Cov xwm txheej tam sim no ntawm Neuroprotection hauv Congenital Heart Disease Part 2

Cov duab microscopic tom qab ntawd raug suav nrog cov duab siv ImageJ software (National Institutes of Health, Bethesda, MD, USA). Lub teeb liab ntawm Hsp60 tau muab faib ua "x".

Hsp60 yog ib qho tseem ceeb ntawm cov protein molecule uas ua lub luag haujlwm tseem ceeb hauv tib neeg lub cev. Kev tshawb fawb nyob rau xyoo tas los no tau pom tias muaj Hsp60 muaj feem cuam tshuam nrog tib neeg kev nco.

Hsp60 yog ib qho molecular chaperone protein uas feem ntau koom nrog hauv cov protein folding, nthuav tawm, ruaj khov, thiab degradation. Cov txheej txheem no tseem ceeb heev rau cov metabolism hauv ib txwm thiab kev ua haujlwm ntawm tes ntawm tib neeg lub cev. Nyob rau tib lub sijhawm, Hsp60 kuj muaj cov tshuaj tiv thaiv kab mob antioxidant, tiv thaiv kab mob, thiab cov tshuaj tiv thaiv kab mob, thiab ua lub luag haujlwm tseem ceeb hauv kev tswj hwm tib neeg kev noj qab haus huv thiab kev sib npaug ntawm kev tiv thaiv kab mob.

Kev tshawb fawb tau pom tias Hsp60 plays lub luag haujlwm tseem ceeb hauv kev tsim kev nco thiab kev saib xyuas. Thaum lub sij hawm kev kawm thiab kev nco, hlwb hlwb tsim ib tug loj tus naj npawb ntawm Hsp60 molecules. Cov molecules no tuaj yeem tiv thaiv cov hlwb los ntawm kev cuam tshuam thiab kev puas tsuaj los ntawm ib puag ncig sab nraud kom ntseeg tau tias kev kawm tau zoo thiab kev nco. Tsis tas li ntawd, Hsp60 tseem tuaj yeem cuam tshuam rau tib neeg kev xav thiab kev coj cwj pwm los ntawm kev hloov cov teeb liab hloov ntawm lub hlwb hlwb thiab kev loj hlob ntawm cov neurons thiab muaj kev cuam tshuam zoo rau kev paub thiab kev ntxhov siab.

Txawm hais tias lub luag haujlwm ntawm Hsp60 tau pom tias muaj txiaj ntsig zoo rau tib neeg lub cev, tib neeg kuj yuav tsum paub tias ob qho tib si ntau dhau thiab tsis txaus ntawm Hsp60 yuav muaj kev cuam tshuam tsis zoo rau tib neeg kev noj qab haus huv. Yog li ntawd, peb yuav tsum nquag tshawb nrhiav thiab kawm txog cov txheej txheem ntawm Hsp60 nyob rau hauv ntau yam physiological thiab pathological mob ntawm tib neeg lub cev kom nkag siab zoo dua txoj hauj lwm thiab lub luag hauj lwm ntawm Hsp60 nyob rau hauv tib neeg noj qab haus huv. Nws tuaj yeem pom tias peb yuav tsum txhim kho kev nco, thiab Cistanche deserticola tuaj yeem txhim kho kev nco, vim Cistanche deserticola muaj antioxidant, tiv thaiv kev mob, thiab tiv thaiv kev laus, uas tuaj yeem pab txo qis oxidation thiab inflammatory tshwm sim hauv lub hlwb, yog li tiv thaiv cov kab mob. kev noj qab haus huv ntawm lub paj hlwb. Tsis tas li ntawd, Cistanche deserticola kuj tseem tuaj yeem txhawb kev loj hlob thiab kho cov paj hlwb, yog li txhim kho kev sib txuas thiab kev ua haujlwm ntawm neural networks. Cov teebmeem no tuaj yeem pab txhim kho kev nco, kev kawm muaj peev xwm, thiab kev xav nrawm, thiab tseem tuaj yeem tiv thaiv kev loj hlob ntawm kev paub tsis meej thiab kab mob neurodegenerative.

Nyem Paub txhawm rau txhim kho lub cim xeeb luv luv

Tus nqi "x" tau txiav txim raws li hauv qab no: rho tawm "x"-folded Hsp60signal los ntawm mitochondrial DNA teeb liab hauv pawg tswj hwm ua rau mitochondrial DNA tus nqi tsawg dua lossis sib npaug rau xoom. Lub teeb liab ntawm histone H2B tau muab faib ua "y".

