PENAMBAHAN INTERLEUKIN-6 PADA MEDIA KULTUR MENINGKATKAN KONSENTRASI PROTEIN PADA MEDIA TERKONDISI SEL PUNCA MESENKIMAL

Marsudi Siburian(1*), Nor Sri Inayati(2)

(1) Institut Teknologi Sumatera
(2) Program Studi Kedokteran, Fakultas Kedokteran, Universitas Jenderal Soedirman
(*) Corresponding Author

Abstract

ABSTRAK

Pengobatan regeneratif menggunakan Media Terkondisi Sel Punca Mesenkimal (MTSPM) merupakan salah satu metode yang dikembangkan untuk pengobatan Diabetes Melitus (DM). Pengembangan metode produksi MTSPM perlu dilakukan untuk meningkatkan efikasi, salah satunya dengan menginduksi sekresi dari sel punca mesenkimal (SPM) menggunakan senyawa penanda dari penyakit DM yaitu Interleukin-6 (IL-6). Penelitian ini bertujuan untuk menguji efek dari penambahan IL-6 pada media kultur terhadap kultur SPM dan konsentrasi protein pada MTSPM yang dihasilkan. Kultur SPM dilakukan pada temperatur 37˚C dan 5% CO2. Kultur SPM dibagi menjadi tiga kelompok yang diberi media tumbuh berbeda, kelompok I dikultur pada media basal, kelompok II dengan media basal + 1pg/mL IL-6, dan kelompok III dengan media basal +10pg/mL IL-6. Pengujian efek IL-6 dilakukan dengan melakukan penghitungan jumlah SPM pada kultur dan pengukuran total protein pada MTSPM menggunakan metode Qubit Fluorometer. Hasil pengukuran dianalisa secara statistik dengan uji One Way Anova, Kruskall Wallis dan Mann Whitney. Hasil pengujian menunjukkan pemberian IL-6 mampu meningkatkan jumlah sel pada kultur SPM. Total protein pada MTSPM mengalami peningkatan hingga 65% dan 262% untuk 24 jam pertama dan kedua ketika diberi penambahan IL-6 dengan konsentrasi 10pg/mL. Hal ini menunjukkan terdapat lebih banyak senyawa sitokin dan faktor tumbuh dalam MTSPM yang dihasilkan sehingga terdapat kemungkinan terjadinya peningkatan efikasi dari MTSPM tersebut saat dipergunakan dalam terapi DM. Tetapi hal tersebut perlu diteliti lebih lanjut dengan melihat profil sitokin dan faktor tumbuh dalam MTSPM dan juga dilanjutkan dengan uji in vivo pada hewan model DM.

 

Kata Kunci: Sel Punca Mesenkimal, Interleukin-6, Media Terkondisi.

 

ABSTRACT

Regenerative medicine using Conditioned Medium-Mesenchymal Stem Cell (CMMSCs) is one of the developed methods for the treatment of Diabetes Mellitus (DM). However, the method to produce CMMSCs still needs to be developed to increase its efficacy, one of which is by inducing secretion from mesenchymal stem cells (MSCs) using marker compounds in DM, such as Interleukin-6 (IL-6). This study aims to examine the effect of Interleukin-6 (IL-6) in the growth medium on the culture of MSCs and the concentration of protein on CMMSCs. The MSCs were cultured at 37oC and 5% CO2. Culture of MSCs grouped into three which were given different growth mediums, group I cultured in basal medium, group II cultured in basal medium + 1pg/mL IL-6, and group III cultured in basal medium +10pg/mL IL-6. Tests were carried out by calculating the amount of MSCs in culture and measuring the total protein on CMMSCs using the Qubit Fluorometer method. Results were analyzed statistically using the One Way Anova test, Kruskall Wallis, and Mann Whitney test. The results showed that the addition of IL-6 was able to increase the number of cells in the MSCs culture. Total protein in CMMSCs increased up to 65% and 262% for the first and second 24 hours when given the addition of IL-6 at a concentration of 10pg/mL. This shows that there are more cytokine compounds and growth factors in the resulting CMMSC, therefore there is a possibility of an increase in efficacy from the CMMSC when used to treat DM. However, it needs further research to evaluate the cytokine and growth factor profile of the CMMSC and followed up with an in vivo trial using the DM animal model.

