Indlela i-methylene blue engena ngayo kwiiseli: ukuncitshiswa, ukuphinda ifakwe kwi-oxidation, nokwahlulelana nenwebu

Xa ubukele iiseli ze-endothelium ezikwisitya zifunxa i-methylene blue, eyona ngcaciso ibonakala icacile ikwayileyo ingachanekanga. Ulwelo ezikhuliswa kulo lulahlekelwa ngumbala walo oluhlaza okwesibhakabhaka, iiseli zifumane amasuntswana aluhlaza okwesibhakabhaka, ngoko kubonakala ngathi idayi idlule kwinwebu ikwimo yemolekyuli eluhlaza okwesibhakabhaka. Imifuniselo ibonisa okwahlukileyo. Imo eluhlaza okwesibhakabhaka iphantse ingaweli kwaphela. Into ewelayo yimo engenambala, yaye umbala oluhlaza okwesibhakabhaka owubonayo ngaphakathi wenziwa ngaphakathi, emva kokufika.

Oku kuguquka kwendlela izinto ezibonakala ngayo kubalulekile kuba kugqiba ngoko ukudayeka kunokukuxelela nangoko kungenakukuxelela. Amasuntswana aluhlaza okwesibhakabhaka abonisa idayi ekwi-oxidation, ethe yaphinda yafakwa kwi-oxidation yaza yavaleleka kwii-organelle. Awabonisi yonke idayi ekwiseli, awabonisi ubungakanani bedayi ekwimo encitshisiweyo, yaye awalinganisi isiphumo sebhayoloji. Ukuze ubone isizathu, landela amanyathelo amathathu: ukuncitshiswa kumphezulu, ukusasazeka kwemo encitshisiweyo, nokuphinda ifakwe kwi-oxidation ize ivaleleke ngaphakathi.

Ezi mo zimbini ziziphatha njengeemolekyuli ezahlukeneyo

I-methylene blue ekwi-oxidation yinkqubo yamakhonkco athe tyaba enentlawulo yombane eyiphozithivu. Iyanyibilika emanzini, ifunxa ukukhanya ngamandla malunga ne-600 ukuya kwi-700 nanometers, yaye ibonakala iluhlaza okwesibhakabhaka. Eyona nto ibalulekileyo yintlawulo yombane. Imolekyuli enentlawulo yombane nenobudlelwane namanzi ayityibiliki lula kwinwebu yee-lipid, ngoko ibinzana elichanileyo elisetyenziswa kuncwadi lwenzululwazi lithi kunzima ngokuthelekisa ukuba igqithe kwinwebu, kungekhona ukuba ayinakugqitha kwaphela.

I-methylene blue encitshisiweyo, ebizwa ngokuba yi-leucomethylene blue, ayinambala, ayinantlawulo yombane, yaye iyanyibilika emafutheni. Xa intlawulo yombane isusiwe, inwebu iyayeka ukuba ludonga. Imolekyuli encitshisiweyo isasazeka lula idlule kuyo, ize yakuba ingaphakathi kwindawo ethanda ngakumbi i-oxidation ilahlekelwe zii-elektroni kwakhona, ibuyele kwimo eluhlaza okwesibhakabhaka, enentlawulo yombane nevalelekileyo. Ngoko ikhemistri ye-redox yale dayi ikwayindlela ethuthwa ngayo. Xa utshintsha inani lee-elektroni, utshintsha nokukwazi kwayo ukugqitha kwinwebu.

Ii-elektroni zenyathelo lokuqala zivela kwiseli ngokwayo. Iiseli zezilwanyana ezanyisayo zithutha ii-elektroni ngaphaya kwenwebu yeplasma, zihambisa amandla okunciphisa avela kubanikeli abangaphakathi kwiseli ziwase kubamkeli abakumphezulu. Lo msebenzi wokudlula kwinwebu yeplasma ngumsebenzi oqhelekileyo weseli, ofumaneka kwiintlobo ezininzi zeeseli. I-enzyme ye-reductase ekumphezulu we-endothelium esebenza ngeedayi ze-thiazine iziphatha njengezinye kwinkalo enye ebalulekileyo: iyaqhubeka nokusebenza xa kukho i-cyanide ne-azide ngemilinganiselo ethintela amanyathelo alandelayo. Lo mahluko ngundoqo wayo yonke le ngcaciso.

