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Toxoplasma gondii waa dulin protozoan intracellular kaas oo wax ka beddela microenvironment ee martida loo yahay cudurka waxaana la og yahay in ay la xiriirto dhacdooyinka koritaanka burada maskaxda.Daraasaddan, waxaan ku qiyaasaynaa in exosomal miRNA-21 ee caabuqa Toxoplasma ay kor u qaaddo koritaanka burada maskaxda.Exosomes-ka Toxoplasma-ku-dhacey BV2 microglia ayaa lagu sifeeyay iyo gudaha unugyada U87 glioma ayaa la xaqiijiyay.Noocyada muujinta microRNA Exosomal ayaa la falanqeeyay iyadoo la adeegsanayo habab microRNA iyo microRNA-21A-5p oo xiriir la leh Toxoplasma gondii iyo kala soocida burada.Waxaan sidoo kale baarnay heerarka mRNA ee hiddo-wadaha ku xiran burada ee unugyada glioma U87 annagoo beddelayna heerarka miR-21 ee exosomes iyo saameynta exosomes ee kororka unugyada glioma ee U87.Unugyada glioma ee U87 ee uu ku dhacay Toxoplasma gondii, muujinta microRNA-21 waa la kordhiyaa waxaana la dhimay waxqabadka hiddo-wadaha antitumor (FoxO1, PTEN, iyo PDCD4).BV2-exosomes-ka-soo-baxa ee uu ku dhacay Toxoplasma waxay keenaan kororka unugyada glioma U87.Exosomes waxay kiciyaan koritaanka unugyada U87 ee qaabka burada jiirka.Waxaan soo jeedinaynaa in kororka exosomal miR-21 ee Toxoplasma-infekshanka BV2 microglia laga yaabo inay door muhiim ah ka ciyaaraan kor u qaadida korriinka unugyada unugyada glioma U87 iyadoo hoos loo dhigayo hiddaha antitumor.
Waxaa lagu qiyaasaa in in ka badan 18.1 milyan oo xaaladood oo kansar heer sare ah laga helay adduunka oo dhan sanadka 2018, iyadoo qiyaastii 297,000 burooyinka habdhiska dhexe ee neerfayaasha la ogaado sannad kasta (1.6% dhammaan burooyinka)1.Cilmi-baaris hore ayaa muujisay in arrimaha khatarta ah ee ku dhaca burooyinka maskaxda ee bini'aadamka ay ka mid yihiin walxo kiimiko oo kala duwan, taariikhda qoyska, iyo shucaaca ionizing ee ka yimaada qalabka daaweynta madaxa iyo baaritaanka.Si kastaba ha ahaatee, lama garanayo sababta dhabta ah ee cudurradan.Ku dhawaad ​​20% dhammaan kansarrada adduunka oo dhan waxaa sababa jeermisyada faafa, oo ay ku jiraan fayrasyada, bakteeriyada iyo dulinnada3,4.Jeermisyada caabuqa ayaa carqaladeeya hababka hidde-sideyaasha unugga martida loo yahay, sida hagaajinta DNA-da iyo wareegga unugga, waxayna u horseedi karaan barar joogto ah iyo dhaawac soo gaadha habka difaaca.
Walaxaha faafa ee la xidhiidha kansarka bini'aadamka ayaa ah cudurada ugu badan ee fayraska, oo ay ku jiraan papillomaviruses iyo fayraska cagaarshow B iyo C.Dulinku waxa kale oo uu door ka ciyaari karaa horumarinta kansarka bini'aadamka.Noocyo dhowr ah oo dulin ah, kuwaas oo kala ah Schistosoma, Opishorchis viverrini, O. felineus, Clonorchis sinensis iyo Hymenolepis nana, ayaa lagu lug yeeshay noocyada kala duwan ee kansarka bini'aadamka 6,7,8.
Toxoplasma gondii waa protozoan intracellular kaas oo maamula microenvironment unugyada martida loo yahay cudurka.Dulinkan waxa lagu qiyaasaa in uu haleelo ku dhawaad ​​30% dadka aduunka, taas oo dadka oo dhan halis galinaysa9,10.Toxoplasma gondii waxay ku dhici kartaa xubnaha muhiimka ah, oo ay ku jiraan habdhiska dhexe ee neerfayaasha (CNS), waxayna sababi kartaa cudurro halis ah sida qoorgooyaha dilaaga ah iyo encephalitis, gaar ahaan bukaanada difaaca jirkooda9.Si kastaba ha noqotee, Toxoplasma gondii waxay sidoo kale bedeli kartaa deegaanka martida loo yahay cudurka iyadoo wax ka beddelaysa koritaanka unugyada iyo jawaabaha difaaca ee shakhsiyaadka difaaca jirka, taasoo horseedaysa dayactirka caabuqa dabadheeraad ah ee asymptomatic9,11.Waxa xiiso leh, marka la eego xidhiidhka ka dhexeeya faafitaanka T. gondii iyo dhacdooyinka buro maskaxda, warbixinnada qaar waxay soo jeedinayaan in vivo martida loo yahay isbeddelka deegaanka sababtoo ah caabuqa T. gondii ee daba-dheeraaday waxay u egtahay buro microenvironment.
