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Toxoplasma gondii ndi tizilombo toyambitsa matenda a intracellular protozoan yomwe imasintha chilengedwe cha munthu yemwe ali ndi kachilomboka ndipo amadziwika kuti amagwirizana ndi kukula kwa chotupa muubongo.Mu kafukufukuyu, tikuganiza kuti exosomal miRNA-21 kuchokera ku matenda a Toxoplasma imalimbikitsa kukula kwa chotupa muubongo.Exosomes kuchokera ku Toxoplasma-infected BV2 microglia adadziwika ndipo kulowetsedwa kwa maselo a glioma a U87 kunatsimikiziridwa.Mafotokozedwe a Exosomal microRNA adawunikidwa pogwiritsa ntchito mitundu ingapo ya microRNA ndi microRNA-21A-5p yolumikizidwa ndi Toxoplasma gondii ndikusankha chotupa.Tidafufuzanso milingo ya mRNA ya majini okhudzana ndi chotupa m'maselo a glioma a U87 posintha miR-21 mu exosomes komanso momwe ma exosomes amakhudzira kuchuluka kwa ma cell a U87 glioma.Mu ma exosomes a U87 glioma maselo omwe ali ndi Toxoplasma gondii, kufotokozera kwa microRNA-21 kumawonjezeka ndipo ntchito ya majini a antitumor (FoxO1, PTEN, ndi PDCD4) imachepetsedwa.Ma exosomes opangidwa ndi BV2 omwe ali ndi Toxoplasma amapangitsa kuchuluka kwa maselo a glioma a U87.Exosomes imapangitsa kukula kwa maselo a U87 mumtundu wa chotupa cha mbewa.Tikuwonetsa kuti kuchuluka kwa exosomal miR-21 mu toxoplasma-infected BV2 microglia kumatha kukhala ndi gawo lofunikira ngati cholimbikitsa kukula kwa ma cell a U87 glioma cell pochepetsa ma gene odana ndi zotupa.
Akuti milandu yopitilira 18.1 miliyoni ya khansa yapamwamba idapezeka padziko lonse lapansi mu 2018, pomwe zotupa zapakati pa 297,000 zomwe zimapezeka chaka chilichonse (1.6% ya zotupa zonse) 1.Kafukufuku wam'mbuyomu wasonyeza kuti zomwe zingayambitse zotupa muubongo wamunthu zimaphatikizapo zinthu zosiyanasiyana zama mankhwala, mbiri yabanja, ndi ma radiation a ionizing ochokera ku zida zochizira komanso zowunikira.Komabe, chomwe chimayambitsa matendawa sichidziwika.Pafupifupi 20% ya khansa zonse padziko lonse lapansi zimayambitsidwa ndi tizilombo toyambitsa matenda, kuphatikizapo mavairasi, mabakiteriya ndi tizilombo toyambitsa matenda3,4.Tizilombo toyambitsa matenda timasokoneza ma genetic a cell, monga kukonza DNA ndi kuzungulira kwa ma cell, ndipo zimatha kuyambitsa kutupa kosatha komanso kuwonongeka kwa chitetezo chamthupi5.
Matenda okhudzana ndi khansa ya anthu ndi omwe amapezeka kwambiri ndi ma virus, kuphatikiza ma virus a papillomavirus ndi ma virus a hepatitis B ndi C.Ma parasite amathanso kutenga nawo gawo pakukula kwa khansa ya anthu.Mitundu ingapo ya tizilombo toyambitsa matenda, yotchedwa Schistosoma, Opishorchis viverrini, O. felineus, Clonorchis sinensis ndi Hymenolepis nana, yakhala ikukhudzidwa ndi mitundu yosiyanasiyana ya khansa yaumunthu 6,7,8.
Toxoplasma gondii ndi protozoan ya intracellular yomwe imayang'anira chilengedwe cha maselo omwe ali ndi kachilomboka.Tizilombo timeneti akuti tikhudza pafupifupi 30% ya anthu padziko lapansi, zomwe zikuyika anthu onse pachiwopsezo9,10.Toxoplasma gondii imatha kupatsira ziwalo zofunika kwambiri, kuphatikiza dongosolo lapakati lamanjenje (CNS), ndikuyambitsa matenda oopsa monga meningitis yakupha ndi encephalitis, makamaka mwa odwala omwe alibe chitetezo chamthupi9.Komabe, Toxoplasma gondii imathanso kusintha chilengedwe cha omwe ali ndi kachilomboka posintha kukula kwa maselo ndi mayankho a chitetezo chamthupi mwa anthu omwe alibe chitetezo chokwanira, zomwe zimapangitsa kuti matenda osachiritsika asamakhalepo9,11.Chochititsa chidwi n'chakuti, chifukwa cha kugwirizana pakati pa kuchuluka kwa T. gondii ndi zochitika za chotupa muubongo, malipoti ena amasonyeza kuti mu vivo khamu kusintha kwa chilengedwe chifukwa cha matenda aakulu T. gondii amafanana ndi chotupa microenvironment.
