ÃÅÐÎÍÒÎËÎÃÈß ÖÈÒÎÃÅÐÎÍÒÎËÎÃÈ×ÅÑÊÈÅ ÈÑÑËÅÄÎÂÀÍÈß ÁÈÎËÎÃÈ×ÅÑÊÎÉ ÀÊÒÈÂÍÎÑÒÈ ÝÔÈÐÍÎÃÎ ÌÀÑËÀ ÎÐÅÃÀÍÎ Å.Ñ. Àëèíêèíà
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ÂÅÑÒÍ. ÌÎÑÊ. ÓÍ-ÒÀ. ÑÅÐ. 16. ÁÈÎËÎÃÈß. 2012. ¹ 2 13 ÃÅÐÎÍÒÎËÎÃÈß ÓÄÊ 576.35:57.017.6:615.322 ÖÈÒÎÃÅÐÎÍÒÎËÎÃÈ×ÅÑÊÈÅ ÈÑÑËÅÄÎÂÀÍÈß ÁÈÎËÎÃÈ×ÅÑÊÎÉ ÀÊÒÈÂÍÎÑÒÈ ÝÔÈÐÍÎÃÎ ÌÀÑËÀ ÎÐÅÃÀÍÎ Å.Ñ. Àëèíêèíà*, À.Ê. Âîðîáüåâà*, Ò.À. Ìèøàðèíà*, Ë.Ä. Ôàòêóëëèíà*, Å.Á. Áóðëàêîâà*, À.Í. Õîõëîâ (ñåêòîð ýâîëþöèîííîé öèòîãåðîíòîëîãèè; e-mail: [email protected]) Äëÿ âûÿñíåíèÿ âîçìîæíûõ öèòîëîãè÷åñêèõ ìåõàíèçìîâ, ëåæàùèõ â îñíîâå áëàãîòâîðíîãî âëèÿíèÿ êàðâàêðîëñîäåðæàùèõ ýôèðíûõ ìàñåë íà çäîðîâüå è óìñòâåííûå ñïîñîáíîñòè, èññëåäîâàëè îäíî èç íèõ (ìàñëî îðåãàíî) â ýêñïåðèìåíòàõ íà êóëüòèâèðóåìûõ òðàíñôîðìèðîâàííûõ êëåòêàõ êèòàéñêîãî õîìÿ÷êà. Ïðåäâàðèòåëüíî îöåíèëè âîçìîæíûå öèòîòîêñè÷åñêèå èëè ìèòîãåííûå ýôôåêòû ïðåïàðàòà â ðàçëè÷íûõ êîíöåíòðàöèÿõ, àíàëèçèðóÿ íàñûùàþùóþ ïëîòíîñòü êóëüòóðû ïîñëå 4 ñóò êóëüòèâèðîâàíèÿ. Êîíöåíòðàöèÿ ïðåïàðàòà â ðîñòîâîé ñðåäå (â ïåðåñ÷åòå íà êàðâàêðîë) âàðüèðîâàëà îò 1 · 10–15 äî 5 · 10–4 M. Ïî ðåçóëüòàòàì òåñòèðîâàíèÿ äëÿ äàëüíåéøèõ ýêñïåðèìåíòîâ âûáðàëè äâå êîíöåíòðàöèè: 2,5 · 10–5 M êàê ìàêñèìàëüíóþ àáñîëþòíî íå òîêñè÷íóþ è 2,5 · 10–4 M êàê êîíöåíòðàöèþ, â êîòîðîé ìàñëî îðåãàíî ñíèæàëî ïëîòíîñòü âûðîñøåé êóëüòóðû êëåòîê ïðèáëèçèòåëüíî â 2 ðàçà. Îêàçàëîñü, ÷òî ïðåïàðàò â êîíöåíòðàöèè 2,5 · 10–5 M íèêàê íå âëèÿåò íè íà ñïîñîáíîñòü êëåòîê ê îáðàçîâàíèþ êîëîíèé, íè íà íàñûùàþùóþ ïëîòíîñòü êóëüòóðû (ÿâëÿþùóþñÿ ìàðêåðîì åå “áèîëîãè÷åñêîãî âîçðàñòà”), íè íà êèíåòèêó åå “ñòàöèîíàðíîãî ñòàðåíèÿ” (ñõîäíàÿ ñ âîçðàñòíûìè èçìåíåíèÿìè êëåòîê ñòàðåþùåãî îðãàíèçìà äåãðàäàöèÿ êóëüòèâèðóåìûõ êëåòîê â ñòàöèîíàðíîé ôàçå ðîñòà). Íàïðîòèâ, ìàñëî îðåãàíî â êîíöåíòðàöèè 2,5 · 10–4 M çíà÷èòåëüíî ïîäàâëÿëî êîëîíèåîáðàçîâàíèå êëåòîê è âëèÿëî êàê ãåðîïðîìîòîð íà íàñûùàþùóþ ïëîòíîñòü êëåòî÷íîé êóëüòóðû è êèíåòèêó ãèáåëè êëåòîê â ðåçóëüòàòå “ñòàöèîíàðíîãî ñòàðåíèÿ”. Îñíîâûâàÿñü íà ñîáñòâåííîé êîíöåïöèè ñòàðåíèÿ è ïîëó÷åííûõ äàííûõ, ìû ïðåäïîëîæèëè, ÷òî âûÿâëåííîå óâåëè÷åíèå ïðîäîëæèòåëüíîñòè æèçíè ìûøåé ïîä äåéñòâèåì ìàñëà îðåãàíî ìîæåò îïðåäåëÿòüñÿ òîëüêî íåêèìè ôóíêöèîíàëüíûìè èçìåíåíèÿìè íà îðãàíèçìåííîì óðîâíå, íî íå ñâÿçàíî ñ êàêîé-ëèáî ãåðîïðîòåêòîðíîé àêòèâíîñòüþ ïðåïàðàòà, ïðîÿâëÿþùåéñÿ íà êëåòî÷íîì óðîâíå è óâåëè÷èâàþùåé æèçíåñïîñîáíîñòü êëåòîê. Êëþ÷åâûå ñëîâà: ìàñëî îðåãàíî, öèòîãåðîíòîëîãèÿ, êëåòî÷íûå êóëüòóðû, “ñòàöèîíàðíîå ñòàðåíèå”, ýôôåêòèâíîñòü êîëîíèåîáðàçîâàíèÿ. Ýôèðíûå ìàñëà (ÝÌ) ÿâëÿþòñÿ ñìåñüþ ëåòó÷èõ âåùåñòâ, âûäåëÿåìûõ èç ïðÿíî-àðîìàòè÷åñêèõ ðàñòåíèé ñ ïîìîùüþ äèñòèëëÿöèè, ýêñòðàêöèè èëè ïðåññîâàíèÿ. Íà ïðîòÿæåíèè ñòîëåòèé ÝÌ èñïîëüçîâàëèñü äëÿ àðîìàòèçàöèè ïèùåâûõ ïðîäóêòîâ è íàïèòêîâ, à òàêæå â ìåäèöèíå è àðîìàòåðàïèè [1]. Îíè ìîãóò îáëàäàòü àíòèìèêðîáíûì, ïðîòèâîãðèáêîâûì, ïðîòèâîâîñïàëèòåëüíûì, îòõàðêèâàþùèì, ñïàçìîëèòè÷åñêèì, ïðîòèâîîïóõîëåâûì, ñåäàòèâíûì, ìî÷åãîííûì è ðåãåíåðèðóþùèì äåéñòâèåì [2]. ÝÌ, ñîäåðæàùèå êàðâàêðîë, ïðåäñòàâëÿþò ñîáîé áèîëîãè÷åñêè àêòèâíûå ïðåïàðàòû, øèðîêî èçâåñòíûå