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Îðèãèíàëüíûå èññëåäîâàíèÿ 57 Èììóíîðåãóëÿòîðíûå ñâîéñòâà êëåòîê ôåòàëüíîé ïå÷åíè ÷åëîâåêà Þ.À. Ïåòðåíêî Èíñòèòóò ïðîáëåì êðèîáèîëîãèè è êðèîìåäèöèíû ÍÀÍ Óêðàèíû, Õàðüêîâ Immunoregulative properties of human fetal hepatic stem cells Yu.A. Petrenko Institute for problems of Cryobiology and Cryomedicine, NAS of Ukraine, Kharkov Ôåòàëüíàÿ ïå÷åíü ÷åëîâåêà (ÔÏ×) ÿâëÿåòñÿ óíèêàëüíûì îðãàíîì, â êîòîðîì ñîäåðæàòñÿ ñòâîëîâûå êðîâåòâîðíûå êëåòêè è èõ êîììèòèðîâàííûå ïîòîìêè ðàçëè÷íîé ñòåïåíè çðåëîñòè.  íàñòîÿùåé ðàáîòå ïðîâåäåíî îïðåäåëåíèå ñîäåðæàíèÿ èììóíîêîìïåòåíòíûõ êëåòîê â ÔÏ× 9-10 íåäåëü ãåñòàöèè ïî ôåíîòèïè÷åñêèì ñâîéñòâàì è ðåàêöèè íà ìèòîãåíû, à òàêæå èññëåäîâàíî âëèÿíèå êëåòîê ÔÏ× íà ïðîëèôåðàöèþ ëèìôîöèòîâ ïåðèôåðè÷åñêîé êðîâè âçðîñëûõ äîíîðîâ ïðè ñîâìåñòíîì êóëüòèâèðîâàíèè. Ñóñïåíçèþ êëåòîê ÔÏ× ïîëó÷àëè íåôåðìåíòàòèâíûì ìåòîäîì ñ èñïîëüçîâàíèåì âèáðàöèè. Êëåòêè êðèîêîíñåðâèðîâàëè ïîä çàùèòîé 5% ÄÌÑÎ ñî ñêîðîñòüþ 1°Ñ/ìèí. Ôåíîòèïè÷åñêèé àíàëèç ïðîâîäèëè ñ èñïîëüçîâàíèåì ôëóîðåñöåíòíîé ìèêðîñêîïèè. Ïðîëèôåðàòèâíóþ àêòèâíîñòü êëåòî÷íûõ êóëüòóð ÔÏ× è ëèìôîöèòîâ ïåðèôåðè÷åñêîé êðîâè (ËÏÊ) âçðîñëûõ äîíîðîâ îïðåäåëÿëè ïî âêëþ÷åíèþ 3H-òèìèäèíà.  ñìåøàííîé êóëüòóðå êëåòêè ÔÏ× ñîâìåñòíî êóëüòèâèðîâàëèñü ñ ËÏÊ â ïðèñóòñòâèè èëè îòñóòñòâèå ìèòîãåíîâ. Èììóíîôåíîòèïè÷åñêèé àíàëèç ñóñïåíçèè êëåòîê ÔÏ× íå âûÿâèë çðåëûõ B- è T-ëèìôîöèòîâ. Êëåòêè ÔÏ×, â îòëè÷èå îò ËÏÊ âçðîñëûõ äîíîðîâ îáëàäàëè âûñîêèì óðîâíåì ñïîíòàííîé ïðîëèôåðàöèè â óñëîâèÿõ êóëüòèâèðîâàíèÿ in vitro. Ìèòîãåíû - ôèòîãåìàããëþòèíèí (ÔÃÀ) è êîíêàíàâàëèí À (Êîí-À) ïîäàâëÿëè ïðîëèôåðàöèþ êëåòîê ÔÏ×. Ïðè ñîâìåñòíîì êóëüòèâèðîâàíèè, êëåòêè ÔÏ× ïîäàâëÿëè êàê ñïîíòàííóþ, òàê è ìèòîãåí-ñòèìóëèðîâàííóþ ïðîëèôåðàöèþ ËÏÊ âçðîñëûõ äîíîðîâ, à òàêæå ïðîëèôåðàöèþ ËÏÊ â äâóíàïðàâëåííîé ñìåøàííîé êóëüòóðå.  ðàáîòå îáñóæäàþòñÿ âîçìîæíûå ìåõàíèçìû äåéñòâèÿ êëåòîê ÔÏ×. Human fetal liver is unique as it contains stem hematopoietic cells and their committed derivatives of different maturation. The present study was aimed to assess the amount of immunocompetent human fetal hepatic cells of 9-10 week gestation by phenotypic properties and their reaction to mitogens as well as to evaluate influence of human fetal hepatic cells to proliferation of peripheral blood lymphocytes of adult donors in a mixed culture. The suspension of human fetal hepatic cells was obtained by a mechanical method using vibration. Cells were cryoconserved under the protection of 5% DMSO at the speed of 1°Ñ per min. Fluorescent microscopy was used for immune marker analysis. The proliferation activity of fetal hepatic cells and adult peripheral blood lymphocytes was assessed according to 3H-thymidine inclusion. In the mixed culture human fetal hepatic cells were co-cultured with peripheral blood lymphocytes in the presence or absence of mitogens. Immune marker analysis of the fetal hepatic cells suspension did not reveal the statistically relevant number of matured B- and Tlymphocytes. Unlike adult peripheral blood lymphocytes human hepatic cells demonstrated high levels of spontaneous proliferation when cultured in vitro. Mitogens, phytohemagglutinine (PHA) or concanavalin À (Con-À) suppressed the proliferation of human fetal hepatic cells. In the mixed culture the fetal hepatic cells suppressed both spontaneous and mitogen-stimulated proliferation of adult peripheral blood lymphocytes as well as peripheral lymphocyte proliferation in a twoforked