I-methylene blue kuphando lwe-zebrafish: isongezelelo sokugcina izilwanyana nomba ophazamisa iziphumo zovavanyo

Amanzi amaqanda e-zebrafish adla ngokuba nombala oluhlaza okwesibhakabhaka ongacacanga, yaye phantse akukho mntu ubhala isizathu soko. Idayi yi-methylene blue, eyongezwa ukuthintela ukungunda, yaye iinkqubo ezininzi ziyiphatha njengento nje ekhoyo engachaphazeli uvavanyo. Oko akulunganga apha. Ingcamango elunge ngakumbi kukuba ngumba wovavanyo ongadweliswanga: into ekhoyo kwizitya ezinyangiweyo nengakhoyo kumaqela olawulo, esebenza ngokwepharmacology kumanqanaba oxinaniso asetyenziswayo, nenokutshintsha kanye izalathisi zesiphumo ezilinganiswa luvavanyo.

Lo mahluko ulungelelanisa yonke into engezantsi. Ukusebenzisa idayi rhoqo ekugcinweni nasekukhulisweni kweentlanzi ukuthintela ukungunda ngumbuzo omnye. Ukuba isongezelelo asichaphazeli uvavanyo lwakho ngumbuzo owahlukileyo. Uncwadi lwakutshanje lwe-zebrafish luphendula owokuqala ngendlela eqinisekisayo, nowesibini ngendlela ebangela inkxalabo.

Oko kumiselwe zizikhokelo ngokwenene

Akukho mgangatho mnye, nto leyo eyinxalenye yengxaki. Isikhokelo se-Canadian Council on Animal Care sivumela i-0.5 mg/L, okanye i-0.00005%, kwisisombululo sokuthutha amaqanda nezibungu ukuze kuncitshiswe ukukhula kokungunda. Isikhokelo saseYurophu sezilwanyana zaselabhoratri sichaza imibungu egcinwa kulwelo lokukhulisa oludla ngokuqulethe kwaloo 0.00005%. Iresiphi ye-Cold Spring Harbor E3 imisela inani eliphindwe kabini kunelo, i-1 mg/L okanye i-0.0001%, njengesithako seresiphi. Ngokwahlukileyo koku kukho into eshiywe ngaphandle ngokuphawulekayo: isikhokelo se-OECD sovavanyo lwetyhefu kwimibungu yeentlanzi simisela amanzi okuxuba abuyiselwe ekubunjweni kwawo kunye nekhemikhali evavanywayo, ngaphandle kwesindululo se-methylene blue kwinguqulelo yaso ka-2013 okanye kuhlaziyo lwamva. Ngoko enye ilabhoratri ikhulisa imibungu ye-zebrafish ene-methylene blue emanzini aluhlaza okwesibhakabhaka ukususela kwiiyure zokuqala zobomi, enye yenze uvavanyo lwemibungu lokuhlangabezana nemimiselo emanzini acacileyo, yaye zombini zibiza ulwelo lwazo ngokuba luqhelekile. Nabani na othelekisa iziphumo phakathi kweelabhoratri, okanye odibanisa idatha yamaqela olawulo yexesha elidlulileyo, ujongene noko kungangqinelani.

Ukuze kucace amanqanaba oxinaniso, i-0.00005% imalunga ne-1.56 micromolar kwaye i-0.0001% imalunga ne-3.13 micromolar. La ngamanani aphantsi, yaye kulula ukucinga ukuba aphantsi kakhulu ukuba angaba nefuthe. Izifundo ezingezantsi zivavanya kanye loo ngcinga, kanye kuloo manqanaba oxinaniso.

Abafundi abavela kwicandelo lokugcina iintlanzi kunokuba bavele kwilabhoratri badibana nale molekyuli inye kwimeko eyahlukileyo, echazwe kwisikhokelo se-methylene blue kwi-aquarium. Ikhemistri iyafana; ingxoxo yendlela idayi ehambisa ngayo ii-electron kwiiseli icacisa ukuba kutheni ikhompawundi esebenzayo kwi-redox ihlala ivela kumanqaku emetabolism. Oku kulandelayo kubhekiselele ngokukodwa kumanzi amaqanda e-zebrafish ane-methylene blue.

