Isihloko
Lapho i-methylene blue iphazamisa ukuhlola okusetshenziselwa ukuyilinganisa
Ilabhorethri ethile ikhulisa imibungu ye-zebrafish ku-methylene blue ngezinga lokugxila elijwayelekile ekunakekelweni kwalezi zinhlanzi, bese ilinganisa imetabolism ngezindlela ezimbili. Ngosuku lokuqala, umhlaziyi we-Seahorse ubika ukuthi ukusetshenziswa komoya-mpilo kuyakhuphuka. Ngosuku lwesine nolwesihlanu, ipuleti le-Alamar Blue libika ukuthi ukusetshenziswa kwamandla kuyehla. Udayi ofanayo, uzalo lwezinhlanzi olufanayo, iziphetho eziphikisanayo. Bese kuvela ukuhlola kokulawula okungabonakali kangako: imigodi equkethe i-methylene blue kanye ne-reagent ye-Alamar Blue kodwa engenazo nhlobo izinhlanzi nayo ikhipha ukukhanya okuncane kune-reagent yodwa. Ingxenye yomphumela yayivela ezintweni eziphilayo. Enye ingxenye yayiwudayi ozimakela isivivinyo sawo. Lokho kuhlukana, okubhalwe ocwaningweni lwe-Communications Biology lwango-2025 nge-methylene blue kuma-zebrafish asakhula, kuyindlela evamile yokuphazamiseka kokuhlolwa nge-methylene blue, futhi yikho okwenza odayi abasebenza ku-redox badinge izilawuli ezingadingeki ezintweni ezivamile ezihlolwayo.
Lokho ukuhlolwa nge-resazurin okubika khona ngempela
I-Alamar Blue yigama lomkhiqizo lesixazululo se-resazurin, ngakho ukuphazamiseka kwe-Alamar Blue okubangelwa yi-methylene blue nokuphazamiseka kokuhlolwa nge-resazurin okubangelwa yi-methylene blue kuchaza ukungqubuzana okufanayo. I-resazurin esesimweni esine-oxidation iluhlaza okwesibhakabhaka futhi ikhipha i-fluorescence encane kakhulu. Amaseli anemetabolism esebenzayo ayinciphisa ngama-electron ibe yi-resorufin, enombala ophinki futhi ekhipha i-fluorescence enamandla lapho ivuswa ngokukhanya okuseduze no-560 nm futhi ikhipha ukukhanya okuseduze no-590 nm. Ngokuvumelana nenqubo yomkhiqizi yokusebenzisa ipuleti elinemigodi emincane, ububanzi obusebenzisekayo bokuvusa buphakathi kuka-530 no-570 nm, kanti obokukhishwa kokukhanya buphakathi kuka-580 no-610 nm. Uma ukuncishiswa ngama-electron kuqhubeka, i-resorufin iguquka ibe yi-dihydroresorufin, ene-fluorescence ebuthakathaka. Ngakho ukugcwalisa umgodi ngamaseli amaningi kakhulu noma ukuyeka ukuhlola isikhathi eside kakhulu kugobisa ijika lokulinganisa liye phansi ekugcineni kwalo okuphezulu.
Ama-electron avela ikakhulukazi ku-NADH naku-NADPH ngokusebenzisa ama-reductase nama-diaphorase amaseli atholakala ku-cytosol, kuma-microsome nakuma-mitochondria. Lokho kwenza ukufundwa kube isilinganiso samandla ahlanganisiwe okunciphisa amakhemikhali, hhayi ukubalwa kwamaseli aphilayo futhi hhayi isilinganiso sama-mitochondria. Isignali ephakeme ingasho amaseli amaningi, ukugeleza okukhulu kwama-electron okunciphisa amakhemikhali kuseli ngalinye, noma kokubili. Into ephambukisa indlela ye-NADH ishintsha isignali ngaphandle kokubulala ngisho neseli elilodwa. Gcina lowo musho engqondweni, ngoba ukuphambukisa i-NADH kuwumsebenzi ojwayelekile we-methylene blue.