Exosomes amadziwika kuti intercellular communicators omwe amapereka zamoyo, kuphatikizapo mapuloteni ndi nucleic acids, kuchokera ku maselo oyandikana nawo16,17.Ma exosomes amatha kukhudza njira zachilengedwe zokhudzana ndi chotupa monga anti-apoptosis, angiogenesis, ndi metastasis mu chotupa microenvironment.Makamaka, ma miRNAs (miRNAs), ma RNA ang'onoang'ono osalemba zilembo za 22 nucleotide m'litali, ndizofunikira zowongolera zamtundu wa post-transcriptal zomwe zimawongolera kuposa 30% ya mRNA yamunthu kudzera mu miRNA-induced silence complex (miRISC).Toxoplasma gondii ikhoza kusokoneza njira zamoyo mwa kuwongolera mafotokozedwe a miRNA mwa omwe ali ndi kachilombo.Host miRNAs ili ndi zizindikiro zofunika zowongolera njira zamoyo zogwirira ntchito kuti tikwaniritse njira yopulumukira.Choncho, kuphunzira kusintha kwa mbiri ya miRNA yolandirayo atadwala ndi T. gondii kungatithandize kumvetsetsa kugwirizana pakati pa wolandirayo ndi T. gondii momveka bwino.Zowonadi, Thirugnanam et al.15 inanena kuti T. gondii imalimbikitsa ubongo wa carcinogenesis mwa kusintha mawu ake pa ma miRNA apadera omwe amagwirizanitsidwa ndi kukula kwa chotupa ndipo anapeza kuti T. gondii angayambitse gliomas mu zinyama zoyesera.
Kafukufukuyu akuyang'ana pa kusintha kwa exosomal miR-21 mu microglia yomwe ili ndi Toxoplasma BV2.Tidawona gawo lotheka la kusintha kwa exosomal miR-21 pakukula kwa ma cell a glioma a U87 chifukwa chosungidwa mumtundu wa FoxO1/p27, womwe ndi chandamale cha miR-21 yochulukitsidwa.
Exosomes yochokera ku BV2 inapezedwa pogwiritsa ntchito kusiyana kwa centrifugation ndikutsimikiziridwa ndi njira zosiyanasiyana kuti ateteze kuipitsidwa ndi zigawo za ma cell kapena ma vesicles ena.SDS-polyacrylamide gel electrophoresis (SDS-PAGE) adawonetsa machitidwe osiyana pakati pa mapuloteni otengedwa ku maselo a BV2 ndi ma exosomes (Chithunzi 1A), ndipo zitsanzo zinayesedwa kuti zikhalepo za Alix, zomwe zinawunikidwa ndi kutsekedwa kwa Kumadzulo kwa zolembera zamapuloteni za exosomal mu .Kulemba kwa Alix kunapezeka m'mapuloteni a exosome koma osati mu BV2 cell lysate mapuloteni (Mkuyu 1B).Kuphatikiza apo, RNA yoyeretsedwa kuchokera ku exosomes yochokera ku BV2 idawunikidwa pogwiritsa ntchito bioanalyzer.18S ndi 28S ribosomal subunits sanali kawirikawiri ankaona mu exosomal RNA kusamuka chitsanzo, kusonyeza chiyero odalirika (Chithunzi 1C).Pomaliza, kufala elekitironi maikulosikopu anasonyeza kuti exosomes anaona anali za 60-150 nm mu kukula ndipo anali chikho ngati dongosolo mmene exosome kapangidwe kazachilengedwe (mkuyu. 1D).
Makhalidwe a exosomes ochokera ku ma cell a BV2.(A) Tsamba latsamba lachitetezo.Mapuloteni anali olekanitsidwa ndi maselo a BV2 kapena ma exosomes ochokera ku BV2.Mapuloteni amasiyana pakati pa maselo ndi exosomes.(B) Kusanthula kwakumadzulo kwa cholembera cha exosomal (Alix).(C) Kuunikira kwa RNA yoyeretsedwa kuchokera ku maselo a BV2 ndi BV2 yochokera ku exosomes pogwiritsa ntchito bioanalyzer.Chifukwa chake, ma subunits a 18S ndi 28S a ribosomal m'maselo a BV2 sanapezeke mu exosomal RNA.(D) Kupatsirana kwa ma electron microscopy kunasonyeza kuti ma exosomes olekanitsidwa ndi maselo a BV2 anali oipitsidwa ndi 2% uranyl acetate.Exosomes ndi pafupifupi 60-150 nm kukula kwake ndi kapu (Nyimbo ndi Jung, deta yosasindikizidwa).
Kuyika kwa ma cell a ma exosomes opangidwa ndi BV2 m'maselo a glioma a U87 adawonedwa pogwiritsa ntchito ma microscopy.PKH26 otchedwa exosomes amapezeka mu cytoplasm ya maselo a U87.Ma nuclei anali odetsedwa ndi DAPI (mkuyu 2A), kusonyeza kuti BV2-derived exosomes akhoza kulowa mkati mwa maselo olandirira ndi kukhudza chilengedwe cha maselo olandira.
Kulowetsedwa kwa ma exosome opangidwa ndi BV2 m'maselo a glioma a U87 ndi ma exosome opangidwa ndi BV2 omwe ali ndi Toxoplasma RH adapangitsa kuchuluka kwa ma cell a U87 glioma.(A) Ma exosomes ozunguliridwa ndi ma cell a U87 oyezedwa ndi confocal microscopy.Maselo a glioma a U87 adakulungidwa ndi ma exosomes olembedwa ndi PKH26 (ofiira) kapena opanda mphamvu kwa maola 24.Ma nuclei anali odetsedwa ndi DAPI (buluu) kenako amawonedwa pansi pa microscope confocal (scale bar: 10 μm, x 3000).(B) Kuchulukitsitsa kwa maselo a glioma a U87 kunatsimikiziridwa ndi kuyesa kwa kuchuluka kwa ma cell.Maselo a glioma a U87 adathandizidwa ndi ma exosomes kwa nthawi yowonetsedwa. *P <0.05 idapezedwa ndi mayeso a Student t. *P <0.05 idapezedwa ndi mayeso a Student t. *P <0,05 получено по t-критерию Стьюдента. *P <0.05 ndi mayeso a Student's. *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 idapezeka pogwiritsa ntchito mayeso a Student's.
