Chinyorwa
Apo methylene blue inovhiringidza muyedzo unoshandiswa kuipima
Imwe rabhoritari inorera maembryo ezebrafish mumethylene blue ine uwandu hunowanzo shandiswa pakurera, yozopima metabolism nenzira mbiri. Pazuva rekutanga, mudziyo weSeahorse unoratidza kuti kushandiswa kweokisijeni kuri kukwira. Pamazuva mana neshanu, plate yeAlamar Blue inoratidza kuti kushandiswa kwesimba kuri kuderera. Dhayi imwe chete, mutsara wehove mumwe chete, asi mhedziso dzakapesana. Zvino panouya muyedzo wekudzora usingashamisiri: makomba ane methylene blue pamwe nereagent yeAlamar Blue, asi asina hove zvachose, anoratidzawo chiedza chakaderera pane ane reagent chete. Chimwe chikamu chemhedzisiro chaibva pakuchinja kwezvipenyu. Chimwe chaibva padhayi yacho ichipa mamakisi kubvunzo yayo pachayo. Kupatsanuka uku, kwakanyorwa muongororo yeCommunications Biology ya2025 yemethylene blue muzebrafish dzichikura, ndiko kunowanzoitika kana methylene blue ichivhiringidza muyedzo, uye ndiko kunoita kuti madhayi anoshanda muredox ade miedzo yekudzora isingadiwi nezvimwe zvinhu zvinowanzoedzwa.
Zvinonyatsoratidzwa nemuyedzo weresazurin
Alamar Blue izita rechiratidzo chekutengesa chemushonga weresazurin wakanyungudutswa, saka kuvhiringidzwa kweAlamar Blue nemethylene blue uye kuvhiringidzwa kwemuyedzo weresazurin nemethylene blue zvinoreva kupindirana kumwe chete. Resazurin yakaitwa oxidation ine ruvara rwebhuruu uye inongobudisa fluorescence shoma kwazvo. Masero ane metabolism inoshanda anoideredza kuti ive resorufin, ine ruvara rwepinki uye inobudisa fluorescence yakasimba pedyo ne560 nm pakumutswa nechiedza uye 590 nm pakubudisa chiedza, iine miganhu inoshandisika iri pa530 kusvika 570 nm pakumutswa uye 580 kusvika 610 nm pakubudisa, maererano nenzira yemugadziri yekushandisa microplate. Kana kuderedzwa uku kukaenderera mberi, resorufin inoshanduka kuita dihydroresorufin, inobudisa fluorescence shoma. Saka kuzadza gomba nemasero akawandisa kana kupa nguva yakawandisa kunokotamisa mutsara wekuenzanisa uchidzika pamucheto wepamusoro.
Maerekitironi anonyanya kubva kuNADH neNADPH kuburikidza nemaenzyme ereductase nediaphorase ari mucytosol, mumicrosome, uye mumitochondria. Izvi zvinoita kuti chiratidzo chinoratidzwe chive kugona kwese kwekuderedza makemikari, kwete kuverengwa kwemasero achiri kurarama uye kwete kuyerwa kwemitochondria. Chiratidzo chakakwirira chinogona kureva masero akawanda, kuyerera kwakawanda kwemaerekitironi ekuderedza pasero rimwe nerimwe, kana zvose zviri zviviri. Chinhu chinoshandura nzira inofambwa neNADH chinoshandura chiratidzo pasina kuuraya kunyange sero rimwe chete. Ramba uchifunga nezvemutsara iwoyo, nokuti methylene blue inogara ichishandura nzira inofambwa neNADH.
Nzira mbiri dzekuvhiringidza, chiratidzo chimwe chakaderera
Methylene blue inokanganisa chiratidzo cheresazurin kuburikidza nenzira mbiri dzakazvimirira, uye imwe neimwe yadzo yoga inoderedza fluorescence inooneka.
Nzira 1 ine chokuita nechiedza. Methylene blue inotora chiedza zvakasimba munharaunda tsvuku, iine bhendi remonomer pedyo ne660 kusvika 665 nm uye chikamu chinobuda padivi chedimer pedyo ne607 kusvika 610 nm, chine muswe unosvika kumashure kuchiedza chinobudiswa neresorufin chiri pa590 nm. Dhayi iri munzira yechiedza inotora chimwe chiedza chekumutsa uye inotorazve imwe fluorescence yakabudiswa. Izvi zvinoita sefirita iri mukati, zvichideredza chiedza chegomba kunyange kugadzirwa kweresorufin kusina kuchinja. Spectrum inoshandukawo zvichienderana nechimiro chedhayi: ongororo yezvinoumba maspectrum emethylene blue mumvura nemumisanganiswa yecyclodextrin inopatsanura monomer, dimer, uye mubatanidzwa wemorekuru rinogamuchira nerinogamuchirwa sezvimiro zvitatu zvine maextinction coefficient akasiyana kubva pa500 kusvika 700 nm. Saka kukura kwekukanganisa kwechiedza kunoshanduka zvichienderana neuwandu hwedhayi, kuungana kwayo, uye kusungana kwayo nezvimwe. Mumuyedzo, patsanura kungoderedzwa kwechiedza kubva pakudzimwa kwefluorescence pamwero wemamorekuru uchishandisa muyedzo wekudzorerwa kwechiratidzo cheresorufin yakawedzerwa unotsanangurwa pasi apa, nokuti nzira dzekugadzirisa dzinosiyana.
