Экспрессия нейротрофических факторов в эмбриональном

13
Óêðà¿íñüêèé íåéðîõ³ðóðã³÷íèé æóðíàë, ¹3, 2003
ÓÄÊ 612.646:612.82:577.95
Ýêñïðåññèÿ íåéðîòðîôè÷åñêèõ ôàêòîðîâ
â ýìáðèîíàëüíîì ìîçãå ÷åëîâåêà 5–9 íåäåëü ãåñòàöèè
Öèìáàëþê Â.È., Âàñèëüåâà È.Ã., ×îïèê Í.Ã., Ãàëàíòà Å.Ñ.,
Öþáêî Î.È., Îëåêñåíêî Í.Ï., Âàøóëåíêî Ò.Í.
Èíñòèòóò íåéðîõèðóðãèè èì. àêàä. À.Ï. Ðîìîäàíîâà ÀÌÍ Óêðàèíû, ã. Êèåâ, Óêðàèíà
 îïðåäåëåíèè êîíå÷íîãî ôåíîòèïà äèôôåðåíöèðóþùèõñÿ íåéðîíîâ, ðåãóëÿöèè èõ
ïðîëèôåðàöèè, à òàêæå ïîääåðæàíèè âûæèâàåìîñòè êàê äèôôåðåíöèðîâàííûõ, òàê è
íåäèôôåðåíöèðîâàííûõ íåðâíûõ êëåòîê îñíîâíóþ ðîëü èãðàåò õèìè÷åñêîå îêðóæåíèå.
Îäíàêî ñöåíàðèé ýêñïðåññèè ñèãíàëüíûõ ìîëåêóë íà ðàçíûõ ñòàäèÿõ ýìáðèîãåíåçà íåðâíîé
òêàíè èçó÷åí äàëåêî íå ïîëíîñòüþ. Öåëüþ íàøåé ðàáîòû ÿâèëîñü èçó÷åíèå ýêñïðåññèè
BDNF, NGF, GDNF, NT-3, NT-4/5 è CNTF â ýìáðèîíàëüíîì ìîçãå ÷åëîâåêà 5–9 íåä
ãåñòàöèè ìåòîäîì ãèáðèäèçàöèè ÄÍÊ–ÐÍÊ íà íèòðîöåëëþëîçíîì ôèëüòðå.
Èññëåäîâàíèÿ ïîêàçàëè, ÷òî íà äàííîì ýòàïå ýìáðèîãåíåçà ýêñïðåññèðóåòñÿ ëèøü
òðè èç âûáðàíûõ íàìè ôàêòîðà: BDNF, NT-3, NT-4/5 â êîëè÷åñòâå, äåòåêòèðóåìîì ñ
ïîìîùüþ äàííîãî ìåòîäà. Åêñïðåññèÿ îñòàëüíûõ òðåõ ôàêòîðîâ íåðâíîé òêàíüþ ëèáî
îòñóòñòâóåò â äàííûé ïåðèîä, ëèáî åå óðîâåíü íàõîäèòñÿ çà ïðåäåëàìè ÷óâñòâèòåëüíîñòè
ÄÍÊ–ÐÍÊ ãèáðèäèçàöèè.
Êëþ÷åâûå ñëîâà: ôàêòîð ðîñòà íåðâîâ, íåéðîòðîôè÷åñêèé ôàêòîð ìîçãîâîãî ïðîèñõîæäåíèÿ,
íåéðîòðîôèí-3, íåéðîòðîôèí-4/5, öèëèàðíûé íåéðîòðîôè÷åñêèé ôàêòîð, ãëèàëüíûé íåéðîòðîôè÷åñêèé ôàêòîð, ýìáðèîíàëüíûé ìîçã ÷åëîâåêà.
Âñòóïëåíèå.  íàñòîÿùåå âðåìÿ ê íåéðîòðîôè÷åñêèì ôàêòîðàì îòíîñÿò áîëåå 20 ðîñòîâûõ
àãåíòîâ, ñîñòàâëÿþùèõ ðàçëè÷íûå ñåìåéñòâà
(íåéðîòðîôèíû, íåéðîëåéêèíû, íåéðîêèíû,
ñåìåéñòâà FGF è TGFb è ò.ä.), â êîòîðûå âõîäÿò
ñòðóêòóðíî-ðîäñòâåííûå áåëêè. Íåêîòîðûå èç
íèõ îáëàäàþò ïëåéîòðîïíîé àêòèâíîñòüþ è ðåàëèçóþò ñâîé ïîòåíöèàë íå òîëüêî â íåðâíîé
ñèñòåìå, íî äëÿ áîëüøèíñòâà ýòèõ ôàêòîðîâ
ìèøåíüþ ÿâëÿþòñÿ íåðâíûå êëåòêè.
