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Toxoplasma gondii is intracellular protozoan parasite inogadzirisa nharaunda yeane hutachiona uye inozivikanwa kuve yakabatana nechiitiko chekukura kwebundu reuropi.Muchidzidzo ichi, isu tinofungidzira kuti exosomal miRNA-21 kubva kuToxoplasma utachiona inosimudzira kukura kwehuropi.Exosomes kubva kuToxoplasma-infected BV2 microglia yakaonekwa uye kuiswa mukati kweU87 glioma masero kwakasimbiswa.Exosomal microRNA kutaura maprofiles akaongororwa uchishandisa arrays e microRNA uye microRNA-21A-5p yakabatana neToxoplasma gondii uye tumor kurongedza.Isu takaongororawo mazinga emRNA emajini ane hukama muU87 glioma masero nekuchinja miR-21 mazinga mune exosomes uye mhedzisiro yemaexosomes pamunhu U87 glioma cell kuwanda.Mune exosomes yeU87 glioma masero ane utachiona neToxoplasma gondii, kutaura kwe microRNA-21 kunowedzera uye basa remajene antitumor (FoxO1, PTEN, uye PDCD4) rakaderedzwa.BV2-yakagadzirwa exosomes inotapukirwa neToxoplasma inokonzera kuwanda kweU87 glioma masero.Exosomes inosimudzira kukura kweU87 masero mune mbeva bundu modhi.Isu tinopa zano kuti yakawedzera exosomal miR-21 muToxoplasma-yakatapukirwa BV2 microglia inogona kuita basa rakakosha sesero kukura kwekusimudzira muU87 glioma masero nekudzikisira antitumor majini.
Zvinofungidzirwa kuti anopfuura mamirioni 18.1 makesi ekenza yepamusoro akaonekwa pasi rese muna 2018, aine zviuru mazana maviri nemakumi mapfumbamwe nenomwe epakati chetsinga yemamota anoongororwa gore rega rega (1.6% yemamota ese) 1.Tsvagiridzo yapfuura yakaratidza kuti njodzi dzekugadzira mamota ehuropi hwemunhu zvinosanganisira akasiyana makemikari zvigadzirwa, nhoroondo yemhuri, uye ionizing radiation kubva mumusoro kurapa uye yekuongorora michina.Zvisinei, chikonzero chaicho chezvirwere izvi hachizivikanwi.Inenge 20% yekenza pasi rose inokonzerwa nehutachiona, kusanganisira mavhairasi, mabhakitiriya nemaparasite3,4.Utachiona hunotapukira hunovhiringidza maitiro eiyo geneti yesero, senge DNA kugadzirisa uye kutenderera kwesero, uye zvinogona kutungamira mukuzvimba kusingaperi uye kukuvadzwa kwe immune system5.
Zvirwere zvinotapukira zvine chekuita negomarara revanhu ndiwo hutachiona hwehutachiona, hunosanganisira vanhu papillomaviruses uye hutachiona hweHepatitis B neC.Maparasite anogona zvakare kuita basa rinogona kuita mukukura kwegomarara revanhu.Mhando dzinoverengeka dzeparasite, dzinoti Schistosoma, Opishorchis viverrini, O. felineus, Clonorchis sinensis uye Hymenolepis nana, dzakabatanidzwa mumhando dzakasiyana dzekenza yevanhu 6,7,8.
Toxoplasma gondii inonzi intracellular protozoan inodzora microenvironment yemasero ane utachiona.Chirwere ichi chinofungidzirwa kuti chinobata chikamu che30% chehuwandu hwepasi rose, izvo zvinoisa huwandu hwevanhu panjodzi9,10.Toxoplasma gondii inogona kukanganisa nhengo dzinokosha, kusanganisira iyo yepakati tsinga system (CNS), uye kukonzera hosha dzakakomba senge inouraya meningitis uye encephalitis, kunyanya muvarwere vasina immunocompromised9.Nekudaro, Toxoplasma gondii inogonawo kushandura nharaunda yeanotapukirwa nekugadzirisa kukura kwesero uye mhinduro dzemuviri muvanhu vasina immunocompetent, zvichitungamira mukuchengetwa kweasymptomatic chronic infection9,11.Sezvineiwo, tichifunga kuwirirana pakati T. gondii kuwanda uye kuitika kwebundu muuropi, mimwe mishumo inoratidza kuti in vivo host shanduko yezvakatipoteredza nekuda kwechirwere cheT. gondii chisingaperi chinofanana nebundu microenvironment.
