Îðèãèíàëüíûå èññëåäîâàíèÿ 39 ÎÐÈÃÈÍÀËÜÍÛÅ ÈÑÑËÅÄÎÂÀÍÈß Ó÷àñòèå êëåòîê Èòî â ãèñòîãåíåçå è ðåãåíåðàöèè ïå÷åíè À.À. Ãóìåðîâà, À.Ï. Êèÿñîâ, Ì.Ñ. Êàëèãèí, È.Ì. Ãàçèçîâ, Ñ.Ð. Àáäóëõàêîâ, Ä.È. Àíäðååâà, Ì.À. Òèòîâà, Ò.Ñ. Ñìåòàííèêîâà Êàôåäðà íîðìàëüíîé àíàòîìèè, Êàçàíñêèé ãîñóäàðñòâåííûé ìåäèöèíñêèé óíèâåðñèòåò Participation of Ito-cells in histogenesis and regeneration of the liver A.A. Gumerova, A.P. Kiasov, M.S. Kaligin, I.M. Gazizov, S.R. Abdulkhakov, D.I. Andreeva, M.A. Titova, T.S. Smetannikova Department of Normal Anatomy, Kazan State Medical University  ïîñëåäíåå äåñÿòèëåòèå îïóáëèêîâàí íå îäèí äåñÿòîê ðàáîò, ñâèäåòåëüñòâóþùèõ î âîçìîæíîñòè îáðàçîâàíèÿ ãåïàòîöèòîâ èç êðîâåòâîðíûõ ñòâîëîâûõ êëåòîê, èìåþùèõ, êàê èçâåñòíî, ìåçåíõèìàëüíîå ïðîèñõîæäåíèå.  íàñòîÿùåé ðàáîòå ïðîâåäåí àíàëèç âåðîÿòíîñòè ðàçâèòèÿ ýïèòåëèÿ ïå÷åíè èç äðóãèõ èñòî÷íèêîâ, íåæåëè ýíòîäåðìà. Êëþ÷åâûå ýòàïû äèôôåðåíöèðîâêè ýïèòåëèàëüíûõ è ñèíóñîèäíûõ êëåòîê ïå÷åíè â õîäå å¸ íîðìàëüíîãî îíòîãåíåçà è ðåãåíåðàöèè ó ÷åëîâåêà è êðûñû èçó÷åíû èììóíîãèñòîõèìè÷åñêè ñ èñïîëüçîâàíèåì øèðîêîé ïàíåëè ìîíîêëîíàëüíûõ àíòèòåë ê ìàðêåðàì ýïèòåëèàëüíûõ, ìåçåíõèìàëüíûõ è ñòâîëîâûõ êëåòîê è àïîïòîçà. Ðåçóëüòàòû èññëåäîâàíèÿ ïîçâîëÿþò ñäåëàòü âûâîä, ÷òî îäíèì èç èñòî÷íèêîâ ðàçâèòèÿ ãåïàòîöèòîâ ìîãóò áûòü äåñìèí-ïîçèòèâíûå ìåçåíõèìàëüíûå êëåòêè, à ñëåäîâàòåëüíî, ÷òî ïåðèñèíóñîèäàëüíûå êëåòêè ïå÷åíè îñíîâíîé ïðåòåíäåíò íà ðîëü ñòâîëîâîé êëåòêè ýòîãî îðãàíà. Êëþ÷åâûå ñëîâà: êëåòêè Èòî, ïå÷åíü, ðåãåíåðàöèÿ. Óñòîÿâøàÿñÿ òåîðèÿ ðàçâèòèÿ ïå÷åíè áàçèðóåòñÿ íà èññëåäîâàíèÿõ, ïðîâåäåííûõ â ñåðåäèíå ÕÕ âåêà. Ñ÷èòàåòñÿ, ÷òî ïðåäøåñòâåííèêè ãåïàòîöèòîâ âûñåëÿþòñÿ â ìåçåíõèìó ïîïåðå÷íîé ïåðåãîðîäêè èç ýïèòåëèÿ äâåíàäöàòèïåðñòíîé êèøêè ýìáðèîíà. Èç ýïèòåëèàëüíûõ êëåòîê îáðàçóþòñÿ ãåïàòîáëàñòû/ãåïàòîöèòû è õîëàíãèîöèòû, à èç êëåòîê ìåçåíõèìû äèôôåðåíöèðóþòñÿ ñèíóñîèäíûå êëåòêè [13]. Íà îïðåäåëåííûõ ýòàïàõ ðàçâèòèÿ ÷àñòü ãåïàòîáëàñòîâ, ðàñïîëîæåííûõ âîêðóã ñîñóäîâ, îáðàçóþò òàê íàçûâàåìóþ ïðîòîêîâóþ ïëàñòèíêó, èç êëåòîê êîòîðîé ôîðìèðóþòñÿ âíóòðèïå÷åíî÷íûå æåë÷íûå ïðîòîêè [4]. Ñóùåñòâîâàíèå ðåãèîíàëüíîé ñòâîëîâîé êëåòêè ïå÷åíè â íàñòîÿùåå âðåìÿ íå âûçûâàåò ñîìíåíèé. Òðàäèöèîííî ñ÷èòàåòñÿ, ÷òî îíà íàõîäèòñÿ ñðåäè êëåòîê ìåëü÷àéøèõ âíóòðèïå÷åíî÷íûõ æåë÷íûõ ïðîòîêîâ êàíàëüöåâ Ãåðèíãà, à èõ íåïîñðåäñòâåííûìè ïîòîìêàìè ÿâëÿþòñÿ òàê íàçûâàåìûå îâàëüíûå êëåòêè, îáëàäàþùèå ñâîéñòâàìè áèïîòåíòíîñòè è äàþùèå íà÷àëî êàê ãåïàòîöèòàì, òàê è õîëàíãèîöèòàì [5]. Îäíàêî â ïîñëåäíèå ãîäû óñòàíîâëåíà âîçìîæíîñòü ðàçâèòèÿ ãåïàòîöèòîâ èç ñòâîëîâîé êðîâåòâîðíîé êëåòêè [68], ò.å. êëåòêè ìåçåíõèìàëüíîé, à íå ýíòîäåðìàëüíîé ïðèðîäû.  ñâÿçè ñ ýòèì âîçíèêàåò âîïðîñ: íå ìîãóò ëè ïîñòîÿííî «ïðîïèñàííûå» â ïå÷åíè êëåòêè ìåçåíõèìàëüíîãî ïðîèñõîæäåíèÿ áûòü èñòî÷íèêîì ãåïàòîöèòîâ â õîäå ðàçâèòèÿ è ðåãåíåðàöèè? Äëÿ îòâåòà íà ýòîò âîïðîñ áûëî ïðåäïðèíÿòî êîìïëåêñíîå èììóíîãèñòîõèìè÷åñêîå èññëåäîâàíèå, â êîòîðîì ñ ïîìîùüþ âûÿâëåíèÿ øèðîêîãî ñïåêòðà ôåíîòèïè÷åñêèõ è äèôôåðåí- There have been a lot of reports about differentiation possibilities of hepaticytes from hematopoitec stem cells or mesenchymal cells in the past decade. The potential of hepatic epithelium differentiation from other sources besides entoderm has been analysed in the present study. A wide variety of monoclonal antibodies of epithelial, mesenchymal and stem cell markers and apoptosis were used to study the main stages of differentiation of epithelial and sinusoidal cells of the