â ñâÿçè ñ èõ áëàãîòâîðíûì âëèÿíèåì íà çäîðîâüå è óìñòâåííûå ñïîñîáíîñòè [3, 4]. Îäíàêî íà äàííûé ìîìåíò ïðàêòè÷åñêè íåò íàó÷íûõ äàííûõ, ïîçâîëÿþùèõ ñ÷èòàòü ÝÌ ãåðîïðîòåêòîðàìè. Ðàíåå ìû ïîêàçàëè, ÷òî îäíî èç íèõ, ÝÌ ÷àáåðà, óâåëè÷èâàåò ñðåäíþþ ïðîäîëæèòåëüíîñòü æèçíè ìûøåé AKR, õàðàêòåðèçóþùèõñÿ âûñîêèì ðèñêîì ðàçâèòèÿ ñïîíòàííûõ ëåéêîçîâ [5]. Êðîìå òîãî, ìû îáíàðóæèëè, ÷òî åùå îäèí êàðâàêðîëñîäåðæàùèé ïðåïàðàò, ÝÌ îðåãàíî (ÝÌÎ), êîòîðîå ïîëó÷àþò èç Origánum vulgáre è ñîñòàâ êîòîðîãî áëèçîê ê ñîñòàâó ÝÌ ÷àáåðà, óâåëè÷èâàåò ñðåäíþþ ïðîäîëæèòåëüíîñòü æèçíè äîëãîæèâóùèõ ìûøåé BALB/c. Äëÿ âûÿñíåíèÿ âîçìîæíûõ öèòîëîãè÷åñêèõ ìåõàíèçìîâ, ëåæàùèõ â îñíîâå òàêîãî äåéñòâèÿ, â íàñòîÿùåé ðàáîòå ìû èññëåäîâàëè ÝÌÎ â ýêñïåðèìåíòàõ íà êóëüòèâèðóåìûõ òðàíñôîðìèðîâàííûõ êëåòêàõ êèòàéñêîãî õîìÿ÷êà, èñïîëüçóÿ íåñêîëüêî ìîäåëüíûõ ñèñòåì, ïðèìåíÿåìûõ â öèòîãåðîíòîëîãè÷åñêèõ èññëåäîâàíèÿõ. Ìàòåðèàëû è ìåòîäû Ñîñòàâ ÝÌÎ (Lionel Hitchen Ltd., Âåëèêîáðèòàíèÿ) îïðåäåëÿëè ìåòîäîì ãàçîæèäêîñòíîé õðîìàòî- * Èíñòèòóò áèîõèìè÷åñêîé ôèçèêè èì. Í.Ì. Ýìàíóýëÿ ÐÀÍ, ã. Ìîñêâà. 14 ãðàôèè íà õðîìàòîãðàôå “Êðèñòàëë 2000 Ì” (Ðîññèÿ) ñ ïëàìåííî-èîíèçàöèîííûì äåòåêòîðîì è êâàðöåâîé êàïèëëÿðíîé êîëîíêîé DB-1 (50 ì ½ 0,32 ìì, ñëîé ôàçû 0,25 ìêM, ôèðìà Supelco, ÑØÀ). Àíàëèç ïðîâîäèëè ïðè ïðîãðàììèðîâàíèè òåìïåðàòóðû êîëîíêè îò 60 äî 250°Ñ ñî ñêîðîñòüþ 4°Ñ/ìèí ïðè òåìïåðàòóðå èíæåêòîðà è äåòåêòîðà 250°Ñ. Ñêîðîñòü ïðîõîæäåíèÿ ãàçà — íîñèòåëÿ ãåëèÿ ÷åðåç êîëîíêó ñîñòàâëÿëà 1,5 ìë/ìèí.  ðàáîòå èñïîëüçîâàëè òðàíñôîðìèðîâàííûå êëåòêè êèòàéñêîãî õîìÿ÷êà ëèíèè B11-dii FAF28 (êëîí 237), ïîëó÷åííûå èç Ìåäèêî-ãåíåòè÷åñêîãî íàó÷íîãî öåíòðà ÐÀÌÍ (Ìîñêâà). Êëåòêè êóëüòèâèðîâàëè â ñòåêëÿííûõ ôëàêîíàõ Êàððåëÿ, èñïîëüçóÿ ñðåäó ÄÌÑÈ (Èíñòèòóò âèðóñîëîãèè èì. Ä.È. Èâàíîâñêîãî ÐÀÌÍ, Ìîñêâà) ñ äîáàâëåíèåì 10% ñûâîðîòêè êðîâè êðóïíîãî ðîãàòîãî ñêîòà (“Áèîëîò”, Ñàíêò-Ïåòåðáóðã), ïåíèöèëëèíà (100 åä/ìë) è ñòðåïòîìèöèíà (100 ìêã/ìë). Ïîääåðæèâàÿ êóëüòóðó, êëåòêè ïåðåñåâàëè â ñîîòíîøåíèè 1 : 10—1 : 3 ÷åðåç 3—4 ñóò. Äëÿ ñíÿòèÿ êëåòîê ñ ôëàêîíîâ èñïîëüçîâàëè ñìåñü (1 : 1) 0,02%-ãî âåðñåíà è 0,25%-ãî òðèïñèíà (Èíñòèòóò âèðóñîëîãèè èì. Ä.È. Èâàíîâñêîãî ÐÀÌÍ, Ìîñêâà). ÝÌÎ ïåðåä ïðîâåäåíèåì öèòîëîãè÷åñêèõ ýêñïåðèìåíòîâ ðàñòâîðÿëè â 96%-ì ýòàíîëå, êîòîðûé ïðåäâàðèòåëüíî ñòåðèëèçîâàëè ôèëüòðîâàíèåì ÷åðåç ìåìáðàííûé ôèëüòð ñ äèàìåòðîì ïîð 0,2 ìêM (Acrodisc® Syringe Filter with Supor® Membrane, Pall Corp., ÑØÀ). Ñòåðèëüíîñòü íåðàçâåäåííîãî ÝÌÎ, îáëàäàþùåãî, êàê èçâåñòíî, âûðàæåííûì áàêòåðèöèäíûì äåéñòâèåì [3], ïîäòâåðäèëè ñ ïîìîùüþ ïîñåâà íà ìÿñîïåïòîííûé àãàð.  ïðåäâàðèòåëüíûõ èññëåäîâàíèÿõ, íàïðàâëåííûõ íà îïðåäåëåíèå öèòîòîêñè÷åñêèõ èëè ìèòîãåííûõ ñâîéñòâ ÝÌÎ, êëåòêè 3—4-äíåâíîãî “âîçðàñòà” (ò.å. âûðàùèâàåìûå áåç ïåðåñåâà â òå÷åíèå 3—4 ñóò) çàñåâàëè â ãåðìåòè÷íî çàêðûâàþùèåñÿ ïåíèöèëëèíîâûå ôëàêîíû ïëîùàäüþ 4,9 ñì2 ñ ïëîòíîñòüþ îêîëî 100 òûñ. êë./ñì2 (ñóñïåíçèÿ â 2 ìë ðîñòîâîé ñðåäû, ñîñòîÿùåé èç 90% ÄÌÑÈ è 10% ñûâîðîòêè êðóïíîãî ðîãàòîãî ñêîòà).  ÷àñòü ôëàêîíîâ äîáàâëÿëè ñïèðòîâîé ðàñòâîð ÝÌÎ äî êîíå÷íîé êîíöåíòðàöèè â ñðåäå (â ïåðåñ÷åòå íà êàðâàêðîë) îò 1 · 10–15 äî 5 · 10–4 M. Ïðè ýòîì èñõîäíûå ðàñòâîðû ÝÌÎ áûëè òàêîé êîíöåíòðàöèè, ÷òîáû îáåñïå÷èòü 1%-å ñîäåðæàíèå ýòàíîëà â ðîñòîâîé ñðåäå (èçâåñòíî, ÷òî â òàêîé êîíöåíòðàöèè ñïèðò íå âëèÿåò íà ðàçìíîæåíèå êóëüòèâèðóåìûõ êëåòîê).  êîíòðîëüíûå ôëàêîíû äîáàâëÿëè ñîîòâåòñòâóþùåå êîëè÷åñòâî ÷èñòîãî ýòàíîëà (20 ìêë íà 2 ìë ñðåäû). Ôëàêîíû ïîìåùàëè íà 4 ñóò â òåðìîñòàò (37°Ñ), ïîñëå ÷åãî êëåòêè ñíèìàëè ñ ïîâåðõíîñòè ðîñòà ñìåñüþ ðàñòâîðîâ âåðñåíà è òðèïñèíà è ïîäñ÷èòûâàëè èõ êîëè÷åñòâî ñ ïîìîùüþ êàìåð Ãîðÿåâà (2—3 ôëàêîíà íà òî÷êó, 4 êàìåðû íà ôëàêîí). Ïîñëå âûáîðà äâóõ ñîîòâåòñòâóþùèõ êîíöåíòðàöèé (ñì. “Ðåçóëüòàòû è îáñóæäåíèå”) îöåíèâàëè âëè- ÂÅÑÒÍ. ÌÎÑÊ. ÓÍ-ÒÀ. ÑÅÐ. 16. ÁÈÎËÎÃÈß. 2012. ¹ 2 ÿíèå ÝÌÎ íà êèíåòèêó ðîñòà êëåòîê è èõ ïîñëåäóþùóþ ãèáåëü â ñòàöèîíàðíîé ôàçå ðîñòà. Äëÿ ýòîãî êëåòêè 3-äíåâíîãî “âîçðàñòà” ñíÿëè ñ ïîâåðõíîñòè ôëàêîíà Êàððåëÿ ñìåñüþ ðàñòâîðîâ âåðñåíà è òðèïñèíà, ñóñïåíäèðîâàëè â ðîñòîâîé ñðåäå (90% ÄÌÑÈ è 10% ñûâîðîòêè êðóïíîãî ðîãàòîãî ñêîòà), ðàçâåëè ñðåäîé äî íåîáõîäèìîé êîíöåíòðàöèè êëåòîê è ïîñåÿëè ïî 2 ìë ñóñïåíçèè â 108 ãåðìåòè÷íî çàêðûâàþùèõñÿ ïåíèöèëëèíîâûõ ôëàêîíîâ ïëîùàäüþ 4,9 ñì2 (ïëîòíîñòü ïîñåâà — 100 òûñ. êë./ñì2). Âñå ôëàêîíû ïîìåñòèëè â òåðìîñòàò (37°Ñ). ×åðåç ñóòêè ïîñëå ïîñåâà ñ ïîìîùüþ ñìåñè ðàñòâîðîâ âåðñåíà è òðèïñèíà ñíÿëè êëåòêè ñî äíà 3 ôëàêîíîâ è ïîäñ÷èòàëè èõ êîëè÷åñòâî â êàìåðå Ãîðÿåâà, ÷òîáû îïðåäåëèòü ïëîòíîñòü ïðèêðåïèâøèõñÿ êëåòîê. Ïîñëå ýòîãî â 35 ôëàêîíîâ ââåëè ïî 20 ìêë ñïèðòîâîãî ðàñòâîðà ÝÌÎ äî êîíöåíòðàöèè 25 ìêM, â 35 ôëàêîíîâ — ïî 20 ìêë ñïèðòîâîãî ðàñòâîðà ÝÌÎ äî êîíöåíòðàöèè 250 ìêM è â 35 ôëàêîíîâ — ïî 20 ìêë ÷èñòîãî ñïèðòà (êîíòðîëü). Çàòåì ôëàêîíû âåðíóëè â òåðìîñòàò. Ïîñëå ýòîãî ÷åðåç îïðåäåëåííûå ïðîìåæóòêè âðåìåíè èçâëåêàëè èç òåðìîñòàòà 9 ôëàêîíîâ (ïî òðè èç êàæäîé ãðóïïû), êëåòêè ñíèìàëè ñ ïîâåðõíîñòè ðîñòà ñìåñüþ âåðñåíà è òðèïñèíà, ñóñïåíäèðîâàëè â ðîñòîâîé ñðåäå áåç ñûâîðîòêè è îöåíèâàëè êîëè÷åñòâî êëåòîê â êàæäîì ôëàêîíå ñ ïîìîùüþ 4 êàìåð Ãîðÿåâà. Íàêîíåö, èññëåäóÿ âëèÿíèå ÝÌÎ íà ýôôåêòèâíîñòü êîëîíèåîáðàçîâàíèÿ, êëåòêè 3-äíåâíîãî “âîçðàñòà” ñíÿëè ñ ïîâåðõíîñòè ôëàêîíà Êàððåëÿ ñìåñüþ ðàñòâîðîâ âåðñåíà è òðèïñèíà, ñóñïåíäèðîâàëè è ðàçâåëè ïîëó÷åííóþ ñóñïåíçèþ ñíà÷àëà â îáû÷íîé ðîñòîâîé ñðåäå ñ ñûâîðîòêîé, à çàòåì (ïîñëåäíåå ðàçâåäåíèå) â ñðåäå, ñîñòîÿâøåé èç 90% ÄÌÑÈ, 5% ñûâîðîòêè êðóïíîãî ðîãàòîãî ñêîòà è 5% ýìáðèîíàëüíîé òåëÿ÷üåé ñûâîðîòêè (FetalClone® III, HyClone, ÑØÀ), äî íåîáõîäèìîé êîíöåíòðàöèè êëåòîê. Ïîñëå ýòîãî ïîñåÿëè ïî 2,5 ìë ñóñïåíçèè â 12 ïëàñòèêîâûõ ÷àøåê Ïåòðè (Nunclon™, Nunc, Äàíèÿ) äèàìåòðîì 35 ìì (250 êëåòîê íà ÷àøêó). ×àøêè ïîìåñòèëè â òåðìîñòàò (37°Ñ, 5% CO2). ×åðåç ñóòêè â 4 ÷àøêè ââåëè ïî 25 ìêë ñïèðòîâîãî ðàñòâîðà ÝÌÎ äî êîíöåíòðàöèè 25 ìêM, â 4 ÷àøêè — ïî 25 ìêë ñïèðòîâîãî ðàñòâîðà ÝÌÎ äî êîíöåíòðàöèè 250 ìêM è â 4 ÷àøêè — ïî 25 ìêë ÷èñòîãî ñïèðòà (êîíòðîëü). Çàòåì ÷àøêè âåðíóëè â òåðìîñòàò åùå íà 6 ñóò, ïîñëå ÷åãî èõ èçâëåêëè è çàôèêñèðîâàëè êëåòî÷íûå êîëîíèè 75%-ì ñïèðòîì (7 ìèí), îêðàñèëè èõ 0,1%-ì âîäíûì ðàñòâîðîì ìåòèëåíîâîãî ñèíåãî (3 ìèí) è ïîäñóøèëè íà âîçäóõå. Ïîñëå ýòîãî ñ ïîìîùüþ ìèêðîñêîïà ïîäñ÷èòàëè êîëè÷åñòâî êëåòîê â êîëîíèÿõ, îáðàçîâàâøèõñÿ íà êàæäîé ÷àøêå. Ðàñïðåäåëåíèå êîëîíèé ïî ðàçìåðàì ñòðîèëè, ðàçáèâàÿ ðåçóëüòàòû ïî 17 êëàññàì, îïðåäåëÿåìûì êîëè÷åñòâîì êëåòîê â êîëîíèè: 1—15, 16—31, 32—47...240—255, 256 è áîëåå. Òàêèì îáðàçîì, ðàç- ÂÅÑÒÍ. ÌÎÑÊ. ÓÍ-ÒÀ. ÑÅÐ. 16. ÁÈÎËÎÃÈß. 