Kuyera mhando dzeokisijeni dzinokurumidza kuita muzviedzo zvemethylene blue

Marabhoritari maviri anoita zviedzo zvinodaidzwa nerimwe nerimwe kuti zviedzo zvekudzivirira oxidation nemethylene blue. Rimwe rinoti dhayi iyi inoderedza mhando dzeokisijeni dzinokurumidza kuita makemikari. Rimwe rinoti inodzigadzira. Ose anoratidza makuruvu akajeka ekuchinja kwemhedzisiro zvichienderana nehuwandu hunoshandiswa. Ose ari kutaura nemwoyo wose. Musiyano uyu haubvi pakunyengera kana kusagona basa. Unobva pakuti mapoka maviri aya akayera zvinhu zvakasiyana ari pasi pechiedza chakasiyana, nezviratidzi zvekuongorora zvinopindura mibvunzo yakasiyana, ndokuzotsanangura mhinduro dzose neshoko rimwe chete: ROS.

Methylene blue imorekuru rakaoma kuyera nemazvo nokuti inotora chikamu mumakemikari ainofanira kunge ichingocherechedza. Murima inotakura maerekitironi pakati pezvinopa nezvinogamuchira maerekitironi, yozodzokera muchimiro chayo chekutanga, yagadzirira kutenderera zvakare. Kana paine chiedza inova chinhu chinokurudzira kuita kwemakemikari nechiedza, ichisvika muchimiro chetriplet chine simba rakawedzerwa. Iri muchimiro ichi inogona kupa simba kuokisijeni kuti igadzire okisijeni ye singlet kana kutamisa maerekitironi kuti igadzire ma radical nehydrogen peroxide. Chero chiratidzi chekuongorora chinoiswa mugadziriro iyi chinogona kuitwa oxidation, reduction, kurasikirwa neruvara, kana kuti chiedza chacho chivharwe nedhayi pachayo. Saka mubvunzo wekutanga pamusoro pechero kuyerwa kweROS hausi wekuti nhamba yacho ndeipi. Ndewekuti bvunzo yacho inonyatsoona chii, uye kuti dhayi inoitei kubvunzo yacho.

Amplex Red: bvunzo yehydrogen peroxide yakaenzaniswa nezviyero zvinozivikanwa

Kubatanidza methylene blue neAmplex Red ndiyo nzira inonyanya kuva nehumbowo hwakasimba muzvinyorwa izvi, kana yakagadzirwa nemazvo. Kuita kwemakemikari kwacho kwakanangana nechinhu chimwe: horseradish peroxidase inoshandisa hydrogen peroxide kuita oxidation yeAmplex substrate kuti ibudise resorufin, chinhu chinobudisa chiedza chefluorescence, nechiyero chemamorekuru chinenge chiri chimwe kuchimwe. Peroxide iri mumuenzaniso inoshanduka kuva fluorescence inoonekwa nemudziyo. Hapana chimwe chiri mumhuri yeROS inowanzozivikanwa chinokonzera kuita uku zvakananga.

Mukana mukuru uri pakuenzanisa nezviyero zvinozivikanwa. Muongorori anongwarira anoedza zviyero zveperoxide zvinozivikanwa mumushonga mumwe chete unochengetedza pH, aine huwandu hwakafanana hweperoxidase nesubstrate, nguva yakafanana yekuchengetwa kwemuenzaniso usati wayerwa, uye marongero mamwe chete emidziyo yechiedza. Anobva aenzanisa mienzaniso nekuruvu iroro. Muchidzidzo chemitochondria dzakaparadzaniswa nehuropi, icho chinotsigira nzira iyi, vanyori vakaita izvozvo chaizvo, vachiwedzera huwandu hweperoxide hunozivikanwa pakupera kwebvunzo imwe neimwe. Nhamba dzacho dzakataurwa semwero mumapicomole paminiti pamilligram yeprotein, kwete semayuniti efluorescence asina chiyero chinozivikanwa. Ndizvo zvinoshandura kupenya kuva kuyerwa.

