11 Íîâîñòè êëåòî÷íûõ òåõíîëîãèé

Íîâîñòè êëåòî÷íûõ òåõíîëîãèé
8. Howell A.N., Sager R. Tumorigenicity and its suppression in cybrids of mouse
and Chinese hamster cell lines. Proc. Natl. Acad. Sci. USA 1978; 75: 2358–62.
9. Abken H., Jungfer H., Albert W.H., Willecke K. Immortalization of human
lymphocytes by fusion with cytoplasts of transformed mouse L cells. J. Cell Biol.
1986; 103: 795–805.
10. Atsumi T., Shirayoshi Y., Takeichi M., Okada T.S. Nullipotent
teratocarcinoma cells acquire the pluripotency for differentiation by fusion with
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somatic cells. Differentiation 1982; 23: 83–6.
11. Do J.T., Scholer H.R. Nuclei of embryonic stem cells reprogram somatic
cells. Stem Cells 2004; 22: 941–9.
12. Takei S., Yamamoto M., Cui L. et al. Phenotype-specific cells with
proliferative potential are produced by polyethylene glycol-induced fusion of mouse
embryonic stem cells with fetal cardiomyocytes. Cell Transplant. 2005; 14(9):
701-8.
Ïîäãîòîâèë À.Â. Áåðñåíåâ
ïî ìàòåðèàëàì Reprod. BioMed Online 2006; 12 (1): 107–11
Ëå÷åíèå ñåðïîâèäíî-êëåòî÷íîé àíåìèè êîìáèíàöèåé
ìåòîäîâ ãåííîé òåðàïèè è ÐÍÊ-èíòåðôåðåíöèè
ßâëåíèå ÐÍÊ-èíòåðôåðåíöèè (RNA interference) áûëî
îòêðûòî â õîäå ýêñïåðèìåíòîâ ïî ïîäàâëåíèþ ýêñïðåññèè
ãåíîâ ïðè ïîìîùè àíòèñìûñëîâîé ÐÍÊ ó C. elegans. Òåðìèí
«ÐÍÊ-èíòåðôåðåíöèÿ» (iRNA) äëÿ ôåíîìåíà ñïåöèôè÷åñêîãî ïîäàâëåíèÿ ýêñïðåññèè ãåíîâ ïðè ââåäåíèè äâóõöåïî÷å÷íîé ÐÍÊ áûë ïðåäëîæåí Andrew Fire â 1998 ãîäó [1,
2]. ÐÍÊ-èíòåðôåðåíöèÿ ïðåäïîëàãàåò ñïåöèôè÷åñêîå íàðóøåíèå ýêñïðåññèè òîëüêî òåõ ãåíîâ, êîòîðûå îáëàäàþò
äîñòàòî÷íî áîëüøîé ñòåïåíüþ ãîìîëîãèè ñ ââåäåííîé äâóõöåïî÷å÷íîé ÐÍÊ) (ðèñ.).
ÐÍÊ-èíòåðôåðåíöèÿ óæå øèðîêî èçó÷àåòñÿ ñ òåðàïåâòè÷åñêîé öåëüþ, íàïðèìåð, äëÿ ïîäàâëåíèÿ ýêñïðåññèè âèðóñíûõ ãåíîâ, îíêîãåíîâ èëè ñïåöèôè÷åñêèõ ãåíîâ, âûçûâàþùèõ çàáîëåâàíèÿ [3]. Äîñòîèíñòâà ýòîãî ìåòîäà – âûñîêàÿ
ñïåöèôè÷íîñòü (ïîäàâëÿåòñÿ ýêñïðåññèÿ òîëüêî òîãî ãåíà,
íóêëåîòèäíàÿ ïîñëåäîâàòåëüíîñòü êîòîðîãî ïîëíîñòüþ ñîîòâåòñòâóåò íóêëåîòèäíîé ïîñëåäîâàòåëüíîñòè ââîäèìîé
äâóõöåïî÷å÷íîé ÐÍÊ); âûñîêàÿ ýôôåêòèâíîñòü (ýêñïðåññèÿ
ãåíà ïîäàâëÿåòñÿ áîëåå ÷åì íà 90%, íåñêîëüêî äåñÿòêîâ
ìîëåêóë äâóíèòåâîé ÐÍÊ ìîãóò ïðèâåñòè ê äåãðàäàöèè íåñêîëüêèõ òûñÿ÷ ìîëåêóë ÐÍÊ-ìèøåíè).