liver during normal ontogenesis and regeneration in human and rat. The results obtained lead to the conclusion that desmin-positive mesenchymal cells might be one of the sources of hepatocyte differentiation, with perisinusoidal cells being the most probable stem cells of the liver. Key words: Ito-cells, liver, regeneration. öèðîâî÷íûõ ìàðêåðîâ ïðîâåäåí äåòàëüíûé àíàëèç âñåõ ýòàïîâ ãèñòî- è îðãàíîãåíåçà ïå÷åíè ÷åëîâåêà è çàêîíîìåðíîñòåé äèôôåðåíöèðîâêè ãåïàòîöèòîâ. Îñîáîå âíèìàíèå áûëî óäåëåíî èçó÷åíèþ âçàèìîäåéñòâèÿ ïå÷åíè ñ îêðóæàþùåé åå ìåçåíõèìîé è ñîïîñòàâëåíèþ ôåíîòèïîâ ýïèòåëèÿ ïåðåäíåé êèøêè è ãåïàòîöèòîâ. Òàêæå áûëà èçó÷åíà äèíàìèêà ìåæêëåòî÷íûõ âçàèìîäåéñòâèé â ïðîöåññå ðåïàðàòèâíîé ðåãåíåðàöèè ïå÷åíè áîëüíûõ õðîíè÷åñêèìè ãåïàòèòàìè è êðûñ ïîñëå ÷àñòè÷íîé ãåïàòýêòîìèè è ïîâðåæäåíèÿ ïå÷åíè íèòðàòîì ñâèíöà. Âûáðàííûå ýêñïåðèìåíòàëüíûå ìîäåëè îòëè÷àþòñÿ òåì, ÷òî íè â òîì, íè â äðóãîì ñëó÷àå íå áûëî îïèñàíî ïîÿâëåíèå îâàëüíûõ êëåòîê â õîäå ðåãåíåðàöèè. Ìàòåðèàë è ìåòîäû Ïå÷åíü ýìáðèîíîâ è ïëîäîâ ÷åëîâåêà ðàçëè÷íûõ ñðîêîâ ãåñòàöèè (ñ 4-é ïî 33-þ íåäåëþ ïðåíàòàëüíîãî ðàçâèòèÿ) ïîëó÷àëè â ðåçóëüòàòå ëåãàëüíûõ ìåäèöèíñêèõ àáîðòîâ, ñàìîïðîèçâîëüíûõ âûêèäûøåé è èíäóöèðîâàííûõ ïðåæäåâðåìåííûõ ðîäîâ ïî ìåäèöèíñêèì ïîêàçàíèÿì (78 îáðàçöîâ). Òàêæå áûëè èçó÷åíû áèîïòàòû ïå÷åíè áîëüíûõ õðîíè÷åñêèìè ãåïàòèòàìè (156 îáðàçöîâ). Âî âñåõ ñëó÷àÿõ èñïîëüçîâàíèÿ äëÿ èññëåäîâàíèé òêàíåé ÷åëîâåêà íàìè áûëî ïîëó÷åíî èíôîðìèðîâàííîå äîáðîâîëüíîå ñîãëàñèå ðîäèòåëåé è áîëüíûõ. Ðåãåíåðàöèÿ ó êðûñû èçó÷åíà íà äâóõ ìîäåëÿõ. Èññëåäîâàíèÿ ïðîâåäåíû íà áåëûõ ëàáîðàòîðíûõ áåñïîðîäíûõ êðûñàõ ñàìöàõ. Îïåðàöèþ ÷àñòè÷íîé ãåïàòýêòîìèè âûïîëíÿëè ïî ìåòîäèêå Õèããåíñà è Àíäåðñîíà [9]. Çàáîé æèâîòíûõ ïîä Êëåòî÷íàÿ òðàíñïëàíòîëîãèÿ è òêàíåâàÿ èíæåíåðèÿ Òîì II, ¹ 4, 2007 40 Îðèãèíàëüíûå èññëåäîâàíèÿ ýôèðíûì íàðêîçîì è âçÿòèå ïå÷åíè ïðîèçâîäèëè ÷åðåç 1, 2, 3, 5, 7 ñóò. ïîñëå îïåðàöèè. Íèòðàò ñâèíöà ââîäèëè âíóòðèâåííî îäíîêðàòíî â õâîñòîâóþ âåíó â äîçå 100 ìêÌ/êã âåñà æèâîòíîãî [10]. Çàáîé æèâîòíûõ ïîä ýôèðíûì íàðêîçîì è èçâëå÷åíèå ïå÷åíè ïðîèçâîäèëè ÷åðåç 24, 48, 72, 96, 120 ÷àñîâ ïîñëå ââåäåíèÿ íèòðàòà ñâèíöà. Ïå÷åíü ÷åëîâåêà è êðûñû ôèêñèðîâàëè â 10% íåéòðàëüíîì ôîðìàëèíå è çàëèâàëè â ïàðàôèí ïî ñòàíäàðòíîé ìåòîäèêå. Èììóíîãèñòîõèìè÷åñêîå îêðàøèâàíèå ñðåçîâ ïå÷åíè ÷åëîâåêà è êðûñû ïðîâîäèëè ñòðåïòàâèäèí-áèîòèíîâûì ìåòîäîì [11] ñ èñïîëüçîâàíèåì êîììåð÷åñêèõ ìîíî- êëîíàëüíûõ àíòèòåë è âèçóàëèçàöèîííûõ ñèñòåì ôèðì DAKO (Äàíèÿ) è NOVOCASTRA (Âåëèêîáðèòàíèÿ).  ðàáîòå èñïîëüçîâàíû àíòèòåëà ê ýïèòåëèàëüíûì ìàðêåðàì öèòîêåðàòèíàì 18, 19, ýïèòåëèàëüíîìó ìåìáðàííîìó àíòèãåíó (Epithelial Membrane Antigen, EMA), ýïèòåëèàëüíîìó ñïåöèôè÷åñêîìó àíòèãåíó (Epithelial Specific Antigen, ESA), ñïåöèôè÷åñêîìó àíòèãåíó ãåïàòîöèòîâ (Hepatocyte Specific Antigen, HSA), áèëèàðíîìó ãëèêîïðîòåèíó CD66à; ê ìåçåíõèìàëüíûì ìàðêåðàì âèìåíòèíó, äåñìèíó, àëüôà-ãëàäêîìûøå÷íîìó àêòèíó (α-SÌÀ); ê ìàðêåðàì ñòâîëîâûõ è ïðîãåíèòîðíûõ êëåòîê Bcl-2, C-kit, C-met. Òàêæå áûëî ïðîâåäåíî äâîé- À Á Â Ã Ä Ðèñ. 1. Èììóíîãèñòîõèìè÷åñêîå îêðàøèâàíèå íà ESA (À, Á), HSA (Â), CD66 (Ã), EMA (Ä). Ïðîäóêò ðåàêöèè êðàñíîãî öâåòà: À ESA â ãåïàòîáëàñòàõ ýìáðèîíà 4 íåäåëü ãåñòàöèè, ×100; Á ESA-ïîçèòèâíûå êëåòêè â çîíå âîñïàëåíèÿ ïðè õðîíè÷åñêîì ãåïàòèòå, õ400;  HSA â ãåïàòîöèòàõ ïëîäà 13,5 íåäåëü ãåñòàöèè, ×400; à CD66 íà áèëèàðíûõ ïîëþñàõ ãåïàòîöèòîâ ïëîäà 13,5 íåäåëü ãåñòàöèè, ×400; Ä ÅÌÀ â ýïèòåëèè êèøêè ýìáðèîíà 4 íåäåëü ãåñòàöèè (â ïå÷åíè (âíèçó ñïðàâà) ÅÌÀ îòñóòñòâóåò), ×200 Êëåòî÷íàÿ òðàíñïëàíòîëîãèÿ è òêàíåâàÿ èíæåíåðèÿ Òîì II, ¹ 4, 2007 Îðèãèíàëüíûå èññëåäîâàíèÿ íîå èììóíîãèñòîõèìè÷åñêîå îêðàøèâàíèå ýìáðèîíàëüíîé è ïëîäíîé ïå÷åíè ÷åëîâåêà íà äåñìèí/öèòîêåðàòèíû 18, 19, äåñìèí/HSA è äåñìèí/Bcl-2. Ðåçóëüòàòû è îáñóæäåíèå Ðàçâèòèå ïå÷åíè ÷åëîâåêà íà÷èíàåòñÿ ñ âûñåëåíèÿ íåáîëüøîãî êîëè÷åñòâà ýïèòåëèàëüíûõ êëåòîê èç ýïèòåëèÿ ïåðåäíåé êèøêè â ìåçåíõèìó ïîïåðå÷íîé ïåðåãîðîäêè. Ðåçóëüòàòû èçó÷åíèÿ çàêîíîìåðíîñòåé ýêñïðåññèè ýïèòåëèàëüíûõ ìàðêåðîâ âûÿâèëè ðàçëè÷íûå ïàòòåðíû èõ ïîÿâëåíèÿ è äèíàìèêè. Òàê, óæå íà 4-é íåäåëå ðàçâèòèÿ ýïèòåëèàëüíûå êëåòêè êèøêè ýêñïðåññèðóþò ýïèòåëèàëüíûé ñïåöèôè÷åñêèé àíòèãåí ESA. Ìîæíî âèäåòü ðÿäû èíòåíñèâíî îêðàøåííûõ êëåòîê, òÿíóùèõñÿ îò äâåíàäöàòèïåðñòíîé êèøêè â ïå÷åíü. ×åðåç íåäåëþ óæå ïðàêòè÷åñêè âñå ýïèòåëèàëüíûå êëåòêè ïå÷åíè ýêñïðåññèðóþò ýòîò áåëîê (ðèñ. 1À). Ïîçäíåå, ïî ìåðå äèôôåðåíöèðîâêè ãåïàòîáëàñòîâ, ýêñïðåññèÿ ESA ïîñòåïåííî ñíèæàåòñÿ, ñìåùàåòñÿ ê ïåðèôåðèè ïå÷åíè è ê êðóïíûì ñîñóäàì, à çàòåì èñ÷åçàåò.  äåôèíèòèâíîé ïå÷åíè äàííûé ïðîòåèí ïðèñóòñòâóåò òîëüêî â êëåòêàõ âíóòðèïå÷åíî÷íûõ æåë÷íûõ ïðîòîêîâ. Òàêèì îáðàçîì, ìîæíî çàêëþ÷èòü, ÷òî ýêñïðåññèÿ ESA õàðàêòåðíà äëÿ ãåïàòîáëàñòîâ è íåçðåëûõ ãåïàòîöèòîâ. Ïîäòâåðæäåíèåì ýòîãî ÿâëÿåòñÿ è óñòàíîâëåííàÿ íàìè ýêñïðåññèÿ ESA â ðåãåíåðèðóþùåé ïå÷åíè ÷åëîâåêà: ìû íàáëþäàëè ìåëêèå ESA+ êëåòêè â çîíàõ íåêðîçîâ è âîñïàëåíèÿ (ðèñ. 1Á), ÷òî ìîæåò îòðàæàòü ïðîöåññ äèôôåðåíöèðîâêè ñòâîëîâîé êëåòêè ïå÷åíè â ãåïàòîöèòû. Ó÷èòûâàÿ, ÷òî ESA ìîæåò ýêñïðåññèðîâàòüñÿ íå òîëüêî êëåòêàìè ýíòîäåðìû, íî è êëåòêàìè ìåçîäåðìàëüíîãî ýïèòåëèÿ [12], íå èñêëþ÷åíî, ÷òî ýòà ïðåäïîëàãàåìàÿ ñòâîëîâàÿ êëåòêà ìîæåò áûòü ëîêàëèçîâàíà â ñèíóñîèäàõ ïå÷åíè. Èçó÷åíèå äèíàìèêè ýêñïðåññèè ñïåöèôè÷åñêîãî àíòèãåíà ãåïàòîöèòîâ è áèëèàðíîãî ãëèêîïðîòåèíà CD66à ïîçâîëèëî óñòàíîâèòü, ÷òî, ïîÿâëÿÿñü íà 4-5 íåäåëÿõ ãåñòàöèè, ýêñïðåññèÿ ýòèõ ïðîòåèíîâ íàðàñòàåò, è ê 1314 íåäåëÿì îíè ïðèñóòñòâóþò âî âñåõ ãåïàòîöèòàõ (ðèñ. 1Â, Ã). Òàêèì îáðàçîì, äàííûå áåëêè ïîÿâëÿþòñÿ óæå â äîñòàòî÷íî äèôôåðåíöèðîâàííûõ ãåïàòîöèòàõ, è èõ ïðèñóòñòâèå õàðàêòåðèçóåò çðåëûé ôåíîòèï ýòèõ êëåòîê. Ïîÿâëåíèå â ðåãåíåðèðóþùèõ ãåïàòîöèòàõ èìåííî ýòèõ áåëêîâ ìîæåò áûòü èñïîëüçîâàíî êàê ïðèçíàê çàâåðøåíèÿ èõ äèôôåðåíöèðîâêè è ïîêàçàòåëü ôóíêöèîíàëüíîé çðåëîñòè äàííûõ êëåòîê ïîñëå ïîâðåæäåíèÿ ïå÷åíè. Ýïèòåëèàëüíûé ìåìáðàííûé àíòèãåí ýêñïðåññèðóåòñÿ â ðàçëè÷íûõ ýïèòåëèÿõ ïðåèìóùåñòâåííî íà àïèêàëüíîé ïîâåðõíîñòè êëåòîê, â çðåëîé ïå÷åíè îí ïðèñóòñòâóåò íà àïèêàëüíîé ïîâåðõíîñòè õîëàíãèîöèòîâ [13]. Ýïèòåëèàëüíûå êëåòêè ïåðåäíåé êèøêè ñ ñàìûõ ðàííèõ ñðîêîâ ýìáðèîãåíåçà ýêñïðåññèðóþò ýòîò áåëîê. Îäíàêî íè íà îäíîì èç èçó÷åííûõ ñðîêîâ ãåñòàöèè EMA íå áûë âûÿâëåí â ãåïàòîöèòàõ ÷åëîâåêà (ðèñ. 1Ä). Òàêèì îáðàçîì, ñ ðàííèõ ñðîêîâ ðàçâèòèÿ îñíîâíîé ýïèòåëèàëüíûé êëåòî÷íûé òèï ïå÷åíè ãåïàòîöèòû èìååò ôåíîòèïè÷åñêèå îòëè÷èÿ îò ýíòîäåðìàëüíîãî ýïèòåëèÿ. Ñ äðóãîé ñòîðîíû, ìåçåíõèìàëüíûå êëåòêè â îáëàñòè çàêëàäêè ïå÷åíè ýêñïðåññèðóþò íå òîëüêî ìàðêåðû ìåçåíõèìàëüíûõ êëåòîê âèìåíòèí è äåñìèí, íî è áåëêè öèòîñêåëåòà ýïèòåëèàëüíûõ êëåòîê öèòîêåðàòèíû 18 è 19. Ïðè÷åì ïîñëåäíèå â êëåòêàõ ìåçåíõèìû è â ñèíóñîèäàõ â ìîìåíò çàêëàäêè ïå÷åíè ýêñïðåññèðóþòñÿ íàìíîãî èíòåíñèâíåå, ÷åì â ãåïàòîáëàñòàõ (ðèñ. 2À). Áîëåå òîãî, ìàðêåð ïåðèñèíóñîèäàëüíûõ êëåòîê ïå÷åíè (èìåþùèõ ìåçîäåðìàëüíîå ïðîèñõîæäåíèå) - äåñìèí íà 47 íåäåëÿõ ãåñòàöèè ïðèñóòñòâóåò íå òîëüêî â ïåðèñèíóñîèäàëüíûõ êëåòêàõ ïå÷åíè (êëåòêàõ Èòî) è â ìåçåíõèìíûõ êëåòêàõ ïîïåðå÷íîé ïåðåãîðîäêè, íî è â ãåïàòîáëàñòàõ. Óðîâåíü ýêñïðåññèè äåñìèíà â ïîñëåäíèõ ñëàáûé, íî 41 îòäåëüíûå ãåïàòîáëàñòû îêðàøèâàþòñÿ äîñòàòî÷íî èíòåíñèâíî (ðèñ. 2Á). Ïîñëå ñåäüìîé íåäåëè ãåñòàöèè ýêñïðåññèÿ äåñìèíà â ìåçåíõèìíûõ è ïåðèñèíóñîèäàëüíûõ êëåòêàõ ÷åëîâåêà ïîñòåïåííî ñíèæàåòñÿ è ê 12-é íåäåëå ïðåêðàùàåòñÿ ïîëíîñòüþ.  