Browsing by Author "Drishya Prakashan"

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    Effect of stingless bee propolis on proliferation of human stem cells
    (Department of Plant Biotechnology, College of Agriculture, Vellayani, 2022-01-17) Drishya Prakashan; Shanas, S
    The present research work entitled “Effect of Stingless bee propolis on the proliferation of human stem cells” was carried out in the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram and Department of Genomic Science, Central University of Kerala during the year 2020 2021, with the objective to study the effect of propolis collected from two different genera of Stingless bees, viz., Lisotrigona and Tetragonula on the proliferation of human induced pluripotent stem cells (hPSCs). Crude propolis samples were collected from the nest of three species of stingless bees Lisotrigona sp. (Kollam), Tetragonula calophyllae (Thiruvananthapuram) and Tetragonula travancorica (Thiruvananthapuram). Propolis samples were macerated at room temperature and extracted with 95% ethanol. MTT assay was performed and IC-50 values were calculated in-order to determine the influence of propolis on the proliferation of hPSCs. Five different concentrations of propolis, viz., 150µg/ml, 300µg/ml, 450µg/ml, 600µg/ml and 900µg/ml respectively, were added to D14/C2 cells for a period of 24 hrs. Cells treated with the propolis extracted from Lisotrigona sp., T. calophyllae and T. travancorica obtained IC50 values of 410.904 ug/ml, 480.097 ug/ml and 215.157 ug/ml respectively. The cells treated with the three different propolis displayed significant proliferation rate after 24 hrs treatment in relative to the control however, higher concentration were observed to be cytotoxic to cells. When the right concentration of propolis was used, marked difference in the morphology of cells was observed. The cells lost its border integrity, uniformity and started to migrate. RNA of the control and propolis treated cell was isolated by QIAzol reagent method. Quality and quantity of the samples were analyzed through nanodrop, which gave an absorbance value of 1.8 to 2.0 for all the samples. The concentration of the samples observed was between 30-200 ngµl-1. The RNA samples were then subjected to 99 cDNA conversion (iScriptTM cDNA Synthesis Kit). The cDNA of control and treated samples were then subjected qRT- PCR. The real-time quantitative PCR (qRT-PCR) was performed with the Applied Biosystems PCR system in a total volume of 10 µl and expression of different genes was studied. Gene expression analysis revealed pluripotency markers (OCT4 and NANOG), important for preserving pluripotency were down regulated upon treatment with propolis, which in turn confirmed early differentiation of hPSCs. Further investigation on the gene expression of early differentiation markers revealed that propolis supported the cells to differentiate into mesendoderm and endoderm lineage, which is a novel finding. To conclude, the result of the study proved that the propolis obtained from stingless bees Tetragonula spp. probably has more therapeutic value in terms of its effect on human pluripotent stem cells compared to the propolis obtained from Lisotrigona sp.