mixed culture. Possible mechanisms of fetal hepatic cells activity have been discussed in the article. Êëþ÷åâûå ñëîâà: ôåòàëüíàÿ ïå÷åíü, èììóíîëîãèÿ, ðåàêöèÿ ñìåøàííîé êóëüòóðû ëèìôîöèòîâ. Key words: fetal liver, immunology, response of mixed lymphocyte culture. Ââåäåíèå Ðåàêöèÿ èììóííîé ñèñòåìû ðåöèïèåíòà íà ââîäèìûå êëåòêè àëëîãåííîãî ïðîèñõîæäåíèÿ ÿâëÿåòñÿ îäíèì èç êëþ÷åâûõ âîïðîñîâ êëåòî÷íîé òåðàïèè. Åñëè òðàíñïëàíòàöèÿ êëåòîê âçðîñëîãî ÷åëîâåêà, íàïðèìåð, êëåòîê êîñòíîãî ìîçãà (ÊÌ), òðåáóåò ïðîâåäåíèÿ òùàòåëüíîãî ïîäáîðà äîíîðà è ðåöèïèåíòà ïî àíòèãåíàì ãèñòîñîâìåñòèìîñòè è âîçìîæíà, â îñíîâíîì, ìåæäó áëèçíåöàìè è ñèáëèíãàìè, òî ñïîñîáíîñòü êëåòîê ôåòàëüíîãî ïðîèñõîæäåíèÿ èíäóöèðîâàòü ñïåöèôè÷åñêèé èììóííûé îòâåò äî íàñòîÿùåãî âðåìåíè èçó÷åíà ñëàáî, à èìåþùèåñÿ äàííûå õàðàêòåðèçóþòñÿ ïðîòèâîðå÷èâîñòüþ [1, 2, 11, 14]. Î÷åâèäíî, ÷òî èììóíîãåííîñòü êëåòîê çàâèñèò îò èõ òèïà è ñòåïåíè ðàçâèòèÿ.  ýòîé ñâÿçè èíòåðåñíûì îáúåêòîì èññëåäîâàíèÿ ÿâëÿþòñÿ êëåòêè ôåòàëüíîé ïå÷åíè ÷åëîâåêà (ÔÏ×) îðãàíà, â êîòîðîì ñîäåðæàòñÿ ñòâîëîâûå êðîâåòâîðíûå êëåòêè è èõ êîììèòèðîâàííûå ïîòîìêè ðàçëè÷íîé ñòåïåíè çðåëîñòè.  òî æå âðåìÿ ðàáîò, ïîñâÿùåííûõ èçó÷åíèþ âëèÿíèÿ êëåòîê ÔÏ× íà ïðîëèôåðàöèþ ëèìôîöèòîâ âçðîñëûõ äîíîðîâ in vitro, äî íàñòîÿùåãî âðåìåíè âûïîëíåíî ÷ðåçâû÷àéíî ìàëî [1 , 2 ].  ÷àñòíîñòè, Ek S. è ñîàâò. [1] íå ñìîãëè ïðèéòè ê çàêëþ÷åíèþ î âëèÿíèè êëåòîê ÔÏ× íà ëèìôîöèòû ïåðèôåðè÷åñêîé êðîâè (ËÏÊ) â ñìåøàííîé êóëüòóðå, òàê êàê â îäíèõ ñëó÷àÿõ ïåðâûå ñòèìóëèðîâàëè ïðîëèôåðàöèþ ËÏÊ, â äðóãèõ èíãèáèðîâàëè, à â òðåòüèõ íå âûçûâàëè çíà÷èòåëüíîãî ýôôåêòà. Ïðîòèâîðå÷èâûå äàííûå, ïîëó÷åííûå â ýòîé ðàáîòå, ìîãóò áûòü îáóñëîâëåíû áîëüøèì äèàïàçîíîì ñðîêîâ ãåñòàöèè èññëåäóåìûõ îáðàçöîâ, êîòîðûå âàðüèðîâàëè â ïðåäåëàõ îò 8 äî 20 íåäåëü.  ñâÿçè ñ ýòèì â íàñòîÿùåé ðàáîòå ïðîâåäåíî îïðåäåëåíèå ñîäåðæàíèÿ èììóíîêîìïåòåíòíûõ êëåòîê â ÔÏ× 9-10 íåäåëü ãåñòàöèè ïî ôåíîòèïè÷åñêèì ñâîéñòâàì è ðåàêöèè íà ìèòîãåíû, à òàêæå èññëåäîâàíî âëèÿíèå êëåòîê ÔÏ× íà ïðîëèôåðàöèþ ëèìôîöèòîâ ïåðèôåðè÷åñêîé êðîâè âçðîñëûõ äîíîðîâ ïðè ñîâìåñòíîì êóëüòèâèðîâàíèè. Ìàòåðèàë è ìåòîäû Ôðàãìåíòû ïå÷åíè ïëîäîâ ÷åëîâåêà 9-10 íåäåëü ãåñòàöèè ïîëó÷àëè ïîñëå èñêóññòâåííîãî ïðåðûâàíèÿ áåðåìåííîñòè ñ ïèñüìåííîãî ñîãëàñèÿ ïðîèíôîðìèðîâàííûõ äîíîðîâ. Ñóñïåíçèþ êëåòîê ÔÏ× ïîëó÷àëè â ñòåðèëüíûõ óñëîâèÿõ íåôåðìåíòàòèâíûì ìåòîäîì ñ èñïîëüçîâàíèåì âèáðàöèè [3 ], ìîäèôèöèðîâàííûì äëÿ ìàëûõ îáúåìîâ. Ïîëó÷åííóþ êëåòî÷íóþ ñóñïåíçèþ ôèëüòðîâàëè ÷åðåç óñòðîéñòâà äëÿ ïåðåëèâàíèÿ êðîâåçàìåíèòåëåé è èñïîëüçîâàëè äëÿ äàëüíåéøèõ èññëåäîâàíèé ëèáî êðèîêîíñåðâèðîâàëè. Ïîäñ÷åò êîëè÷åñòâà êëåòîê ïðîâîäèëè â êàìåðå Ãîðÿåâà, æèçíåñïîñîáíîñòü äî è ïîñëå êðèîêîíñåðâèðîâàíèÿ îïðåäåëÿëè ïî îêðàøèâàíèþ òðèïàíîâûì ñèíèì. Îïðåäåëåíèå α-ôåòîïðîòåèíà (ÀÔÏ) ïðîâîäèëè èììóíîôåðìåíòíûì Êëåòî÷íàÿ òðàíñïëàíòîëîãèÿ è òêàíåâàÿ èíæåíåðèÿ Òîì II, ¹ 3, 2007 Îðèãèíàëüíûå èññëåäîâàíèÿ ìåòîäîì ñ ïîìîùüþ íàáîðà «Âåêòîð-Áåñò», Íîâîñèáèðñê, Ðîññèÿ) ñîãëàñíî èíñòðóêöèè ïðîèçâîäèòåëÿ íà ïëàíøåòíîì ñïåêòðîôîòîìåòðå Tecan GENios (Àâñòðàëèÿ). Äëÿ êðèîêîíñåðâèðîâàíèÿ ñóñïåíçèþ êëåòîê ðàçâîäèëè äî êîíöåíòðàöèè 2×106/ìë è ïî êàïëÿì äîáàâëÿëè ðàâíûé îáúåì êðèîçàùèòíîé ñðåäû, ñîäåðæàùåé 10% äèìåòèëñóëüôîêñèäà (ÄÌÑÎ). Ñóñïåíçèþ ýêâèëèáðèðîâàëè ñ êðèîïðîòåêòîðîì ïðè 4°Ñ â òå÷åíèå 15 ìèí., ïîñëå ÷åãî çàìîðàæèâàëè â êîíòåéíåðå Nalgene Mr. Frosty (Sigma, ÑØÀ) òðè ýòàïà: äî òåìïåðàòóðû -40°Ñ ñî ñêîðîñòüþ 1°/ìèí, çàòåì äî -80°Ñ ñî ñêîðîñòüþ 10°/ìèí. Ñóñïåíçèè êëåòîê õðàíèëè â æèäêîì àçîòå íå ìåíåå 3-õ ìåñÿöåâ, à íåïîñðåäñòâåííî