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I-Toxoplasma gondii yi-intracellular protozoan parasite emodareyitha i-microenvironment yenginginya eyosulelekileyo kwaye yaziwa ngokuba inyanyaniswa nesehlo sokukhula kwethumba lobuchopho.Kolu phononongo, siqikelela ukuba i-exosomal miRNA-21 yosulelo lwe-Toxoplasma ikhuthaza ukukhula kwethumba lobuchopho.I-Exosomes evela kwi-Toxoplasma-esulelwe yi-BV2 microglia ibonakaliswe kwaye i-internalization ye-U87 iiseli ze-glioma zaqinisekiswa.Iiprofayili zokubonisa i-Exosomal microRNA zahlalutywa kusetyenziswa uluhlu lwe-microRNA kunye ne-microRNA-21A-5p ehambelana ne-Toxoplasma gondii kunye nokuhlelwa kwe-tumor.Sikwaphande amanqanaba e-mRNA eejene ezihambelana nethumba kwiiseli ze-glioma ze-U87 ngokuguqula amanqanaba e-miR-21 kwi-exosomes kunye nefuthe le-exosomes ekwandeni kweeseli ze-U87 ze-glioma.Kwii-exosomes ze-U87 iiseli ze-glioma ezisuleleke nge-Toxoplasma gondii, ukubonakaliswa kwe-microRNA-21 kwanda kwaye umsebenzi we-antitumor genes (FoxO1, PTEN, kunye ne-PDCD4) iyancitshiswa.I-BV2-derived exosomes esulelwe yi-Toxoplasma yenza ukwanda kweeseli ze-glioma ze-U87.I-Exosomes ikhuthaza ukukhula kweeseli ze-U87 kwimodeli yethumba lempuku.Sicebisa ukuba ukunyuka kwe-exosomal miR-21 kwi-Toxoplasma-infected BV2 microglia inokudlala indima ebalulekileyo njengomgqugquzeli wokukhula kweeseli kwiiseli ze-glioma ze-U87 ngokunciphisa i-antitumor genes.
Kuqikelelwa ukuba ngaphezulu kwe-18.1 yezigidi zeemeko zomhlaza ophambili zafunyaniswa kwihlabathi liphela ngo-2018, malunga ne-297,000 yenkqubo ye-nervous central tumors efunyenwe ngonyaka (1.6% yazo zonke iithumba)1.Uphando lwangaphambili lubonise ukuba izinto ezinobungozi ekuphuhliseni amathumba obuchopho bomntu zibandakanya iimveliso ezahlukeneyo zemichiza, imbali yosapho, kunye nemitha ye-ionizing evela kunyango lwentloko kunye nezixhobo zokuxilonga.Noko ke, oyena nobangela wezi zifo awukaziwa.Malunga ne-20% yazo zonke ii-cancer kwihlabathi jikelele zibangelwa zizifo ezosulelayo, kubandakanywa iintsholongwane, iibhaktheriya kunye ne-parasites3,4.Iintsholongwane ezosulelayo ziphazamisa iindlela zofuzo zeseli yomkhosi, ezifana nokulungiswa kweDNA kunye nomjikelo weseli, kwaye kunokukhokelela ekudumbeni okungapheliyo kunye nomonakalo kumajoni omzimba5.
Iintsholongwane ezosulelayo ezinxulumene nomhlaza womntu zezona zifo zixhaphakileyo zentsholongwane egazini, kubandakanywa iintsholongwane zepilloma yabantu kunye neentsholongwane ze-hepatitis B kunye no-C.Izifunxi-gazi zinokudlala indima enokubakho ekuphuhliseni umhlaza womntu.Iintlobo ezininzi ze-parasite, ezizezi, i-Schistosoma, i-Opishorchis viverrini, i-O. felineus, i-Clonorchis sinensis kunye ne-Hymenolepis nana, ziye zabandakanyeka kwiindidi ezahlukeneyo zomhlaza womntu 6,7,8.
I-Toxoplasma gondii yiprotozoan ye-intracellular elawula i-microenvironment yeeseli zomkhosi osulelekileyo.Esi sifunxi-gazi kuqikelelwa ukuba sosulela malunga nama-30% abemi behlabathi, nto leyo ebeka bonke abantu emngciphekweni9,10.I-Toxoplasma gondii inokosulela amalungu abalulekileyo, kubandakanywa nenkqubo ye-nervous central (CNS), kwaye ibangele izigulo ezinobungozi ezifana ne-meningitis ebulalayo kunye ne-encephalitis, ngakumbi kwizigulane ezingenayo i-immunocompromised9.Nangona kunjalo, i-Toxoplasma gondii inokutshintsha kwakhona indawo engqongileyo yomkhosi osulelekileyo ngokumodareyitha ukukhula kweeseli kunye neempendulo ze-immune kubantu abangenamandla omzimba, okukhokelela ekugcinweni kosulelo olungapheliyo lwe-asymptomatic9,11.Okubangel 'umdla kukuba, kunikwe unxulumano phakathi T. gondii ukuxhaphaka kunye izehlo ithumba ebuchotsheni, ezinye iingxelo zibonisa ukuba in vivo host utshintsho lokusingqongileyo ngenxa yezifo ezingapheliyo T. gondii usulelo zifana microenvironment thumba.