2012. ¹ 2 15 ìåðû âñåõ êëàññîâ, êðîìå ïåðâîãî è ïîñëåäíåãî, áûëè îäèíàêîâûìè è ñîñòàâëÿëè 16 êëåòîê. Ñðåäíåâçâåøåííûé íîìåð êëàññà (ÑÂÍÊ) äëÿ êàæäîãî ðàñïðåäåëåíèÿ ðàññ÷èòûâàëè ïî ôîðìóëå: n ÑÂÍÊ = æC i ç å è i =1 M ö × i÷ , ø ãäå i — íîìåð êëàññà, n — êîëè÷åñòâî êëàññîâ, Ci — êîëè÷åñòâî êîëîíèé â êëàññå “i”, M — îáùåå êîëè÷åñòâî êîëîíèé. ×åì ìåíüøå ÑÂÍÊ, òåì ìåíüøå â ïîïóëÿöèè äîëÿ áîëüøèõ êîëîíèé. Èñïîëüçîâàíèå ýòîãî ïîêàçàòåëÿ ïîçâîëÿåò îöåíèòü òîíêèå èçìåíåíèÿ ïðîëèôåðàòèâíîé àêòèâíîñòè êëåòîê äàæå ïðè íåèçìåííîé ýôôåêòèâíîñòè êîëîíèåîáðàçîâàíèÿ (ÝÊÎ). ÝÊÎ ðàññ÷èòûâàëè ïî ôîðìóëå: ÝÊÎ = K × 100%, N ãäå N — êîëè÷åñòâî ïîñåÿííûõ êëåòîê, à K — êîëè÷åñòâî âûðîñøèõ êîëîíèé. Ìàòåìàòè÷åñêèå ðàñ÷åòû è ñòàòèñòè÷åñêóþ îáðàáîòêó ðåçóëüòàòîâ ïðîèçâîäèëè ñ ïîìîùüþ ïðîãðàìì Microsoft Excel 2007 è SigmaPlot 11. Ðåçóëüòàòû è îáñóæäåíèå  òàáëèöå ïðåäñòàâëåíû ðåçóëüòàòû ãàçîõðîìàòîãðàôè÷åñêîãî àíàëèçà èñïîëüçîâàííîãî â ðàáîòå ÝÌÎ. Ñ ó÷åòîì ýòèõ äàííûõ âî âñåõ ïðîâîäèìûõ ýêñïåðèìåíòàõ êîíöåíòðàöèÿ ïðåïàðàòà â êóëüòóðàëüíîé ñðåäå ðàññ÷èòûâàëàñü â ïåðåñ÷åòå íà êàðâàêðîë, êîòîðûé íàðÿäó ñ òèìîëîì, ñóäÿ ïî âñåìó, è îïðåäåëÿåò â îñíîâíîì áèîëîãè÷åñêóþ àêòèâíîñòü ÝÌÎ [3, 6]. Ñîñòàâ ýôèðíîãî ìàñëà îðåãàíî, îïðåäåëåííûé ñ ïîìîùüþ ãàçîæèäêîñòíîé õðîìàòîãðàôèè Ñîåäèíåíèå Ñîäåðæàíèå, % Êàðâàêðîë 65,89 ï-Öèìåí 12,52 g-Òåðïèíåí 9,22 Òèìîë 4,51 a-Ïèíåí 1,67 Ëèíàëîîë 1,53 b-Êàðèîôèëëåí 1,52 b-Ïèíåí 1,43 Êàìôåí 0,42 Ñàáèíåí 0,56 a-Òóéåí 0,38 Ëèìîíåí 0,23 b-Ìèðöåí 0,12 Ðèñ. 1. Âëèÿíèå ýôèðíîãî ìàñëà îðåãàíî â êîíöåíòðàöèè 25 è 250 ìêM (â ïåðåñ÷åòå íà êàðâàêðîë) íà ïëîòíîñòü êóëüòóðû êëåòîê êèòàéñêîãî õîìÿ÷êà íà 7-å ñóò ðîñòà (ïîñåâ ñ ïëîòíîñòüþ 100 òûñ. êë./ñì2). Ïðèâåäåíû ñòàíäàðòíûå îøèáêè ñðåäíåãî  ïðåäâàðèòåëüíûõ ýêñïåðèìåíòàõ ìû îöåíèëè âîçìîæíûå öèòîòîêñè÷åñêèå èëè ìèòîãåííûå ýôôåêòû ïðåïàðàòà â ðàçëè÷íûõ êîíöåíòðàöèÿõ, àíàëèçèðóÿ íàñûùàþùóþ ïëîòíîñòü êóëüòóðû íà 4-å ñóò êóëüòèâèðîâàíèÿ. Êîíöåíòðàöèÿ ÝÌÎ â ðîñòîâîé ñðåäå (â ïåðåñ÷åòå íà êàðâàêðîë) âàðüèðîâàëà îò 1 · 10–15 äî 5 · 10–4 M. Ïî ðåçóëüòàòàì òåñòèðîâàíèÿ äëÿ äàëüíåéøèõ ýêñïåðèìåíòîâ ìû âûáðàëè äâå êîíöåíòðàöèè: 2,5 · 10–5 M êàê ìàêñèìàëüíóþ àáñîëþòíî íåòîêñè÷íóþ è 2,5 · 10–4 M êàê êîíöåíòðàöèþ, â êîòîðîé ÝÌÎ ñíèæàëî ïëîòíîñòü êóëüòóðû êëåòîê 4-äíåâíîãî “âîçðàñòà” ïðèáëèçèòåëüíî â 2 ðàçà (ðèñ. 1). Íàø âûáîð ïåðâîé êîíöåíòðàöèè îïðåäåëèëî òàêæå òî, ÷òî, êàê áûëî ïîêàçàíî â ðàáîòå ñëîâàöêèõ èññëåäîâàòåëåé [7], èìåííî ýòà êîíöåíòðàöèÿ êàðâàêðîëà ÿâëÿåòñÿ ìèíèìàëüíî íåîáõîäèìîé äëÿ âûÿâëåíèÿ åãî çàùèòíîãî ýôôåêòà ïðè äåéñòâèè íà êóëüòèâèðóåìûå êëåòêè HepG2 (ãåïàòîöåëëþëÿðíàÿ êàðöèíîìà ÷åëîâåêà) ïåðîêñèäîì âîäîðîäà â êîíöåíòðàöèè 2,5 · 10–4 M. Íà ðèñ. 2 ïðèâåäåíû äàííûå î