Igihe methylene blue ibangamira igerageza rikoreshwa mu kuyipima

Laboratwari imwe yororera insoro z'amafi ya zebrafish muri methylene blue ku bwinshi busanzwe bukoreshwa mu bworozi, hanyuma igapima ihinduranyabintu mu buryo bubiri. Ku munsi wa mbere, igikoresho cya Seahorse cyerekana ko ikoreshwa rya ogisijeni ryiyongereye. Ku minsi ya kane n'iya gatanu, isahani y'ibipimo bya Alamar Blue yerekana ko ikoreshwa ry'ingufu ryagabanutse. Irangi rimwe, umurongo umwe w'amafi, ariko imyanzuro irahabanye. Hanyuma hakaza igenzura ritagaragara cyane: utwobo turimo methylene blue n'umuti wa Alamar Blue, nta mafi na gato, na two dutanga urumuri ruke kurusha uturimo umuti wonyine. Igice cy'ibisubizo cyaturukaga ku binyabuzima. Ikindi gice cyaturukaga ku irangi ryihaga amanota mu kizamini cyaryo. Iryo tandukaniro, ryagaragajwe mu bushakashatsi bwa Communications Biology bwo mu 2025 kuri methylene blue mu mafi ya zebrafish akura, ni bwo buryo methylene blue isanzwe ibangamiramo ibipimo. Ni na yo mpamvu amarangi agira uruhare mu guhererekanya electron asaba igenzura ibinyabutabire bisanzwe bipimwa bidakenera.

Icyo igipimo cya resazurin cyerekana koko

Alamar Blue ni izina ry'ubucuruzi ry'umuti wa resazurin. Bityo, kuvuga ko methylene blue ibangamira Alamar Blue cyangwa ko ibangamira igipimo cya resazurin ni ukuvuga imikoranire imwe. Resazurin yambuwe electron iba ubururu kandi itanga floresansi nkeya cyane. Uturemangingo dukora ihinduranyabintu tuyihereza electron, tukayihindura resorufin, iba iroza kandi itanga floresansi nyinshi hafi y'uburebure bw'umuraba bwa 560 nm bwo kuyibyutsa na 590 nm bwo gusohora urumuri. Hakurikijwe amabwiriza y'uruganda yo gukoresha isahani y'utwobo duto, uburebure bw'umuraba bukoreshwa bushobora kuba hafi ya 530 kugeza kuri 570 nm mu kuyibyutsa na 580 kugeza kuri 610 nm mu gusohora urumuri. Iyo ikomeza guhabwa electron, resorufin ihinduka dihydroresorufin, itanga floresansi nkeya. Bityo, gushyira uturemangingo twinshi cyane mu kobo cyangwa kurekera igerageza igihe kirekire cyane bituma umurongo wo kugena ibipimo ugana hasi ku mpera yawo yo hejuru.

Electron ziva ahanini kuri NADH na NADPH, zinyuze kuri reductase na diaphorase byo mu turemangingo, biri muri cytosol, microsome na mitokondiriya. Ibyo bituma igipimo cyerekana ubushobozi rusange bwo gutanga electron, aho kuba umubare w'uturemangingo tukiri tuzima cyangwa igipimo cya mitokondiriya. Ikimenyetso kiri hejuru gishobora gusobanura uturemangingo twinshi, umuvuduko munini wo gutanga electron kuri buri karemangingo, cyangwa byombi. Ikibinyabutabire gihindura inzira NADH inyuramo gihindura ikimenyetso kitishe n'akaremangingo kamwe. Ujye wibuka iyo nteruro, kuko guhindura inzira NADH inyuramo ari kimwe mu byo methylene blue ikora.

Inzira ebyiri zo kubangamira igipimo, ikimenyetso kimwe kigabanutse

Methylene blue igoreka igipimo cya resazurin binyuze mu nzira ebyiri zigenga, kandi buri imwe muri zo yonyine igabanya floresansi igaragara.

Inzira ya 1 ijyanye n'urumuri. Methylene blue yinjiza cyane urumuri rutukura: umurongo w'iyinjizurumuri w'imolekile yayo imwe uba hafi ya 660 kugeza kuri 665 nm, naho agace k'umurongo k'imolekile ebyiri zifatanye kaba hafi ya 607 kugeza kuri 610 nm, umurizo wako ukagera mu cyerekezo cy'urumuri resorufin isohora hafi ya 590 nm. Irangi riri mu nzira y'urumuri ryinjiza igice cy'urumuri rubyutsa floresansi kandi rikongera kwinjiza igice cy'urumuri rwa floresansi rwasohotse. Uko kuyungurura urumuri imbere mu gisukika gutuma akobo gatanga urumuri ruke n'iyo umusaruro wa resorufin utahindutse. Imiterere y'urumuri rwinjizwa na yo ihinduka bitewe n'uko irangi rimeze: isesengura ry'ibice bigize ibipimo by'urumuri rwa methylene blue mu mazi no mu miti ya cyclodextrin ritandukanya imolekile imwe, imolekile ebyiri zifatanye, n'urusobe rw'imolekile yakira n'iyakirwamo nk'ubwoko butatu bufite ibipimo bitandukanye byo kuzimya urumuri kuva kuri 500 kugeza kuri 700 nm. Bityo, ubunini bw'ikosa rijyanye n'urumuri buhindukana n'ubwinshi bw'irangi, ukwikusanyiriza hamwe kwaryo n'ukwifatanya kwaryo n'izindi molekile. Mu igerageza, tandukanya kugabanya urumuri bisanzwe no kuzimya floresansi ku rwego rw'imolekile ukoresheje igerageza ryo kugaruza igipimo cy'inyongera risobanurwa hasi, kuko uburyo bwo gukosora butandukanye.

Inzira ya 2 ni uguhatanira electron. Methylene blue yambuwe electron yakira electron za NAD(P)H, igahinduka leuco methylene blue itagira ibara. Ogisijeni yo mu rwego rw'imolekile yongera kuyambura electron, ikayisubiza mu buryo bw'ubururu, ari na ko hakorwa ibikomoka kuri ogisijeni yahawe electron, birimo perogiside ya hydrogène. Buri cyiciro cy'urwo ruziga gikoresha ubushobozi bwo gutanga electron bwari kuba bwahaye resazurin electron, kandi kigakoresha ogisijeni ari na ko gikora perogiside ku ruhande. Ikimenyetso cya Alamar Blue cyagabanutse gishobora rero gusobanura ko electron zanyuze mu ruziga rwa methylene blue aho kujya muri resorufin. Ku isahani, ibyo bigaragara nk'ihinduranyabintu ryagabanutse, hatitawe ku byo uturemangingo twakoze. Iyo mikoranire y'ibinyabutabire inasobanura umutego uhabanye n'uwo mu bikoresho bipima ogisijeni: irangi ryahawe electron, iyo ogisijeni yongeye kuzivana kuri ryo, rituma ogisijeni ikoreshwa n'ibinyabutabire nta kinyabuzima gihari. Ni yo mpamvu n'ibipimo by'ikoreshwa rya ogisijeni bikenera igenzura ririmo irangi ariko nta binyabuzima, nk'uko igereranya ry'ibipimo bya ogisijeni, ATP n'itandukaniro ry'amashanyarazi ku gahu ribisobanura ku bijyanye na mitokondiriya.

Ikimenyetso cyahindutse n'imikorere y'ibinyabuzima yahindutse bishobora kuba ari ukuri byombi

Ubushakashatsi ku mafi ya zebrafish bufite agaciro cyane kuko butanga nta mwanzuro woroshye wo guhitamo kimwe ngo uhakane ikindi. Impinduka z'ibinyabuzima zari nyakuri: ikoreshwa rya ogisijeni mu mimerere isanzwe ryiyongereye mu nsoro zifite umunsi 1 ku bwinshi bwombi bw'irangi bukoreshwa mu bworozi; ikoreshwa rya ogisijeni rijyanye no gukora ATP ryiyongereye ku bwinshi bwo hasi; itandukaniro ry'amashanyarazi ku gahu ryapimwe na JC-10 ryiyongereye ku munsi wa kabiri; kandi inyandiko za RNA z'urusobe rw'ibinyabutabire bya mitokondiriya zaragabanutse ku minsi ya mbere n'iya kane. Ikosa ry'igerageza na ryo ryari nyakuri: amafi yogejwe inshuro eshatu mbere y'intambwe ya Alamar Blue yari agifite ibara ry'ubururu rigaragara ku mutwe, mu kanwa no mu gice cy'impumuro, kandi utwobo tutarimo uturemangingo ariko turimo irangi n'umuti twatakaje floresansi nkeya ariko ifite ubusobanuro mu ibarurishamibare, nta kindi kibigizemo uruhare. Kugabanuka kwa Alamar Blue kwapimwe ku binyabuzima ku minsi ya kane n'iya gatanu kwari kunini kurusha ingaruka zapimwe nta turemangingo. Ni yo mpamvu abanditsi babonye ibisubizo nk'impinduka nyakuri z'ihinduranyabintu zireberwa mu ndorerwamo igoreka gato. Uwo ni wo mwumvire ukwiye. Ikibinyabutabire gihererekanya electron mu ruziga, kandi kikinjiza urumuri nk'irangi ry'ubururu, akenshi kibangamira icyarimwe inzira iri kwigwa n'imikoranire y'ibinyabutabire itanga ikimenyetso. Intego y'igenzura ni ugupima uruhare rwa buri kimwe, si ukwemeza ko kimwe kitigeze kibaho. Irindi somo rifitanye isano n'iryo riva mu bushakashatsi ku kwifatanya na cyclodextrin: igihe imolekile yakira yafungiraga irangi muri yo, gusiga ibara no kongera iyinjizwa rya glucose byagabanutse icyarimwe. Ibyo byerekanye ko ingaruka ku ihinduranyabintu zasabaga imikoranire itaziguye n'irangi, aho guterwa n'imikoranire rusange yo guhererekanya electron. Guhuza igipimo cy'irangi gishingiye ku miterere yaryo n'igipimo cy'imikorere ni byo bitandukanya uburyo igikorwa kibamo n'ikosa ry'igerageza. Imfashanyigisho yo gusoma ibivugwa ku ihinduranyabintu bishingiye ku marangi ikomeza gutsimbataza uwo mwitozo.

