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

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0,7

3 24 48 72time (hour)

optic

al d

ensi

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Cells Cells + EBV

Cells (clinorotation) Cells + EBV (clinorotation)

0

2

4

6

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10

12

3 24 48 72

time gompling

exti

nct

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co

effi

cien

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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)