ïåðåä èññëåäîâàíèÿìè îòîãðåâàëè íà âîäÿíîé áàíå ïðè 37°Ñ. Äëÿ óäàëåíèÿ êðèîïðîòåêòîðà ñóñïåíçèè ðàçâîäèëè ñðåäîé êóëüòèâèðîâàíèÿ, ñîäåðæàùåé 10% ýìáðèîíàëüíîé ñûâîðîòêè êðóïíîãî ðîãàòîãî ñêîòà (ÝÑ), è öåíòðèôóãèðîâàëè ïðè 200 g â òå÷åíèå 10 ìèí. Îñàäîê ñóñïåíäèðîâàëè â 1 ìë ñðåäû. Ëèìôîöèòû ïåðèôåðè÷åñêîé êðîâè (ËÏÊ) ÷åëîâåêà âûäåëÿëè â ãðàäèåíòå Ficoll-Paque (Pharmacia, Øâåöèÿ) ñîãëàñíî ïðîòîêîëó [4 ]. Äëÿ ïðîâåäåíèÿ ôåíîòèïè÷åñêîãî àíàëèçà êëåòêè èììîáèëèçèðîâàëè íà ïðåäìåòíîì ñòåêëå ñ ïîìîùüþ 10% ðàñòâîðà ïîëè-L-ëèçèíà, îêðàøèâàëè ìîíîêëîíàëüíûìè àíòèòåëàìè è, ïî ìåðå íåîáõîäèìîñòè, ôèêñèðîâàëè 4% ðàñòâîðîì ïàðàôîðìàëüäåãèäà.  ðàáîòå èñïîëüçîâàëè ìîíîêëîíàëüíûå àíòèòåëà ê: CD3, CD4, CD8, CD20, CD45, HLF-DR, HLA-ABC. Íàáëþäåíèå è êîëè÷åñòâåííûé àíàëèç êëåòîê, ìå÷åííûõ âòîðè÷íûìè àíòèòåëàìè, êîíúþãèðîâàííûìè ñ FITC, ïðîâîäèëè ïîä ôëóîðåñöåíòíûì ìèêðîñêîïîì Jenaval (Karl Zeiss, Éåíà, Ãåðìàíèÿ). Äëÿ èññëåäîâàíèÿ ïðîëèôåðàòèâíîé àêòèâíîñòè êëåòêè ñóñïåíäèðîâàëè â ñðåäå RPMI-1640, ñîäåðæàùåé 10% ÝÑ, 50 åä/ìë ïåíèöèëëèíà è 50 ìã/ìë ñòðåïòîìèöèíà è ïî 2×105 êëåòîê ïîìåùàëè â ëóíêè 96-ëóíî÷íîãî ïëàíøåòà. Êëåòêè êóëüòèâèðîâàëè ïðè 37°Ñ, 95% âëàæíîñòè è 5% ÑÎ2 â òå÷åíèå 48 ÷àñîâ, ïîñëå ÷åãî â ëóíêè äîáàâëÿëè ïî 1µCi ðàñòâîðà 3H-òèìèäèíà. Îáðàçöû èíêóáèðîâàëè â òåõ æå óñëîâèÿõ â òå÷åíèå 24 ÷àñîâ. Âêëþ÷åíèå 3H-òèìèäèíà â ÄÍÊ êëåòîê îïðåäåëÿëè íà ñöèíòèëëÿöèîííîì β-ñïåêòðîìåòðå S-7800 Beckman (ÑØÀ). Äàííûå âûðàæàëè â èìïóëüñàõ â ìèíóòó (èìï/ìèí). Äëÿ îïðåäåëåíèÿ âëèÿíèÿ ìèòîãåíîâ íà èíòåíñèâíîñòü ïðîëèôåðàöèè êëåòîê ÔÏ× êëåòêè êóëüòèâèðîâàëè ñ ôèòîãåìàããëþòèíèíîì (ÔÃÀ, 20 ìêã/ìë) èëè êîíêàíàâàëèíîì À (Êîí-À, 10 ìêã/ìë).  íåêîòîðûõ ýêñïåðèìåíòàõ ïðîëèôåðàöèþ êëåòîê ÔÏ× èíãèáèðîâàëè îáðàáîòêîé ìèòîìèöèíîì Ñ (ÌÒÌ-Ñ) â êîíå÷íîé êîíöåíòðàöèè 25 ìêã/ìë, ïîñëå ÷åãî ÌÒÌ-Ñ òðèæäû îòìûâàëè ñðåäîé RPMI-1640. Äëÿ ïîñòàíîâêè ñìåøàííîé êóëüòóðû êëåòêè ÔÏ× è ëèìôîöèòû ïåðèôåðè÷åñêîé êðîâè ñîâìåñòíî êóëüòèâèðîâàëè â ñîîòíîøåíèè 1:1 â òå÷åíèå 48 ÷àñîâ, ïîñëå ÷åãî â ëóíêè âíîñèëè 3H-òèìèäèí è îïðåäåëÿëè èíòåíñèâíîñòü åãî âêëþ÷åíèÿ ÷åðåç 24 ÷àñà. Ïîëó÷åííûå ðåçóëüòàòû îáðàáàòûâàëè ñòàòèñòè÷åñêè; â çàâèñèìîñòè îò õàðàêòåðà ðàñïðåäåëåíèÿ èñïîëüçîâàëè ïàðàìåòðè÷åñêèé ìåòîä (t êðèòåðèé Ñòüþäåíòà) èëè íåïàðàìåòðè÷åñêèé - Âèëêîêñîíà [5 ]. Ðåçóëüòàòû è îáñóæäåíèå Æèçíåñïîñîáíîñòü ñâåæåâûäåëåííîé ïåðâè÷íîé ñóñïåíçèè êëåòîê ÔÏ×, îöåíåííàÿ ïî îêðàøèâàíèþ òðèïàíîâûì ñèíèì, ñîñòàâëÿëà 85±2%. Èììóíîôåíîòèïè÷åñêèé àíàëèç ïåðâè÷íîé ñóñïåíçèè íå âûÿâèë çðåëûõ B- è T- ëèìôîöèòîâ (CD3, CD4, CD8, CD20). Åäèíñòâåííûìè ïðåäñòàâèòåëÿìè êëåòîê ëèìôîèäíîãî ðÿäà áûëè CD45 ïîçèòèâíûå êëåòêè, êîëè÷åñòâî êîòîðûõ ñîñòàâëÿëî 2,09±0,32%. Îäíàêî Êëåòî÷íàÿ òðàíñïëàíòîëîãèÿ è òêàíåâàÿ èíæåíåðèÿ Òîì II, ¹ 3, 2007 î çðåëîñòè ýòèõ êëåòîê ñëîæíî ñóäèòü, ó÷èòûâàÿ, ÷òî CD45 ýêñïðåññèðóåòñÿ íå òîëüêî íà çðåëûõ ëèìôîöèòàõ, íî è íà ëèìôîèäíî êîììèòèðîâàííûõ êëåòêàõ, à òàêæå íà êðîâåòâîðíûõ êëåòêàõ-ïðåäøåñòâåííèêàõ [6 ]. Ïðîâåäåííûé àíàëèç íå èñêëþ÷àåò ïðèñóòñòâèÿ â ñóñïåíçèè êëåòîê ÔÏ× íåçðåëûõ ïðåäøåñòâåííèêîâ Ò-ëèìôîöèòîâ, ñïîñîáíûõ äèôôåðåíöèðîâàòüñÿ òèìóñ-íåçàâèñèìûì ïóòåì ïðè êóëüòèâèðîâàíèè èëè òðàíñïëàíòàöèè [14].  ñâÿçè ñ ýòèì áûëà ïðåäïðèíÿòà ïîïûòêà âûÿâèòü ôóíêöèîíàëüíî àêòèâíûå Ò-ëèìôîöèòû â ôåòàëüíîé ïå÷åíè ÷åëîâåêà ïðè ñîâìåñòíîì êóëüòèâèðîâàíèè êëåòîê ÔÏ× ñ ìèòîãåíàìè è àëëîàíòèãåíàìè. Èçâåñòíî, ÷òî ôèòîãåìàããëþòèíèí (ÔÃÀ) è êîíêàíàâàëèí À (Êîí-À) àêòèâèðóþò Ò-ëèìôîöèòû, èçáèðàòåëüíî ñâÿçûâàÿñü ñ óãëåâîäíûìè êîìïîíåíòàìè êëåòî÷íûõ ìåìáðàí [7 ].  