Zvinhu zvitatu muchidzidzo ichocho zvinofanira kutevedzerwa. Chekutanga, vanyori vakasimbisa chiratidzo necatalase, iyo inoparadza peroxide. Kuwedzera kwefluorescence kwakakonzerwa nemethylene blue kwakamira pakawedzerwa catalase, zvichisimbisa kuti chiratidzo chaiva cheperoxide, kwete chikanganiso chinobva pachiedza. Chechipiri, vakaedza chiedza zvakananga nekuvhara danda rechiedza chinomutsa fluorescence kuitira kuti mienzaniso iwane chikamu chimwe chezvisere chechiedza chainowanzowana, uye chiratidzo chakaderera nezvipfumbamwe kubva muzana chete. Inopfuura makumi mapfumbamwe kubva muzana yeperoxide yavakataura yakanga isingabvi pachiedza chekuyera. Chechitatu, vakayera kubviswa kweperoxide kwakazvimirira vachishandisa electrode, vakaona kuti dhayi yaikwidza kugadzirwa kwayo uye yaidzikisa kukurumidza kubviswa kwayo. Chiratidzi chinongoratidza fluorescence inosara chingadai chakasanganisa mhedzisiro mbiri idzodzo.

Bvunzo iyi ichine nzira dzainogona kukanganisika nadzo, uye methylene blue inokonzera dzinoverengeka. Dhayi inomedza chiedza mumhando dzakawanda dzewavelength munharaunda tsvuku imwe chete inobudiswa chiedza neresorufin. Saka kana huwandu hwedhayi hwakanyanya, muenzaniso unogona kuvhara chiratidzo chawo pachawo kuburikidza nemhedzisiro yekusefa chiedza mukati. Naizvozvo zviyero zvekuenzanisa zvinofanira kuva nehuwandu hwedhayi hwakafanana nehuri mumienzaniso. Amplex Red inogonawo kuita oxidation nekuda kwechiedza iri yega, uye resorufin yainobudisa inogona kupinda mukutenderera kwephotoredox kunogadzira superoxide neperoxide. Izvi zvinokosha zvikuru kana chiedza muchiedzo chichivhenekerawo chinhu chinokurudzira kuita kwemakemikari nechiedza. Nzira inoshanda yekugadzirisa izvi ndeyekutanga kuvhenekera mienzaniso yemethylene blue, wozoyera zvikamu zvayo nemishonga yeAmplex murima, pane kuvhenekera dhayi nechiratidzi pamwe chete. Pakupedzisira, superoxide dismutase haingoderedzi chiratidzo cheAmplex: kushandura superoxide kuva peroxide kunogona kuchikwidza. Chidzoro cheSOD chinowedzera fluorescence hachina kukundikana. Chinenge charatidza kuti superoxide ndiyo imwe yemhando dzinotangira peroxide munzira yekuita kwemakemikari.

DCFDA: chiratidzi cheoxidation chisina kunangana nechinhu chimwe chete

Kubatanidza methylene blue neDCFDA ndiko kunobva kuvhiringidzika kwakawanda. DCFH-DA chinhu chinotangira chimwe, chinopinzwa musero. Maesterase ari mukati mesero anobvisa mapoka ayo eacetate, achisiya DCFH isingabudisi fluorescence, uye oxidation yeDCFH inogadzira DCF inobudisa fluorescence. Zvinogona kuita oxidation yayo zvakawanda: ma radical ehydroxyl, zvinhu zvepakati zvinobva muperoxidase neheme, nitrogen dioxide, hypochlorous acid, uye ma radical anobva muperoxynitrite. Hydrogen peroxide iri yega haiiti oxidation yayo zvakananga pamwero unobatsira pakuyera; fluorescence inoenderana neperoxide inowanzoda iron, maprotein ane heme, kana kuita kwemakemikari kweperoxidase mugadziriro yacho.