âëèÿíèè ÝÌÎ â êîíöåíòðàöèè 25 è 250 ìêM (â ïåðåñ÷åòå íà êàðâàêðîë) íà êèíåòèêó ðîñòà è íàñûùàþùóþ ïëîòíîñòü êóëüòóðû êëåòîê êèòàéñêîãî õîìÿ÷êà. Êðèâûå àïïðîêñèìèðîâàíû 4-ïàðàìåòðè÷åñêèì óðàâíåíèåì Ãîìïåðòöà. Âèäíî, ÷òî ÝÌÎ â ìåíüøåé êîíöåíòðàöèè íèêàê íå âëèÿåò íà õîä êðèâîé ðîñòà, à â áîëüøåé êîíöåíòðàöèè çíà÷èòåëüíî ñíèæàåò íàñûùàþùóþ ïëîòíîñòü êëåòî÷íîé êóëüòóðû. Îñíîâûâàÿñü íà ïîëîæåíèÿõ êëåòî÷íî-êèíåòè÷åñêîé ìîäåëè äëÿ èñïûòàíèÿ ãåðîïðîòåêòîðîâ è ãåðîïðîìîòîðîâ [8, 9], ìû çàêëþ÷èëè, ÷òî ïðåïàðàò â êîíöåíòðàöèè 250 ìêM âûçûâàåò óâåëè÷åíèå “áèîëîãè÷åñêîãî âîçðàñòà” èññëåäîâàííûõ êëåòîê, ò.å. äåéñòâóåò êàê ãåðîïðîìîòîð, à â êîíöåíòðàöèè 25 ìêM íèêàê íå ïðîÿâëÿåò ñåáÿ â äàííîé òåñò-ñèñòåìå. 16 Ðèñ. 2. Âëèÿíèå ýôèðíîãî ìàñëà îðåãàíî â êîíöåíòðàöèè 25 è 250 ìêM (â ïåðåñ÷åòå íà êàðâàêðîë) íà êèíåòèêó ðîñòà è íàñûùàþùóþ ïëîòíîñòü êóëüòóðû êëåòîê êèòàéñêîãî õîìÿ÷êà (£ — êîíòðîëü, — 25 ìêM, s — 250 ìêM). Ïðèâåäåíû êðèâûå, ïîëó÷åííûå ñ ïîìîùüþ àïïðîêñèìàöèè 4-ïàðàìåòðè÷åñêèì óðàâíåíèåì Ãîìïåðòöà (–––– êîíòðîëü, –– –– 25 ìêM, –– · –– 250 ìêM) ÂÅÑÒÍ. ÌÎÑÊ. ÓÍ-ÒÀ. ÑÅÐ. 16. ÁÈÎËÎÃÈß. 2012. ¹ 2 Ðèñ. 4. Âëèÿíèå ýôèðíîãî ìàñëà îðåãàíî â êîíöåíòðàöèè 25 è 250 ìêM (â ïåðåñ÷åòå íà êàðâàêðîë) íà ýôôåêòèâíîñòü îáðàçîâàíèÿ êîëîíèé êóëüòèâèðóåìûìè êëåòêàìè êèòàéñêîãî õîìÿ÷êà (ó÷èòûâàëèñü òîëüêî êîëîíèè, ñîñòîÿùèå èç 16 è áîëåå êëåòîê). Ïðèâåäåíû ñòàíäàðòíûå îøèáêè ñðåäíåãî. * — ðàçëè÷èÿ äîñòîâåðíû (p < 0,05) Íà ðèñ. 3 ïðèâåäåíû ðåçóëüòàòû èññëåäîâàíèÿ âëèÿíèÿ ÝÌÎ íà êèíåòèêó ãèáåëè êëåòîê â ìîäåëè “ñòàöèîíàðíîãî ñòàðåíèÿ” [10—14], ïðåäïîëàãàþùåé ñõîäñòâî èçìåíåíèé, êîòîðûå ïðåòåðïåâàþò êóëüòèâèðóåìûå êëåòêè ïðè îãðàíè÷åíèè èõ ïðîëèôåðàöèè âñëåäñòâèå êîíòàêòíîãî òîðìîæåíèÿ, ñ èçìåíåíèÿìè êëåòîê ñòàðåþùåãî îðãàíèçìà. Ýòè äàííûå òàêæå àïïðîêñèìèðîâàíû 4-ïàðàìåòðè÷åñêèì óðàâíåíèåì Ãîìïåðòöà. Î÷åâèäíî, ÷òî ÝÌÎ â ìåíüøåé êîíöåíòðàöèè îïÿòü-òàêè íå âëèÿåò íà õîä ýêñïåðèìåíòàëüíîé êðèâîé, â òî âðåìÿ êàê â áîëüøåé êîíöåíòðàöèè îùóòèìî óñêîðÿåò “ñòàöèîíàðíîå ñòàðåíèå” êëåòîê. Ñóäÿ ïî âñåìó, è â ðàìêàõ ýòîé ìîäåëüíîé ñèñòåìû ÝÌÎ â êîíöåíòðàöèè 250 ìêM âåäåò ñåáÿ êàê ãåðîïðîìîòîð, à â êîíöåíòðàöèè 25 ìêM íå âëèÿåò íà æèçíåñïîñîáíîñòü êëåòîê. Èç äàííûõ, ïðåäñòàâëåííûõ íà ðèñ. 4 è 5, âèäíî, ÷òî æèçíåñïîñîáíîñòü èçó÷åííûõ êëåòîê, îïðåäåëÿåìàÿ ïî èõ ñïîñîáíîñòè ê êîëîíèåîáðàçîâàíèþ (ñ ïîìîùüþ îöåíêè êàê ÝÊÎ, òàê è ÑÂÍÊ), òàêæå óìåíüøàåòñÿ òîëüêî ïîä äåéñòâèåì ÝÌÎ â êîíöåíòðàöèè 250 ìêM è íèêàê íå ìåíÿåòñÿ ïðè êîíöåíòðàöèè 25 ìêM. Äîïîëíèòåëüíîé èëëþñòðàöèåé èíãèáèðóþùåãî äåéñòâèÿ ÝÌÎ â êîíöåíòðàöèè 250 ìêM íà ïðîëèôåðàòèâíóþ àêòèâíîñòü êëåòîê êèòàéñêîãî õîìÿ÷êà ìîãóò ñëóæèòü ôîòîãðàôèè, ïðèâåäåííûå íà ðèñ. 6. Ïðåäñòàâëåíû ôîòîãðàôèè íàèáîëåå òè- Ðèñ. 3. Âëèÿíèå ýôèðíîãî ìàñëà îðåãàíî â êîíöåíòðàöèè 25 è 250 ìêM (â ïåðåñ÷åòå íà êàðâàêðîë) íà êèíåòèêó ãèáåëè ñòàöèîíàðíîé êóëüòóðû êëåòîê êèòàéñêîãî õîìÿ÷êà (£ — êîíòðîëü, — 25 ìêM, s — 250 ìêM). Ïðèâåäåíû êðèâûå, ïîëó÷åííûå ñ ïîìîùüþ àïïðîêñèìàöèè 4-ïàðàìåòðè÷åñêèì óðàâíåíèåì Ãîìïåðòöà (–––– êîíòðîëü, –– –– 25 ìêM, –– · –– 250 ìêM) Ðèñ. 5. Âëèÿíèå ýôèðíîãî ìàñëà îðåãàíî â êîíöåíòðàöèè 25 è 250 ìêM (â ïåðåñ÷åòå íà êàðâàêðîë) íà ñðåäíåâçâåøåííûé íîìåð êëàññà ðàñïðåäåëåíèÿ ïî ðàçìåðàì êîëîíèé, îáðàçîâàííûõ êóëüòèâèðóåìûìè êëåòêàìè êèòàéñêîãî õîìÿ÷êà (ïîÿñíåíèÿ — â òåêñòå). Ïðèâåäåíû ñòàíäàðòíûå îøèáêè ñðåäíåãî. * — ðàçëè÷èÿ äîñòîâåðíû (p < 0,05) ÂÅÑÒÍ. ÌÎÑÊ. ÓÍ-ÒÀ. ÑÅÐ. 16. ÁÈÎËÎÃÈß. 