Nzira 2 kukwikwidzana muredox. Methylene blue yakaitwa oxidation inogamuchira maerekitironi kubva kuNAD(P)H kuti igadzire leuco methylene blue isina ruvara. Okisijeni yemamorekuru inozoidzorera nechimwe chiitiko cheoxidation kuchimiro chebhuruu, ichigadzira zvigadzirwa zveokisijeni yakaderedzwa, zvinosanganisira hydrogen peroxide. Kutenderera kumwe nekumwe kunoshandisa zvinopa maerekitironi ekuderedza zvaizodai zvaderedza resazurin, uye kunoshandisa okisijeni uku kuchigadzirawo peroxide. Naizvozvo chiratidzo cheAlamar Blue chakaderera chinogona kureva kuti maerekitironi akafamba mukutenderera kwemethylene blue pane kupinda muresorufin. Izvi zvinoverengwa pa plate semetabolism yakaderera, pasinei nezvakaitwa nemasero. Makemikari mamwe chete aya anotsanangura musungo wakapesana uri mumidziyo inopima okisijeni: dhayi yakaderedzwa painodzorerwa neokisijeni kuchimiro cheoxidation, inoita kuti okisijeni ishandiswe nemakemikari kunyange pasina zvipenyu. Saka miedzo yedhayi isina zvipenyu inodiwawo pakupima kufema, sezvinotsanangurwa mukuenzanisa zviratidzo zveokisijeni, ATP, uye musiyano wemagetsi pamembrane panyaya yemitochondria.
Chiratidzo chakachinja nekushanda kwezvipenyu kwakachinja zvinogona kuva zvechokwadi zvose
Ongororo yezebrafish inokosha nokuti hairerutsi nyaya ichiti zvinofanira kuva chimwe chete kana chimwe. Kuchinja kwezvipenyu kwaiva kwechokwadi: kushandiswa kweokisijeni kwepakutanga kwakakwira mumaembryo ane zuva 1 pauwandu hwose huviri hunoshandiswa pakurera; kufema kwakabatana neATP kwakakwira pauwandu hwakaderera; musiyano wemagetsi pamembrane wakayerwa neprobe yeJC-10 wakakwira pazuva rechipiri; uye zvinyorwa zveRNA zvemacomplex emitochondria zvakaderera pamazuva rekutanga nerechina. Kukanganiswa kwemuyedzo kwaivawo kwechokwadi: hove dzakagezwa katatu dzisati dzasvika padanho reAlamar Blue dzakanga dzichiri kuratidza ruvara rwebhuruu mumusoro, mumuromo, uye munharaunda yekunzwa hwema. Makomba asina masero, ane dhayi nereagent, akarasikirwa oga nefluorescence shoma asi yaikosha pachiverengero. Kudzikira kweAlamar Blue kwakayerwa muhove pamazuva mana neshanu kwaiva kukuru kupfuura kukanganiswa kwakawanikwa pasina masero, saka vanyori vakaona mhedzisiro sekuchinja chaiko kwemetabolism kwaionekwa nomulenzi wakakanganiswa zvishoma. Ndiyo nzira yakarurama yekufunga nezvazvo. Chinhu chinotenderera muredox uye chinotorawo chiedza chiri bhuruu chinowanzovhiringidza nzira yezvipenyu iri kuongororwa pamwe nemakemikari echinoratidza mhedzisiro panguva imwe chete. Basa remiedzo yekudzora nderekuona kukura kwechikamu chimwe nechimwe, kwete kuti chimwe chacho ndechekufungidzira. Chidzidzo chakafanana chinobva mubasa rekusungana necyclodextrin: morekuru rinogamuchira parakavharira dhayi mukati maro, kupenda nekuwedzerwa kwekutora glucose zvakaderera pamwe chete. Izvi zvairatidza kuti kukanganiswa kwemetabolism kwaida kudyidzana kwakananga nedhayi, kwete kungovhiringidzika kweredox kwakapararira. Kubatanidza kuverenga chimiro chedhayi nekuverenga mashandiro ezvipenyu ndiyo nzira chaiyo inopatsanura mashandiro echinhu kubva pakukanganiswa kwemuyedzo, uye gwara rekuverenga zvinotaurwa nezvemetabolism zvichibva pamadhayi rinowedzera tsika iyoyo.