Ïîêà òåðàïåâòè÷åñêîå èñïîëüçîâàíèå ÐÍÊ-èíòåðôåðåíöèè îãðàíè÷åíî, âî-ïåðâûõ, æåñòêèìè óñëîâèÿìè âûáîðà
ãåíà, ðàáîòó êîòîðîãî íàäî ïîäàâèòü, âî-âòîðûõ, èíäóêöèåé
îòâåòà èììóííîé ñèñòåìû íà ýêçîãåííóþ ÐÍÊ, êîòîðàÿ ìîæåò ïðèâåñòè ê ïîëíîìó ïîäàâëåíèþ ñèíòåçà áåëêà è àïîïòîçó [4, 5]. Ðåãóëÿöèÿ ñèíòåçà siRNA â îïðåäåëåííîå âðåìÿ
è â îïðåäåëåííûõ êëåòêàõ ïîçâîëèò ìèíèìèçèðîâàòü âîçìîæíîå ïîâðåæäàþùåå äåéñòâèå ÐÍÊ-èíòåðôåðåíöèè.
Ïðèìåíåíèå ÐÍÊ-èíòåðôåðåíöèè â êëåòî÷íîé òåðàïèè òðåáóåò òî÷íîãî, âûñîêîñïåöèôè÷íîãî îïðåäåëåíèÿ ãåíà, èãðàþùåãî ãëàâíóþ ðîëü â ðàçâèòèè çàáîëåâàíèÿ, òàê êàê
ÐÍÊ-èíòåðôåðåíöèÿ çàñòàâëÿåò ýòîò ãåí «çàìîë÷àòü».
Ó÷åíûå èç Sloan-Kettering Institute (New York, NY, USA)
âïåðâûå ïîêàçàëè âîçìîæíîñòü èñïîëüçîâàíèÿ ÐÍÊ-èíòåðôåðåíöèè âìåñòå ñ òðàíñãåíåçîì ïðè ëå÷åíèè ñåðïîâèäíîêëåòî÷íîé àíåìèè (ÑÊÀ).
Ïðè÷èíà ÑÊÀ çàêëþ÷àåòñÿ â îäíîíóêëåîòèäíîé çàìåíå
óðàöèëà íà àäåíèí, â ðåçóëüòàòå ÷åãî ñèíòåçèðóåòñÿ öåïü
ìîëåêóëû ãëîáèíà ñ ãëþòàìèíîì, âìåñòî âàëèíà. Çàìåíà
îäíîé àìèíîêèñëîòû îêàçûâàåòñÿ äîñòàòî÷íîé, ÷òîáû èçìåíèòü ôóíêöèîíàëüíûå ñâîéñòâà ãåìîãëîáèíà (ïîíèæåííàÿ ðàñòâîðèìîñòü, ïîâûøåííàÿ ïîëèìåðèçàöèÿ). Ïðè ýòîì
ãåìîãëîáèí óæå íå ìîæåò âûïîëíÿòü êèñëîðîäàêöåïòîðíóþ
ôóíêöèþ è êðèñòàëëèçóåòñÿ ïðè íåäîñòàòêå êèñëîðîäà, à
ýðèòðîöèòû ïðèîáðåòàþò ñåðïîâèäíóþ ôîðìó, ñêëåèâàþòñÿ,
òðîìáèðóþò êàïèëëÿðû è ò. ä. Ìóòàíòíûé ãåí ïîëó÷èë íàçâàíèå βs, â îòëè÷èå îò íîðìàëüíîãî β-ãëîáèíà.