äàëüíåéøåì äåñìèí â ïå÷åíè ÷åëîâåêà ìîæíî âûÿâèòü òîëüêî â ãëàäêîìûøå÷íûõ êëåòêàõ êðîâåíîñíûõ ñîñóäîâ. Èíòåðåñíåéøåé íàõîäêîé íàøåãî èññëåäîâàíèÿ ñòàëî âûÿâëåíèå êëåòîê, îäíîâðåìåííî ýêñïðåññèðóþùèõ äåñìèí è öèòîêåðàòèíû (ðèñ. 2Â). Êðîìå òîãî, êëåòêè, ýêñïðåññèðóþùèå äåñìèí, è HSA-ïîçèòèâíûå êëåòêè íàõîäÿòñÿ â íåïîñðåäñòâåííîé áëèçîñòè äðóã ê äðóãó (Ðèñ. 2Ã). Ïîñêîëüêó ÍSA áåëîê, êîòîðûé ðàíåå îáíàðóæèâàëñÿ òîëüêî â ôåòàëüíûõ è çðåëûõ ãåïàòîöèòàõ [14, 15], òàêàÿ êàðòèíà ïîçâîëÿåò ïðåäïîëîæèòü, ÷òî äåñìèí-ïîçèòèâíûå êëåòêè ìîãóò äèôôåðåíöèðîâàòüñÿ â ãåïàòîöèòû. Óñòàíîâëåííûé ôàêò ýêñïðåññèè êëåòêàìè ìåçåíõèìû è ñèíóñîèäíûìè êëåòêàìè ïå÷åíè öèòîêåðàòèíîâ, à ãåïàòîöèòàìè äåñìèíà â îäíè è òå æå ñðîêè ãåñòàöèè óêàçûâàåò íà îáùíîñòü ýòèõ êëåòîê è âîçìîæíîñòü òðàíñäèôôåðåíöèðîâêè îäíîãî êëåòî÷íîãî òèïà â äðóãîé. Ïîõîæèå ðåçóëüòàòû áûëè ïîëó÷åíû íàìè ðàíåå [16] ïðè èçó÷åíèè ïðåíàòàëüíîãî ðàçâèòèÿ ïå÷åíè êðûñû íà 1113 äíè ãåñòàöèè âñå êëåòêè æåëåçû ýêñïðåññèðóþò äåñìèí, çàòåì â öèòîïëàçìå áîëüøåé ÷àñòè äåñìèí+ êëåòîê ïîÿâëÿþòñÿ öèòîêåðàòèíû 8 è 18, à äåñìèí ïîñòåïåííî èñ÷åçàåò, è êëåòêè ñòàíîâÿòñÿ òèïè÷íûìè ãåïàòîáëàñòàìè. Îñòàâøàÿñÿ ÷àñòü äåñìèí + êëåòîê íèêóäà íå èñ÷åçàåò, îíè îñòàþòñÿ â êîíòàêòå ñ ãåïàòîáëàñòàìè, à çàòåì ñ ãåïàòîöèòàìè, è ïðåäñòàâëÿþò ñîáîé äåñìèí+ êëåòêè Èòî. Èñõîäÿ èç ïîëó÷åííûõ ðåçóëüòàòîâ, ìîæíî ïðåäïîëîæèòü, ÷òî ñóùåñòâóåò àëüòåðíàòèâíûé êèøå÷íîé ýíòîäåðìå èñòî÷íèê ðàçâèòèÿ ãåïàòîáëàñòîâ, à èìåííî ìåçåíõèìà ïîïåðå÷íîé ïåðåãîðîäêè. Áîëåå òîãî, íàëè÷èå â ãåïàòîáëàñòàõ è ñèíóñîèäíûõ êëåòêàõ ìàðêåðà ïåðèñèíóñîèäàëüíûõ êëåòîê ïå÷åíè äåñìèíà ïîçâîëÿåò âûäåëèòü êîíêðåòíóþ ïîïóëÿöèþ êëåòîê, ðàçâèâàþùèõñÿ èç ìåçåíõèìû, à çàòåì äàþùèõ íà÷àëî ãåïàòîöèòàì. Òàêîé ïîïóëÿöèåé, î÷åâèäíî, ÿâëÿþòñÿ ïåðèñèíóñîèäàëüíûå êëåòêè.  ëèòåðàòóðå èìåþòñÿ äàííûå î òåñíîé ñâÿçè ïðîëèôåðàöèè îâàëüíûõ êëåòîê, êîòîðûå, ñ îäíîé ñòîðîíû, ðàññìàòðèâàþòñÿ êàê ïîòîìêè ñòâîëîâûõ êëåòîê ïå÷åíè [5], à ñ äðóãîé íè÷åì íå îòëè÷àþòñÿ îò ãåïàòîáëàñòîâ [17], ñ ïðîëèôåðàöèåé ïåðèñèíóñîèäàëüíûõ êëåòîê [18, 19], ÷òî óêàçûâàåò íà òåñíóþ ôóíêöèîíàëüíóþ âçàèìîñâÿçü ìåæäó íèìè. Áîëåå òîãî, â ïîñëåäíèå ãîäû îïóáëèêîâàíû ðàáîòû, ïðÿìî èëè êîñâåííî ñâèäåòåëüñòâóþùèå î òîì, ÷òî ïåðèñèíóñîèäàëüíûå êëåòêè ìîãóò äèôôåðåíöèðîâàòüñÿ â ýïèòåëèé. Òàê, óñòàíîâëåíî [20], ÷òî â ïåðèîä ïå÷åíî÷íîãî ãåìîïîýçà ó ìûøåé è ó ÷åëîâåêà ñòðîìàëüíûå ýëåìåíòû ìèêðîîêðóæåíèÿ, îñíîâíóþ ìàññó êîòîðûõ ñîñòàâëÿþò ïåðèñèíóñîèäàëüíûå êëåòêè, îäíîâðåìåííî ýêñïðåññèðóþò ìàðêåðû ìåçåíõèìíûõ è ýïèòåëèàëüíûõ êëåòîê in vitro. Ìåòîäàìè äâîéíîãî èììóíîãèñòîõèìè÷åñêîãî îêðàøèâàíèÿ áûëî ïîêàçàíî, ÷òî ïîêîÿùèåñÿ ïåðèñèíóñîèäàëüíûå êëåòêè êðûñû è ÷åëîâåêà ýêñïðåññèðóþò ñïåöèôè÷åñêèé ìàðêåð ýïèòåëèàëüíûõ êëåòîê Å-êàäãåðèí êàê in vivo, òàê è in vitro [21]. Òàêæå áûëî îáíàðóæåíî, ÷òî ïåðèñèíóñîèäàëüíûå êëåòêè ÷åëîâåêà in vitro [22] è ïåðèñèíóñîèäàëüíûå êëåòêè òðåñêè [23] ýêñïðåññèðóþò öèòîêåðàòèíû. Ðåçóëüòàòû ðàíåå ïðîâåäåííûõ íàìè ýêñïåðèìåíòîâ ïî îðãàíîòèïè÷åñêîìó êóëüòèâèðîâàíèþ ýìáðèîíàëüíîé ïå÷åíè êðûñ è ïî êóëüòèâèðîâàíèþ ÷èñòîé ïîïóëÿöèè ïåðèñèíóñîèäàëüíûõ êëåòîê, â êîòîðûõ áûëî ïîêàçàíî, ÷òî in vitro îíè ïðèîáðåòàþò ôåíîòèï ãåïàòîáëàñòîâ/ãåïàòîöèòîâ, ïîçâîëèëè âïåðâûå îïèñàòü ìåçåíõèìàëüíî-ýïèòåëèàëüíóþ òðàíñôîðìàöèþ ïåðèñèíóñîèäàëüíûõ êëåòîê [24], ÷òî åùå áîëüøå ïîäòâåðæäàåò íàøè ïðåäïîëîæåíèÿ. Êëåòî÷íàÿ òðàíñïëàíòîëîãèÿ è òêàíåâàÿ èíæåíåðèÿ Òîì II, ¹ 4, 2007 42 Îðèãèíàëüíûå èññëåäîâàíèÿ À Á Â Ã Ä Äëÿ âûÿñíåíèÿ ñòâîëîâûõ ïîòåíöèé ìåçåíõèìíûõ êëåòîê ïå÷åíè áûëè èçó÷åíû çàêîíîìåðíîñòè ýêñïðåññèè ìàðêåðîâ ñòâîëîâûõ è ïðîãåíèòîðíûõ êëåòîê â ïðåíàòàëüíîì îíòîãåíåçå ïå÷åíè ÷åëîâåêà è â õîäå ðåãåíåðàöèè ïå÷åíè. Ðåçóëüòàòû èçó÷åíèÿ ýêñïðåññèè ìàðêåðîâ ïðîãåíèòîðíûõ êëåòîê ïîêàçàëè, ÷òî ïî÷òè âî âñåõ êëåòêàõ ìåçåíõèìû, îêðóæàþùåé ïå÷åíü, è â áîëüøèíñòâå å¸ ñèíóñîèäíûõ êëåòîê, â òîì ÷èñëå è â äåñìèí+ (ðèñ. 2Ä), ïðîèñõîäèò ýêñïðåññèÿ àíòèàïîïòîòè÷åñêîãî ïðîòåèíà Bcl-2 ñ ÷åòâåðòîé äî ñåäüìîé íåäåëè ãåñòàöèè âêëþ÷èòåëüíî. Ýòîò áåëîê ïðèñóòñòâîâàë òàêæå â åäèíè÷íûõ ãåïàòîöèòàõ. Î÷åâèäíî, ÷òî äàííûé ïàòòåðí ýêñïðåññèè Bcl-2 (ÿâëÿþùåãîñÿ îäíèì èç ìàðêåðîâ ïðîãåíèòîðíûõ êëåòîê) ìîæåò ñâèäåòåëüñòâîâàòü î Êëåòî÷íàÿ òðàíñïëàíòîëîãèÿ è òêàíåâàÿ èíæåíåðèÿ Òîì II, ¹ 4, 2007 Ðèñ. 2. Èììóíîãèñòîõèìè÷åñêîå îêðàøèâàíèå íà öèòîêåðàòèí 19 (À) è äåñìèí (Á). Ïðîäóêò ðåàêöèè êðàñíîãî öâåòà. Äâîéíîå èììóíîãèñòîõèìè÷åñêîå îêðàøèâàíèå íà äåñìèí (ïðîäóêò ðåàêöèè - êðàñíîãî öâåòà) è öèòîêåðàòèí 19 (Â), HSA (Ã), Bcl-2 (Ä) - ïðîäóêò ðåàêöèè ñèíåãî öâåòà: À öèòîêåðàòèí19 â ñèíóñîèäíûõ êëåòêàõ (ñòðåëêè) ýìáðèîíà 4-5 íåäåëü ãåñòàöèè, ×400; Á äåñìèí â ãåïàòîáëàñòàõ ýìáðèîíà 4 íåäåëü ãåñòàöèè, ×1000;  äåñìèí+/öèòîêåðàòèí 19+ êëåòêè (ñòðåëêè) â ïå÷åíè ýìáðèîíà 4 íåäåëü ãåñòàöèè, ×1000; à äåñìèí è HSA â êëåòêàõ (ñòðåëêè) ïå÷åíè ýìáðèîíà 6 íåäåëü ãåñòàöèè, ×1000; Ä äåñìèí+/Bcl-2+ êëåòêè (ñòðåëêè) â ïå÷åíè ýìáðèîíà 4 íåäåëü ãåñòàöèè, ×1000 ñòâîëîâûõ ïîòåíöèÿõ ñèíóñîèäíûõ êëåòîê ïå÷åíè è êëåòîê îêðóæàþùåé å¸ ìåçåíõèìû, â òîì ÷èñëå è äåñìèí-ïîçèòèâíûõ êëåòîê. C-met ÿâëÿåòñÿ ðåöåïòîðîì ê ôàêòîðó ðîñòà ãåïàòîöèòîâ (ñèëüíîìó ìèòîãåíó äëÿ ýòèõ êëåòîê) è ðàññìàòðèâàåòñÿ â êà÷åñòâå îäíîãî èç ìàðêåðîâ ñòâîëîâûõ êëåòîê [25].  êîíöå ÷åòâåðòîé íà÷àëå ïÿòîé íåäåëè âíóòðèóòðîáíîãî ðàçâèòèÿ âûÿâëåíà ñëàáàÿ ýêñïðåññèÿ C-met â ãåïàòîáëàñòàõ, ïðè÷¸ì íåìíîãî èíòåíñèâíåå îêðàøèâàëèñü êëåòêè íà ïåðèôåðèè ïå÷åíè (ðèñ. 3À), îñîáåííî â îáëàñòè ìåçåíõèìû ïîïåðå÷íîé ïåðåãîðîäêè. Íà 68 íåäåëÿõ ãåñòàöèè ýêñïðåññèÿ Ñ-met â ãåïàòîöèòàõ ñòàíîâèëàñü åùå áîëåå íåîäíîðîäíîé: áîëåå ÿðêî îêðàøèâàëèñü ãåïàòîáëàñòû (ðèñ. 3Á) Îðèãèíàëüíûå èññëåäîâàíèÿ è ñâîáîäíî ëåæàùèå ãåïàòîöèòû, òîãäà êàê â «ïëîòíî óïàêîâàííûõ» ãåïàòîöèòàõ ýêñïðåññèÿ áûëà íàìíîãî ñëàáåå.  öåíòðàëüíûõ îòäåëàõ ïå÷åíè òàêæå áûëè âûÿâëåíû Ñ-met+ êëåòêè. Îíè áûëè áëèçêè ïî ñâîèì ìîðôîëîãè÷åñêèì ïðèçíàêàì ê ãåïàòîöèòàì, èíòåíñèâíîñòü èõ îêðàøèâàíèÿ áûëà óìåðåííîé.  ýòè æå ñðîêè (4-9 íåäåëü ãåñòàöèè) ýêñïðåññèÿ C-met áûëà âûÿâëåíà â ìåëêèõ, î÷åâèäíî, ñèíóñîèäíûõ, êëåòêàõ, è â íèõ îíà áûëà íàìíîãî áîëåå èíòåíñèâíîé, ÷åì â ãåïàòîáëàñòàõ è ãåïàòîöèòàõ. Ñèíóñîèäíûå C-met+ êëåòêè áûëè ëîêàëèçîâàíû â îñíîâíîì íà ïåðèôåðèè ïå÷åíè, à â íåêîòîðûõ ìåñòàõ êëåòêè îáðàçîâûâàëè êëàñòåðû (ðèñ. 3Â). Òàêèå æå êëåòêè âíóòðè ïå÷åíè ìîæíî áûëî âèäåòü îêîëî ñîñóäîâ, íî îíè áûëè åäèíè÷íûìè. C-met+ êëåòêè âûÿâëÿëèñü â ñèíóñîèäàõ ïå÷åíè è â áîëåå ïîçäíèå ñðîêè ïðåíàòàëüíîãî ðàçâèòèÿ (êàê ìèíèìóì, äî 14-é íåäåëè ãåñòàöèè).  õîäå ðåãåíåðàöèè ïå÷åíè ÷åëîâåêà ìû òàêæå âûÿâèëè C-met-ïîçèòèâíûå ñèíóñîèäíûå êëåòêè â çîíàõ íåêðîâîñïàëèòåëüíûõ èçìåíåíèé (ðèñ. 3Ã). Êðîìå òîãî, êëåòêè òàêîé æå ëîêàëèçàöèè è ìîðôîëîãèè ýêñïðåññèðîâàëè ESA (ñì. ðèñ. 1Á). 