Exosomes waxaa loo yaqaanaa isgaadhsiinta intercellular kuwaas oo keena maaddooyinka noolaha, oo ay ku jiraan borotiinno iyo asiidhyada nucleic, unugyada deriska ah16,17.Exosomes waxay saameyn ku yeelan karaan hababka bayooloji ee la xiriira buro sida anti-apoptosis, angiogenesis, iyo metastasis ee microenvironment burada.Gaar ahaan, miRNA-yada (miRNAs), RNA-yada yaryar ee aan codeeyn ee ku saabsan 22 nucleotide oo dhererkoodu yahay, ayaa muhiim u ah xakameynaya hidda-wadaha transcriptional ka dib kuwaas oo xakameynaya in ka badan 30% mRNA bini'aadamka iyada oo loo marayo isku-dhafka aamusnaanta miRNA-ku-abuuray (miRISC).Toxoplasma gondii waxay carqaladayn kartaa hababka noolaha iyadoo la xakameynayo muujinta miRNA ee martida cudurka qaba.MiRNA-yada martida loo yahay waxay ka kooban yihiin calaamado muhiim ah oo loogu talagalay habaynta hababka noolaha martida loo yahay si loo gaaro istiraatiijiyadda badbaadada dulinka.Haddaba, barashada isbeddellada ku dhaca astaanta miRNA-ga martida loo yahay marka caabuqa laga qaado T. gondii waxay naga caawin kartaa inaan si cad u fahanno isdhexgalka ka dhexeeya martida loo yahay iyo T. gondii.Runtii, Thirugnanam et al.15 waxay soo jeedisay in T. gondii ay kor u qaaddo kansarka maskaxda iyada oo beddeleysa muujinteeda miRNA-yada gaarka ah ee martida loo yahay ee la xidhiidha koritaanka burada oo la ogaaday in T. gondii ay keeni karto gliomas xayawaanka tijaabada ah.
Daraasadani waxay diiradda saaraysaa beddelka exosomal miR-21 ee microglia martida loo yahay ee qaba Toxoplasma BV2.Waxaan aragnay doorka suurtagalka ah ee miR-21 ee isbeddelka ah ee koritaanka unugyada glioma U87 sababtoo ah haynta xudunta FoxO1 / p27, taas oo ah bartilmaameedka miR-21 ee xad-dhaafka ah.
Exosomes oo laga soo qaatay BV2 ayaa la helay iyadoo la adeegsanayo centrifugation kala duwan waxaana lagu ansixiyay habab kala duwan si looga hortago wasakheynta qaybaha gacanta ama xuubka kale.SDS-polyacrylamide gel electrophoresis (SDS-PAGE) waxay muujisay qaabab kala duwan oo u dhexeeya borotiinnada laga soo saaro unugyada BV2 iyo exosomes (Jaantus 1A), iyo muunado ayaa lagu qiimeeyay joogitaanka Alix, kaas oo lagu falanqeeyay xoqida Galbeedka ee calaamadaha borotiinka exosomal ee .Calaamadaynta Alix waxaa laga helay borotiinada exosome laakiin maaha borotiinka lysate unugga BV2 (Jaantus. 1B).Intaa waxaa dheer, RNA la safeeyey oo ka soo baxay exosomes oo laga soo qaatay BV2 ayaa la falanqeeyay iyadoo la adeegsanayo bioanalyzer.18S iyo 28S ribosomal subunits ayaa si naadir ah loogu arkay qaabka socdaalka RNA exosomal, taasoo muujinaysa nadiifnimo la isku halayn karo (Jaantuska 1C).Ugu dambeyntii, gudbinta mikroskoobyada elektarooniga ah waxay muujisay in exosomes-ka la arkay ay ahaayeen qiyaastii 60-150 nm oo cabbir ah waxayna lahaayeen qaab-dhismeedka koob-u eg ee caadiga ah ee morphology exosome (Jaantus. 1D).
Astaamaha exosomes ee ka soo jeeda unugyada BV2.(A) bogga xaashida xogta badbaadada.Barootiinnada ayaa laga soocay unugyada BV2 ama exosomes laga soo qaatay BV2.Qaababka borotiinku way ku kala duwan yihiin unugyada iyo exosomes.(B) Falanqaynta Western blot ee calaamadeeyaha exosomal (Alix).(C) Qiimaynta RNA la safeeyey ee unugyada BV2 iyo BV2 exosomes laga soo qaatay iyadoo la isticmaalayo bioanalyzer.Sidaa darteed, 18S iyo 28S ribosomal subunits ee unugyada BV2 ayaa dhif ah laga helay RNA exosomal.(D) Mikroskoobyada elektarooniga ah ee gudbinta ayaa muujisay in exosomes ka go'doomay unugyada BV2 ay si xun u wasakheeyeen 2% uranyl acetate.Exosomes waa qiyaastii 60-150 nm cabbir ahaan iyo qaab koob leh (Hees iyo Jung, xog aan la daabicin).
Dhexdhexaadinta unugyada ee BV2-ka-soo-baxa exosomes ee U87 unugyada glioma bini'aadamka ayaa lagu arkay iyadoo la adeegsanayo microscopy confocal.PKH26 calaamadeysan exosomes waxay ku yaalliin cytoplasm ee unugyada U87.Nukleiyada waxaa lagu dheehday DAPI (Jaantus. 2A), taas oo muujinaysa in BV2-ka-soo-baxa exosomes ay gudaha u geli karaan unugyada martida loo yahay oo ay saameyn ku yeeshaan deegaanka unugyada qaataha.
Dhexdhexaadinta BV2-ka-soo-baxa exosomes ee unugyada glioma U87 iyo BV2-derived exosomes oo qaba Toxoplasma RH waxay keentay kororka unugyada glioma U87.(A) Exosomes oo ay qariyeen unugyo U87 oo lagu cabbiray mikroskoobyada konfocal.Unugyada glioma ee U87 waxaa lagu dhex galiyay exosomes oo lagu calaamadeeyay PKH26 (casaan) ama iyada oo aan la xakameynin 24 saacadood.Nukleiyada waxaa lagu dheehday DAPI (buluug) ka dibna lagu arkay mikroskoob koofiyad ah (cabir: 10 μm, x 3000).(B) U87 fidinta unugyada glioma waxaa lagu go'aamiyay baaritaanka unugyada.Unugyada glioma U87 waxaa lagu daaweeyay exosomes wakhtiga la tilmaamay. *P <0.05 waxa lagu helay imtixaanka Student's t. *P <0.05 waxa lagu helay imtixaanka Student's t. P <0,05 получено по t-критерию Стьюдента. *P <0.05 by t-tijaabka Ardayga. P <0.05 通过学生t 检验获得。 P <0.05 P <0,05, полученный с помощью t-критерия Стьюдента. P <0.05 oo la helay iyadoo la isticmaalayo t-tijaabka Ardayga.