Exosomes inozivikanwa se intercellular communicators iyo inopa zvinyorwa zvehupenyu, kusanganisira mapuroteni uye nucleic acids, kubva kumasero akavakidzana16,17.Exosomes inogona kukanganisa tumor-inoenderana nebiological maitiro akadai anti-apoptosis, angiogenesis, uye metastasis mune bundu microenvironment.Kunyanya, miRNAs (miRNAs), maRNA madiki asina-coding anenge makumi maviri nemaviri nucleotides pakureba, akakosha post-transcriptional gene regulators anodzora kupfuura 30% yemRNA yevanhu kuburikidza ne miRNA-induced silence complex (miRISC).Toxoplasma gondii inogona kukanganisa maitiro ehupenyu nekudzora miRNA kutaura mune vane hutachiona.Host miRNAs ine masiginecha akakosha ekudzora maitirwo ezvipenyu kuti awane nzira yekupona kweparasite.Nokudaro, kudzidza kuchinja kwemugadziri miRNA profile pane kutapukirwa neT. gondii kunogona kutibatsira kunzwisisa kupindirana pakati pemubati naT. gondii zvakajeka.Chokwadi, Thirugnanam et al.15 yakaratidza kuti T. gondii inosimudzira brain carcinogenesis nekushandura matauriro ayo pane chaiyo host miRNAs inobatanidza nekukura kwebundu uye yakawana kuti T. gondii inogona kukonzera gliomas mumhuka dzekuedza.
Ichi chidzidzo chinotarisa pakushandurwa kwe exosomal miR-21 mune inotambira microglia ine hutachiona neToxoplasma BV2.Isu takaona basa rinogoneka rekushandurwa exosomal miR-21 mukukura kweU87 glioma masero nekuda kwekuchengetwa kweiyo nucleus yeFoxO1 / p27, inova iyo inovavarira yakanyanyisa miR-21.
Exosomes inotorwa kubva kuBV2 yakawanikwa ichishandiswa kusiyanisa centrifugation uye yakasimbiswa nenzira dzakasiyana-siyana kudzivirira kusvibiswa nemasero emasero kana mamwe mavhesi.SDS-polyacrylamide gel electrophoresis (SDS-PAGE) yakaratidza maitiro akasiyana pakati pemapuroteni akatorwa kubva muBV2 masero uye exosomes (Mufananidzo 1A), uye sampuli dzakaongororwa kuvapo kweAlix, iyo yakaongororwa neWestern blotting ye exosomal protein markers mu.Alix labeling yakawanikwa mune exosome mapuroteni asi kwete muBV2 cell lysate mapuroteni (Fig. 1B).Pamusoro pezvo, RNA yakacheneswa kubva kune exosomes inotorwa kubva kuBV2 yakaongororwa uchishandisa bioanalyzer.18S uye 28S ribosomal subunits zvaisawanzoonekwa mune exosomal RNA migration pateni, zvichiratidza kuchena kwakavimbika (Mufananidzo 1C).Pakupedzisira, kutapurirana erekitironi maikorosikopu kwakaratidza kuti akacherekedza exosomes akanga anenge 60-150 nm muhukuru uye aiva nekapu-kufanana marongerwo akafanana exosome morphology (Fig. 1D).
Hunhu hwe exosomes inotorwa kubva kuBV2 masero.(A) Chengetedzo data sheet peji.Mapuroteni akaparadzaniswa kubva kuBV2 masero kana exosomes akatorwa kubva kuBV2.Mapuroteni maitiro anosiyana pakati pemasero uye exosomes.(B) Western blot kuongororwa kweiyo exosomal marker (Alix).(C) Kuongororwa kweRNA yakacheneswa kubva kuBV2 masero uye BV2 yakatorwa exosomes uchishandisa bioanalyzer.Nokudaro, 18S uye 28S ribosomal subunits muBV2 masero akanga asingawanzo kuwanikwa mu exosomal RNA.(D) Transmission electron microscopy yakaratidza kuti exosomes yakaparadzaniswa neBV2 masero akanga asina kusvibiswa ne2% uranyl acetate.Exosomes angangoita 60-150 nm muhukuru uye mukombe-akaumbwa (Rwiyo naJung, isina kuburitswa data).
