Isihloko
I-methylene blue nokuguga kwamaseli: lokho okukalwa ukuhlolwa kwama-fibroblast
Ukufunda ngendlela engafanele okuvamile kuhamba kanje. Iphepha locwaningo libika ukuthi i-methylene blue yengeze ukuphindeka kabili okungaphezu kuka-20 kwesibalo samaseli kuma-fibroblast akhuliswa elabhorethri, bese umfundi ephetha ngokuthi isusa amaseli asegugile noma yelula isikhathi sokuphila kwabantu ngezinga elifanayo. Leso siphetho sibonakala sinengqondo. Kodwa futhi asifanele ngezindlela ezimbili ezihlukene.
Inkinga eyisisekelo imayelana nemiqondo, hhayi izinombolo. Ukuphindeka kabili kwesibalo samaseli, izimpawu zokuguga kwamaseli, nezilinganiso zama-telomere ngakunye kuphendula umbuzo ohlukile. Ucwaningo lwama-fibroblast nge-methylene blue luqondakala kangcono njengocwaningo lokubambezeleka kokungena ekugugeni kohlobo olulodwa lwamaseli esitsheni selabhorethri, olusekelwa izilinganiso zama-mitochondria. Lokho kuhlukile ekususweni okukhethiwe kwamaseli asegugile kakade, futhi kuhlukile nasekugugeni komzimba wonke. Umongo obanzi walowo mehluko uyingxenye yendlela ukuthuthwa kwama-electron kuma-mitochondria okulolonga ngayo amandla eseli nendlela yokuhumusha izimangalo zocwaningo nge-methylene blue.
Ukuphindeka kabili kwesibalo samaseli kubala umlando, hhayi ubusha
Imiphumela eminingi ebalulekile ivela kumaseli e-IMR90, uhlobo lwama-fibroblast avamile aphuma emaphashini ombungu womuntu, anamandla alinganiselwe okuziphindaphinda. Ocwaningweni lokuqala lwe-methylene blue nesikhathi sokuphila sokuziphindaphinda sama-fibroblast e-IMR90, amaseli akhulisiwe ayedluliselwa ezitsheni ezintsha ngokohlelo, futhi ukuphindeka kabili okuqoqekile kwesibalo samaseli kwakurekhodwa kuze kube yilapho eyeka ukuhlukana. Ukwelashwa ngamazinga e-nanomolar, agxile ku-100 nM, kwavumela amaseli akhulisiwe ukuba aqongelele ukuphindeka kabili okwengeziwe okungaphezu kuka-20 ngaphambi kokuma kokuhlukana.
Ukuphindeka kabili kwesibalo samaseli kuyiyunithi yokubala. Kurekhoda ukuthi isibalo samaseli siphindeke cishe kabili izikhathi ezingaki. Akubiki ngokuqondile ukuthi iseli ngalinye seligugile, lithule okwesikhashana, seliguqukele ohlotsheni olukhethekile, noma lifile. Amaseli akhulisiwe angazuza ukuphindeka kabili okwengeziwe ngoba ahlala ekwazi ukuhlukana isikhathi eside, ngoba mancane amisa ukuhlukana kusenesikhathi ngenxa yengcindezi, noma ngoba izimo zivuna ingxenye yamaseli eqhubeka nokwanda. Inani lodwa alikwazi ukuhlukanisa lezo zindlela.
Yingakho izibalo zokuphindeka kabili zihambisana nezimpawu. Isibalo sisho ukuthi ukwanda kwaqhubeka isikhathi esingakanani. Izimpawu zisho ukuthi amaseli ayeke ukuhlukana noma asindile angena kusiphi isimo.
Umhlahlandlela wezilinganiso zemiphumela: lokho okubikwa uphawu ngalunye
Ukuguga kwamaseli akhuliswa elabhorethri kuyisimo esibonakala ngenhlanganisela yezilinganiso eziningana ezizimele. Akukho kudaya okukodwa noma iphrotheni eyodwa ekufakazelayo.
