Gupima ubwoko bwa ogisijeni bukora cyane mu igerageza rya methylene blue

Laboratwari ebyiri zikora icyo buri imwe yita igerageza ryo kurwanya okisidasiyo hakoreshejwe methylene blue. Imwe ivuga ko iryo rangi rigabanya ubwoko bwa ogisijeni bukora cyane mu myitwarire ya shimi. Indi ivuga ko ribubyara. Zombi zerekana imirongo isobanutse y’uko igisubizo gihinduka uko ingano y’irangi ihinduka. Zombi zivuga ibyo zabonye nta buryarya. Itandukaniro ntiriterwa n’uburiganya cyangwa kutagira ubushobozi. Ahubwo ayo matsinda yombi yapimye ibintu bitandukanye, mu rumuri rutandukanye, akoresheje ibinyabutabire bipima bisubiza ibibazo bitandukanye, hanyuma asobanura ibyo bisubizo byombi akoresheje ijambo rimwe: ROS.

Methylene blue ni molekile igoye gupima neza kuko igira uruhare mu myitwarire ya shimi iba igomba gukurikirana. Mu mwijima yimura elegitoroni hagati y’abazitanga n’abazakira, hanyuma igasubira mu miterere yayo ya mbere, yiteguye kongera gukora uwo muzenguruko. Mu rumuri ihinduka ikinyabutabire gituma habaho imyitwarire ya shimi iterwa n’urumuri, ikagera mu rwego rwa triplet rufite ingufu ziyongereye. Muri urwo rwego ishobora guha ogisijeni ingufu ikabyara ogisijeni ya singlet, cyangwa kwimura elegitoroni ikabyara radikali na perogiside ya hidirojeni. Ikinyabutabire icyo ari cyo cyose gipima gishyizwe muri iyo sisitemu gishobora gukorerwa okisidasiyo, kugabanyirizwa, gucibwa ibara, cyangwa ikimenyetso cyacyo cy’urumuri kigapfukiranwa n’irangi ubwaryo. Bityo, ikibazo cya mbere ku gipimo icyo ari cyo cyose cya ROS si ukumenya umubare wabonetse. Ni ukumenya icyo isuzuma ritahura koko, n’icyo irangi rikora kuri iryo suzuma.

Amplex Red: isuzuma rya perogiside ya hidirojeni rifite ibipimo fatizo

Guhuza methylene blue na Amplex Red ni bwo buryo bufite ishingiro rikomeye kurusha ubundi muri izi nyandiko z’ubushakashatsi, iyo buteguwe neza. Imyitwarire ya shimi yabwo irasobanutse: perogisidaze yo mu gihingwa cya horseradish ikoresha perogiside ya hidirojeni mu gukorera okisidasiyo ikinyabutabire Amplex ikagihindura resorufin, ikinyabutabire gitanga urumuri rwa fluorescence, ku kigereranyo cya shimi hafi ya kimwe kuri kimwe. Perogiside iri mu cyitegererezo ihinduka urumuri rwa fluorescence rufatwa n’igikoresho gitahura ibimenyetso. Nta kindi kinyabutabire cyo mu muryango usanzwe wa ROS gitera iyo myitwarire mu buryo butaziguye.

Inyungu y’ingenzi ni ukugena ibipimo fatizo. Umushakashatsi witonda apima ingano zizwi za perogiside mu gisubizo kimwe kigumana pH, akoresheje ingano zimwe za perogisidaze n’ikinyabutabire ikoreraho, igihe kimwe cyo kubireka bigakorana, n’imiterere imwe y’igikoresho gipima urumuri, hanyuma akagereranya ibipimo by’ibyitegererezo n’uwo murongo fatizo. Mu bushakashatsi kuri mitokondiriya zatandukanijwe n’ubwonko, ari bwo shingiro ry’ubu buryo, abanditsi bakoze ibyo neza, bongeramo ingano zizwi za perogiside ku musozo wa buri gerageza. Imibare yatanzwe nk’umuvuduko muri pikomole ku munota kuri miligarama ya poroteyine, aho kuba ibipimo bya fluorescence bidafite igipimo fatizo. Ibyo ni byo bihindura urumuri rukaba igipimo.