43 Ïðèñóòñòâèå Ñ-met â ìåçåíõèìíûõ êëåòêàõ (èíòåíñèâíàÿ ýêñïðåññèÿ), ãåïàòîáëàñòàõ (óìåðåííàÿ ýêñïðåññèÿ) è ãåïàòîöèòàõ (ñëàáàÿ ýêñïðåññèÿ) ïîçâîëÿåò ãîâîðèòü î ñóùåñòâîâàíèè êëåòîê ñ ïåðåõîäíûì ôåíîòèïîì è ïðåäïîëàãàòü, ÷òî ñèíóñîèäíûå êëåòêè, ýêñïðåññèðóþùèå Ñ-met, ìîãóò áûòü ïðåäøåñòâåííèêàìè ãåïàòîöèòîâ, òî åñòü äàííûé ïðîòåèí ïîÿâëÿåòñÿ â êëåòêàõ, êîòîðûå, èìåÿ ìåçåíõèìíîå ïðîèñõîæäåíèå, ñòàíîâÿòñÿ íà ïóòü äèôôåðåíöèðîâêè â ýïèòåëèé. Ó÷èòûâàÿ òî, ÷òî Ñ-met ÿâëÿåòñÿ ðåöåïòîðîì ê ôàêòîðó ðîñòà ãåïàòîöèòîâ, êîòîðûé òàêæå íàçûâàþò ðàññåèâàþùèì ôàêòîðîì [26, 27], ìîæíî ïðåäïîëîæèòü òàêæå, ÷òî ýêñïðåññèðóþùèå äàííûé áåëîê êëåòêè ÿâëÿþòñÿ ïîäâèæíûìè è ìîãóò äàëåå ðàñïðîñòðàíÿòüñÿ â äðóãèå îáëàñòè ïå÷åíè. Ïî ìåðå äèôôåðåíöèðîâêè êëåòîê ýêñïðåññèÿ ðåöåïòîðà ê ôàêòîðó ðîñòà ãåïàòîöèòîâ ïîñòåïåííî óìåíüøàåòñÿ (î ÷åì ñâèäåòåëüñòâóåò ñíèæåíèå èíòåíñèâíîñòè îêðàøèâàíèÿ äèôôåðåíöèðîâàííûõ êëåòêîê ãåïàòîöèòîâ). Íàëè÷èå åäèíè÷íûõ C-met + êëåòîê îêîëî ñîñóäîâ ïîçâîëÿåò ïðåäïîëàãàòü, ÷òî ýòè êëåòêè ìîãóò ïîñòóïàòü â ïå÷åíü íå òîëüêî èç ìåçåíõèìû ïîïåðå÷íîé ïåðåãîðîäêè, íî è ñ êðîâüþ. À Á  à Ðèñ. 3. Ýêñïðåññèÿ C-met â ýìáðèîãåíåçå (À, Á, Â) è ïðè ðåãåíåðàöèè (Ã). Ïðîäóêò èììóíîãèñòîõèìè÷åñêîé ðåàêöèè êðàñíîãî öâåòà, ×400: À â ãåïàòîáëàñòàõ ýìáðèîíà 4 íåäåëü ãåñòàöèè; Á â ãåïàòîáëàñòàõ ýìáðèîíà 7 íåäåëü ãåñòàöèè;  âèäíû êëàñòåðû êëåòîê â ñèíóñîèäàõ è íà ïåðèôåðèè ïå÷åíè ýìáðèîíà 7 íåäåëü ãåñòàöèè; à â çîíå âîñïàëåíèÿ ïðè õðîíè÷åñêîì ãåïàòèòå Êëåòî÷íàÿ òðàíñïëàíòîëîãèÿ è òêàíåâàÿ èíæåíåðèÿ Òîì II, ¹ 4, 2007 44 Îðèãèíàëüíûå èññëåäîâàíèÿ Ñ 4,55 íåäåëü ãåñòàöèè âñå ãåïàòîáëàñòû ñëàáî ýêñïðåññèðîâàëè Ñ-kit (CD117) ðåöåïòîð ê ôàêòîðó ñòâîëîâûõ êëåòîê, ìàðêåð êðîâåòâîðíûõ ñòâîëîâûõ êëåòîê. ×åðåç íåäåëþ ïåðèôåðèÿ ïå÷åíè íà ãðàíèöå ñ ìåçåíõèìîé îêðàøèâàëàñü ñèëüíåå, à ñ 5,56 íåäåëè îêîëî ñîñóäîâ ïîÿâëÿëèñü ÿðêî îêðàøåííûå åäèíè÷íûå ãåïàòîöèòû (ìàêñèìàëüíîå ÷èñëî òàêèõ êëåòîê âûÿâëåíî íà 7-é íåäåëå ãåñòàöèè) è åäèíè÷íûå îòðîñò÷àòûå ñèíóñîèäíûå êëåòêè (ðèñ. 4À). Ïîñëåäíèå ñîõðàíÿëèñü â ïå÷åíè íà âñåõ èçó÷åííûõ íàìè ñðîêàõ ãåñòàöèè. Óäèâèòåëüíîé íàõîäêîé ñòàëî îáíàðóæåíèå ýêñïðåññèè Ñ-kit â ãåïàòîöèòàõ ðåãåíåðèðóþùåé ïå÷åíè êðûñû. ×åðåç ñóòêè ïîñëå ÷àñòè÷íîé ãåïàòýêòîìèè ìîæíî áûëî âèäåòü åäèíè÷íûå è ñãðóïïèðîâàííûå ãåïàòîöèòû. Èõ ÷èñëî îñòàâàëîñü íåèçìåííûì äî êîíöà ýêñïåðèìåíòà (ðèñ. 4Á). ×åðåç äâîå ñóòîê ïîñëå ââåäåíèÿ íèòðàòà ñâèíöà ïîÿâëÿëèñü åäèíè÷íûå è ñãðóïïèðîâàííûå Ñ-kit-ïîçèòèâíûå ãåïàòîöèòû, ÷åðåç 3 ñóòîê èõ ÷èñëî ñíèæàëîñü, è ê 5 ñóòêàì òàêèå êëåòêè èñ÷åçàëè. Ìû òàêæå îáíàðóæèëè ýêñïðåññèþ Ñ-kit â ñèíóñîèäíûõ êëåòêàõ: ÷åðåç ñóòêè ïîñëå íà÷àëà ýêñïåðèìåíòîâ âûÿâëÿëèñü ìíîãî÷èñëåííûå ìåëêèå âûòÿíóòûå êëåòêè, ýêñïðåññèðóþùèå ýòîò áåëîê (ðèñ. 4Â). Ïî ìåðå âîññòàíîâëåíèÿ ïàðåíõèìû ïå÷åíè â õîäå ðåãåíåðàöèè ÷èñëî èõ íåñêîëüêî óìåíüøàëîñü. Ïîõîæèå çàêîíîìåðíîñòè áûëè óñòàíîâëåíû è ïðè èçó÷åíèè ðåãåíåðàöèè ïå÷åíè ÷åëîâåêà C-kit-ïîçèòèâíûå êëåòêè âûÿâëåíû â çîíàõ ïîðòàëüíîãî íåêðîçà è âîñïàëåíèÿ, òàêæå ýêñïðåññèÿ ýòîãî ðåöåïòîðà îáíàðóæåíà â ñèíóñîèäíûõ êëåòêàõ (ðèñ. 4Ã). ×èñëî C-kit+ êëåòîê çàâèñåëî îò òÿæåñòè ãåïàòèòà: îíî íàðàñòàëî ïðîïîðöèîíàëüíî òÿæåñòè çàáîëåâàíèÿ, äîñòèãàëî ìàêñèìóìà ïðè óìåðåííîì ãåïàòèòå, à çàòåì ïîñòåïåííî ñíèæàëîñü. Ïîëó÷åííûå ðåçóëüòàòû, òàêèì îáðàçîì, òàêæå ïîäòâåðæäàþò ñòâîëîâûå ïîòåíöèè ñèíóñîèäíûõ êëåòîê ïå÷åíè. Ïðèñóòñòâèå C-kit â ãåïàòîáëàñòàõ íà ðàííèõ ñðîêàõ ãåñòàöèè (4,55 íåäåëè), ïî-âèäèìîìó, óêàçûâàåò íà ÷óâñòâèòåëüíîñòü ýòèõ êëåòîê ê ôàêòîðó ñòâîëîâûõ êëåòîê. Óñòàíîâëåíî, ÷òî âûäåëåííûå èç ïå÷åíè C-kit+ êëåòêè äåëÿòñÿ â êóëüòóðå, è â íèõ ïîÿâëÿåòñÿ àëüáóìèí [28], òî åñòü îíè ìîãóò áûòü ïðåäøåñòâåííèêàìè ãåïàòîöèòîâ. Ó÷èòûâàÿ, ÷òî C-kit ÿâëÿåòñÿ ìàðêåðîì êðîâåòâîðíûõ ñòâîëîâûõ êëåòîê, ìîæíî ïðåäïîëîæèòü, ÷òî â ïåðèîä ñòàíîâëåíèÿ ïå÷åíî÷íîãî êðîâåòâîðåíèÿ À Á  à Ðèñ. 4. Ýêñïðåññèÿ C-kit â ýìáðèîãåíåçå (À) è ïðè ðåãåíåðàöèè (Á, Â, Ã). Ïðîäóêò èììóíîãèñòîõèìè÷åñêîé ðåàêöèè êðàñíîãî öâåòà, ×400: À â ñèíóñîèäíûõ êëåòêàõ ýìáðèîíà 5-6 íåäåëü ãåñòàöèè; Á â ãåïàòîöèòàõ êðûñû ÷åðåç 3 ñóòîê ïîñëå ÷àñòè÷íîé ãåïàòýêòîìèè;  â ñèíóñîèäíûõ êëåòêàõ (ñòðåëêè) ïå÷åíè ÷åðåç 3 ñóòîê ïîñëå ÷àñòè÷íîé ãåïàòýêòîìèè; à â âîñïàëèòåëüíîì èíôèëüòðàòå ïðè õðîíè÷åñêîì ãåïàòèòå Êëåòî÷íàÿ òðàíñïëàíòîëîãèÿ è òêàíåâàÿ èíæåíåðèÿ Òîì II, ¹ 4, 2007 Îðèãèíàëüíûå èññëåäîâàíèÿ (67 íåäåëè ãåñòàöèè), à òàêæå ïðè ðåãåíåðàöèè ïå÷åíè ÷àñòü ãåïàòîöèòîâ ìîæåò îáðàçîâûâàòüñÿ èç êðîâåòâîðíûõ ñòâîëîâûõ êëåòîê. Îäíàêî ìàññîâîå ó÷àñòèå êðîâåòâîðíûõ ñòâîëîâûõ êëåòîê â ðàçâèòèè ãåïàòîöèòîâ âûçûâàåò ñîìíåíèÿ, ïîñêîëüêó â ãèñòîãåíåçå ïå÷åíè ÷èñëî Ñ-kit+ êëåòîê íå áûëî äîñòàòî÷íî áîëüøèì, ÷òîáû îïðåäåëÿòü òå÷åíèå è èñõîä äàííûõ ñîáûòèé. Áîëåå âåðîÿòíî, íà íàø âçãëÿä, ÷òî êðîâåòâîðíûå ñòâîëîâûå êëåòêè èãðàþò îïðåäåëåííóþ ðîëü â âîññòàíîâëåíèè ïóëà ðåãèîíàëüíûõ ñòâîëîâûõ êëåòîê, ïîâèäèìîìó ñèíóñîèäíûõ. Ýòî ïîäòâåðæäàþò è äàííûå äðóãèõ àâòîðîâ. Ïîêàçàíî, ÷òî â 38% ÑD34+ êëåòîê (CD34 îäèí èç ìàðêåðîâ êðîâåòâîðíûõ ñòâîëîâûõ êëåòîê), èçîëèðîâàííûõ èç ïå÷åíè ÷åëîâåêà 14-22 íåäåëü ãåñòàöèè, ìîãóò ýêñïðåññèðîâàòüñÿ öèòîêåðàòèíû, äåñìèí è íåêîòîðûå äðóãèå ìàðêåðû ïåðèñèíóñîèäàëüíûõ êëåòîê [29], ÷òî íå èñêëþ÷àåò âîçìîæíîñòè äèôôåðåíöèðîâêè CD34+ ñòâîëîâîé êðîâåòâîðíîé êëåòêè â äåñìèí-ïîçèòèâíóþ ïåðèñèíóñîèäàëüíóþ êëåòêó, à çàòåì â öèòîêåðàòèí-ïîçèòèâíûé ãåïàòîáëàñò. Îäíàêî, ó÷èòûâàÿ ìàëî÷èñëåííîñòü C-kit-ïîçèòèâíûõ êëåòîê â ýìáðèîíàëüíîé ïå÷åíè, î÷åâèäíî, ÷òî ÷èñëî òàêèõ ãåïàòîöèòîâ âðÿä ëè áóäåò çíà÷èòåëüíûì. Îáíàðóæåíèå ýêñïðåññèè Ñ-kit â ìíîãî÷èñëåííûõ ñèíóñîèäíûõ êëåòêàõ ïå÷åíè è â åäèíè÷íûõ ãåïàòîöèòàõ â õîäå ðåãåíåðàöèè òàêæå ïîçâîëÿåò ñäåëàòü âûâîä, ÷òî, íåñìîòðÿ íà îòñóòñòâèå â ïå÷åíè îâàëüíûõ êëåòîê, ìîæåò ïðîèñõîäèòü àêòèâàöèÿ ðåãèîíàëüíîãî ñòâîëîâîãî êîìïàðòìåíòà, ëîêàëèçîâàííîãî â ñèíóñîèäàõ. Ìîæíî ïðåäïîëîæèòü, ÷òî Ñ-kit+ ñèíóñîèäíûå êëåòêè äàþò íà÷àëî ãåïàòîáëàñòàì, òàêæå ýêñïðåññèðóþùèì Ñ-kit. Ïîñêîëüêó ãåïàòîáëàñòû ÿâëÿþòñÿ êëåòêàìè äèôôåðåíöèðóþùèìèñÿ, â õîäå ýòîãî ïðîöåññà îíè ïîñòåïåííî óòðà÷èâàþò ñïîñîáíîñòü ýêñïðåññèðîâàòü ðåöåïòîð ê ôàêòîðó ñòâîëîâûõ êëåòîê, è ìû âèäèì ëèøü åäèíè÷íûå C-kit+ ãåïàòîöèòû. ËÈÒÅÐÀÒÓÐÀ: 1. Le Douarin N.M. An experimental analysis of liver development. Med. Biol. 1975; 53: 427-55. 2. Houssaint E. Differentiation of the mouse hepatic primordium. I. An analysis of tissue interactions in hepatocyte differentiation. Cell Differ. 1980; 9: 26979. 3. Desmet V.J. Embryology of the liver and intrahepatic biliary tract, and an overview of malformations of the bile duct. In: Oxford textbook of clinical hepatology. OxfordNew YorkTokyo: Oxford University Press; 1992: 497519. 4. Van Eyken P., Sciot R., Desmet V. Intrahepatic bile duct development in the rat. Lab. Invest. 1988; 59: 529. 5. Fausto N. Hepatocyte differentiation and liver progenitor cells. Curr. Opin. Cell Biol. 1990; 2: 103642. 