Titatsimikizira kulowetsedwa kwa ma exosome opangidwa ndi BV2 m'maselo a glioma a U87, tidayesa kuchuluka kwa maselo kuti tifufuze ntchito ya BV2 yotengedwa ndi Toxoplasma-yochokera ku exosomes pakupanga ma cell a glioma.Kuchiza kwa maselo a U87 okhala ndi ma exosomes ochokera ku T. gondii omwe ali ndi kachilombo ka BV2 maselo amasonyeza kuti T. gondii yochokera ku BV2 yochokera ku T.
Kuonjezera apo, kukula kwa maselo a U118 kunali ndi zotsatira zofanana ndi U87, monga Toxoplasma inalimbikitsa exosomes inachititsa kuti chiwerengero chapamwamba chichuluke (deta yosasonyezedwa).Kutengera izi, titha kuwonetsa kuti ma exosomes opangidwa ndi BV2 opangidwa ndi Toxoplasma amakhala ndi gawo lofunikira pakuchulukira kwa maselo a glioma.
Kuti tifufuze zotsatira za ma exosomes opangidwa ndi Toxoplasma opangidwa ndi BV2 pakukula kwa chotupa, tidabaya ma cell a glioma a U87 mu mbewa zamaliseche za mtundu wa xenograft ndikubaya ma exosome opangidwa ndi BV2 kapena ma exosomes opangidwa ndi RH opangidwa ndi BV2.Zotupa zitawonekera pambuyo pa sabata la 1, gulu lililonse loyesera la mbewa za 5 lidagawika molingana ndi kukula kwa chotupa kuti lidziwe komwe kumayambira, ndipo kukula kwa chotupa kunayesedwa kwa masiku 22.
Mu mbewa ndi chitsanzo cha U87 xenograft, kukula kwakukulu kwa chotupa ndi kulemera kwake kunawonedwa mu BV2-derived RH-infected exosome gulu pa tsiku 22 (mkuyu 3A, B).Kumbali inayi, panalibe kusiyana kwakukulu mu kukula kwa chotupa pakati pa gulu lochokera ku BV2 lochokera ku exosome ndi gulu lolamulira pambuyo pa chithandizo chambiri.Komanso, mbewa jekeseni ndi glioma maselo ndi exosomes zowoneka anasonyeza yaikulu chotupa buku mu gulu la RH kachilombo BV2-anachokera exosomes (mkuyu. 3C).Zotsatira izi zikuwonetsa kuti ma exosome opangidwa ndi BV2 opangidwa ndi Toxoplasma amapangitsa kukula kwa glioma mumtundu wa chotupa cha mbewa.
Oncogenesis (AC) ya ma exosome opangidwa ndi BV2 mu mtundu wa mbewa wa U87 xenograft.Kukula kwa chotupa (A) ndi kulemera (B) kunawonjezeka kwambiri mu BALB / c mbewa zamaliseche zomwe zimagwiritsidwa ntchito ndi RH-infected exosomes yochokera ku BV2.BALB/c mbewa zamaliseche (C) anabayidwa pansi pa khungu ndi 1 x 107 U87 maselo kuyimitsidwa mu osakaniza Matrigel.Patatha masiku asanu ndi limodzi jekeseni, 100 μg ya ma exosomes opangidwa ndi BV2 adathandizidwa mu mbewa.Kukula kwa chotupa ndi kulemera kwake zinali kuyezedwa pa masiku osonyezedwa ndi pambuyo pa nsembe, motsatana. *P <0.05. *P <0.05. r <0.05. *P <0.05. *P <0.05. *P <0.05. r <0.05. *P <0.05.
Deta inasonyeza kuti 37 miRNAs (16 overexpressed ndi 21 downexpressed) yokhudzana ndi chitetezo cha mthupi kapena chitukuko cha chotupa chinasinthidwa kwambiri mu microglia pambuyo pa matenda a Toxoplasma RH strain (mkuyu 4A).Miyezo yofananira ya miR-21 pakati pa ma miRNA osinthidwa idatsimikiziridwa ndi nthawi yeniyeni RT-PCR mu exosomes yochokera ku BV2, exosomes yothandizidwa ndi BV2 ndi ma U87 cell.Kufotokozera kwa miR-21 kunasonyeza kuwonjezeka kwakukulu kwa exosomes kuchokera ku maselo a BV2 omwe ali ndi Toxoplasma gondii (RH strain) (mkuyu. 4B).Wachibale misinkhu miR-21 mu BV2 ndi U87 maselo kuchuluka pambuyo kutengeka za kusinthidwa exosomes (mkuyu. 4B).Wachibale misinkhu miR-21 mawu mu ubongo zimakhala za chotupa odwala ndi mbewa kachilombo Toxoplasma gondii (ME49 kupsyinjika) anali apamwamba kuposa amazilamulira, motero (mkuyu. 4C).Zotsatira izi zimagwirizana ndi kusiyana pakati pa kuchuluka kwa mawu omwe adanenedweratu ndi ma microRNA otsimikizika mu vitro ndi mu vivo.