Kuisa mukati meserura yeBV2-yakatorwa exosomes muU87 yemunhu glioma masero akaonekwa achishandisa confocal microscopy.PKH26 yakanyorwa exosomes inowanikwa mu cytoplasm yeU87 masero.Nuclei yakasvibiswa neDAPI (Fig. 2A), zvichiratidza kuti BV2-yakagadzirwa exosomes inogona kuiswa mukati nemasero ekugadzirisa uye inokonzera nzvimbo yemasero anogamuchira.
Internalization yeBV2-yakatorwa exosomes muU87 glioma masero uye BV2-yakatorwa exosomes inotapukirwa neToxoplasma RH yakakonzera kuwanda kweU87 glioma masero.(A) Exosomes akaputirwa neU87 masero akayerwa neconfocal microscopy.U87 glioma masero akaiswa exosomes akanyorwa kuti PKH26 (tsvuku) kana asina kutonga kwemaawa makumi maviri nemana.Iyo nuclei yakasvibiswa neDAPI (blue) uye yakazoonekwa pasi peiyo confocal microscope (scale bar: 10 μm, x 3000).(B) U87 glioma cell proliferation yakatemwa nesero proliferation assay.U87 glioma masero akabatwa ne exosomes kwenguva yakatarwa. *P <0.05 yakawanikwa neMudzidzi t bvunzo. *P <0.05 yakawanikwa neMudzidzi t bvunzo. *P < 0,05 получено по t-критерию Стьюдента. *P <0.05 neMudzidzi t-bvunzo. *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 yakawanikwa uchishandisa t-bvunzo yeMudzidzi.
Mushure mekusimbisa kuiswa kwemukati kweBV2-yakatorwa exosomes muU87 glioma masero, takaita masero ekuwedzera bvunzo kuti tiongorore basa reBV2-yakatorwa Toxoplasma-yakatorwa exosomes mukuvandudza masero eglioma evanhu.Kurapa kwemasero eU87 ane exosomes kubva kuT. gondii-infected BV2 masero akaratidza kuti T. gondii-inotapukirwa neBV2-yakagadzirwa exosomes yakakonzera kupararira kwakanyanya kwemasero eU87 achienzaniswa nekutonga (Fig. 2B).
Mukuwedzera, kukura kwemasero eU118 kwaiva nemigumisiro yakafanana neU87, sezvo Toxoplasma yakakurudzira exosomes yakakonzera kuwanda kwepamusoro (data isina kuratidzwa).Kubva pane idzi data, tinogona kuratidza kuti BV2-yakatorwa Toxoplasma-inotapukirwa exosomes inoita basa rakakosha mukuwanda kwe glioma cell.
Kuti tiongorore mashandiro eToxoplasma-infected BV2-derived exosomes pakukura kwebundu, takabaya masero eU87 glioma mumakonzo asina kusimira emuenzaniso wexenograft uye takabaya maexosomes anotorwa neBV2 kana maRH-akatapukirwa neBV2-anotorwa exosomes.Mushure mekunge mapundu ave pachena mushure mevhiki ye1, boka rega rega rekuedza remakonzo mashanu rakakamurwa zvichienderana nekukura kwebundu kuti rione nzvimbo imwechete yekutangira, uye saizi yebundu yakayerwa kwemazuva makumi maviri nemaviri.
Mumakonzo ane U87 xenograft modhi, zvakakura zvakanyanya kukura bundu uye uremu zvakaonekwa muBV2-yakatorwa RH-infected exosome boka pazuva 22 (Fig. 3A, B).Kune rimwe divi, pakanga pasina mutsauko wakakosha muhukuru hwebundu pakati peBV2-yakatorwa exosome boka neboka rekutonga mushure mekurapa exosome.Uyezve, mbeva jekiseni pamwe glioma masero uye exosomes nemaziso airatidza yakakura bundu vhoriyamu muboka RH-ine utachiona BV2-yakatorwa exosomes (Fig. 3C).Mhedzisiro iyi inoratidza kuti BV2-yakatorwa Toxoplasma-yakatapukirwa exosomes inoita kuti glioma ikure mune yembeva tumor modhi.