Ongororo inotsoropodza iyi iyo chikamu chimwe nechimwe chenzira dzekutsvakurudza chinofanira kureva inobva pakuongororwa kweDCFH sechiratidzi naKalyanaraman nevamwe vake, sezvakapfupikiswa muchidzidzo chemitochondria dzehuropi chakaramba DCFDA nokuda kwechikonzero ichi. Vakaratidza kuti dhayi inopindura zvishoma nezvishoma, nechiyero chemamorekuru chisina kutsanangurwa, isina kuenzaniswa nenzira yakakodzera nezviyero zvinozivikanwa, uye ichipindurawo kuzvinhu zvisiri ROS. Chiratidzi chacho chinowedzerawo zvachinoyera: oxidation yeDCFH inobvisa erekitironi rimwe chete inogadzira radical yeDCF inopindura neokisijeni kugadzira superoxide, iyo inozoshanduka nekuita kwedismutation kuva imwe peroxide. Kuyerwa kwacho kunenge kuri kugadzira chikamu chemakemikari akuri kuratidza.

Izvi hazviiti kuti DCFDA ishaye basa. Zvinoita kuti ive bvunzo yekutanga yekuona kana pane shanduko, kwete yekuziva kuti chinhu chacho chii. Kukwira kwefluorescence yeDCF kunoreva kuti oxidation yechiratidzi inokonzerwa nezvinhu zvinoita oxidation yawedzera. Hazvirevi kuti peroxide yawedzera, uye hazvitomborevi kuti ROS yose yawedzera, nokuti hapana chiratidzi chimwe chete chinoyera huwandu ihwohwo. Tsvakurudzo yekutanga yemethylene blue yaivimba neoxidation yeDCF pahuwandu hwaipfuura micromolar imwe yakataura mhedzisiro inokurudzira oxidation, ukuwo dzimwe gadziriro dzenguva iyoyo dzichitaura kudzivirirwa kwerufu rwemasero rwakakonzerwa neperoxide. Zvose zviri zviviri zvinogona kuva zvechokwadi padhayi imwe chete mumamiriro eredox akasiyana, uye mhedzisiro yeDCF yoga haigoni kutaura kuti ndechipi chinhu chinoita oxidation chakakonzera chiratidzo. Kukosha kwetsvakurudzo yekutanga yemitrochondria dzehuropi kuri pakuti yakatsiva bvunzo iyi yekutanga nekuverengwa kweperoxide kwakaenzaniswa nezviyero zvinozivikanwa, ikaona huwandu hwacho zvakananga: kunyange dhayi iri pahuwandu hwenanomolar zana yakawedzera kubudiswa kweperoxide zvakapetwa kakawanda pane substrate imwe neimwe uye mamiriro ekufema emasero ose akaedzwa.

DCFDA ine mumwe musungo wakanangana nemethylene blue. Dhayi iyi ine ruvara rwakasimba uye inoshanda muredox, saka inogona kudzima fluorescence, kuderedza chiedza chinobudiswa, kana kuita oxidation yechiratidzi pasina kubatanidzwa kweROS yemasero. Chidzoro chisina masero chine dhayi nechiratidzi hachisi chokuwedzera chinosarudzwa kana zvichidiwa chete. Ndicho chiedzo chinokuudza kana dhayi ichipindura nechiratidzi.