Kusintha kwa mawu a exosomal miP-21a-5p mu microglia yomwe ili ndi Toxoplasma gondii (RH).(A) Amasonyeza kusintha kwakukulu mu siRNA yokhudzana ndi chitetezo cha mthupi kapena chitukuko cha chotupa chotsatira T. gondii RH matenda.(B) Miyezo yofananira ya miR-21 idazindikirika ndi RT-PCR yeniyeni mu ma exosomes opangidwa ndi BV2, ma exosomes opangidwa ndi BV2, ndi ma cell a U87.(C) Milingo ya mawu achibale a miR-21 inapezeka mu minyewa yaubongo ya odwala chotupa (N=3) ndi mbewa zomwe zili ndi Toxoplasma gondii (ME49 strain) (N=3). *P <0.05 idapezedwa ndi mayeso a Student t. *P <0.05 idapezedwa ndi mayeso a Student t. *P <0,05 было получено с помощью t-критерия Стьюдента. *P <0.05 idapezedwa pogwiritsa ntchito mayeso a Student's. *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 idapezeka pogwiritsa ntchito mayeso a Student's.
Exosomes kuchokera ku maselo a BV2 omwe ali ndi RH adayambitsa kukula kwa gliomas mu vivo ndi mu vitro (Mkuyu 2, 3).Kuti tizindikire ma mRNA ofunikira, tidasanthula milingo ya mRNA yamtundu wa antitumor, bokosi la forkhead O1 (FoxO1), PTEN, ndi ma cell kufa 4 (PDCD4) m'maselo a U87 omwe ali ndi ma exosomes ochokera ku BV2 kapena RH BV2.Kusanthula kwa Bioinformatics kwawonetsa kuti majini angapo okhudzana ndi chotupa, kuphatikiza mitundu ya FoxO1, PTEN, ndi PDCD4, ali ndi malo omangira a miR-2121,22.MRNA milingo ya antitumor chandamale jini anachepetsedwa RH-infected BV2-derived exosomes poyerekeza BV2-derived exosomes (Fig. 5A).FoxO1 inawonetsa kuchepa kwa mapuloteni mu RH-infected BV2-derived exosomes poyerekeza ndi BV2-derived exosomes (Chithunzi 5B).Kutengera zotsatirazi, titha kutsimikizira kuti ma exosomes ochokera ku RH-infected BV2 amatsitsa ma gene odana ndi oncogenic, ndikusunga gawo lawo pakukula kwa chotupa.
Toxoplasma RH-yochokera ku BV2 yotengedwa ndi exosomes imapangitsa kuponderezedwa kwa majini a antitumor mu maselo a glioma a U87 ndi Toxoplasma RH-infected BV2-derived exosomes.(A) PCR yeniyeni ya FoxO1, PTEN ndi PDCD4 kufotokoza mu exosomes yochokera ku T. gondii RH-infected BV2 poyerekeza ndi PBS exosomes.β-actin mRNA idagwiritsidwa ntchito ngati kuwongolera.(B) Mafotokozedwe a FoxO1 adatsimikiziridwa ndi Western blotting ndi densitometry deta idawunikidwa motsatira pulogalamu ya ImageJ. *P <0.05 idapezedwa ndi mayeso a Student t. *P <0.05 idapezedwa ndi mayeso a Student t. *P <0,05 было получено с помощью t-критерия Стьюдента. *P <0.05 idapezedwa pogwiritsa ntchito mayeso a Student's. *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 idapezeka pogwiritsa ntchito mayeso a Student's.
Kuti timvetse zotsatira za miP-21 mu exosomes pa chotupa-associated gene regulation, maselo a U87 anasamutsidwa ndi inhibitor ya miP-21 pogwiritsa ntchito Lipofectamine 2000 ndipo maselo anakololedwa maola 24 atapatsirana.Mawonekedwe a FoxO1 ndi p27 m'maselo opatsirana ndi miR-21 inhibitors ankafanizidwa ndi maselo opangidwa ndi BV2-derived exosomes pogwiritsa ntchito qRT-PCR (Mkuyu 6A, B).Kusintha kwa miR-21 inhibitor mu maselo a U87 kumachepetsa kwambiri FoxO1 ndi mawu a p27 (FIG. 6).
RH-infected exosomal BV2-derived miP-21 inasintha FoxO1/p27 m'maselo a glioma a U87.Ma cell a U87 adasinthidwa ndi miP-21 inhibitor pogwiritsa ntchito Lipofectamine 2000 ndipo ma cell adakololedwa patatha maola 24 atapatsirana.Mawonekedwe a FoxO1 ndi p27 m'maselo omwe amapatsirana ndi miR-21 inhibitors anafanizidwa ndi ma cell omwe amathandizidwa ndi ma exosome opangidwa ndi BV2 pogwiritsa ntchito qRT-PCR (A, B).