Ibintu bitatu byo muri ubwo bushakashatsi bikwiye kwiganwa. Icya mbere, abanditsi bagenzuye ikimenyetso bakoresheje katalaze, isenya perogiside. Ukwiyongera kwa fluorescence kwatewe na methylene blue kwarahagaze igihe hongewemo katalaze, byemeza ko ikimenyetso cyaturukaga kuri perogiside aho kuba ikimenyetso kitari nyakuri gitewe n’imiterere y’urumuri. Icya kabiri, bapimye ingaruka z’urumuri mu buryo butaziguye bafungura kandi bagafunga inzira y’umurase utera fluorescence, ku buryo ibyitegererezo byabonaga kimwe cya munani cy’ingano isanzwe y’urumuri. Ikimenyetso cyagabanutseho icyenda ku ijana gusa. Hejuru ya mirongo cyenda ku ijana bya perogiside batangaje ntibyaterwaga n’urumuri rwakoreshwaga mu gupima. Icya gatatu, bapimye ukwikuraho perogiside ukwako bakoresheje elekturode, basanga irangi ryarongeraga ikorwa ryayo kandi rikagabanya umuvuduko wo kuyikuraho. Ikinyabutabire gipima fluorescence isigaye gusa kiba cyarafashe izo ngaruka zombi nk’ikintu kimwe.

Iri suzuma riracyafite uburyo rishobora gutanga ibipimo bitari byo, kandi methylene blue ituma bwinshi muri bwo bugaragara. Irangi rinyunyuza urumuri mu gice kinini cy’akarere k’urumuri rutukura resorufin isohoreramo urumuri. Bityo, iyo ingano y’irangi ari nyinshi, icyitegererezo gishobora gupfukirana ikimenyetso cyacyo ubwacyo kubera ingaruka zo kuyungurura urumuri imbere mu cyitegererezo. Ni yo mpamvu ibisubizo bikoreshwa mu kugena ibipimo fatizo bigomba kuba birimo ingano y’irangi ingana n’iyo mu byitegererezo. Amplex Red na yo ishobora gukorerwa okisidasiyo n’urumuri yonyine, kandi resorufin itanga ishobora kwinjira mu muzenguruko wa okisidasiyo no kugabanyirizwa uterwa n’urumuri, ubyara superogiside na perogiside. Ibyo ni ingenzi cyane iyo igerageza rinamurikira ikinyabutabire gituma habaho imyitwarire ya shimi iterwa n’urumuri. Igisubizo gifatika ni ukubanza kumurikira ibyitegererezo bya methylene blue, hanyuma ugapima ibice byabyo hakoreshejwe ibinyabutabire bya Amplex mu mwijima, aho kumurikira irangi n’ikinyabutabire gipima icyarimwe. Hanyuma, superogiside disimutaze ntigabanya gusa ikimenyetso cya Amplex: guhindura superogiside mo perogiside bishobora kucyongera. Igenzura rikoresheje SOD ryongera fluorescence ntiriba ryananiwe. Riba ryagaragaje ko superogiside ari ikinyabutabire kibanziriza perogiside muri iyo myitwarire.

DCFDA: ikimenyetso cya okisidasiyo kidatandukanya ibiyitera

Guhuza methylene blue na DCFDA ni byo bitera urujijo rwinshi. DCFH-DA ni ikinyabutabire kibanziriza igipimisho, cyinjizwa mu ngirabuzimafatizo. Esiteraze ziri imbere mu ngirabuzimafatizo zikuraho amatsinda yacyo ya asetate, zigasiga DCFH idatanga fluorescence. Okisidasiyo ya DCFH itanga DCF itanga fluorescence. Ibiyikorera okisidasiyo ni byinshi: radikali za hydroxyl, ibinyabutabire by’agateganyo bya perogisidaze na heme, diyogiside ya azote, aside hypochlorous, na radikali zikomoka kuri perogisinitirite. Perogiside ya hidirojeni yonyine ntishobora kuyikorera okisidasiyo mu buryo butaziguye ku muvuduko w’ingirakamaro; fluorescence ishingiye kuri perogiside muri rusange ikenera ubutare, poroteyine za heme, cyangwa imyitwarire ya shimi ya perogisidaze muri uwo muzenguruko.