Izindlela ezimbili zokuphazamiseka, isignali eyodwa eyehlisiwe
I-methylene blue ihlanekezela ukufundwa kwe-resazurin ngezindlela ezimbili ezizimele, futhi ngayinye iyodwa yehlisa i-fluorescence ebonakalayo.
Indlela 1 iphathelene nokukhanya. I-methylene blue imunca kakhulu ukukhanya okubomvu, ibhande layo le-monomer liseduze no-660 kuya ku-665 nm, kanti ihlombe le-dimer liseduze no-607 kuya ku-610 nm, nomsila walo ufinyelela emuva ekukhanyeni okukhishwa yi-resorufin ngaku-590 nm. Udayi osendleleni yokukhanya umunca ingxenye yokukhanya okuvusa i-fluorescence futhi uphinde umunce ingxenye ye-fluorescence ekhishiwe. Lo mphumela wokuhlunga ngaphakathi wenza umgodi ubonakale ufiphele ngisho noma ukukhiqizwa kwe-resorufin kungashintshile. Ububanzi bokumuncwa kokukhanya buyashintsha futhi ngokwesimo sikadayi: ukuhlaziywa kwezici zemibukwane ye-methylene blue emanzini nasezixazululweni ze-cyclodextrin kuhlukanisa i-monomer, i-dimer nenhlanganisela yomamukeli nesivakashi njengezinhlobo ezintathu ezinama-coefficient ahlukene okubuthaka kokukhanya kusukela ku-500 kuya ku-700 nm. Ngakho ubukhulu bephutha lokukhanya buyashintsha ngokugxila, ukuhlangana kwama-molecule nokubophana. Ekuhloleni, hlukanisa ukubuthaka kokukhanya okuvamile nokucishwa kwe-fluorescence ngama-molecule ngokusebenzisa ukuhlolwa kokubuyiselwa kwesignali yesengezo okuchazwe ngezansi, ngoba izindlela zokulungisa ziyahluka.
Indlela 2 iwukuncintisana ku-redox. I-methylene blue esesimweni esine-oxidation yamukela ama-electron avela ku-NAD(P)H ukuze yakhe i-leuco methylene blue engenambala. Umoya-mpilo wama-molecule uphinde uyifake ku-oxidation, uyibuyisele esimweni esiluhlaza okwesibhakabhaka, ngesikhathi ikhiqiza imikhiqizo yokuncishiswa komoya-mpilo, kuhlanganise ne-hydrogen peroxide. Umjikelezo ngamunye usebenzisa ama-equivalent okunciphisa amakhemikhali abenganciphisa i-resazurin, futhi usebenzisa umoya-mpilo ngesikhathi ukhiqiza ne-peroxide. Ngakho isignali eyehlisiwe ye-Alamar Blue ingasho ukuthi ama-electron adlule emjikelezweni we-methylene blue kunokuba aye ku-resorufin. Lokhu kufundeka njengokwehla kwemetabolism epuletini kungakhathaliseki ukuthi amaseli enzeni. Amakhemikhali afanayo achaza ugibe oluphambene kumathuluzi okulinganisa umoya-mpilo: udayi oncishiswe ngama-electron futhi ophinde afakwe ku-oxidation ngumoya-mpilo ubangela ukusetshenziswa kwamakhemikhali komoya-mpilo ngaphandle kwezinto eziphilayo. Ngakho amasampula kadayi okulawula angenazo izinto eziphilayo ayadingeka nasekulinganiseni ukuphefumula, njengoba ukuqhathaniswa kokufundwa komoya-mpilo, kwe-ATP nokomehluko wamandla kagesi kulwelwesi kuchaza endabeni yama-mitochondria.
