (LX-2) yang ditandai dengan penurunan ekspresi TGF-beta dan inhibisi deposisi matriks ekstrasel yang ditandai penurunan ekspresi kolagen tipe 1A1.
Kata kunci: sel punca CD34+ asal darah tali pusat, sel stelata hepatik, liver fibrosis, TGF-beta, tenascin-C, kolagen 1A1.


Background : The development of stem cell therapy antifibrotik placing as one of the promising therapy. Umbilical cord blood CD34+ stem cells has been widely used in the study antifibrosis. This study describes the effect of co-culture between hepatic stellate cells (HSC) LX-2 and umbilical cord blood CD34 stem cells on cell morphology and expression of TGF-β, tenascin-C and collagen type 1A1. Method : CD34+ cells were isolated from thawed cryopreserved human umbilical cord blood cells using magnetic separation. LX-2 cells culture were harvested and counted. CD34+ and LX-2 cells were mixed in suspension with 1:1 ratio (v/v). Cell suspension divided into 2 sets: 2D co-culture plated in standard well plate and 3D co-culture as hanging drops. LX-2 monoculture, CD34 dan LX-2 coculture were harvested on day 1, 2 and 3 as sample for further analysis. Tenascin-C expression was analysed by imunocytochemistry techniques. TGFBeta and collagen type 1A1 expression was analysed by qPCR. Result : The result showed different morphology between co-culture and monoculture on 2D and 3D hanging drop. The 2D co-culture showed micromass formation, instead of no micromass formation on monoculture. The 3D hanging drop showed smaller spheroid formation (spheroid formation inhibition) compared with monoculture. CD34+ cells showed direct effect on hepatic stellate cell signalling activity represented by the decrease in TGF-beta expression, inhibition of extracellular matrix deposition represented by a decrease in Collagen type 1A1 expression. Conclusion : UCB CD34+ cells showed direct effect on cell morphology, inhibition of hepatic stellate cell (LX-2) activity represented by a decrease in TGF-beta expression, inhibition of extracellular matrix deposition represented by a decrease in collagen type 1A1 expression.
Keywords : UCB CD34+ stem cells, hepatic stellate cells, liver fibrosis, TGFbeta, tenascin-C, collagen 1A1.