Xa i-methylene blue iphazamisa uvavanyo olusetyenziselwa ukuyilinganisa

Enye ilabhoratri ikhulisa iimbumba ze-zebrafish kwi-methylene blue exinaniswe ngokomgangatho wokukhulisa, ize ilinganise imetabolism ngeendlela ezimbini. Ngosuku lokuqala, isihlalutyi se-Seahorse sibonisa ukwanda kokusetyenziswa kweoksijini. Ngeentsuku zesine nezesihlanu, ipleyiti ye-Alamar Blue ibonisa ukwehla kokusetyenziswa kwamandla. Idayi enye, inzala enye yeentlanzi, izigqibo ezichaseneyo. Emva koko kuvela uvavanyo lolawulo olungatsali ngqalelo: amaqula ane-methylene blue kunye nesixhobo sokusabela se-Alamar Blue, kodwa engenazo kwaphela iintlanzi, nawo abonisa ukukhanya okuncinane kunesixhobo sokusabela sodwa. Inxalenye yesiphumo yayibangelwa ziinkqubo zebhayoloji. Enye inxalenye yayiyidayi izimakela uvavanyo lwayo. Oku kwahlukana, okubhalwe kuphononongo luka-2025 lwe-Communications Biology nge-methylene blue kwi-zebrafish ezikhulayo, yindlela eqhelekileyo yokuphazamiseka kovavanyo yi-methylene blue, kwaye kungenxa yoko le nto iidayi ezisebenzayo kwi-redox zifuna iimvavanyo zolawulo ezingafunekiyo kwiikhompawundi eziqhelekileyo ezivavanywayo.

Oko uvavanyo lwe-resazurin lukubonisayo ngokwenene

I-Alamar Blue ligama lophawu lesisombululo se-resazurin, ngoko ukuphazamiseka kwe-Alamar Blue yi-methylene blue nokuphazamiseka kovavanyo lwe-resazurin yi-methylene blue kuchaza ungquzulwano olunye. I-resazurin ekwimo eneoksideshini iluhlaza okwesibhakabhaka kwaye ikhupha ukukhanya kwe-fluorescence okuncinane kakhulu. Iiseli ezinemetabolism esebenzayo ziyinciphisa ngokwamkela ii-elektroni ibe yi-resorufin, epinki kwaye ikhupha i-fluorescence enamandla kufutshane nokuvuselelwa ku-560 nm nokukhutshwa ku-590 nm, inemida enokusetyenziswa emalunga no-530 ukuya ku-570 nm wokuvuselelwa no-580 ukuya ku-610 nm wokukhutshwa, ngokwe-prothokholi yomenzi yeepleyiti ezinamaqula amancinci. Ukuba ukuncitshiswa ngee-elektroni kuqhubeka ngakumbi, i-resorufin iguquka ibe yi-dihydroresorufin, ene-fluorescence ebuthathaka, ngoko ukuxinanisa iiseli equleni okanye ukulinda ixesha elide kakhulu kugobela ezantsi isiphelo esiphezulu segophe lokulinganisa.

Ii-elektroni zivela ikakhulu kwi-NADH ne-NADPH ngee-reductase nee-diaphorase zeeseli ezikwi-cytosol, ii-microsome, nakwi-mitochondria. Oko kwenza ufundo lube ngumlinganiselo odityanisiweyo wobuchule bokunciphisa ngokudlulisa ii-elektroni, kungekhona ukubalwa kweeseli eziphilayo okanye umlinganiselo we-mitochondria. Umqondiso ophezulu usenokuthetha iiseli ezininzi, ukuhamba okungakumbi kwee-elektroni zokunciphisa kwiseli nganye, okanye zombini. Ikhompawundi ejika indlela ye-NADH itshintsha umqondiso ngaphandle kokubulala neseli enye. Gcina loo ngcinga isondele, kuba ukujika indlela ye-NADH yinto eyenziwa yi-methylene blue rhoqo.

Iindlela ezimbini zokuphazamiseka, umqondiso omnye owehlileyo

I-methylene blue igqwetha ufundo lwe-resazurin ngeendlela ezimbini ezizimeleyo, kwaye nganye kwezi yodwa yehlisa i-fluorescence ebonakalayo.

Indlela 1 yeyokukhanya. I-methylene blue ifunxa ngamandla kummandla obomvu, inommandla wokufunxa we-monomer kufutshane no-660 ukuya ku-665 nm kunye negxalaba le-dimer kufutshane no-607 ukuya ku-610 nm, elinomsila ofikelela ngasemva ekukhutshweni kwe-resorufin malunga no-590 nm. Idayi ekwindlela yokukhanya ifunxa inxalenye yokukhanya okuvuselelayo ize iphinde ifunxe inxalenye ye-fluorescence ekhutshiweyo; esi sisiphumo sesihluzi sangaphakathi esenza iqula likhanye kancinci naxa ukuveliswa kwe-resorufin kungatshintshanga. Ispektram nayo iyatshintsha ngokwemeko yedayi: uhlalutyo lwezinto ezichaphazela iispektram ze-methylene blue emanzini nakwizisombululo ze-cyclodextrin lwahlula i-monomer, i-dimer, kunye nekhompleksi yomamkeli nondwendwe njengeentlobo ezintathu ezineekhoyefishiyenti zokuncipha kokukhanya ezahlukileyo ukusuka ku-500 ukuya ku-700 nm, ngoko ubungakanani bempazamo yokukhanya butshintsha ngokuxinana, ukuhlanganisana, nokubophelela. Kuvavanyo, yahlula ukuncipha kokukhanya okuqhelekileyo ekucinyweni kokukhanya kweemolekyuli ngovavanyo lokubuyiswa komqondiso owongeziweyo oluchazwe ngezantsi, kuba indlela yokulungisa iyahluka.

Indlela 2 lukhuphiswano lwe-redox. I-methylene blue ekwimo eneoksideshini yamkela ii-elektroni kwi-NAD(P)H yenze i-leuco methylene blue engenambala, ethi ioksijini yeemolekyuli iphinde iyiguqule ngeoksideshini ibuyele kwimo eluhlaza okwesibhakabhaka, ngoxa ivelisa iimveliso zeoksijini ezincitshisiweyo kuquka i-hydrogen peroxide. Umjikelo ngamnye usebenzisa amandla okudlulisa ii-elektroni awayenokunciphisa i-resazurin, kwaye usebenzisa ioksijini ngoxa uvelisa ne-peroxide. Umqondiso we-Alamar Blue owehlileyo ke unokuthetha ukuba ii-elektroni zidlule kumjikelo we-methylene blue endaweni yokungena kwi-resorufin, nto leyo ebonakala njengemetabolism eyehlileyo epleyitini kungakhathaliseki ukuba iiseli zenze ntoni. Kwale khemistri ichaza umgibe ojikelezileyo kwizixhobo zeoksijini: idayi encitshisiweyo ephinda ifumane ioksideshini ngeoksijini ibangela ukusetyenziswa kweoksijini ngekhemistri ngaphandle kweenkqubo zebhayoloji, ngoko iisampulu zedayi ezingenanto ziphinde zifuneke kwimilinganiselo yokuphefumla, njengoko uthelekiso lofundo lweoksijini, i-ATP, kunye nomahluko wombane kwinwebu lucacisa kwimeko ye-mitochondria.

Umqondiso otshintshileyo neenkqubo zebhayoloji ezitshintshileyo zinokuba yinyani zombini

Uphononongo lwe-zebrafish lubalulekile kanye kuba alwamkeli ukukhetha okulula phakathi kwenye okanye kwenye. Utshintsho lwebhayoloji lwaluyinyani: ukusetyenziswa kweoksijini okusisiseko kwenyuka kwiimbumba ezinemini 1 kuwo omabini amanqanaba oxinaniso lokukhulisa, ukuphefumla okunxulumene ne-ATP kwenyuka kwelisezantsi, umahluko wombane kwinwebu olinganiswe ngesivamvo se-JC-10 wenyuka ngosuku lwesibini, kwaye imibhalo ye-RNA yeekhompleksi ze-mitochondria yehla ngeentsuku zokuqala nezesine. Impazamo yovavanyo nayo yayiyinyani: iintlanzi ezihlanjwe kathathu phambi kwenyathelo le-Alamar Blue zazisenamabala aluhlaza okwesibhakabhaka abonakalayo entloko, emlonyeni, nakummandla wokujoja, kwaye amaqula angenazo iiseli anedayi kunye nesixhobo sokusabela aphulukana ngokwawo ne-fluorescence encinane kodwa ebalulekileyo. Ukwehla kwe-Alamar Blue kwisilwanyana esipheleleyo ngeentsuku zesine nezesihlanu kwakukhulu kunesiphumo esingenazo iiseli, ngoko ababhali bachaza isiphumo njengotshintsho lokwenene lwemetabolism olubonwa ngelensi egqwethwe kancinane. Leyo yindlela echanekileyo yokucinga ngalo. Into ejikeleza kwi-redox, ekwafunxa ukukhanya okuluhlaza okwesibhakabhaka, idla ngokuphazamisa indlela ephandwayo kunye nekhemistri yesixhobo sokubonisa ngaxeshanye, kwaye umsebenzi weemvavanyo zolawulo kukulinganisa igalelo ngalinye, kungekhona ukubhengeza elinye njengelingekhoyo. Isifundo esinxulumeneyo sivela kumsebenzi wokubophelela kwe-cyclodextrin: xa imolekyuli engumamkeli yabamba idayi, ukushiya amabala nokuvuselelwa kokuthathwa kweglucose kwehla kunye, nto leyo yabonisa ukuba isiphumo kwimetabolism sasifuna unxibelelwano oluthe ngqo nedayi kunokuphazamiseka kwe-redox okusasazekileyo. Ukunxulumanisa ufundo lomlinganiselo wedayi nofundo lomsebenzi kanye yindlela yokwahlula indlela esebenza ngayo kwimpazamo yovavanyo, kwaye isikhokelo sokufunda amabango emetabolism asekelwe kwidayi siphuhlisa loo mkhwa ngakumbi.