Tus nqi "y" tau txiav txim siab raws li hauv qab no: rho tawm "y"-folded histone-H2B teeb liab los ntawm lub teeb liab nuclear-DNA hauv pawg tswj hwm ua rau cov nqi hluav taws xob-DNA tsawg dua orequal rau xoom. Yog tias lub teeb liab tom qab dhau los ntawm qhov sib npaug, qhov sib piv ntawm cov duab multiplied tuaj yeem hloov kho rau tib yam xwm txheej hauv txhua qhov qauv.

Cov duab ntawm dsDNA staining, rho tawm cov duab hloov kho ntawm multiplied Hsp60 thiab histone H2B, tau siv rau kev tshuaj xyuas tom qab. Siv qhov "Kho-Threshold", cov duab no tau binarized. Peb xaiv dsDNA puncta qhov loj ntawm 2 mus rau 2 0 µm2 (circularity0.1–1.0), thiab lawv tau suav nrog siv "Tshawb Xyuas-Tshaj Particles" muaj nuj nqi.

2.3. Electron Microscopy

Ib hnub ua ntej transfection, HeLa hlwb tau plated ntawm qhov ceev ntawm 600 hlwb / µL.Qhov kev qhia ntawm cov noob paj hlwb tau ntsiag to hauv HeLa hlwb siv Stealth RNAi siRNA (Thermo Fisher Scientific) hauv Lipofectamine RNAiMAX Transfection Reagent (ThermoFisher Scientific) raws li kev tshawb fawb. chaw tsim tshuaj paus cov lus qhia (nrog rau qhov kawg RNA concentration ntawm 10 nM).

Plaub hnub tom qab siRNA knockdown, cov hlwb raug kho.Cell-culture kuaj tau kho nrog 2% PFA thiab 2% glutaraldehyde hauv 0.1 M phosphatebuffer, pH 7.4, ntawm 37 ◦C, ces muab tso rau hauv 4. ◦C lub tub yees rau 30 min. Cov qauv raug kho hauv 2% glutaraldehyde hauv 0.1 M phosphate tsis nyob ib hmos ntawm 4 ◦C. Cov qauv raug ntxuav peb zaug nrog 0.1 M phosphate tsis rau 30 min thiab tom qab kho nrog 2% osmiumtetroxide hauv 0.1 M phosphate tsis nyob ntawm 4 ◦C rau 1 h.

Cov qauv no tau dehydrated ingraded ethanol daws, hloov mus rau resin (Quetol-812, Nisshin EM Co., Tokyo, Nyiv), thiab polymerized ntawm 60 ◦C rau 48 h. Cov polymerized resins raug txiav rau hauv ultrathin 70-nmsections nrog lub pob zeb diamond riam siv ultramicrotome (Ultracut UCT, Leica Microsystems, Wetzlar, Lub teb chaws Yelemees) thiab tom qab ntawd tau mounted rau ntawm daim phiaj tooj liab.

Cov ntu tau stained nrog 2% uranyl acetate ntawm RT rau 15 feeb thiab ntxuav nrog dej distilled, tom qab ntawd los ntawm kev sib xyaw nrog cov hmoov lead staining tov (Sigma-Aldrich, Tokyo, Nyiv, Nyiv) ntawm RT rau 3 min.

Cov kab hluav taws xob tau pom nyob rau hauv lub tshuab hluav taws xob xa hluav taws xob (JEM-1400Plus, JEOL Ltd., Tokyo, Nyiv) ntawm qhov nrawm nrawm ntawm 80 kV (zebrafish hlwb) lossis 100 kV (cell), thiab cov duab tau sau tseg siv lub koob yees duab charge-coupled-device (CCD) (EM-14830RUBY2,JEOL Ltd.) .

2.4. Immunoelectron Microscopy

Cov qauv ntawm cov pob zeb kub tau khov rau hauv cov kua propane ntawm −175 ◦C. Tom qab cov qauv tau khov lawm, lawv tau khov-hloov pauv nrog 1% tannic acid hauv ethanol thiab 2% dej distilled ntawm −80 ◦C rau 24 teev.

Tom qab ntawd cov qauv tau khaws cia ntawm −20 ◦C rau 4 hr, tom qab incubation ntawm 4 ◦C rau 1 hr. Tom qab ntawd, cov qauv tau qhuav dej hauv cov dej tsis muaj dej ethanol 3 zaug rau 30 feeb. txhua lub sij hawm, ua raws li infiltration nrog 50:50 sib tov ntawm ethanoland resin (LR dawb: London Resin Co. Ltd., Berkshire, UK) ntawm 4 ◦C rau 1 hr. Cov qauv raug xa mus rau tshiab 100% resin thiab incubated ntawm 4 ◦C rau 30 min, thiab cov txheej txheem no tau rov ua peb zaug.