ñâÿçè ñ ýòèì, èññëåäîâàëè âëèÿíèå ýòèõ ìèòîãåíîâ íà âêëþ÷åíèå 3Í-òèìèäèíà â ÄÍÊ êëåòîê ÔÏ× (ðèñ. 1). Èç ðèñ.1. âèäíî, ÷òî ÔÃÀ è Êîí-À íå òîëüêî íå àêòèâèðîâàëè êëåòêè ÔÏ×, íî äàæå ïîäàâëÿëè èõ ïðîëèôåðàöèþ. Ïðè ýòîì ÔÃÀ äîñòîâåðíî (ð<0,05) èíãèáèðîâàë âêëþ÷åíèå 3 Í-òèìèäèíà â ÄÍÊ êëåòîê ÔÏ×, òîãäà êàê çíà÷åíèÿ, ïîëó÷åííûå â ïðèñóòñòâèè ýòèõ äâóõ ìèòîãåíîâ, äîñòîâåðíî íå îòëè÷àëèñü ìåæäó ñîáîé. Âêëþ÷åíèå 3Í-òèìèäèíà, èìï/ìèí 58  îòñóòñòâèè ìèòîãåíà ÔÃÀ Êîí À Ïðèìå÷àíèÿ: * - ðàçëè÷èÿ äîñòîâåðíû ïî ñðàâíåíèþ ñ îáðàçöàìè áåç ìèòîãåíà Ðèñ. 1. Âëèÿíèå ìèòîãåíîâ ôèòîãåìàããëþòèíèíà (ÔÃÀ) è êîíêàíàâàëèíà À (Êîí-À) íà âêëþ÷åíèå 3Í-òèìèäèíà â ÄÍÊ êëåòîê ÔÏ× Â ëèòåðàòóðå îòñóòñòâóåò åäèíîå ìíåíèå î äåéñòâèè ìèòîãåíîâ íà êëåòêè ÔÏ×. Íàëè÷èå àíòèãåí-ñâÿçûâàþùèõ êëåòîê â ÔÏ× áûëî óñòàíîâëåíî 30 ëåò íàçàä Roberts I.M. è ñîàâò. [8 ]. Îäíàêî ñòèìóëÿöèè êëåòîê ÔÏ× â îòâåò íà äîáàâêó ÔÃÀ â ýòîé ðàáîòå îáíàðóæèòü íå óäàëîñü. Ñòèìóëèðóþùèé ýôôåêò ÔÃÀ íà êëåòêè ÔÏ× ÷åëîâåêà ïîêàçàí â ðàáîòå [9 ]. Îäíàêî â ýòîé ðàáîòå èññëåäîâàëèñü êëåòêè, ïîëó÷åííûå èç ïëîäîâ áîëåå ïîçäíèõ ñðîêîâ ðàçâèòèÿ (15-26 íåäåëü ãåñòàöèè).  ðàáîòàõ [2, 10 ], ïðîâåäåííûõ íà êëåòêàõ ÔÏ× 8-12 íåäåëü ãåñòàöèè, áûëî îáíàðóæåíî èíãèáèðóþùåå äåéñòâèå ìèòîãåíîâ (ÔÃÀ èëè Êîí-À) íà ïðîëèôåðàöèþ êëåòîê ÔÏ×. Áûëî ïîêàçàíî, ÷òî â ïðîöåññå âçàèìîäåéñòâèÿ ìèòîãåíà ñ êëåòêàìè ÔÏ× â ñðåäå îáíàðóæèâàþòñÿ ôàêòîðû, èíãèáèðóþùèå ñèíòåç ÄÍÊ è ïîäàâëÿþùèå ïðîëèôåðàöèþ ëèìôîöèòîâ, èíäóöèðîâàííóþ àëëîàíòèãåíîì. 59 Îðèãèíàëüíûå èññëåäîâàíèÿ Ðåçóëüòàòû, ñâèäåòåëüñòâóþùèå îá îòñóòñòâèè ôåíîòèïè÷åñêè çðåëûõ è ôóíêöèîíàëüíî àêòèâíûõ Ò-ëèìôîöèòîâ â ñóñïåíçèè êëåòîê ÔÏ× 1-ãî òðèìåñòðà ãåñòàöèè, ñîîòâåòñòâóþò äàííûì ðàáîòû [11 ], â êîòîðîé ïðèñóòñòâèå ôåíîòèïè÷åñêè çðåëûõ Ò-ëèìôîöèòîâ îáíàðóæåíî â ïå÷åíè ÷åëîâåêà â áîëåå ïîçäíèå ñðîêè (13-22 íåäåëè âíóòðèóòðîáíîãî ðàçâèòèÿ). Ïðåäïîëàãàþò, ÷òî ïðåäøåñòâåííèêè Ò-ëèìôîöèòîâ ïåðåñåëÿþòñÿ â òèìóñ èç ôåòàëüíîé ïå÷åíè [6]. Íà÷èíàÿ ñ 8-é íåäåëè ãåñòàöèè â ôåòàëüíîì òèìóñå Ò-ëèìôîöèòû ïðîõîäÿò ñëîæíóþ öåïü äèôôåðåíöèðîâî÷íûõ ñîáûòèé [12 ].  ðåçóëüòàòå ýòèõ ñîáûòèé ê 13 íåäåëå ãåñòàöèè â êðîâü ïîñòóïàþò çðåëûå Ò-êëåòêè ñî ñôîðìèðîâàâøèìñÿ TCR (T-cell receptor) êîìïëåêñîì, êîòîðûå ìîãóò êîëîíèçèðîâàòü ïåðèôåðè÷åñêèå îðãàíû, â òîì ÷èñëå è ïå÷åíü. Íàðÿäó ñ ýòèì, ñóùåñòâóåò ìíåíèå, ÷òî ïå÷åíü ìîæåò ÿâëÿòüñÿ è ñàìîñòîÿòåëüíûì, òèìóñ-íåçàâèñèìûì ìåñòîì ðàçâèòèÿ è ñîçðåâàíèÿ Ò-êëåòîê [13 ]. Äëÿ âûÿñíåíèÿ ýòîãî âîïðîñà â ðàáîòå [14 ] áûë ïðîâåäåí ìîëåêóëÿðíûé, ôåíîòèïè÷åñêèé è ôóíêöèîíàëüíûé àíàëèç êëåòîê ÔÏ×. Ìåòîäîì ÏÖÐ ñ îáðàòíîé òðàíñêðèïòàçîé áûëà èññëåäîâàíà ýêñïðåññèÿ ãåíîâ, êîäèðóþùèõ öåïè ÒCR. Ìîëåêóëÿðíûé àíàëèç ïîêàçàë, ÷òî, íåñìîòðÿ íà äîñòàòî÷íî ðàííåå (íà÷èíàÿ ñ 7 íåäåëè ãåñòàöèè) ðàçâèòèå îòäåëüíûõ öåïåé, çðåëûé ÒÑR êîìïëåêñ îêîí÷àòåëüíî ôîðìèðóåòñÿ òîëüêî ïîñëå 13,5 íåäåëü ãåñòàöèè. Òðàíñêðèïöèÿ ÒÑR êîìïëåêñà óâåëè÷èâàåòñÿ ïðîïîðöèîíàëüíî ñðîêó ðàçâèòèÿ ïëîäà, íî äàæå â âîçðàñòå 16 íåäåëü çðåëûé ÒÑR êîìïëåêñ áûë îáíàðóæåí òîëüêî â 6 èç 10 èññëåäîâàííûõ îáðàçöîâ ÔÏ× [14]. Êðîìå òîãî, ïðîâåäåííûå â ðàáîòå [14] èññëåäîâàíèÿ ïîêàçàëè, ÷òî ïóòåì íàïðàâëåííîé äèôôåðåíöèðîâêè â ïðèñóòñòâèè ìèòîãåíîâ, öèòîêèíîâ è àëëîàíòèãåíîâ ïðè êóëüòèâèðîâàíèè êëåòîê ÔÏ× 2-ãî òðèìåñòðà ãåñòàöèè óäàåòñÿ ïîëó÷èòü çðåëûå è ôóíêöèîíàëüíî àêòèâíûå Ò-ëèìôîöèòû. Îäíàêî êîëè÷åñòâî êëåòîê, ñïîñîáíûõ îáðàçîâûâàòü êëîíû Ò-ëèìôîöèòîâ, ÷ðåçâû÷àéíî ìàëî ëèøü 2 êëåòêè èç 106 îáëàäàëè òàêîé ñïîñîáíîñòüþ. Ñîâîêóïíîñòü íàøèõ è ëèòåðàòóðíûõ äàííûõ ñâèäåòåëüñòâóþò â ïîëüçó òîãî, ÷òî