 

Keywords: Mesenchymal Stem Cells, Interleukin-6, Conditioned Medium.

References

da Silva Meirelles, L., Fontes, A.M., Covas, D.T., Caplan, A.I., 2009. Mechanisms involved in the therapeutic properties of mesenchymal stem cells. Cytokine Growth Factor Rev. 20, 419–427. https://doi.org/10.1016/j.cytogfr.2009.10.002

Dahlan, S.M., 2018. Statistik untuk kedokteran dan kesehatan, 5th ed. Salemba Medica, Jakarta.

Khairani, Kurniasih, N., Maula, R., 2019. InfoDATIN 2018. Pus. Data dan Inf. Kementeri. Kesehat. RI 1–10.

Kwon, Y.W., Heo, S.C., Jeong, G.O., Yoon, J.W., Mo, W.M., Lee, M.J., Jang, I.H., Kwon, S.M., Lee, J.S., Kim, J.H., 2013. Tumor necrosis factor-α-activated mesenchymal stem cells promote endothelial progenitor cell homing and angiogenesis. Biochim. Biophys. Acta - Mol. Basis Dis. 1832, 2136–2144. https://doi.org/10.1016/j.bbadis.2013.08.002

Li, P., Zhou, H., Di, G., Liu, J., Liu, Y., Wang, Z., Sun, Y., Duan, H., Sun, J., 2017. Mesenchymal stem cell-conditioned medium promotes MDA-MB-231 cell migration and inhibits A549 cell migration by regulating insulin receptor and human epidermal growth factor receptor 3 phosphorylation. Oncol. Lett. 13, 1581–1586. https://doi.org/10.3892/ol.2017.5641

Lin, H., Joehanes, R., Pilling, L.C., Dupuis, J., Lunetta, K.L., Ying, S.-X., Benjamin, E.J., Hernandez, D., Singleton, A., Melzer, D., Munson, P.J., Levy, D., Ferrucci, L., Murabito, J.M., 2014. Whole Blood Gene Expression and Interleukin-6 Levels. Genomics 104, 490–495. https://doi.org/10.1016/j.ygeno.2014.10.003.

Ljungberg, L.U., Zegeye, M.M., Kardeby, C., Fälker, K., Repsilber, D., Sirsjö, A., 2020. Global Transcriptional Profiling Reveals Novel Autocrine Functions of Interleukin 6 in Human Vascular Endothelial Cells. Mediators Inflamm. 2020, 1–12. https://doi.org/10.1155/2020/4623107

Luo, Y., Zheng, S.G., 2016. Hall of fame among pro-inflammatory cytokines: Interleukin-6 gene and its transcriptional regulation mechanisms. Front. Immunol. 7, 1–7. https://doi.org/10.3389/fimmu.2016.00604

Madrigal, M., Rao, K.S., Riordan, N.H., 2014. A review of therapeutic effects of mesenchymal stem cell secretions and induction of secretory modification by different culture methods. J. Transl. Med. 12, 1–14. https://doi.org/10.1186/s12967-014-0260-8

Noone, C., Kihm, A., English, K., O’Dea, S., Mahon, B.P., 2013. IFN-γ stimulated human umbilical-tissue-derived cells potently suppress NK activation and resist NK-mediated cytotoxicity in vitro. Stem Cells Dev. 22, 3003–3014. https://doi.org/10.1089/scd.2013.0028

Nunemaker, C.S., 2016. Considerations for Defining Cytokine Dose, Duration, and Milieu That Are Appropriate for Modeling Chronic Low-Grade Inflammation in Type 2 Diabetes. J. Diabetes Res. 2016, 1–9. https://doi.org/10.1155/2016/2846570

