Maƙala
Methylene blue da tsufar ƙwayoyin halitta: abin da gwaje-gwajen fibroblast suke aunawa
Wata fahimta marar kyau da ake yawan samu ita ce kamar haka. Wata takardar bincike ta bayyana cewa methylene blue ya ƙara fiye da 20 na ninkewar yawan fibroblast da ake renawa a dakin gwaje-gwaje, sai mai karatu ya ɗauka cewa yana cire ƙwayoyin halittar da suka tsufa ko kuma yana ƙara tsawon rayuwar ɗan Adam da irin wannan adadi. Wannan fahimtar tana iya zama kamar mai ma'ana. Amma tana da kuskure a wurare biyu daban-daban.
Babbar matsalar fahimtar ma'anoni ce, ba ta lambobi ba. Ninkewar yawan ƙwayoyin halitta, alamomin tsufar ƙwayoyin halitta, da ma'aunin telomere kowannensu yana amsa tambaya dabam. Ya fi dacewa a karanta aikin fibroblast kan methylene blue a matsayin nazarin jinkirta shiga yanayin tsufa a nau'in ƙwayar halitta guda ɗaya cikin tasa, tare da goyon bayan ma'aunan mitochondria. Wannan ya bambanta da cire ƙwayoyin halittar da suka riga suka tsufa ta hanyar zaɓe, kuma ya sake bambanta da tsufar cikakken halitta. Faɗaɗɗen bayanin wannan bambanci yana cikin yadda jigilar electron a mitochondria ke tsara makamashin ƙwayoyin halitta da yadda ake fassara ikirarin bincike kan methylene blue.
Ninkewar yawan ƙwayoyin halitta tana ƙidaya tarihin rarrabuwa, ba ƙuruciya ba
Yawancin muhimman sakamakon sun fito ne daga ƙwayoyin IMR90, wani nau'in fibroblast na al'ada daga huhun ɗan tayin mutum, wanda ƙarfin rarrabuwarsa yana da iyaka. A nazarin farko kan methylene blue da tsawon rayuwar rarrabuwa a fibroblast na IMR90, an riƙa mayar da ƙwayoyin halittar da ake renawa zuwa sabbin tasoshi bisa jadawali, ana kuma rubuta jimillar ninkewar yawansu har sai sun daina rarrabuwa. Amfani da methylene blue a matakan nanomolar, musamman 100 nM, ya ba su damar tara fiye da 20 na ƙarin ninkewa kafin tsayawar rarrabuwa.
Ninkewar yawan ƙwayoyin halitta guda ɗaya naúrar ƙididdiga ce. Tana rubuta sau nawa yawansu ya kusan ninka. Ba ta bayyana kai tsaye ko wata ƙwayar halitta guda ɗaya ta tsufa, tana hutawa, ta ƙware don wani aiki, ko ta mutu. Ƙwayoyin halittar da ake renawa na iya samun ƙarin ninkewa saboda sun daɗe suna da ikon rarrabuwa, saboda kaɗan daga cikinsu sun daina rarrabuwa da wuri sakamakon damuwa, ko kuma saboda yanayin renon ya fifita wani ƙaramin rukuni mai saurin yawaita. Ƙidayar kaɗai ba za ta iya bambance waɗannan hanyoyin ba.
Shi ya sa ake haɗa ƙidayar ninkewa da alamomi. Ƙidayar tana nuna tsawon lokacin da yawaita ta ci gaba. Alamomin kuma suna nuna yanayin da ƙwayoyin da suka daina rarrabuwa ko suka tsira suka shiga.
Jagorar ma'aunan ƙarshe: abin da kowace alama ke nunawa
Tsufar ƙwayoyin halitta a yanayin reno wani yanayi ne da ake tantancewa ta ma'aunai masu zaman kansu da dama. Babu gwajin rini ko furotin guda ɗaya da yake tabbatar da ita.
Tsayawar girma tare da daina haɗa DNA. Ana bincika tsayawar rarrabuwa mai ɗorewa ta hanyar raguwar shigar BrdU ko EdU, siffa mai faɗi da lebur, da kasa sake girma bayan mayar da ƙwayoyin zuwa wata tasa. Wannan yana bambance tsayawar rarrabuwa saboda tsufa da hutu na ɗan lokaci.
Beta galactosidase mai alaƙa da tsufar ƙwayoyin halitta. Rinin SA-beta-gal yana nuna aikin lysosome kusa da pH 6.0. Fibroblast da suka tsufa sukan nuna rinin yayin da jimillar lysosome ke ƙaruwa. Ba alama ce da ta keɓanta da tsufa ba, don haka ana fassara ta tare da alamomin yawaita da na matakin kwayoyin sinadarai.