I-Exosomes iyaziwa ngokuba yi-intercellular communicators ehambisa umxholo we-biological, kuquka iiprotheni kunye ne-nucleic acids, ukusuka kwiiseli ezingabamelwane16,17.I-Exosomes inokuchaphazela iinkqubo zebhayoloji ezinxulumene ne-tumor ezifana ne-anti-apoptosis, i-angiogenesis, kunye ne-metastasis kwi-tumor microenvironment.Ngokukodwa, i-miRNAs (i-miRNAs), i-RNAs ezincinci ezingenayo ikhowudi malunga ne-nucleotides ye-22 ubude, zibalulekile i-post-transcriptal gene regulators ezilawula ngaphezu kwe-30% ye-mRNA yabantu ngokusebenzisa i-miRNA-induced silence complex (miRISC).I-Toxoplasma gondii inokuphazamisa iinkqubo zebhayoloji ngokulawula ukubonakaliswa kwe-miRNA kwimikhosi eyosulelekileyo.I-MiRNAs ebambayo iqulethe imiqondiso ebalulekileyo yokulawula iinkqubo zebhayoloji ezibambayo ukuze kuphunyezwe isicwangciso sokusinda se-parasite.Ngaloo ndlela, ukufunda utshintsho kwiprofayile ye-miRNA yomamkeli phezu kosulelo nge-T. gondii kunokusinceda siqonde intsebenziswano phakathi komkhosi kunye no-T. gondii ngokucacileyo ngakumbi.Ewe, uThirugnanam et al.I-15 iphakamise ukuba i-T. gondii ikhuthaza i-brain carcinogenesis ngokuguqula intetho yayo kwi-miRNAs ethile yomkhosi ehambelana nokukhula kwethumba kwaye yafumanisa ukuba i-T. gondii ingabangela i-gliomas kwizilwanyana zokulinga.
Olu phononongo lujolise ekuguqulweni kwe-exosomal miR-21 kwi-host microglia eyosulelwe yi-Toxoplasma BV2.Siye sabona indima enokwenzeka yokuguqulwa kwe-exosomal miR-21 ekukhuleni kweeseli ze-glioma ze-U87 ngenxa yokugcinwa kwi-nucleus ye-FoxO1 / p27, ekujoliswe kuyo kwi-miR-21.
I-Exosomes ephuma kwi-BV2 ifunyenwe ngokusebenzisa i-centrifugation eyahlukileyo kwaye iqinisekiswe ngeendlela ezahlukeneyo zokuthintela ukungcoliseka ngamacandelo eselula okanye ezinye ii-vesicles.I-SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ibonise iipatheni ezahlukileyo phakathi kweeprotheyini ezikhutshwe kwiiseli ze-BV2 kunye ne-exosomes (Umfanekiso 1A), kunye neesampuli zavavanywa ubukho be-Alix, eyahlalutywa ngokuchithwa kwe-Western kweempawu zeprotheni exosomal kwi.Ukubhalwa kwe-Alix kwafunyanwa kwiiprotheni eziphumayo kodwa kungekhona kwi-BV2 ye-cell lysate proteins (Fig. 1B).Ukongeza, i-RNA ecocekileyo evela kwi-exosomes evela kwi-BV2 yahlalutywa kusetyenziswa i-bioanalyzer.I-18S kunye ne-28S i-ribosomal subunits ayizange ibonwe kwi-exosomal RNA iphethini yokufuduka, ebonisa ukucoceka okuthembekileyo (Umfanekiso 1C).Ekugqibeleni, i-electron microscopy yokudluliselwa ibonise ukuba i-exosomes ebonwayo yayimalunga ne-60-150 nm ngobukhulu kwaye yayinesakhiwo esifana nekomityi eqhelekileyo ye-exosome morphology (Fig. 1D).
Ukubonakaliswa kwee-exosomes ezivela kwiiseli ze-BV2.(A) Iphepha lephepha ledatha yokhuseleko.Iiprotheyini zaye zahlukaniswa kwiiseli ze-BV2 okanye i-exosomes evela kwi-BV2.Iipateni zeprotheyini ziyahluka phakathi kweeseli kunye ne-exosomes.(B) Uhlalutyo lweblot lwaseNtshona lwe-exosomal marker (Alix).(C) Ukuvavanywa kwe-RNA ecocekileyo evela kwiiseli ze-BV2 kunye ne-BV2 ephuma exosomes usebenzisa i-bioanalyzer.Ngaloo ndlela, i-18S kunye ne-28S i-ribosomal subunits kwiiseli ze-BV2 azifane zifumaneke kwi-exosomal RNA.(D) Ukuhanjiswa kwe-electron microscopy kubonise ukuba i-exosomes ehlukanisiwe kwiiseli ze-BV2 zaye zangcoliswa kakubi nge-2% ye-acetate ye-uranyl.I-Exosomes imalunga ne-60-150 nm ngobukhulu kunye ne-cup-shaped (Ingoma kunye neJung, idatha engapapashwa).
Ukufakwa ngaphakathi kweeseli ze-BV2 eziphuma kwiiseli ze-glioma zomntu ze-U87 zabonwa kusetyenziswa i-confocal microscopy.I-PKH26 ebhalwe i-exosomes ifakwe kwi-cytoplasm yeeseli ze-U87.I-Nuclei yahlanjululwa nge-DAPI (Umfanekiso we-2A), ebonisa ukuba i-exosomes ephuma kwi-BV2 inokuthi ifakwe ngaphakathi ngamaseli abamba kwaye ichaphazele indawo yeeseli zabamkeli.
Ukufakwa ngaphakathi kwee-exosomes ezivela kwi-BV2 kwiiseli ze-glioma ze-U87 kunye ne-BV2-derived exosomes ezosulelwe yi-Toxoplasma RH zenza ukwanda kweeseli ze-glioma ze-U87.(A) Ii-exosomes ezigutyungelwe ziiseli ezi-U87 ezilinganiswa nge-confocal microscopy.Iiseli ze-glioma ze-U87 zifakwe kwi-exosomes ezibhalwe nge-PKH26 (ezibomvu) okanye ngaphandle kokulawula iiyure ze-24.I-nuclei yahlanjululwa nge-DAPI (eluhlaza okwesibhakabhaka) kwaye emva koko yabonwa phantsi kwe-microscope ye-confocal (i-scale bar: 10 μm, x 3000).(B) Ukwanda kweeseli ze-glioma ze-U87 kwagqitywa ngovavanyo lokwandisa iiseli.Iiseli ze-glioma ze-U87 zaphathwa nge-exosomes ngexesha elibonisiweyo. *P <0.05 ifunyenwe ngovavanyo loMfundi. *P <0.05 ifunyenwe ngovavanyo loMfundi. *P < 0,05 получено по t-критерию Стьюдента. *P <0.05 ngovavanyo loMfundi. *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 efunyenwe kusetyenziswa Uvavanyo loMfundi.