Miedzo yekudzora inozvipatsanura
Ita izvi usati wadudzira chero mhedzisiro yemethylene blue kubva pa plate yeresazurin. Uwandu hwezvinhu, medium, nguva yekuchengetwa mumamiriro emuyedzo, uye kuvhenekerwa zvinofanira kunyatsofanana nemuyedzo une masero, nokuti nzira dzose dziri pamusoro dzinoenderana nazvo.
| # | Muyedzo wekudzora | Zvaunoona | Chiyero chekuti wapasa | Kana ukasapasa |
|---|---|---|---|---|
| 1 | Medium nedhayi, pasina reagent, pasina masero | Chiratidzo chedhayi chekumashure pawavelength dzemuyedzo | Chiratidzo chekumashure chidiki kana chichienzaniswa nechiratidzo chemasero | Bvisa chiratidzo chemuyedzo usina masero pauwandu humwe nehumwe, kwete kushandisa muyedzo mumwe chete kune zvose |
| 2 | Medium nereagent, pasina dhayi, pasina masero | Kuderedzwa kwereagent kunongoitika kwega | Chiratidzo chepakutanga chakaderera uye chakadzikama munguva yekuchengetwa | Pfupisa nguva yekuchengetwa, chinja medium, kana kubvuma muganhu wepasi wakakwirira |
| 3 | Medium, dhayi nereagent, pasina masero | Kudyidzana kwakananga kwemakemikari edhayi nereagent | Zvakafanana nemuyedzo wekudzora 2 mukati mekusiyana kudiki kwekuverenga | Taura mutsauko wacho, usauti unobva kumasero |
| 4 | Kuwedzera resorufin mumakomba ane chinotakura dhayi uchienzanisa neane dhayi | Kuderedzwa kwechiedza kana kudzimwa kwefluorescence | Kudzorerwa kwechiratidzo kwakaenzana mumisanganiswa yose iri miviri | Pima kurasikirwa pauwandu humwe nehumwe hwedhayi wogadzirisa, kana kuchinja chiratidzo chaunoverenga |
| 5 | Dhayi yasara mumiedzo isina masero yakagezwa ichienzaniswa neisina kugezwa | Dhayi yasara mushure mematanho ako ekugeza | Kugeza kunobvisa kukanganiswa kunoitika pasina masero | Dhayi yakapinda mukati inoramba iripo, saka miedzo yekudzora 3 ne4 ichiri kudiwa |
Miedzo yekudzora 3 ne4 ndiyo miviri inosiiwa nemapepa akawanda uye inodiwa nemapepa akawanda anotaura nezvemethylene blue. Muyedzo wekudzora 3 unoona kukanganiswa kwemakemikari, we4 unoona kukanganiswa kwechiedza, uye pamwe chete inoisa miganhu yekukanganiswa kwemuyedzo pasati pataurwa chinhu chipi nechipi pamusoro pezvipenyu.
Zviratidzo zvinoverengwa nenzira dzakazvimirira, chimwe nechimwe chine zvokungwarira zvacho
Hapana muyedzo mumwe wechipiri unosimbisa kuti wekutanga wakarurama. Chiratidzo chimwe nechimwe chakazvimirira chinonzvenga imwe nzira yekukanganiswa asi chichiramba chiri pachena kune imwe. Saka sarudza mubatanidzwa une nzvimbo dzausingaoni dzisingapindirani. Tafura yekusarudza miedzo yezvinhu zvinoshanda muredox inotsanangura kufunga kunodiwa pakubatanidza miedzo, uye gwara remaprobe emusiyano wemagetsi pamembrane rinotsanangura zvakadzama zvokungwarira pafluorescence.
Kuyerwa kwekushandiswa kweokisijeni neSeahorse kunotevera maerekitironi achienda kuokisijeni pane kupinda muresorufin. Izvi zvinonzvenga zvachose makemikari echinoratidza mhedzisiro, asi dhayi inoshandisawo okisijeni nemakemikari. Naizvozvo miedzo yedhayi isina mitochondria mumedium yakakwana inotofanira kuitwa. Miedzo yeATP inoshandisa luciferase inobudisa chiedza kubva mukudyidzana kwezvinhu zvezvipenyu pane fluorescence yeresorufin, saka inonzvenga zvose kupindirana kwechiedza nekukwikwidzana kwemareductase. Asi inoratidza mwero weATP iripo uchibva pakuenzana kwekugadzirwa nekushandiswa kwayo, kwete kuyerera kwekugadzirwa kwayo. Maprobe emusiyano wemagetsi pamembrane akadai seTMRM kana JC-10 anopindura kuti musiyano wechaji unoramba uripo here, asi anobudisa fluorescence munharaunda imwe chete yechiedza chinooneka inotorwa nedhayi. Saka ruvara rwebhuruu rwakasara runoaomeserawo, uye anoda miedzo yedhayi chete pauwandu humwe nehumwe hwedhayi. Kuyerwa kwezvinyorwa zveRNA zvezvikamu zvemacomplex ekufema kunonzvenga nyaya dzechiedza zvachose uye kunounganidza zvakaitika kwemaawa, zvichibatsirana nezviratidzo zvepa plate zvinoratidza zvakaitika mumaminitsi.
