Inyandiko
Methylene blue no gusaza kw’ingirabuzimafatizo: icyo ubushakashatsi kuri fibroblaste bupima
Kumva nabi ibyavuye mu bushakashatsi bikunze kugenda gutya. Inyandiko y’ubushakashatsi ivuga ko methylene blue yatumye fibroblaste zororerwa muri laboratwari zikuba kabiri inshuro zirenga 20 ziyongera ku zari zisanzwe, maze umusomyi akanzura ko ikuraho ingirabuzimafatizo zamaze gusaza cyangwa ko yongera igihe abantu babaho ku rugero rusa n’urwo. Uwo mwanzuro ushobora kumvikana nk’uwihuta kuza mu mutwe. Ariko urimo amakosa abiri atandukanye.
Ikibazo nyamukuru kiri mu gusobanukirwa ibyo bipimo, si mu mibare. Inshuro umubare w’ingirabuzimafatizo wikuba kabiri, ibimenyetso byo gusaza kwazo n’ibipimo bya telomere, buri kimwe gisubiza ikibazo gitandukanye. Ubushakashatsi bwa methylene blue kuri fibroblaste bukwiye kumvikana nk’ubureba gutinza kwinjira mu gusaza kw’ubwoko bumwe bw’ingirabuzimafatizo zororerwa mu gasahani ka laboratwari, bushyigikiwe n’ibipimo bya mitokondiriya. Ibyo bitandukanye no gukuraho mu buryo bwihariye ingirabuzimafatizo zamaze gusaza, kandi na byo bitandukanye no gusaza kw’ikinyabuzima cyose. Ibisobanuro byagutse by’iryo tandukaniro biri mu nyandiko ivuga uko ihererekanya rya elegitoroni muri mitokondiriya rigena ingufu z’ingirabuzimafatizo no mu gusobanukirwa ibivugwa mu bushakashatsi kuri methylene blue.
Kwikuba kabiri k’umubare w’ingirabuzimafatizo kubara ibyabaye, ntigupima ubusore bwazo
Ibyinshi mu bisubizo by’ingenzi biva mu ngirabuzimafatizo za IMR90, zikaba ari ubwoko bwa fibroblaste zisanzwe zo mu bihaha by’urusoro rw’umuntu, zifite ubushobozi ntarengwa bwo kwigabanya. Mu bushakashatsi bwa mbere kuri methylene blue n’igihe fibroblaste za IMR90 zimara zishobora kwigabanya, ingirabuzimafatizo zororwaga zajyanwaga mu bindi bikoresho byo kuzororeramo kuri gahunda yagenwe, kandi inshuro zose umubare wazo wikubaga kabiri zarabarwaga kugeza zihagaritse kwigabanya. Gukoresha methylene blue ku rugero rwa nanomolar, cyane cyane kuri 100 nM, byatumye zigeraho ziyongeraho inshuro zirenga 20 zo kwikuba kabiri mbere yo guhagarara.
Kwikuba kabiri k’umubare w’ingirabuzimafatizo ni igipimo cyo kubara. Cyandika inshuro umubare wazo wikubye hafi kabiri. Ntikivuga mu buryo butaziguye niba ingirabuzimafatizo imwe yashaje, yarahagaritse kwigabanya by’igihe gito, yarahindutse ikagira umurimo wihariye cyangwa yarapfuye. Itsinda ry’ingirabuzimafatizo rishobora kwikuba kabiri inshuro nyinshi kubera ko zigumana ubushobozi bwo kwigabanya igihe kirekire, kubera ko nke muri zo zihagarara hakiri kare bitewe n’ihungabana, cyangwa kubera ko uburyo zororwamo butonesha itsinda rito ryazo rishobora gukomeza kwiyongera. Umubare wonyine ntushobora gutandukanya izo nzira.
Ni yo mpamvu kubara inshuro zo kwikuba kabiri bijyana no gupima ibimenyetso. Umubare uvuga igihe kwiyongera kwakomeje. Ibimenyetso bivuga uko ingirabuzimafatizo zahagaritse kwigabanya cyangwa izakomeje kubaho zari zimeze.
Amabwiriza ku bipimo by’ibisubizo: icyo buri kimenyetso kigaragaza
Gusaza kw’ingirabuzimafatizo zororerwa muri laboratwari ni uruhurirane rw’imiterere igaragazwa n’ibipimo byinshi bitandukanye. Nta rangi rimwe ripima cyangwa poroteyine imwe bibihamya byonyine.