Imetabolism: ifuthe litshintsha icala ngokwesigaba

Olona phando lunolwazi oluninzi lwabeka ii-zebrafish ezikhula ngokuqhelekileyo kwidayi ukususela kusuku lokuqala lobomi ukuya kwiintsuku eziyi-5 emva kokuchunyiswa, lwaza lwalinganisa imetabolism ye-oxidative kwisigaba ngasinye, njengoko kuchaziwe kwingxelo epapashiweyo yovavanyo lwemetabolism yemibungu. Kubalulekile ukuwuchaza ngononophelo lo mkhwa kuba utshintsha icala.

Ngosuku oluyi-1 emva kokuchunyiswa, ukusetyenziswa kweoksijini okusisiseko kwenyuka kwimibungu eyayikwidayi kuwo omabini amanqanaba oxinaniso asetyenziselwa ukugcina nokukhulisa iintlanzi. Kwiintsuku eziyi-2, umahluko wombane kwinwebu ye-mitochondria kumaleko ongaphezulu wezibungu wawunyukile xa zibekwe kumanqanaba e-0.00005%, i-0.0001%, ne-0.0003%. Ngeentsuku eziyi-4 ukuya kweziyi-5, emva kokuqandusela, icala lalitshintshile: ukusetyenziswa kwamandla nge-oxidation kwehla, nokuba ukubekwa kwidayi kwakusoloko kuqhubeka ukususela ekuchunyisweni okanye kwakukokwexesha elifutshane kuphela ukusuka kusuku lwe-3 ukuya kolwe-4. Kwangaxeshanye, imibhalo ye-RNA yamacandelwana eekhompileksi zokuphefumla ze-mitochondria yehla kusuku lwe-1 nolwe-4, kunye nemibhalo enxulumene nokwenziwa kweesteroid nokwenziwa kweglucose kwimithombo engeyiyo icarbohydrate.

Ezinye izinto ezimbini eziqatshelweyo ziwenza ucace ngakumbi lo mfanekiso. Ukuqandusela kwenzeka ngaphambi kancinane kwexesha kwinqanaba eliphezulu loxinaniso olusetyenziselwa ukugcina nokukhulisa iintlanzi, imibungu eyayikwidayi ihambele phambili kwiintsuku eziyi-2, aza amaqela olawulo ayifumana ngosuku lwe-3. Yaye inkqubo yokuphendula kuxinzelelo ngokwayo yabonakala isasebenza kakuhle: xa izibungu ezineentsuku eziyi-5 zabekwa phantsi koxinzelelo lokujikeleziswa kwamanzi, icortisol yomzimba wonke yenyuka ngokuqhelekileyo kuwo omabini amaqela. Ngoko idayi ayiyotyhefu eyonakalisa ukukhula ngokubanzi kwezi zixa, yaye ayicinezeli yonke impendulo yomzimba. Itshintsha imetabolism yamandla ngokwesigaba, iyinyusa ngaphambi kokuqandusela ize iyihlise emva koko.

Ukuziphatha: kutheni izifundo ezimbini zingavumelani, nokuba kutheni zombini zinokuba zichanekile

Uncwadi lokuziphatha lubonakala luphikisana de uyilo lokubeka izilwanyana kwidayi lubekwe ecaleni kolunye. Olunye uphando lokhuseleko ekukhuleni nasekuziphatheni lwabeka imibungu kwidayi ukususela malunga neeyure eziyi-6 emva kokuchunyiswa, kumanqanaba oxinaniso afikelela kwi-10 micromolar, lwatshintsha izisombululo yonke imihla ukuya kusuku lwe-4, lwaza lwafudusela izibungu kulwelo olungenadayi ngeentsuku ze-5 neze-6, lwavavanya ukuhamba kuvavanyo lokukhanya nobumnyama ngosuku lwe-6. Isiphumo: akukho futhe libalulekileyo ekufeni, ekuqanduseleni, okanye ekwakhekeni komzimba, kwaye akukho futhe lokunyangwa ekuhambeni. Ababhali bacebisa ngokufanelekileyo i-10 micromolar njengomda ophezulu phantsi kweemeko zabo.