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Íîâîñòè êëåòî÷íûõ òåõíîëîãèé
Íà ïåðâîì ýòàïå ýêñïåðèìåíòà, àâòîðû çàêîäèðîâàëè â
èíòðîí γ-ãëîáèíîâîãî ãåíà øïèëüêó ÐÍÊ (small hairpin RNA,
shRNA), êîòîðàÿ ïîçâîëÿåò ïîñòðîèòü ìàëûå èíòåðôåðèðóþùèå ÐÍÊ (small interfering RNA, siRNA), êîìïëåìåíòàðíûå ãåíó-ìèøåíè. Ñîâìåñòíàÿ ýêñïðåññèÿ ãåíà è siRNA
ïîçâîëÿåò ñïåöèôè÷åñêè ïîäàâëÿòü óðîâåíü òðàíñêðèïòîâ
ãåíà-ìèøåíè äëÿ siRNA ñòðîãî â îïðåäåëåííûõ êëåòêàõ è
íà îïðåäåëåííîé ñòàäèè. Ïîëîæåíèå shRNA â èíòðîíå ïîçâîëÿåò äîñòèãíóòü ñèíõðîííîãî óðîâíÿ ýêñïðåññèè ýêçîãåíà
è siRNA.
Ïî ðàñ÷¸òó ó÷¸íûõ, òðàíñôåêöèÿ ãåìîïîýòè÷åñêèõ ñòâîëîâûõ êëåòîê ñ ìóòàöèåé β> βs, ïðèâîäÿùåé ê ðàçâèòèþ ÑÊÀ,
äîëæíà ïðèâåñòè ê òðàíñêðèïöèè γ-ãëîáèíà ñ îäíîâðåìåííîé
ðåäóêöèåé ýêñïðåññèè βs-ãëîáèíà – áåëêà, îòâåòñòâåííîãî
çà âîçíèêíîâåíèå ýòîé áîëåçíè.
Äëÿ èññëåäîâàíèÿ âîçìîæíîñòè èñïîëüçîâàíèÿ ýòîãî
ìåòîäà â òåðàïèè ó÷åíûå âûáðàëè ãåíû, ýêñïðåññèðóþùèåñÿ â êëåòêàõ ýðèòðîëåéêåìèè êðûñû (murine erythroleukemia,
MEL): green fluorescent protein (GFP) è murine β-major (Mβ).
Êëåòêè MEL áûëè òðàíñôåöèðîâàíû êîíñòðóêöèåé ñ γ-ãëîáèíîì è shRNA, à çàòåì ïîäâåðãíóòû äèôôåðåíöèðîâêå äëÿ
çàïóñêà ýíäîãåííîé ýêñïðåññèè ãëîáèíà.
 êëåòêàõ, òðàíñôåöèðîâàííûõ âåêòîðîì, ñîäåðæàùèì
shRNA, êîìïëåìåíòàðíóþ Ìβ, ñîäåðæàíèå òðàíñêðèïòîâ
ýòîãî ãåíà ñíèçèëîñü íà 86% óæå íà øåñòîé äåíü ïîñëå
òðàíñôåêöèè. Ïðè òðàíñôåêöèè âåêòîðîì ñ äðóãîé âñòàâêîé shRNA óðîâåíü òðàíñêðèïöèè ãåíà Mβ íå ìåíÿëñÿ, ÷òî
äîêàçûâàåò âûñîêóþ ñïåöèôè÷íîñòü ïîäàâëåíèÿ ýêñïðåññèè.  ñëó÷àå òðàíñôåêöèè êëåòîê, ýêñïðåññèðóþùèõ GFP áåëîê, âåêòîðîì ñ êîìïëåìåíòàðíîé âñòàâêîé, óðîâåíü ôëóîðåñöåíöèè òîæå ïîíèæàëñÿ íà 85%. Ýòî äîêàçûâàåò âîçìîæíîñòü
èçáèðàòåëüíîãî, âûñîêîñïåöèôè÷íîãî ïîäàâëåíèÿ ýêñïðåññèè ãåíîâ â îïðåäåëåííîå âðåìÿ. Òàêæå áûëè ïîêàçàíû ðàçëè÷èÿ ýòîãî ïîäàâëåíèÿ â çàâèñèìîñòè îò ïîëîæåíèÿ âñòàâêè
shRNA âíóòðè èíòðîíà.
Èçâåñòíî, ÷òî ââåäåíèå ýêçîãåííîé ÐÍÊ âûçûâàåò èììóííûé îòâåò, à èìåííî àêòèâàöèþ ñèñòåìû èíòåðôåðîíà [4,
5]. Àâòîðû èçó÷èëè èììóííûé îòâåò íà òðàíñôåêöèþ ñîçäàííîé èìè êîíñòðóêöèè, à èìåííî ïðîàíàëèçèðîâàëè óðîâåíü
ËÈÒÅÐÀÒÓÐÀ:
1. Fire A., Xu S., Montgomery M.K. et al. Potent and specific genetic
interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998;
391(6669): 806-11.