Guhagarara kw’ubwiyongere no kubura kw’ikorwa rya DNA. Guhagarara kurambye gusuzumwa harebwa igabanuka ry’iyinjizwa rya BrdU cyangwa EdU, imiterere irambuye y’ingirabuzimafatizo no kutongera kwiyongera nyuma yo kuzimura mu kindi gikoresho cyo kuzororeramo. Ibi bitandukanya guhagarara bitewe no gusaza no guhagarara by’igihe gito.
Beta galactosidase ijyanye no gusaza kw’ingirabuzimafatizo. SA-beta-gal ikoresha irangi rigaragaza ibikorwa bya lizozome hafi ya pH 6.0. Fibroblaste zamaze gusaza akenshi zigaragaza icyo kimenyetso uko ubwinshi bwa lizozome bwiyongera. Ntikigaragaza gusaza gusa, bityo gisobanurwa hamwe n’ibipimo by’ukwiyongera kw’ingirabuzimafatizo n’ibimenyetso byo ku rwego rwa molekile.
Inzira za p16 na p21. p16 ikora inyuze muri CDK4 na CDK6 no mu nzira ya RB. p21 akenshi iterwa gukora binyuze muri p53. Kwiyongera kwazo gushyigikira ko habaye guhagarara kw’ukwigabanya, ariko nta n’imwe muri izo poroteyine iboneka gusa mu gusaza kw’ingirabuzimafatizo.
Imiterere y’isohorwa ry’ibintu. Imiterere y’isohorwa ry’ibintu ijyanye no gusaza kw’ingirabuzimafatizo, cyangwa SASP, ivuga ihinduka mu gusohora cytokine, chemokine, ibintu bitera gukura na protease. Ibipimo bya RNA cyangwa poroteyine by’ibigize SASP bisobanura uko ingirabuzimafatizo zohereza ibimenyetso ku zindi zegeranye na zo. Ntibipima mu buryo butaziguye ubushobozi bwo kwigabanya.
Ibintu bitera okisidasiyo n’ibipimo bya mitokondiriya. Inyandiko z’ubushakashatsi kuri methylene blue zongeraho ibipimo bya mitokondiriya: ubwinshi n’imikorere bya complex IV, ikoreshwa rya ogisijeni, ikorwa rya heme, ubwinshi bw’uduhu, ikinyuranyo cy’amashanyarazi ku ruhu, ATP n’ibimenyetso by’ibintu bitera okisidasiyo. Ibi bisobanura uko ihindurabintu rimeze mu gihe guhagarara kw’ukwigabanya gutinze. Byonyine ntibisobanura gusaza kw’ingirabuzimafatizo, ariko bigenzura uburyo bw’imikorere bwatanzwe nk’ibisobanuro. Uburyo bw’umuzenguruko wa redox, aho NAD(P)H iha methylene blue elegitoroni maze cytochrome c ikongera kuyikuraho izo elegitoroni, busobanurwa muri ubwo bushakashatsi bwa IMR90 buhuza umuzenguruko wa redox n’igabanuka ry’ikorwa ry’ibintu bitera okisidasiyo. Ibisobanuro bifitanye isano n’ingano ikoreshwa n’uko ikwirakwira biri mu nyandiko yerekeye imikorere ya methylene blue mu mubiri n’uko igera mu ngirangingo.
Umuvuduko w’igabanuka rya telomere. Igipimo gikwiye ni umubare w’ibice shingiro bya DNA bibura kuri buri kwikuba kabiri mu gihe cy’ibyumweru, si uburebure bupimwe rimwe gusa. Kugabanuka buhoro bivuga ko telomere zigabanukaho bike kuri buri kwigabanya. Ntibivuga ko ziba ndende kurushaho, kandi ihungabana rishobora guhagarika ingirabuzimafatizo za IMR90 telomere zahindutseho gato gusa.
