Uko ubururu bwa metilene bwinjira mu turemangingo: kwakira elegitoroni, kongera okisidasiyo no kwigabanya mu gahu

Iyo witegereje ubururu bwa metilene bwinjira mu turemangingo twa endoteliyumu turi mu gasahani, igisobanuro gihita kiza mu mutwe ni na cyo kitari cyo. Amazi turemeramo atakaza ibara ry'ubururu, uturemangingo tukagira utubumbe tw'ubururu, bityo umuntu akibwira ko irangi ryambutse agahu rikiri molekile y'ubururu. Ubushakashatsi bwerekana ibinyuranye n'ibyo. Uburyo bw'ubururu ntibwambuka hafi na gato. Ubwambuka ni uburyo butagira ibara, naho ubururu ubona imbere bukorerwa imbere, nyuma yo kwinjira.

Uku guhinduka kw'ibyari byitezwe ni ingenzi kuko kugena icyo gusiga ibara bishobora kukubwira n'icyo bidashobora kukubwira. Utubumbe tw'ubururu tugaragaza irangi ryanyuze muri okisidasiyo, ryongeye kuyinyuramo maze rigafatirwa mu duce twihariye two mu karemangingo. Ntitugaragaza irangi ryose riri mu karemangingo, ntitugaragaza igice cyakiriye elegitoroni, kandi ntitupima ingaruka ku mikorere y'ibinyabuzima. Kugira ngo wumve impamvu, kurikira intambwe eshatu: kwakira elegitoroni ku buso, gukwirakwira k'uburyo bwakiriye elegitoroni, no kongera okisidasiyo no gufatirwa imbere.

Uburyo bubiri bwitwara nka molekile zitandukanye

Ubururu bwa metilene bwanyuze muri okisidasiyo ni urusobe rw'impeta rutambitse kandi rufite chaji ya pozitif. Bushonga mu mazi, bukanyunyuza cyane urumuri ruri hafi ya 600 kugeza kuri 700 nanometers, kandi bukagaragara nk'ubururu. Chaji ni yo y'ingenzi hano. Molekile ifite chaji kandi ikunda amazi ntiyambuka byoroshye agahu kagizwe na lipide; ni yo mpamvu inyandiko z'ubushakashatsi zikoresha imvugo yitondewe ivuga ko itambuka agahu ku rugero ruto, aho kuvuga ko itakambuka na gato.

Ubururu bwa metilene bwakiriye elegitoroni, bwitwa leucomethylene blue, ntibugira ibara, ntibugira chaji, kandi bushonga mu binure. Iyo chaji ikuweho, agahu ntigakomeza kuba urukuta. Molekile yakiriye elegitoroni yambuka byoroshye binyuze mu gukwirakwira; iyo igeze imbere mu gice gifite ubushobozi bwinshi bwo gutera okisidasiyo, ishobora kongera gutakaza elegitoroni maze igasubira mu buryo bw'ubururu, bufite chaji kandi bufatirwa imbere. Bityo ubutabire bwo kwakira no gutakaza elegitoroni bw'irangi ni na bwo buryo ritwarwamo. Iyo uhinduye umubare wa elegitoroni, uhindura n'ubushobozi bwo kwambuka agahu.

Elegitoroni zikoreshwa mu ntambwe ya mbere zituruka mu karemangingo ubwako. Uturemangingo tw'inyamabere dutwara elegitoroni zambuka agahu k'akaremangingo, tukavana ubushobozi bwo gutanga elegitoroni ku bizitanga biri imbere tukabwerekeza ku bizakira biri ku buso. Uyu murimo ukorerwa hakurya no hakuno y'agahu ni imikorere isanzwe y'akaremangingo, iboneka mu moko menshi y'uturemangingo. Enzime ya reductase yo ku buso bwa endoteliyumu ikora ku marangi ya thiazine yitwara nk'izindi mu buryo bumwe bw'ingenzi: ikomeza gukora n'iyo hari cyanide na azide mu ngano zihagarika intambwe zikurikiraho. Iri tandukaniro ni ryo shingiro ry'ibi bisobanuro byose.