Pofuna kupewa chitetezo cha mthupi, tizilombo toyambitsa matenda toxoplasma timasandulika kukhala chotupa.Amasokoneza minyewa yosiyanasiyana, kuphatikiza ubongo, mtima, ndi chigoba, nthawi yonse ya moyo wa wolandirayo ndikuwongolera momwe chitetezo cha mthupi chimathandizira.Kuphatikiza apo, amatha kuwongolera ma cell ndi ma apoptosis a cell host, kulimbikitsa kuchuluka kwawo14,24.Toxoplasma gondii nthawi zambiri imayambitsa maselo a dendritic, neutrophils, ndi mzere wa monocyte/macrophage, kuphatikizapo ubongo wa microglia.Toxoplasma gondii imapangitsa kusiyana kwa macrophages a M2 phenotype, imakhudza machiritso a zilonda pambuyo pa matenda a tizilombo toyambitsa matenda, komanso imagwirizanitsidwa ndi hypervascularization ndi granulomatous fibrosis.Matenda a Toxoplasma amatha kukhala okhudzana ndi zolembera zokhudzana ndi kukula kwa chotupa.Malo ankhanza oyendetsedwa ndi Toxoplasma angafanane ndi khansa yofananira.Choncho, tingaganize kuti matenda a Toxoplasma ayenera kuthandizira kukula kwa zotupa za ubongo.M'malo mwake, kuchuluka kwa matenda a Toxoplasma kumanenedwa mu seramu ya odwala omwe ali ndi zotupa zosiyanasiyana muubongo.Komanso, Toxoplasma gondii akhoza kukhala carcinogenic effecter ndi kuchita synergistically kuthandiza ena matenda carcinogens kukhala zotupa mu ubongo.Pankhani imeneyi, ndi bwino kudziwa kuti P. falciparum ndi Epstein-Barr HIV synergistically amathandiza mapangidwe Burkitt a lymphoma.
Udindo wa exosomes monga owongolera pa kafukufuku wa khansa wafufuzidwa mozama.Komabe, ntchito ya exosomes pakati pa tizilombo toyambitsa matenda ndi tizilombo toyambitsa matenda sitikudziwikabe.Pakalipano, olamulira osiyanasiyana, kuphatikizapo mapuloteni obisika, afotokoza njira zamoyo zomwe tizilombo toyambitsa matenda timakanira kumenyana ndi tizilombo toyambitsa matenda ndi kupitiriza matenda.Posachedwapa, pakhala pali lingaliro lomwe likukula loti ma microvesicles ogwirizana ndi protozoan ndi ma microRNA awo amalumikizana ndi ma cell omwe amawalandira kuti apange malo abwino kuti apulumuke.Chifukwa chake, maphunziro owonjezera akufunika kuti apeze ubale pakati pa ma exosomal miRNA osinthidwa ndi kuchuluka kwa ma cell a glioma.Kusintha kwa MicroRNA (ma gene gene miR-30c-1, miR-125b-2, miR-23b-27b-24-1 ndi miR-17-92) kumangiriza kwa STAT3 wolimbikitsa mu macrophages omwe ali ndi kachilombo ka toxoplasma, amawongolera ndikupangitsa anti -apoptosis poyankha matenda a Toxoplasma gondii 29.Toxoplasma matenda kumawonjezera mawu a miR-17-5p ndi miR-106b-5p, amene amagwirizana ndi angapo hyperproliferative matenda 30 .Izi zikusonyeza kuti ma miRNA omwe amayendetsedwa ndi matenda a Toxoplasma ndi mamolekyu ofunikira kuti tizilombo toyambitsa matenda tipulumuke komanso tizilombo toyambitsa matenda.
Ma miRNA osinthidwa amatha kukhudza mitundu yosiyanasiyana ya machitidwe pakuyambika ndi kufalikira kwa maselo oyipa, kuphatikiza ma gliomas: kudzikwanira kwa ma siginecha akukula, kusakhudzidwa ndi ma siginecha oletsa kukula, kuthawa kwa apoptosis, kuthekera kobwerezabwereza kopanda malire, angiogenesis, kuwukira ndi metastasis, ndi kutupa.Mu glioma, ma miRNA osinthidwa adadziwika m'maphunziro angapo ofotokozera.
Mu kafukufuku wapano, tidatsimikizira kuchuluka kwa mawu a miRNA-21 m'maselo okhala ndi toxoplasma.miR-21 yadziwika kuti ndi imodzi mwama microRNA omwe amachulukirachulukira mu zotupa zolimba, kuphatikiza ma gliomas, 33 ndipo mawu ake amalumikizana ndi giredi ya glioma.Umboni wochulukirachulukira ukuwonetsa kuti miR-21 ndi buku la oncogene lomwe limagwira ntchito ngati anti-apoptotic factor pakukula kwa glioma ndipo limachulukitsidwa kwambiri mu minofu ndi plasma ya zilonda zaubongo wamunthu.Chosangalatsa ndichakuti, kusakhazikika kwa miR-21 m'maselo a glioma ndi minyewa kumayambitsa kuletsa kuchulukana kwa maselo chifukwa cha apoptosis yodalira caspase.Kusanthula kwa bioinformatics kwa miR-21 yomwe idanenedweratu milingo idavumbulutsa majini angapo opondereza chotupa okhudzana ndi njira za apoptosis, kuphatikiza ma cell kufa 4 (PDCD4), tropomyosin (TPM1), PTEN, ndi bokosi lamphumphu O1 (FoxO1), yokhala ndi malo omangira a miR-2121..22.38.