Igenzura ritandukanya izo ngaruka

Kora aya magenzura mbere yo gusobanura igisubizo icyo ari cyo cyose cya methylene blue kivuye ku isahani ya resazurin. Ubwinshi bw'ibinyabutabire, umuti uturemangingo tubamo, igihe cyo kubirekera hamwe n'urumuri bigomba guhura neza n'iby'igerageza ririmo uturemangingo, kuko buri nzira yavuzwe haruguru ibishingiraho.

Igenzura rya 3 n'irya 4 ni yo magenzura abiri inyandiko nyinshi zisimbuka kandi inyandiko nyinshi kuri methylene blue zikenera. Igenzura rya 3 ritahura imikoranire y'ibinyabutabire, irya 4 rigatahura ingaruka z'urumuri. Hamwe, agena imbibi z'ikosa ry'igerageza mbere yo kuvuga umwanzuro uwo ari wo wose ku binyabuzima.

Ibipimo byigenga, buri kimwe gifite ibyo kwitondera

Nta gipimo kimwe cya kabiri cyonyine cyemeza icya mbere. Buri gipimo cyigenga kirinda ingaruka z'inzira imwe ariko kigakomeza guhura n'iz'indi. Hitamo rero ibipimo ibyo bidashobora gutahura bitahurirana. Imbonerahamwe yo guhitamo ibipimo by'ibinyabutabire bigira uruhare mu guhererekanya electron isobanura uburyo bwo kubihuza, naho imfashanyigisho y'ibikoresho bipima itandukaniro ry'amashanyarazi ku gahu igasobanura mu buryo burambuye ibyo kwitondera kuri floresansi.

Igipimo cya Seahorse cy'ikoreshwa rya ogisijeni gikurikirana electron zigana kuri ogisijeni aho kujya muri resorufin. Ibyo birinda burundu imikoranire y'ibinyabutabire itanga ikimenyetso, ariko irangi na ryo rikoresha ogisijeni binyuze mu mikoranire y'ibinyabutabire. Ni yo mpamvu igenzura ririmo irangi, ridafite mitokondiriya, mu muti wuzuye uturemangingo tubamo ari ngombwa. Ibipimo bya ATP bikoresha luciferase bishingira ku rumuri rukomoka ku mikoranire y'ibinyabuzima aho kuba floresansi ya resorufin. Bityo, birinda guhurirana kw'urumuri n'uguhatanira electron binyuze kuri reductase, ariko bikagaragaza uburinganire bw'ibigega bya ATP aho kuba umuvuduko wo kuyikora. Ibikoresho bipima itandukaniro ry'amashanyarazi ku gahu, nka TMRM cyangwa JC-10, byerekana niba itandukaniro ry'umuriro rikomeza kubaho. Nyamara, bitanga floresansi mu gice kimwe cy'urumuri rugaragara irangi ryinjizamo urumuri. Bityo, ibara ry'ubururu risigaye na ryo ribigora, kandi bikenera igenzura ririmo irangi gusa kuri buri bwinshi bw'irangi. Ibipimo by'inyandiko za RNA z'ibice bigize urusobe rw'ibinyabutabire by'ubuhumekero birinda burundu ibibazo by'urumuri kandi bigakusanya impinduka zibaho mu masaha, bikuzuza ibipimo byo ku isahani bikorwa mu minota.