Ukwahlulelana kwezigaba kwafundwa kuqala, kwaphantse kwachaza ukufunxwa

Ngo-1929, uMarian Irwin kwiRockefeller Institute wabuza umbuzo olula ngakumbi. Mhlawumbi ukungena kwedayi kukwahlulelana nje phakathi kwamanzi neengqimba ezinamafutha zeseli, yaye ungqimba lwe-chloroform lunokumela ezo ngqimba zinamafutha kwilinge laselabhoratri. Izifundo zakhe zokungena ezisebenzisa i-spectrophotometry zashukumisa izinyibiliko zedayi ezisekelwe emanzini kunye ne-chloroform, zaza zathelekisa oko kwangena kwisigaba se-organic noko kwangena kwiiseli eziphilayo ze-algae.

Izinto ezimbini ezafunyanwa kulo msebenzi zisafanele ukuqwalaselwa. Okokuqala, izinyibiliko ze-methylene blue ezithengiswayo zaziqulethe i-trimethyl thionine, ngoku ebizwa ngokuba yi-azure B, yaye obu bumdaka bangena ngokukhawuleza kune-methylene blue ngokwayo, ingakumbi kwi-pH ene-alkali apho eyona mo ininzi iyi-free base enyibilikayo kwii-lipid. Okunye okwakubonakala kukungena kwe-methylene blue eneneni yayikukungena ngokukhethwayo kwe-azure B. Okwesibini, i-pH yayigqiba isiphumo ngokumisela imo ye-ionization, yaye iiseli ezonzakeleyo zazingenisa idayi eninzi kakhulu kuneeseli ezingonakalanga. Ii-coefficient zokwahlulelana kwezigaba nemeko yentlawulo yombane zazilawula oko kufunxwa yinkqubo eyenziweyo naloko kufunxwa yiseli ephilayo, yaye ukufana kwezi zimbini kwaxhasa ingcamango yokuba kukho iingqimba ezingasekelwanga emanzini kumda weseli.

Lo mzekelo wawungaphambi kwexesha lawo ekuqondeni ii-lipid ne-ionization, kodwa wawungenakwahlula phakathi kwedayi eyahlulelana phakathi kwezigaba ingatshintshanga nedayi eguqulwa ngokwekhemistri kumphezulu, iwele ikwenye imo, ize iguqulwe ibuyele kweyokuqala ngaphakathi. Ukwenza loo mahluko kwakufuna omnye umfuniselo, owafika kwiminyaka engamashumi asixhenxe kamva kwi-endothelium yemiphunga.

Ukuncitshiswa ngaphandle, ukuvaleleka ngaphakathi

UMerker noogxa bakhe basebenza ngeeseli ze-endothelium zomthambo wemiphunga yenkomo ezikhuliswe elabhoratri, baza bacebisa ulandelelwano lwamanyathelo amathathu: idayi ekwi-oxidation incitshiswa kumphezulu weseli ibe yimo yayo engenambala nethanda amafutha, imo encitshisiweyo isasazeka ingene ngaphakathi, kuze ukuphinda ifakwe kwi-oxidation ngaphakathi kwiseli kubuyisele imo ekwi-oxidation engakwaziyo ukugqitha kwinwebu, ethi iqokelelane. Uphononongo lwabo lokufunxwa kwe-thiazine yi-endothelium lwavavanya olo landelelwano ngokuzama ukuluphazamisa kwiindawo ezithile.