2012. ¹ 2 17 è ïîëó÷åííûõ äàííûõ, ìû ïðåäïîëîæèëè, ÷òî âûÿâëåííîå óâåëè÷åíèå ïðîäîëæèòåëüíîñòè æèçíè ìûøåé ïîä äåéñòâèåì ÝÌÎ ìîæåò îïðåäåëÿòüñÿ òîëüêî íåêèìè ôóíêöèîíàëüíûìè èçìåíåíèÿìè íà îðãàíèçìåííîì óðîâíå, íî íå ñâÿçàíî ñ êàêîé-ëèáî ãåðîïðîòåêòîðíîé àêòèâíîñòüþ ïðåïàðàòà, ïðîÿâëÿþùåéñÿ íà êëåòî÷íîì óðîâíå è âûçûâàþùåé ïîâûøåíèå æèçíåñïîñîáíîñòè êëåòîê. Âîçìîæíî, ãåðîïðîòåêòîðíîå äåéñòâèå ÝÌÎ è äðóãèõ êàðâàêðîëñîäåðæàùèõ ïðåïàðàòîâ in vivo ñâÿçàíî ñ èõ áàêòåðèöèäíûì èëè ïðîòèâîîïóõîëåâûì äåéñòâèåì [2, 3, 6]. Êðîìå òîãî, íå èñêëþ÷åíî, ÷òî îíè ëèøü çàïóñêàþò íåêîòîðûå âòîðè÷íûå çàùèòíûå ìåõàíèçìû îðãàíèçìà [14, 15], Ðèñ. 6. Âëèÿíèå ýôèðíîãî ìàñëà îðåãàíî â êîíöåíòðàöèè 25 è 250 ìêM (â ïåðå- íå âëèÿÿ íåïîñðåäñòâåííî íà æèçíåñ÷åòå íà êàðâàêðîë) íà ôîðìó è ðàçìåð êîëîíèé, îáðàçîâàííûõ êóëüòèâèðóåìûìè ñïîñîáíîñòü êîíêðåòíûõ êëåòîê-ìèøåêëåòêàìè êèòàéñêîãî õîìÿ÷êà íåé. È íàêîíåö, ìîæíî òàêæå äîïóñòèòü, ÷òî ìû ïðîñòî íå ñìîãëè íàéòè ïè÷íûõ êîëîíèé. Âèäíî, ÷òî è ðàçìåð, è ôîðìà êî- íóæíóþ êîíöåíòðàöèþ ÝÌÎ, íåîáõîäèìóþ äëÿ ïðîëîíèé çíà÷èòåëüíî èçìåíÿþòñÿ ëèøü ïîä âîçäåéñò- ÿâëåíèÿ ãåðîïðîòåêòîðíîãî äåéñòâèÿ íà êëåòî÷íîì óðîâíå. Âïîëíå âîçìîæíî, ÷òî ïðèìåíåíèå ÝÌÎ âèåì ÝÌÎ â áîëüøåé êîíöåíòðàöèè. â ñâåðõìàëûõ äîçàõ ïîçâîëèëî áû îáíàðóæèòü òàêîå Çàêëþ÷åíèå äåéñòâèå [16, 17]. Íà âûÿñíåíèå ýòîãî è áóäóò íàïðàâëåíû íàøè äàëüíåéøèå èññëåäîâàíèÿ. Òàêèì îáðàçîì, îñíîâûâàÿñü íà ñôîðìóëèðîâàííîé îäíèì èç íàñ êîíöåïöèè ñòàðåíèÿ [10—14] ÑÏÈÑÎÊ ËÈÒÅÐÀÒÓÐÛ 1. Bauer K., Garbe D., Surburg H. Common fragrance and flavor materials. Preparation, properties and uses. Weinheim: Wiley-VCH Verlag GmbH, 2001. 282 p. 2. Koroch A.R., Juliani H.R., Zygadlo J.A. Bioactivity of essential oils and their components // Flavours and fragrances. Chemistry, bioprocessing and sustainability / Ed. R.G. Berger. Berlin; Heidelberg: Springer-Verlag, 2007. P. 87115. 3. Baser K.H.C. Biological and pharmacological activities of carvacrol and carvacrol bearing essential oils // Curr. Pharm. Des. 2008. Vol. 14. N 29. P. 31063119. 4. El Babili F., Bouajila J., Souchard J.P., Bertrand C., Bellvert F., Fouraste I., Moulis C., Valentin A. Oregano: chemical analysis and evaluation of its antimalarial, antioxidant, and cytotoxic activities // J. Food Sci. 2011. Vol. 76. N 3. P. C512C518. 5. Áóðëàêîâà Å.Á., Åðîõèí Â.Í., Ìèøàðèíà Ò.À., Ôàòêóëëèíà Ë.Ä., Êðåìåíöîâà À.Â., Ñåìåíîâ Â.À., Òåðåíèíà Ì.Á., Âîðîáüåâà À.Ê., Ãîëîùàïîâ À.Í. Âëèÿíèå ëåòó÷èõ àíòèîêñèäàíòîâ ðàñòèòåëüíîãî ïðîèñõîæäåíèÿ íà ðàçâèòèå ñïîíòàííîãî ëåéêîçà ó ìûøåé // Èçâ. ÐÀÍ. Ñåð. áèîë. 2010. ¹ 6. Ñ. 18. 6. Edris A.E. Pharmaceutical and therapeutic potentials of essential oils and their individual volatile constituents: a review // Phytother. Res. 2007. Vol. 21. N 4. P. 308323. 