Hanyoyin p16 da p21. p16 yana aiki ta CDK4 da CDK6 da hanyar RB. Sau da yawa ana tayar da p21 ta hanyar p53. Ƙaruwar matakansu tana goyon bayan tsayawar rarrabuwa, amma babu ɗaya daga cikin furotin ɗin da ya keɓanta da tsufar ƙwayoyin halitta.
Yanayin fitar da sinadarai. Yanayin fitar da sinadarai mai alaƙa da tsufar ƙwayoyin halitta, ko SASP, yana nufin sauyin fitar cytokines, chemokines, abubuwan haɓaka girma, da proteases. Gwaje-gwajen RNA ko furotin na abubuwan SASP suna bayyana yadda ƙwayoyin halitta ke aika saƙonni ga maƙwabtansu. Ba sa auna ƙarfin rarrabuwa kai tsaye.
Sinadaran oxidation da ma'aunan mitochondria. Takardun methylene blue suna ƙara wani ɓangaren bincike na mitochondria: yawan complex IV da aikinsa, amfani da oxygen, haɗa heme, jimillar membrane, ƙarfin lantarkin membrane, ATP, da alamun sinadaran oxidation. Waɗannan suna bayyana yanayin metabolism da ke tafiya tare da jinkirin tsayawar rarrabuwa. Su kaɗai ba sa bayyana tsufar ƙwayoyin halitta, amma suna gwada tsarin aikin da aka gabatar. Tsarin zagayawar redox, inda NAD(P)H ke rage methylene blue sannan cytochrome c ya sake oxidize shi, an bayyana shi a wannan nazarin IMR90 da ke danganta zagayawar redox da raguwar samar da sinadaran oxidation. An tattauna ka'idojin adadin da ake amfani da shi da rarrabuwarsa masu alaƙa da wannan a ilimin aikin methylene blue da yadda yake kaiwa ga kyallen jiki.
Ƙimar raguwar telomere. Naúrar da ta dace ita ce adadin base pairs da ake rasa a kowace ninkewa a tsawon makonni, ba tsawon telomere a lokaci guda ba. Raguwar da ke tafiya a hankali tana nufin ƙarancin gajertawa a kowace rarrabuwa. Ba ta nufin ƙaruwa a tsawo ba, kuma damuwa na iya sa ƙwayoyin IMR90 su daina rarrabuwa ba tare da babban sauyi a telomere ba.
Kwatanta tsare-tsaren gwajin fibroblast guda uku
Abin da aka samar musamman domin wannan shafi shi ne kwatancen da ke ƙasa. Ya haɗa nau'in ƙwayar halitta, abin da aka yi amfani da shi, ma'auni, da iyaka a wuri ɗaya.
| Nazari | Ƙwayoyin halitta da abin da aka yi amfani da shi | Abin da aka auna | Abin da ba zai iya nunawa ba |
|---|---|---|---|
| Atamna da abokan aiki, FASEB J 2008 | Fibroblast na IMR90 na al'ada, amfani da methylene blue na nanomolar na dogon lokaci, 100 nM a matsayin yanayin gwaji na yau da kullum | Jimillar ninkewa, yawan complex IV, ƙaruwar amfani da oxygen da kashi 37 zuwa 70, haɗa heme, kariya daga tsufar ƙwayoyin halitta kafin lokaci da hydrogen peroxide ko cadmium suka jawo, enzymes na phase 2 a ƙwayoyin HepG2 | Babu gwajin cire ƙwayoyin halittar da suka tsufa. Babu ma'aunin tsawon rayuwar dabba ko mutum. Tsawon rayuwar rarrabuwa a nan yana nufin ninkewa a cikin tasa. |
| Atamna da abokan aiki, Redox Biology 2015, wanda aka bayyana a binciken tsarin aiki da ya biyo baya kan AMPK da samuwar complex IV | Fibroblast na IMR90, galibi 100 nM na methylene blue, tare da AICAR a matsayin mai kunna AMPK na dogon lokaci domin kwatantawa | Sauyin rabon NAD zuwa NADH a tsawon lokaci, phosphorylation na AMPK na ɗan lokaci, tayar da PGC-1alpha da SURF1, ƙaruwar aikin complex IV da fiye da kashi 100, raguwar sinadaran oxidation da kashi 28, raguwar telomere a hankali, kusan 18 na ƙarin ninkewa idan aka kwatanta da kusan 2 da AICAR, babu tasiri a zagayen ƙwayar halitta | Adadin AICAR da aka yi amfani da shi ya bambanta da kusan ninki 1000, haka kuma keɓancewar aikinsa ta bambanta, don haka kwatancen yana gwada tsarin kunnawa maimakon ƙarfin aiki a daidaitaccen mataki. Sakamakon telomere ya shafi ƙwayoyin halittar da suke rarrabuwa a tsawon makonni. |