â ñóñïåíçèè êëåòîê ÔÏ× 1-ãî òðèìåñòðà ãåñòàöèè îòñóòñòâóþò êëåòêè, ñïîñîáíûå âûçûâàòü ðåàêöèþ òðàíñïëàíòàò ïðîòèâ õîçÿèíà (ÐÒÏÕ) ïðè òðàíñïëàíòàöèè. Ñêîðîñòü îòòîðæåíèÿ êëåòîê ïîñëå èõ òðàíñïëàíòàöèè çàâèñèò, ãëàâíûì îáðàçîì, îò ýêñïðåññèè àíòèãåíîâ ÃÊÑ êëàññà II. Ôåíîòèïè÷åñêèé àíàëèç êëåòîê ÔÏ× íà ïðåäñòàâëåííîñòü àíòèãåíîâ ÃÊÑ ïîêàçàë, ÷òî â ñóñïåíçèè ñâåæåâûäåëåííûõ êëåòîê ÔÏ× ñîäåðæèòñÿ 22,3±5,4% êëåòîê, ýêñïðåññèðóþùèõ HLA-À,Â,Ñ (W6/32) è 7,5±1,4% êëåòîê, ïîçèòèâíûõ íà HLA-DR. Ïîñëå êðèîêîíñåðâèðîâàíèÿ ñîäåðæàíèå êëåòîê, ýêñïðåññèðóþùèõ àíòèãåíû ÃÊÑ êëàññà I è II, íå èçìåíÿëîñü è ñîñòàâëÿëî 23,6±4,1 è 6,1±1,8%, ñîîòâåòñòâåííî. Ñëåäîâàòåëüíî, êëåòêè, ñîäåðæàùèå àíòèãåíû ÃÊÑ, ñîõðàíÿþòñÿ â ïðîöåññå êðèîêîíñåðâèðîâàíèÿ è äîëæíû ðàñïîçíàâàòüñÿ èììóííîé ñèñòåìîé ðåöèïèåíòà ïðè ââåäåíèè. Èììóííûé îòâåò îðãàíèçìà âêëþ÷àåò àêòèâàöèþ Ò-ëèìôîöèòîâ, êîòîðàÿ çàïóñêàåò êàñêàä ñîáûòèé, âåäóùèõ ê îòòîðæåíèþ ââåäåííûõ êëåòîê.  ñèñòåìå in vitro ïðîöåññ àêòèâàöèè ëèìôîöèòîâ ìîæåò áûòü èññëåäîâàí ïðè ñîâìåñòíîì êóëüòèâèðîâàíèè èññëåäóåìûõ êëåòîê ñ ëèìôîöèòàìè ïåðèôåðè÷åñêîé êðîâè âçðîñëîãî ÷åëîâåêà.  òàáë. 1 ïðèâåäåíû äàííûå ïî âêëþ÷åíèþ 3Í-òèìèäèíà â ÄÍÊ êëåòîê ÔÏ× è ëèìôîöèòîâ âçðîñëûõ äîíîðîâ. Óðîâåíü ñïîíòàííîé ïðîëèôåðàòèâíîé àêòèâíîñòè ñâåæåâûäåëåííûõ êëåòîê ÔÏ×, îöåíåííûé ïî âêëþ÷åíèþ 3Í-òèìèäèíà ñîñòàâëÿë 7787±820 èìï/ìèí. Êðèîêîíñåðâèðîâàíèå ïðèâîäèëî ê ñíèæåíèþ æèçíåñïîñîáíîñòè êëåòîê ÔÏ× íà 30%. Ïðè ýòîì óðîâåíü ïðîëèôåðàòèâíîé àêòèâíîñòè òàêæå ñíèæàëñÿ íà 30% è ñîñòàâëÿë 5344±694 èìï/ìèí (ñì. òàáë. 1). Òàáëèöà 1. Âêëþ÷åíèå 3Í-òèìèäèíà (èìï/ìèí) â êëåòêè ÔÏ× è ËÏÊ âçðîñëûõ äîíîðîâ ïðè ðàçäåëüíîì èëè ñîâìåñòíîì êóëüòèâèðîâàíèè (n = 5-8) Óñëîâèÿ ýêñïåðèìåíòà Êëåòêè ÔÏ× Êëåòêè ÔÏ×,îáðàáîòàííûå ÌÒÌ-Ñ Âêëþ÷åíèå 3Í-òèìèäèíà, èìï/ìèí/ëóíêó 5344±694 241±42 ËÏÊ 1069±112 ËÏÊ1 + ËÏÊ2 6548±285 ËÏÊ+ÔÏ× 5421±582 ÔÏ×1+ ÔÏ× 2 4125±628  èññëåäîâàíèÿõ ïî ñîâìåñòíîìó êóëüòèâèðîâàíèþ èñïîëüçîâàëè êðèîêîíñåðâèðîâàííûå êëåòêè ÔÏ×, ñâîáîäíûå îò âèðóñíîé è áàêòåðèàëüíîé êîíòàìèíàöèè. Îáðàáîòêà êëåòîê ÔÏ× ÌÒÌ-Ñ ïðàêòè÷åñêè ïîëíîñòüþ ïîäàâëÿëà ïðîëèôåðàòèâíóþ àêòèâíîñòü. ËÏÊ âçðîñëûõ äîíîðîâ îáëàäàëè íèçêîé ñïîíòàííîé ïðîëèôåðàòèâíîé àêòèâíîñòüþ.  äâóñòîðîííåé ñìåøàííîé êóëüòóðå, ïðè ñîâìåñòíîì êóëüòèâèðîâàíèè ËÏÊ äâóõ ðàçëè÷íûõ äîíîðîâ (ËÏÊ1+ ËÏÊ2) óðîâåíü âêëþ÷åíèÿ 3Í-òèìèäèíà ñîñòàâëÿë 6548±285 èìï/ìèí (ñì. òàáë. 1). Ýòè ðåçóëüòàòû ïîêàçûâàþò, ÷òî ëèìôîöèòû âçðîñëûõ äîíîðîâ, èñïîëüçîâàííûå â íàñòîÿùåé ðàáîòå, ôóíêöèîíàëüíî àêòèâíû è àäåêâàòíî ðåàãèðóþò íà àêòèâàöèþ, ñòèìóëèðîâàííóþ ìèòîãåíîì è àëëîãåíîì. Íàïðîòèâ, ñîâìåñòíîå êóëüòèâèðîâàíèå ïîïàðíî îáðàçöîâ àëëîãåííûõ êëåòîê ÔÏ× (ÔÏ×1 + ÔÏ×2) íå ïðèâîäèëî ê ñòèìóëÿöèè ñèíòåçà ÄÍÊ (ñì. òàáë. 1). Ïðè ñîâìåñòíîì êóëüòèâèðîâàíèè êëåòîê ÔÏ× è ËÏÊ áûëè ïîëó÷åíû çíà÷åíèÿ âêëþ÷åíèÿ 3Í-òèìèäèíà â ÄÍÊ, çíà÷èòåëüíî ïðåâûøàþùèå óðîâåíü ñïîíòàííîé ïðîëèôåðàöèè ëèìôîöèòîâ (ñì. òàáë. 1). Ïîäîáíûé æå óðîâåíü ñèíòåçà ÄÍÊ íàáëþäàëè ïðè êóëüòèâèðîâàíèè êëåòîê ÔÏ× â îòñóòñòâèå ËÏÊ. Ïîýòîìó ïðåäïîëîæèëè, ÷òî îí ìîæåò áûòü îáóñëîâëåí ïðîëèôåðàöèåé ôåòàëüíûõ êëåòîê. Âíåñåíèå â êóëüòóðàëüíóþ ñðåäó ÔÃÀ (ËÏÊ+ÔÃÀ) ïðèâîäèëî ê 7-9-êðàòíîé ñòèìóëÿöèè âêëþ÷åíèÿ 3Í-òèìèäèíà â ËÏÊ (ðèñ. 2). Äëÿ èñêëþ÷åíèÿ âêëàäà ñïîíòàííîé ïðîëèôåðàöèè êëåòîê ÔÏ× âî âêëþ÷åíèå 3Í-òèìèäèíà â ñìåøàííîé êóëüòóðå, êëåòêè ÔÏ× ïðåäâàðèòåëüíî îáðàáàòûâàëè ìèòîìèöèíîì Ñ. Ïîñëå 3-õ êðàòíîé îòìûâêè ìèòîìèöèíà Ñ, ñîâìåñòíîå êóëüòèâèðîâàíèå êëåòîê ÔÏ× è ËÏÊ íå âûçûâàëî ñòèìóëÿöèè âêëþ÷åíèÿ 3Í-òèìèäèíà â ËÏÊ. Áîëåå òîãî, íàáëþäàëîñü