Isihloko
I-methylene blue ocwaningweni lwe-zebrafish: isengezo sokunakekela nesici esiphazamisa ukuhlola
Amanzi amaqanda e-zebrafish avame ukuba nombala oluhlaza okwesibhakabhaka ofiphele, futhi cishe akekho obhala ukuthi kungani. Udayi yi-methylene blue, efakwa ukulwa nesikhunta, futhi izinqubo eziningi ziyithatha njengengxenye engabalulekile yesimo sokuhlola. Lowo akuwona umqondo ofanele lapha. Kungcono ukuyibheka njengesici sokuhlola esingabhalwanga: into ekhona ezitsheni ezifakwe into ehlolwayo kodwa engekho emaqenjini okulawula, esebenza ngokwesayensi yemithi emazingeni okugxila asetshenziswayo, futhi ekwazi ukushintsha zona izilinganiso zemiphumela ukuhlola okuzikalayo.
Lo mehluko uqondisa konke okulandelayo. Ukunakekela okuvamile kokulwa nesikhunta kungumbuzo owodwa. Ukuthi isengezo asinamphumela yini esivivinyweni sakho kungomunye umbuzo. Ucwaningo lwakamuva lwe-zebrafish luphendula owokuqala ngendlela eqinisekisayo, kodwa owesibili ngendlela engakhululeki.
Lokho imihlahlandlela ekunqumayo ngempela
Alikho izinga elilodwa elisetshenziswa yibo bonke, okuyingxenye yenkinga. Umhlahlandlela we-Canadian Council on Animal Care uvumela u-0.5 mg/L, noma u-0.00005%, esixazululweni sokuthutha amaqanda nezibungu ukuze kuncishiswe ukukhula kwesikhunta. Umhlahlandlela waseYurophu wezilwane zaselabhorethri uchaza imibungu egcinwa esixazululweni esivame ukuba nalowo 0.00005% ofanayo. Indlela yokwenza isixazululo se-E3 yase-Cold Spring Harbor icacisa inani eliphindwe kabili, u-1 mg/L noma u-0.0001%, njengesithako sesixazululo. Ngokuphambene nalokhu, kukhona okushiywe ngaphandle okuphawulekayo: umhlahlandlela we-OECD wokuhlola ubuthi emibungwini yezinhlanzi ucacisa amanzi okuhlambulula ahlanganiswe kabusha kanye nekhemikhali ehlolwayo, kodwa awunaso isincomo se-methylene blue kungaba enguqulweni yawo yango-2013 noma ekubuyekezweni kwakamuva. Ngakho enye ilabhorethri ikhulisa imibungu ye-zebrafish emanzini aluhlaza ane-methylene blue kusukela emahoreni okuqala okuphila, enye yenze isivivinyo semibungu sokuhlangabezana nemithetho emanzini acwebile, futhi zombili zibiza isixazululo sazo ngokuthi sijwayelekile. Noma ubani oqhathanisa imiphumela phakathi kwamalabhorethri, noma ohlanganisa idatha yamaqembu okulawula omlando, ubhekana nalokho kungahambisani.
Ukuze kubekwe amazinga okugxila esimweni sawo, u-0.00005% ucishe ube ngu-1.56 micromolar kanti u-0.0001% ucishe ube ngu-3.13 micromolar. Lawa ngamanani aphansi, futhi kulula ukuwathatha njengaphansi kakhulu ukuba abe nomthelela. Ucwaningo olungezansi luhlola wona kanye lowo mcabango, kuwo kanye lawo mazinga okugxila.
Abafundi abavela emkhakheni wokugcina izinhlanzi kunasemalabhorethri bahlangana nale molekhuli endaweni ehlukile, echazwe emhlahlandleleni we-methylene blue kuma-aquarium. Ikhemistri iyafana; ingxoxo yokuthi udayi uthutha kanjani ama-electron phakathi kwamaseli ichaza ukuthi kungani isakhi esisebenza ku-redox silokhu sivela emibhalweni yocwaningo lwemetabolism. Okulandelayo kugxile emanzini amaqanda e-zebrafish ane-methylene blue.
Imetabolism: umphumela ushintsha indlela ngokuya ngesigaba
Ucwaningo olunikeza ulwazi oluningi kakhulu lwachaya i-zebrafish ekhula ngokujwayelekile kusukela osukwini lokuqala lokuphila kuze kube yizinsuku ezingu-5 ngemva kokuvundiswa, lwalinganisa imetabolism ye-oxidative esigabeni ngasinye, njengoba kuchazwe embikweni oshicilelwe wokuhlola imetabolism yemibungu. Iphethini idinga ukuchazwa ngokucophelela ngoba ishintsha indlela.