FoxO1, monga imodzi mwazinthu zolembera (FoxO), imakhudzidwa ndikukula kwa mitundu yosiyanasiyana ya khansa yaumunthu ndipo imatha kuwongolera mafotokozedwe amtundu wopondereza chotupa monga p21, p27, Bim, ndi FasL40.FoxO1 imatha kumanga ndi kuyambitsa ma cell cycle inhibitors monga p27 kuti achepetse kukula kwa maselo.Kuphatikiza apo, FoxO1 ndiyomwe imakhudza kwambiri kusaina kwa PI3K/Akt ndipo imayang'anira njira zambiri zachilengedwe monga kupita patsogolo kwa ma cell ndi kusiyanitsa kwa ma cell kudzera pakutsegula kwa p2742.
Pomaliza, timakhulupirira kuti exosomal miR-21 yochokera ku Toxoplasma-infected microglia ikhoza kukhala ndi gawo lofunikira monga kukula kwa maselo a glioma (mkuyu 7).Komabe, maphunziro owonjezera akufunika kuti apeze kulumikizana kwachindunji pakati pa exosomal miR-21, matenda osinthika a Toxoplasma, ndi kukula kwa glioma.Zotsatirazi zikuyembekezeredwa kupereka poyambira kuphunzira za ubale pakati pa matenda a Toxoplasma ndi zochitika za glioma.
Chithunzi chojambula cha makina a glioma (ubongo) carcinogenesis akuperekedwa mu kafukufukuyu.Wolembayo amajambula mu PowerPoint 2019 (Microsoft, Redmond, WA).
Ndondomeko zonse zoyesera mu phunziroli, kuphatikizapo kugwiritsa ntchito nyama, zinali zogwirizana ndi Seoul National University Animal Care ndi User Committee Standard Ethical Guidelines ndipo zinavomerezedwa ndi Institutional Review Board ya Seoul National University School of Medicine (IRB nambala SNU- 150715).-2).Njira zonse zoyesera zidachitika motsatira malingaliro a ARRIVE.
BV2 mouse microglia ndi ma cell a U87 human glioma adapangidwa mu Dulbecco's Modified Eagle's Medium (DMEM; Welgene, Seoul, Korea) ndi Roswell Park Memorial Institute's Medium (RPMI; Welgene), motsatana, iliyonse ili ndi 10% fetal bovine serum, 4 mM l- glutamine, 0.2 mM penicillin ndi 0.05 mM streptomycin.Maselo adapangidwa mu chofungatira chokhala ndi 5% CO2 pa 37°C.Mzere wina wa cell wa glioma, U118, unagwiritsidwa ntchito poyerekeza ndi ma cell a U87.
Kupatula ma exosomes kuchokera ku T. gondii-infected RH ndi ME49 strains, T. gondii tachyzoites (RH strain) adakololedwa kuchokera m'mimba ya 6 masabata a BALB / c mbewa jekeseni masiku 3-4 asanafike.Tachyzoites adatsukidwa katatu ndi PBS ndikuyeretsedwa ndi centrifugation mu 40% Percoll (Sigma-Aldrich, St. Louis, MO, USA)43.Kuti mupeze ma tachyzoite a kupsyinjika kwa ME49, mbewa za BALB / c zidalowetsedwa m'mimba ndi ma cysts amtundu wa 20 ndipo kusintha kwa tachyzoite mu cysts kunasonkhanitsidwa ndikutsuka pamimba pamimba pa tsiku la 6-8 pambuyo pa matenda (PI).Makoswe omwe ali ndi PBS.ME49 tachyzoites anakulira m'maselo owonjezera 100 μg/ml penicillin (Gibco/BRL, Grand Island, NY, USA), 100 μg/ml streptomycin (Gibco/BRL), ndi 5% seramu ya bovine ya fetal (Lonza, Walkersville, MD) .., USA) pa 37 °C ndi 5% carbon dioxide.Atalimidwa m'maselo a Vero, ma tachyzoite a ME49 adadutsa kaŵirikaŵiri kudzera mu singano ya 25 ya singano ndiyeno kupyolera mu fyuluta ya 5 µm kuchotsa zinyalala ndi maselo.Atatha kutsuka, ma tachyzoite adayimitsidwanso mu PBS44.Tissue cysts of Toxoplasma gondii strain ME49 adasungidwa ndi jakisoni wa intraperitoneal wa cysts olekanitsidwa ndi ubongo wa C57BL/6 mbewa (Oriental Bio Animal Center, Seongnam, Korea).Ubongo wa mbewa zomwe zili ndi ME49 zidakololedwa patatha miyezi 3 ya PI ndi minced pansi pa maikulosikopu kuti azipatula ma cysts.Makoswe omwe ali ndi kachilomboka amasungidwa m'malo apadera opanda tizilombo toyambitsa matenda (SPF) ku Seoul National University School of Medicine.
RNA yonse idachotsedwa ku ma exosomes opangidwa ndi BV2, ma cell a BV2 ndi minyewa yogwiritsa ntchito miRNeasy Mini Kit (Qiagen, Hilden, Germany) molingana ndi malangizo a wopanga, kuphatikiza nthawi yofikira pa sitepe ya elution.Kukhazikika kwa RNA kudatsimikiziridwa pa NanoDrop 2000 spectrophotometer.Ubwino wa ma RNA microarrays adayesedwa pogwiritsa ntchito Agilent 2100 bioanalyzer (Agilent Technologies, Amstelveen, Netherlands).