äîñòîâåðíîå (ð<0,05) ñíèæåíèå ñïîíòàííîé ïðîëèôåðàöèè ËÏÊ. Ïðè àêòèâàöèè ËÏÊ ìèòîãåíîì, ïðèñóòñòâèå êëåòîê ÔÏ× òàêæå çíà÷èòåëüíî óãíåòàëî âêëþ÷åíèå 3Í-òèìèäèíà â ÄÍÊ (ñì. ðèñ. 2). Óãíåòåíèå ïðîëèôåðàöèè ËÏÊ êëåòêàìè ÔÏ× ìîæåò áûòü îáóñëîâëåíî ïðèñóòñòâèåì â ñóñïåíçèè êëåòîê ÔÏ× ìåçåíõèìàëüíûõ ñòâîëîâûõ êëåòîê (ÌÑÊ) [15 , 16 ]. Óñòàíîâëåíî [15, 17 ], ÷òî ÌÑÊ, âûäåëåííûå èç ÊÌ âçðîñëûõ ëþäåé, õàðàêòåðèçóþòñÿ óìåðåííîé ýêñïðåññèåé ìîëåêóë ÃÊÑ êëàññà I è ñëàáîé ýêñïðåññèåé ÃÊÑ êëàññà II. Ñëåäîâàòåëüíî, àëëîãåííûå ëèìôîöèòû óçíàâàëè èõ, íî â ñìåøàííîé êóëüòóðå ýòèõ êëåòîê íàáëþäàëè ýôôåêò, ïîäîáíûé îòìå÷åííîìó íàìè ïîäàâëåíèå ïðîëèôåðàöèè ëèìôîöèòîâ ïðè ñîâìåñòíîì êóëüòèâèðîâàíèè [17,18 ]. Áîëåå òîãî, óãíåòåíèå èììóííîãî îòâåòà âçðîñëûõ ÌÑÊ áûëî ïîäòâåðæäåíî â óñëîâèÿõ in vivo ó ïðèìàòîâ ïðè òðàíñôóçèè àëëîãåííûõ ÌÑÊ îòìå÷åíî óëó÷øåíèå ïðèæèâëåíèÿ àëëîãåííîãî êîæíîãî òðàíñïëàíòàòà [19 ]. Êëåòî÷íàÿ òðàíñïëàíòîëîãèÿ è òêàíåâàÿ èíæåíåðèÿ Òîì II, ¹ 3, 2007 60 Âêëþ÷åíèå 3Í-òèìèäèíà, èìï/ìèí/ëóíêà Îðèãèíàëüíûå èññëåäîâàíèÿ 1 2 3 4 Ïðèìå÷àíèÿ: * - ðàçëè÷èÿ äîñòîâåðíû (p<0,05) ïî-îòíîøåíèþ ê ËÏÊ áåç äîáàâîê (1); $ - ðàçëè÷èÿ äîñòîâåðíû ïî-îòíîøåíèþ ê ãðóïïå 3 (p<0,05). Ðèñ. 2. Âêëþ÷åíèå 3Í-òèìèäèíà â ËÏÊ âçðîñëîãî äîíîðà â îäíîñòîðîííåé ñìåøàííîé êóëüòóðå: 1 ñïîíòàííàÿ ïðîëèôåðàöèÿ ËÏÊ; 2 ñìåøàííàÿ êóëüòóðà ËÏÊ è êëåòîê ÔÏ×, îáðàáîòàííûõ ÌÒÌ-Ñ; 3 ïðîëèôåðàöèÿ ËÏÊ ïîä âîçäåéñòâèåì ÔÃÀ; 4 ïðîëèôåðàöèÿ ËÏÊ ïîä âîçäåéñòâèåì ÔÃÀ â ïðèñóòñòâèè êëåòîê ÔÏ×, îáðàáîòàííûõ ÌÒÌ-Ñ Íåäàâíî â íàøåé ëàáîðàòîðèè èç ñóñïåíçèè ÔÏ× áûëè âûäåëåíû ïðåòåíäåíòû íà ÌÑÊ, êîòîðûå îáðàçîâûâàëè êîëîíèè ôèáðîáëàñòîïîäîáíûõ êëåòîê è áûëè ñïîñîáíû äèôôðåíöèðîâàòüñÿ â îñòåîãåííîì è àäèïîãåííîì íàïðàâëåíèÿõ [20 , 21 ]. Äîëåâîå ñîäåðæàíèå êîëîíèåîáðàçóþùèõ êëåòîê â ôåòàëüíîé ïå÷åíè îêàçàëîñü â 5-7 ðàç âûøå, ÷åì â ÊÌ âçðîñëûõ: îäíà êîëîíèåîáðàçóþùàÿ êëåòêà ïðèõîäèëàñü íà 30 000-50 000 êëåòîê ÔÏ×, â òî âðåìÿ êàê â ÊÌ îäíà ÌÑÊ íàéäåíà ñðåäè 250 000 êëåòîê [22 ]. Ôåíîòèïè÷åñêèé àíàëèç, ïðîâåäåííûé â ðàáîòàõ [21, 22, 23 ], ïîêàçàë, ÷òî ìåçåíõèìàëüíûå êëåòêè ÔÏ× íå îòëè÷àþòñÿ îò ÌÑÊ ÊÌ: è òå, è äðóãèå ïîçèòèâíû ïî ìîëåêóëàì CD29, CD44, CD166, CD105 è íåãàòèâíû ïî CD14, CD34, CD45. Ìîæíî ïðåäïîëîæèòü, ÷òî ìåõàíèçì äåéñòâèÿ ìåçåíõèìàëüíûõ ñòâîëîâûõ êëåòîê îáóñëîâëåí èõ ñïîñîáíîñòüþ ïðîäóöèðîâàòü ñïåöèôè÷åñêèå ôàêòîðû, ñïîñîáíûå îêàçûâàòü èììóíîñóïðåññîðíîå äåéñòâèå. Òàê, íà ïðèìàòàõ ïîêàçàíî, ÷òî äîáàâêà IL-2 ïðåäîòâðàùàåò èíãèáèðîâàíèå ìåçåíõèìàëüíûìè êëåòêàìè ìèòîãåí-èíäóöèðîâàííîé àêòèâàöèè ËÈÒÅÐÀÒÓÐÀ: 1. Ek S., Ringden O., Markling L., Westgren M. Immunological capacity of human fetal liver cells. Bone Marrow Transpl. 1994;14: 9-14. 2. Lindton B., Markling L., Ringden O. Mixed lymphocyte culture of human fetal liver cells. Fetal Diagnosis and Therapy. 2000; 15: 71-8. 3. Petrenko A.Yu., Sukach A.N. Isolation of intact mitochondria and hepatocytes using vibration. Analytical Biochem. 1991; 194: 325-9. 4. Recktenwald D., Radbruch A. Cell separation methods and applications / NewYork: Marcel Dekker. Inc.,1998: 246. 5. Ïëàòîíîâ À.Å. Ñòàòèñòè÷åñêèé àíàëèç â áèîëîãèè è ìåäèöèíå: çàäà÷è, òåðìèíîëîãèÿ, êîìïüþòåðíûå ìåòîäû. 2000; Ì.: Èçäàòåëüñòâî ÐÀÌÍ: 52. 6. ßðèëèí À.À. Îñíîâû èììóíîëîãèè. 1999; Ì.: Ìåäèöèíà: 607. 7. Greaves M., Janossy G., Doenhoff M. Selective triggering of human T and B lymphocytes in vitro by polyclonal mitogens. J. Exp. Med. 1974; 140: 1-18. 8. Roberts I.M., Whittingham S., Cowling D.C., Mackay I.R. Antigen-binding cells in human fetal liver. Int. Arch. Allergy Appl. Immunol. 