Naizvozvo, zvinodiwa zvishoma kuti zvinotaurwa nezvekukanganiswa kwemetabolism nemethylene blue zvivimbike ndeizvi: mhedzisiro yeresazurin ine miedzo yekudzora 1 kusvika 4, pamwe nechimwe chiyero chisiri cheresazurin chinoshandisa musimboti wefizikisi wakasiyana, pamwe netsananguro yakajeka yekuti kuchinja kwacho kunoratidza uwandu hwemasero, kuyerera kwemaerekitironi ekuderedza pasero rimwe nerimwe, kana zvose zviri zviviri. Kana izvi zvisina kukwana, muverengi haakwanisi kupatsanura metabolism yakaderera kubva pagomba rinongobudisa chiedza chakaderera.
Pepa rekushandira rekudzora kuvhiringidzwa
Kopa pepa rekushandira iri mubhuku rerabhoritari pamuyedzo mumwe nemumwe unosanganisa dhayi nechinoratidza redox. Ichi ndicho chishandiso chepakutanga chechinyorwa chino: zadza mutsara mumwe pauwandu humwe nehumwe hwedhayi.
A. Nzira dzechiratidzo. Pauwandu humwe nehumwe, nyora: chimiro chinotarisirwa chemonomer chichienzaniswa nedimer (micromolar yakaderera ichienzaniswa nemakumi emicromolar zvichikwira), kutorwa kwechiedza pa590 nm nezviri mugomba pakupedzisira mugomba risina masero, uye chikamu muzana chekudzorerwa kwechiratidzo cheresorufin yakawedzerwa. Kana kudzorerwa kwechiratidzo kukadzika pasi peinenge 90 muzana, tora zviyero zvose zvefluorescence pauwandu ihwohwo sezvakaderedzwa nekukanganiswa kwechiedza, kwete sezvinonyatsoratidza kushanda kwezvipenyu.
B. Kuongorora makemikari. Panguva imwe neimwe yekuverenga, nyora fluorescence yedhayi nereagent pasina masero sechikamu muzana chechiratidzo chepakutanga chereagent chete. Kana mutsauko uchikura nenguva, dhayi iri kudyidzana nemakemikari echinoratidza mhedzisiro panguva yemuyedzo, uye kuverenga pakupedzisira chete kunotsausa. Funga nezvekuverenga kunotevedza kuchinja nenguva kana kushandisa chimwe chinoratidza mhedzisiro.
C. Kuenzanisa kushanda kwezvipenyu. Taura chiratidzo chakazvimirira chakasarudzwa pamuyedzo uyu, muyedzo wacho wedhayi usina zvipenyu, uye mhedzisiro inotarisirwa kana mhedzisiro yeresazurin iri yechokwadi. Kudzikira chaiko kwemetabolism kunofanira kufambisa chiyero chakazvimirira munzira inoenderana nazvo. Kana chiyero chakazvimirira chikasawirirana nacho, usavimba neimwe yemhedzisiro iyi parizvino; gadzirazve muyedzo pane kuverenga avhareji yezviviri izvi.
D. Mutsara wemhedziso. Nyora mutsara mumwe muchimiro ichi: pauwandu nedanho zvakataurwa, chiratidzo cheresazurin chakachinja nechiyero chakataurwa, icho chine chikamu chakataurwa chinobva pakukanganiswa kunoitika pasina masero, uye chiyero chakazvimirira (chinowirirana kana kuti hachiwirirani). Kana chimwe chikamu chisina kuzadzwa, muyedzo hausati wapera.
Methylene blue inoramba iri chishandiso chinobatsira pakuongorora metabolism, uye basa rezebrafish rinoratidza kuchinja chaiko kwemitochondria pauwandu hunodururwa munzira dzemvura dzinoraswa nezvivako zvakawanda zuva nezuva. Dhayi yacho inongoda kupimwa nemidziyo yaisingagoni kukanganisa. Ipe makomba ane dhayi chete, muyedzo wekudzorerwa kwechiratidzo chezvakawedzerwa, uye chiratidzo chimwe chinoverengwa pachishandiswa fizikisi yakasiyana, uye kuvhiringidzwa kunorega kuva musungo kwotanga kuva chinhu chinogona kupimwa chikapiwa nhamba.