Kwigabanya hagati y'ibice byabanje, kandi byari hafi gusobanura uko irangi ryinjira

Mu 1929, Marian Irwin wo muri Rockefeller Institute yabajije ikibazo cyoroshye kurushaho. Yibajije niba kwinjira kw'irangi kutaba gusa kwigabanya hagati y'amazi n'ibice birimo ibinure by'akaremangingo, kandi niba igice cya chloroform kitashobora gusimbura ibyo bice birimo ibinure mu igerageza rya laboratwari. Mu bushakashatsi bwe bwo kwinjira kw'irangi hakoreshejwe gupima iyinjizwa ry'urumuri, yakunkumuriye ibisubizo by'amazi birimo irangi hamwe na chloroform, agereranya ibyimukiye mu gice cy'umusemburo udakomoka ku mazi n'ibyinjiraga mu turemangingo tuzima twa algue.

Ibintu bibiri byabonetse muri ubwo bushakashatsi biracyakwiriye kwitabwaho. Icya mbere, ibisubizo by'ubururu bwa metilene byacuruzwaga byarimo trimethyl thionine, ubu yitwa azure B, kandi icyo kinyabutabire kitari kigamijwe cyinjiraga vuba kurusha ubururu bwa metilene ubwabwo, cyane cyane kuri pH ya alkalini, aho uburyo bwa baze idafite chaji kandi ishonga muri lipide ari bwo bwiganje. Bimwe mu byagaragaraga nko kwinjira kw'ubururu bwa metilene mu by'ukuri byari kwinjira kwihariye kwa azure B. Icya kabiri, pH yagenaga ibyavaga mu igerageza kuko yagenaga imiterere ya ionizasiya, kandi uturemangingo twangiritse twinjirwagamo n'irangi ryinshi cyane kurusha utwangiritse. Ibipimo byo kwigabanya hagati y'ibice n'imiterere ya chaji byagengaga ibyo uburyo bwakozwe muri laboratwari n'akaremangingo kazima byinjizaga. Kuba byaragaragazaga imyitwarire isa byashyigikiraga igitekerezo cy'uko ku mbibi z'akaremangingo hari ibice bitari iby'amazi.

Ubu buryo bwo gusobanura ibintu bwari bwarabonye hakiri kare uruhare rwa lipide na ionizasiya, ariko ntibwashoboraga gutandukanya irangi ryigabanya hagati y'ibice rikiri uko ryari rimeze n'irangi rihindurwa mu buryo bwa shimi ku buso, rikambuka mu bundi buryo, hanyuma rigasubizwa uko ryari rimeze imbere. Kugira ngo iryo tandukaniro rigaragare, hari hakenewe irindi gerageza; ryaje nyuma y'imyaka mirongo irindwi, rikorerwa kuri endoteliyumu y'ibihaha.

Kwakira elegitoroni hanze, gufatirwa imbere

Merker na bagenzi be bakoresheje uturemangingo twa endoteliyumu y'imiyoboro y'amaraso iva mu mutima ijya mu bihaha by'inka, twakujijwe muri laboratwari. Batanze igitekerezo cy'intambwe eshatu zikurikirana: irangi ryanyuze muri okisidasiyo ryakira elegitoroni ku buso bw'akaremangingo rigahinduka uburyo butagira ibara kandi bukunda ibinure; ubwo buryo bwakiriye elegitoroni bukinjira binyuze mu gukwirakwira; hanyuma kongera okisidasiyo imbere mu karemangingo bigasubizaho uburyo bwanyuze muri okisidasiyo butambuka agahu, maze bukirundanya. Ubushakashatsi bwabo ku kwinjira kwa thiazine muri endoteliyumu bwagerageje uru ruhererekane bushaka kuruhagarika ku ntambwe zihariye.