Oncogenesis (AC) yeBV2-yakatorwa exosomes muU87 xenograft mouse modhi.Tumor size (A) uye huremu (B) yakawedzera zvakanyanya muBALB / c nude mice yakabatwa neRH-infected exosomes yakabva kuBV2.BALB/c nude mbeva (C) dzakaiswa jekiseni pasi peganda ne 1 x 107 U87 masero akamiswa mumusanganiswa weMatrigel.Mazuva matanhatu mushure mejekiseni, 100 μg yeBV2-yakatorwa exosomes yakarapwa mumakonzo.Kukura kwebundu uye kuyerwa kwaiyerwa pamazuva airatidzwa uye mushure mechibayiro, zvichiteerana. *P <0.05. *P <0.05. *Р <0,05. *P <0.05. *P <0.05. *P <0.05. *Р <0,05. *P <0.05.
Dhiyabhorosi yakaratidza kuti 37 miRNAs (16 yakanyanyisa uye 21 yakaderera) yakabatana nehutachiona kana kukura kwechirwere yakashandurwa zvakanyanya mu microglia mushure mekutapukirwa neToxoplasma RH strain (Fig. 4A).Hukama hwekutaura huwandu hwe miR-21 pakati pemiRNAs yakashandurwa yakasimbiswa nechaiyo-nguva RT-PCR mune exosomes inotorwa kubva kuBV2, exosomes inobatwa neBV2 uye U87 masero.Kutaura kwe miR-21 kwakaratidza kuwedzera kukuru kwe exosomes kubva kuBV2 masero ane utachiona neToxoplasma gondii (RH strain) (Fig. 4B).Relative kutaura mazinga e miR-21 muBV2 uye U87 masero akawedzera mushure mekutora kwakashandurwa exosomes (Fig. 4B).The hama mwero miR-21 kutaura muuropi zvinyama bundu varwere uye mbeva utachiona Toxoplasma gondii (ME49 strain) akanga akakwirira kupfuura zvidzoro, ukuwo (Fig. 4C).Mhedzisiro iyi inoenderana nekusiyana pakati pematanho ekutaura eakafanotaurwa uye akasimbiswa mamicroRNAs mu vitro uye mu vivo.
Shanduko mukutaura kwe exosomal miP-21a-5p mu microglia inotapukirwa neToxoplasma gondii (RH).(A) Inoratidza kuchinja kukuru muSiRNA inobatanidza nehutachiona kana kukura kwechirwere chinotevera T. gondii RH chirwere.(B) Relative miR-21 kutaura mazinga akaonekwa nechaiyo-nguva RT-PCR muBV2-yakatorwa exosomes, BV2-yakabatwa exosomes, uye U87 masero.(C) Relative miR-21 kutaura mazinga akawanikwa muuropi matishu emota varwere (N = 3) uye mbeva dzakatapukirwa neToxoplasma gondii (ME49 strain) (N = 3). *P <0.05 yakawanikwa neMudzidzi t bvunzo. *P <0.05 yakawanikwa neMudzidzi t bvunzo. *P < 0,05 было получено с помощью t-критерия Стьюдента. *P <0.05 yakawanikwa pachishandiswa t-test yeMudzidzi. *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 yakawanikwa uchishandisa t-bvunzo yeMudzidzi.
Exosomes kubva kuRH-infected BV2 masero akatungamirira kukura kwegliomas mu vivo uye in vitro (Fig. 2, 3).Kuti tione mRNAs akakodzera, takaongorora mRNA mazinga eantitumor target genes, forkhead box O1 (FoxO1), PTEN, uye programmed cell death 4 (PDCD4) mumasero eU87 ane hutachiona hweexosomes anobva kuBV2 kana RH BV2.Ongororo yeBioinformatics yakaratidza kuti akati wandei ane tumarara emajini, kusanganisira iyo FoxO1, PTEN, uye PDCD4 genes, ine miR-2121,22 inosunga nzvimbo.mRNA mazinga e-antitumor target genes akaderedzwa muRH-infected BV2-derived exosomes achienzaniswa neBV2-derived exosomes (Fig. 5A).FoxO1 yakaratidza kuderedzwa kweprotein mazinga muRH-yakatapukirwa neBV2-yakatorwa exosomes yakaenzaniswa neBV2-yakatorwa exosomes (Mufananidzo 5B).Zvichienderana nemhedzisiro iyi, tinogona kusimbisa kuti exosomes inotorwa kubva kuRH-yakatapukirwa BV2 inoderedza anti-oncogenic genes, kuchengetedza basa rayo mukukura kwebundu.