Ukuma kokukhula okuhambisana nokuphela kokwakhiwa kwe-DNA. Ukuma okuhlala isikhathi eside kuhlolwa ngokuncipha kokufakwa kwe-BrdU noma kwe-EdU, ukuma kweseli okuyisicaba, nokwehluleka ukuphinde likhule ngemva kokufakwa esitsheni esisha. Lokhu kuhlukanisa ukuma okubangelwa ukuguga kwamaseli nokuthula kwesikhashana.
I-beta galactosidase ehlobene nokuguga kwamaseli. I-SA-beta-gal idaya umsebenzi wama-lysosome eduze kwe-pH 6.0. Ama-fibroblast asegugile avame ukubonisa ukudayeka njengoba inani lama-lysosome likhula. Ayigcini ekugugeni kwamaseli kuphela, ngakho ihunyushwa kanye nezimpawu zokwanda nezimpawu zamangqamuzana.
Izindlela ze-p16 ne-p21. I-p16 isebenza nge-CDK4 ne-CDK6 nangendlela ye-RB. I-p21 ivame ukuvuselelwa nge-p53. Amazinga akhuphukayo asekela ubufakazi bokuma kokuhlukana, kodwa akukho neyodwa kula maphrotheni ehlobene nokuguga kwamaseli kuphela.
Isimo sokukhishwa kwezinto yiseli. Isimo sokukhishwa kwezinto esihlobene nokuguga kwamaseli, noma i-SASP, sisho ushintsho ekukhishweni kwama-cytokine, ama-chemokine, izici zokukhula, nama-protease. Ukuhlolwa kwe-RNA noma kwamaphrotheni ezici ze-SASP kuchaza indlela amaseli athumela ngayo izimpawu kumaseli angomakhelwane. Akukali ngokuqondile amandla okuziphindaphinda.
Ama-oxidant nezilinganiso zama-mitochondria. Amaphepha ocwaningo nge-methylene blue engeza ungqimba lwezilinganiso zama-mitochondria: inani nomsebenzi we-complex IV, ukusetshenziswa komoya-mpilo, ukwakhiwa kwe-heme, inani lolwelwesi, umehluko wamandla kagesi olwelwesini, i-ATP, nezimpawu zama-oxidant. Lokhu kuchaza isimo sokuguqulwa kwezinto namandla esihambisana nokubambezeleka kokuma kokuhlukana. Akuchazi ukuguga kwamaseli kukodwa, kodwa kuhlola indlela yokusebenza ehlongozwayo. Imodeli yokujikeleza kwe-redox, lapho i-NAD(P)H yehlisa isimo se-oxidation se-methylene blue bese i-cytochrome c iphinde isinyuse, ichazwe kulolo cwaningo lwe-IMR90 oluxhumanisa ukujikeleza kwe-redox nokukhiqizwa okuphansi kwama-oxidant. Umqondo ohlobene namanani asetshenziswayo nokusabalala kwento emzimbeni uchazwa kusayensi yokusebenza kwe-methylene blue emzimbeni nasekuchayekeni kwezicubu kuyo.
Izinga lokuncipha kwama-telomere. Iyunithi efanele ngamapheya ezisekelo alahlekayo ekuphindekeni kabili ngakunye phakathi namasonto, hhayi ubude ngesikhathi esisodwa. Ukuncipha okuhamba kancane kusho ukufinyela okuncane ekuziphindaphindeni ngakunye. Akusho ukweluleka, futhi ingcindezi ingamisa ukuhlukana kwamaseli e-IMR90 kube noshintsho oluncane kuma-telomere.
Ukuqhathaniswa kwezindlela ezintathu zokuhlola ama-fibroblast
Okuqukethwe okuyingqayizivele kwaleli khasi ukuqhathanisa okungezansi. Kuhlanganisa uhlobo lwamaseli, ukuchayeka, izilinganiso, nemikhawulo endaweni eyodwa.