Íàèáîëåå èíòåíñèâíî èçó÷àåìûìè òðîôè÷åñêèìè ôàêòîðàìè íà ñåãîäíÿøíèé äåíü ÿâëÿþòñÿ ôàêòîð ðîñòà íåðâîâ–nerve growth
factor (NGF), íåéðîòðîôè÷åñêèé ôàêòîð ìîçãîâîãî ïðîèñõîæäåíèÿ–brain derived neurotrophic factor (BDNF), íåéðîòðîôèí-3–
neurotrophin three (NT-3), íåéðîòðîôèí-4/5–
neurotrophin four/five (NT-4/5) (ñåìåéñòâî íåéðîòðîôèíîâ), öèëèàðíûé íåéðîòðîôè÷åñêèé
ôàêòîð–ciliary neurotrophic factor (CNTF) (ñåìåéñòâî íåéðîêèíîâ) è ãëèàëüíûé íåéðîòðîôè÷åñêèé ôàêòîð–glia derived neurotrophic
factor(GDNF) (ñåìåéñòâî TGFb). Ìíîãî÷èñëåííûìè èññëåäîâàíèÿìè íà æèâîòíûõ áûëà ïðîäåìîíñòðèðîâàíà èõ ðîëü â ýìáðèîãåíåçå è
ïîñòíàòàëüíîì ïåðèîäå êàê ôàêòîðîâ äèôôåðåíöèðîâêè, ñîçðåâàíèÿ è ïîääåðæàíèÿ âûæèâàåìîñòè êëåòîê ïåðèôåðè÷åñêîé è öåíòðàëüíîé íåðâíîé ñèñòåìû [3, 7, 8, 10,]. Îäíàêî, íåñìîòðÿ íà òî ÷òî çà ïÿòèäåñÿòèëåòíèé
ïåðèîä èíòåíñèâíîãî èçó÷åíèÿ âûøåíàçâàííûõ
ãîðìîíîïîäîáíûõ ðîñòîâûõ àãåíòîâ ñîáðàí îáøèðíûé ôàêòè÷åñêèé ìàòåðèàë, îòêðûòûìè îñòàþòñÿ âîïðîñû îá èõ ýêñïðåññèè, ìåõàíèçìàõ
äåéñòâèÿ, îáúåêòàõ ïðèëîæåíèÿ è ôóíêöèîíàëüíîì âçàèìîäåéñòâèè â ýìáðèîãåíåçå è ôèëîãåíåçå ÷åëîâåêà.
Èçó÷åíèå ñïåöèàëüíîé ëèòåðàòóðû ïîêàçàëî, ÷òî íàèáîëüøèé ìàññèâ ýêñïåðèìåíòàëüíûõ äàííûõ îá ýêñïðåññèè íåéðîòðîôè÷åñêèõ
ôàêòîðîâ è âûïîëíÿåìûõ èìè ôóíêöèÿõ îòðàæàåò ñîáûòèÿ âòîðîé ïîëîâèíû ýìáðèîíàëüíîãî ðàçâèòèÿ ïîçâîíî÷íûõ, êîãäà óñòàíàâëèâàåòñÿ èííåðâàöèÿ òêàíåé-ìèøåíåé è, íàðÿäó
ñ áëàñòíûìè ôîðìàìè íåðâíûõ êëåòîê, óæå
ñóùåñòâóþò ìîëîäûå íåéðîíû è êëåòêè ãëèè
[1]. Tàê, ñèíòåç ìÐÍÊ NT-4/5 îòìå÷àëñÿ â áîëüøèíñòâå òêàíåé íà 17–21-å ñóòêè ðàçâèòèÿ ýìáðèîíîâ êðûñ [19], à ìÐÍÊ BDNF îáíàðóæèâàëàñü â ñóáïîïóëÿöèè êëåòîê êîõëåîâåñòèáóëÿðíîãî ãàíãëèÿ â ïåðèîä Å11.5 ó ìûøåé [6].