Emva kokuqinisekisa ukufakwa kwangaphakathi kwee-exosomes ezivela kwi-BV2 kwiiseli ze-glioma ze-U87, senze i-cell proliferation assays ukuphanda indima ye-BV2-derived Toxoplasma-derived exosomes ekuphuhliseni iiseli ze-glioma zabantu.Unyango lweeseli ze-U87 ezine-exosomes ezivela kwiiseli ze-BV2 ezine-T. gondii ezosulelekileyo zibonise ukuba i-T. gondii-esulelwe yi-BV2-derived exosomes yabangela ukwanda okukhulu kweeseli ze-U87 xa kuthelekiswa nokulawula (Umfanekiso 2B).
Ukongezelela, ukukhula kweeseli ze-U118 kuneziphumo ezifanayo njenge-U87, njengoko i-Toxoplasma ivuselela i-exosomes ibangele amanqanaba aphezulu okunyuka (idatha engaboniswa).Ngokusekelwe kule datha, sinokubonisa ukuba i-BV2-derived Toxoplasma-exosomes-infected exosomes idlala indima ebalulekileyo kwi-glioma cell proliferation.
Ukuphanda isiphumo se-Toxoplasma-esulelwe yi-BV2-ephuma kwi-exosomes ekuphuhlisweni kwethumba, safaka iiseli ze-glioma ze-U87 kwiimpuku ezinqunu zemodeli ye-xenograft kunye ne-exosomes ephuma kwi-BV2 okanye i-RH-esulelwe yi-BV2 ephumayo.Emva kokuba i-tumor ibonakale emva kweveki ye-1, iqela ngalinye lokulinga leempuku ze-5 lahlulwa ngokwesayizi ye-tumor ukumisela indawo yokuqala efanayo, kwaye ubukhulu be-tumor bulinganiswe kwiintsuku ze-22.
Kwiigundane kunye ne-U87 imodeli ye-xenograft, ubukhulu becala obukhulu be-tumor kunye nobunzima babonwa kwi-BV2-derived RH-infected exosome group ngosuku lwe-22 (Fig. 3A, B).Kwelinye icala, kwakungekho mahluko ubalulekileyo kubungakanani bethumba phakathi kweqela le-exosome eliphuma kwi-BV2 kunye neqela lolawulo emva konyango oluphumayo.Ukongeza, iimpuku zitofwe ngeeseli ze-glioma kunye ne-exosomes ebonakalayo ebonisa umthamo omkhulu wethumba kwiqela le-RH-esulelwe yi-BV2-derived exosomes (Fig. 3C).Ezi ziphumo zibonisa ukuba i-BV2-derived Toxoplasma-exosomes-exosomes ibangela ukukhula kwe-glioma kwimodeli yethumba lempuku.
I-Oncogenesis (AC) ye-BV2-derived exosomes kwi-U87 xenograft model mouse.Ubungakanani be-Tumor (A) kunye nobunzima (B) bonyuswe kakhulu kwi-BALB / c iigundane ze-nude eziphathwe nge-RH-exosomes exosomes evela kwi-BV2.I-BALB/c iimpuku ezinqunu (C) zatofwa ngaphantsi kwe-1 x 107 iiseli ze-U87 zixhonywe kumxube weMatrigel.Kwiintsuku ezintandathu emva kokutofa, i-100 μg ye-BV2-derived exosomes yaphathwa kwiigundane.Ubungakanani bethumba kunye nobunzima balinganiswa ngeentsuku ezichaziweyo nasemva kombingelelo, ngokulandelelanayo. *P <0.05. *P <0.05. * Р < 0,05. *P <0.05. *P <0.05. *P <0.05. * Р < 0,05. *P <0.05.
Idatha ibonise ukuba i-37 miRNAs (i-16 i-overexpressed kunye ne-21 ephantsi) ehambelana nokukhuseleka okanye ukuphuhliswa kwe-tumor yatshintshwa kakhulu kwi-microglia emva kokusuleleka kwi-Toxoplasma RH strain (Fig. 4A).Amanqanaba okuthetha ahambelanayo we-miR-21 phakathi kwe-miRNAs etshintshiweyo yaqinisekiswa yi-RT-PCR yexesha langempela kwii-exosomes ezivela kwi-BV2, i-exosomes ephathwa nge-BV2 kunye neeseli ze-U87.Ukubonakaliswa kwe-miR-21 kubonise ukwanda okukhulu kwee-exosomes ezivela kwiiseli ze-BV2 ezosulelwe yi-Toxoplasma gondii (i-RH strain) (umzobo 4B).Amanqanaba okubonakaliswa okuhambelanayo kwe-miR-21 kwi-BV2 kunye neeseli ze-U87 zonyuka emva kokuthathwa kwee-exosomes ezitshintshileyo (umzobo 4B).Amanqanaba ahlobeneyo we-miR-21 intetho kwizicubu zobuchopho zezigulane ezinethumba kunye neempuku ezosulelwe yi-Toxoplasma gondii (i-ME49 strain) yayiphezulu kunolawulo, ngokulandelelana (umzobo 4C).Ezi ziphumo zinxibelelana nomahluko phakathi kwamanqanaba okuchazwa kwe-microRNAs eqikelelweyo kunye neqinisekisiweyo kwi-vitro nakwi-vivo.
