Q: Kodi ndiyenera kusankha motani ma microsomes ndi hepatocytes ya kagayidwe kafukufuku? Kodi ndingasankhe imodzi yokha mwa iwo kuti ikwaniritse zofunika?
A: Mu kafukufuku wokhazikika, kusankha kwa ma microsomes a chiwindi kapena hepatocytes makamaka kumatengera kagayidwe kazinthu, komwe kumakhala ndi kuchuluka kwa kagayidwe ka kagayidwe kachakudya. Mwambiri, ma microsis a chiwindi amakondedwa, makamaka pamene mamolekyu ophatikizika ndi madzi ambiri - kusungunuka ndi metabolism ndiye njira yayikulu ya kagayidwe ka metabolic (makamaka pa cyp). Hepatocytes akhoza kugwiritsidwa ntchito poyesa pakakhala umboni wa awiri - Msonkhano wa metabolism monga njira yayikulu ya ma mitima ya chiwindi, ndipo kagayidwe kambiri ka chiwindi sikuwonekera. Nthawi zambiri, dongosolo limodzi la kagayidwe limatha kusankhidwa kuti likwaniritse zosowa; Ngati mikhalidwe mu mbali zonse zimalola, ndibwino kusankha madeji nthawi yomweyo.
Q: Chifukwa chiyani kuli kofunikira kugwiritsa ntchito hepatocytes yoyamba ipatocytes ya enzyme itay?
Yankho: Cyp enzyme - Chiwonetsero cha Assicy chikuyenera kuchoka ku "kulembedwa" kutanthauza "post - kusinthasintha kwa enp enzyme protein. Chifukwa chake, makina oyeserera azikhala maselo a chiwindi, osati ma microsomes kapena chiwindi S9.
Q: Chifukwa chiyani ndikufunika kusankha itatu ya IPONCECYSH CIPACCYTE cha Enzyme Itay?
Yankho: Malinga ndi kuyambitsa kwa malangizo omwe mwa kugwiritsa ntchito mankhwala osokoneza bongo, opereka atatu ayenera kugwiritsidwa ntchito, ndipo zotsatira za wopereka aliyense ziyenera kuyesedwa payokha. Kuphatikiza pa kupanga ziwerengero zazikulu, kusankha kwa opereka atatu kuti ayesedwenso ndikofunikira kwambiri pakuwunika maere - kusinthana payekha. Ngati zotsatira zake kuchokera pa wopereka imodzi zimaposa chisanachitike, wosankhidwa mankhwalawa akhoza kukhala osayenera ndikutsatira - kuwunika kumafunikira.
Q: Chifukwa chiyani kuyang'ana pa ep enzyme inzyment ndipo osati pa enzyme ija?
Yankho: Chifukwa chongoganizira za cypase ku cypase kukhala kuti makina a cypasetion aphunziridwa bwino. Mwanjira ina, chifukwa chosafuna kuti phunziroli lizikhala ndi chiwonetsero cha Ugtase ndichakuti makinawa samvekabe; Komabe, izi sizitanthauza kuti ugtase siyikukakamizidwa. Maupangiriwo ananenanso kuti palibe njira yokhazikika yothandizira kapena zotupa za zoyendera ndi gawo la meigabolism enzyme.
Q: Kodi otsatira oyamba a hepatocytes angapulumutsidwe mpaka liti?
A: Parteurtive the hepatocytes nthawi zambiri amagwiritsidwa ntchito kwa enzyme ikuluikulu. Pambuyo pakubwezeretsa ma cell, maselo oyimitsidwa mu mbale akufalikira, kusinthidwa kukhala malo oyenera, komanso otukuka mu collagen - Mimba yophimba; Kenako maselo amatha kukhala osakwana maola 4 ~ 6. Maselo amaphatikizidwa ndi khoma, sing'anga yokonzayo yasinthidwa ndikusungidwa kwa 18 h kuwonetsetsa kubwezeretsa kwa maselo. Chotsatira, enzyme ikuluikulu imatha kuchitidwa kuti azindikire zochita za kagayidwe ndi mrna. Kuchokera pakuwona konsekonse, hepatocytes amatha kusungidwa patatha masiku 6 ~ 7 pambuyo potsatira khoma. Pakapita nthawi, khoma la cell lamess limawonongeka zimawonongeka, ndipo maselowo amasakaniza ndi kuyimitsa.