Ka dib markii aan xaqiijinay gudaha gudaha BV2-derived exosomes ee unugyada glioma U87, waxaan sameynay baaritaanno kordhinta unugyada si aan u baarno doorka BV2-derived Toxoplasma-derived exosomes ee horumarinta unugyada glioma bini'aadamka.Daaweynta unugyada U87 oo leh unugyo ka soo jeeda unugyada BV2 ee T. gondii-ku-dhacey waxay muujiyeen in T. gondii-ku-dhacey BV2-ka-soo-baxa ay sababtay kororka unugyada U87 oo aad u sarreeya marka la barbardhigo kantaroolka (Jaantus 2B).
Intaa waxaa dheer, koritaanka unugyada U118 waxay heleen natiijooyin isku mid ah U87, sida Toxoplasma kicisay exosomes waxay sababtay heerarka ugu sarreeya ee faafinta (xogta aan la muujin).Iyada oo ku saleysan xogtan, waxaan muujin karnaa in BV2-ka-soo-baxa Toxoplasma-ka-soo-baxa ee exosomes ay door muhiim ah ka ciyaaraan kororka unugyada glioma.
Si loo baadho saamaynta Toxoplasma-cudurada BV2-ka-soo-baxa exosomes ee horumarinta burooyinka, waxaan ku durinay unugyada glioma U87 jiirarka qaawan ee qaabka xenograft iyo duritaan BV2-derived exosomes ama RH-cudur-qaadka BV2-derived exosomes.Ka dib markii ay burooyinku soo shaac baxeen 1 usbuuc ka dib, koox kasta oo tijaabo ah oo 5 jiir ah ayaa loo qaybiyay iyadoo loo eegayo cabbirka buro si loo go'aamiyo isla barta bilawga ah, iyo cabbirka buro ayaa la qiyaasay 22 maalmood.
Jiirarka leh qaabka U87 xenograft, cabbirka buro iyo miisaan aad u weyn ayaa lagu arkay BV2-ku-soo-saaray kooxda exosome-ka ee RH ee maalintii 22 (Jaantus. 3A,B).Dhanka kale, ma jirin farqi weyn oo u dhexeeya xajmiga burooyinka ee u dhexeeya kooxda BV2 ee ka soo jeeda kooxda exosome iyo kooxda xakamaynta ka dib daaweynta exosome.Intaa waxaa dheer, jiirarka lagu duray unugyada glioma iyo exosomes muuqaal ahaan waxay muujiyeen mugga burada ugu weyn ee kooxda RH-cudurka BV2-derived exosomes (Jaantus. 3C).Natiijooyinkani waxay muujinayaan in BV2-ka-soo-baxa Toxoplasma-infekshanka exosomes ay keento koritaanka glioma ee qaabka burada jiirka.
Oncogenesis (AC) ee exosomes-ka BV2 ee ku jira moodalka jiirka U87 xenograft.Cabbirka burada (A) iyo miisaanka (B) ayaa si aad ah u kordhay BALB/c jiirarka qaawan ee lagu daweeyay xayndaabyo cudurka RH ah oo laga soo qaatay BV2.BALB/c jiirarka qaawan (C) ayaa si hoose loogu duray iyada oo 1 x 107 U87 unugyo laga hakiyay isku dhafka Matrigel.Lix maalmood ka dib duritaanka, 100 μg ee exosomes-ka BV2 ayaa lagu daweeyey jiir.Cabbirka burada iyo miisaanka ayaa la qiyaasay maalmaha la tilmaamay iyo ka dib allabariga, siday u kala horreeyaan. P <0.05. P <0.05. *R <0,05. P <0.05. P <0.05. P <0.05. *R <0,05. P <0.05.
Xogta ayaa muujisay in 37 miRNAs (16 si xad dhaaf ah iyo 21 hoos loo dhigay) oo la xidhiidha difaaca ama horumarinta burooyinka ayaa si weyn u bedelay microglia ka dib markii uu ku dhacay xanuunka Toxoplasma RH (Jaantus. 4A).Heerarka muujinta qaraabada ee miR-21 ee ka mid ah miRNA-yada la beddelay waxaa lagu xaqiijiyay RT-PCR-waqtiga-dhabta ah ee exosomes laga soo qaatay BV2, exosomes lagu daweeyay unugyada BV2 iyo U87.Muujinta miR-21 waxay muujisay koror weyn oo ah exosomes unugyada BV2 ee qaba Toxoplasma gondii (RH stress) (Jaantus. 4B).Heerarka muujinta qaraabada ee miR-21 ee unugyada BV2 iyo U87 ayaa kordhay ka dib qaadashada exosomes-ka beddelka ah (Jaantus. 4B).Heerarka qaraabada ah ee muujinta miR-21 ee unugyada maskaxda ee bukaannada buro iyo jiirarka qaba Toxoplasma gondii (nooca ME49) ayaa ka sarreeyay kantaroolayaasha, siday u kala horreeyaan (Jaantus. 4C).Natiijooyinkani waxay la xiriiraan kala duwanaanshaha u dhexeeya heerarka muujinta ee la saadaaliyay iyo kuwa la xaqiijiyay ee microRNAs in vitro iyo in vivo.