| Xiong da abokan aiki, Aging Cell 2016, wanda aka ruwaito a nazarin fibroblast na progeria kan gyaran mitochondria da tsakiya | Fibroblast na fata da aka samo kai tsaye daga masu Hutchinson-Gilford progeria tare da ƙwayoyin al'ada masu dacewa domin kwatantawa, galibi 100 nM na makonni 5 zuwa 12 | Siffar mitochondria ta microscopy na fluorescence da electron, motsi ta hoton ƙwayoyin masu rai, superoxide, ƙarfin lantarkin membrane, ATP, PGC-1alpha, alamomin yawaita da tsufar ƙwayoyin halitta, da kumburan membrane na tsakiya, wurin progerin, heterochromatin, da bayyanar aikin genes | Samfurin tsufa kafin lokaci da ya keɓanta da wata cuta, ba tsufa na al'ada ba. Gyaran siffa da alamomi ba cire ƙwayoyin halittar da suka tsufa ba ne. Babu ma'aunin tasiri a dabba ko ma'aunin gwajin asibiti. |
| Bitar Xue da abokan aiki, Cells 2021, wadda aka taƙaita a bitar methylene blue a matsayin sinadarin da ake nazari don yaƙi da tsufa | Haɗa bayanai daga rubuce-rubucen bincike kan tsufar kwakwalwa, lalacewar tsarin jijiyoyi, fata, warkewar rauni, da progeria | Tana bayyana methylene blue a matsayin sinadarin da ke zagayawar redox ta hanyar catalysis, wanda zai iya tsallake wasu sassan jigilar electron a complex I da III, tare da kwatanta wannan da ka'idojin free radicals na tsufa | Haɗa bayanai ta hanyar bayani kawai. Tana tsara hasashe da nuna inda shaidar gwajin asibiti ba ta kai ga yanke hukunci ba. |
Tsarin da yake bayyana a duk layukan ya fi muhimmanci fiye da kowane layi guda. Ƙaramin adadin methylene blue yana da alaƙa akai-akai da tsawon lokacin yawaita, ƙarin aikin complex IV, ƙarancin alamun sinadaran oxidation, da ingantacciyar siffa. Tsarin gwajin yana bin sauyi a tsawon lokaci: a fara da ƙwayoyin halitta masu rarrabuwa, a yi amfani da sinadarin yayin da suke ƙaruwa, sannan a rubuta tsawon lokacin da rarrabuwar ta ci gaba. Wannan tsarin na iya tallafa wa ikirarin jinkirtawa. Ba zai iya tallafa wa ikirarin cirewa ba sai da wani tsarin gwaji dabam.
Jinkirta tsufar ƙwayoyin halitta ba cire waɗanda suka tsufa ba ne
Jinkirtawa yana nufin ƙwayoyin halitta masu rarrabuwa suna ci gaba da rarrabuwa na tsawon lokaci kafin su tsaya. Cirewa, wanda ake yawan kira tasirin senolytic, yana nufin kashe ko rage ƙwayoyin halittar da suka riga suka tsufa ta hanyar zaɓe, yayin da maƙwabtansu masu yawaita suke rayuwa.
Rubuce-rubucen binciken methylene blue a fibroblast suna gwada tsarin farko. Ƙwayoyin halittar da ake renawa suna farawa suna yawaita, suna samun sinadarin yayin da ake mayar da su zuwa sabbin tasoshi a jere, sannan su kai ga tsayawar rarrabuwa daga baya. Saboda haka, alamomi kamar SA-beta-gal, p16, p21, da abubuwan SASP suna bayyana daga baya ko a ƙananan matakai a wani zagayen mayarwa zuwa sabuwar tasa. Ana sa ran wannan tsarin duk lokacin da farkon tsufa ya koma baya. Ba ya tabbatar da cewa an cire wata ƙwayar halitta da aka riga aka tabbatar ta tsufa.
Ikirarin tasirin senolytic zai buƙaci wani tsari dabam: da farko a sa ƙwayoyin halittar da ake renawa su shiga tsufa da aka tabbatar, sannan a yi amfani da sinadarin, sannan a nuna asarar waɗannan ƙwayoyin masu tsufa ta hanyar zaɓe idan aka kwatanta da ƙwayoyin kwatanci masu yawaita, tare da ma'aunin rayuwa da mutuwa kai tsaye. Raguwar wata alama kaɗai ba ta isa ba domin matakin bayyana alamar na iya sauyawa ba tare da asarar ƙwayoyin halitta ba, kuma ragowar ƙwayoyin masu yawaita na iya cika tasar. Babu ɗaya daga cikin tushe huɗu da aka ambata da ya yi amfani da wannan tsarin rage ƙwayoyin halitta ta hanyar zaɓe don methylene blue, saboda haka sunan da ya dace har yanzu shi ne aikin jinkirta ko hana tsufar ƙwayoyin halitta a dakin gwaje-gwaje, ba senolytic ba. An ƙara bayyana ɓangaren damuwar oxidation na wannan bambanci a yadda ƙwayoyin halitta ke sarrafa nau'o'in oxygen masu saurin yin martani.