Ngati Photocyte wotsatira sakhala wotukuka, tatsimikizira kuti akhoza kukhalabe m'magulu abwino pasanathe maola 4 ~ 6. Sitinachitebe chitsimikizo kwa nthawi yayitali.
Q: Hepatocytes akhoza kugwiritsidwa ntchito ngati ma cell olumikizidwa ndi ma cell otengera chikhalidwe zosiyanasiyana
Yankho ndi ma cell okhazikika minofu ndi ziwalo zokhazikika zimayendetsedwa, koma si maselo onse omwe amayendetsedwa atazunguliridwa mu - Vitro. Kaya maselo ndi khoma - chomatira kapena ayi chimatengera mkhalidwe wamaselo; Nthawi yomweyo, Khoma - Maselo ogwirira ntchito amafunikira mawonekedwe apadera ndi ena apadera pro - Ma cell a cell fortiogenu, zinthu za seramogeninogen) zomwe zingatenge nawo gawo pakugwirira cell. Pomaliza, ma cell oweluza amagwiritsidwa ntchito ngati maselo oyimitsidwa, koma maselo oyimitsidwa siofunikira kugwirira ntchito.
Q: Ndi phindu liti / zovuta zomwe zimagwiritsidwa ntchito pogwiritsa ntchito ma vesi olumikizidwa a hepacyte? Kodi ndi chiyani chomwe chimasankhidwa ndi chiyani?
A: Pambuyo pa hepatocytes ndiotalikirana, zimatha kuyerekezera pakuyimitsidwa kapena khoma lotsatira. Ntchito ya cytochrome p450 enzyme yochokera ku chilumba cha hepatocyte ndizogwirizana ndi - Vivo wa 4 ~ 6h, kenako amachepetsa mwachangu ndi kutalika kwa nthawi. Chifukwa chake, hepatocytes imagwiritsidwa ntchito pophunzira kagayidwe ka metabolic kapena kafukufuku wa metabolite. Hepatocytes wamkulu wolumikizidwa khoma ili ndi nthawi yokwanira kuti muchepetse kuwonongeka kuti muchepetse mikhalidwe yachilengedwe ndi kagayidwe kake ka hepatocyte wamba. Chifukwa chake, nthawi zambiri amagwiritsidwa ntchito pophunzira za eyzyme, maphunziro a cytotoxity, kukhazikika kwa kagayidwe ka kachakudya kachakudya pang'onopang'ono kapena mankhwala osokoneza bongo.
Pakadali pano, sitimapereka ntchito yogula pa intaneti. Zogulitsa zomwe zimawonjezeredwa pagalimoto yogula pambuyo polembetsa ndikulowera ndikungotchulira kokha. Ngati mukufuna kugula zinthu zathu, chonde siyani chidziwitso chanu polumikizana ndi ife, ndipo tikulumikizani nthawi kuti titsirize ntchito zathu.
Q: Kodi No - Makina Ogwirizanitsa Anzake Amakhudza Zotsatira Zoyesedwa?
Yankho: Ngati mankhwala ogwiritsa ntchito omwe sakhala - Mapuloteni a Microomal, izi zimapangitsa magawo a kinetic. Pamene mapuloteni amakhazikika pamadzi ambiri amawonjezeka, mtengo wa Km umawonjezeka, womwe umapangitsa kuti wotsika upangiriric. Komanso oyang'anira mankhwala osokoneza bongo a ma protein m'ma mitimayomes amatha kukhala osiyanasiyana chifukwa cha maloboritotories osiyanasiyana komanso machitidwe osiyanasiyana.
Q: Kodi muli ndi mapuloteni a chiwindi microsomal mu bukhu la gawo la gawo la gawo la gawo la metabolic to plat ndi 0.1 mg / ml, kodi izi zikutanthauza kuti akangowathamangitsana ndi chiwindi musanawonjezere dongosolo?
Y: Palibe chifukwa chochepetsera ma microsomes a Hepatic poyamba; Kukhazikika kwa microsomes microsomes mu Kit ndi 20 mg / ml ndi mic /1 - 1 ml, yomwe imatha kuphatikizidwa mofatsa.
Q: Kodi kachulukidwe kachulukidwe ka masenti ndi chiyani kuti ayesetse ma metabolic? Kodi kuwerengetsa kokwanira monga ma microroomes a chiwindi?