Kugereranya uburyo butatu bw’ubushakashatsi kuri fibroblaste
Igice cy’umwimerere cy’iyi paji ni imbonerahamwe yo kugereranya iri hasi. Ishyira hamwe ubwoko bw’ingirabuzimafatizo, ibyo zahawe, ibyapimwe n’imbibi z’ibyo ubushakashatsi bushobora kwerekana.
| Ubushakashatsi | Ingirabuzimafatizo n’ibyo zahawe | Ibyapimwe | Ibyo budashobora kwerekana |
|---|---|---|---|
| Atamna n’abandi, FASEB J 2008 | Fibroblaste zisanzwe za IMR90, methylene blue ku rugero rwa nanomolar ikoreshwa igihe kirekire, 100 nM nk’urugero rusanzwe | Inshuro zose zo kwikuba kabiri, ubwinshi bwa complex IV, ikoreshwa rya ogisijeni ryiyongereyeho 37 kugeza kuri 70 ku ijana, ikorwa rya heme, gukumira gusaza hakiri kare guterwa na hydrogen peroxide cyangwa cadmium, anzime zo mu cyiciro cya 2 mu ngirabuzimafatizo za HepG2 | Nta kugerageza gukuraho ingirabuzimafatizo zamaze gusaza. Nta gipimo cy’igihe inyamaswa cyangwa abantu babaho. Igihe cyo kwigabanya hano kivuga inshuro zo kwikuba kabiri mu gasahani ka laboratwari. |
| Atamna n’abandi, Redox Biology 2015, busobanurwa mu bushakashatsi bwakurikiyeho ku mikorere ya AMPK n’ikorwa rya complex IV | Fibroblaste za IMR90, cyane cyane methylene blue kuri 100 nM, hamwe na AICAR nk’ikigereranyo gikomeza gukangura AMPK igihe kirekire | Uko igipimo cya NAD ugereranyije na NADH gihinduka mu gihe, iyongerwa ry’agatsinda ka fosifate kuri AMPK ry’igihe gito, gukangurwa kwa PGC-1alpha na SURF1, ukwiyongera kurenga 100 ku ijana kw’imikorere ya complex IV, igabanuka rya 28 ku ijana ry’ibintu bitera okisidasiyo, igabanuka ritinze rya telomere, hafi inshuro 18 ziyongereye zo kwikuba kabiri ugereranyije na hafi 2 kuri AICAR, nta ngaruka ku ruzenguruko rw’ingirabuzimafatizo | Ingano ya AICAR ikoreshwa mu kugereranya itandukanyeho hafi inshuro 1000, kandi n’ibyo ikoraho mu buryo bwihariye biratandukanye; bityo igereranya risuzuma uburyo bwo gukangura aho kugereranya ububasha bungana. Ibyavuye ku bipimo bya telomere bireba ingirabuzimafatizo zikomeza kwigabanya mu gihe cy’ibyumweru. |
| Xiong n’abandi, Aging Cell 2016, bwatangajwe mu bushakashatsi kuri fibroblaste za progeria no gusubiza mitokondiriya n’intima mu mimerere myiza | Fibroblaste z’ibanze zo mu ruhu rw’abafite progeria ya Hutchinson-Gilford hamwe n’izisanzwe zatoranyijwe ngo zigereranywe, ahanini 100 nM mu gihe cy’ibyumweru 5 kugeza kuri 12 | Imiterere ya mitokondiriya hakoreshejwe floresansi na mikorosikopi y’elegitoroni, uko zigenda hakoreshejwe amashusho y’ingirabuzimafatizo zikiri nzima, superoxide, ikinyuranyo cy’amashanyarazi ku ruhu, ATP, PGC-1alpha, ibimenyetso by’ukwiyongera no gusaza kw’ingirabuzimafatizo, hamwe n’udubyimba ku ntima, aho progerin iri, heterochromatin n’uko genes zikora | Ni uburyo bwo kwiga gusaza hakiri kare bujyanye n’indwara yihariye, si gusaza bisanzwe. Gusubiza imiterere n’ibimenyetso mu mimerere myiza si ugukuraho ingirabuzimafatizo zamaze gusaza. Nta gipimo cy’imikorere ku nyamaswa cyangwa mu bantu mu rwego rw’ubuvuzi. |
| Xue n’abandi, isesengura muri Cells 2021, ryavuzwe mu isesengura rya methylene blue nk’ikinyabutabire gishobora kwigwa ku kurwanya gusaza | Guhuriza hamwe inyandiko ku gusaza k’ubwonko, kwangirika kw’ingirabuzimafatizo z’imyakura, uruhu, gukira kw’ibikomere na progeria | Risobanura methylene blue nk’ikinyabutabire cyihutisha umuzenguruko wa redox gishobora kunyura ku ruhande rw’ibice bimwe by’ihererekanya muri complex I na III, rikabigereranya n’ibitekerezo bihuza gusaza n’ibice byigenga bifite elegitoroni itagira iyunganira | Ni isesengura rihuza inyandiko gusa. Ritegura ibitekerezo bigomba kugenzurwa kandi rikagaragaza aho ibimenyetso byo mu bushakashatsi ku bantu bitaratanga umwanzuro. |
Icyerekezo rusange kigaragara muri iyo mirongo gifite akamaro kurusha umurongo umwe uwo ari wo wose. Methylene blue ikoreshwa ku rugero ruto ihora ihuzwa n’igihe kirekire cyo kwiyongera kw’ingirabuzimafatizo, imikorere ya complex IV yiyongereye, ibimenyetso by’ibintu bitera okisidasiyo byagabanutse n’imiterere yarushijeho kuba myiza. Uburyo bw’ubushakashatsi bukomeza gukurikirana impinduka mu gihe: gutangirana n’ingirabuzimafatizo zigabanya, kuzishyiraho ikinyabutabire mu gihe ziyongera no kwandika igihe kwigabanya bikomeza. Ubwo buryo bushobora gushyigikira ko gusaza gutinze. Ntibushobora gushyigikira ko ingirabuzimafatizo zamaze gusaza zakuwemo hatabaye ubundi buryo bw’ubushakashatsi.