Iimvavanyo zolawulo ezizahlulayo

Yenza ezi mvavanyo phambi kokutolika nasiphi na isiphumo se-methylene blue esivela kwipleyiti ye-resazurin. Uxinano, indawo yokukhulisela, ixesha lokufukama, kunye nokukhanyiswa kufuneka zingqinelane ncam novavanyo lweeseli, kuba zonke iindlela ezichazwe ngasentla zixhomekeke kuzo.

Iimvavanyo zolawulo 3 no-4 zezona zimbini amaphepha amaninzi azishiyayo neziphantse zifuneke kuwo onke amaphepha e-methylene blue. Uvavanyo lolawulo 3 lufumana ikhemistri, uvavanyo lolawulo 4 lufumana iziphumo zokukhanya, kwaye kunye zibeka imida yempazamo yovavanyo phambi kokuba kwenziwe naliphi na ibango lebhayoloji.

Ufundo oluzimeleyo, ngalunye lunemiqathango yalo

Alukho uvavanyo olunye lwesibini oluqinisekisa olokuqala. Ufundo ngalunye oluzimeleyo luyayiphepha enye indlela luze luhlale luchanabeke kwenye, ngoko khetha indibaniselwano eneendawo ezingaboniyo ezingadibaniyo. Itheyibhile yokukhetha iimvavanyo zeekhompawundi ezisebenzayo kwi-redox ichaza ingqiqo yokudibanisa, kwaye isikhokelo sezivamvo zomahluko wombane kwinwebu sicacisa ngokweenkcukacha izilumkiso ze-fluorescence.

Umlinganiselo wokusetyenziswa kweoksijini we-Seahorse ulandela ii-elektroni eziya kwioksijini endaweni yokungena kwi-resorufin, nto leyo ephepha ngokupheleleyo ikhemistri yesixhobo sokubonisa, kodwa idayi nayo isebenzisa ioksijini ngekhemistri, ngoko iisampulu zedayi ezingenanto nezingenayo i-mitochondria kwindawo yokukhulisela epheleleyo ziyimfuneko. Iimvavanyo ze-ATP ezisebenzisa i-luciferase zisebenzisa ukukhanya okuveliswa yinkqubo yebhayoloji endaweni ye-fluorescence ye-resorufin, ngoko ziphepha ukudibana kokukhanya nokhuphiswano lwee-reductase, kodwa zona zibonisa ibhalansi yobungakanani be-ATP ekhoyo kunesantya sokuyivelisa. Izivamvo zomahluko wombane kwinwebu ezifana ne-TMRM okanye i-JC-10 ziphendula ukuba umahluko wentlawulo yombane uyagcinwa na, kodwa zikhupha i-fluorescence kwakuloo mmandla wokukhanya okubonakalayo ufunxwa yidayi, ngoko amabala aluhlaza okwesibhakabhaka aseleyo nawo ayazidida kwaye zifuna iisampulu ezingenanto ezinedayi kuphela kwisixa ngasinye sedayi. Imilinganiselo yemibhalo ye-RNA yamacandelo eekhompleksi zokuphefumla iphepha ukukhanya ngokupheleleyo kwaye idibanisa iziphumo kwiiyure, nto leyo ehambelana nofundo lweepleyiti olwenziwa kwimizuzu.