Èíôîðìàöèÿ îá ýêñïðåññèè èçó÷àåìûõ íàìè
ðîñòîâûõ àãåíòîâ íà ðàííèõ ýòàïàõ íåéðîîíòîãåíåçà äîñòàòî÷íî ñêóäíà. Èçâåñòíî, ÷òî ìÐÍÊ
BDNF è NT-3 â ðàçâèâàþùåéñÿ ïåðâè÷íîé ïîëîñêå äåòåêòèðóåòñÿ ñ ïåðâîãî äíÿ ýìáðèîãåíåçà ïòèö, à íåáîëüøèå êîëè÷åñòâà ìÐÍÊ
BDNF, NT-3 è NGF â ãàíãëèîíàðíîé ïëàñòèíêå, íåðâíîé òðóáêå, ñîìèòàõ è íîòîõîðäå – ñî
2-ãî äíÿ [18]. Ýêñïðåññèÿ GDNF â íåéðîýêòî-
14
Öèìáàëþê Â.È., Âàñèëüåâà È.Ã., ×îïèê Í.Ã., Ãàëàíòà Å.Ñ., Öþáêî Î.È., Îëåêñåíêî Í.Ï., Âàøóëåíêî Ò.Í.
äåðìå ðàçâèâàþùåãîñÿ ìûøèíîãî ýìáðèîíà
íàáëþäàëàñü â ïåðèîä 7,5–10,5 ñóò ïîñëå îïëîäîòâîðåíèÿ [6]. Óíèêàëüíîé ÿâëÿåòñÿ èíôîðìàöèÿ îá ýêñïðåññèè ðåöåïòîðîâ íåéðîòðîôèíîâ â ðàçâèâàþùåìñÿ êîõëåîâåñòèáóëÿðíîì
ãàíãëèè ýìáðèîíàëüíîãî âíóòðåííåãî óõà ÷åëîâåêà 5–24 íåä ãåñòàöèè, ÷òî óêàçûâàåò íà
çàâèñèìîñòü êëåòîê ãàíãëèÿ îò ýòèõ ôàêòîðîâ
íà äàííîì ýòàïå ýìáðèîãåíåçà. Trk-B è Trk-C
(ðåöåïòîðû BDNF è NT-3 ñîîòâåòñòâåííî) îáíàðóæèâàëè èììóíîðåàêòèâíîñòü, íà÷èíàÿ ñ
5-é íåäåëè, ïðè÷åì ïèê ýêñïðåññèè ïðèõîäèëñÿ
íà 7–12-þ íåäåëþ. Trk-A (ðåöåïòîð NGF) äåìîíñòðèðîâàë ñëàáóþ èììóíîðåàêòèâíîñòü
ëèøü íà 5–7-é íåäåëå [16].
Öåëüþ ðàáîòû ÿâèëîñü êîëè÷åñòâåííîå îïðåäåëåíèå ýêñïðåññèè NGF, BDNF, NT-3, NT4/5, GDNF è CNTF â öåëüíîì ýìáðèîíàëüíîì
ìîçãå ÷åëîâåêà 5–9 íåä ãåñòàöèè.
Ìàòåðèàëû è ìåòîäû.  ýêñïåðèìåíòå èñïîëüçîâàëè ìîçã 18 ïÿòèíåäåëüíûõ, 16 øåñòèíåäåëüíûõ, 16 ñåìèíåäåëüíûõ, 12 âîñüìèíåäåëüíûõ è 12 äåâÿòèíåäåëüíûõ ýìáðèîíîâ ÷åëîâåêà (àáîðòèâíûé ìàòåðèàë).
ìÐÍÊ áûëà ïîëó÷åíà ìåòîäîì ôåíîëüíîé
ýêñòðàêöèè ñîãëàñíî Ìàíèàòèñ Ò. [13]
Óðîâåíü ýêñïðåññèè ôàêòîðîâ îïðåäåëÿëè
ìåòîäîì ãèáðèäèçàöèè ÄÍÊ–ÐÍÊ íà íèòðîöåëëþëîçûõ ôèëüòðàõ, ñîãëàñíî Ìàíèàòèñ Ò.,
Ôðàíöèñêî Ã. [4,13] ñ íåêîòîðûìè ìîäèôèêàöèÿìè.
