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Abstrak :
Oct-4 dan Klf4 merupakan dua gen penyandi pluripotensi yang terdapat pada manusia. Kedua gen tersebut dapat dideteksi pada mRNA sel kanker manusia dengan menggunakan rancangan primer spesifik pada metode RT-PCR. Proses RT-PCR akan mengubah mRNA menjadi cDNA yang kemudian hasilnya dilakukan purifikasi dari gel agarosa. Hasil purifikasi gel agarosa diamplifikasi dengan PCR. Selanjutnya, dilakukan proses sekuensing untuk memeriksa keakuratan sekuen gen yang nantinya akan dikloning. Hasil sekuensing dianalisis dengan BLASTN untuk pencarian homologi pada database asam nukleat. Ligasi dilakukan menggunakan vektor pET101/D-TOPO®. Rancangan primer yang spesifik dan penambahan basa CACC ATG pada primer forward merupakan syarat untuk melakukan ligasi DNA ke vektor pET101/D-TOPO®, dimana proses ligasi ini tidak menggunakan enzim ligase. Selanjutnya, produk hasil ligasi ditransformasi ke inang Escherichia coli DH5α yang selnya sudah dibuat kompeten dengan metode heat shock. Koloni yang tumbuh di media LB agar dengan ampisilin diisolasi untuk mendapatkan plasmid yang selanjutnya dianalisis dengan menggunakan teknik PCR. Vektor plasmid yang terdapat pada E.coli DH5α kompeten mengandung sisipan gen Oct-4 dan Klf4.

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Oct-4 and Klf4 are two genes coding of pluripotency in human. Both of the genes can be detected in human cancer cell mRNA by using specifically designed primers in RT-PCR method. RT-PCR process changed the mRNA into cDNA. The result from this process was purified from agarose gel fragment and amplified by PCR method. Then, sequencing of the purified products were done and analyzed using BLASTN feature in NCBI site to search the homology of the sequence compared to the sequences in nucleic acid database. The vector used in ligation process was pET101/D-TOPO® vector. Specifically designed primers and adding CACC ATG base in the beginning of forward primer are the requirements of using this vector. The ligation process using this TOPO® vector does not require the presence of DNA ligase. The next process is transformation by using the heat shock method. Ligation product is transformed into competent Escherichia coli DH5α host. Colonies that grows in LB agar media with ampicillin are treated by plasmid isolation protocol in order getting the plasmids for further anlysis using PCR method. The result from PCR analysis shows that the plasmids in competent E.coli DH5α contain Oct-4 and Klf4 inserts.

Abstrak :
[ABSTRAK
Glioma adalah tumor otak primer yang sampai saat ini sering timbul resistensi terapi. Sel punca glioma diduga berperan penting dalam resistensi dan rekurensi sel tumor. Sel punca glioma memiliki penanda permukaan CD133 dan mampu berpluripotensi dengan mengekspresikan Oct4. Kondisi hipoksia tumor juga berperan dalam self renewal sel punca glioma. Tujuan dari penelitian ini adalah untuk mengetahui hubungan keberadaan sel punca glioma dengan keganasan, pluripotensi dan kondisi hipoksia. Cross sectional digunakan sebagai desain penelitian dengan jumlah sampel sebanyak 35 jaringan, terdiri atas 15 glioma derajat keganasan tinggi dan 20 glioma derajat keganasan rendah. Pengukuran ekspresi relatif mRNA CD133, Oct4 dan HIF-1α menggunakan metode qRTPCR. Protein HIF-1α dilihat ekspresinya melalui teknik imunohistokimia. Ekspresi relatif mRNA CD133 dan Oct4 lebih tinggi bermakna (p < 0.05, Mann- Whitney) pada glioma derajat keganasan tinggi dibanding glioma derajat keganasan rendah. Protein HIF-1α lebih tinggi bermakna (p < 0,01, Mann- Whitney) pada glioma derajat keganasan tinggi dibanding glioma derajat keganasan rendah. Terdapat hubungan ekspresi sel punca glioma CD133 dengan pluripotensi serta kondisi hipoksia (r = 0,518, r = 0,339; Spearman?s rho) serta pluripotensi dengan kondisi hipoksia pada derajat keganasan tinggi (r = 0,749; Spearman?s rho). Ekspresi relatif mRNA CD133, Oct4 dan HIF-1α meningkat seiring dengan peningkatan derajat keganasan. Terdapat hubungan yang bermakna antara keberadaan penanda sel punca glioma CD133 dengan pluripotensi dan kondisi hipoksia pada glioma derajat keganasan tinggi.

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ABSTRACT
Glioma is primary brain tumor with frequent therapeutic resistance. Glioma cancer stem cells were considered to play a role in resistance and recurrence of tumor cells. Glioma cancer stem cells expressed CD133 on their surface and capable of pluripotency as expressed by Oct4 positive. Tumor hypoxic condition also play a role in glioma cancer stem cells self renewal. Aim of this study is to investigate correlation between glioma cancer stem cells, degree of malignancy, pluripotency and hypoxia. Design of this study is cross sectional with 35 glioma samples comprises of 20 low grade malignant glioma and 15 high grade malignant glioma. Expression of mRNA CD133, Oct4 and HIF-1α were measured using qRT-PCR. HIF-1α protein expression was detected by immunohistochemistry from glioma sample. mRNA CD133 and Oct4 expression significantly higher (p < 0.05, Mann-Whitney) in high grade malignant glioma compared to low grade malignant glioma. HIF-1α tissue expression significantly higher (p < 0,01, Mann- Whitney) in high grade malignant glioma compared to low grade malignant glioma. There was correlation between expression of CD133 glioma cancer stem cells marker with pluripotency and hypoxia (r = 0,518, r = 0,543; Spearman?s rho) and pluripotency with hypoxia in high grade malignant glioma (r = 0,749; Spearman?s rho). mRNA CD133, Oct4 and HIF-1α expression increased with high grade malignant glioma. There was significant correlation between CD133 glioma cancer stem cell marker with pluripotency and hypoxia in high grade malignant glioma, Glioma is primary brain tumor with frequent therapeutic resistance. Glioma cancer stem cells were considered to play a role in resistance and recurrence of tumor cells. Glioma cancer stem cells expressed CD133 on their surface and capable of pluripotency as expressed by Oct4 positive. Tumor hypoxic condition also play a role in glioma cancer stem cells self renewal. Aim of this study is to investigate correlation between glioma cancer stem cells, degree of malignancy, pluripotency and hypoxia. Design of this study is cross sectional with 35 glioma samples comprises of 20 low grade malignant glioma and 15 high grade malignant glioma. Expression of mRNA CD133, Oct4 and HIF-1α were measured using qRT-PCR. HIF-1α protein expression was detected by immunohistochemistry from glioma sample. mRNA CD133 and Oct4 expression significantly higher (p < 0.05, Mann-Whitney) in high grade malignant glioma compared to low grade malignant glioma. HIF-1α tissue expression significantly higher (p < 0,01, Mann- Whitney) in high grade malignant glioma compared to low grade malignant glioma. There was correlation between expression of CD133 glioma cancer stem cells marker with pluripotency and hypoxia (r = 0,518, r = 0,543; Spearman’s rho) and pluripotency with hypoxia in high grade malignant glioma (r = 0,749; Spearman’s rho). mRNA CD133, Oct4 and HIF-1α expression increased with high grade malignant glioma. There was significant correlation between CD133 glioma cancer stem cell marker with pluripotency and hypoxia in high grade malignant glioma]