Isbeddellada muujinta exosomal miP-21a-5p ee microglia ee qaba Toxoplasma gondii (RH).(A) Waxay muujisaa isbeddelada muhiimka ah ee siRNA ee la xidhiidha difaaca ama horumarinta burada ka dib caabuqa T. gondii RH.(B) Heerarka muujinta miR-21 ee qaraabada ah waxaa lagu ogaaday RT-PCR-waqtiga-dhabta ah ee BV2-ka-soo-baxa, exosomes-BV2-la daweeyay, iyo unugyada U87.(C) Heerarka muujinta miR-21 ee qaraabada ah ayaa laga helay unugyada maskaxda ee bukaannada buro (N=3) iyo jiirarka qaba Toxoplasma gondii (ME49 strain) (N=3). *P <0.05 waxa lagu helay imtixaanka Student's t. *P <0.05 waxa lagu helay imtixaanka Student's t. P <0,05 было получено с помощью t-критерия Стьюдента. *P <0.05 waxa lagu helay iyadoo la isticmaalayo t-tijaabka Ardayga. P <0.05 通过学生t 检验获得。 P <0.05 P <0,05, полученный с помощью t-критерия Стьюдента. P <0.05 oo la helay iyadoo la isticmaalayo t-tijaabka Ardayga.
Exosomes ee unugyada BV2 ee caabuqa RH waxay horseedeen koritaanka gliomas ee vivo iyo in vitro (Jaantus. 2, 3).Si loo ogaado mRNA-yada khuseeya, waxaanu baarnay heerarka mRNA ee hiddo-wadaha bartilmaameedka antitumor, sanduuqa fargeetada O1 (FoxO1), PTEN, iyo dhimashada unugyada barnaamijka 4 (PDCD4) ee unugyada U87 ee qaba exosomes laga soo qaatay BV2 ama RH BV2.Falanqaynta bioinformatics ayaa muujisay in dhowr hidde-sideyaal buro, oo ay ku jiraan FoxO1, PTEN, iyo PDCD4, ay leeyihiin goobaha miR-2121,22.Heerarka mRNA ee hiddo-wadaha bartilmaameedka antitumor ayaa lagu yareeyay RH-ku-dhacsan BV2-derived exosomes marka la barbar dhigo exosomes-ka BV2 (Jaantus. 5A).FoxO1 waxay muujisay hoos u dhac ku yimid heerarka borotiinka ee BV2-ka-soo-baxyada ka soo jeeda RH marka la barbar dhigo exosomes-ka BV2 (Jaantus 5B).Iyada oo ku saleysan natiijooyinkaan, waxaan xaqiijin karnaa in exosomes-ka laga soo qaatay RH-cudurka BV2 ay hoos u dhigaan hiddo-wadaha anti-oncogenic, iyaga oo ilaalinaya doorkooda koritaanka burada.
Toxoplasma RH-ku-dhacsan BV2-ka-soo-baxa exosomes waxay keenaysaa xakamaynta hiddo-wadaha antitumor ee unugyada U87 glioma by Toxoplasma RH-cudur-qaadka BV2-derived exosomes.(A) PCR-waqtiga-dhabta ah ee FoxO1, PTEN iyo PDCD4 ee muujinta exosomes ee ka soo jeeda T. gondii RH-cudurka BV2 marka la barbar dhigo PBS exosomes.β-actin mRNA ayaa loo isticmaalay xakameyn ahaan.(B) Muujinta FoxO1 waxaa lagu go'aamiyay xannibaadda Galbeedka iyo xogta densitometry ayaa si tirakoob ah loo qiimeeyay iyadoo la adeegsanayo barnaamijka ImageJ. *P <0.05 waxa lagu helay imtixaanka Student's t. *P <0.05 waxa lagu helay imtixaanka Student's t. P <0,05 было получено с помощью t-критерия Стьюдента. *P <0.05 waxa lagu helay iyadoo la isticmaalayo t-tijaabka Ardayga. P <0.05 通过学生t 检验获得。 P <0.05 P <0,05, полученный с помощью t-критерия Стьюдента. P <0.05 oo la helay iyadoo la isticmaalayo t-tijaabka Ardayga.
Si loo fahmo saamaynta miP-21 ee exosomes ee habaynta hidda-wadaha-la xidhiidha burada, unugyada U87 waxaa lagu beddelay xannibaadaha miP-21 iyadoo la adeegsanayo Lipofectamine 2000 iyo unugyada ayaa la goostay 24 saacadood ka dib gudbinta.FoxO1 iyo p27 heerarka muujinta ee unugyada lagu wareejiyay miR-21 inhibitors ayaa la barbardhigay unugyada lagu daaweeyay BV2-ka soo jeeda exosomes iyadoo la adeegsanayo qRT-PCR (Jaantus. 6A, B).Ku wareejinta miR-21 inhibitor ee unugyada U87 ayaa si weyn hoos ugu dhigay FoxO1 iyo muujinta p27 (FIG. 6).
RH-cudur-ku-takoorka exosomal BV2-ka soo jeeda miP-21 ayaa beddelay muujinta FoxO1/p27 ee unugyada glioma U87.Unugyada U87 waxaa lagu beddelay miP-21 inhibitor iyadoo la adeegsanayo Lipofectamine 2000 iyo unugyada ayaa la goostay 24 saacadood ka dib gudbinta.FoxO1 iyo p27 heerarka muujinta ee unugyada lagu wareejiyay miR-21 inhibitors ayaa la barbardhigay heerarka unugyada lagu daaweeyay BV2-exosomes iyadoo la adeegsanayo qRT-PCR (A, B).