Iqela lesibini, elalinabanye ababhali ababekwakuphando lwemetabolism, lasebenzisa idayi ngendlela eyenzeka ngayo ngokwenene ekugcinweni nasekukhulisweni kweentlanzi: idayi yayikhona ukususela kwiiyure eziyi-2 ukuya kweziyi-3 emva kokuchunyiswa, inxalenye yolwelo itshintshwa yonke imihla, yaye ukuziphatha kulinganiswa ngoxa izilwanyana zisekwidayi okanye zisandula ukukhutshwa kuyo. Kwakuloo manqanaba oxinaniso asetyenziswa rhoqo, izibungu zabonisa ukuncipha kokuhamba kuvavanyo lweeyure eziyi-24, okwakuphinda kufumaneke kwiintlobo zofuzo nakwiilabhoratri ezahlukeneyo, yaye uphando lokuphazamiseka kweziphumo zokuziphatha lwalatha kwinqanaba lomsebenzi wenkqubo yeserotonin njengonobangela, ngokuhambelana nokuthintela kwe-monoamine oxidase okwaziwayo ngale dayi. Eyona nto yayicacisa oku kukuba ulwelo lokukhulisa imibungu olune-methylene blue lwafihla impendulo yokuhamba eyayilindelwe kwi-SSRI fluoxetine. Ngoko uvavanyo lokuhlola olwenziwa emanzini aluhlaza okwesibhakabhaka lunokungafumani ifuthe lechiza elinokubonakala emanzini acacileyo.

Akukho kuphikisana kokwenene. Ikhompawundi isenokungashiyi futhe linokufunyanwa kwiintsuku ezimbini emva kokuhlanjwa kwayo kolunye uvavanyo, kodwa isasebenza ngoxa ikhona kolunye. Intsingiselo esebenzisekayo kukuba isiphumo esingabonisi mahluko sivumela ukumiselwa komda ophezulu woxinaniso, kungekhona ibango lokuba idayi ayichaphazeli uvavanyo, ngoxa isiphumo esibonisa umahluko sichaza umngcipheko obalulekileyo: ukubekwa kwidayi esebenzayo ngexesha lokulinganisa, kanye kwindlela esetyenziswa ziilabhoratri ezininzi.

Idayi ye-redox ngaphakathi kovavanyo lwe-redox

Omnye umgibe ufanele umhlathi wawo kuba uphazamisa isixhobo sokulinganisa kunokuba uphazamise isilwanyana. Uvavanyo lokusetyenziswa kwamandla lwe-Alamar Blue ngokwalo yikhemistri ye-redox: iresazurin iyancitshiswa ibe yiresorufin ekhupha ukukhanya kwefluorescence. I-methylene blue nayo yikhemistri ye-redox. Kulawulo olungenaseli, omabini amanqanaba oxinaniso asetyenziselwa ukugcina nokukhulisa iintlanzi anciphisa kancinane kodwa ngokuphawulekayo ukukhanya kwefluorescence kwe-Alamar Blue emva kweeyure eziyi-24. Kuvavanyo lwezilwanyana, imibungu eyayikwidayi yahlanjwa ubuncinane kathathu kulwelo olungenadayi ngaphambi kovavanyo, yaye ifuthe lebhayoloji kwizibungu eziqanduselweyo lalilikhulu kunokuphazamiseka okwakubonakala ngaphandle kweeseli. Ngoko zombini ezi zinto ziyinyaniso: izilwanyana ezihlanjiweyo zisenefuthe lokwenene kwimetabolism, kwaye idayi engahlanjwanga yongeza ukuphazamiseka okubangelwa yikhemistri ngokuthe ngqo. Amaqela olawulo kufuneka ahlule ezi zinto zimbini, kungenjalo akukho nani kuzo zombini elithetha nto. Isikhokelo ngokubanzi sokufunda iziphumo zovavanyo olusekelwe kwidayi sisebenza apha ngokubaluleka okungaqhelekanga.

Umgca wexesha wezigaba zokukhula

Isixhobo esiyintsusa seli nqaku. Amanqanaba oxinaniso ngamaxabiso amabini aqhelekileyo asetyenziselwa ukugcina nokukhulisa iintlanzi, i-0.00005% ne-0.0001%, ngaphandle kokuba kuchazwe ngenye indlela.

Umkhwa omele ukhunjulwe kukunyuka kwangoko nokuhla kamva kwezibonisi zeziphumo zemetabolism, kunye neziphumo zokuziphatha ezikhoyo ngexesha lokubekwa kwidayi nezingafumanekiyo emva kokukhutshwa kwayo kolunye uvavanyo olwakuvavanya oko.