Cov qauv raug xa mus rau tshiab 100% resin thiab raug polymerized ntawm 50 ◦CO / N. Cov polymerized resins yog ultra-thin sectioned ntawm 90 nm nrog pob zeb diamond riam siv ib qho ultramicrotome (Ultracut UCT; Leica), thiab cov seem tau muab tso rau ntawm daim phiaj npib tsib xee. Cov kab sib txuas tau tsim nrog cov tshuaj tiv thaiv dsDNA antibody (35I9 DNA) (1/800, Abcam, Cat# ab27156, RRID: AB_470907) hauv 1% BSA/PBS ntawm 4 ◦CO/N, ua raws li peb zaug yaug nrog 1% BSA / PBS rau 1 min txhua.

Tom qab ntawd lawv tau incubated nrog cov tshuaj tiv thaiv kab mob no txuas mus rau {{{0}}nm kub hais (Tshis los tiv thaiv nas IgG polyclonalantibody) rau 2 hr ntawm RT. Tom qab yaug nrog PBS, cov kab sib txuas tau muab tso rau hauv 2% glutaraldehydein 0.1 M cacodylate tsis.

Tom qab ntawd, cov kab sib txuas tau qhuav thiab tom qab ntawd stained nrog 2% uranyl acetate rau 15 min thiab nrog cov tshuaj lead stain (Sigma-Aldrich) ntawm RT rau 3 min.Cov kab sib txuas tau pom los ntawm kev xa hluav taws xob hluav taws xob (JEM-1400) ntxiv; JEOLLtd.) ntawm qhov nrawm nrawm ntawm 100 kV. Cov duab digital tau txais nrog lub koob yees duab CCD (EM-14830RUBY2, JEOL Ltd.).

3. Cov txiaj ntsig

3.1. Txo TFAM lossis GBA Induces nce hauv Ectopic Mitochondrial DNA

Ua ntej, peb tau pom ib qho piv txwv ntawm kev tshawb pom ntawm mitochondria-derived cytoplasmicDNA hauv HeLa hlwb uas cov TFAM protein raug tsoo los ntawm siRNA (Daim duab 1A).TFAM yog ib qho tseem ceeb mitochondrial transcription factor thiab kuj ua haujlwm hauv mitochondrialDNA replication thiab kho [20] .

Sequence polymorphisms hauv cov noob no tau tshaj tawm tias muaj feem cuam tshuam nrog Parkinson tus kab mob [21,22]. Tom qab 3 hnub ntawm TFAM protein knockdown, ntau ob-stranded DNA dots tau kuaj pom nyob rau hauv lub cytoplasm ntawm HeLa hlwb.

Histones yog cov cim zoo rau nuclear- keeb kwm DNA nyob rau hauv cytoplasm, thiab cov cim tsis zoo rau mitochondrial DNA thiab mitochondrial-origin DNA nyob rau hauv cytoplasm [23].Cov dots no tsis co-localize nrog histones, qhia tias lawv tsis yog nuclear-derivedDNA. Tsis tas li ntawd, qee qhov ntawm cov cytoplasmic, ectopic DNA dots tsis koom nrog Hsp60, ib qho cim rau mitochondrial matrix, qhia tias lawv nyob sab nraum mitochondria (Daim duab 1B, C).

Electron microscopy kuj qhia tau tias ectopic DNA leaked los ntawm mitochondria (Daim duab 2A, B). Hauv cov ntsiab lus, qhov nce hauv ectopic DNAdots nyob rau hauv cytoplasm los ntawm TFAM knockdown yog vim mitochondrial DNA to los ntawm mitochondria. Thib ob, peb tau pom lwm qhov piv txwv ntawm kev tshawb pom ntawm mitochondria-derived cytoplasmic DNA hauv HeLa hlwb uas GBA protein tau tsoo los ntawm siRNA. (Daim duab 1A).

GBA yog cov noob caj noob ces rau tus kab mob Gaucher, tab sis kev hloov pauv ntawm GBA kuj tseem muaj feem cuam tshuam rau Parkinson tus kab mob [24]. Ntau qhov ob-stranded DNA dots tau kuaj pom hauv cytoplasm ntawm HeLa hlwb tom qab 3 hnub ntawm GBA protein knockdown. Qee cov cim DNA tau sib koom ua ke nrog histones, tawm tswv yim tias lawv yog nuclear-derivedDNA.

Lwm cov DNA dots tsis tau sib koom ua ke nrog histones, qhia tias lawv tsis yog DNA los ntawm nuclear tab sis mitochondria-derived DNA (Daim duab 1D). Hauv cov ntsiab lus, qhov nce hauv ectopic DNA dots nyob rau hauv cytoplasm los ntawm GBA knockdown yog tsawg kawg yog ib feem ntawm mitochondrial DNA los ntawm mitochondria.

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