 êà÷åñòâå çîíäîâ èñïîëüçîâàëè ñëåäóþùèå ñèíòåòè÷åñêèå îëèãîíóêëåîòèäû (Amersham Pharmacia Biotech):
1. 5’-cgccttgacaaaggtgtgagtcgtggtacaatatgagttc3’ NGF.
2. 5’-gtttgcttctttcatgggggcagccttcatgcaaccaaag3’ BDNF.
3. 5’-gcggtgtggagcagcaccaggcagacagccacgacatccc3’ GDNF.
4. 5’- ggaattgagcgagtcttctggcaaactcctttgatccatg3’ NT-3.
5. 5’- tcgcccaacacctccacctcgcgcccacgcaagtccacag3’ NT-4/5.
6. 5’-gagcagtcaggtctgaacgaatcttccttgctagccagat3’ CNTF.
Ìå÷åíèå çîíäîâ [γ-32Ð]ATP (>3000Ci/mM)
(Amersham Pharmacia Biotech) îñóùåñòâëÿëè
ìåòîäîì êîíöåâîãî ôåðìåíòàòèâíîãî ìå÷åíèÿ
ñîãëàñíî Ìàíèàòèñ Ò. [13].
Î÷èñòêó çîíäîâ ïðîâîäèëè â ïîëèàêðèëàìèäíîì ãåëå â ïðèñóòñòâèè 7Ì ìî÷åâèíû.
Ïðåãèáðèäèçàöèþ ôèëüòðîâ âûïîëíÿëè â
ïðèñóòñòâèè 50% ôîðìàìèäà / 5õ SSPE
(1xSSPE=15mM öèòðàò íàòðèÿ / 0,15M NaCl /
10ìÌ NaH2ÐÎ4 / 1ìÌ EDTA ) / 5x ðåàêòèâà
Äåíõàðäòà (1x ðåàêòèâ Äåíõàðäòà= 0,02% BSA
/ 0,02%Ficoll / 0,02% ïîëèâèíèëïèðîëèäîí)
/ 0,1% SDS ïðè 42°Ñ â òå÷åíèå 1÷. Ãèáðèäèçàöèþ ñ ìå÷åííûì 32Ð-çîíäîì (50 000-100 000
ðàñ/ìèí) ïðîâîäèëè â òå÷åíèå 24 ÷ ïðè òåìïåðàòóðå 42°Ñ.Ôèëüòðû îòìûâàëè îäíîêðàòíî
â 1õSSC ñ 0,1%SDS ïðè êîìíàòíîé òåìïåðàòóðå è äâàæäû â 0,2xSSC ñ 0,1%SDS ïðè òåìïåðàòóðå 42°Ñ. Ðàäèîàêòèâíîñòü ôèëüòðà îïðåäåëÿëè íà ñöèíòèëëÿöèîííîì ñ÷åò÷èêå “ Áåòà1” (Óêðàèíà).
Ðåçóëüòàòû è èõ îáñóæäåíèå. Ïîëó÷åííûå
äàííûå ïîêàçàëè, ÷òî â èçó÷àåìûé ïåðèîä íåéðîîíòîãåíåçà ÷åëîâåêà â ýìáðèîíàëüíîì ãîëîâíîì ìîçãå ýêñïðåññèðóþòñÿ ëèøü òðè èç èññëåäîâàííûõ ôàêòîðîâ: BDNF, NT-3, NT-4/5,
ïðè÷åì BDNF äåìîíñòðèðîâàë áîëåå âûñîêèé
óðîâåíü ýêñïðåññèè,÷åì NT-3 è NT-4/5 (òàáëèöà).