Utshintsho kwintetho ye-exosomal miP-21a-5p kwi-microglia esulelwe yi-Toxoplasma gondii (RH).(A) Ubonisa utshintsho oluphawulekayo kwi-siRNA ehambelana nokukhuseleka okanye ukuphuhliswa kwe-tumor emva kokusuleleka kwe-T. gondii RH.(B) Amanqanaba e-Relative miR-21 expression afunyenwe yi-RT-PCR yexesha langempela kwii-exosomes ezivela kwi-BV2, i-BV2-treated exosomes, kunye neeseli ze-U87.(C) Amanqanaba okuchazwa kwe-miR-21 afunyenwe kwizicubu zengqondo zezigulane ze-tumor (N = 3) kunye neegundane ezosulelwe yi-Toxoplasma gondii (i-ME49 strain) (N = 3). *P <0.05 ifunyenwe ngovavanyo loMfundi. *P <0.05 ifunyenwe ngovavanyo loMfundi. *P < 0,05 было получено с помощью t-критерия Стьюдента. *P <0.05 ifunyenwe kusetyenziswa uvavanyo loMfundi. *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 efunyenwe kusetyenziswa Uvavanyo loMfundi.
I-Exosomes evela kwiiseli ze-BV2 ezine-RH eziye zakhokelela ekukhuleni kwe-gliomas kwi-vivo kunye ne-vitro (Umfanekiso 2, 3).Ukubona i-mRNA echaphazelekayo, sihlolisise amanqanaba e-mRNA ye-antitumor target genes, ibhokisi ye-forkhead O1 (FoxO1), i-PTEN, kunye ne-programmed cell death 4 (PDCD4) kwiiseli ze-U87 ezisuleleke nge-exosomes ezivela kwi-BV2 okanye i-RH BV2.Uhlalutyo lwe-Bioinformatics lubonise ukuba iintlobo ezininzi ezinxulumene ne-tumor, kuquka i-FoCO1, i-PTEN, kunye ne-PDCD4 genes, zineendawo zokubopha i-miR-2121,22.Amanqanaba e-mRNA ye-antitumor target genes aye ancitshiswa kwi-RH-esulelwe yi-BV2-ephuma kwi-exosomes xa kuthelekiswa ne-BV2-derived exosomes (Fig. 5A).I-FoxO1 ibonise amanqanaba eprotheyini ancitshisiweyo kwi-RH-esulelwe yi-BV2 ephuma kwi-exosomes xa kuthelekiswa ne-BV2-derived exosomes (Figure 5B).Ngokusekelwe kwezi ziphumo, sinokuqinisekisa ukuba i-exosomes ephuma kwi-RH-eyosulelwe yi-BV2 inciphisa i-anti-oncogenic genes, igcina indima yabo ekukhuleni kwethumba.
I-Toxoplasma ye-RH-esulelwe yi-BV2-ephuma kwi-exosomes ibangela ukunyanzeliswa kwee-antitumor genes kwiiseli ze-glioma ze-U87 nge-Toxoplasma RH-esulelwe yi-BV2-ephuma e-exosomes.(A) I-PCR yexesha langempela le-Foxo1, i-PTEN kunye ne-PDCD4 ibonakaliso kwii-exosomes ezivela kwi-T. gondii i-RH-esulelekileyo ye-BV2 xa kuthelekiswa ne-PBS exosomes.I-β-actin mRNA yasetyenziswa njengolawulo.(B) Inkcazo ye-FoxO1 inqunywe yi-Western blotting kunye nedatha ye-densitometry ihlolwe ngokwezibalo kusetyenziswa inkqubo ye-ImageJ. *P <0.05 ifunyenwe ngovavanyo loMfundi. *P <0.05 ifunyenwe ngovavanyo loMfundi. *P < 0,05 было получено с помощью t-критерия Стьюдента. *P <0.05 ifunyenwe kusetyenziswa uvavanyo loMfundi. *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 efunyenwe kusetyenziswa Uvavanyo loMfundi.
Ukuqonda umphumo we-miP-21 kwi-exosomes kwi-tumor-associated gene regulation, iiseli ze-U87 zadluliselwa kunye ne-inhibitor ye-miP-21 usebenzisa i-Lipofectamine 2000 kwaye iiseli zavunwa iiyure ze-24 emva kokudluliselwa.I-FoxO1 kunye ne-p27 amanqanaba okuchazwa kwiiseli ezidluliselwe nge-miR-21 inhibitors zafaniswa neeseli eziphathwa nge-BV2-derived exosomes usebenzisa i-qRT-PCR (Fig. 6A, B).Ukutshintshwa kwe-miR-21 inhibitor kwiiseli ze-U87 zinciphise kakhulu i-FoxO1 kunye ne-p27 inkcazo (FIG. 6).
I-RH-esulelwe yi-exosomal ye-BV2-ephuma kwi-miP-21 iguqule i-FoxO1 / p27 intetho kwiiseli ze-glioma ze-U87.Iiseli ze-U87 zadluliselwa nge-miP-21 inhibitor usebenzisa i-Lipofectamine 2000 kwaye iiseli zavunwa iiyure ze-24 emva kokudluliselwa.I-FoxO1 kunye ne-p27 amanqanaba okuchaza kwiiseli ezidluliselwe nge-miR-21 inhibitors zafaniswa namanqanaba kwiiseli eziphathwa nge-BV2-derived exosomes usebenzisa i-qRT-PCR (A, B).