| Ucwaningo | Amaseli nokuchayeka | Okwakukalwa | Lokho olungakwazi ukukubonisa |
|---|---|---|---|
| Atamna nabanye, FASEB J 2008 | Ama-fibroblast avamile e-IMR90, ukuchayeka okuqhubekayo ku-methylene blue yezinga le-nanomolar, u-100 nM uyisimo esijwayelekile | Ukuphindeka kabili okuqoqekile, inani le-complex IV, ukusetshenziswa komoya-mpilo okukhuphuke ngamaphesenti angu-37 kuya ku-70, ukwakhiwa kwe-heme, ukuvikelwa ekugugeni kwamaseli ngaphambi kwesikhathi okubangelwa i-hydrogen peroxide noma i-cadmium, ama-enzyme esigaba 2 kumaseli e-HepG2 | Akukho ukuhlolwa kokususwa kwamaseli asegugile. Akukho silinganiso sokuphila isikhathi eside kwezilwane noma kwabantu. Isikhathi sokuphila sokuziphindaphinda lapha sisho ukuphindeka kabili esitsheni selabhorethri. |
| Atamna nabanye, Redox Biology 2015, okuchazwe ocwaningweni olulandelayo lwezindlela zokusebenza ze-AMPK nokwakhiwa kwe-complex IV | Ama-fibroblast e-IMR90, ikakhulukazi i-methylene blue engu-100 nM, kuqhathaniswa ne-AICAR njengesivuseleli se-AMPK esisebenza ngokuqhubekayo | Ukushintsha kwesilinganiso se-NAD ne-NADH ngokuhamba kwesikhathi, ukufakwa kweqembu le-phosphate ku-AMPK okwesikhashana, ukuvuselelwa kwe-PGC-1alpha ne-SURF1, ukukhuphuka komsebenzi we-complex IV ngamaphesenti angaphezu kuka-100, ama-oxidant anciphe ngamaphesenti angu-28, ukuncipha okuhamba kancane kwama-telomere, ukuphindeka kabili okwengeziwe okungaba ngu-18 uma kuqhathaniswa nokungaba ngu-2 nge-AICAR, akukho mphumela emjikelezweni weseli | Ukuqhathaniswa kwe-AICAR kuhlukile ngenani elisetshenziswayo cishe ngokuphindwe ka-1000 nangokuqondisa kwayo, ngakho kuhlola iphethini yokuvuselela kunamandla afaniswe ngokulinganayo. Umphumela wama-telomere usebenza kumaseli akhulisiwe ahlukana phakathi namasonto. |
| Xiong nabanye, Aging Cell 2016, okubikwe ocwaningweni lwama-fibroblast e-progeria lokubuyiselwa kwesimo sama-mitochondria nesama-nucleus | Ama-fibroblast esikhumba e-Hutchinson-Gilford progeria athathwe ngokuqondile ezicutshini, kanye namaseli avamile okuqhathanisa afaniswe nawo, ikakhulukazi u-100 nM amasonto angu-5 kuya ku-12 | Ukuma kwama-mitochondria ngezibonakhulu ze-fluorescence nezama-electron, ukuhamba kwawo ngezithombe zamaseli aphilayo, i-superoxide, umehluko wamandla kagesi olwelwesini, i-ATP, i-PGC-1alpha, izimpawu zokwanda nokuguga kwamaseli, kanye nokuvuvukala kwezingxenye zolwelwesi lwe-nucleus, indawo ye-progerin, i-heterochromatin, nokusebenza kofuzo | Imodeli yokuguga ngaphambi kwesikhathi ehlobene nesifo esithile, hhayi ukuguga okuvamile. Ukubuyiselwa kokuma kwamaseli nezimpawu akukhona ukususwa kwamaseli asegugile. Akukho silinganiso sokusebenza ezilwaneni noma sesivivinyo sasemtholampilo. |
| Xue nabanye, ukubuyekezwa ku-Cells 2021, okufingqwe ekubuyekezweni kwe-methylene blue njengento engase ilwe nokuguga | Ukuhlanganiswa kwemibhalo ephathelene nokuguga kobuchopho, ukuwohloka kwamangqamuzana ezinzwa, isikhumba, ukuphola kwamanxeba, ne-progeria | Kuchaza i-methylene blue njengento ejikeleza ku-redox ngokusebenza kwe-catalyst, engadlula izingxenye zokuthuthwa kwama-electron ku-complex I naku-III, kuqhathaniswa nemibono yokuguga ehlobene nama-free radical | Ukuhlanganiswa okuchazayo kuphela. Kuhlela imibono ehlongozwayo futhi kuphawula lapho ubufakazi basemtholampilo bungakafinyeleli esiphethweni esiqinisekile. |
Iphethini kuyo yonke imigqa ibaluleke kakhulu kunomugqa owodwa. I-methylene blue ngamanani aphansi ihlotshaniswa kaningi nesikhathi eside sokwanda kwamaseli, umsebenzi ophakeme we-complex IV, izimpawu eziphansi zama-oxidant, nokuma kwamaseli okuthuthukile. Indlela yokuhlola ihlala ilandelela ngokuhamba kwesikhathi: kuqalwa ngamaseli ahlukanayo, asetshenziswe into ehlolwayo ngesikhathi anda, bese kurekhodwa ukuthi ukuhlukana kuqhubeka isikhathi esingakanani. Leso sakhiwo singasekela isimangalo sokubambezeleka. Asikwazi ukusekela isimangalo sokususwa ngaphandle kwendlela yokuhlola ehlukile.