Gutinza gusaza kw’ingirabuzimafatizo si ugukuraho izamaze gusaza
Gutinza bivuga ko ingirabuzimafatizo zigabanya zikomeza kwigabanya igihe kirekire mbere yo guhagarara. Gukuraho, bikunze kwitwa igikorwa cya senolytic, bivuga ko ingirabuzimafatizo zamaze gusaza zicwa cyangwa zigabanywa mu buryo bwihariye, mu gihe izindi zegeranye na zo zikiyongera zisigara ari nzima.
Inyandiko z’ubushakashatsi bwa methylene blue kuri fibroblaste zisuzuma ubwo buryo bwa mbere. Ingirabuzimafatizo zororwa zitangira zishobora kwiyongera, zigahabwa ikinyabutabire mu gihe zikomeza kwimurirwa mu bindi bikoresho byo kuzororeramo, maze zigahagarika kwigabanya nyuma. Bityo ibimenyetso nka SA-beta-gal, p16, p21 n’ibigize SASP bigaragara nyuma cyangwa ku rugero ruto mu gihe runaka cyo kwimura ingirabuzimafatizo. Icyo cyerekezo kiba cyitezwe igihe cyose itangira ryo gusaza ryimukiye inyuma. Ntigihamya ko hari ingirabuzimafatizo yari yaramaze gusaza yakuwemo.
Kuvuga ko ikinyabutabire gifite igikorwa cya senolytic byasaba uburyo butandukanye: kubanza gutuma ingirabuzimafatizo zororwa zigera mu gusaza kwemejwe n’ibipimo, hanyuma gushyiraho ikinyabutabire, hanyuma kwerekana ko izo ngirabuzimafatizo zamaze gusaza zigabanuka mu buryo bwihariye ugereranyije n’izikiyongera zo kugereranya, hakoreshejwe ibipimo bitaziguye by’ubuzima n’urupfu rwazo. Kugabanuka kw’ikimenyetso kimwe gusa ntibihagije, kuko uko kigaragazwa bishobora guhinduka ingirabuzimafatizo zitagabanutse, kandi izisigaye zikiyongera zishobora kugwira zikuzura agasahani. Nta na rimwe muri ayo masoko ane yavuzwe rikoresha ubwo buryo bwo kugabanya mu buryo bwihariye ingirabuzimafatizo zamaze gusaza hakoreshejwe methylene blue; bityo ibisobanuro nyabyo bikomeza kuba igikorwa cyo gutinza cyangwa kurwanya gusaza kw’ingirabuzimafatizo muri laboratwari, si igikorwa cya senolytic. Uruhare rw’ihungabana riterwa na okisidasiyo muri iryo tandukaniro rusobanurwa kurushaho mu nyandiko yerekeye uko ingirabuzimafatizo zigenzura amoko ya ogisijeni akora cyane mu miti.
Ibyavuye ku bimenyetso by’igihe gito birushaho gusobanura icyo gitekerezo. Mu bushakashatsi bwa IMR90 bwo muri 2015, methylene blue yatumye igipimo cya NAD ugereranyije na NADH cyiyongera igihe gito, inatera iyongerwa ry’agatsinda ka fosifate kuri AMPK ry’igihe gito, bikurikirwa no gukangurwa kwa PGC-1alpha na SURF1, byombi bifitanye isano n’ikorwa rya complex IV. Gukomeza gukangura AMPK igihe kirekire hakoreshejwe AICAR byatanze inshuro nke cyane ziyongereye zo kwikuba kabiri.