Si looga baxsado jawaab celinta difaaca qofka martida loo yahay, dulinka Toxoplasma waxa uu isu beddelaa fiix unug ah.Waxay ku dulmaneeyaan unugyo kala duwan, oo ay ku jiraan maskaxda, wadnaha, iyo murqaha lafaha, inta ay nool yihiin martida loo yahay waxayna beddelaan jawaabta difaaca ee martida loo yahay.Intaa waxaa dheer, waxay xakameyn karaan wareegga unugyada iyo apoptosis ee unugyada martida loo yahay, iyaga oo kor u qaadaya fidintooda14,24.Toxoplasma gondii waxay inta badan ku dhacdaa unugyada dendritic-ka, neutrophils, iyo monocyte/macrophage lineage, oo ay ku jiraan microglia maskaxda.Toxoplasma gondii waxay keentaa kala duwanaanshaha macrophages ee M2 phenotype, waxay saamaysaa bogsashada boogta ka dib caabuqa pathogene, iyo sidoo kale waxay la xiriirtaa hypervascularization iyo granulomatous fibrosis.Cudurkan hab-dhaqanka ee caabuqa Toxoplasma waxa laga yaabaa inuu la xidhiidho calaamadaha la xidhiidha horumarka burada.Deegaanka colaadeed ee ay maamusho Toxoplasma waxa laga yaabaa inuu u ekaado kansarka hore ee u dhigma.Sidaa darteed, waxaa loo qaadan karaa in caabuqa Toxoplasma uu gacan ka geysto horumarinta burooyinka maskaxda.Dhab ahaantii, heerar sare oo caabuqa Toxoplasma ayaa lagu soo warramey serumka bukaanada qaba burooyinka maskaxda ee kala duwan.Intaa waxaa dheer, Toxoplasma gondii waxay noqon kartaa saameyn kale oo kansarka leh oo u dhaqmo si isku mid ah si ay u caawiso kansarka kale ee faafa inay yeeshaan burooyinka maskaxda.Marka tan la eego, waxaa xusid mudan in P. falciparum iyo fayraska Epstein-Barr ay si wada jir ah uga qaybqaataan samaynta lymphoma Burkitt.
Doorka exosomes sida nidaamiyayaal ee goobta cilmi baarista kansarka ayaa si weyn loo baaray.Si kastaba ha ahaatee, doorka exosomes ee u dhexeeya dulinnada iyo martida cudurka qaba ayaa weli ah mid si liidata loo fahmay.Ilaa hadda, nidaamiyayaal kala duwan, oo ay ku jiraan borotiinnada qarsoodiga ah, ayaa sharraxay hababka bayooloji ee ay dulinnada protozoanku iska caabiyaan weerarka martida iyo sii wadida caabuqa.Dhawaan, waxaa jirtay fikrad sii kordheysa oo ah in microvesicles-ku-xiran protozoan-yada iyo microRNA-yadooda ay la falgalaan unugyada martida loo yahay si ay u abuuraan jawi wanaagsan oo ay ku badbaadaan.Sidaa darteed, daraasado dheeraad ah ayaa loo baahan yahay si loo ogaado xiriirka ka dhexeeya miRNA-yada exosomal ee la beddelay iyo kororka unugyada glioma.Beddelka MicroRNA (Hidoskoobyada kutlada miR-30c-1, miR-125b-2, miR-23b-27b-24-1 iyo miR-17-92) waxay ku xidhan tahay dhiirigeliyaha STAT3 ee macrophages-ka bini'aadamka ee cudurka toxoplasma, waa la habeeyey oo keena anti -apoptosis oo ka jawaabaya caabuqa Toxoplasma gondii 29.Caabuqa toxoplasma wuxuu kordhiyaa muujinta miR-17-5p iyo miR-106b-5p, kuwaas oo la xiriira dhowr cudur oo hyperproliferative 30.Xogtaasi waxay soo jeedinaysaa in miRNA-yada martida loo yahay ee uu maamulo caabuqa Toxoplasma ay yihiin unugyo muhiim u ah badbaadada dulinka iyo cudur-sidaha hab-dhaqanka noole ee martida loo yahay.
MiRNA-yada la beddelo waxay saameyn ku yeelan karaan noocyada kala duwan ee dabeecadaha inta lagu jiro bilawga iyo horumarka unugyada malignantiga ah, oo ay ku jiraan gliomas: isku filnaanta calaamadaha koritaanka, dareen la'aanta calaamadaha koritaanka-hortagga, dhuumashada apoptosis, awoodda taranka ee aan xadidnayn, angiogenesis, duullaan iyo metastasis, iyo caabuq.Glioma, miRNA-yada la beddelay ayaa lagu aqoonsaday dhowr daraasadood oo muujinta muujinta.
Daraasadda hadda jirta, waxaan xaqiijinay heerar sare oo miRNA-21 muujinta unugyada martida loo yahay toxoplasma.miR-21 ayaa loo aqoonsaday mid ka mid ah microRNA-yada ugu badan ee si xad dhaaf ah loo buufiyay ee burooyinka adag, oo ay ku jiraan gliomas, 33 iyo muujinteedu waxay la xiriirtaa heerka glioma.Caddaynta ururintu waxay soo jeedinaysaa in miR-21 uu yahay novel oncogene kaas oo u dhaqma sidii cunsur ka-hortagga apoptotic ee korriinka glioma oo aad u xad-dhaaf ah unugyada iyo balaasmaha cudurrada maskaxda aadanaha.Waxa xiisaha lihi leh, miR-21 la'aanta unugyada glioma iyo unugyadu waxay kicisaa xannibaadda kordhinta unugyada sababtoo ah apoptosis-ku-tiirsan.Falanqaynta bioinformatic ee bartilmaameedyada la saadaaliyay ee miR-21 ayaa daaha ka qaaday hiddo-sideyaal badan oo xakameynaya burooyinka oo la xiriira dariiqyada apoptosis, oo ay ku jiraan dhimashada unugyada barnaamijka 4 (PDCD4), tropomyosin (TPM1), PTEN, iyo sanduuqa fargeetada O1 (FoxO1), oo leh goobta miR-2121..22.38.