Isesengura rinenga ubu buryo, buri gice gisobanura uburyo bw’ubushakashatsi cyagombye kwifashisha, rikomoka ku bushakashatsi bwa Kalyanaraman na bagenzi be kuri DCFH nk’ikinyabutabire gipima. Nk’uko bivunaguye mu bushakashatsi kuri mitokondiriya z’ubwonko bwanze gukoresha DCFDA kubera iyo mpamvu, berekanye ko icyo kinyabutabire gikora buhoro, ku kigereranyo cya shimi kitaramenyekana, kidafite ibipimo fatizo byemewe, kandi gikorana n’ibinyabutabire bitari ROS. Ikinyabutabire gipima kinongera ibyo gipima: okisidasiyo ya DCFH ikuraho elegitoroni imwe itanga radikali ya DCF ikorana na ogisijeni ikabyara superogiside, na yo igahinduka ikabyara indi perogiside. Igipimo kiboneka kiba gifite uruhare mu gukora igice cy’imyitwarire ya shimi kivuga ko cyapimye.

Nta na kimwe muri ibyo gituma DCFDA iba idafite akamaro. Bituma iba uburyo bwo gushakisha ikimenyetso, aho kuba uburyo bwo kumenya ikinyabutabire nyacyo. Ukwiyongera kwa fluorescence ya DCF bivuga ko okisidasiyo y’ikinyabutabire gipima, iterwa n’ibinyabutabire bikora okisidasiyo, yiyongereye. Ntibivuga ko perogiside yiyongereye, kandi rwose ntibivuga ko ROS zose hamwe ziyongereye, kuko nta kinyabutabire kimwe gipima uwo mubare wose. Ubushakashatsi bwa mbere kuri methylene blue bwashingiye kuri okisidasiyo ya DCF ku ngano irenga mikromolari imwe bwatangaje ingaruka zo guteza okisidasiyo, mu gihe izindi sisitemu zo muri icyo gihe zatangaje kurindwa urupfu rw’ingirabuzimafatizo rwatewe na perogiside. Ibyo byombi bishobora kuba ukuri ku irangi rimwe mu mimerere itandukanye ya okisidasiyo no kugabanyirizwa, kandi igisubizo cya DCF cyonyine ntigishobora kuvuga ikinyabutabire gitera okisidasiyo cyatanze ikimenyetso. Akamaro k’ubushakashatsi bwa mbere kuri mitokondiriya z’ubwonko ni uko bwasimbuje ubu buryo bwo gushakisha ikimenyetso igipimo cya perogiside gifite ibipimo fatizo, bukabona ubwiyongere mu buryo butaziguye: ndetse na nanomolari ijana gusa z’irangi zongereye inshuro nyinshi isohoka rya perogiside ku binyabutabire byose byagaburiraga iyo sisitemu no mu mimerere yose y’ubuhumekero bw’ingirabuzimafatizo byageragejwe.

DCFDA ifite undi mutego wihariye kuri methylene blue. Irangi rifite ibara rikomeye kandi rigira uruhare muri okisidasiyo no kugabanyirizwa, bityo rishobora guhanagura fluorescence, kugabanya urumuri rusohoka, cyangwa gukorera okisidasiyo ikinyabutabire gipima bitagendeye kuri ROS zo mu ngirabuzimafatizo. Igenzura ririmo irangi n’ikinyabutabire gipima ariko ritarimo ingirabuzimafatizo si inyongera ushobora kureka. Ni ryo gerageza rikwereka niba irangi rikorana n’ikinyabutabire gipima.