Ukuze ubaleke kwimpendulo yokhuselo lomkhosi, isifunxi-gazi seToxoplasma siguquka sibe sisihlunu sethishu.Benza ii-parasitize izihlunu ezahlukeneyo, kubandakanya ingqondo, intliziyo, kunye nesihlunu samathambo, kubo bonke ubomi bomsingathi kwaye bamodareyitha impendulo yokhuselo lomkhosi.Ukongeza, banokulawula umjikelo weseli kunye ne-apoptosis yeeseli ezibambayo, ukukhuthaza ukwanda kwazo14,24.I-Toxoplasma gondii kakhulu yosulela iiseli ze-dendritic, i-neutrophils, kunye ne-monocyte / macrophage lineage, kuquka i-brain microglia.I-Toxoplasma gondii ibangela ukuhlukana kwe-macrophages ye-phenotype ye-M2, ichaphazela ukuphulukiswa kwesilonda emva kokusuleleka kwe-pathogen, kwaye idibene ne-hypervascularization kunye ne-granulomatous fibrosis.Le pathogenesis yokuziphatha yosulelo lwe-Toxoplasma inokunxulumana namanqaku anxulumene nophuhliso lwethumba.Imekobume enobutshaba elawulwa yiToxoplasma inokufana ne-precancer ehambelana nayo.Ngoko ke, kunokucingelwa ukuba usulelo lwe-Toxoplasma kufuneka lube negalelo ekuphuhlisweni kwamathumba ebuchosheni.Ngapha koko, amazinga aphezulu osulelo lwe-Toxoplasma axelwe kwiserum yezigulana ezinamathumba obuchopho ahlukeneyo.Ukongeza, i-Toxoplasma gondii inokuba yenye i-carcinogenic effect kwaye isebenze ngokubambisana ukunceda ezinye ii-carcinogens ezosulelayo ziphuhlise amathumba engqondo.Kule nkalo, kuyafaneleka ukuba uqaphele ukuba i-P. falciparum kunye ne-Epstein-Barr virus synergistically igalelo ekubunjweni kwe-lymphoma ye-Burkitt.
Indima ye-exosomes njengabalawuli kwicandelo lophando lomhlaza sele iphandwe ngokubanzi.Nangona kunjalo, indima ye-exosomes phakathi kwee-parasites kunye neenginginya ezosulelekileyo zihlala zingaqondwa kakuhle.Ukuza kuthi ga ngoku, abalawuli abahlukeneyo, kubandakanywa neeprotheyini ezifihliweyo, baye bachaza iinkqubo zebhayoloji apho i-protozoan parasites imelana nokuhlaselwa komkhosi kwaye iqhubekisela phambili usulelo.Kutshanje, kukho ingcamango ekhulayo yokuba i-microvesicles ehambelana ne-protozoan kunye nee-microRNAs zazo zinxibelelana neeseli ezibambayo ukuze zenze indawo efanelekileyo yokuphila kwazo.Ke ngoko, izifundo ezongezelelweyo ziyafuneka ukufumanisa ubudlelwane phakathi kwe-exosomal miRNAs etshintshiweyo kunye nokwanda kweeseli ze-glioma.Ukuguqulwa kwe-MicroRNA (i-cluster gene miR-30c-1, miR-125b-2, miR-23b-27b-24-1 kunye ne-miR-17-92) ibophelela kumgqugquzeli we-STAT3 kwi-macrophages yabantu abosulelwe yi-toxoplasma, ilawulwa kwaye ibangela i-anti. -i-apoptosis ekuphenduleni ukusuleleka kwe-Toxoplasma gondii 29.Ukusuleleka kwe-Toxoplasma kwandisa ukubonakaliswa kwe-miR-17-5p kunye ne-miR-106b-5p, ehambelana nezifo ezininzi ze-hyperproliferative 30.Ezi datha zibonisa ukuba ii-miRNAs ezibambayo zilawulwa lusulelo lwe-Toxoplasma ziamolekyuli ezibalulekileyo zokusinda kwi-parasite kunye ne-pathogenesis kwindlela yokuziphatha yebhayoloji.
Ii-miRNA ezitshintshileyo zinokuphembelela iintlobo ezahlukeneyo zokuziphatha ngexesha lokuqaliswa kunye nokuqhubekeka kweeseli ezinobungozi, kubandakanya i-gliomas: ukuzanelisa kweempawu zokukhula, ukungabi naluvelwano kwimiqondiso ethintela ukukhula, ukuphepha kwe-apoptosis, amandla okuphindaphinda okungapheliyo, i-angiogenesis, ukuhlasela kunye ne-metastasis, kunye nokudumba.Kwi-glioma, ii-miRNA ezitshintshiweyo zichongiwe kwizifundo ezininzi zokuchaza iprofayili.
Kuphononongo lwangoku, siqinisekisile amanqanaba aphezulu e-miRNA-21 intetho kwiiseli ze-toxoplasma ezosulelwe yi-toxoplasma.I-miR-21 ichongiwe njengenye yezona zinto zixhaphake kakhulu kwi-microRNAs kumathumba aqinileyo, kubandakanya i-gliomas, i-33 kunye nokubonakaliswa kwayo kuhambelana nebakala le-glioma.Ubungqina obuninzi bubonisa ukuba i-miR-21 yinoveli ye-oncogene esebenza njenge-anti-apoptotic factor ekukhuleni kwe-glioma kwaye igxininiswe kakhulu kwizicubu kunye neplasma yobuchopho bomntu.Okubangela umdla kukuba, ukungasebenzi kwe-miR-21 kwiiseli ze-glioma kunye nezicubu zibangela uthintelo lokwanda kweeseli ngenxa ye-apoptosis exhomekeke kwi-caspase.Uhlalutyo lwe-Bioinformatic ye-miR-21 eqikelelweyo ekujoliswe kuyo ibonise i-tumor suppressor genes ezininzi ezinxulumene neendlela ze-apoptosis, kubandakanywa ukufa kweseli ehleliweyo 4 (PDCD4), i-tropomyosin (TPM1), i-PTEN, kunye nebhokisi ye-O1 (FoxO1), kunye ne-miR-2121 indawo yokubopha..22.38.
