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Development of apoptosis in Namalwa lymphoblastoid
cells in conditions of clinorotation
Nesterova N.V., Zagorodnya S.D.,
Golovan A.V., Baranova G.V.
D.K. Zabolotny Institute of Microbiology and Virology NAS of Ukraine
Laboratory of viruses reproductionLaboratory of viruses reproduction
Infectious mononucleosis
Clinical presentations of infectious
mononucleosis (IM) are lymphadenitis,
lymphadenopathy, adenoid disease, hepatitis
(hepatosplenomegaly), splenomegaly, angina,
hematological changes.
EBV-associated malignant lymphoproliferative diseases
Burkitt's lymphomaNon- Hodgkin's lymphoma
Nasopharyngeal carcinoma, Adenocarcinoma
lymphogranulomatosis Oral cells lymphoplakia
X-linked lymphoproliferative
syndrome.
Autoimmune malignant
Syndrome of a chronic fatigue. Autoimmune
lymphoproliferative syndrome (ALPS)
Pseudorheumatism
Tasks of research:
1. Study of influence of microgravitation on Namalwa lymphoid cells viability and functional condition2. Study of effect microgravitation on Epstein-Barr virus reproduction in Namalwa cell culture.3. Investigation of influence of microgravitation on apoptosis processes in Namalwa cell - Epstein-Barr virus system.
Suspension cell culture of B-cell line of the Burkitt's lymphoma – Namalwa - human malignant lymphoid cells were used as a test model in experiment with microgravitation. This cell line was given by bank of cell cultures of R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of NAS of Ukraine. The cells were cultivated at the temperature of 370С in media consisted of medium 1640 with supplement of 10 % heated embryonic serum, 2mM L-glutamine and antibiotics.
Namalwa cells were infected by Epstein-Barr virus (virus was purified by dextran sulphate density-gradient centrifugation, quantity of viral protein was determined using a «QuantiPro BCA ASSAY Kit», Sigma, USA). Quantity of DNA genome-equivalents was determined by PCR method in a system "AMPLY-Senc-100R" (Russia) and Programme " Biotest A ".
Horizontal clinorotation of cells was carried out at 370С with speed 4
rpm (device «Clin - 1", "«Respirator", Donetsk) for
modeling of microgravitation in ground conditions.
Influence of microgravitationon proliferate activity cells (MTT- test)
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
3 24 48 72time (hour)
optic
al d
ensi
ty
Cells Cells + EBV
Cells (clinorotation) Cells + EBV (clinorotation)
0
2
4
6
8
10
12
3 24 48 72
time gompling
exti
nct
ion
co
effi
cien
t
Cells+EBV Cells+EBV+microgravitation
Level of Epstein-Barr virus core protein accumulation in Namalwa cell culture under the
influence of microgravitation
Detection of apoptic manifestation in Namalwa cells using Hoechst 33342
(fluorescence microscopy)
Apoptic
fragmentation
of nuclear cells
Influence of microgravitation on development of apoptosis in Epstein-Barr virus-infected
Namalwa lymphoid cells (in percent).
Time samplings
24 h 48 h 72 h
Cells 10 7 3
Cells+EBV 8 9 7
Cells+microgravitation 15 19 20
Cells+EBV+microgravitation
26 27 36
Detection of apoptic manifestation in Namalwa cells using Annexin V and 6-Carboxyfluorescein
(fluorescence microscopy)
Annexin V - RED 6-Carboxyfluorescein – Green
Detection of apoptic manifestation in Namalwa cells using Acridine Orange and Ethidium Bromide
(fluorescence microscopy)