Kubata ma radical nekutora kweokisijeni: kuziva mhando uye mwero wekuyerera

Apo zviratidzi zvefluorescence zvinoratidza kuti pane chinhu chinogona kuitwa oxidation chabuda, electron spin resonance ine zvinobata ma radical inoratidza zviri pedyo nekuti chinhu ichocho chaiva chii. Chidzidzo chemethylene blue chinozivikanwa pamusoro pekuita kwemakemikari nechiedza chakashandisa ESR kuona radical yeascorbate yaibva mudhayi, ascorbate nechiedza, ndokuratidza kugadzirwa kweperoxide muzviedzo zvekutora kweokisijeni, uye kugadzirwa kweradical yehydroxyl kwakakurudzirwa neiron muzviedzo zvekubata ma radical. Nzira imwe neimwe yakapindura mubvunzo wainge usingagoni kupindurwa nedzimwe: ESR yakaratidza mhando yeradical, kutorwa kweokisijeni kwakaratidza mwero wekuita kwemakemikari pasina kushandisa chiratidzi chefluorescence, uye kubatanidza nzira idzi kwakaratidza kuti nzira dzose dzesinglet oxygen nedzema radical dzaishanda mugadziriro imwe chete yaivhenekerwa.

Mhedzisiro iyoyo ya1984 ichiri kunzwika seyambiro. Kana paine chiedza nechinhu chinoita reduction, dhayi yakagadzira peroxide, uye kana paine iron gadziriro yacho yakagadzira radical yehydroxyl, inova ndiyo mhando inokurumidza kuita zvikuru muboka iri. Vanyori vakabatanidza izvi neascorbate iri muziso, vakabvunza kukodzera kwekushandiswa kwayo muziso pasina kunyatsochenjerera. Vaverengi vemazuva ano vanofanira kucherechedza mamiriro acho: chiedza pamwe nechinhu chinoita reduction pamwe neiron. Kana chimwe chete chezvitatu izvi chikabviswa, nzira yacho inopera simba kana kutsakatika.

Maelectrode eokisijeni anoratidza kukosha kwawo neimwe nzira. Electrode yemhando yeClark inoyera huwandu hweokisijeni hunodyiwa mushure mekubvisa hunodzorerwa. Izvi zvinosimbisa kuti kuita kwemakemikari eredox kunofambiswa nedhayi kuri kushandisa mamorekuru eokisijeni, uye zvinopa mwero wekuita usingagoni kutevedzerwa nechikanganiso chefluorescence. Haizivi kuti chigadzirwa chacho chii. Kugadzirwa kweperoxide, superoxide nemvura, pamwe nekupera kwesinglet oxygen, zvose zvinoshandisa okisijeni. Kuziva mwero wekuyerera usingazivi mhando yechinhu hakuna kukwana, sezvakangoita kuziva mhando usingazivi mwero wekuyerera. Ndokusaka zvinyorwa zvine humbowo hwakasimba zvichibatanidza electrode nebvunzo yeperoxide yakaenzaniswa nezviyero zvinozivikanwa kana nechinhu chinobata ma radical.

Nei mhedzisiro yekudzivirira oxidation ichipesana

Kana zvinoyerwa nenzira nhatu idzi zvikaiswa pamwe chete, zvinoita sekupesana muzvinyorwa zvemethylene blue zvinojekeswa nemisiyano yenzira dzekuyera pamwe nezvinhu zvitatu zvechokwadi pamusoro pezvinoitika muzvinhu zvipenyu.

Chekutanga, dhayi inotakura maerekitironi uye inogadzira peroxide. Nekutsausa maerekitironi kubva panzvimbo dzekufema kwemasero apo okisijeni yaizoshanduka kuva superoxide, inogona kuderedza imwe ROS, ukuwo chimiro chayo chakaitwa reduction chichishandura okisijeni zvakananga kuva peroxide, chichiwedzera imwe. Naizvozvo rabhoritari iri kuongorora kuita kwemakemikari eredox mumitochondria nebasa rekupfuudza maerekitironi neimwe nzira inofanira kutarisira mhinduro dzinoenderana nechiratidzi chinoshandiswa: kuyerwa kunonzwa superoxide kunoderera uku kuyerwa kweperoxide kuchikwira mucuvette imwe chete.