DMEM yokhala ndi 10% exosome-foor FBS inakonzedwa ndi ultracentrifugation pa 100,000g kwa maola 16 pa 4°C ndikusefedwa kudzera mu fyuluta ya 0.22 µm (Nalgene, Rochester, NY, USA).Maselo a BV2, 5 × 105, adakula mu DMEM yomwe ili ndi 10% ya FBS ya exosome-depleted ndi 1% maantibayotiki pa 37 ° C ndi 5% CO2.Pambuyo pa maola a 24 a incubation, tachyzoites of strain RH kapena ME49 (MOI = 10) anawonjezeredwa m'maselo ndipo tizilombo toyambitsa matenda tinachotsedwa mkati mwa ola limodzi ndikudzazidwa ndi DMEM.Ma exosomes ochokera ku ma cell a BV2 adasiyanitsidwa ndi kusinthidwa kosiyana kwa centrifugation, njira yomwe imagwiritsidwa ntchito kwambiri.Yambitsaninso pellet ya exosome mu 300 µl PBS pa RNA kapena kusanthula mapuloteni.Kuchuluka kwa ma exosomes akutali kudatsimikiziridwa pogwiritsa ntchito zida zoyesa mapuloteni a BCA (Pierce, Rockford, IL, USA) ndi NanoDrop 2000 spectrophotometer.
Madzi ochokera ku ma cell a BV2 kapena ma exosomes otengedwa ku BV2 adapangidwa mu PRO-PREP™ protein extraction solution (iNtRon Biotechnology, Seongnam, Korea) ndipo mapuloteni adayikidwa pa Coomassie buluu wonyezimira wothimbirira 10% SDS polyacrylamide gels.Kuphatikiza apo, mapuloteni adasamutsidwa ku nembanemba ya PVDF kwa maola awiri.Ma blots akumadzulo adatsimikiziridwa pogwiritsa ntchito antibody ya Alix (Cell Signaling Technology, Beverly, MA, USA) ngati chizindikiro cha exosomal.HRP-conjugated goat anti-mouse IgG (H + L) (Bethyl Laboratories, Montgomery, TX, USA) ndi LAS-1000 plus luminescent image analyzer (Fuji Photographic Film, Tokyo, Japan) adagwiritsidwa ntchito ngati antibody yachiwiri..Transmission electron microscopy anachitidwa kuti aphunzire kukula ndi morphology ya exosomes.Ma exosomes olekanitsidwa ndi ma cell a BV2 (6.40 µg/µl) adakonzedwa pamiyala yokhala ndi kaboni ndipo amadetsedwa ndi 2% uranyl acetate kwa 1 min.Zitsanzo zokonzedwazo zidawonedwa pamagetsi othamanga a 80 kV pogwiritsa ntchito JEOL 1200-EX II (Tokyo, Japan) yokhala ndi kamera ya ES1000W Erlangshen CCD (Gatan, Pleasanton, CA, USA).
Ma exosomes opangidwa ndi BV2 adadetsedwa pogwiritsa ntchito PKH26 Red Fluorescent Linker Kit (Sigma-Aldrich, St. Louis, MO, USA) kwa mphindi 15 kutentha.Maselo a U87, 2 × 105, okhala ndi PKH26-otchedwa exosomes (ofiira) kapena opanda ma exosomes monga kulamulira koipa, adayikidwa pa 37 ° C kwa maola 24 mu 5% CO2 chofungatira.Ma cell a U87 anali odetsedwa ndi DAPI (buluu), ma cell a U87 adayikidwa mu 4% paraformaldehyde kwa mphindi 15 pa 4 ° C ndikuwunikidwa mu Leica TCS SP8 STED CW confocal microscope system (Leica Microsystems, Mannheim, Germany).zowoneka.
cDNA idapangidwa kuchokera ku siRNA pogwiritsa ntchito Mir-X siRNA yoyamba strand synthesis ndi SYBR qRT-PCR kit (Takara Bio Inc., Shiga, Japan).PCR yochulukira nthawi yeniyeni idachitidwa pogwiritsa ntchito iQ5 nthawi yeniyeni yodziwira PCR (Bio-Rad, Hercules, CA, USA) pogwiritsa ntchito zoyambira ndi ma templates osakanikirana ndi SYBR Premix.DNA inakulitsidwa kwa mikombero 40 ya denaturation pa 95 ° C kwa 15 s ndi annealing pa 60 ° C kwa 60 s.Deta kuchokera pamachitidwe aliwonse a PCR adawunikidwa pogwiritsa ntchito gawo losanthula deta la iQ™5 optical system software (Bio-Rad).Zosintha zofananira za jini pakati pa jini zomwe zasankhidwa ndi β-actin/siRNA (ndi U6) zidawerengedwa pogwiritsa ntchito njira yokhazikika yokhotakhota.Njira zoyambira zomwe zimagwiritsidwa ntchito zikuwonetsedwa mu Table 1.