Ngosuku olungu-1 ngemva kokuvundiswa, ukusetshenziswa komoyampilo okuyisisekelo kwanda emibungwini echayekile kuwo womabili amazinga okugxila asetshenziswa ekunakekeleni. Ngezinsuku ezingu-2, amandla kagesi kulwelwesi lwama-mitochondria ongqimbeni olungaphandle lwezibungu ayenyukile lapho zichayeke ku-0.00005%, 0.0001%, naku-0.0003%. Ngezinsuku ezingu-4 kuya kwezingu-5, ngemva kokuchanyuselwa, indlela yomphumela yayisiphendukile: ukusetshenziswa kwamandla nge-oxidative metabolism kwehla, kungakhathaliseki ukuthi ukuchayeka kwakuqhubeke kusukela ekuvundisweni noma kwakungokwesikhashana kusukela osukwini lwesi-3 kuya kolwesi-4 kuphela. Ngesikhathi esifanayo, imibhalo ye-RNA yezingxenye zezinkimbinkimbi zokuphefumula zama-mitochondria yehla ngezinsuku ezingu-1 nezingu-4, kanye nemibhalo ye-RNA ehlobene nokwakhiwa kwama-steroid nokwakhiwa kweglucose ngemithombo engewona ama-carbohydrate.
Okunye okubili okwabonwa kwenza isithombe sicace kakhudlwana. Ukuchanyuselwa kwenzeka kusenesikhathi kancane ezingeni eliphakeme lokugxila elisetshenziswa ekunakekeleni, lapho imibungu echayekile yayihamba phambili ngezinsuku ezingu-2, amaqembu okulawula asefike ezingeni elifanayo ngosuku lwesi-3. Futhi uhlelo lokuphendula ekucindezelekeni ngokwalo lwalubonakala lusebenza kahle: lapho izibungu ezinezinsuku ezingu-5 zifakwa esimweni esicindezelayo sokuzungeziswa, i-cortisol yomzimba wonke yakhuphuka ngokujwayelekile kuwo womabili amaqembu. Ngakho udayi awuwona ushevu ophazamisa ukukhula ngokubanzi kula manani, futhi awucindezeli zonke izindlela umzimba ophendula ngazo. Ushintsha imetabolism yamandla ngendlela encike esigabeni, uyikhuphule ngaphambi kokuchanyuselwa bese uyehlisa ngemva kwalokho.
Ukuziphatha: kungani izifundo ezimbili zingavumelani, nokuthi kungani zombili zingaba yiqiniso
Imibhalo yocwaningo ngokuziphatha ibonakala iphikisana kuze kube yilapho kuqhathaniswa izindlela zokuchayeka. Olunye ucwaningo lokuphepha ekukhuleni nasekuziphatheni lwachaya imibungu kusukela cishe emahoreni angu-6 ngemva kokuvundiswa emazingeni okugxila afinyelela ku-10 micromolar, lwavuselela izixazululo nsuku zonke kuze kube usuku lwesi-4, lwase luthuthela izibungu esixazululweni esingenadayi ngezinsuku zesi-5 nezesi-6, lwahlola ukunyakaza esivivinyweni sokukhanya nobumnyama ngosuku lwesi-6. Umphumela: awukho umphumela obalulekile ngokwezibalo ekufeni, ekuchanyuselweni, noma ekwakhekeni komzimba, futhi awukho umphumela wokuchayeka ekunyakazeni. Ababhali bancoma ngokufanele u-10 micromolar njengomkhawulo ophezulu ngaphansi kwezimo zabo.
Iqembu lesibili, elalinababhali abathile abafanayo nocwaningo lwemetabolism, lenza ukuchayeka ngendlela ukunakekela okwenzeka ngayo empeleni: udayi wawukhona kusukela emahoreni angu-2 kuya kwangu-3 ngemva kokuvundiswa, ingxenye yesixazululo ivuselelwa nsuku zonke, futhi ukuziphatha kulinganiswa ngesikhathi ukuchayeka kusaqhubeka noma kusanda kuphela. Kuwo lawo mazinga okugxila avamile, izibungu zabonisa ukuncipha kokunyakaza esivivinyweni samahora angu-24, okwaphinde kwabonakala ezinhlotsheni zofuzo nasemalabhorethri ahlukene, futhi ucwaningo lokuphazamiseka kokuziphatha lwalandela umphumela lwawuhlobanisa nezinga lokusebenza kohlelo lwe-serotonin, okuhambisana nokuvinjelwa kwe-monoamine oxidase okwaziwayo ngalodayi. Okubonisa lokhu ngokucace kakhulu ukuthi isixazululo semibungu esine-methylene blue safihla impendulo elindelekile yokunyakaza emuthini i-fluoxetine oyi-SSRI. Ngakho isivivinyo sokuhlunga esenziwa emanzini aluhlaza singaphuthelwa umphumela womuthi obungabonakala emanzini acwebile.