2. Timmons L., Fire A. Specific interference by ingested dsRNA. Nature 1998;
395(6705): 854.
3. Jiang M., Rubbi C.P., Milner J. Gel-based application of siRNA to human
epithelial cancer cells induces RNAi-dependent apoptosis. Oligonucleotides 2004;
14(4): 239-48.
4. Farrell P.J., Sen G.C., Dubois M.F. et al. Interferon action: two distinct
pathways for inhibition of protein synthesis by double-stranded RNA. Proc. Natl.
Acad. Sci. USA 1978; 75(12): 5893-7.
5. Miyamoto N.G., Samuel C.E. Mechanism of interferon action. Interferonmediated inhibition of reovirus mRNA translation in the absence of detectable
mRNA degradation but in the presence of protein phosphorylation. Virology 1980;
107(2): 461-475.
6. May R.C., Plasterk R.H. RNA interference spreading in C. elegans. Methods
Enzymol. 2005; 392: 308-15.
7. Delgado R., Regueiro B.J. The future of HIV infection: gene therapy and
RNA interference. Enferm. Infecc. Microbiol. Clin. 2005; 23(Supl. 2): 76-83.
òðàíñêðèïöèè ãåíîâ, âîâëå÷åííûõ â ñèñòåìó èíòåðôåðîíà.
Âûÿñíèëîñü, ÷òî ýêñïðåññèÿ ýòèõ ãåíîâ óâåëè÷èâàåòñÿ âìåñòå ñ óâåëè÷åíèåì ýêñïðåññèè âåêòîðà êëîíèðîâàíèÿ. Îäíàêî, ïðè óìåíüøåíèè ðàçìåðà òðàíñêðèáèðóåìîé âñòàâêè,
à òàêæå ïðè íåêîòîðûõ ïîëîæåíèÿõ âñòàâêè shRNA, ýòîò
ýôôåêò óìåíüøàåòñÿ. Ýòè ðåçóëüòàòû ìîæíî ó÷åñòü ïðè
ïðèìåíåíèè ÐÍÊ-èíòåðôåðåíöèè in vivo.
Ó÷¸íûå òðàíñôåöèðîâàëè ÷åëîâå÷åñêèå ãåìîïîýòè÷åñêèå ñòîëîâûå êëåòêè (CD34+) îò çäîðîâûõ ëþäåé (ãåíîòèï
β/β) è îò ïàöèåíòîâ - ãîìî- (βs/βs) è ãåòåðîçèãîò (β/βs) ñ
ÑÊÀ. Ïîñëå äèôôåðåíöèðîâêè ýòèõ êëåòîê, èíäóöèðîâàííîé
ýðèòðîïîýòèíîì, ïîäòâåðäèëîñü ñòðîãî ñïåöèôè÷íîå ñíèæåíèå óðîâíÿ òðàíñêðèïòîâ βs, òîãäà êàê óðîâåíü β-ãëîáèíà
ñóùåñòâåííî íå ñíèçèëñÿ. Â òî æå âðåìÿ ìîæíî áûëî äåòåêòèðîâàòü òðàíñêðèïöèþ ãåíà γ-ãëîáèíà íà ñðàâíèòåëüíî
âûñîêîì óðîâíå. Ýòè ðåçóëüòàòû ïîêàçûâàþò, ÷òî â êëåòêàõ
ñèíõðîííî îäèí òðàíñêðèïò (βs) çàìåíÿåòñÿ äðóãèì (γ-ãëîáèí),
çà ñ÷åò ÷åãî è äîñòèãàåòñÿ òåðàïåâòè÷åñêèé ýôôåêò, òàê
êàê ôåòàëüíûé γ-ãëîáèí ñíèæàåò óðîâåíü ïîëèìåðèçàöèè
βs-ãëîáèíà â ýðèòðîöèòàõ. Âàæíûì äîñòîèíñòâîì ìåòîäà
ÿâëÿåòñÿ âîçìîæíîñòü èñïîëüçîâàíèÿ ñîáñòâåííûõ êëåòîê
ïàöèåíòà, à íå äîíîðñêîãî ìàòåðèàëà.