6. Theise N.D., Badve S., Saxena R. et al. Derivation of hepatocytes from bone marrow cells of mice after radiation-induced myeloablation. Hepatology. 2000a; 31: 23540. 7. Theise N.D., Nimmakayalu M., Gardner R. et al. Liver from bone marrow in humans. Hepatology. 2000b; 32: 116. 8. Petersen B.E., Grossbard B., Hatch H. et al. Mouse A6-positive hepatic oval cells also express several hematopoietic stem cell markers. Hepatology. 2003; 37: 63240. 9. Higgins G.M., Anderson R.M. Experimental pathology of the liver: I. Restoration of the liver of the white rat following partial surgical removal. Arch. Pathol. 1931; 12: 186-202. 10. Shinozuka H., Kubo Y., Katyal S.L. et al. Roles of growth factors and of tumor necrosis factor-a on liver cell proliferation induced in rats by lead nitrate Lab. Invest. 1994; 71(1): 3541. 11. Êèÿñîâ À.Ï. Ñîâðåìåííûå òåõíîëîãèè ìîðôîëîãè÷åñêèõ èññëåäîâàíèé. Ìåòîäè÷åñêîå ïîñîáèå äëÿ ñòóäåíòîâ, àñïèðàíòîâ è âðà÷åéïàòîëîãîâ. Êàçàíü: ÊÃÌÓ, 2001. 12. Latza U., Niedobitek G., Schwarting R. et al. Ber-EP4: new monoclonal antybody which distinguishes epithelia from mesothelia. J. Clin. Pathol. 1990; 43: 21319. 13. Pinkus G.S, Kurtin P.J. Epithelial membrane antigen a diagnostic discriminant in surgical pathology: immunohistochemical profile in epithelial, mesenchymal, and hematopoietic neoplasms using paraffin sections and monoclonal antibodies. Hum. Pathol. 1985; 16(9): 92940. 14. Kakar S., Muir T., Murphy L.M. et al. Immunoreactivity of Hep Par 1 in hepatic and extrahepatic tumors and its correlation with albumin in situ hybridization in hepatocellular carcinoma. Am. J. Clin. Pathol. 2003; 119(3): 3616. 45 Âûäâèíóòàÿ íàìè ãèïîòåçà î âîçìîæíîñòè ðàçâèòèÿ ãåïàòîöèòîâ èç ìåçåíõèìíûõ êëåòîê ïîïåðå÷íîé ïåðåãîðîäêè è î ïåðèñèíóñîèäàëüíûõ êëåòêàõ, êàê ðåãèîíàëüíûõ ñòâîëîâûõ êëåòêàõ ïå÷åíè, íàõîäèò â ïîñëåäíèå ãîäû âñå áîëüøå ïîäòâåðæäåíèé â ðàáîòàõ äðóãèõ èññëåäîâàòåëåé. Òàê, óñòàíîâëåíà âîçìîæíîñòü îáðàçîâàíèÿ ãåïàòîöèòîâ èç ìåçåíõèìíûõ êëåòîê, âûäåëåííûõ èç æèðîâîé òêàíè ÷åëîâåêà in vitro è in vivo [30] è èç ìåçåíõèìíûõ ñòâîëîâûõ êëåòîê in vitro [31, 32]. Áûëî ïîêàçàíî, ÷òî ïîñëå òðàíñïëàíòàöèè ëåòàëüíî îáëó÷åííûì êðûñàì êëåòîê êðàñíîãî êîñòíîãî ìîçãà ïîñëåäíèå ìîãóò äàâàòü íà÷àëî îâàëüíûì êëåòêàì è ãåïàòîöèòàì [33]. Ïîçäíåå ïîäîáíûå ýêñïåðèìåíòû ñ àíàëîãè÷íûìè ðåçóëüòàòàìè áûëè ïðîâåäåíû íà ìûøàõ [6, 34, 35]. Ïîÿâèëèñü äàííûå îá îáðàçîâàíèè ïåðèñèíóñîèäàëüíûõ êëåòîê èç ñòâîëîâûõ êðîâåòâîðíûõ êëåòîê ó ìûøåé [36]. È, íàêîíåö, â 2006 ãîäó îïóáëèêîâàíà ñòàòüÿ, â êîòîðîé âïåðâûå ïåðèñèíóñîèäàëüíûå êëåòêè ðàññìàòðèâàþòñÿ êàê ñòâîëîâûå êëåòêè, ïîñêîëüêó ýêñïðåññèðóþò îäèí èç ìàðêåðîâ êðîâåòâîðíûõ ñòâîëîâûõ êëåòîê CD133 [37]. Ïîäâîäÿ èòîã, ìîæíî ñêàçàòü, ÷òî óñòàíîâëåííûé íàìè ôàêò ñóùåñòâîâàíèÿ â ïå÷åíè è îêðóæàþùåé åå ìåçåíõèìå êëåòîê, ñî÷åòàþùèõ ôåíîòèïè÷åñêèå ïðèçíàêè ïðîãåíèòîðíûõ êëåòîê, ïåðèñèíóñîèäàëüíûõ êëåòîê ïå÷åíè è ãåïàòîöèòîâ, ñâèäåòåëüñòâóåò î âîçìîæíîñòè ðàçâèòèÿ ãåïàòîöèòîâ èç äåñìèí-ïîçèòèâíûõ êëåòîê ìåçåíõèìû ïîïåðå÷íîé ïåðåãîðîäêè, ÷òî âïëîòíóþ ïðèáëèæàåò íàñ ê ðàçãàäêå âîïðîñà î ðåãèîíàëüíîé ñòâîëîâîé êëåòêå ïå÷åíè, êîòîðîé ìîæåò ÿâëÿòüñÿ ïîïóëÿöèÿ çâ¸çä÷àòûõ ïåðèñèíóñîèäàëüíûõ êëåòîê (êëåòîê Èòî). Ðàáîòà âûïîëíåíà ïðè ôèíàíñîâîé ïîääåðæêå ÐÔÔÈ (ãðàíò 04-04-49164) è Ôåäåðàëüíîé öåëåâîé ïðîãðàììû (ãîñóäàðñòâåííûé êîíòðàêò 02.512.11.2052 îò 27.02.2007). 15. Ramos-Vara J.A., Miller M.A., Johnson G.C. 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