Akukho ukuphikisana kwangempela. Isakhi singashiya kungekho nsalela yomphumela etholakalayo ezinsukwini ezimbili ngemva kokusigeza sisuswe kwesinye isivivinyo, kodwa sibe sisasebenza lapho sikhona kwesinye. Incazelo esebenzisekayo ukuthi umphumela ongakhombisi mehluko uvumela umkhawulo ophezulu wokugxila, hhayi isimangalo sokuthi isakhi asinamphumela, kanti umphumela okhombisa umehluko uchaza ingozi ebalulekile: ukuchayeka entweni esasebenza ngesikhathi sokulinganisa, ngaphansi kwazo kanye izimo ezisetshenziswa amalabhorethri amaningi.
Udayi we-redox ngaphakathi kwesivivinyo se-redox
Olunye ugibe ludinga isigaba salo ngoba luphazamisa ithuluzi lokulinganisa kunesilwane. Isivivinyo se-Alamar Blue sokusetshenziswa kwamandla ngokwaso sisebenzisa ikhemistri ye-redox: i-resazurin yehliswa ibe yi-resorufin ekhipha ukukhanya kwe-fluorescence. I-methylene blue nayo isebenza ngekhemistri ye-redox. Emaqenjini okulawula angenamaseli, womabili amazinga okugxila asetshenziswa ekunakekeleni anciphisa kancane kodwa ngokubalulekile ngokwezibalo ukukhanya kwe-fluorescence kwe-Alamar Blue ngemva kwamahora angu-24. Ekuhlolweni kwezilwane, imibungu echayekile yagezwa okungenani kathathu esixazululweni esingenadayi ngaphambi kwesivivinyo, futhi umphumela webhayoloji ezibungwini ezichanyuselwe wawungaphezu kokuphazamiseka okubonwe lapho kungekho maseli. Ngakho zombili lezi zinto ziyiqiniso: izilwane ezigeziwe zinomphumela wangempela kumetabolism, futhi udayi ongasuswanga ngokugeza wengeza ukuphazamiseka kwamakhemikhali okuqondile. Amaqembu okulawula kufanele ahlukanise lokhu okubili, ngaphandle kwalokho alikho inani elisho okuthile. Umhlahlandlela ojwayelekile wokufunda imiphumela yezivivinyo ezisebenzisa udayi usebenza lapha ngokubaluleka okungajwayelekile.
Umugqa wesikhathi wezigaba zokukhula
Ithebula lokuqala elenzelwe lesi sihloko. Amazinga okugxila yilawo amabili avamile ekunakekeleni, u-0.00005% no-0.0001%, ngaphandle kwalapho kuphawulwe okuhlukile.
| Isigaba | Okwahlolwa | Okutholakele emazingeni okugxila asetshenziswa ekunakekeleni |
|---|---|---|
| 0 kuya ku-1 dpf, umbungu ongakachanyuselwa | Ukusetshenziswa komoyampilo okuyisisekelo, imibhalo ye-RNA yama-mitochondria | Ukusetshenziswa kwenyuka; imibhalo ye-RNA yezinkimbinkimbi I no-IV yehla |
| 2 dpf, isikhathi sokuchanyuselwa | Amandla kagesi kulwelwesi; inqubekela-phambili yokuchanyuselwa | Amandla enyuka ku-0.00005% kuya ku-0.0003%; ukuchanyuselwa kwashesha okwesikhashana ku-0.0001% |
| 3 kuya ku-4 dpf, ukuchayeka kwesikhashana ngemva kokuchanyuselwa | Ukusetshenziswa kwamandla ngemva kokuchayeka okungu-24 h kuphela | Ukusetshenziswa kwehla, kufana nokuchayeka okuqhubekayo |
| 4 kuya ku-5 dpf, izibungu ezichanyuselwe | Ukusetshenziswa kwamandla; imibhalo ye-RNA yama-mitochondria | Ukusetshenziswa kwehla ku-24 naku-48 h; imibhalo ye-RNA yezinkimbinkimbi I, II, III, ne-ATP synthase yehla |
| 5 kuya ku-6 dpf, ukuziphatha kwezibungu | Ukunyakaza kwamahora angu-24; impendulo ku-fluoxetine | Ukunyakaza kwancipha; impendulo ku-SSRI yafihlwa |
| 6 dpf ngemva kwezinsuku ezingu-2 zokususa udayi | Ukunyakaza ekukhanyeni nasebumnyameni; ukwakheka komzimba | Awukho umphumela osalayo ofinyelela ku-10 micromolar |
Iphethini okufanele ikhunjulwe ukwenyuka ekuqaleni nokwehla kamuva kwezilinganiso zemiphumela yemetabolism, nemiphumela yokuziphatha ekhona ngesikhathi sokuchayeka kodwa engatholakali ngemva kokukhishwa kukadayi emzimbeni esivivinyweni esisodwa esahlola lokho.