Isignali eshintshile nokusebenza kwezinto eziphilayo okushintshile kungaba yiqiniso kokubili
Ucwaningo lwe-zebrafish lubalulekile ngoba alwamukeli umbono olula wokuthi kufanele kube okukodwa kuphela. Ushintsho ezintweni eziphilayo lwalungokoqobo: ukusetshenziswa okuyisisekelo komoya-mpilo kwakhuphuka emibungwini enosuku olu-1 kuwo womabili amazinga okugxila asetshenziswa ekunakekelweni; ukuphefumula okuhlobene ne-ATP kwakhuphuka ezingeni eliphansi; umehluko wamandla kagesi kulwelwesi owalinganiswa ngesihloli se-JC-10 wakhuphuka ngosuku lwesibili; futhi imibhalo ye-RNA yezinhlanganisela zama-mitochondria yehla ngosuku lokuqala nolwesine. Umphumela wokuhlola ongaveli ezintweni eziphilayo nawo wawungokoqobo: izinhlanzi ezagezwa kathathu ngaphambi kwesinyathelo se-Alamar Blue zazisenombala oluhlaza okwesibhakabhaka obonakalayo ekhanda, emlonyeni nasendaweni yokuzwa iphunga. Imigodi engenamaseli kodwa enodayi ne-reagent yalahlekelwa yingxenye encane kodwa ebalulekile ye-fluorescence ngokwayo. Ukwehla kwe-Alamar Blue ezilwaneni ngosuku lwesine nolwesihlanu kwakukhulu kunomphumela ongenamaseli. Ngakho ababhali bahumusha umphumela njengoshintsho lwangempela lwemetabolism olubonwe ngelensi ehlanekezelwe kancane. Leyo yindlela efanele yokucabanga ngalokhu. Into ejikeleza ku-redox futhi emunca ukukhanya ngenxa yombala wayo oluhlaza okwesibhakabhaka ngokuvamile izophazamisa indlela efundwayo kanye namakhemikhali esibiki ngesikhathi esisodwa. Umsebenzi wezilawuli uwukulinganisa umnikelo ngamunye, hhayi ukumemezela ukuthi omunye awukho. Isifundo esihlobene nalokhu sivela ocwaningweni lokubophana ne-cyclodextrin: lapho i-molecule engumamukeli ibamba udayi, umbala nokuvuselelwa kokungeniswa kwe-glucose kwehla ndawonye. Lokhu kwasekela umqondo wokuthi umphumela kwimetabolism wawudinga ukuxhumana okuqondile nodayi kunomsindo we-redox osakazekile. Ukuhlobanisa ukufundwa kwesici sikadayi nokufundwa kokusebenza kuyisinyathelo esihlukanisa indlela esebenzayo nomphumela wokuhlola ongaveli ezintweni eziphilayo. Umhlahlandlela wokufunda izimangalo zemetabolism ezisekelwe kudayi uthuthukisa lo mkhuba ngokwengeziwe.
Izilawuli ezikuhlukanisayo
Yenza lokhu ngaphambi kokuhumusha noma yimuphi umphumela we-methylene blue ovela epuletini le-resazurin. Amazinga okugxila, indawo yokukhulisela, isikhathi sokufukamela nokukhanyiswa kufanele kufane ncamashi nokuhlolwa okunamaseli, ngoba zonke izindlela ezingenhla zincike kukho.