Isixhobo sokuqala yayiyi-ferricyanide, umamkeli wee-elektroni ongakwaziyo ukungena kwiseli. Xa ii-enzyme ezikumphezulu zinciphisa idayi eluhlaza okwesibhakabhaka ibe yimo engenambala ngaphandle kweseli, i-ferricyanide inokuthatha ezo elektroni, ibuyisele idayi eluhlaza okwesibhakabhaka kulwelo ngaphambi kokuba iwele. Logama i-ferricyanide yayikho ngobuninzi obugqithisileyo (10 micromolar yedayi xa kuthelekiswa ne-50 ukuya kwi-500 micromolar ye-ferricyanide), ukufunxwa kwedayi kwaphantse kwema, kwaza kwaqala kwakhona ngesantya sako esiqhelekileyo kuphela emva kokuba i-ferricyanide iphelile. Izantya zotshintsho zabalisa ibali elifanayo ngenye indlela. I-ferricyanide yanyamalala ngesantya esiphantse singaguquki, nto leyo ehambelana nokuqhubeka kokuncitshiswa kumphezulu okondla umthetheli onokuphinda asetyenziswe, ngoxa idayi engaphandle kweseli yanyamalala ngesantya esilandela igophe le-exponential njengoko ubungakanani bayo babuncipha. Ukuhamba okungaguqukiyo kunye nobungakanani obunciphayo kuthetha ukuba umqondiso we-ferricyanide ubonisa ukuncitshiswa okuqhubekayo, kungekhona iimolekyuli zedayi eziphuma kwisinyibiliko.

Isixhobo sesibini sasusa ngokupheleleyo inyathelo lokuwela. Iqela lancamathelisa i-toluidine blue O, isalamane esisondeleyo se-thiazine, kwikhonkco elikhulu le-polyacrylamide elalilikhulu kakhulu ukuba lingangena kwiseli kuyo nayiphi na imo ye-oxidation. Iiseli zaqhubeka nokunciphisa loo dayi ibotshelelweyo. Ngoko ukuncitshiswa akufuni kungena ngaphakathi kwiseli. Oomatshini abenza oku bafikeleleka kumphezulu okanye badityaniswe nawo ngombane.

Kwalandela ukwahlulwa kwamanyathelo okwenza eli phepha lifumane isihloko salo. I-cyanide ne-azide kwi-2 millimolar zathintela kakhulu ukuqokelelana kwedayi, yaye phantsi kwemikroskopu iziqulatho eziluhlaza okwesibhakabhaka nzulu nezimfusa ezidla ngokurhangqa i-nucleus zaphantse zanyamalala. Kodwa akukho nasinye kwezi zithinteli esacothisa ukuncitshiswa ngaphandle kweseli: umjikelo we-ferricyanide waqhubeka unganciphanga, yaye i-polymer engangeniyo yayisancitshiswa. Isithintelo sasikwinqanaba elilandelayo. I-cyanide ne-azide zazimisa ukuphinda kufakwe idayi kwi-oxidation nokuvalelwa kwayo ngaphakathi kwiseli, kungekhona ukuncitshiswa kumphezulu. Uvavanyo lokulawula lokuqinisekisa ukuba iiseli zisaphila lwayixhasa le ngcaciso, kuba iiseli zazigcine i-enzyme yazo esisiphawuli se-cytosol, i-lactate dehydrogenase, kulo lonke olu nyango. Ngoko ukuncipha kokufunxwa kwakungeyonto nje yokutyhefeka okanye yokugqabhuka kwenwebu.

