Inqaku
Imethylene blue nokuguga kweeseli: oko kulinganiswa luvavanyo lweefibroblast
Ukutolika okungachanekanga okuqhelekileyo kuhamba ngolu hlobo. Iphepha lophando lixela ukuba imethylene blue yongeze izihlandlo ezingaphezu kwe-20 zokuphindeka kabini kwenani leefibroblast ezikhuliswe elabhoratri, aze umfundi agqibe kwelokuba isusa iiseli esele zigugile okanye yandisa ubomi bomntu ngomlinganiselo ofanayo. Esi sigqibo sibonakala sisengqiqweni. Kodwa asichanekanga kwiindawo ezimbini ezahlukeneyo.
Ingxaki esisiseko ikwindlela yokuqonda, hayi kumanani. Ukuphindeka kabini kwenani leeseli, iimpawu zokuguga kweeseli, nemilinganiselo yeetelomere nganye iphendula umbuzo owahlukileyo. Uphando lweefibroblast ngemethylene blue luqondakala kakuhle njengesifundo sokulibaziseka kokungena kwimeko yokuguga kuhlobo olunye lweeseli kwisitya selabhoratri, luxhaswa yimilinganiselo yeemitochondria. Oku kwahlukile ekususweni okukhethiweyo kweeseli esele zigugile, kwaye kwahlukile kwakhona ekugugeni kwesidalwa siphela. Umxholo obanzi walo mahluko uyinxalenye yendlela ukuthuthwa kwee-electron kwiimitochondria okubumba ngayo amandla eeseli nendlela yokutolika amabango ophando ngemethylene blue.
Ukuphindeka kabini kwenani leeseli kubala imbali, hayi ubutsha
Uninzi lweziphumo eziphambili luvela kwiiseli ze-IMR90, uhlobo oluqhelekileyo lweefibroblast zemiphunga yosana olungekazalwa lomntu, olunomthamo wokuphindaphindeka onomda. Kwisifundo sokuqala semethylene blue nobude bobomi bokuphindaphindeka kwiifibroblast ze-IMR90, iiseli ezikhuliswayo zazidluliselwa kwizitya ezintsha ngokweshedyuli, kuze kubhalwe ukuphindeka kabini kwenani leeseli okuqokelelweyo de ziyeke ukwahlukana. Ukusetyenziswa kwimilinganiselo ye-nanomolar, kugxininiswe kwi-100 nM, kwavumela iiseli ezikhuliswayo ukuba ziqokelele izihlandlo ezongezelelweyo ezingaphezu kwe-20 zokuphindeka kabini ngaphambi kokuyeka ukwahlukana.
Ukuphindeka kabini kwenani leeseli yiyunithi yokugcina ingxelo. Kubhala ukuba inani leeseli liphindeke phantse kabini kangaphi. Akuxeli ngokuthe ngqo ukuba iseli nganye igugile, ikwimeko yokuphumla, itshintshele kumsebenzi okhethekileyo, okanye ifile. Iiseli ezikhuliswayo zinokufumana izihlandlo ezingakumbi zokuphindeka kabini ngenxa yokuba zigcina amandla okwahlukana ixesha elide, ngenxa yokuba zimbalwa eziyeka kwangethuba ngenxa yoxinzelelo, okanye ngenxa yokuba iimeko zikhetha iqela elincinci leeseli ezikwaziyo ukwanda. Ubalo lodwa alukwazi ukwahlula ezi ndlela.
Yiloo nto ubalo lokuphindeka kabini ludityaniswa neempawu. Ubalo luxela ukuba ukwanda kuqhubeke ixesha elingakanani. Iimpawu zixela imeko iiseli eziyekileyo ukwahlukana okanye ezisindileyo ezingene kuyo.
Isikhokelo seziphumo ezilinganisiweyo: oko kuxelwa luphawu ngalunye
Ukuguga kweeseli ezikhuliswe elabhoratri yimeko yeempawu ezibonakalayo echongwa ngemilinganiselo emininzi ezimeleyo. Akukho bala linye okanye proteni enye eyingqina yodwa.