| # | Isilawuli | Lokho esikutholayo | Umbandela wokuphumelela | Uma singaphumeleli |
|---|---|---|---|---|
| 1 | Indawo yokukhulisela kanye nodayi, ngaphandle kwe-reagent namaseli | Isignali yangemuva kadayi kubude bamagagasi asetshenziswa ekuhloleni | Isignali yangemuva incane uma iqhathaniswa nesignali yamaseli | Susa isignali yesampula yokulawula yezinga ngalinye lokugxila, ungalokothi usebenzise eyodwa kukho konke |
| 2 | Indawo yokukhulisela kanye ne-reagent, ngaphandle kukadayi namaseli | Ukuncishiswa kwe-reagent okuzenzekelayo | Isignali eyisisekelo ephansi futhi ezinzile phakathi nesikhathi sokufukamela | Nciphisa isikhathi sokufukamela, shintsha indawo yokukhulisela, noma wamukele umkhawulo ophansi ophakeme kakhulu |
| 3 | Indawo yokukhulisela kanye nodayi ne-reagent, ngaphandle kwamaseli | Ukusabela kwamakhemikhali okuqondile phakathi kukadayi ne-reagent | Kufana nesilawuli 2 ngaphakathi komkhawulo womsindo | Bika umehluko, ungawuniki amaseli njengembangela |
| 4 | Isengezo se-resorufin emigodini enoketshezi oluthwalayo uma kuqhathaniswa nemigodi enodayi | Ukubuthaka kokukhanya noma ukucishwa kwe-fluorescence | Ukubuyiselwa kwesignali okulinganayo kuzo zombili izingxube | Linganisa ukulahleka kwesignali ngenani ngalinye likadayi bese ukulungisa, noma ushintshe indlela yokufunda |
| 5 | Insalela engenamaseli egeziwe uma iqhathaniswa nengageziwe | Udayi osele ngemva kwezinyathelo zakho zokugeza | Ukugeza kususa umphumela ongenamaseli | Udayi ongene ngaphakathi usala, ngakho izilawuli 3 no-4 zisadingeka |
Izilawuli 3 no-4 yilezi ezimbili amaphepha amaningi azishiyayo futhi amaphepha amaningi nge-methylene blue azidingayo. Isilawuli 3 sithola ukusabela kwamakhemikhali, isilawuli 4 sithola imiphumela yokukhanya, futhi ndawonye zibeka imingcele yomphumela wokuhlola ongaveli ezintweni eziphilayo ngaphambi kokuba kwenziwe noma yisiphi isimangalo ngazo.
Ukufundwa okuzimele, ngakunye kunemibandela yako
Akukho ukuhlola kwesibili okukodwa okuqinisekisa okokuqala. Ukufundwa ngakunye okuzimele kugwema enye indlela yokuphazamiseka kodwa kusachayeka kwenye, ngakho khetha inhlanganisela enezindawo ezingaboni ezingagqagqani. Ithebula lokukhetha ukuhlola ezintweni ezisebenza ku-redox lichaza indlela yokukhetha okubili okuhambisanayo, kanti umhlahlandlela wezihloli zomehluko wamandla kagesi kulwelwesi uchaza ngokuningiliziwe izixwayiso ze-fluorescence.
Ukulinganisa ukusetshenziswa komoya-mpilo nge-Seahorse kulandela ama-electron aya emoyeni-mpilo kunokuba aye ku-resorufin, okugwema ngokuphelele amakhemikhali esibiki. Kodwa udayi nawo usebenzisa umoya-mpilo ngokusabela kwamakhemikhali, ngakho amasampula kadayi okulawula angenawo ama-mitochondria futhi akwindawo yokukhulisela ephelele ayimpoqo. Ukuhlolwa kwe-ATP nge-luciferase kusebenzisa ukukhanya okukhiqizwa ukusabela kwezinto eziphilayo esikhundleni se-fluorescence ye-resorufin. Ngakho kugwema kokubili ukugqagqana kokukhanya nokuncintisana kwama-reductase, kodwa kubika ibhalansi yenqolobane ye-ATP esikhundleni sokugeleza kokukhiqizwa kwayo. Izihloli zomehluko wamandla kagesi kulwelwesi ezifana ne-TMRM noma i-JC-10 ziphendula ukuthi umehluko weshaji ugcinwa yini. Nokho, zikhipha i-fluorescence ebangeni elifanayo lokukhanya okubonakalayo elimuncwa udayi, ngakho umbala oluhlaza okwesibhakabhaka osele nazo uyaziphazamisa, futhi zidinga amasampula okulawula anodayi kuphela ngenani ngalinye likadayi. Ukulinganiswa kwemibhalo ye-RNA yezingxenye zezinhlanganisela zokuphefumula kugwema imiphumela yokukhanya ngokuphelele futhi kuhlanganisa izinguquko ezenzeka emahoreni, okuhambisana nokufundwa kwamapuleti okuthatha imizuzu.