Esinye isilumkiso esikweli phepha sisasebenza. Iziqulatho eziluhlaza okwesibhakabhaka zazijikeleze i-nucleus kodwa zingekho ngaphakathi kuyo, nto leyo ehambelana ne-oxidation ngaphakathi kwii-organelle zeseli, kodwa uphononongo aluzange luchonge ukuba zeziphi ezo organelle. Iipateni ezifanayo kwezinye iintlobo zeeseli zazibizwa ngokuba zezee-lysosome nangona ubungqina babuncinci. Ukuvaleleka makuthathwe njengento eqinisekisiweyo, kodwa indawo okwenzeka kuyo mayithathwe njengeqikelelo elingekaqinisekiswa. Abafundi abalandela apho ii-elektroni ziya khona emva koku bangaqhubeka bafunde ngokuthuthwa kwee-elektroni kwi-mitochondria nokulawulwa kwe-redox nangombono obanzi wokufunxwa nokusasazwa ngokwendlela yokungena.

Ukudayeka akubonisi umlinganiselo ofike kwizicubu

Uphononongo lwesithathu lubonisa okwenzekayo xa le ndlela yokufunxwa idibana nomlinganiselo womsebenzi webhayoloji. UScott noogxa bakhe bafumanisa ukuba i-methyl beta cyclodextrin, iswekile emile okwesangqa, ibophelela ngqo kwi-methylene blue, inomlinganiselo ongaguqukiyo wokudibana omalunga ne-310 per molar wesakhiwo esenziwa kukuvalelwa kwedayi ngaphakathi kwayo, xa kuthelekiswa nomalunga ne-5846 per molar wokudibana kweemolekyuli ezimbini zedayi. Kwiiseli ze-fibroblast ze-L929, oku kubophelela kwathintela ukudayeka kweeseli kunye nokukhuthazwa kokufunxwa kweglucose: ukugcina iiseli kunye ne-cyclodextrin ngaphambi kokongeza idayi kwathintela ukudayeka okuluhlaza okwesibhakabhaka, yaye ukuyongeza emva koko kwakhawulezisa ukuphela kokudayeka. Njengoko umqondiso wokukhanya oluhlaza okwesibhakabhaka wawushiya iiseli, ukuthuthwa kweglucose okwakuvuselelwe nako kwancipha ngokuhambelana okusondeleyo.

Ababhali bawutolika lo mlinganiselwano njengoxhasa ukudibana ngqo phakathi kwedayi neprotheyini ekwindlela yokuvuselela umthuthi weglucose, kunendlela yoxinzelelo lwe-oxidation ehlala ixesha elide. Eso sigqibo sisengqiqweni kodwa sinemida. Umfuniselo awuyichongi iprotheyini, awubonisi ukubophelela kumthuthi ngokwakhe, yaye ukuhambelana kokuphela kokudayeka nokulahleka kwesiphumo akunakuzikhupha zonke iindlela ezilawulwa yi-redox.

Isifundo esihlala sibalulekile simalunga nokulinganisa. Ukudayeka okubonwa ngokukhanya kubonisa idayi ebonakalayo, ekwi-oxidation nenxulumene neseli. Akubonisi kwaphela idayi ekwimo encitshisiweyo engenambala, ngoko iseli engadayekanga kakhulu isenokuba isenayo idayi, yaye iseli edayeke nzulu ibonisa idayi evalelekileyo ekwi-oxidation kunokuba ibonise umlinganiselo uwonke ofike kwizicubu okanye ubungakanani besiphumo. Esi silumkiso sisebenza nakwimilinganiselo emikhulu: ukuthelekisa ukudayeka phakathi kweentlobo zeeseli, okanye ukuthatha isiqwenga sesicubu esidayeke nzulu njengobungqina bokuba sifumene idayi eninzi, kuphinda impazamo yokuqala yesitya. Ukufunxwa ngumjikelo wokuncitshiswa, ukusasazeka nokuphinda kufakwe kwi-oxidation, yaye nawuphi na umfanekiso wemeko yemo eluhlaza okwesibhakabhaka ngexesha elinye ubona inxalenye encinci kuphela yaloo mjikelo. Iziphumo zoko ekudayeni nasekuphatheni idayi elabhoratri nasekutolikeni amabango ngedosi nangomlinganiselo ofike kwizicubu zilandela ngokuthe ngqo.