Ukuyeka ukukhula nokuphela kokwenziwa kwe-DNA. Ukuyeka okuhlala ixesha elide kuhlolwa ngokuncipha kokufakwa kwe-BrdU okanye i-EdU, ubume obusicaba, nokungakwazi ukuphinda zikhule emva kokufakwa kwizitya ezintsha. Oku kwahlula ukuyeka okubangelwa kukuguga kweeseli kwimeko yokuphumla yethutyana.
I-beta galactosidase enxulumene nokuguga kweeseli. I-SA-beta-gal ibonisa umsebenzi weelysosome ngokubala kufutshane ne-pH 6.0. Iifibroblast esele zigugile zidla ngokubonisa ibala njengoko ubuninzi beelysosome busanda. Olu phawu aluphelelanga ekugugeni kweeseli, ngoko lutolikwa kunye nokwanda kweeseli neempawu zemolekyuli.
Iindlela ze-p16 ne-p21. I-p16 isebenza nge-CDK4 ne-CDK6 nangendlela ye-RB. I-p21 idla ngokukhuthazwa nge-p53. Ukunyuka kwamanqanaba azo kuxhasa ubungqina bokuyeka ukwahlukana, kodwa akukho nanye kwezi proteni enxulumene kuphela nokuguga kweeseli.
Imeko yokukhupha izinto. Iphenotype yokukhupha izinto enxulumene nokuguga kweeseli, okanye i-SASP, ibhekisa kutshintsho ekukhutshweni kwee-cytokine, ii-chemokine, izinto ezikhuthaza ukukhula, neeprotease. Uvavanyo lwe-RNA okanye lweeproteni lwezinto ze-SASP luchaza indlela iiseli ezithumela ngayo imiqondiso kwiiseli ezikufuphi. Alulinganisi ngokuthe ngqo umthamo wokuphindaphindeka.
Izinto ezibangela i-oxidation nemilinganiselo yeemitochondria. Amaphepha emethylene blue ongeza inkalo yeemitochondria: ubuninzi nomsebenzi wekhompleksi IV, ukusetyenziswa kweoksijini, ukwenziwa kwe-heme, ubuninzi beenwebu, amandla ombane kwinwebu, i-ATP, nemiqondiso yezinto ezibangela i-oxidation. Ezi zinto zichaza imeko yemetabolism ehamba nokulibaziseka kokuyeka ukwahlukana. Azizichazi ngokwazo ukuguga kweeseli, kodwa zivavanya indlela yokusebenza ecetywayo. Imodeli yokujikeleza kwe-redox, apho i-NAD(P)H inciphisa imethylene blue ize i-cytochrome c iphinde iyenze i-oxidation, icaciswa kwakweso sifundo se-IMR90 esidibanisa ukujikeleza kwe-redox nokuncipha kokuveliswa kwezinto ezibangela i-oxidation. Ingqiqo enxulumene needosi nokusasazeka ichazwa kwifamakholoji yemethylene blue nokuchanabeka kwezicubu kuyo.
Izinga lokuncipha kweetelomere. Iyunithi echanekileyo zizibini zeziseko ezilahlekileyo ngokuphindeka kabini ngakunye kwiiveki ezininzi, hayi ubude ngexesha elinye. Ukuncipha okucothayo kuthetha ukuncipha kobude okuncinci ngokuphindaphindeka ngakunye. Akuthethi ukwanda kobude, kwaye uxinzelelo lunokwenza iiseli ze-IMR90 ziyeke ukwahlukana zingatshintshanga kangako iitelomere.
Ukuthelekiswa kwezicwangciso ezintathu zovavanyo lweefibroblast
Umxholo wokuqala owenziwe kweli phepha luthelekiso olungezantsi. Lubeka uhlobo lweeseli, ukuchanabeka, umlinganiselo, nomda kumbono omnye.