Chechipiri, peroxide pahuwandu hushoma chiratidzo chekufambiswa kwemashoko, kwete kungokuvadza chete. Chidzidzo chemitochondria dzehuropi hwembeva chakatevera tsvakurudzo yekutanga chakawana kugadzirwa kweperoxide kubva padhayi shoma senanomolar makumi mashanu, pasina kukuvadzwa kweDNA yemitochondria pahuwandu hunoshandiswa pakurapa, uye kudzivirirwa pakukuvadzwa kunokonzerwa nerotenone mushure mekunge dzambotanga dzaiswa mudhayi. Dhayi yakakuvadza zvikamu zvakasarudzwa zveDNA chete pahuwandu hwemicromolar zana, huri kure pamusoro pehuwandu hunoshandiswa pakurapa. Chiedza chitsvuku pamwe nedhayi iri pahuwandu hwemicromolar makumi maviri zvakagadzira singlet oxygen yakakuvadza zvikamu zvose zvakaedzwa. Nzira inofungidzirwa ndeyehormesis: kugadzirwa kweperoxide kushoma kunomutsa nzira dzekudzivirira oxidation dzine chekuita neNrf2 uye kugadzirwa kwemitochondria mitsva, saka kuyerwa kunoratidza kukurudzirwa kweoxidation munguva pfupi kunotangira mhedzisiro yekudzivirira oxidation munguva refu. Bvunzo inoverengwa paawa imwe neinoverengwa pazuva rimwe dzinogona kusiyana nokuti zvinoitika muzvinhu zvipenyu zvashanduka pakati penguva idzodzo.

Chechitatu, chiedza ndicho chinosarudza kuti imorekuru ripi rinenge riripo. Chiedza chinobvisa mapoka emethyl mudhayi, chichiiendesa kuAzure B nezvimwe zvigadzirwa zvine hukama nayo, uye mwenje wemaikorosikopu uri pamusoro pendiro unokwana kuita kuti dhayi ikurudzire kuita kwemakemikari nechiedza. Zviedzo zvinoitwa murima nezvinoitwa muchiedza zviedzo zvakasiyana, uye chikamu chezvidzoro zvechiedza nekubata dhayi chechero gwara rechiedzo chinofanira kuzvibata saizvozvo.

Zvidzoro zvishoma zvinodiwa, uye kuverenga chero chirevo

Kuenzanisa kuri pasi apa ndiyo tafura yepakutanga yekurevera pachinyorwa chino. Usati wagamuchira chero mhedzisiro yeROS pamethylene blue, tarisa kuti vanyori vakaita mutsara upi uye vakasvetuka upi.

Pane mutemo unoshanda wekuverenga unobva pane izvi. Amplex ine peroxidase, yakaenzaniswa nezviyero zvinozivikanwa uye yakaongororwa necatalase, inopa humbowo pamusoro peperoxide. DCFDA inopa humbowo pamusoro peoxidation yechiratidzi isina kunangana nechinhu chimwe chete, uye hapana chimwe. Kubata ma radical, paine zvidzoro zvezvinhu zvinobvisa ma radical, kunopa humbowo pamusoro pemhando yeradical. Kutorwa kweokisijeni kunopa humbowo pamusoro pemwero wekuyerera. Mutsara unotaura zvinopfuura zvinogona kutsigirwa nebvunzo yawo, pasinei nekuti wakanyorwa nechivimbo chakadini, mutsara unotaura pamusoro pechiratidzi, kwete pamusoro pedhayi. Hurukuro dzine hukama pamusoro pekuenderana nehuwandu hunoshandiswa uye muganhu wehormesis, kufamba kwemaerekitironi mumitochondria, kukanganiswa kwebvunzo dzekuyera mumienzaniso yakaiswa dhayi, uye zvikanganiso zvinokonzerwa nechiedza chemaikorosikopu dzinotsanangura imwe neimwe yepfungwa idzi zvakadzama, uye imwe neimwe inoramba ichishanda kana chinongedzo chayo chenguva pfupi chatsiviwa.