Toxoplasma RH-infected BV2-derived exosomes inokonzera kudzvinyirirwa kwemajene antitumor mumasero eU87 glioma neToxoplasma RH-infected BV2-derived exosomes.(A) Real-time PCR yeFoxO1, PTEN uye PDCD4 kutaura mune exosomes yakabva kuT. gondii RH-infected BV2 kana ichienzaniswa nePBS exosomes.β-actin mRNA yakashandiswa sekutonga.(B) FoxO1 kutaura kwakatemwa neWestern blotting uye densitometry data yakaongororwa nenhamba uchishandisa iyo ImageJ chirongwa. *P <0.05 yakawanikwa neMudzidzi t bvunzo. *P <0.05 yakawanikwa neMudzidzi t bvunzo. *P < 0,05 было получено с помощью t-критерия Стьюдента. *P <0.05 yakawanikwa pachishandiswa t-test yeMudzidzi. *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 yakawanikwa uchishandisa t-bvunzo yeMudzidzi.
Kuti unzwisise mhedzisiro ye miP-21 mune exosomes pane tumor-inosanganiswa gene regulation, masero eU87 akachinjirwa ne inhibitor ye miP-21 achishandisa Lipofectamine 2000 uye maseru akakohwewa 24 maawa mushure mekutapurirana.FoxO1 uye p27 mazinga ekutaura mumasero akachinjwa ne miR-21 inhibitors akafananidzwa nemasero anobatwa neBV2-derived exosomes achishandisa qRT-PCR (Fig. 6A, B).Kuchinja kweiyo miR-21 inhibitor mumasero eU87 zvakanyanya kuderedza FoxO1 uye p27 kutaura (FIG. 6).
RH-infected exosomal BV2-derived miP-21 yakashandura FoxO1/p27 kutaura muU87 glioma masero.Masero eU87 akatapurirwa ne miP-21 inhibitor achishandisa Lipofectamine 2000 uye masero akakohwewa maawa makumi maviri nemana mushure mekutapurirana.FoxO1 uye p27 mazinga ekutaura mumaseru akachinjirwa ane miR-21 inhibitors akafananidzwa nemazinga mumaseru anobatwa neBV2-yakatorwa exosomes vachishandisa qRT-PCR (A, B).
Kuti utize mhinduro yomugadziri, utachiona hweToxoplasma hunoshanduka kuita cyst.Ivo vanoparadzisa zvinyama zvakasiyana-siyana, kusanganisira uropi, moyo, uye tsandanyama, kwehupenyu hwese hwemugamuchiri uye vanogadzirisa maitiro ekudzivirira emuviri.Mukuwedzera, ivo vanogona kudzora kutenderera kwesero uye apoptosis yemasero anotambira, vachikurudzira kuwanda kwavo14,24.Toxoplasma gondii inonyanya kukanganisa masero edendritic, neutrophils, uye monocyte/macrophage mutsara, kusanganisira brain microglia.Toxoplasma gondii inokonzera kusiyanisa kwema macrophages yeM2 phenotype, inokanganisa kuporesa kweronda mushure mehutachiona hwehutachiona, uye inobatanidzawo ne hypervascularization uye granulomatous fibrosis.Iyi maitiro ekuita pathogenesis yeToxoplasma hutachiona inogona kunge yakabatana nemakasi ane chekuita nekukura kwebundu.Iyo nharaunda ine hutsinye inodzorwa neToxoplasma inogona kufanana neinofanana precancer.Nokudaro, zvinogona kufungidzirwa kuti utachiona hweToxoplasma hunofanira kubatsira mukukura kwemamota europi.Muchokwadi, huwandu hwepamusoro hwehutachiona hweToxoplasma hwakataurwa muserum yevarwere vane mamota akasiyana ehuropi.Pamusoro pezvo, Toxoplasma gondii inogona kunge iri imwe carcinogenic effect uye kuita synergistically kubatsira mamwe macarcinogens anotapukira kukudziridza mapundu europi.Panyaya iyi, zvakakosha kuziva kuti P. falciparum uye Epstein-Barr virus synergistically inobatsira pakuumbwa kweBurkitt's lymphoma.