Êðîìå òîãî, ê 7-é íåäåëå ýìáðèîãåíåçà íàáëþäàëè òåíäåíöèþ ê óâåëè÷åíèþ ñèíòåçà
ìÐÍÊ äàííîãî íåéðîòðîôèíà. Ýòîò ðåçóëüòàò
êîððåëèðóåò ñ îïèñàíûìè â ëèòåðàòóðå íàáëþäåíèÿìè îá àêòèâàöèè ýêñïðåññèè ðåöåïòîðà äàííîãî íåéðîòðîôèíà [16]. Àíàëîãè÷íóþ
äèíàìèêó îïðåäåëÿëè òàêæå ïðè èçó÷åíèè ýêñïðåññèè íåéðîòðîôè÷åñêèõ ôàêòîðîâ â íåéðîîíòîãåíåçå ïòèö. Òàê, ïèê ñèíòåçà BDNF ïðèõîäèëñÿ íà ïåðèîä Å4,5 [7], êîãäà ó ïòèö, ñîãëàñíî äàííûì ëèòåðàòóðû, îáðàçóþòñÿ êëåòêè ïðåäøåñòâåííèêè íåéðîíîâ è íà÷èíàåòñÿ èõ
äèôôåðåíöèðîâêà [5]. Îäíàêî, ñîãëàñíî äðóãèì
Таблица. Экспрессия нейротрофических факторов в эмбриональном мозге человека
5–9 нед гестации
Срок
гестации
фмоль мРНК
BDNF/мг
мРНК
фмоль мРНК
NGF/мг
мРНК
фмоль мРНК
NT-3/мг
мРНК
фмоль мРНК
NT-4/5/мг
мРНК
фмоль мРНК
GDNF/мг
мРНК
фмоль мРНК
CNTF/мг
мРНК
5 нед
n=6
2,974±0,544
–
0,581±0,141
0,884±0,202
–
–
6 нед
n=6
2,123±0,649
–
–
0,178±0,094
–
–
7 нед
n=8
3,191±0,749
–
–
0,670±0,038
–
–
8 нед
n=8
3,237±0,743
–
0,884±0,306
0,170±0,028
–
–
9 нед
n=10
3,818±0,212
–
–
0,453±0,082
–
–
Ýêñïðåññèÿ íåéðîòðîôè÷åñêèõ ôàêòîðîâ â ýìáðèîíàëüíîì ìîçãå ÷åëîâåêà 5–9 íåäåëü ãåñòàöèè
äàííûì [10], ïîëó÷åíûì íà êðûñàõ, BDNF èìååò íèçêèé óðîâåíü ýêñïðåññèè â íåçðåëûõ ðåãèîíàõ ÖÍÑ è ïîâûøàåòñÿ îí ëèøü ñ èõ ñîçðåâàíèåì. À â ïåðèîä ïðîëèôåðàöèè, ìèãðàöèè
è äèôôåðåíöèðîâêè êëåòîê ïðåäøåñòâåííèêîâ
â ìîçãå êðûñ ïðåèìóùåñòâåííî ýêñïðåññèðóåòñÿ ðîäñòâåííûé íåéðîòðîôèí – NT-3 [10].
Ñîãëàñíî ðåçóëüòàì äàííîé ðàáîòû ýòîò áåëîê
íå äåìîíñòðèðóåò ñòàáèëüíóþ ýêñïðåññèþ â
ýìáðèîíàëüíîì ìîçãå ÷åëîâåêà â èçó÷àåìûé
ïåðèîä. Ïîäîáíûå ðàñõîæäåíèÿ, âîçìîæíî, ñâÿçàíû ñ âèäîâûìè îñîáåííîñòÿìè ôîðìèðîâàíèÿ ÖÍÑ.  ëþáîì ñëó÷àå ïðèñóòñòâèå ìÐÍÊ
ýòèõ ðîäñòâåííûõ áåëêîâ â ðàçâèâàþùåìñÿ
ìîçãå îáóñëîâëåíî âûïîëíÿåìûìè èìè ôóíêöèÿìè. Òàê, in vitro áûëà ïîêàçàíà ðîëü BDNF
â äèôôåðåíöèðîâêå êëåòîê ïðåäøåñòâåííèêîâ,
ïîëó÷åííûõ èç ýìáðèîíàëüíîãî ñòðèàòóìà, â
ñòîðîíó ìóëüòèïîëÿðíûõ, à NT-3 – â ñòîðîíó
áèïîëÿðíûõ íåéðîíîâ [8]. Êðîìå òîãî, íà êóëüòóðå íåéðîíîâ ãèïïîêàìïà áûë ïðîäåìîíñòðèðîâàí èõ ñòèìóëèðóþùèé ýôôåêò â îáðàçîâàíèè ñèíàïñîâ [17].