7. Slameòová D., Horváthová E., Šramková M., Maršálková L. DNA-protective effects of two components of essential plant oils carvacrol and thymol on mammalian cells cultured in vitro // Neoplasma. 2007. Vol. 54. N 2. P. 108112. 8. ×èðêîâà Å.Þ., Ãîëîâèíà Ì.Ý., Íàäæàðÿí Ò.Ë., Õîõëîâ À.Í. Êëåòî÷íî-êèíåòè÷åñêàÿ ìîäåëü äëÿ èçó÷åíèÿ ãåðîïðîòåêòîðîâ è ãåðîïðîìîòîðîâ // Äîêë. ÀÍ ÑÑÑÐ. 1984. Ò. 278. ¹ 6. Ñ. 14741476. 9. Khokhlov A.N. The cell kinetics model for determination of organism biological age and for geroprotectors or geropromoters studies // Biomarkers of aging: expression and regulation. Proceeding / Ed. F. Licastro, C.M. Caldarera. Bologna: CLUEB, 1992. P. 209216. 10. Õîõëîâ À.Í. Ïðîëèôåðàöèÿ è ñòàðåíèå // Èòîãè íàóêè è òåõíèêè ÂÈÍÈÒÈ ÀÍ ÑÑÑÐ. Ñåð. Îáùèå ïðîáëåìû ôèçèêî-õèìè÷åñêîé áèîëîãèè. Ò. 9. Ì.: ÂÈÍÈÒÈ, 1988. 176 ñ. 11. Khokhlov A.N. Stationary cell cultures as a tool for gerontological studies // Ann. N.Y. Acad. Sci. 1992. Vol. 663. P. 475476. 12. Õîõëîâ À.Í. Öèòîãåðîíòîëîãèÿ â íà÷àëå òðåòüåãî òûñÿ÷åëåòèÿ: îò êîððåëÿòèâíûõ ê ñóùíîñòíûì ìîäåëÿì // Îíòîãåíåç. 2003. Ò. 34. ¹ 5. Ñ. 382389. 13. Khokhlov A.N. In search of gist cytogerontological models // Longevity, aging and degradation models in reliability, public health, medicine and biology. Vol. 1 / Ed. V. Antonov, C. Huber, M. Nikulin, V. Polischook. St. Petersburg: SPbSPU, 2004. P. 8492. 14. Khokhlov A.N. From Carrel to Hayflick and back, or what we got from the 100-year cytogerontological studies // Biophysics. 2010. Vol. 55. N 5. P. 859864. 18 ÂÅÑÒÍ. ÌÎÑÊ. ÓÍ-ÒÀ. ÑÅÐ. 16. ÁÈÎËÎÃÈß. 2012. ¹ 2 15. Khokhlov A.N. Does aging need an own program or the existing development program is more than enough? // Russ. J. Gen. Chem. 2010. Vol. 80. N 7. P. 1507—1513. 16. Áóðëàêîâà Å.Á., Êîíðàäîâ À.À., Ìàëüöåâà Å.Ë. Äåéñòâèå ñâåðõìàëûõ äîç áèîëîãè÷åñêè àêòèâíûõ âåùåñòâ è íèçêîèíòåíñèâíûõ ôèçè÷åñêèõ ôàêòîðîâ // Ïðîáëåìû ðåãóëÿöèè â áèîëîãè÷åñêèõ ñèñòåìàõ. Áèîôèçè÷åñêèå àñ- ïåêòû / Ïîä îáù. ðåä. À.Á. Ðóáèíà. Ì.; Èæåâñê: ÍÈÖ ÐÕÄ, ÈÊÈ, 2007. Ñ. 390—423. 17. Áóðëàêîâà Å.Á., Êîíðàäîâ À.À., Ìàëüöåâà Å.Ë. Ñâåðõñëàáûå âîçäåéñòâèÿ õèìè÷åñêèõ ñîåäèíåíèé è ôèçè÷åñêèõ ôàêòîðîâ íà áèîëîãè÷åñêèå ñèñòåìû // Áèîôèçèêà. 2004. Ò. 49. ¹ 3. Ñ. 551—564. Ïîñòóïèëà â ðåäàêöèþ 15.11.11 CYTOGERONTOLOGICAL STUDIES OF OREGANO ESSENTIAL OIL BIOLOGICAL ACTIVITY E.S. Alinkina, A.K. Vorobyova, T.A. Misharina, L.D. Fatkullina, E.B. Burlakova, A.N. Khokhlov Carvacrol-bearing essential oils (EO) are well known biologically active preparations widely used to improve health, mental abilities, and well-being. Recently we found that one of them, the savory EO, had a positive effect on the average life-span of AKR mice with the high incidence of spontaneous leukemia. Besides, we have shown that one more carvacrol-bearing preparation, the oregano EO obtained from Origanum vulgare (its composition is very similar to the composition of the savory EO), increased the average life-span of