I-FoxO1, njengenye yezinto ezishicilelweyo (i-FoxO), ibandakanyeka kuphuhliso lweentlobo ezahlukeneyo zomhlaza womntu kwaye inokulawula ukubonakaliswa kwe-tumor suppressor genes ezifana ne-p21, p27, Bim, kunye ne-FasL40.I-FoxO1 inokubopha kwaye isebenze ii-inhibitors ze-cell cycle ezifana ne-p27 ukucinezela ukukhula kweeseli.Ngaphezu koko, i-FoxO1 iyimpembelelo ephambili ye-PI3K / Akt yokubonisa kwaye ilawula iinkqubo ezininzi zebhayoloji ezifana nokuqhubela phambili komjikelo weseli kunye nokwahlulahlula kweeseli ngokusebenzisa i-activation ye-p2742.
Ekugqibeleni, sikholelwa ukuba i-exosomal miR-21 evela kwi-microglia ene-Toxoplasma-infected ingadlala indima ebalulekileyo njengokulawula ukukhula kweeseli ze-glioma (umzobo 7).Nangona kunjalo, uphononongo olongezelelweyo luyafuneka ukufumana ikhonkco elithe ngqo phakathi kwe-exosomal miR-21, olutshintshileyo usulelo lwe-Toxoplasma, kunye nokukhula kwe-glioma.Ezi ziphumo kulindeleke ukuba zinike isiqalo sokufunda ubudlelwane phakathi kosulelo lwe-Toxoplasma kunye nesiganeko se-glioma.
Umzobo oqingqiweyo wendlela ye-glioma (ingqondo) ye-carcinogenesis icetywayo kolu phononongo.Umbhali uzoba kwiPowerPoint 2019 (Microsoft, Redmond, WA).
Zonke iiprothokholi zokulinga kolu phononongo, kubandakanywa nokusetyenziswa kwezilwanyana, zihambelana neSeoul National University Animal Care kunye neKomiti yeKomiti yoMsebenzi yeSikhokelo sokuziphatha kwaye zavunywa yiBhodi yokuHlola yeZiko yeSeoul National University School of Medicine (inombolo ye-IRB SNU- 150715).-2).Zonke iinkqubo zovavanyo zenziwe ngokuhambelana neengcebiso ze-ARRIVE.
I-BV2 mouse microglia kunye ne-U87 human glioma cells zakhuliswa kwi-Dulbecco's Modified Eagle's Medium (DMEM; Welgene, Seoul, Korea) kunye neRoswell Park Memorial Institute's Medium (RPMI; Welgene), ngokulandelelanayo, nganye iqulethe i-10% ye-fetus bovine serum, 4 mM l- glutamine, 0.2 mM penicillin kunye 0.05 mM streptomycin.Iiseli zakhuliswa kwi-incubator ene-5% CO2 kuma-37°C.Enye i-glioma cell line, i-U118, isetyenziselwe ukuthelekisa neeseli ze-U87.
Ukwahlula i-exosomes kwi-T. gondii-esulelwe yi-RH kunye ne-ME49 strains, i-T. gondii tachyzoites (i-RH strain) yavunwa kwi-cavity yesisu se-6-iveki ye-BALB / c iigundane ezifakwe kwii-3-4 iintsuku ngaphambili.I-Tachyzoites yahlanjwa kathathu nge-PBS kwaye yahlanjululwa yi-centrifugation kwi-40% Percoll (Sigma-Aldrich, St. Louis, MO, USA)43.Ukufumana i-tachyzoites yoxinzelelo lwe-ME49, i-BALB / c iigundane zifakwe kwi-intraperitoneally kunye ne-20 ye-tissue cysts kunye nokuguqulwa kwe-tachyzoite kwi-cysts yaqokelelwa ngokuhlamba isisu esiswini ngosuku lwe-6-8 emva kokusuleleka (PI).Iimpuku ezosulelwe yi-PBS.I-ME49 tachyzoites yakhuliswa kwiiseli zongezwa nge-penicillin ye-100 μg / ml (Gibco/BRL, Grand Island, NY, USA), 100 μg/ml streptomycin (Gibco/BRL), kunye ne-5% ye-fetal bovine serum (Lonza, Walkersville, MD) .., USA) kwi-37 °C kunye ne-5% ye-carbon dioxide.Emva kokulinywa kwiiseli zeVero, i-ME49 tachyzoites yagqithiswa kabini ngenaliti yegeji ye-25 kwaye emva koko nge-5 µm yokucoca ukususa inkunkuma kunye neeseli.Emva kokuhlamba, i-tachyzoites yaphinda yamiswa kwi-PBS44.I-cysts ye-tissue ye-Toxoplasma gondii strain ME49 igcinwe ngenaliti ye-intraperitoneal ye-cysts ehlukanisiwe kwingqondo ye-C57BL / i-6 yeegundane ezichaphazelekayo (i-Orient Bio Animal Centre, i-Seongnam, eKorea).Ubuchopho beempuku ezosulelwe yi-ME49 zavunwa emva kweenyanga ezi-3 ze-PI kwaye zancinwa phantsi kwe-microscope ukwahlula ama-cysts.Iimpuku ezosulelekileyo zagcinwa phantsi kweemeko ezikhethekileyo ezingenasifo (i-SPF) kwiSikolo seYunivesithi yaseSeoul yeSizwe yezoNyango.
I-RNA iyonke ikhutshwe kwi-BV2-derived exosomes, iiseli ze-BV2 kunye nezicubu ezisebenzisa i-miRNeasy Mini Kit (Qiagen, Hilden, eJamani) ngokwemiyalelo yomenzi, kubandakanywa nexesha lokufakwa kwisinyathelo se-lution.I-concentration ye-RNA inqunywe kwi-NanoDrop 2000 spectrophotometer.Umgangatho we-RNA microarrays uhlolwe kusetyenziswa i-Agilent 2100 bioanalyzer (Agilent Technologies, Amstelveen, Netherlands).