| Isifundo | Iiseli nokuchanabeka | Oko kwalinganiswa | Oko singenakukubonisa |
|---|---|---|---|
| Atamna nabanye, FASEB J 2008 | Iifibroblast eziqhelekileyo ze-IMR90, imethylene blue kwimilinganiselo ye-nanomolar esetyenziswa ixesha elide, i-100 nM njengemeko esemgangathweni | Ukuphindeka kabini okuqokelelweyo, ubuninzi bekhompleksi IV, ukusetyenziswa kweoksijini okunyuke nge-37 ukuya kuma-70 ekhulwini, ukwenziwa kwe-heme, ukukhuselwa ekugugeni kweeseli kwangaphambi kwexesha okubangelwa yi-hydrogen peroxide okanye yi-cadmium, ii-enzyme zesigaba 2 kwiiseli ze-HepG2 | Akukho vavanyo lokususwa kweeseli esele zigugile. Akukho siphumo silinganisa ubude bobomi bezilwanyana okanye abantu. Ubude bobomi bokuphindaphindeka apha buthetha ukuphindeka kabini kwisitya selabhoratri. |
| Atamna nabanye, Redox Biology 2015, esichazwe kuphononongo olulandelayo lwendlela yokusebenza kwe-AMPK nokwakhiwa kwekhompleksi IV | Iifibroblast ze-IMR90, ubukhulu becala i-100 nM yemethylene blue, kusetyenziswa i-AICAR njengesivuseleli se-AMPK sexesha elide sokuthelekisa | Utshintsho ekuhambeni kwexesha kumlinganiselo we-NAD ne-NADH, ukufakwa kweqela le-phosphate kwi-AMPK okwethutyana, ukukhuthazwa kwe-PGC-1alpha ne-SURF1, ukunyuka komsebenzi wekhompleksi IV ngaphezu kwe-100 ekhulwini, ukuncipha kwezinto ezibangela i-oxidation ngama-28 ekhulwini, ukuncipha okucothayo kweetelomere, malunga ne-18 lezihlandlo ezongezelelweyo zokuphindeka kabini xa kuthelekiswa nezimalunga ne-2 nge-AICAR, akukho mpembelelo kumjikelo weseli | Uthelekiso lwe-AICAR lwahlukile kwidosi malunga namaxesha ayi-1000 nakwindlela ejolise ngayo kwizinto ezithile, ngoko luvavanya ipateni yokuvuselela kunokuba luthelekise amandla okusebenza alinganayo. Isiphumo seetelomere sisebenza kwiiseli ezikhuliswayo ezahlukanayo kwiiveki ezininzi. |
| Xiong nabanye, Aging Cell 2016, esixelwe kwisifundo seefibroblast zeprogeria sokubuyiselwa kwemeko yeemitochondria neenucleus | Iifibroblast zolusu ezithathwe ngokuthe ngqo kubantu abane-Hutchinson-Gilford progeria kunye neeseli zolawulo eziqhelekileyo ezihambelanayo, ubukhulu becala i-100 nM kangangeeveki ezi-5 ukuya kwezi-12 | Ubume beemitochondria nge-fluorescence nangemikroskopu yee-electron, ukuhamba kwazo ngemifanekiso yeeseli eziphilayo, i-superoxide, amandla ombane kwinwebu, i-ATP, i-PGC-1alpha, iimpawu zokwanda nokuguga kweeseli, kunye nokuphuma kwamaqhuma kwi-nucleus, indawo ye-progerin, i-heterochromatin, nokusebenza kweejini | Imodeli yokuguga kwangaphambi kwexesha enxulumene nesifo esithile, hayi ukuguga okuqhelekileyo. Ukubuyiselwa kobume neempawu akukokususwa kweeseli esele zigugile. Akukho siphumo sokusebenza kwizilwanyana okanye siphumo sovavanyo lwezonyango. |
| Xue nabanye, uphononongo lwe-Cells 2021, olushwankathelwe kuphononongo lwemethylene blue njengento enokuchasa ukuguga | Ukudityaniswa koncwadi lokuguga kwengqondo, ukonakala kweeseli zemithambo-luvo, ulusu, ukuphiliswa kwamanxeba, neprogeria | Lubeka imethylene blue njengento ekhuthaza ukujikeleza kwe-redox enokudlula kwezinye iinxalenye zokuthuthwa ngekhompleksi I ne-III, xa kuthelekiswa neethiyori zokuguga ezisekelwe kwiiradical ezikhululekileyo | Kukudityaniswa okuchazayo kuphela. Luququzelela iingcamango zophando luze luphawule apho ubungqina bezonyango bungakafikeleli kwisigqibo esicacileyo. |
Ipateni kuyo yonke imiqolo ibaluleke ngaphezu kwawo nawuphi na umqolo omnye. Imethylene blue kwidosi ephantsi iphinda-phinda ukunxulunyaniswa nexesha elide lokwanda kweeseli, umsebenzi ophezulu wekhompleksi IV, imiqondiso esezantsi yezinto ezibangela i-oxidation, nobume obuphucukileyo. Isicwangciso sihlala silandela iiseli ekuhambeni kwexesha: kuqalwa ngeeseli ezahlukanayo, kusetyenziswe into evavanywayo ngexesha lokwanda, kuze kubhalwe ukuba ukwahlukana kuqhubeka ixesha elingakanani. Eso sakhiwo sinokuxhasa ibango lokulibazisa. Asinakuxhasa ibango lokususa ngaphandle kwesicwangciso esahlukileyo.