Ukubambezela ukuguga kwamaseli akukhona ukususa amaseli asegugile
Ukubambezeleka kusho ukuthi amaseli ahlukanayo aqhubeka nokuhlukana isikhathi eside ngaphambi kokuma. Ukususwa, okuvame ukubizwa ngokuthi umphumela we-senolytic, kusho ukuthi amaseli asegugile kakade abulawa noma ancishiswa ngokukhetha, kuyilapho amaseli angomakhelwane aqhubeka nokwanda esinda.
Imibhalo yocwaningo lwama-fibroblast nge-methylene blue ihlola isakhiwo sokuqala. Amaseli akhulisiwe aqala ekwazi ukwanda, athole into ehlolwayo ngesikhathi edluliselwa ezitsheni ezintsha ngokulandelana, bese eyeka ukuhlukana kamuva. Ngakho izimpawu ezifana ne-SA-beta-gal, i-p16, i-p21, nezici ze-SASP zivela kamuva noma ngamazinga aphansi esigabeni esithile sokudluliselwa esitsheni esisha. Leyo phethini ilindelekile noma nini lapho isikhathi sokuqala kokuguga siguquka. Ayifakazeli ukuthi kukhona iseli elase ligugile elisusiwe.
Isimangalo se-senolytic singadinga indlela yokuhlola ehlukile: kuqala kwenziwe amaseli akhulisiwe afinyelele esimweni sokuguga esiqinisekisiwe, bese kusetshenziswa into ehlolwayo, bese kuboniswa ukuncipha okukhethiwe kwalawo maseli asegugile uma kuqhathaniswa namaseli okulawula andayo, kusetshenziswa izilinganiso eziqondile zokuphila nokufa kwamaseli. Ukwehla kophawu olulodwa akwanele ngoba ukukhiqizwa kwalo kungashintsha ngaphandle kokulahleka kwamaseli, futhi amaseli andayo asele angagcwala isitsheni. Akukho neyodwa yemithombo emine ecashuniwe esebenzisa leyo ndlela yokunciphisa okukhethiwe ekuhloleni i-methylene blue, ngakho incazelo enembile isalokhu ingumsebenzi wokubambezela noma wokulwa nokuguga kwamaseli elabhorethri, hhayi umsebenzi we-senolytic. Uhlangothi lwengcindezi ye-oxidation kulo mehluko luchazwa kabanzi endleleni amaseli alawula ngayo izinhlobo zomoya-mpilo ezisebenza kalula.
Umphumela wokuthunyelwa kwezimpawu okwesikhashana ucacisa leli phuzu. Ocwaningweni lwe-IMR90 lwango-2015, i-methylene blue yabangela ukukhuphuka okufushane kwesilinganiso se-NAD ne-NADH nokufakwa kweqembu le-phosphate ku-AMPK okwesikhashana, okwalandelwa ukuvuselelwa kwe-PGC-1alpha ne-SURF1, zombili ezihlotshaniswa nokwakhiwa kwe-complex IV. Ukuvuselelwa okuqhubekayo kwe-AMPK nge-AICAR kwaba nemiphumela ephansi kakhulu ekuphindekeni kabili okwengeziwe.