FoxO1, oo ah mid ka mid ah qodobbada qoraalka (FoxO), waxay ku lug leedahay horumarinta noocyada kala duwan ee kansarka bini'aadamka waxayna nidaamin kartaa muujinta hiddo-wadaha xakameynaya burooyinka sida p21, p27, Bim, iyo FasL40.FoxO1 waxay xidhi kartaa oo dhaqaajin kartaa horjoogayaasha wareegga unugyada sida p27 si loo xakameeyo koritaanka unugyada.Waxaa intaa dheer, FoxO1 waa raadiye muhiim ah oo tilmaamaya PI3K/Akt waxayna nidaamisaa habab badan oo bayooloji ah sida horumarka wareegga unugyada iyo kala-soocidda unugyada iyada oo loo marayo hawlgelinta qoraalka p2742.
Gabagabadii, waxaan aaminsanahay in exosomal miR-21 oo ka soo jeeda Toxoplasma-infekshanka microglia laga yaabo inay door muhiim ah ka ciyaaraan xakameynta koritaanka unugyada glioma (Jaantus. 7).Si kastaba ha ahaatee, daraasado dheeraad ah ayaa loo baahan yahay si loo helo xiriir toos ah oo ka dhexeeya exosomal miR-21, caabuqa Toxoplasma oo beddelay, iyo korriinka glioma.Natiijooyinkan ayaa la filayaa inay bixiyaan bar bilow ah oo lagu baranayo xiriirka ka dhexeeya caabuqa Toxoplasma iyo dhacdooyinka glioma.
Jaantuska naqshadaynta habka glioma (maskaxda) kansarka kansarka ayaa lagu soo jeediyay daraasaddan.Qoraagu waxa uu ku sawiray PowerPoint 2019 (Microsoft, Redmond, WA).
Dhammaan borotokoollada tijaabada ah ee daraasaddan, oo ay ku jiraan isticmaalka xoolaha, waxay ahaayeen kuwo waafaqsan Xeerka Ilaalinta Xayawaanka ee Jaamacadda Qaranka ee Seoul iyo Guddiga Adeegga ee Tilmaamaha Anshaxa waxaana ansixiyay Guddiga Dib-u-eegista Hay'adaha ee Dugsiga Caafimaadka ee Jaamacadda Qaranka ee Seoul (Lambarka IRB SNU- 150715).-2).Dhammaan habraacyada tijaabada waxaa loo fuliyay si waafaqsan talooyinka ARRIVE.
BV2 mouse microglia iyo U87 unugyada glioma bini'aadamka ayaa lagu dhaqay Dulbecco's Modified Eagle's Medium (DMEM; Welgene, Seoul, Korea) iyo Roswell Park Memorial Institute's Medium (RPMI; Welgene), siday u kala horreeyaan, mid kasta oo ka kooban 10% serum bovine uurjiifka, 4 mM l- glutamine, 0.2 mm penicillin iyo 0.05 mM streptomycin.Unugyada waxaa lagu beeray unugyo leh 5% CO2 at 37°C.Khad kale oo unug glioma ah, U118, ayaa loo isticmaalay marka la barbardhigo unugyada U87.
Si loo go'doomiyo exosomes ee T. gondii-cuduray RH iyo noocyada ME49, T. gondii tachyzoites (RH stress) ayaa laga soo goostay godka caloosha ee jiirarka BALB/c 6-usbuuc jira oo lagu duray 3-4 maalmood ka hor.Tachyzoites waxaa lagu maydhay saddex jeer PBS waxaana lagu nadiifiyay centrifugation 40% Percoll (Sigma-Aldrich, St. Louis, MO, USA)43.Si loo helo tachyzoites ee cadaadiska ME49, jiirarka BALB/c waxaa lagu duray intraperitoneal 20 unug oo unug ah iyo isbeddelka tachyzoite ee cysts ayaa la ururiyey iyada oo la dhaqay godka caloosha ee maalinta 6-8aad ka dib caabuqa (PI).Jiirarka ayaa ku dhacay PBS.ME49 tachyzoites ayaa lagu koray unugyo lagu kabay 100 μg/ml penicillin (Gibco/BRL, Grand Island, NY, USA), 100 μg/ml streptomycin (Gibco/BRL), iyo 5% serum bovine (Lonza, Walkersville, MD) .., USA) at 37 °C iyo 5% carbon dioxide.Ka dib beerista unugyada Vero, ME49 tachyzoites ayaa la marsiiyay laba jeer irbad 25 ka dibna iyada oo loo marayo shaandhada 5 µm si looga saaro qashinka iyo unugyada.Ka dib markii la dhaqay, tachyzoites ayaa dib loo hakiyay PBS44.Fiixyada unugyada Toxoplasma gondii cadaadiska ME49 waxaa lagu habeeyay cirbadeynta intraperitoneal ee cysts oo ka go'ay maskaxda jiirarka C57BL/6 cudurka (Xarunta Xayawaanka ee Orient Bio, Seongnam, Korea).Maskaxda jiirarka qaba ME49 ayaa la goostay 3 bilood ka dib PI waxaana lagu jarjaray mikroskoob hoostooda si loo go'doomiyo fiix.Jiirarka cudurka qaba waxaa lagu hayaa xaalado gaar ah oo ka xor ah cudur-sidaha (SPF) ee Dugsiga Caafimaadka ee Jaamacadda Qaranka ee Seoul.
Wadarta guud ee RNA waxaa laga soo saaray exosomes-ka BV2, unugyo BV2 iyo unugyo la isticmaalayo MiRNeasy Mini Kit (Qiagen, Hilden, Germany) sida ku cad tilmaamaha soo saaraha, oo ay ku jirto wakhtiga soo saarista ee talaabada sareynta.Diiradda RNA waxaa lagu go'aamiyay NanoDrop 2000 spectrophotometer.Tayada microarrays RNA waxaa lagu qiimeeyay iyadoo la isticmaalayo Agilent 2100 bioanalyzer (Agilent Technologies, Amstelveen, Nederland).