Ukulibazisa ukuguga kweeseli akukokususa iiseli esele zigugile
Ukulibazisa kuthetha ukuba iiseli ezahlukanayo ziqhubeka zisenza njalo ixesha elide ngaphambi kokuba ziyeke. Ukususa, okuhlala kubizwa ngokuba yimpembelelo ye-senolytic, kuthetha ukuba iiseli esele zigugile ziyabulawa okanye ziyancitshiswa ngokukhethiweyo ngoxa ezikufuphi ezandayo zisinda.
Uncwadi lweefibroblast ngemethylene blue luvavanya isakhiwo sokuqala. Iiseli ezikhuliswayo ziqala zisanda, zifumane into evavanywayo ngexesha lokudluliselwa ngokulandelelana kwizitya ezintsha, zize ziyeke ukwahlukana kamva. Ngoko iimpawu ezifana ne-SA-beta-gal, i-p16, i-p21, nezinto ze-SASP zivela kamva okanye kumanqanaba asezantsi kwinqanaba elithile lokudluliselwa. Loo pateni ilindelekile xa ukuqala kokuguga kushenxisiwe. Ayingqini ukuba kukho nasiphi na iseli esele ikwimeko yokuguga esisusiweyo.
Ibango le-senolytic beliya kufuna isicwangciso esahlukileyo: kuqala kubekwe iiseli ezikhuliswayo kwimeko eqinisekisiweyo yokuguga, emva koko kusetyenziswe ikhompawundi, kuze kuboniswe ukulahleka okukhethiweyo kwezo seli zigugileyo xa kuthelekiswa neeseli zolawulo ezandayo, ngemilinganiselo ethe ngqo yokuphila nokufa kweeseli. Ukwehla kophawu lodwa akwanelanga kuba ukubonakaliswa kwalo kunokutshintsha ngaphandle kokulahleka kweeseli, kwaye iiseli ezandayo eziseleyo zinokukhula zizalise isitya. Akukho namnye kwimithombo emine ecatshuliweyo osebenzisa eso sicwangciso sokunciphisa ngokukhethiweyo xa kuvavanywa imethylene blue, ngoko igama elichanekileyo lihlala ikukusebenza kokulibazisa okanye kokuchasa ukuguga kweeseli elabhoratri, hayi ukusebenza kwe-senolytic. Icala loxinzelelo olubangelwa yi-oxidation kulo mahluko lichazwa ngakumbi kwindlela iiseli ezilawula ngayo iintlobo zeoksijini ezisebenzayo.
Isiphumo semiqondiso yethutyana siyayicacisa ngakumbi le ngongoma. Kuphando lwe-IMR90 lowama-2015, imethylene blue yavelisa ukunyuka okufutshane komlinganiselo we-NAD ne-NADH nokufakwa kweqela le-phosphate kwi-AMPK okwethutyana, kwalandela ukukhuthazwa kwe-PGC-1alpha ne-SURF1, zombini ezinxulumene nokwakhiwa kwekhompleksi IV. Ukuvuselelwa kwe-AMPK ixesha elide nge-AICAR kwaba neziphumo ezisezantsi kakhulu kwizihlandlo ezongezelelweyo zokuphindeka kabini.