Basa re exosomes sevanodzora mumunda wekutsvagisa cancer rakaongororwa zvakanyanya.Nekudaro, basa re exosomes pakati pemaparasite neane hutachiona rinoramba risinganzwisisike.Kusvika pari zvino, vatongi vakasiyana-siyana, kusanganisira mapuroteni akavanzika, vakatsanangura maitiro ebhayoloji ayo maparasite eprotozoan anoramba kurwiswa kwevatambi uye kuenderera mberi nehutachiona.Munguva pfupi yadarika, kwave neruzivo rwuri kukura rwekuti maprotozoan-akabatana mamicrovesicles uye mamicroRNAs awo anodyidzana neanogamuchira maseru kugadzira nharaunda yakanaka yekurarama kwavo.Naizvozvo, zvimwe zvidzidzo zvinodiwa kuti uwane hukama pakati peakachinjika exosomal miRNAs uye glioma cell kuwanda.MicroRNA alteration (cluster genes miR-30c-1, miR-125b-2, miR-23b-27b-24-1 uye miR-17-92) inosunga kune STAT3 inosimudzira mu toxoplasma-inotapukirwa nevanhu macrophages, inodzorwa uye induces anti. -apoptosis mukupindura Toxoplasma gondii chirwere 29.Utachiona hweToxoplasma hunowedzera kutaura kwe miR-17-5p uye miR-106b-5p, iyo inosanganiswa nezvirwere zvakawanda zve hyperproliferative 30.Idzi data dzinoratidza kuti miRNAs inodzorwa neToxoplasma utachiona mamorekuru akakosha ekupona kweparasite uye pathogenesis mune inotambira biological maitiro.
Yakashandurwa miRNAs inogona kukanganisa marudzi akasiyana-siyana ehunhu panguva yekutanga uye kufambira mberi kwemasero akashata, anosanganisira gliomas: kuzvikwanira kwezviratidzo zvekukura, kusanzwa kune masaini-anodzivirira masaini, apoptosis evasion, unlimited kudzokorora kugona, angiogenesis, kupinda uye metastasis, uye kuzvimba.Mu glioma, yakashandurwa miRNAs yakaonekwa mune akati wandei ekutaura profiling zvidzidzo.
Muchidzidzo chazvino, takasimbisa mazinga epamusoro e miRNA-21 kutaura mune toxoplasma-ine hutachiona hwemaseru maseru.miR-21 yakaonekwa seimwe yeanonyanya kudzvinyirirwa microRNAs mumamota akasimba, anosanganisira gliomas, 33 uye kutaura kwayo kunopindirana negiredhi reglioma.Kuunganidza humbowo hunoratidza kuti miR-21 inyowani oncogene inoshanda senge anti-apoptotic chinhu mukukura kweglioma uye yakanyanya kuwedzeredzwa mumatishu uye plasma yehuropi hwemunhu.Sezvineiwo, miR-21 inactivation mumaseru eglioma uye matishu inokonzeresa kuvharirwa kwesero kuwanda nekuda kwecaspase-inotsamira apoptosis.Ongororo yeBioinformatic ye miR-21 yakafanotaurwa zvinangwa yakaratidza akawanda bundu suppressor genes ane chekuita neapoptosis nzira, kusanganisira programmed cell death 4 (PDCD4), tropomyosin (TPM1), PTEN, uye forkhead bhokisi O1 (FoxO1), ine miR-2121 inosunga saiti..22.38.
FoxO1, seimwe yezvinyorwa zvinyorwa (FoxO), inobatanidzwa mukugadzirwa kwemhando dzakasiyana dzegomarara revanhu uye inokwanisa kudzora mataurirwo ejene rebundu suppressor senge p21, p27, Bim, uye FasL40.FoxO1 inogona kusunga uye kumisa cell cycle inhibitors senge p27 kudzvanya kukura kwesero.Uyezve, FoxO1 ndiyo yakakosha mhedzisiro yePI3K/Akt kusaina uye inoronga akawanda maitiro ebhayoloji akadai sekufambira mberi kwesero uye kusiyanisa kwesero kuburikidza ne activation yep2742 transcription.
Mukupedzisa, tinotenda kuti exosomal miR-21 yakabva kuToxoplasma-infected microglia inogona kuita basa rinokosha semugadziri wekukura kwemasero eglioma (Fig. 7).Nekudaro, zvimwe zvidzidzo zvinodikanwa kuwana chinongedzo chakananga pakati peexosomal miR-21, yakashandurwa hutachiona hweToxoplasma, uye kukura kweglioma.Mhedzisiro iyi inotarisirwa kupa pekutangira pakudzidza hukama pakati peToxoplasma utachiona uye chiitiko cheglioma.