DMEM oo leh 10% exosome- saboolka ah FBS waxaa lagu diyaariyey ultracentrifugation at 100,000g 16 saacadood at 4°C waxaana lagu sifeeyey 0.22 µm filter (Nalgene, Rochester, NY, USA).Unugyada BV2, 5 × 105, ayaa lagu dhaqay DMEM oo ay ku jiraan 10% FBS exosome-depleted iyo 1% antibiyootiga 37 ° C iyo 5% CO2.Ka dib 24 saacadood oo ku-soo-bax ah, tachyzoites of stress RH ama ME49 (MOI = 10) ayaa lagu daray unugyada iyo dulin aan soo weerarin ayaa laga saaray saacad gudaheed waxaana lagu buuxiyey DMEM.Exosomes ee unugyada BV2 waxaa lagu go'doomiyay centrifugation kala duwan oo wax laga beddelay, habka ugu isticmaalka badan.Dib ugu celi pellet-ka exosome 300 µl PBS ee RNA ama falanqaynta borotiinka.Isku-duubnaanta exosomes-ka go'doonsan waxaa lagu go'aamiyay iyadoo la adeegsanayo xirmada baaritaanka borotiinka BCA (Pierce, Rockford, IL, USA) iyo NanoDrop 2000 spectrophotometer.
Roobab ka soo da'ay unugyada BV2 ama exosomes oo laga soo qaatay BV2 ayaa lagu daadiyay xalka soo saarista borotiinka ee PRO-PREP ™ (iNtRon Biotechnology, Seongnam, Korea) iyo borotiinada waxaa lagu raray Coomassie buluug dhalaalaya oo leh 10% SDS polyacrylamide gels.Intaa waxaa dheer, borotiinada waxaa loo wareejiyay xuubka PVDF 2 saacadood.Burooyinka reer galbeedka ayaa la ansixiyay iyadoo la isticmaalayo Alix antibody (Tiknoolajiyada Signaling Cell, Beverly, MA, USA) oo ah calaamade exosomal.HRP-conjugated ari-ka-hortagga jiirka IgG (H + L) (Bethyl Laboratories, Montgomery, TX, USA) iyo LAS-1000 oo lagu daray sawir qaadaha sawirka luminescent (Fuji Photographic Film, Tokyo, Japan) ayaa loo isticmaalay sidii unug sare..Gudbinta mikroskoobyada elektarooniga ah ayaa la sameeyay si loo barto cabbirka iyo qaab-dhismeedka exosomes.Exosomes ka go'doonsan unugyada BV2 (6.40 µg/µl) ayaa lagu diyaariyey meshes kaarboon-dahaarka leh oo si xun u wasakheeyay 2% uranyl acetate 1 min.Tijaabooyin la diyaariyey ayaa lagu arkay korantada dardargelinta ee 80 kV iyadoo la adeegsanayo JEOL 1200-EX II (Tokyo, Japan) oo ku qalabaysan kamarad ES1000W Erlangshen CCD (Gatan, Pleasanton, CA, USA).
Exosomes-ka BV2 ka soo jeeda ayaa lagu walaaqay iyadoo la isticmaalayo PKH26 Red Fluorescent Linker Kit (Sigma-Aldrich, St. Louis, MO, USA) 15 daqiiqo heerkulka qolka.Unugyada U87, 2 × 105, oo leh PKH26-calaamadeysan exosomes (casaan) ama aan lahayn exosomes sida xakamaynta xun, ayaa lagu dhajiyay 37 ° C saacadaha 24 ee 5% CO2 incubator.Unugyada unugyada U87 waxaa lagu dhajiyay DAPI (buluug), unugyada U87 waxaa lagu hagaajiyay 4% paraformaldehyde ee 15 min at 4 ° C ka dibna lagu falanqeeyay Leica TCS SP8 STED CW nidaamka microscope confocal (Leica Microsystems, Mannheim, Germany).la arki karo.
cDNA waxaa laga soo saaray siRNA iyadoo la adeegsanayo Mir-X siRNA synthesis first strand synthesis iyo SYBR qRT-PCR gunt (Takara Bio Inc., Shiga, Japan).PCR-ga tirada dhabta ah ee wakhtiga dhabta ah waxaa la sameeyay iyadoo la isticmaalayo iQ5 nidaamka ogaanshaha PCR ee waqtiga dhabta ah (Bio-Rad, Hercules, CA, USA) iyadoo la isticmaalayo asal-raacyo iyo qaabab lagu qasay SYBR Premix.DNA-da waxaa lagu kordhiyey 40 wareeg oo denaturation at 95°C 15s iyo 60°C 60s.Xogta falcelis kasta oo PCR ah ayaa la lafa guray iyadoo la isticmaalayo habka falanqaynta xogta ee iQ™5 software nidaamka indhaha (Bio-Rad).Isbeddellada u dhigma ee muujinta hidde-sidaha ee u dhexeeya hiddo-wadaha la xushay iyo β-actin/siRNA (iyo U6) ayaa la xisaabiyay iyadoo la adeegsanayo habka qalooca caadiga ah.Taxanaha asaasiga ah ee la isticmaalay waxa lagu muujiyay shaxda 1.
Unugyada glioma 3 x 104 U87 ayaa lagu beeray taarikada 96-si fiican waxaana lagu qasi jiray Toxoplasma-cudurka caabuqa ee ka soo jeeda BV2 (50 μg / mL) ama exosomes aan garaacin oo laga soo qaatay BV2 (50 μg / mL) sida kontaroolada 12, 18 iyo 36 .Heerka faafinta unugyada waxaa lagu go'aamiyay iyadoo la isticmaalayo Kit-8 (Dojindo, Kumamoto, Japan) (Tirooyinka dheeriga ah S1-S3) 46.