Uluhlu lokujonga uyilo lwamaqela olawulo

Isixhobo sesibini esiyintsusa. Sisebenzise xa iinkqubo ze-zebrafish ezine-methylene blue zidibana novavanyo.

1. Xela idayi njenge-reagent, kungekhona njengamanzi. Chaza uxinaniso ngepesenti nange-micromolar, ukuqala nokuphela kwiiyure okanye kwiintsuku emva kokuchunyiswa, ishedyuli yokutshintsha ulwelo, kunye nexesha lokukhutshwa kwidayi ngaphambi komlinganiselo ngamnye.
2. Lungelelanisa amaqela olawulo nombuzo. Uphando lwekhemikhali evavanywayo ludinga amaqela olawulo angenadayi; uphando lokugcina nokukhulisa iintlanzi ludinga amaqela angenadayi namaqela anedayi, kubhalwe isiphumo sokuthintela ukungunda, kuba akukho nalunye kwezi zifundo zimbini zikhankanyiweyo olwalinganisa ukusebenza kokuthintela ukungunda.
3. Hlamba ngaphambi kovavanyo lwe-redox, uze uqinisekise. Pula ubuncinane kathathu kulwelo olungenadayi uze wenze ecaleni uvavanyo lolawulo olungenaseli olunedayi ne-reagent, kuba idayi inciphisa umqondiso we-Alamar Blue ngokuthe ngqo.
4. Phatha izalathisi zeziphumo zokuziphatha nezemonoamine njengezisengozini enkulu yokuphazamiseka. Izifundo zokuhamba, zokusabela ngokothuka, zokuziphatha okufana nexhala, zamachiza okulwa nokudakumba, nezokuthintela i-MAO kufuneka ziphephe idayi ngexesha lokulinganisa okanye zibonise ukuba ayichaphazeli isalathisi sesiphumo esithile; ukukhutshwa kwidayi kangangosuku olunye ukuya kwezimbini kwasusa ifuthe elinye ekuhambeni kodwa oko akukaqinisekiswa kwezinye iindlela zovavanyo.
5. Phatha izalathisi zeziphumo zemetabolism neze-mitochondria njengezisengozini enkulu yokuphazamiseka kwisigaba ngasinye. Ukusetyenziswa kweoksijini, ukusetyenziswa kwamandla, umahluko wombane kwinwebu, kunye nemibhalo ye-RNA yekhonkco lokuphefumla kuyatshintsha kumanqanaba oxinaniso asetyenziselwa ukugcina nokukhulisa iintlanzi, kuhamba kumacala achaseneyo ngaphambi nasemva kokuqandusela.
6. Musa ukugqithisela ukungabikho kwefuthe ukusuka kolunye uyilo ukuya kolunye. Isiphumo esingabonisi mahluko emva kokukhutshwa kwidayi asibandakanyi ukubekwa kwidayi okuqhubekayo; isiphumo esingabonisi mahluko ekwakhekeni komzimba asibandakanyi imetabolism okanye ukusebenzisana namachiza; isiphumo kuhlobo olunye lofuzo, kubushushu obuthile, okanye kubude obuthile bovavanyo asibandakanyi ezinye iimeko.
7. Chaza isikhokelo osilandeleyo kunye nenye indlela. Xela ngokucacileyo amanqanaba oxinaniso e-CCAC, aseYurophu, okanye e-Cold Spring Harbor, uze uqaphele apho uvavanyo lwemibungu lwe-OECD lushiya ngaphandle idayi, ukuze umfundi azi ukuba izilwanyana zakho zikhuliswe phantsi kweziphi iimeko.

Akukho nanye koku echasene nokusebenzisa idayi ukugcina amaqanda ephila. Kuchasene nokuvumela isigqibo sokugcina nokukhulisa iintlanzi singene kwidatha singakhange sihlolwe. Umbala oluhlaza okwesibhakabhaka esityeni yimolekyuli enesenzo sepharmacology, yaye iimvavanyo ezinokwenzeka ukuba ziphazamiseke zezo kanye zokuhlola imetabolism nokuziphatha okunxulumene nenkqubo yemithambo-luvo ngomthamo omkhulu, ekukhethwa ii-zebrafish eziselula kuzo ngoku. Yibhale phantsi, yilawule, yikhuphe ngokuhlamba ngaphambi kokulinganisa, uze ujonge uvavanyo ekusenokwenzeka ukuba ithatha inxaxheba kulo ngokungaqaphelekiyo.