Участие клеток Ито в гистогенезе и регенерации печени

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. Â 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 [1–3]. 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
ïðîòîêî⠖ 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 [6–8], 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 13–14 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
4–7 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 11–13 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. Â 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 6–8 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
Â
Ã
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 4,5–5 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 5,5–6 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 (4,5–5 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
Â
Ã
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
(6–7 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 3–8% 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:
269–79.
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.
Oxford–New York–Tokyo: Oxford University Press; 1992: 497–519.
4. Van Eyken P., Sciot R., Desmet V. Intrahepatic bile duct development in
the rat. Lab. Invest. 1988; 59: 52–9.
5. Fausto N. Hepatocyte differentiation and liver progenitor cells. Curr. Opin.
Cell Biol. 1990; 2: 1036–42.
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: 235–40.
7. Theise N.D., Nimmakayalu M., Gardner R. et al. Liver from bone marrow
in humans. Hepatology. 2000b; 32: 11–6.
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: 632–40.
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): 35–41.
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:
213–19.
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): 929–40.
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): 361–6.
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. Immunohistochemical
characterization of canine hyperplastic hepatic lesions and hepatocellular and
biliary neoplasms with monoclonal antibody hepatocyte paraffin 1 and a
monoclonal antibody to cytokeratin 7. Vet. Pathol. 2001; 38(6): 636–43.
16. Kiassov A.P., Van Eyken P., van Pelt J.F. et al. Desmin expressing
nonhematopoietic liver cells during rat liver development: An immunohistochemical
and morphometric study. Differentiation. 1995; 59: 253–8.
17. Êèÿñîâ À.Ï., Ãóìåðîâà À.À., Òèòîâà Ì.À. Îâàëüíûå êëåòêè –
ïðåäïîëàãàåìûå ñòâîëîâûå êëåòêè ïå÷åíè èëè ãåïàòîáëàñòû? Êëåòî÷íàÿ
òðàíñïëàíòîëîãèÿ è òêàíåâàÿ èíæåíåðèÿ. 2006; 2(4): 55–8.
18. Evarts R.P., Nakatsukasa H., Marsden E.R. et al. Cellular and molecular
changes in the early stages of chemical hepatocarcinogenesis in the rat. Cancer
Res. 1990; 50: 3439–44.
19. Paku S., Schnur J., Nagy P. et al. Origin and structural evolution of the
early proliferating oval cells in rat liver. Am. J. Hepathol. 2001; 158: 1313-23.
20. Chagraoui J., Lepage-Noll A., Anjo A. et al. Fetal liver consists of cells in
epithelial-to-mesenchimal transition. Blood. 2003; 101: 2973-82.
21. Lim Y.S., Lee H.S. The expression of E-cadherin in human and rat hepatic
stellate cells: evidence of epithelial-mesenchymal transition. Taechan. Kan.
Hakhoe. Chi. 2002; 8: 90-9.
22. Lim Y.S., Kim K.A., Jung J.O. et al. Modulation of cytokeratin expression
during in vitro cultivation of human hepatic stellate cells: evidence of
transdifferentiation from epithelial to mesenchymal phenotype. Histochem. Cell
Biol. 2002; 118: 127-36.
23. Senda T., Nomura R. The expression of cytokeratin in hepatic stellate
cells of the cod. Arch. Histochem. Cytochem. 2003; 66: 437-44.
24. Êèÿñîâ À.Ï., Ãóìåðîâà À.À., Òèòîâà Ì.À. Ìåçåíõèìàëüíîýïèòåëèàëüíàÿ òðàíñôîðìàöèÿ êëåòîê Èòî in vitro. Êëåòî÷íûå òåõíîëîãèè â
áèîëîãèè è ìåäèöèíå. 2006; 3: 150-3.
25. Ljubimova J.Y., Petrovic L.M., Wilson S.E et al. Expression of HGF, its
receptor c-met, c-myc, and albumin in cirrhotic and neoplastic human liver tissue.
J. Histochem. Cytochem. 1997; 45(1): 79-87.
26. Borowiak M., Garratt A.N., Wüstefeld T. et al. Met provides essential signals
for liver regeneration. Proc. Natl. Acad. Sci. USA. 2004; 101(29): 10608-13.
27. Huh C.G., Factor V.M., Sánchez A. et al. Hepatocyte growth factor/cmet signaling pathway is required for efficient liver regeneration and repair. Proc.
Natl. Acad. Sci. USA. 2004; 101(13): 4477-82.
28. Corcelle V., Stieger B., Gjinovci A. et al. Characterization of two distinct
liver progenitor cell subpopulations of hematopoietic and hepatic origins. Exp.Ñell
Res. 2006; 312(15): 2826-36.
Êëåòî÷íàÿ òðàíñïëàíòîëîãèÿ è òêàíåâàÿ èíæåíåðèÿ Òîì II, ¹ 4, 2007
46
Îðèãèíàëüíûå èññëåäîâàíèÿ
29. Suskind D.L., Muench M.O. Searching for common stem cells of the
hepatic and hematopoietic systems in the human fateal liver: CD34+ cytokeratin
7/8+ cells express markers for stellate cells. J. Hepatol. 2004; 40: 261-8.
30. Seo M.J., Suh S.Y., Bae Y.C. et al. Differentiation of human adipose stromal
cells into hepatic lineage in vitro and in vivo. Biochem. Biophys. Res. Commun.
2005; 328: 258-64.
31. Lee K.D., Kuo T.K., Whang-Peng J. et al. In vitro hepatic differentiation of
human mesenchymal stem cells. Hepatology. 2004; 40: 1256-9.
32. Lange C., Bruns H., Kluth D. et al. Hepatocytic differentiation of
mesenchymal stem cells in cocultures with fetal liver cells. World J. Gastroenterol.
2006; 12(15): 2394-97.
33. Petersen B.E., Bowen W.C., Patrene K.D. et al. Bone marrow as a
potential source of hepatic oval cells. Science. 1999; 284: 1168-70.
34. Lagasse E., Connors H., Al-Dhalimy M. et al. Purified hematopoietic stem
cells can differentiate into hepatocytes in vivo. Nat. Med. 2000; 6: 1229-34.
35. Krause D.S., Theise N.D., Collector M.I. et al. Multiorgan, multi-lineage
engraftment by a single bone marrow-derived stem cell. Cell. 2001; 105:
369-77.
36. Baba S., Fujii H., Hirose T. et al. Commitment of bone marrow cells to
hepatic stellate cells in mouse. J. Hepatol. 2004; 40: 255-60.
37. Kordes C., Sawitza I., Müller-Marbach A. et al. CD133+ hepatic stellate cells
are progenitor cells. Biochem. Biophys. Res. Commun. 2007; 352(2): 410-17.
Ïîñòóïèëà 16.06.2007
Êëåòî÷íàÿ òðàíñïëàíòîëîãèÿ è òêàíåâàÿ èíæåíåðèÿ Òîì II, ¹ 4, 2007