Igikoresho cya mbere cyari ferricyanide, ikinyabutabire cyakira elegitoroni ariko kidashobora kwinjira mu karemangingo. Iyo enzime zo ku buso zituma irangi ry'ubururu ryakira elegitoroni rigahinduka uburyo butagira ibara hanze y'akaremangingo, ferricyanide ishobora kuryambura izo elegitoroni, igasubizaho irangi ry'ubururu mu mazi turemeramo mbere y'uko ryambuka. Igihe cyose ferricyanide yabaga irenze ikenewe (irangi rya 10 micromolar ugereranyije na ferricyanide ya 50 kugeza kuri 500 micromolar), kwinjira kw'irangi kwarahagararaga hafi burundu. Kwinjira kwasubukuraga ku muvuduko usanzwe ari uko ferricyanide imaze gukoreshwa yose. Imigendekere y'umuvuduko w'ibi bikorwa yatanze ibisobanuro bimwe mu bundi buryo. Ferricyanide yagabanukaga ku muvuduko hafi udahinduka, bihuye no kwakira elegitoroni ku buso ku rugero rudahinduka, bigakomeza guha elegitoroni umuhuza ushobora kongera gukoreshwa. Naho irangi ryo hanze y'uturemangingo ryagabanukaga mu buryo bwa eksiponansiyeli uko ingano yaryo yagendaga ishira. Urujya n'uruza rudahinduka hamwe n'ingano igenda ishira bivuze ko ikimenyetso cya ferricyanide kigaragaza kwakira elegitoroni bikomeje, aho kugaragaza molekile z'irangi ziva mu gisubizo.

Igikoresho cya kabiri cyakuyeho burundu intambwe yo kwambuka. Itsinda ryahambiriye toluidine blue O, ikinyabutabire cya thiazine gifitanye isano ya hafi n'ubururu bwa metilene, ku munyururu munini wa polyacrylamide utashoboraga kwinjira mu karemangingo uko imiterere ya okisidasiyo yaba iri kose. Uturemangingo twakomeje guha elegitoroni irangi ryari rihambiriye kuri uwo munyururu. Bityo, kwakira elegitoroni ntibisaba ko irangi ribanza kwinjira mu karemangingo. Uburyo bubikora buri ku buso cyangwa buhujwe na bwo mu buryo bw'amashanyarazi.

Hakurikiyeho itandukaniro riha inyandiko y'ubushakashatsi umutwe wayo. Cyanide na azide kuri 2 millimolar byagabanyije cyane kwirundanya kw'irangi. Kandi munsi ya mikorosikopi, utubumbe tw'ubururu bwijimye n'ubw'umwura dusanzwe dukikiza intima twarazimye hafi burundu. Nyamara nta na kimwe muri ibyo bibuza cyagabanyije umuvuduko wo kwakira elegitoroni hanze y'uturemangingo: uruzenguruko rwa ferricyanide rwakomeje nta kugabanuka, kandi polimeri itambuka agahu yakomeje kwakira elegitoroni. Ihagarikwa ryari ku ntambwe zo nyuma. Cyanide na azide byahagarikaga kongera okisidasiyo no gufatirwa imbere mu karemangingo, aho guhagarika kwakira elegitoroni ku buso. Ibizamini byo kugenzura niba uturemangingo twari tukiri tuzima byashyigikiye ibyo bisobanuro: muri ibi bikorwa, uturemangingo twagumanye lactate dehydrogenase, enzime ikoreshwa nk'ikimenyetso cy'ibiri mu gice cy'amazi cy'imbere mu karemangingo. Bityo, kubura kwinjira kw'irangi ntikwari gusa ingaruka z'uburozi cyangwa guturika kw'agahu.

Hari ingingo imwe yo kwitondera muri iyo nyandiko igikwiye kubahirizwa. Utubumbe tw'ubururu twari dukikije intima ariko tutari muri yo, bihuye na okisidasiyo ibera mu duce twihariye two imbere mu karemangingo, ariko ubushakashatsi ntibwagaragaje utwo duce utwo ari two. Imiterere isa n'iyo mu yandi moko y'uturemangingo yari yariswe iyo muri lizosome, hashingiwe ku bimenyetso bidakomeye. Gufatirwa imbere gufatwe nk'ikimaze kugaragazwa, ariko aho nyaho bibera hafatwe nk'ahatarahamya. Abasomyi bakurikirana aho elegitoroni zijya nyuma bashobora gukomeza ku itwarwa rya elegitoroni muri mitokondiriya n'imicungire yo kwakira no gutakaza elegitoroni no ku bisobanuro byagutse by'iyinjizwa n'ikwirakwizwa hakurikijwe inzira byanyuzemo.