Ngakho ubufakazi obuncane obuthembekile besimangalo semetabolism esiphathelene ne-methylene blue buyilobu: umphumela we-resazurin onezilawuli 1 kuya ku-4, kanye nomphumela wokugcina owodwa ongasebenzisi i-resazurin futhi osekelwe esimisweni esihlukile sezinto ezibonakalayo, kanye nencazelo ecacile yokuthi ushintsho lubonisa inani lamaseli, ukugeleza kwama-electron okunciphisa amakhemikhali kuseli ngalinye, noma kokubili. Okungaphansi kwalokho kushiya umfundi engakwazi ukuhlukanisa imetabolism eyehlile nomgodi okhipha ukukhanya okufiphele.
Ishidi lokulawula ukuphazamiseka
Kopisha leli shidi encwadini yamanothi yelabhorethri ekuhloleni ngakunye okuhlanganisa udayi nesibiki se-redox. Liyithuluzi lokuqala elenzelwe lesi sihloko: gcwalisa umugqa owodwa ngezinga ngalinye lokugxila kukadayi.
A. Izindlela zesignali. Ngezinga ngalinye lokugxila, bhala: isimo esilindelekile se-monomer uma siqhathaniswa nese-dimer (amazinga aphansi e-micromolar uma kuqhathaniswa namashumi e-micromolar nangaphezulu), ukumuncwa kokukhanya okuqukethwe kokugcina komgodi ku-590 nm emgodini ongenamaseli, kanye nephesenti lokubuyiselwa kwesignali yesengezo se-resorufin. Uma ukubuyiselwa kwehla ngaphansi kwamaphesenti angaba ngu-90, phatha wonke amanani e-fluorescence kulelo nani likadayi njengancishiswe yimiphumela yokukhanya, hhayi njengezilinganiso ezinembile zezinto eziphilayo.
B. Ukuhlola amakhemikhali. Bhala i-fluorescence yodayi ne-reagent ngaphandle kwamaseli njengephesenti lesignali eyisisekelo ye-reagent yodwa ngesikhathi ngasinye sokufunda. Uma umehluko ukhula ngokuhamba kwesikhathi, udayi usabela nohlelo lwesibiki phakathi nokuhlola, futhi ukufunda ekugcineni kuphela kuzodukisa. Cabanga ngokufunda okuqhubekayo ngokuhamba kwesikhathi noma ngesibiki esihlukile.
C. Ukuqinisekisa imiphumela yezinto eziphilayo. Yisho indlela yokufunda ezimele ekhethwe kulokhu kuhlola, isampula yayo kadayi yokulawula, kanye nomphumela olindelekile uma umphumela we-resazurin ungokoqobo. Ukwehla kwangempela kwemetabolism kufanele kuhambise umphumela wokugcina ozimele ngendlela ehambisanayo. Uma umphumela wokugcina ozimele ungavumelani, ungathembi nowodwa okwamanje, bese uhlela ukuhlola kabusha kunokuba uthole isilinganiso esimaphakathi sayo yomibili.
D. Umusho wesiphetho. Bhala umusho owodwa onalesi simo: ngenani nangesigaba esishiwo, isignali ye-resazurin yashintsha ngenani elishiwo, ingxenye eshiwo yalolo shintsho iwumphumela wokuhlola ongenamaseli, futhi umphumela wokugcina ozimele (uyavumelana noma awuvumelani). Uma kukhona okushiywe kungagcwalisiwe, ukuhlola akukapheli.
I-methylene blue isalokhu iyisihloli esiwusizo semetabolism, futhi umsebenzi nge-zebrafish ubonisa imiphumela yangempela kuma-mitochondria ngamanani izikhungo eziningi eziwachitha emapayipini nsuku zonke. Udayi umane uphoqelela ukuthi ulinganiswe ngamathuluzi ongenakuwaphazamisa. Wunike imigodi enodayi kuphela, ukuhlolwa kokubuyiselwa kwesignali yesengezo, nokufundwa okukodwa okusekelwe esimisweni esihlukile sezinto ezibonakalayo, bese ukuphazamiseka kuyeka ukuba ugibe kube yinto elinganisekayo enenani elicacile.