Igihe ingirabuzimafatizo zimara zishobora kwigabanya si igihe abantu babaho
Igihe ingirabuzimafatizo zimara zishobora kwigabanya kibarwa mu nshuro umubare wazo wikuba kabiri mbere yo guhagarara, mu gasahani ka laboratwari gafite ogisijeni, uruvange ruzitunga n’inshuro zo kuzimura bigenzurwa kandi biguma ku rugero rumwe. Igihe abantu babaho gikubiyemo imyaka myinshi yo gukura, ubudahangarwa, imiyoboro y’amaraso, ahantu ingirabuzimafatizo shingiro ziba, imyitwarire n’indwara. Ikinyabutabire gishobora kongera igihe cya mbere kidahinduye icya kabiri, kandi ibyinshi ni uko bigenda.
Fibroblaste za progeria zifite ihinduka ryihariye muri gene ya LMNA rituma hakorwa progerin, zikagira mitokondiriya zicagaguye kandi zitagenda cyane, ATP nkeya, intima zifite imiterere idasanzwe n’igabanuka rya heterochromatin. Methylene blue ku rugero rwa nanomolar yatumye ibipimo bya mitokondiriya biba byiza kurushaho kandi isubiza ku rugero rukomeye ibipimo by’intima mu mimerere myiza, harimo no kurekura progerin ikava ku ruhu. Ibyo ni impinduka zikomeye ku rwego rw’ingirabuzimafatizo. Ariko bikomeza kuba ibisubizo byo kuri fibroblaste, kandi abanditsi bashyize kugerageza ku nyamaswa mu bushakashatsi buzaza. Ubwo buryo bwo kwiga indwara busobanurwa kurushaho mu nyandiko yerekeye methylene blue muri progeria no mu buryo bwo kwiga gusaza hakiri kare.
Ibi bitanga urutonde rw’ibibazo bifasha gusoma ubushakashatsi.
- Baza niba ikinyabutabire cyatangiye gukoreshwa mbere yo guhagarara kw’ukwigabanya cyangwa nyuma y’uko uko guhagarara kwemejwe. Gukoreshwa mbere bishyigikira gutinza. Gukoreshwa nyuma, hamwe n’ibipimo by’urupfu rw’ingirabuzimafatizo rwihariye, birakenewe kugira ngo hemezwe gukuraho izamaze gusaza.
- Baza uko umurongo werekana kwikuba kabiri kw’ingirabuzimafatizo zo kugereranya wahindutse. Hatabaye kubara inshuro zose zo kwikuba kabiri, impinduka mu bimenyetso ntizigira aho zishingira.
- Saba nibura ibyiciro bibiri by’ibimenyetso, nk’ikimenyetso cy’ukwiyongera hamwe na p16 cyangwa p21 hamwe na SA-beta-gal, aho kwishingikiriza ku irangi rimwe gusa.
- Baza uko ibipimo bya telomere byatangajwe. Umuvuduko w’igabanuka kuri buri kwikuba kabiri mu gihe runaka ufite icyo usobanura. Uburebure bupimwe rimwe gusa ntibuhagije.
- Baza ibyagumishijwe ku rugero rumwe. Ubucucike bw’ingirabuzimafatizo igihe zimurwa, ogisijeni, guhindura uruvange ruzitunga no gukoresha imvange za methylene blue nshya cyangwa zimaze igihe, byose bihindura guhagarara kw’ukwigabanya kwa fibroblaste.
Iyo zisomwe muri ubu buryo, inyandiko z’ubushakashatsi kuri fibroblaste ziha methylene blue uruhare rwumvikana ariko rufite imbibi: ikinyabutabire gikora muri redox ku rugero ruto, gitinza gusaza kujyanye n’ukwigabanya kwa fibroblaste z’umuntu zororerwa muri laboratwari kandi kigatuma ibipimo bya mitokondiriya biba byiza kurushaho. Ibisobanuro bikomeye kurusha ibindi by’imikorere yacyo biri ku bimenyetso by’ingufu by’igihe gito no ku ikorwa rya complex IV, kandi nta gihamya muri izi nyandiko yerekana gukuraho ingirabuzimafatizo zamaze gusaza cyangwa kongera igihe abantu babaho.