Gusiga ibara si igipimo cy'ingano yageze mu ngirangingo

Ubushakashatsi bwa gatatu bwerekana uko bigenda iyo ubu buryo bwo kwinjira buhujwe n'igipimo cy'imikorere y'ibinyabuzima. Scott na bagenzi be basanze methyl beta cyclodextrin, isukari ifite ishusho y'impeta, yifatanya n'ubururu bwa metilene mu buryo butaziguye. Igipimo kidahinduka cyo kwifatanya cyari hafi 310 per molar ku rusobe rurimo irangi imbere, ugereranyije na hafi 5846 per molar ku kwifatanya kwa molekile ebyiri z'irangi. Mu turemangingo twa fibroblaste twa L929, uku kwifatanya kwahagaritse gusiga uturemangingo ibara no gushishikariza iyinjizwa rya gilikose: kubanza kubika irangi hamwe na cyclodextrin mbere yo kurikoresha byabujije ibara ry'ubururu kugaragara, naho kuyongeramo nyuma byihutishije kuvaho kw'ibara. Uko ikimenyetso cy'ubururu kigaragara mu rumuri cyavaga mu turemangingo, ni ko n'itwarwa rya gilikose ryari ryongerewe ryagabanukaga ku buryo bujyanye cyane na cyo.

Abanditsi babonye ko iri sano rishyigikira kurushaho igitekerezo cy'imikoranire itaziguye hagati y'irangi na poroteyine iri mu nzira ituma umutwazi wa gilikose ukora, aho gushyigikira uburyo bw'imikorere buterwa n'ihungabana rya okisidasiyo rimara igihe kirekire. Uwo mwanzuro urumvikana, ariko ufite imbibi. Igerageza ntirigaragaza poroteyine iyo ari yo, ntiryerekana ko irangi ryifatanya n'umutwazi ubwawo, kandi isano hagati yo kuvaho kw'ibara no kubura kw'ingaruka ntishobora guhakana inzira zose zinyura mu kwakira no gutakaza elegitoroni.

Isomo riramba rirebana no gupima. Ibara ripimwa hakoreshejwe urumuri rigaragaza irangi riboneka, ryanyuze muri okisidasiyo kandi rifitanye isano n'akaremangingo. Ntiryerekana na gato igice kitagira ibara cyakiriye elegitoroni. Bityo, akaremangingo gafite ibara ryoroshye gashobora kuba kagifite ingano ihwanye n'irangi, naho akaremangingo gafite ibara rikomeye kakagaragaza irangi ryanyuze muri okisidasiyo ryafatiwe imbere, aho kugaragaza ingano yose yageze mu ngirangingo cyangwa ubunini bw'ingaruka. Uko kwitondera ni ngombwa no ku rwego rwagutse: kugereranya ibara mu moko atandukanye y'uturemangingo, cyangwa gufata igice cy'ingirangingo gifite ibara ryijimye nk'ikimenyetso cy'uko cyagezweho n'ingano nini kurushaho, bisubiramo ikosa rya mbere ryo mu gasahani. Kwinjira ni uruzenguruko rwo kwakira elegitoroni, gukwirakwira no kongera okisidasiyo; ishusho iyo ari yo yose ifashwe rimwe y'uburyo bw'ubururu ibona igice kimwe gusa cyarwo. Ingaruka zifatika ku gusiga ibara no gukoresha irangi muri laboratwari no ku gusobanura ibivugwa ku ngano ikoreshwa n'ingano igera mu ngirangingo zikomoka kuri ibyo mu buryo butaziguye.