5-usbuuc jir dheddig BALB/c jiirar qaawan ayaa laga soo iibiyay Orient Bio (Seongnam-si, South Korea) waxaana si gaar ah loogu hayaa qafis nadiif ah heerkulka qolka (22± 2°C) iyo qoyaan (45± 15°C).%) heerkulka qolka (22± 2°C) iyo qoyaan (45± 15%).Wareeg iftiin 12-saac ah iyo wareeg mugdi ah oo 12-saac ah ayaa lagu sameeyay hoosta SPF (Seoul National University School of Medicine Animal Center).Jiirarka ayaa si aan kala sooc lahayn loo kala qaybiyay saddex kooxood oo midkiiba 5 jiir ah waxaana dhammaan kooxaha lagu duray si hoose oo 400 ml PBS ah oo ka kooban 1 x 107 U87 unugyo glioma ah iyo qodobka kobaca ee BD Matrigel ™ (BD Science, Miami, FL, USA).Lix maalmood ka dib duritaanka burada, 200 mg oo exosomes ah oo laga helay unugyada BV2 (oo aan lahayn caabuqa Toxoplasma) ayaa lagu duray goobta burada.Laba iyo labaatan maalmood ka dib caabuqa buro, xajmiga burada ee jiirarka ee koox kasta waxaa lagu qiyaasaa caliper saddex jeer toddobaadkii, mugga burada waxaa lagu xisaabiyay caanaha: 0.5 × (ballaaran) × 2 × dhererka.
Falanqaynta muujinta MicroRNA iyadoo la adeegsanayo miRCURYTM LNA miRNA array, jiilka 7aad wuxuu leeyahay, mmu iyo rno arrays (EXIQON, Vedbaek, Denmark) oo daboolaya 1119 jiirar si wanaagsan loo sifeeyay oo ka mid ah 3100 bini'aadam, jiir iyo jiir miRNA baarayaal qabsaday.Inta lagu jiro nidaamkan, 250 ilaa 1000 ng ee wadarta RNA ayaa laga saaray 5'-fosfate iyadoo lagu daawaynayo kubka mindhicirka fosfatase ee fosfatase oo ay ku xigto calaamadaynta Hy3 dheeha cagaarka ah.Muunadaha la calaamadeeyay ayaa markaa la isku daray iyada oo la raray sawiro yar yar oo la isticmaalayo qalab isku-dhafan (Agilent Technologies, Santa Clara, CA, USA) iyo xirmada iskudhafan (Agilent Technologies).Isku-dhafka ayaa la sameeyay 16 saacadood 56 ° C, ka dibna microarrays ayaa la dhaqay si waafaqsan talooyinka soo-saaraha.Sawirada microarray-ga la farsameeyay ayaa markaa la sawiray iyada oo la isticmaalayo Agilent G2565CA microarray system (Agilent Technologies).Sawirada la sawiray ayaa la soo dhoofiyay iyadoo la adeegsanayo Agilent Feature Extraction software nooca 10.7.3.1 (Agilent Technologies) iyo xoojinta iftiinka sawir kasta ayaa la qiyaasay iyadoo la adeegsanayo faylka GAL ee u dhigma ee borotokoolka Exiqon ee la beddelay.Xogta Microarray ee daraasadda hadda jirta waxaa lagu kaydiyaa xogta GEO ee hoos timaada lambarka gelitaanka GPL32397.
Tilmaamaha muujinta miRNA-yada qaan-gaarka ah ee exosomal ee microglia ee noocyada RH ama ME49 ee qaba Toxoplasma ayaa la falanqeeyay iyadoo la adeegsanayo qalabyo shabakadeed oo kala duwan.miRNA-yada la xidhiidha korriinka burada ayaa lagu aqoonsaday iyadoo la isticmaalayo miRWalk2.0 (http://mirwalk.umm.uni-heidelberg.de) waxaana lagu sifeeyay xoojinta signalka caadiga ah (log2) oo ka weyn 8.0.MiRNA-yada dhexdooda, miRNA-yada kala duwan ayaa la ogaaday inay ka badan yihiin 1.5-laab oo lagu beddelay falanqaynta shaandhaynta miRNA-yada ay beddeleen noocyada RH ama ME49 ee qaba T. gondii.
Unugyada waxaa lagu beeray taarikada lix ceel (3 x 105 unug / ceel) ee opti-MEM (Gibco, Carlsbad, CA, USA) iyadoo la adeegsanayo Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).Unugyada la beddelay ayaa la dhaqan geliyay 6 saacadood ka dibna dhexdhexaadinta ayaa loo beddelay dhexdhexaad cusub oo dhammaystiran.Unugyada ayaa la goostay 24 saacadood ka dib wareejinta.
Falanqaynta tirakoobka waxaa inta badan lagu sameeyay iyadoo la isticmaalayo t-tijaabka Ardayga oo wata software Excel (Microsoft, Washington, DC, USA).Falanqaynta xayawaanka tijaabada ah, ANOVA laba-geesood ah ayaa la sameeyay iyadoo la adeegsanayo software Prism 3.0 (Software GraphPad, La Jolla, CA, USA). P-qiimaha <0.05 ayaa loo tixgaliyay inay yihiin kuwo muhiim ah. P-qiimaha <0.05 ayaa loo tixgaliyay inay yihiin kuwo muhiim ah. Значения P <0,05 считались статистически значимыми. Qiimaha P <0.05 ayaa loo tixgaliyay inay muhiim tahay. P 值< 0.05 被认为具有统计学意义。 P 值 <0.05 Значения P <0,05 считались статистически значимыми. Qiimaha P <0.05 ayaa loo tixgaliyay inay muhiim tahay.
Dhammaan borotokoollada tijaabada ah ee loo adeegsaday daraasaddan waxaa ansixiyay Guddiga Dib-u-eegista Hay'adaha ee Dugsiga Caafimaadka ee Jaamacadda Qaranka ee Seoul (lambarka IRB SNU-150715-2).
The data used in this study are available upon reasonable request from the first author (BK Jung; mulddang@snu.ac.kr). And the microarray data for the current study is deposited in the GEO database under registration number GPL32397.
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