I-DMEM ene-10% ye-exosome-poor FBS yalungiswa nge-ultracentrifugation kwi-100,000g kwiiyure ze-16 kwi-4 ° C kwaye ihluzwe ngesihluzo se-0.22 µm (Nalgene, Rochester, NY, USA).Iiseli ze-BV2, i-5 × i-105, zikhuliswe kwi-DMEM equkethe i-10% ye-FBS exosome kunye ne-1% ye-antibiotics kwi-37 ° C kunye ne-5% CO2.Emva kweeyure ezingama-24 zokufukamela, i-tachyzoites yoxinzelelo lwe-RH okanye i-ME49 (MOI = 10) yongezwa kwiiseli kunye nee-parasites ezingahlaseli zisuswe kwiyure kwaye zizaliswe nge-DMEM.Ii-exosomes ezivela kwiiseli ze-BV2 zaye zahlukaniswa ngokuguqulwa kwe-centrifugation eguquliweyo, eyona ndlela isetyenziswa kakhulu.Phinda umise i-pellet exosome kwi-300 µl PBS ye-RNA okanye uhlalutyo lweprotheni.Ukuxinwa kwee-exosomes ezizimeleyo kwagqitywa ngokusebenzisa i-BCA protein assay kit (Pierce, Rockford, IL, USA) kunye ne-NanoDrop 2000 spectrophotometer.
Amanzi aphuma kwiiseli ze-BV2 okanye ii-exosomes ezithathwe kwi-BV2 zaye zatsalwa kwisisombululo seprotein ye-PRO-PREP™ (iNtRon Biotechnology, Seongnam, Korea) kwaye iiproteni zalayishwa kwi-Coomassie eluhlaza okwesibhakabhaka enemibala eyi-10% yeejeli ze-SDS polyacrylamide.Ukongezelela, iiprotheni zathunyelwa kwiimbrane ze-PVDF kwiiyure ze-2.Amachaphaza aseNtshona aye aqinisekiswa kusetyenziswa i-antibody ye-Alix (iTekhnoloji yokuSawula iCell, i-Beverly, i-MA, i-USA) njengesiphawuli se-exosomal.I-HRP-conjugated ibhokhwe ye-anti-mouse IgG (H + L) (Bethyl Laboratories, Montgomery, TX, USA) kunye ne-LAS-1000 kunye ne-luminescent image analyzer (iFuji Photographic Film, Tokyo, Japan) yasetyenziswa njenge-antibody yesibini..Ukudluliselwa kwe-electron microscopy kwenziwa ukufunda ubungakanani kunye ne-morphology ye-exosomes.I-Exosomes ehlukanisiwe kwiiseli ze-BV2 (i-6.40 µg / µl) zalungiswa kwi-carbon-coated meshes kunye ne-2% ye-uranyl acetate ye-1 min.Iisampulu ezilungisiweyo zabonwa kumbane okhawulezayo we-80 kV kusetyenziswa iJEOL 1200-EX II (eTokyo, eJapan) exhotyiswe ngekhamera ye-ES1000W Erlangshen CCD (Gatan, Pleasanton, CA, USA).
I-BV2-derived exosomes yachithwa kusetyenziswa i-PKH26 Red Fluorescent Linker Kit (Sigma-Aldrich, St. Louis, MO, USA) imizuzu ye-15 kwiqondo lokushisa.Iiseli ze-U87, i-2 × 105, kunye ne-PKH26 ebhalwe i-exosomes (ebomvu) okanye ayikho i-exosomes njengolawulo olubi, zifakwe kwi-37 ° C kwiiyure ezingama-24 kwi-5% CO2 incubator.I-nuclei ye-cell ye-U87 yahlanjululwa nge-DAPI (eluhlaza okwesibhakabhaka), iiseli ze-U87 zilungiswe kwi-4% ye-paraformaldehyde ye-15 min kwi-4 ° C kwaye emva koko ihlalutye kwi-Leica TCS SP8 STED CW confocal microscope system (Leica Microsystems, Mannheim, Germany).ebonakalayo.
I-cDNA yenziwe yavela kwi-siRNA isebenzisa i-Mir-X siRNA yokuqala ye-strand synthesis kunye ne-SYBR qRT-PCR kit (Takara Bio Inc., Shiga, Japan).Ixesha langempela lobungakanani be-PCR lwenziwa kusetyenziswa inkqubo ye-iQ5 yexesha langempela lokufumanisa i-PCR (i-Bio-Rad, i-Hercules, i-CA, i-USA) isebenzisa ii-primers kunye neetemplates ezixutywe kunye ne-SYBR Premix.I-DNA yandiswe kwimijikelo engama-40 ye-denaturation kwi-95 ° C kwi-15 s kunye ne-annealing kwi-60 ° C kwi-60 s.Idatha evela kwimpendulo ye-PCR nganye yahlalutywa kusetyenziswa imodyuli yocazululo lwedatha ye-iQ™5 isoftware yenkqubo ye-optical system (Bio-Rad).Utshintsho oluhambelanayo kwi-gene expression phakathi kwe-target target genes kunye ne-β-actin/siRNA (kunye ne-U6) zibalwe kusetyenziswa indlela eqhelekileyo yegophe.Ulandelelwano lwe-primer olusetyenzisiweyo luboniswe kwiThebhile 1.
I-3 x 104 i-U87 iiseli ze-glioma zifakwe kwi-96-well plates kwaye zixutywe kunye ne-Toxoplasma-infected exosomes evela kwi-BV2 (50 μg / mL) okanye i-nonpulse exosomes evela kwi-BV2 (50 μg / mL) njengolawulo kwi-12, iiyure ze-18 kunye ne-36 .Izinga lokunyuka kweeseli linqunywe ngokusebenzisa i-Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) (Amanani abongezelelweyo S1-S3) 46.