3 x 104 U87 ma cell a glioma adabzalidwa m'mbale za 96-chitsime ndikusakanikirana ndi ma exosomes okhala ndi Toxoplasma ochokera ku BV2 (50 μg/mL) kapena nonpulse exosomes ochokera ku BV2 (50 μg/mL) monga zowongolera pa 12, 18 ndi 36 .Kuchulukitsa kwa maselo kunatsimikiziridwa pogwiritsa ntchito Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) (Supplementary Figures S1-S3) 46.
mbewa zazikazi za 5 zazaka zakubadwa za BALB/c zamaliseche zidagulidwa kuchokera ku Orient Bio (Seongnam-si, South Korea) ndipo zimasungidwa m'zipinda zowuma kutentha (22 ± 2 ° C) ndi chinyezi (45 ± 15 ° C).%) pa kutentha (22 ± 2 ° C) ndi chinyezi (45±15%).Kuzungulira kwa maola a 12 ndi mdima wamdima wa 12 kunachitidwa pansi pa SPF (Seoul National University School of Medicine Animal Center).Mbewa zinagawidwa mwachisawawa m'magulu atatu a mbewa za 5 aliyense ndipo magulu onse anabayidwa pansi pa khungu ndi 400 ml ya PBS yomwe ili ndi 1 x 107 U87 maselo a glioma ndi kukula kwa BD Matrigel ™ (BD Science, Miami, FL, USA).Masiku asanu ndi limodzi pambuyo pa jekeseni ya chotupa, 200 mg ya exosomes yochokera ku maselo a BV2 (opanda / opanda matenda a Toxoplasma) adabayidwa pamalo otupa.Patatha masiku makumi awiri ndi awiri kuchokera ku matenda a chotupa, kukula kwa mbewa pagulu lililonse kumayesedwa ndi caliper katatu pa sabata, ndipo voliyumu ya chotupacho inawerengedwa ndi ndondomekoyi: 0.5 × (m'lifupi) × 2 × kutalika.
Kusanthula kwa mawu a MicroRNA pogwiritsa ntchito miRCURYTM LNA miRNA array, 7th generation ili ndi, mmu ndi rno arrays (EXIQON, Vedbaek, Denmark) yophimba 1119 mbewa zodziwika bwino pakati pa 3100 anthu, mbewa ndi makoswe a miRNA yojambula.Munthawi imeneyi, 250 mpaka 1000 ng ya RNA yonse idachotsedwa ku 5'-phosphate pothandizidwa ndi matumbo am'mimba amchere phosphatase ndikutsatiridwa ndi utoto wa Hy3 wobiriwira wa fulorosenti.Zitsanzo zolembedwazo zidasakanizidwa pokweza ma slide ang'onoang'ono pogwiritsa ntchito zida zophatikizira chipinda (Agilent Technologies, Santa Clara, CA, USA) ndi zida zosakanikirana (Agilent Technologies).Kuphatikizika kunachitika kwa maola 16 pa 56 ° C, ndiye ma microarrays adatsukidwa motsatira malingaliro a wopanga.Ma slide opangidwa ndi ma microarray adasinthidwa pogwiritsa ntchito Agilent G2565CA microarray scanner system (Agilent Technologies).Zithunzi zojambulidwa zinatumizidwa pogwiritsa ntchito pulogalamu ya Agilent Feature Extraction version 10.7.3.1 (Agilent Technologies) ndipo mphamvu ya fluorescence ya chithunzi chilichonse inatsimikiziridwa pogwiritsa ntchito fayilo yofanana ya GAL ya protocol ya Exiqon yosinthidwa.Zambiri za Microarray za kafukufuku wapano zasungidwa mu nkhokwe ya GEO pansi pa nambala ya GPL32397.
Mafotokozedwe a ma exosomal miRNA okhwima mu ma microglia a RH kapena ME49 strains omwe ali ndi Toxoplasma adawunikidwa pogwiritsa ntchito zida zosiyanasiyana zama network.ma miRNA okhudzana ndi kukula kwa chotupa adadziwika pogwiritsa ntchito miRWalk2.0 (http://mirwalk.umm.uni-heidelberg.de) ndikusefedwera ndi mphamvu yazidziwitso yokhazikika (log2) yopitilira 8.0.Pakati pa miRNAs, ma miRNA omwe amasonyezedwa mosiyana adapezeka kuti ndi oposa 1.5 osinthidwa ndi fyuluta ya miRNAs yosinthidwa ndi RH kapena ME49 tizilombo toyambitsa matenda T. gondii.
Maselo adabzalidwa mu mbale zisanu ndi imodzi (3 x 105 maselo / bwino) mu opti-MEM (Gibco, Carlsbad, CA, USA) pogwiritsa ntchito Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).Ma cell opatsirana adakulitsidwa kwa maola 6 kenako sing'angayo idasinthidwa kukhala sing'anga yatsopano.Maselo adakololedwa patatha maola 24 atapatsirana.
Kusanthula kowerengera kunkachitika makamaka pogwiritsa ntchito t-test ya Student ndi pulogalamu ya Excel (Microsoft, Washington, DC, USA).Poyesa kuyesa nyama, njira ziwiri za ANOVA zidapangidwa pogwiritsa ntchito pulogalamu ya Prism 3.0 (GraphPad Software, La Jolla, CA, USA). P-makhalidwe <0.05 adawonedwa ngati ofunika kwambiri. P-makhalidwe <0.05 adawonedwa ngati ofunika kwambiri. Значения P <0,05 считались статистически значимыми. Makhalidwe a P <0.05 adawonedwa ngati ofunika kwambiri. P 值< 0.05 被认为具有统计学意义. P ku <0.05 Значения P <0,05 считались статистически значимыми. Makhalidwe a P <0.05 adawonedwa ngati ofunika kwambiri.
Ndondomeko zonse zoyesera zomwe zimagwiritsidwa ntchito mu phunziroli zinavomerezedwa ndi Bungwe Loyang'anira Institutional Review la Seoul National University School of Medicine (IRB nambala 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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