long-living BALB/c mice. To clarify the possible cytological mechanisms underlying the effects we studied the oregano EO in experiments on transformed cultured Chinese hamster cells. In the preliminary investigations we evaluated various concentrations of the oil in terms of its cytotoxic or mitogenic effects by analyzing the cell culture density on the 4th day of growth. The concentrations in the growth medium studied (on carvacrol basis) ranged from 1 · 10–15 to 5 · 10–4 M. As a result the concentrations of 2.5 · 10–5 and 2,5 · 10–4 M were chosen for the further cytogerontological experiments because the former seemed to be the maximal 100% non-toxic concentration and the latter induced just about 50% reducing of the final cell density. The preparation at 2,5 · 10–5 M was shown to have no effect neither on colony-forming ability of the cells nor on saturation density (an index of cell culture “biological age”) or “stationary phase aging” of the culture (aging-like degradation of cells in the stationary phase of growth). In contrast, oregano EO at 2,5 · 10–4 M abruptly diminished colony-forming ability of the cells and influenced as a “pro-aging” factor on the saturation density and the death rate in the stationary phase modifying respectively the culture survival curve. Basing on our conception of aging and the data obtained we assumed that the beneficial effect of oregano EO on the mice life-span could be realized at the organism level only but not related to any anti-aging activity manifesting at the cellular level and improving cell viability. Key words: oregano essential oil, cytogerontology, cell culture, “stationary phase aging”, colonyforming ability. Ñâåäåíèÿ îá àâòîðàõ Àëèíêèíà Åêàòåðèíà Ñåðãååâíà — àñïèðàíòêà ëàáîðàòîðèè ôèçèêî-õèìè÷åñêèõ îñíîâ ðåãóëÿöèè áèîëîãè÷åñêèõ ñèñòåì Èíñòèòóòà áèîõèìè÷åñêîé ôèçèêè èì. Í.Ì. Ýìàíóýëÿ ÐÀÍ. Òåë.: 8-495-939-71-81; e-mail: [email protected] Âîðîáüåâà Àíàñòàñèÿ Êîíñòàíòèíîâíà — àñïèðàíòêà ëàáîðàòîðèè ôèçèêî-õèìè÷åñêèõ îñíîâ ðåãóëÿöèè áèîëîãè÷åñêèõ ñèñòåì Èíñòèòóòà áèîõèìè÷åñêîé ôèçèêè èì. Í.Ì. Ýìàíóýëÿ ÐÀÍ. Òåë.: 8-495-939-71-81; e-mail: [email protected] Ìèøàðèíà Òàìàðà Àðñåíüåâíà — äîêò. õèì. íàóê, çàâ. ëàáîðàòîðèåé ôëåéâîõèìèè Èíñòèòóòà áèîõèìè÷åñêîé ôèçèêè èì. Í.Ì. Ýìàíóýëÿ ÐÀÍ. Òåë.: 8-495-939-73-43; e-mail: [email protected] Ôàòêóëëèíà Ëþäìèëà Äìèòðèåâíà — êàíä. áèîë. íàóê, ñò. íàó÷. ñîòð. ëàáîðàòîðèè ôèçèêî-õèìè÷åñêèõ îñíîâ ðåãóëÿöèè áèîëîãè÷åñêèõ ñèñòåì Èíñòèòóòà áèîõèìè÷åñêîé ôèçèêè èì. Í.Ì. Ýìàíóýëÿ ÐÀÍ. Òåë.: 8-495-939-71-81; e-mail: [email protected] Áóðëàêîâà Åëåíà Áîðèñîâíà — äîêò. áèîë. íàóê, ïðîô., çàâ. îòäåëîì êèíåòèêè õèìè÷åñêèõ è áèîëîãè÷åñêèõ ïðîöåññîâ, çàâ. ëàáîðàòîðèåé ôèçèêî-õèìè÷åñêèõ îñíîâ ðåãóëÿöèè áèîëîãè÷åñêèõ ñèñòåì Èíñòèòóòà áèîõèìè÷åñêîé ôèçèêè èì. Í.Ì. Ýìàíóýëÿ ÐÀÍ. Òåë.: 8-499-137-31-27; e-mail: [email protected] Õîõëîâ Àëåêñàíäð Íèêîëàåâè÷ — äîêò. áèîë. íàóê, çàâ. ñåêòîðîì ýâîëþöèîííîé öèòîãåðîíòîëîãèè áèîëîãè÷åñêîãî ôàêóëüòåòà ÌÃÓ. Òåë.: 8-495-939-15-90; e-mail: [email protected]