Îæèäàåìûì ðåçóëüòàòîì äëÿ íàñ ÿâèëîñü
îáíàðóæåíèå ýêñïðåññèè ìÐÍÊ NT-4/5 â èçó÷àåìûé ïåðèîä. Äàííûé ôàêòîð îáåñïå÷èâàåò
âûæèâàíèå è ôîðìèðîâàíèå íåéðîíàëüíîãî
ôåíîòèïà äèôôåðåíöèèðóþùèõñÿ êëåòîê ïðåäøåñòâåííèêîâ, ïîëó÷åííûõ èç ýìáðèîíàëüíîãî
ìîçãà ÷åëîâåêà è êóëüòèâèðóåìûõ in vitro [3],
à òàêæå èìååò çíà÷åíèå â ôîðìèðîâàíèè ìàðãèíàëüíîé çîíû íåîêîðòåêñà ãðûçóíîâ [2].
Îòñóòñòâèå ýêñïðåññèè òêàíüþ ýìáðèîíàëüíîãî ãîëîâíîãî ìîçãà 5–9 íåä GDNF, NGF, CNTF
ìîæåò ñâèäåòåëüñòâîâàòü ëèáî î íåâîñòðåáîâàííîñòè äàííûõ ôàêòîðîâ íà ýòàïå ïðîëèôåðàöèè, äèôôåðåíöèðîâêè è ìèãðàöèè êëåòîê
ïðåäøåñòâåííèêîâ íåéðîíîâ, ëèáî î ïðåèìóùåñòâåííîì çíà÷åíèè â ýòîò ïåðèîä äàííûõ
ðîñòîâûõ àãåíòîâ ýêçîãåííîãî ïðîèñõîæäåíèÿ
(èñòî÷íèêîì ìîãóò áûòü îêðóæàþùèå òêàíè),
òàê êàê èõ ðåöåïòîðû äåòåêòèðóþòñÿ â ÖÍÑ
äîâîëüíî ðàíî [9,12], à äåëåòèðîâàíèå ãåíà
ïðèâîäèò ê àíîìàëèè ðàçâèòèÿ öåíòðàëüíîé è
ïåðèôåðè÷åñêîé íåðâíîé ñèñòåìû [14 ].
Òàêèì îáðàçîì, ïîëó÷åííûå íàìè äàííûå
ïîäòâåðæäàþò ñóùåñòâîâàíèå ýíäîãåííîãî ñèíòåçà BDNF, NT-3, NT-4/5 ðàçâèâàþùèìñÿ ãîëîâíûì ìîçãîì ÷åëîâåêà 5–9 íåä ãåñòàöèè, ÷òî
åùå ðàç ïîä÷åðêèâàåò ó÷àñòèå ýòèõ ôàêòîðîâ
â ïðîöåññå äèôôåðåíöèðîâêè íåðâíîé òêàíè.
Äàííûé ôàêò ìîæåò îáúÿñíÿòü òîò òðîôè÷åñêèé ýôôåêò, êîòîðûé äàåò ýìáðèîíàëüíàÿ íåðâíàÿ òêàíü ïðè åå òðàíñïëàíòàöèè, ÷òî ëèøíèé ðàç ïîäòâåðæäàåò âîçìîæíîñòü èñïîëüçîâàíèÿ ýìáðèîíàëüíîãî äîíîðñêîãî ìàòåðèàëà â
òåðàïåâòè÷åñêèõ öåëÿõ.
15
Ñïèñîê ëèòåðàòóðû
1. Íåéðîîíòîãåíåç. –Ì: Íàóêà, 1985. – 267 c.
2. Brunstrom J.E., Gray-Swain M.R., Osborne P.A.,
Pearlman A.L. Neuron al heterotopias in the
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3. Caldwell M.A., He X., Wilkie N., Pollak S., Marshall
G., Wafford K.A., Svendsen C.N. Growth factors
regulate the syrvival and fate of cells derived from
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Tzagoloff. A rapid Method for detecting specific
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6. Hellmich H.L., Kos L., Cho E.S., Mahon K.A., Zimmer
A. Embryonic expression of glial cell-line derived
neurotrophic factor (GDNF) suggests multi ple
developmental roles in neural differentiation and
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–1996. – V.54, ¹1. – P. 95–105.
7. Howard L., Weiner M.D. The pole of growth factors
receptors in central nervous system development
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8. Lachyankar Ì.B., Condon P.J., Quesenberry P.J.,
Litofsky N.S., Recht L.D., Ross A.H. Embryonic
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D.L., Pfeifer M., Lee N. Immunohistochemical
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10. Maisonpierre P.C., Bellusio L., Friedman B., Alderson
R.F., Wiegand S.J, Furth M.E, Lindsay R.M,
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11. Mariano B. Neurotrophic factors and their receptors
// Curr. Opin. in Cell Biol. – 1995. –¹7.–P. 148–155.