Uhlu lokuhlola ukuhlelwa kwamaqembu okulawula
Insiza yesibili yokuqala eyenzelwe lesi sihloko. Yisebenzise lapho izinqubo ze-zebrafish ezisebenzisa i-methylene blue zihlangana nokuhlola.
- Bika udayi njengesithako sokuhlola, hhayi njengamanzi. Chaza izinga lokugxila ngamaphesenti nange-micromolar, isikhathi sokuqala nesokuphela ngamahora noma ngezinsuku ngemva kokuvundiswa, uhlelo lokuvuselela isixazululo, nesikhathi sokususa udayi ngaphambi kwesilinganiso ngasinye.
- Qondanisa amaqembu okulawula nombuzo. Ucwaningo lwekhemikhali ehlolwayo ludinga amaqembu okulawula angenadayi; ucwaningo lokunakekela ludinga amaqembu angenadayi aqhathaniswa nanadayi, kubhalwe umphumela wokulwa nesikhunta, njengoba kungekho kulolu cwaningo olubili olucashuniwe olwalinganisa ukusebenza kokulwa nesikhunta.
- Geza ngaphambi kwezivivinyo ze-redox, bese uqinisekisa. Hlanza okungenani kathathu esixazululweni esingenadayi bese wenza ngokuhambisana iqembu lokulawula elingenamaseli kodwa elinodayi nesithako sesivivinyo, ngoba udayi unciphisa isignali ye-Alamar Blue ngokuqondile.
- Phatha izilinganiso zemiphumela yokuziphatha neyama-monoamine njengezisengozini enkulu yokuphazamiseka. Ucwaningo lokunyakaza, ukwethuka okusheshayo, ukuziphatha okufana nokukhathazeka, izinto ezilwa nokudangala, nezivimbeli ze-MAO kufanele lugweme udayi ngesikhathi sokulinganisa noma lubonise ukuthi awunamphumela esilinganisweni esithile; ukususa udayi isikhathi sosuku olulodwa kuya kwezimbili kwasusa umphumela owodwa wokunyakaza, kodwa lokhu akuqinisekisiwe kwezinye izindlela zokuhlola.
- Phatha izilinganiso zemiphumela yemetabolism neyama-mitochondria njengezisengozini enkulu yokuphazamiseka kuzo zonke izigaba. Ukusetshenziswa komoyampilo, ukusetshenziswa kwamandla, amandla kagesi kulwelwesi, nemibhalo ye-RNA yochungechunge lokuphefumula kuyashintsha emazingeni okugxila asetshenziswa ekunakekeleni, kuya ezindaweni eziphambene ngaphambi nangemva kokuchanyuselwa.
- Ungadluliseli ukungabi namphumela kusuka kolunye uhlelo lokuhlola kuya kolunye. Umphumela ongakhombisi mehluko ngemva kokususa udayi awusebenzi ekuchayekeni okuqhubekayo; umphumela ongakhombisi mehluko ekwakhekeni komzimba awusebenzi kumetabolism noma ekusebenzelaneni nomuthi; umphumela ohlotsheni olulodwa lofuzo, ezingeni elilodwa lokushisa, noma esikhathini esisodwa sesivivinyo awusebenzi kwezinye.
- Chaza umhlahlandlela owulandelile nokunye ongakulandela esikhundleni sawo. Yisho ngokucacile amazinga okugxila e-CCAC, aseYurophu, noma ase-Cold Spring Harbor, futhi uphawule lapho isivivinyo semibungu se-OECD singawufaki khona udayi, ukuze umfundi aqonde izimo izilwane zakho ezigcinwe ngaphansi kwazo.
Akukho kulokhu okuphikisa ukusetshenziswa kukadayi ukugcina amaqanda ephila. Kuphikisa ukuvumela ukukhetha kokunakekela kungene kudatha kungahloliwe. Okuluhlaza okwesibhakabhaka esitsheni kuyimolekhuli esebenza ngokwesayensi yemithi, futhi ukuhlola okungenzeka kuphazamiseke kakhulu yikho kanye ukuhlunga kwemetabolism nokuziphatha okuhlobene nohlelo lwezinzwa okucubungula amasampula amaningi, i-zebrafish encane esikhethwa kukho manje. Kubhale phansi, kulawule, kususe ngokugeza ngaphambi kokulinganisa, futhi uhlole isivivinyo okungenzeka kube yingxenye yaso ngaphandle kokuba kuqashelwe.