Yankho: Chibwenzi cholimbikitsidwa cha sepacyte metabolic okhazikika pa 0,5 mpaka 2 × 106 Maselo / ml, ndipo chivomerezo chowerengera komanso kukonza zotsatira za hepatic ma microsomel okhazikika a metabolic.
Q: Ndi ziti zazikulu mu buffer yanu? Kodi ili ndi KCL ndi Nacl?
A: Zigawo zikuluzikulu za Pss Terffer ndi k2Mbutsani4 ndi h2PO4, ndipo ndi mfulu ya KCL ndi Nacl.
Q: Kodi cholinga cha phosphate biffer ndi chiani? Chifukwa chiyani mukufuna phosphate?
A: Zingwe za phosphate zimasankhidwa chifukwa chotseka zachilengedwe, ndipo phosphate ndi amodzi mwa awiri ofunikira kwambiri kuti azikhala ndi madzi.
Q: Kodi ma prezyme - - olandidwa mokhazikika? Kodi pali yankho lililonse ngati kusungunuka kwa dongosolo liyenera kuyesedwa ndi kusauka?
Yankho: A Enzyme iyenera kukhazikitsidwa ndi mitundu itatu yosiyanasiyana, mulingo wovuta kwambiri, ndipo ndende yayikulu kwambiri imasankhidwa kukhala dongosolo limodzi la kuchuluka kwa magazi okwera kwambiri mwa anthu. Ngati kusungunuka mu gawo lamadzi kumakhala wosauka, zosungunulira zokhazokha zimatha kusankhidwa ngati zosungunulira, e.g. DMSO, koma kuchuluka kwa zosungunulira zopangidwa ndi dongosolo kumafunikira kuti ziziyendetsedwa.
Q: M'maphunziro okhazikika a metabolic, kodi ndikofunikira kugwiritsa ntchito njira ziwiri zoyeserera, ma microsomes a chiwindi ndi hepatocyte, poyesa?
A: Microsomes microsomes ali ndi ma membrane vets - monga zojambula zopangidwa ndi Interrisugation Retabiction Makina oyambira (ma phcs) ndi hepatocytes olekanitsidwa atadzipatula pachimodzimodzi kuchokera ku ziwindi, zomwe zimasunga ntchito za mbozi za chiwindi, zomwe zimasunga ntchito za chiwindi za chiwindi, zomwe zimasungidwa bwino kwambiri chiwindi, makamaka kusungitsa ma enzyme omwe amagwirizana ndi omwe ali ku Vivo. Mu kafukufuku wokhazikika wa metabolic, palibe chifukwa choganizira mtengo, ndipo njira ziwiri zoyeserera zitha kusankhidwa kuti muyesedwe nthawi yomweyo; Kapenanso mayeso oyenerera amatha kusankhidwa malinga ndi kagayidwe kachakudya kambiri, ndipo mfundo yake ndi yomwe ili ndi dongosolo lazinthu zambiri lomwe limasankhidwa. Mwambiri, ma microsomes a chiwindi ndiye njira yabwino kwambiri pamene mamolekyu ophatikizika ndi madzi ambiri - kusungunuka ndi metabolism ndiye njira yayikulu ya metabolic (makamaka pa cyp); Ngati awiri - metabolism ndiye njira yayikulu, hydrolysis ndi njira yayikulu ya metabolic, yopanda ma protein imadziwika kwambiri, mayesowo amatha kuchitika pogwiritsa ntchito hepatocyte woyamba.
Q: Kodi kupsinjika kwa ma microsomes kumayesedwa mu mayeso okhazikika kagayidwe? Kodi zotsatira za kuchuluka kwambiri kapena zotsika kwambiri?
Yankho: Kuyeserera kwa kagayidwe ka metabolic, komwe katswiri wochita ma protein amakhudzanso kuchuluka kwa ma microsomal kukhazikika kwa 0,1 mg / ml, kusankha komwe kumachitika molingana ndi kachakudya. Kukwera kwambiri kwa mapuloteni microsomal kumabweretsa kulibe - kumangiriza kwa mankhwalawa ku ma protein a Microsomal; Ngakhale otsika kwambiri ndende mapuloteni a Microsomal atha kubweretsa kagayidwe ka mankhwala.