1975; 49: 743-53. 9. Sirianni M.C., Fiorilli M., Casadei A.M., Aiuti F. Response to B and T cell mitogens and surface markers in human fetal lymphoid tissues. Thymus1980; 1: 257-63. Êëåòî÷íàÿ òðàíñïëàíòîëîãèÿ è òêàíåâàÿ èíæåíåðèÿ Òîì II, ¹ 3, 2007 ëèìôîöèòîâ [24 ].  íåäàâíî ïðîâåäåííîé ðàáîòå [25 ] áûëî óñòàíîâëåíî, ÷òî äåéñòâèå ÌÑÊ ïðè òðàíñïëàíòàöèè ñâÿçàíî íå ñ äèôôåðåíöèðîâêîé êëåòîê, à ñ èçìåíåíèåì öèòîêèíîâîãî ïðîôèëÿ â îðãàíå-ìèøåíè. Ïðè ýòîì íàáëþäàëè ñíèæåíèå óðîâíÿ ïðîâîñïàëèòåëüíûõ öèòîêèíîâ è ñòèìóëÿöèþ îáðàçîâàíèÿ àíòèâîñïàëèòåëüíûõ öèòîêèíîâ è àíòèàïîïòè÷åñêèõ ôàêòîðîâ.  äðóãîì èññëåäîâàíèè [15] ïðè äîáàâëåíèè ê ñìåøàííîé êóëüòóðå ÌÑÊ è Ò-ëèìôîöèòîâ àíòèòåë ïðîòèâ òðàíñôîðìèðóþùåãî ôàêòîðà ðîñòà-â (TGF-â1) è ôàêòîðà ðîñòà ãåïàòîöèòîâ (HGF) íàáëþäàëè âìåñòî óãíåòàþùåãî äåéñòâèÿ ÌÑÊ îáóñëîâëåííóþ èìè àëëîãåííóþ ñòèìóëÿöèþ.  ýòîé ñâÿçè ñëåäóåò îòìåòèòü, ÷òî âûøåóêàçàííûå ðîñòîâûå ôàêòîðû îáíàðóæèâàþòñÿ â ÔÏ× 1-ãî òðèìåñòðà ãåñòàöèè [26 , 27 ]. Åùå îäíèì ïðåòåíäåíòîì íà ðîëü ñóïðåññîðà èììóííîãî îòâåòà ÿâëÿåòñÿ α-ôåòîïðîòåèí (ÀÔÏ), êîòîðûé íå âëèÿåò íà àêòèâíîñòü Ò- è Â-ëèìôîöèòîâ, íî ïîäàâëÿåò ïðîëèôåðàòèâíûé îòâåò ëèìôîöèòîâ íà ìèòîãåíû [28 ]. Äëÿ îöåíêè âîçìîæíîñòè ðåàëèçàöèè òàêîãî ìåõàíèçìà óãíåòåíèÿ ïðîëèôåðàöèè ëèìôîöèòîâ íàìè áûëî îïðåäåëåíî ñîäåðæàíèå ÀÔÏ â ñóñïåíçèÿõ êëåòîê ÔÏ×. Áûëî ïîêàçàíî, ÷òî ñîäåðæàíèå ÀÔÏ â ñóñïåíçèè ñâåæåâûäåëåííûõ êëåòîê ÔÏ× ñîñòàâëÿëî 1044±205 ÌÅ/106 êëåòîê. Ýòîò óðîâåíü ÀÔÏ â ðåàêöèè ÑÊË çíà÷èòåëüíî ïðåâûøàåò ôèçèîëîãè÷åñêèå çíà÷åíèÿ, ÷òî è ìîæåò ÿâèòüñÿ ïðè÷èíîé óãíåòåíèÿ ïðîëèôåðàòèâíîé àêòèâíîñòè ËÏÊ. Ñëåäóåò îòìåòèòü, ÷òî ÀÔÏ íå âûðàáàòûâàåòñÿ ÌÑÊ è ìîæåò ÿâëÿòüñÿ äîïîëíèòåëüíûì ñóïðåññîðíûì ôàêòîðîì ïðè èñïîëüçîâàíèè òîòàëüíîé ñóñïåíçèè êëåòîê ÔÏ×. Çàêëþ÷åíèå Ïîëó÷åííûå ðåçóëüòàòû ñâèäåòåëüñòâóþò î òîì, ÷òî â ñîñòàâå ÔÏ× 9-10 íåäåëü ãåñòàöèè îòñóòñòâóþò çðåëûå, ôóíêöèîíàëüíî àêòèâíûå ëèìôîèäíûå êëåòêè, ñïîñîáíûå ó÷àñòâîâàòü â ðåàêöèÿõ èììóííîãî îòâåòà. Âûÿâëåííîå â ðàáîòå ñâîéñòâî êëåòîê ÔÏ× ïîäàâëÿòü ñïîíòàííóþ è ìèòîãåí-ñòèìóëèðîâàííóþ ïðîëèôåðàöèþ ëèìôîöèòîâ ïðè ñîâìåñòíîì êóëüòèâèðîâàíèè îáóñëîâëåíî, î÷åâèäíî, ïðèñóòñòâèåì â ÔÏ× ýìáðèîñïåöèôè÷åñêèõ ôàêòîðîâ, ñïîñîáíûõ ïîäàâëÿòü èììóííûé îòâåò. Îòñóòñòâèå â ñîñòàâå ñóñïåíçèè êëåòîê ÔÏ× èììóíîêîìïåòåíòíûõ êëåòîê, à òàêæå ñïîñîáíîñòü óãíåòàòü ðåàêöèè èììóííîãî îòâåòà ïîçâîëÿþò ðàññìàòðèâàòü êëåòêè ÔÏ× â êà÷åñòâå èììóíîìîäóëÿòîðà, ñïîñîáíîãî ñíèæàòü ñêîðîñòü îòòîðæåíèÿ êëåòîê ïðè òðàíñïëàíòàöèè. Àâòîð âûðàæàåò áëàãîäàðíîñòü Ïàâëþêó Àëåêñàíäðó Ñåðãååâè÷ó çà ïîìîùü â ïîäãîòîâêå ñòàòüè ê îïóáëèêîâàíèþ. 10. Rabinowich H., Bahary C., Ben-Aderet N., Klajman A. Cellular and humoral suppressor activity induced by concanavalin A-stimulated human fetal liver cells. Transpl.1983;35:452-8. 11. Sanchez M.J., Gutierrez-Amos J.C., Fernandez E. Putative prethymic T cell precursors within the early human embryonic liver: a molecular and functional analysis. J. Exp. Med.1993;177: 1933. 12. Barton F.H., Heinly C.S. Early human T cell development: analysis of the human thymus at the time of initial entry of hematopoietic stem cells into the fetal thymic microenvironment. J. Exp. Med. 1995;181:144558. 13. Aguila H.L., Akashi K., Domen J. From stem cells to lymphocytes: biology and transplantation. Immunol. Rev. 1997; 157:135. 14. Renda M.C., Fecarotta E., Dieli F. et al. Evidence of alloreactive T lymphocytes in fetal liver: implications for fetal hematopoietic stem cell transplantation. Bone Marrow Transpl.2000;25:13541. 