Gufata radikali hakoreshejwe spin no gupima ikoreshwa rya ogisijeni: ubwoko n’umuvuduko

Aho ibinyabutabire bipima bitanga fluorescence byerekana ko hari ikintu gishobora gukorerwa okisidasiyo cyagaragaye, rezonansi ya spin ya elegitoroni ikoresheje ibifata radikali itanga amakuru yegera cyane kumenya icyo ari cyo. Ubushakashatsi bw’ingenzi ku myitwarire ya shimi ya methylene blue iterwa n’urumuri bwakoresheje ESR mu gutahura radikali ya asikorubate ikomoka ku irangi, asikorubate n’urumuri. Nyuma bwerekanye ikorwa rya perogiside mu magerageza yo gupima ikoreshwa rya ogisijeni, n’ikorwa rya radikali ya hydroxyl ryihutishwa n’ubutare mu magerageza yo gufata radikali hakoreshejwe spin. Buri buryo bwasubizaga ikibazo ubundi butashoboraga gusubiza: ESR yamenyeshaga ubwoko bwa radikali, ikoreshwa rya ogisijeni rikagaragaza umuvuduko w’imyitwarire ya shimi utagendeye ku kinyabutabire icyo ari cyo cyose gipima fluorescence, naho kubihuza bikerekana ko inzira ya ogisijeni ya singlet n’iza radikali zombi zakoraga muri sisitemu imwe yamurikwaga.

Icyo gisubizo cyo mu 1984 kiracyasomwa nk’umuburo. Iyo habaga hari urumuri n’ikinyabutabire gitanga elegitoroni, irangi ryabyaraga perogiside; iyo habaga harimo n’ubutare, sisitemu yabyaraga radikali ya hydroxyl, ari yo ikora cyane kurusha ibindi binyabutabire byo muri iryo tsinda. Abanditsi babihuje na asikorubate iri mu jisho, bashidikanya ku mikoreshereze yaryo mu kuvura amaso idafite ubwitonzi buhagije. Abasomyi b’ubu bakwiye kuzirikana imimerere y’igerageza: urumuri hamwe n’ikinyabutabire gitanga elegitoroni hamwe n’ubutare. Iyo ukuyeho kimwe muri ibyo, iyo nzira iracika intege cyangwa igashira.

Elekturode zipima ogisijeni zifite akamaro mu bundi buryo. Elekturode yo mu bwoko bwa Clark ipima ikoreshwa rusange rya ogisijeni, bikemeza ko imyitwarire ya okisidasiyo no kugabanyirizwa iterwa n’irangi ikoresha ogisijeni ya molekile, kandi igatanga amakuru y’umuvuduko adashobora kwiganwa n’ikimenyetso kitari nyakuri cya fluorescence. Ntimenya ikinyabutabire kibyara. Ikorwa rya perogiside, superogiside n’amazi, ndetse no gushira kwa ogisijeni ya singlet, byose bishobora kujyana n’ikoreshwa rya ogisijeni. Kumenya umuvuduko utazi ubwoko bw’ibiboneka ntibihagije, kimwe no kumenya ubwoko bwabyo utazi umuvuduko. Ni yo mpamvu inyandiko z’ubushakashatsi zikomeye zihuza elekturode n’isuzuma rya perogiside rifite ibipimo fatizo cyangwa n’igifata radikali.

Impamvu ibisubizo ku kurwanya okisidasiyo bivuguruzanya

Iyo ugereranyije ibyo bipimo bitatu, ibisa n’ibivuguruzanya mu nyandiko z’ubushakashatsi kuri methylene blue bisobanuka nk’itandukaniro ry’uburyo bwakoreshejwe hamwe n’ibintu bitatu by’ukuri mu binyabuzima.

Icya mbere, irangi ryimura elegitoroni kandi rikaba isoko ya perogiside. Iyo riyoboreye elegitoroni kure y’ahantu hakorerwa ubuhumekero bw’ingirabuzimafatizo aho ogisijeni iba yarahindutse superogiside, rishobora kugabanya ubwoko bumwe bwa ROS, mu gihe imiterere yaryo yakiriye elegitoroni ihindura ogisijeni mo perogiside mu buryo butaziguye, ikongera ubundi bwoko. Laboratwari yiga imyitwarire ya okisidasiyo no kugabanyirizwa muri mitokondiriya n’uruhare rwa methylene blue mu gutanga indi nzira rero ikwiye kwitega ibisubizo biterwa n’ikinyabutabire gipima: igipimo cyumva superogiside kiragabanuka, mu gihe igipimo cya perogiside cyiyongera mu gaheha kamwe ko gupimiramo.