Pricola, K.L., Kuhn, N.Z., Haleem-Smith, H., Song, Y., Tuan, R.S., 2009. Interleukin-6 maintains bone marrow-derived mesenchymal stem cell stemness by an ERK1/2-dependent mechanism. J. Cell. Biochem. 108, 577–588. https://doi.org/10.1002/jcb.22289

Redondo-Castro, E., Cunningham, C., Miller, J., Martuscelli, L., Aoulad-Ali, S., Rothwell, N.J., Kielty, C.M., Allan, S.M., Pinteaux, E., 2017. Interleukin-1 primes human mesenchymal stem cells towards an anti-inflammatory and pro-trophic phenotype in vitro. Stem Cell Res. Ther. 8, 1–11. https://doi.org/10.1186/s13287-017-0531-4

Rickard, M., 2002. Key Ethical Issues in Embryonic Stem Cell Research. Inf. Res. Serv. 1–15.

Rodriguez, L.A., Mohammadipoor, A., Alvarado, L., Kamucheka, R.M., Asher, A.M., Cancio, L.C., Antebi, B., 2019. Preconditioning in an Inflammatory Milieu Augments the Immunotherapeutic Function of Mesenchymal Stromal Cells. Cells 8, 1–31. https://doi.org/10.3390/cells8050462

Ryan, J.M., Barry, F., Murphy, J.M., Mahon, B.P., 2007. Interferon-γ does not break, but promotes the immunosuppressive capacity of adult human mesenchymal stem cells. Clin. Exp. Immunol. 149, 353–363. https://doi.org/10.1111/j.1365-2249.2007.03422.x

Ryu, Y.J., Cho, T.J., Lee, D.S., Choi, J.Y., Cho, J., 2013. Phenotypic characterization and in vivo localization of human adipose-derived mesenchymal stem cells. Mol. Cells 35, 557–564. https://doi.org/10.1007/s10059-013-0112-z

Shin, J.J., Lee, E.K., Park, T.J., Kim, W., 2015. Damage-associated molecular patterns and their pathological relevance in diabetes mellitus, Ageing Research Reviews. Elsevier B.V. https://doi.org/10.1016/j.arr.2015.06.004

Sun, L., Li, D., Song, K., Wei, J., Yao, S., Li, Z., Su, X., Ju, X., Chao, L., Deng, X., Kong, B., Li, L., 2017. Exosomes derived from human umbilical cord mesenchymal stem cells protect against cisplatin-induced ovarian granulosa cell stress and apoptosis in vitro. Sci. Rep. 7, 1–13. https://doi.org/10.1038/s41598-017-02786-x

Tie, R., Li, H., Cai, S., Liang, Z., Shan, W., Wang, B., Tan, Y., Zheng, W., Huang, H., 2019. Interleukin-6 signaling regulates hematopoietic stem cell emergence. Exp. Mol. Med. 51, 1–12. https://doi.org/10.1038/s12276-019-0320-5

Tiwari, P., 2015. Recent trends in therapeutic approaches for diabetes management: A comprehensive update. J. Diabetes Res. 2015, 1–12. https://doi.org/10.1155/2015/340838

Tsalamandris, S., Antonopoulos, A.S., Oikonomou, E., Papamikroulis, G.A., Vogiatzi, G., Papaioannou, S., Deftereos, S., Tousoulis, D., 2019. The role of inflammation in diabetes: Current concepts and future perspectives. Eur. Cardiol. Rev. 14, 50–59. https://doi.org/10.15420/ecr.2018.33.1

Vizoso, F.J., Eiro, N., Cid, S., Schneider, J., Perez-Fernandez, R., 2017. Mesenchymal stem cell secretome: Toward cell-free therapeutic strategies in regenerative medicine. Int. J. Mol. Sci. 18.

DOI: 10.23917/biomedika.v13i2.12881

Article Metrics

Abstract view(s): 203 time(s)
PDF (Bahasa Indonesia): 276 time(s)

Refbacks

  • There are currently no refbacks.