Isikhathi sokuphila sokuziphindaphinda asikona ukuphila kwabantu isikhathi eside
Isikhathi sokuphila sokuziphindaphinda kweseli sibala ukuphindeka kabili ngaphambi kokuma kokuhlukana esitsheni esilawulwayo esinomoya-mpilo, uketshezi lokukhulisa amaseli, nesigqi sokudluliselwa ezitsheni ezintsha okungaguquki. Ukuphila kwabantu isikhathi eside kuhlanganisa amashumi eminyaka okukhula, ukuzivikela komzimba, imithambo yegazi, izindawo ezihlala amaseli ayisiqu, ukuziphatha, nezifo. Into ethile ingelula okokuqala ngaphandle kokushintsha okwesibili, futhi iningi lenza kanjalo.
Ama-fibroblast e-progeria aphethe ushintsho oluthile kufuzo lwe-LMNA olukhiqiza i-progerin, ahambisana nama-mitochondria ahlukene aba yizicucu nangahambi kakhulu, i-ATP ephansi, ama-nucleus angakhekile kahle, nokulahleka kwe-heterochromatin. I-methylene blue yezinga le-nanomolar yathuthukisa izilinganiso zama-mitochondria futhi yabuyisela kakhulu izilinganiso zama-nucleus, kuhlanganise nokukhululwa kwe-progerin olwelwesini. Lokho kuwukubuyiselwa okuphawulekayo ezingeni leseli. Noma kunjalo kusengumphumela wama-fibroblast, futhi ababhali babeka ukuhlolwa kwezilwane njengomsebenzi wesikhathi esizayo. Imodeli yesifo uqobo ichazwa kabanzi ku-methylene blue kumamodeli e-progeria nokuguga ngaphambi kwesikhathi.
Kulokhu kuvela uhlu oluwusizo lokuhlola lapho kufundwa.
- Buza ukuthi ukusetshenziswa kwento ehlolwayo kwaqala ngaphambi kokuma kokuhlukana noma ngemva kokuba ukuma sekuqinisekisiwe. Ngaphambi kusekela ukubambezeleka. Ngemva, kanye nedatha yokufa okukhethiwe, kuyadingeka ukuze kusekelwe ukususwa.
- Buza ukuthi ijika lokuphindeka kabili lamaseli okulawula lenzani. Ngaphandle kokuphindeka kabili okuqoqekile, izinguquko zezimpawu azinasisekelo sokuhunyushwa.
- Cela okungenani imikhakha emibili yezimpawu, njengophawu lokwanda kanye ne-p16 noma i-p21 kanye ne-SA-beta-gal, kunokudaya okukodwa kuphela.
- Buza ukuthi ama-telomere abikwa kanjani. Izinga lokuncipha ekuphindekeni kabili ngakunye ngokuhamba kwesikhathi linenjongo. Isilinganiso esisodwa sobude ngesikhathi esithile asinayo.
- Buza ukuthi yini eyagcinwa ingaguquki. Ukuminyana kwamaseli ngesikhathi sokudluliselwa, umoya-mpilo, ukushintshwa koketshezi lokukhulisa amaseli, nezixazululo ezigciniwe ze-methylene blue ezintsha uma kuqhathaniswa nezindala, konke kushintsha ukuma kokuhlukana kwama-fibroblast.
Uma ifundwa ngale ndlela, imibhalo yocwaningo lwama-fibroblast inikeza i-methylene blue indima ecacile kodwa enemikhawulo: into esebenza ku-redox ngamanani aphansi ebambezela ukuguga okuhlobene nokuziphindaphinda kwama-fibroblast omuntu akhuliswa elabhorethri, kuyilapho ithuthukisa izilinganiso zama-mitochondria. Imininingwane eqine kakhulu yendlela yokusebenza igxile ekuthunyelweni kwezimpawu zamandla okwesikhashana nasekwakhiweni kwe-complex IV, futhi kula maphepha akukho okubonisa ukususwa kwamaseli asegugile noma ukwelulwa kwesikhathi sokuphila kwabantu.