Karnovsky's Fixative tiv thaiv cov khoom cuav tshwm sim raws li Vacuolation tau los ntawm cov txheej txheem hauv lub raum kho nrog Antisense Oligonucleotide Ⅱ

Macroscopically, tsis muaj kev hloov pauv txawv txav tau pom. Tsis tas li ntawd, hauv cov ntshav chemistry, lub raum tsis ua haujlwm (piv txwv li, BUN thiab CRE) tsis pom muaj kev hloov pauv tseem ceeb hauv ib pawg (Table 1).

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Cov ntsiab lus ntawm cov kev tshawb pom histopathological hauv ob lub raum kho nrog 10% NBF yog qhia hauv Table 2. Microscopically, basophilic granules thiab vacuoles nyob rau hauv cov tubules ze ze, tshwj xeeb tshaj yog nyob rau hauv S1 thiab S2 ntu, tau pom nyob rau hauv ob qho tib si ASO-1W thiab ASO-2W pawg rau lub raum kho nrog 10% NBF (Fig. 1A). Cov kev hloov pauv no hnyav dua hauv ASO-2W pawg dua li hauv ASO-1W pawg. Kev sib sau ntawm basophilic granules feem ntau tau pom nyob rau ntawm txhuam ciam teb ntawm cov tubules ze ze li kev sib sau ua ke thiab / lossis punctate cov qauv. Raws li basophilic granules tau tshaj tawm tias muaj nyob rau hauv cov endosomes los yog lysosomes thiab dawb do nyob rau hauv lub cytoplasm ntawm proximal tubular epithelium14, cov ntaub ntawv sib txawv no yuav muaj kev cuam tshuam rau lawv qhov chaw nyob; uas yog, cov khoom sib sau ua ke thiab cov cim cim tuaj yeem sib sau hauv lysosomes thiab cytoplasm, feem. Cov vacuoles nyob rau hauv cov tubules ze ze yog puag ncig thiab ntxhib hauv cov duab, thiab feem ntau tau fused nrog cov vacuoles nyob ib sab. Tsis tas li ntawd, qee qhov vacuoles muaj faint basophilic granules (Daim duab 1E), qhia tias cov vacuoles muaj feem cuam tshuam rau kev sib sau ntawm ASOs. Immunohistochemically, LAMP-2 tau zoo rau ntawm daim nyias nyias ntawm lub vacuoles (Daim duab 1G thiab 1H). Cov txiaj ntsig tau qhia tias qhov vacuolation yog qhov dilatation ntawm lysosomes. Qhov sib txawv ntawm qhov hnyav ntawm qhov nqus ntawm ASO-1W thiab ASO-2W pawg yuav qhia tau qhov txawv ntawm cov ASOs uas muaj nyob hauv lysosomes. Peb kwv yees tias tus nqi ntawm ASOs tau sau rau hauv lysosomes nce nrog nce lub sij hawm raug rau ASOs. Yog li ntawd, lysosomal expansion tshwm sim raws li vacuolation vim yog cov ntaub so ntswg ua.

Table 1. Cov ntaub ntawv ntshav Chemistry

Table 2. Histopathological tshawb pom nyob rau hauv lub raum kho nrog 10% N

Txawm hais tias qhov nruab nrab vacuolation tau pom, qhov sib thooj tubular degeneration tsis tshwm sim, thiab feem ntau ntawm cov vacuolated epithelia yog qhov tsis zoo rau KIM-1 nyob rau hauv immunohistochemical staining (Fig. 1D). Nyob rau hauv sib piv, rau cov qauv kho nyob rau hauv Karnovsky lub fixative, vacuoles nyob rau hauv lub proximal tubules tsis tuaj nyob rau hauv tag nrho cov tib neeg, whereas basophilic granules tau nquag pom (Fig. 1B). Cov basophilic granules yog qhov tseem ceeb tshaj li cov hauv cov qauv uas raug 10% NBF fixation vim tias cov qauv ntawm tes tau khaws cia zoo (Daim duab 1F). Hauv cov qauv raug rau 4% PFA fixation, vacuoles thiab basophilic granules tau pom; Txawm li cas los xij, qhov hnyav ntawm vacuolation qis dua me ntsis hauv cov qauv raug rau 10% NBF fixation (Fig. 1C).