Ngoko iphakheji encinane enokuthenjwa yebango lemetabolism ye-methylene blue yile: isiphumo se-resazurin esineemvavanyo zolawulo 1 ukuya ku-4, kunye nomlinganiselo omnye wesiphumo ongasebenzisi i-resazurin nosekelwe kumgaqo wefiziksi owahlukileyo, kunye nenkcazo yokuba utshintsho lubonisa inani leeseli, ukuhamba kwee-elektroni zokunciphisa kwiseli nganye, okanye zombini. Nantoni na engaphantsi koko ishiya umfundi engakwazi ukwahlula imetabolism ethobileyo kwiqula elikhanya kancinci.

Iphepha lomsebenzi lokulawula ukuphazamiseka

Khuphela eli phepha lomsebenzi kwincwadi yelabhoratri kuvavanyo ngalunye oludibanisa idayi nesixhobo sokubonisa i-redox. Sisixhobo sokuqala esilungiselelwe eli nqaku: gcwalisa umqolo omnye ngoxinaniso ngalunye lwedayi.

A. Iindlela zomqondiso. Kuxinaniso ngalunye, bhala: imeko elindelekileyo ye-monomer xa ithelekiswa ne-dimer (uxinaniso oluphantsi lwe-micromolar xa luthelekiswa namashumi e-micromolar nangaphezulu), ukufunxwa kokukhanya kwezinto ezikwiqula lokugqibela ku-590 nm kwiqula elingenazo iiseli, kunye nepesenti yokubuyiswa komqondiso we-resorufin owongeziweyo. Ukuba ukubuyiswa komqondiso kwehla ngaphantsi kweepesenti ezimalunga no-90, phatha onke amaxabiso e-fluorescence kweso sixa njengancitshiswe ziziphumo zokukhanya, kungekhona njengachanekileyo ngokwebhayoloji.

B. Ukuhlolwa kwekhemistri. Bhala i-fluorescence yedayi kunye nesixhobo sokusabela ngaphandle kweeseli njengepesenti yomgca osisiseko wesixhobo sokusabela kuphela ngexesha ngalinye lokufunda. Ukuba umahluko uyakhula ngokuhamba kwexesha, idayi isabela kwinkqubo yesixhobo sokubonisa ngexesha lovavanyo, kwaye ufundo olwenziwa ekupheleni kuphela luya kulahlekisa. Cinga ngofundo olulandelela utshintsho ngokuhamba kwexesha okanye ngesixhobo sokubonisa esahlukileyo.

C. Ukuhlolwa kwebhayoloji ngendlela ezimeleyo. Chaza ufundo oluzimeleyo olukhethwe kolu vavanyo, isampulu yalo engenanto yedayi, kunye nesiphumo esiqikelelweyo ukuba isiphumo se-resazurin siyinyani. Ukwehla kokwenene kwemetabolism kufanele kutshintshe umlinganiselo wesiphumo ozimeleyo ngendlela ehambelanayo. Ukuba umlinganiselo wesiphumo ozimeleyo awuvumelani nalo, sukuthemba nasiphi na isiphumo okwangoku, uze uyile uvavanyo ngokutsha endaweni yokwenza umndilili wazo zombini.

D. Isivakalisi sesigqibo. Bhala isivakalisi esinye esikule ndlela: kwisixa nakwinqanaba elichaziweyo, umqondiso we-resazurin utshintshe ngesixa esichaziweyo, apho isixa esichaziweyo siyimpazamo yovavanyo engenazo iiseli, kwaye umlinganiselo wesiphumo ozimeleyo (uyavumelana okanye awuvumelani). Ukuba kukho indawo engazaliswanga, uvavanyo alukagqitywa.

I-methylene blue ihlala isisivamvo esiluncedo semetabolism, kwaye umsebenzi we-zebrafish ubonisa iziphumo zokwenene kwi-mitochondria kwizixa ezithululwa ngamaziko amaninzi kwimisele yonke imihla. Idayi ifuna nje ukulinganiswa ngezixhobo engenakuzichaphazela. Yinike amaqula anedayi kuphela, uvavanyo lokubuyiswa komqondiso owongeziweyo, kunye nofundo olunye olwakhelwe kwimigaqo yefiziksi eyahlukileyo, kuze ukuphazamiseka kuyeke ukuba ngumgibe kuqale ukuba bubungakanani obunenani obulinganiswe ngalo.