Icya kabiri, perogiside iri mu ngano nto iba ikimenyetso, si ibyangiza gusa. Ubushakashatsi kuri mitokondiriya z’ubwonko bw’imbeba bwakurikiye ubwa mbere bwagaragaje ikorwa rya perogiside ritangirira ku irangi ringana na nanomolari mirongo itanu gusa, nta kwangirika kwa DNA ya mitokondiriya ku ngano zikoreshwa mu buvuzi, kandi gukoresha irangi mbere byarindaga kwangirika guterwa na rotenone. Ku ngano ya mikromolari ijana gusa, iri kure cyane hejuru y’urwego rukoreshwa mu buvuzi, ni ho irangi ryangije ibice bimwe bya DNA. Urumuri rutukura hamwe n’irangi ringana na mikromolari makumyabiri byabyaye ogisijeni ya singlet yangije ibice byose byageragejwe. Uburyo bwatanzwe nk’igisobanuro ni ubwa hormesis: ikorwa rito rya perogiside rikangura uburyo bwo kwirinda okisidasiyo bufitanye isano na Nrf2 n’ikorwa rya mitokondiriya nshya. Bityo, igipimo cy’igihe gito cyerekana guteza okisidasiyo kibanziriza igisubizo cy’igihe kirekire cyo kuyirwanya. Isuzuma risomwe nyuma y’isaha imwe n’isuzuma risomwe nyuma y’umunsi umwe bishobora kutavuga rumwe kuko imikorere y’ibinyabuzima iba yahindutse hagati yabyo.

Icya gatatu, urumuri rugena na molekile iba ihari. Kumurika bikuraho amatsinda ya metile ku irangi, bikariganisha kuri Azure B n’ibindi binyabutabire bifitanye isano na yo, kandi itara rya mikorosikopi riri hejuru y’agasahani k’igerageza rirahagije kugira ngo rikangure imyitwarire yaryo iterwa n’urumuri. Amagerageza yo mu mwijima n’ayo mu rumuri ni amagerageza atandukanye, kandi igice kivuga ku igenzura ry’urumuri n’uburyo bwo gufata irangi muri amabwiriza ayo ari yo yose y’igerageza kigomba kuyafata gityo.

Igenzura ry’ibanze rikenewe n’uburyo bwo gusoma icyemezo icyo ari cyo cyose

Igereranya rikurikira ni imbonerahamwe y’umwimerere yifashishwa muri iyi nyandiko. Mbere yo kwemera igisubizo icyo ari cyo cyose cya ROS kijyanye na methylene blue, reba umurongo abanditsi bujuje n’uwo basimbutse.

Hari ihame rifatika ryo gusoma rikurikira. Amplex ikoreshejwe hamwe na perogisidaze, ifite ibipimo fatizo kandi yagenzuwe na katalaze, itanga gihamya kuri perogiside. DCFDA itanga gihamya ya okisidasiyo y’ikinyabutabire gipima idatandukanya ibiyitera, nta kindi. Gufata radikali hakoreshejwe spin, hamwe n’igenzura rikoresha ibinyabutabire bifata radikali bikazikuraho, bitanga gihamya y’ubwoko bwa radikali. Ikoreshwa rya ogisijeni ritanga gihamya y’umuvuduko w’imyitwarire ya shimi. Interuro yemeza ibirenze ibyo isuzuma ryayo rishobora gushyigikira, uko yaba yanditswe yizeye kose, iba ari interuro ivuga ku kinyabutabire gipima, aho kuvuga ku irangi. Ibiganiro bifitanye isano ku ngaruka ziterwa n’ingano no ku rwego rwa hormesis, ku rugendo rwa elegitoroni muri mitokondiriya, ku kubangamira amasuzuma mu byitegererezo byasizwe irangi, no ku bimenyetso bitari nyakuri biterwa n’urumuri rwa mikorosikopi bisobanura buri imwe muri izi ngingo mu buryo bwagutse, kandi buri ngingo ikomeza kugira agaciro iyo icyifashisho cy’agateganyo kiyijyanyeho gisimbujwe icyifashisho nyacyo.