12. Mikaels A., Livet J., Westphal H., De Lapeyriere O.,
Errfors P. A dyn amic regulation of GDNF-family
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16
Öèìáàëþê Â.È., Âàñèëüåâà È.Ã., ×îïèê Í.Ã., Ãàëàíòà Å.Ñ., Öþáêî Î.È., Îëåêñåíêî Í.Ï., Âàøóëåíêî Ò.Í.
16. Vega J.A., San Jose I., Cabo R., Rodriquez S., Represa
J. Trks and p75 genes are differentially expressed
in the inner ear of human embryos. What may Trks
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¹2. – P. 103–106.
17. Vicario-Abejon C., Collin C., McKay R.D., Segal M.
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– V. 165, ¹2. – P. 727–730.
19. Zhang S.H, Zhou X.F., Deng Y.S., Rush R.A.
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– 1999. – V.89, ¹1.– P. 69–74.
Åêñïðåñ³ÿ íåéðîòðîô³÷íèõ ôàêòîð³â
ó åìáð³îíàëüíîìó ìîçêó ëþäèíè
5–9 òèæí³â ãåñòàö³¿
Öèìáàëþê Â.², Âàñèëüºâà ².Ã., ×îïèê Í.Ã., Ãàëàíòà Î.Ñ.,
Öþáêî Î.²., Îëåêñåíêî Í.Ï., Âàøóëåíêî Ò.Í.
Ó âèçíà÷åíí³ ê³íöåâîãî ôåíîòèïó íåéðîí³â, ùî äèôåðåíö³þþòüñÿ, ðåãóëÿö³¿ ¿õ ïðîë³ôåðàö³¿, à òàêîæ
ï³äòðèìö³ âèæèâàííÿ ÿê äèôåðåíö³éîâàíèõ, òàê ³ íåäèôåðåíö³éîâàíèõ íåðâîâèõ êë³òèí ãîëîâíó ðîëü â³ä³ãðàº
õ³ì³÷íå îòî÷åííÿ. Ïðîòå ñöåíàð³é åêñïðåñ³¿ ñèãíàëüíèõ
ìîëåêóë íà ð³çíèõ ñòàä³ÿõ åìáð³îãåíåçó íåðâîâî¿ òêàíè-
íè âèâ÷åíèé ùå äàëåêî íå ïîâí³ñòòþ. Ìåòîþ íàøî¿ ðîáîòè ñòàëî âèâ÷åííÿ åêñïðåñ³¿ BDNF, NGF, GDNF, NT3, NT-4/5 òà CNTF ó åìáð³îíàëüíîìó ìîçêó ëþäèíè 5–
9 òèæ ãåñòàö³¿ ìåòîäîì ã³áðèäèçàö³¿ ÄÍÊ–ÐÍÊ íà í³òðîöåëþëîçíîìó ô³ëüòð³.
Äîñë³äæåííÿ ïîêàçàëè. ùî íà öüîìó åòàï³ åìáð³îãåíåçó åêñïðåñóþòüñÿ ëèøå òðè ç îáðàíèõ íàìè ôàêòîð³â:
BDNF, NT-3 òà NT-4/5 ó ê³ëüêîñò³, ùî ìîæå áóòè âèçíà÷åíà öèì ìåòîäîì. Åêñïðåñ³ÿ ³íøèõ ôàêòîð³â íåðâîâîþ òêàíèíîþ àáî â³äñóòíÿ ó öåé ïåð³îä àáî ¿¿ ð³âåíü
çíàõîäèòüñÿ çà ìåæàìè ÷óòëèâîñò³ ÄÍÊ–ÐÍÊ ã³áðèäèçàö³¿.
Neurotrophins expression in human brain from
embryonic weeks 5–9
Tsymbaliuk V.I, Vasilyeva I.G., Galanta E.S., Chopik N.G.,
Tsyubko O.I., Olexenko N.P., Vaschulenko T.N.
The chemical surrounding play a crucial role in
determin ation of phenotyping differentiation during
neurogenesis and ðromotion of survival as differentiated
so as undifferentiated neural cells. But the scen ario of
sign al molecules expression in early embryonic stage is
poorly understood. Therefore we examined expression
of neurotrophic factors BDNF, NGF, GDNF, NT-3, NT4/5 and CNTF in human brain from emryonic weeks 59 by DNA-RNA hybridization on nitrocellulose filter.