Òàêèì îáðàçîì, àâòîðû âïåðâûå ïîêàçàëè îñóùåñòâèìîñòü êîìáèíèðîâàííîãî òåðàïåâòè÷åñêîãî ïîäõîäà ãåííîé
è siRNA òåðàïèè íà ìîäåëè ÑÊÀ ñ êëåòêàìè ÷åëîâåêà. Òåðàïåâòè÷åñêèå âîçìîæíîñòè ÐÍÊ èíòåðôåðåíöèè ïîòåíöèàëüíî
î÷åíü âåëèêè, íåñìîòðÿ íà ðèñê èììóííîãî îòâåòà è íåîáõîäèìîñòü ñòðîãèõ óñëîâèé âûáîðà ãåíà-ìèøåíè.  íàñòîÿùåå âðåìÿ âåäóòñÿ ðàáîòû ïî èçó÷åíèþ òðàíñïîðòà ÐÍÊ èç
êëåòêè â êëåòêó [6], îïóáëèêîâàíû ñòàòüè îá èññëåäîâàíèÿõ
ÐÍÊ- èíòåðôåðåíöèè â áîðüáå ñî ÑÏÈÄîì [7-9], ãåïàòèòîì [10, 11] è ðàêîì [12-14]. Âñå ýòî äàåò íàäåæäó íà ðàçðàáîòêó íîâîãî, áîëåå ýôôåêòèâíîãî ìåòîäà (÷åì îáû÷íàÿ
ãåííàÿ èëè êëåòî÷íàÿ òåðàïèÿ) â áèîìåäèöèíå. Òåì íå ìåíåå,
ìåòîä ìîæåò èìåòü è ðÿä íåäîñòàòêîâ - íàðóøåíèå åñòåñòâåííîãî õîäà òðàíñëÿöèè, èììóííûé îòâåò, íåñïåöèôè÷åñêîå ïîäàâëåíèå ýêñïðåññèè [15, 16]. Íåñîìíåííî, ðàçâèòèå ýòîãî ïåðñïåêòèâíîãî ìåòîäà ïîìîæåò ðåøèòü
íåêîòîðûå èç ýòèõ ïðîáëåì.
8. Huelsmann P.M., Rauch P., Allers K. et al. Inhibition of drug-resistant HIV1 by RNA interference. Antiviral. Res. 2006; 69(1): 1-8.
9. Cullen B.R. Does RNA interference have a future as a treatment for HIV-1
induced disease? AIDS Rev. 2005; 7(1): 22-5.
10. Ying R.S., Fan X.G., Zhu C. et al. [Inhibition of hepatitis B virus replication
and expression by RNA interference in vivo.]. Zhonghua Gan Zang Bing Za Zhi,
2006; 14(1): 15-8.
11. Wu Y., Huang A.L., Tang N. et al. Specific anti-viral effects of RNA
interference on replication and expression of hepatitis B virus in mice. Chin. Med.
J. 2005; 118(16): 1351-6.
12. Sun Y.L., Zhou G.Y., Li K.N. et al., Suppression of glucosylceramide synthase
by RNA interference reverses multidrug resistance in human breast cancer cells.
Neoplasma 2006; 53(1): 1-8.
13. Charames G.S., Bapat B. Cyclooxygenase-2 knockdown by RNA
interference in colon cancer. Int. J. Oncol. 2006; 28(2): 543-9.
14. Pai S.I., Lin Y.Y., Macaes B. et al. Prospects of RNA interference therapy
for cancer. Gene Ther. 2006; 13(6): 464-77.
15. Rutz S., Scheffold A. Towards in vivo application of RNA interference new toys, old problems. Arthritis Res. Ther. 2004; 6(2): 78-85.
16. Shimamoto A. [Therapeutic application of RNA interference]. Nippon
Rinsho 2005; 63(7): 1291-7.
Ïîäãîòîâèëà Ò.Â. Ëîïàòèíà
ïî ìàòåðèàëàì Nat. Biotechnol. 2006; 24(1): 89-94
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