15. Di Nicola M., Carlo-Stella C., Magni M. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 2002; 99:3838-43. 16. Campagnoli C., Roberts I.A., Kumar S. Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. Blood 2001;98: 2396-402. 61 Îðèãèíàëüíûå èññëåäîâàíèÿ 17. Young H.E., Steele T.A., Bray R.A. Human pluripotent and progenitor cells display cell surface cluster differentiation markers CD10, CD13, CD56, and MHC class-I. Proc. Soc. Exp. Biol. Med.1999; 221: 63-71. 18. Le Blanc K., Tammik C., Sundberg B. Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompability complex. Scand. J. Immunol. 2003; 57: 11-20. 19. Devine S.M., Bartholomew A.M., Mahmud N. Mesenchymal stem cells are capable of homing to the bone marrow of non-human primates following systemic infusion. Exp. Hematol. 2001; 29: 244-55. 20. Ãðèùåíêî Â.È., Ïåòðåíêî À.Þ., Âîëêîâà Í.À., Ñêîðîáîãàòîâà Í.Ã. Êîëîíèåîáðàçóþùàÿ àêòèâíîñòü ôèáðîáëàñòîïîäîáíûõ êëåòîê ïðåäøåñòâåííèêîâ èç ýìáðèîíàëüíîé ïå÷åíè ÷åëîâåêà â óñëîâèÿõ in vitro. Äîïîâ³ä³ ÍÀÍ Óêðà¿íè 2005; 2: 138-41. 21. Petrenko Yu.A., Skorobogatova N.G., Jones R.E., Petrenko A.Yu. Cryosensitivity of hematopoietic and mesenchymal stem/progenitor cells derived from human fetal liver. Cryobiology 2006; 53 : 381-2. 22. Pittenger M.F., Mackay A.M., Beck S.C. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284: 143-7. 23. Campagnoli C., Roberts I.A., Kumar S. Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. Blood 2001; 98: 2396-402. 24. Bartholomew A., Sturgeon C., Siatskas M. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp. Hematol. 2002; 30: 42-8. 25. Togel F., Hu Z., Weiss K. Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms. Am. J. Physiol. Renal. Physiol. 2005; 289: 3142. 26. Sennikov S.V., Krysov S.V., Injelevskaya T.V. Production of cytokines by immature erythroid cells derived from human embryonic liver. Eur. Cytokine Netw. 2001; 12(2): 274-9. 27. Sennikov S.V., Krysov S.V., Silkov A.N. Production of IL-10, TNF-alpha, IFN-gamma, TGF-beta1 by different populations of erythroid cells derived from human embryonal liver. Cytokine 2002;17: 221-5. 28. Czokalo M., Wishnewski L. Culture conditions modify the effect exerted by human fetal AFP on some lymphocyte functions in vitro. Exp. Pathol. 1981; 20: 233-8. Ïîñòóïèëà 29.04.2007 Êàôåäðà «Êëåòî÷íîé âîññòàíîâèòåëüíîé ìåäèöèíû» Ðîññèéñêîãî Ãîñóäàðñòâåííîãî Ìåäèöèíñêîãî Óíèâåðñèòåòà ñ 2007 ã. íà÷èíàåò ïîäãîòîâêó ñïåöèàëèñòîâ â îáëàñòè êëåòî÷íîé ìåäèöèíû Êàôåäðà áàçèðóåòñÿ íà áàçå êëèíèêè âîññòàíîâèòåëüíîé èíòåðâåíöèîííîé íåâðîëîãèè è òåðàïèè «ÍåéðîÂèòà» ïðè Ðîññèéñêîì Îíêîëîãè÷åñêîì Íàó÷íîì Öåíòðå èì. Í.Í. Áëîõèíà ÐÀÌÍ Ñïèñîê ëåêöèé ( â öèêëàõ) Öèêë 1. 1. Áàíêè ïóïîâèííîé êðîâè â Ðîññèè è çà ðóáåæîì èñòîðèÿ, çàêîíîäàòåëüñòâî, îñîáåííîñòè ôóíêöèîíèðîâàíèÿ. 2. Âûäåëåíèå è êðèîêîíñåðâèðîâàíèå ãåìîïîýòè÷åñêèõ ñòâîëîâûõ êëåòîê èç ïóïîâèííîé êðîâè (ÃÑÊ ÏÊ). 3. Ïåðñïåêòèâû èñïîëüçîâàíèÿ ÃÑÊ ÏÊ äëÿ òåðàïèè íåãåìàòîëîãè÷åñêèõ çàáîëåâàíèé íà ìîäåëÿõ æèâîòíûõ. Öèêë 2. 1. Ïîíÿòèå ÝÑÊ, èõ ìîëåêóëÿðíî-ãåíåòè÷åñêå îñîáåííîñòè, îáåñïå÷èâàþùèå ñàìîïîääåðæàíèå è ïëþðèïîòåíòíîñòü. 2. Ñïîñîáíîñòè ÝÑÊ ê äèôôåðåíöèðîâêå in vitro, ìîëåêóëÿðíî-ãåíåòè÷åñêèå ìåõàíèçìû, îáåñïå÷èâàþùèå ýòî. 3. Ïîâåäåíèå ÝÑÊ in vivo, âîçìîæíîñòè ïðàêòè÷åñêîãî ïðèìåíåíèÿ. Àäðåñ: 115478, Ìîñêâà Êàøèðñêîå øîññå, ä. 23, ñòð. À. Òåëåôîíû äëÿ ñïðàâîê: (495) 324-93-39. Êëåòî÷íàÿ òðàíñïëàíòîëîãèÿ è òêàíåâàÿ èíæåíåðèÿ Òîì II, ¹ 3, 2007