I-BALB / c iigundane ezinqunu zabasetyhini zathengwa kwi-Orient Bio (Seongnam-si, South Korea) kwaye zigcinwe nganye kwiikheji ezinyumba kwiqondo lokushisa (22 ± 2 ° C) kunye nokufuma (45 ± 15 ° C).%) kwiqondo lobushushu begumbi (22±2°C) kunye nokufuma (45±15%).Umjikelezo wokukhanya weeyure ze-12 kunye ne-12-umjikelezo omnyama weeyure zenziwa phantsi kwe-SPF (i-Seoul National University School of Medicine Animal Centre).Iigundane zahlulwe ngokungaqhelekanga zibe ngamaqela amathathu eegundane ze-5 nganye kwaye onke amaqela afakwe ngaphantsi kwe-400 ml ye-PBS equkethe iiseli ze-glioma ze-1 x 107 ze-U87 kunye nokukhula kwe-BD Matrigel ™ (BD Science, Miami, FL, USA).Kwiintsuku ezintandathu emva kokutofa kwethumba, i-200 mg ye-exosomes ephuma kwiiseli ze-BV2 (kunye / ngaphandle kosulelo lwe-Toxoplasma) zafakwa kwindawo yethumba.Kwiintsuku ezingamashumi amabini anesibini emva kokusuleleka kwe-tumor, ubukhulu besisu seegundane kwiqela ngalinye bulinganiswa ne-caliper kathathu ngeveki, kwaye umthamo we-tumor ubalwa ngefomula: 0.5 × (ububanzi) × 2 × ubude.
Uhlalutyo lwentetho ye-MicroRNA kusetyenziswa i-miRCURY TM LNA miRNA array, isizukulwana se-7 sine, i-mmu kunye ne-rno arrays (EXIQON, Vedbaek, Denmark) egquma i-1119 yeempuku ezinophawu oluhle phakathi kwe-3100 yabantu, i-mouse kunye ne-rat miRNA yokubamba i-probes.Ngethuba le nkqubo, i-250 ukuya kwi-1000 ng ye-RNA iyonke yasuswa kwi-5'-phosphate ngonyango ngethole le-alkaline yamathumbu e-alkaline phosphatase elandelwa ngokubhala ngedayi ye-Hy3 eluhlaza.Iisampulu ezilebhile zithe zaxutywa ngokulayisha izilayidi ze-microarray kusetyenziswa ikhithi yegumbi lokuxutywa (Agilent Technologies, Santa Clara, CA, USA) kunye nekhithi yesilayidi esixutyiweyo (Agilent Technologies).I-Hybridization yenziwa kwiiyure ze-16 kwi-56 ° C, ngoko i-microarrays yahlanjwa ngokuhambelana neziphakamiso zomenzi.Izilayidi ze-microarray ezicutshungulweyo zaye zaskenwa kusetyenziswa inkqubo ye-Agilent G2565CA microarray scanner (Agilent Technologies).Imifanekiso eskeniweyo iye yangeniswa ngaphandle kusetyenziswa i-Agilent Feature Extraction software version 10.7.3.1 (Agilent Technologies) kunye nobunzulu be-fluorescence bomfanekiso ngamnye bubalwe kusetyenziswa ifayile ye-GAL ehambelanayo yeprotocol ye-Exiqon elungisiweyo.Idatha ye-Microarray yophando lwangoku ifakwe kwi-database ye-GEO phantsi kwenombolo yokungena GPL32397.
Iiprofayili zokubonakaliswa kwe-exosomal miRNAs evuthiweyo kwi-microglia ye-RH okanye i-ME49 strains ezosulelwe yi-Toxoplasma zahlalutywa kusetyenziswa izixhobo ezahlukeneyo zenethiwekhi.I-miRNAs enxulumene nophuhliso lwe-tumor ichongiwe kusetyenziswa i-miRWalk2.0 (http://mirwalk.umm.uni-heidelberg.de) kwaye yahluzwa ngaphandle kokuqina kophawu oluqhelekileyo (log2) ngaphezulu kwe-8.0.Phakathi kwe-miRNAs, ii-miRNA ezibonakaliswe ngokwahlukileyo zifunyenwe zingaphezulu kwe-1.5-zitshintshwe ngohlalutyo lokucoca lwe-miRNAs ezitshintshwe yi-RH okanye i-ME49 strains ezosulelwe yi-T. gondii.
Iiseli zaye zahlwaywa kwiiplate ze-6-well (3 x 105 iiseli / kakuhle) kwi-opti-MEM (Gibco, Carlsbad, CA, USA) usebenzisa i-Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).Iiseli ezosulelekileyo zakhuliswa iiyure ezi-6 emva koko i-medium yatshintshwa yaba yinto entsha epheleleyo.Iiseli zavunwa kwiiyure ezingama-24 emva kokudluliselwa.
Uhlalutyo lweenkcukacha-manani lwenziwa ikakhulu kusetyenziswa uvavanyo loMfundi nge-Excel software (Microsoft, Washington, DC, USA).Uhlalutyo lwezilwanyana zovavanyo, iindlela ezimbini ze-ANOVA zenziwe kusetyenziswa isoftware yePrism 3.0 (iGraphPad Software, La Jolla, CA, USA). Ii-P-values ​​<0.05 zithathwe njengezibalo ezibalulekileyo. Ii-P-values ​​<0.05 zithathwe njengezibalo ezibalulekileyo. Значения P <0,05 считались статистически значимыми. Amaxabiso e-P <0.05 athathwa njengebalulekile ngokwezibalo. P 值< 0.05 被认为具有统计学意义。 P <0.05 Значения P <0,05 считались статистически значимыми. Amaxabiso e-P <0.05 athathwa njengebalulekile ngokwezibalo.
Zonke iiprothokholi zokulinga ezisetyenzisiweyo kolu phononongo zivunyiwe yiBhodi yokuHlola yeziko leSeoul National University School of Medicine (inombolo ye-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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