Ubude bobomi bokuphindaphindeka abufani nobude bobomi bomntu
Ubude bobomi bokuphindaphindeka kweeseli bubala izihlandlo zokuphindeka kabini ngaphambi kokuyeka ukwahlukana kwisitya esilawulwayo, esineoksijini, ulwelo lokukhulisa, nesingqi sokudluliselwa ezingaguqukiyo. Ubude bobomi bomntu budibanisa amashumi eminyaka okukhula, ukhuseleko lomzimba, imithambo yegazi, iindawo ezihlala iiseli eziyintsusa, ukuziphatha, nezifo. Ikhompawundi inokwandisa obokuqala ngaphandle kokutshintsha obesibini, kwaye uninzi lwenza njalo.
Iifibroblast zeprogeria zinotshintsho oluthile kwi-LMNA oluvelisa i-progerin, zineemitochondria eziqhekekileyo nezingahambi kangako, i-ATP ephantsi, iinucleus ezinobume obungaqhelekanga, nokulahleka kwe-heterochromatin. Imethylene blue kwimilinganiselo ye-nanomolar yaphucula imilinganiselo yeemitochondria yaza yabuyisela kakhulu imilinganiselo yeenucleus, kuquka ukukhululwa kwe-progerin kwinwebu. Oko kukubuyiselwa kwemeko okumangalisayo kwinqanaba leseli. Sekunjalo sisiphumo seefibroblast, kwaye ababhali babeka uvavanyo lwezilwanyana njengomsebenzi wexesha elizayo. Imodeli yesifo ngokwayo ixoxwa ngakumbi kwimethylene blue kwiimodeli zeprogeria nokuguga kwangaphambi kwexesha.
Oku kukhokelela kolu luhlu lokuhlola olusebenzisekayo ekufundeni.
- Buza ukuba ukusetyenziswa kuqale ngaphambi kokuyeka ukwahlukana okanye emva kokuba ukuyeka kuqinisekisiwe. Ukuqala ngaphambi koko kuxhasa ukulibazisa. Ukuqala emva koko kunye nedatha yokufa okukhethiweyo kuyafuneka ukuze kuxhaswe ukususa.
- Buza ukuba ijika lokuphindeka kabini leeseli zolawulo lenze ntoni. Ngaphandle kobalo lokuphindeka kabini oluqokelelweyo, utshintsho lweempawu alunasiseko somxholo.
- Cela ubuncinane iindidi ezimbini zeempawu, ezifana nesalathisi sokwanda kunye ne-p16 okanye i-p21 kunye ne-SA-beta-gal, kunokuba kusetyenziswe ibala elinye kuphela.
- Buza ukuba iitelomere zixelwe njani. Izinga ngokuphindeka kabini ngakunye ekuhambeni kwexesha linentsingiselo. Umfanekiso wobude ngexesha elinye awunayo.
- Buza ukuba yintoni ebigcinwe ingaguquki. Ukuxinana kweeseli xa zidluliselwa, ioksijini, ukutshintshwa kolwelo lokukhulisa, nezisombululo ezigciniweyo zemethylene blue ezisandul’ ukwenziwa xa kuthelekiswa nezindala zonke zitshintsha ukuyeka ukwahlukana kweefibroblast.
Xa lufundwa ngolu hlobo, uncwadi lweefibroblast lunika imethylene blue indima ehambelanayo kodwa enemida: ikhompawundi ekwidosi ephantsi nesebenzayo kwi-redox elibazisa ukuguga okunxulumene nokuphindaphindeka kwiifibroblast zabantu ezikhuliswe elabhoratri ngoxa iphucula imilinganiselo yeemitochondria, ineenkcukacha ezomeleleyo zendlela yokusebenza malunga nemiqondiso yamandla yethutyana nokwakhiwa kwekhompleksi IV, kwaye la maphepha awabonisi ukususwa kweeseli esele zigugile okanye ukwandiswa kobomi bomntu.