Sakamakon aika saƙonni na ɗan lokaci yana ƙara fayyace batun. A aikin IMR90 na 2015, methylene blue ya haifar da ƙaruwar rabon NAD zuwa NADH na ɗan gajeren lokaci da phosphorylation na AMPK na ɗan gajeren lokaci, sannan ya tayar da PGC-1alpha da SURF1, waɗanda dukansu suke da alaƙa da samuwar complex IV. Kunna AMPK na dogon lokaci da AICAR ya samar da ƙarin ninkewa ƙasa sosai.
Tsawon rayuwar rarrabuwar ƙwayar halitta ba tsawon rayuwar ɗan Adam ba ne
Tsawon rayuwar rarrabuwar ƙwayar halitta yana ƙidaya ninkewa kafin tsayawa a cikin tasa da aka sarrafa yanayinta, tare da daidaitaccen oxygen, sinadaran reno, da tsarin mayarwa zuwa sabuwar tasa. Tsawon rayuwar ɗan Adam kuwa yana haɗa shekarun da dama na ci gaba, garkuwar jiki, tsarin jijiyoyin jini, muhallan ƙwayoyin tushe, halayya, da cututtuka. Sinadari na iya ƙara na farko ba tare da sauya na biyu ba, kuma yawancinsu haka suke.
Fibroblast na progeria suna ɗauke da wani sanannen sauyin LMNA wanda yake samar da progerin, tare da mitochondria da suka kakkarye kuma ba sa motsi sosai, ƙarancin ATP, tsakiya masu karkatacciyar siffa, da asarar heterochromatin. Methylene blue a matakin nanomolar ya inganta ma'aunan mitochondria kuma ya gyara ma'aunan tsakiya sosai, ciki har da sakin progerin daga membrane. Wannan gyara ne mai ban mamaki a matakin ƙwayar halitta. Har yanzu dai sakamakon fibroblast ne, kuma marubutan sun sanya gwajin dabbobi a matsayin aikin nan gaba. An ƙara tattauna samfurin cutar kansa a methylene blue a progeria da samfuran tsufa kafin lokaci.
Daga wannan, ga jerin abubuwan dubawa domin karatu mai amfani.
- Tambayi ko an fara amfani da sinadarin kafin tsayawar rarrabuwa ko bayan an tabbatar da ita. Fara kafin tsayawa yana goyon bayan jinkirtawa. Don tabbatar da cirewa, ana buƙatar amfani da shi bayan tsayawa tare da bayanan mutuwa ta hanyar zaɓe.
- Tambayi abin da jadawalin ninkewar ƙwayoyin kwatanci ya nuna. Ba tare da jimillar ninkewa ba, sauyin alamomi ba shi da mahallin da za a danganta shi da shi.
- Nemi aƙalla rukunin alamomi guda biyu, kamar alamar yawaita tare da p16 ko p21 da SA-beta-gal, maimakon rini guda ɗaya kawai.
- Tambayi yadda aka ruwaito telomeres. Ƙimar sauyi a kowace ninkewa a tsawon lokaci tana da ma'ana. Ma'aunin tsawo a lokaci guda kaɗai ba ya da wannan ma'anar.
- Tambayi abin da aka riƙe bai sauya ba. Cunkoson ƙwayoyin halitta lokacin mayarwa zuwa sabuwar tasa, oxygen, sauya sinadaran reno, da sabbin ko tsoffin ruwan methylene blue da aka tanada duk suna sauya tsayawar rarrabuwar fibroblast.
Idan aka karanta su ta wannan hanya, rubuce-rubucen binciken fibroblast suna ba methylene blue wani matsayi mai ma'ana amma mai iyaka: sinadari da ake amfani da shi a ƙaramin adadi kuma yake aiki a redox, wanda yake jinkirta tsufa sakamakon rarrabuwa a fibroblast na mutum da ake renawa tare da inganta ma'aunan mitochondria. Mafi ƙarfafa bayanin tsarin aikinsa yana kan saƙonnin makamashi na ɗan lokaci da samuwar complex IV, kuma waɗannan takardun ba su nuna cire ƙwayoyin halittar da suka tsufa ko ƙara tsawon rayuwar ɗan Adam ba.