These study revealed that only BDNF, NT-3 and
NT4/5 were expressed in detectable rate in these period.
Other factors demonstrated no expression ore its mRNK
rate was aut of sensitiveness of these method.
Êîììåíòàðèé
ê ñòàòüå Öèìáàëþêà Â.È., Âàñèëüåâîé È.Ã., ×îïèê Í.Ã., Ãàëàíòû Å.Ñ., Öþáêî Î.È., Îëåêñåíêî Í.Ï., Âàøóëåíêî
Ò.Í. « Ýêñïðåññèÿ íåéðîòðîôè÷åñêèõ ôàêòîðîâ â ýìáðèîíàëüíîì ìîçãå ÷åëîâåêà 5–9 íåä ãåñòàöèè»
Òðàíñïëàíòàöèÿ ýìáðèîíàëüíîé íåðâíîé òêàíè îñòàåòñÿ øèðîêî äèñêóòèðóåìûì, àëüòåðíàòèâíûì ìåäèêàìåíòîçíîìó íàïðàâëåíèåì â òåðàïèè ìíîãèõ çàáîëåâàíèé ÖÍÑ. Ïîëîæèòåëüíîå äåéñòâèå, îêàçûâàåìîå èìïëàíòèðóåìûìè ýìáðèîíàëüíûìè êëåòêàìè, èçó÷àåòñÿ ìíîãèìè ëàáîðàòîðèÿìè ìèðà, îäíàêî îñòàåòñÿ áîëüøîå êîëè÷åñòâî îòêðûòûõ âîïðîñîâ îòíîñèòåëüíî ìåõàíèçìîâ èõ áèîëîãè÷åñêîãî äåéñòâèÿ. Îäíèì èç îáúÿñíåíèé íàáëþäàåìîãî òðîôè÷åñêîãî ýôôåêòà ìîæåò ñëóæèòü ñèíòåç èìïëàíòàòîì îïðåäåëåííûõ ñèãíàëüíûõ ìîëåêóë. Ïðåäñòàâëåííàÿ ãðóïïîé àâòîðîâ ðàáîòà îïðåäåëÿåò òðè íåéðîòðîôèíà (BDNF, NT-3, NT-4/5), ýêñïðåññèðóåìûõ ýìáðèîíàëüíûì ìîçãîì ÷åëîâåêà â ïðèîðèòåòíûé äëÿ çàáîðà äîíîðñêîãî ìàòåðèàëà ïåðèîä. Èñõîäÿ èç ïîëó÷åííûõ
ðåçóëüòàòîâ, ìîæíî ñäåëàòü âûâîä, ÷òî äåâÿòèíåäåëüíûé ýìáðèîíàëüíûé ìîçã èìååò ïðåèìóùåñòâî ïî ñðàâíåíèþ ñ äðóãèìè ñðîêàìè ãåñòàöèè. Ïîýòîìó äàííàÿ ðàáîòà ïðåäñòàâëÿåò íå òîëüêî íàó÷íóþ, íî è ïðàêòè÷åñêóþ
öåííîñòü.
Îáðàùàåò íà ñåáÿ âíèìàíèå áîëüøîå êîëè÷åñòâî ýêñïåðèìåíòàëüíîãî ìàòåðèàëà, à òàêæå òùàòåëüíî ïîäîáðàííûé îáçîð ëèòåðàòóðíûõ äàííûõ, òàê êàê èíôîðìàöèÿ îá ýíäîãåííîì ñèíòåçå òðîôè÷åñêèõ ôàêòîðîâ â
ýìáðèîíàëüíûõ òêàíÿõ ÷åëîâåêà è æèâîòíûõ ñêóäíà è íîñèò ðàçðîçíåííûé ñïîðàäè÷åñêèé õàðàêòåð.
À.Ò.Íîñîâ
äîêòîð ìåä. íàóê, ïðîôåññîð,
çàâåäóþùèé ëàáîðàòîðèåé ýëåêòðîííîé ìèêðîñêîïèè
Èíñòèòóòà íåéðîõèðóðãèè
èì. àêàä. À.Ï.Ðîìîäàíîâà ÀÌÍ Óêðàèíû