ANGGRAINI DWI S., 091070107 (2013) DEPOSISI KALSIUM PADA NEURON BERASAL DARI SEL PUNCA NEURON KULIT MANUSIA YANG DIINOKULASI TOXOPLASMA GONDII MELALUI SINYAL KALSIUM, NEKROPTOSIS DAN MENGEKSPRESIKAN Hsp70. Disertasi thesis, UNIVERSITAS AIRLANGGA.

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Abstract

Background:The neuronal calcium deposition in cerebral congenital toxoplasmosis calcification, is not well understood. It was thought that calcium signalling during necrosis and the expression of protein Hsp70 play an important role. The main purpose of this study was to discover the mechanism of calcium deposition in fully differentiated neuron culture from human skin-derived stem cell inoculated by Toxoplasma gondii was composed. Methods: Twenty four CD 133 and III tubulin validated neuronal culture of human-skin-derived neural stem cell were divided into six groups, consisting of three uninfected groups and three infected groups. Each group was further divided into three groups according to the time interval of inoculation, 2 hours, 24 hours and 48 hours. The infected groups were inoculated by virulent strain of Toxoplasma gondii. The neuron-Toxoplasma gondii ratio was 1 : 5. Intracellular calcium of neuron and tachyzoite,the evidence of necrosis, apoptosis, calcium signalling, and the expression of Hsp 70 of neuron were examined by confocal microscope. Uninfected groups also undergone the same examination except for intracellular calcium of the tachyzoite. For immunocytochemical staining cell was fixed with methanol and incubate with the chemical probe calcium indicator Fura-2 for calcium measurement, propidium iodide for necrosis, TACS-Annexin-V for apoptosis and calcium channel protein and rabbit anti Hsp70 polyclonal antibody primary antibody for Hsp70. The normality of the data data was analyzed by Kolmogorov-Smirnov Test, differentiation test was tested by t2 Test, and Anova, for correlation test was done by Pearson Correlation Test. Result: From the data showed that calcium intensity of cytosolic neuron and T. gondii was significanly different from control groups (p<0.05). There is also significant correlation between calcium intensity with the evidence of necrosis and Hsp70 expression at 2 hours after infection, but not at the 24 and 48 hours groups. It means that necrosis was very high at the early moment of infection compare to the 24 and 48 hours groups. Conclusion : Apoptosis and necrosis were simultaneously shown with calcium contribution in this study, and it is proposed a death receptor-induced necroptosis category. Necrosis is high at 2 hours after infection, decreased at 24 hours and increased again at 48 hours. It show that T. gondii provide capability against cell death and the increament of necrosis at 48 hours may be cause of egress of T. gondii that makes neuronal lysis. Calcium signalling plays an important role in the mechanism of neuronal cell death, there is significant correlation between calcium intensity, evidence of necrosis and the expression of Hsp70. In the nearby future, the different intracellular signalling events that contribute to necroptosis should be proven.

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