Oligonucleotides yog cov dej-soluble molecules heev thiab tau muab rho tawm los ntawm cov ntaub so ntswg thaum ua haujlwm vim tsis ua tiav crosslinks cellular Cheebtsam.

Raws li cov txiaj ntsig no, peb tau txiav txim siab tias cov vacuoles hauv cov tubules ze ze tau cuam tshuam los ntawm txoj kev kho thiab cov kev hloov morphological pom nyob rau hauv cov ntaub so ntswg raug rau 10% NBF thiab 4% PFA fixation tej zaum yuav yog cov khoom cuav hauv thaj tsam ntawm ASO tsub zuj zuj thaum lub sij hawm ua cov ntaub so ntswg. Hauv lwm lo lus, vacuolation tej zaum yuav ua rau toxicologically insignificant vim tsis muaj tubular degeneration, nce qhia ntawm KIM-1, los yog txawv txav nyob rau hauv lub raum tsis. Yog li, peb xav kom siv Karnovsky's fixative raws li kev xaiv kho kom raug rau kev kuaj mob ntawm lub raum hauv ASO-kho tsiaj.

Hauv kev xaus, peb qhov kev tshawb pom tam sim no tau qhia txog qhov muaj peev xwm overestimation ntawm vacuolation thaum siv cov qauv NBF fixation txheej txheem. Karnovsky's fixative tej zaum yuav yog ib qho cuab yeej muaj txiaj ntsig rau kev paub qhov txawv ntawm cov vacuoles los ntawm qhov tseeb nephrotoxicity.

Fig. 1. Cov neeg sawv cev photomicrographs ntawm lub raum kho nrog antisense oligonucleotide (ASO). Histopathological hloov nyob rau hauv lub 2-lub lim tiam kaw nucleic acid uas muaj antisense oligonucleotide (LNA-ASO)-kho pab pawg (ASO-2W) rau lub raum kho nrog (A) 10% neutralbuffered formaldehyde tov (NBF), ( B) Karnovsky's fixative, los yog (C) 4% paraformaldehyde phosphate buffer (PFA). (D) Immunohistochemical staining rau lub raum raug mob molecule-1 (KIM-1) ​​ntawm ASO-2W rau lub raum kho nrog 10% NBF.Histopathological hloov pauv hauv 1-lub lim tiam LNA-ASO-kho pab pawg (ASO-1W) rau lub raum kho nrog (E) 10% NBF thiab (F) Karnovsky's fixative. Immunohistochemical staining rau lysosomal-associated membrane protein 2 (LAMP-2) ntawm (G) ASO-1W thiab (H) ASO-2W rau lub raum kho nrog 10% NBF. (A) Basophilic granules (dub xub) thiab vacuoles (dub xub taub hau) tau pom nyob rau hauv cov tubules ze. Hematoxylin thiab eosin (H&E) staining. Bar =20 μm. (B) Vacuoles tsis pom, tab sis basophilic granules (dub xub) tau pom. H&E staining. Bar =20 μm. (C) Basophilic granules (dub xub) thiab vacuoles (dub xub taub hau) raug pom. Txawm li cas los xij, qhov hnyav ntawm qhov nqus tsev vacuolation qis dua qhov piv txwv uas raug rau 10% NBF fixation. H&E staining. Bar =20 μm. (D) Feem ntau ntawm cov vacuolated proximal tubules tsis zoo rau KIM-1. Bar =20 μm. (E, F) Qee qhov vacuoles pom nyob rau hauv cov tubules ze ze muaj basophilic granules faintly (ntsuab xub xub). Cov basophilic granules tau pom meej meej, thiab cov qauv ntawm tes yog khaws cia (ntsuab xub taub). H&E staining. Bars =20 μm. (G, H) Lub membrane nto ntawm lub vacuoles yog qhov zoo rau LAMP-2 (xub xub xiav). Bars =20 hli.

Kev Tshaj Tawm Txog Kev Muaj Peev Xwm Tsis Txaus Siab: Cov kws sau ntawv tshaj tawm tias lawv tsis muaj teeb meem ntawm kev txaus siab.

Kev lees paub: Cov kws sau ntawv ua tsaug rau Keisuke Yoshikawa thiab Tetsuya Ohta rau lawv cov lus qhia zoo.

Cov ntaub ntawv

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