Bityo, ibimenyetso bike bishoboka byizewe byo kuvuga impinduka z'ihinduranyabintu zatewe na methylene blue ni ibi: igisubizo cya resazurin gifite igenzura rya 1 kugeza ku rya 4, hiyongereyeho nibura igipimo kimwe kidakoresha resazurin kandi gishingiye ku rindi hame ry'ubugenge, hamwe no kugaragaza niba impinduka zituruka ku mubare w'uturemangingo, ku muvuduko wo gutanga electron kuri buri karemangingo, cyangwa kuri byombi. Ibiri munsi y'ibyo bisiga umusomyi adashobora gutandukanya ihinduranyabintu ryagabanutse n'akobo gatanga urumuri ruke.

Urupapuro rwo kugenzura ibangamirwa ry'ibipimo

Andukura uru rupapuro mu ikayi ya laboratwari kuri buri gerageza rihuza irangi n'ikimenyetso gishingiye ku guhererekanya electron. Ni igikoresho cy'umwimerere cy'iyi ngingo: uzuza umurongo umwe kuri buri bwinshi bw'irangi.

A. Inzira z'ikimenyetso. Kuri buri bwinshi bw'irangi, andika: niba witeze ko higanje imolekile imwe cyangwa imolekile ebyiri zifatanye (ubwinshi buke bwo ku rwego rwa micromolar ugereranyije na mirongo ya micromolar no hejuru yayo), igipimo cyo kwinjiza urumuri kuri 590 nm cy'ibiri mu kobo nyuma yo gutegura byose, mu kobo katarimo uturemangingo, n'ijanisha ry'igipimo cya resorufin yongewemo cyagarujwe. Niba igipimo cyagarujwe kiri munsi ya hafi 90 ku ijana, fata ibipimo byose bya floresansi kuri ubwo bwinshi bw'irangi nk'ibyagabanyijwe n'ingaruka z'urumuri, aho kubifata nk'ibigaragaza neza imikorere y'ibinyabuzima.

B. Kugenzura imikoranire y'ibinyabutabire. Kuri buri gihe cyo gusoma igipimo, andika floresansi y'irangi n'umuti w'igerageza bitarimo uturemangingo nk'ijanisha ry'igipimo fatizo cy'umuti w'igerageza wonyine. Niba itandukaniro ryiyongera uko igihe kigenda, irangi ririmo gukorana n'uburyo butanga ikimenyetso mu gihe cy'igerageza, kandi gusoma igipimo cya nyuma gusa bizayobya. Tekereza ku gusoma ibipimo bikurikirana uko igihe kigenda cyangwa ku gukoresha ikindi kimenyetso.

C. Kugenzura impinduka z'ibinyabuzima mu buryo bwigenga. Vuga igipimo cyigenga wahisemo kuri iri gerageza, igenzura ryacyo ririmo irangi ariko nta binyabuzima, n'igisubizo witeze niba igisubizo cya resazurin ari nyakuri. Kugabanuka nyakuri kw'ihinduranyabintu kugomba gutuma igipimo cyigenga gihinduka mu cyerekezo gihuje na ko. Niba igipimo cyigenga kidahuye na cyo, ntugire igisubizo na kimwe wizera kugeza ubu; ongera utegure igerageza aho gufata impuzandengo y'ibyo byombi.

D. Interuro y'umwanzuro. Andika interuro imwe muri ubu buryo: ku bwinshi bw'irangi no ku cyiciro byavuzwe, ikimenyetso cya resazurin cyahindutse ku rugero rwavuzwe, muri rwo urugero rwavuzwe rukaba ari ikosa ry'igerageza riboneka nta turemangingo, kandi igipimo cyigenga (kirahuye cyangwa ntigihuye na cyo). Niba hari ahatuzuye, igerageza ntirirarangira.

Methylene blue ikomeje kuba igikoresho cy'ingirakamaro mu kwiga ihinduranyabintu, kandi ubushakashatsi ku mafi ya zebrafish bwerekana ingaruka nyakuri kuri mitokondiriya ku bwinshi ibigo byinshi bisuka mu miyoboro y'amazi buri munsi. Iri rangi risaba gusa gupimwa n'ibikoresho ridashobora kubangamira. Rihe utwobo turimo irangi gusa, igerageza ryo kugaruza igipimo cy'inyongera n'igipimo kimwe gishingiye ku mahame y'ubugenge atandukanye, maze ibangamirwa ry'ibipimo rireke kuba umutego, ribe ikintu gipimwa kikagaragazwa n'umubare.