A schematic diagram yemaitiro eglioma (brain) carcinogenesis inotsanangurwa muchidzidzo ichi.Iye munyori anodhirowa muPowerPoint 2019 (Microsoft, Redmond, WA).
Mitemo yose yekuedza muchidzidzo ichi, kusanganisira kushandiswa kwemhuka, yaienderana neSeoul National University Animal Care uye User Committee Standard Ethical Guidelines uye yakabvumirwa neInstitutional Review Board yeSeoul National University School of Medicine (IRB nhamba SNU- 150715).-2).Ese maitiro ekuyedza akaitwa zvinoenderana neARRIVE kurudziro.
BV2 mouse microglia uye U87 human glioma masero akagadzirwa muDulbecco's Modified Eagle's Medium (DMEM; Welgene, Seoul, Korea) uye Roswell Park Memorial Institute's Medium (RPMI; Welgene), zvichiteerana, imwe neimwe iine 10% fetal bovine serum, 4 mM l- glutamine, 0.2 mM penicillin uye 0.05 mM streptomycin.Masero akagadzirwa muincubator ine 5% CO2 pa37°C.Imwe glioma cell line, U118, yakashandiswa kuenzanisa neU87 masero.
Kuparadzanisa exosomes kubva kuT. gondii-infected RH uye ME49 strains, T. gondii tachyzoites (RH strain) yakakohwewa kubva mudumbu remudumbu re6-vhiki yeBALB / c mice injected 3-4 mazuva apfuura.Tachyzoites yakashambidzwa katatu nePBS uye yakacheneswa ne centrifugation mu40% Percoll (Sigma-Aldrich, St. Louis, MO, USA)43.Kuti uwane tachyzoites of strain ME49, BALB / c mbeva dzakaiswa intraperitoneally intraperitoneally ne 20 tissue cysts uye tachyzoite shanduko muma cysts yakaunganidzwa nekugeza dumbu remudumbu pazuva re6-8 mushure mekutapukirwa (PI).Mbeva dzakabatwa nePBS.ME49 tachyzoites yakakura mumasero akawedzerwa ne100 μg/ml penicillin (Gibco/BRL, Grand Island, NY, USA), 100 μg/ml streptomycin (Gibco/BRL), uye 5% fetal bovine serum (Lonza, Walkersville, MD) .., USA) pa37 °C uye 5% carbon dioxide.Mushure mekurima muVero masero, ME49 tachyzoites yakapfuudzwa kaviri kuburikidza ne25 gauge tsono uyezve kuburikidza ne5 µm sefa yekubvisa marara nemaseru.Mushure mekugeza, ma tachyzoite akamiswa zvakare muPBS44.Tissue cysts yeToxoplasma gondii strain ME49 yakachengetwa nejekiseni remukati rema cysts akaparadzaniswa kubva kuuropi hwehutachiona hweC57BL / 6 mice (Orient Bio Animal Center, Seongnam, Korea).Uropi hweME49-ane hutachiona mbeva dzakakohwewa mushure memwedzi mitatu yePI uye yakachekwa pasi pemaikorosikopu kuti iparadzanise macysts.Makonzo ane hutachiona akachengetwa pasi peyakakosha pathogen-isina mamiriro (SPF) paSeoul National University Chikoro cheMishonga.
Yese RNA yakatorwa kubva kuBV2-yakatorwa exosomes, BV2 maseru uye matishu achishandisa iyo miRNeasy Mini Kit (Qiagen, Hilden, Germany) maererano nemirairo yemugadziri, kusanganisira iyo incubation nhanho yerution.Iyo RNA yakagadziriswa yakatemerwa paNanoDrop 2000 spectrophotometer.Hunhu hweRNA microarrays hwakaongororwa uchishandisa Agilent 2100 bioanalyzer (Agilent Technologies, Amstelveen, Netherlands).
DMEM ine 10% exosome-varombo FBS yakagadzirwa neultracentrifugation pa100,000g kwemaawa gumi nematanhatu pa4°C uye yakasefa kuburikidza ne0.22 µm sefa (Nalgene, Rochester, NY, USA).BV2 masero, 5 × 105, akagadzirwa muDMEM ine 10% exosome-depleted FBS uye 1% mishonga inorwisa mabhakitiriya pa37 ° C uye 5% CO2.Mushure memaawa makumi maviri nemana ekuputira, tachyzoites yekutambudzika RH kana ME49 (MOI = 10) yakawedzerwa kumasero uye zvipembenene zvisingapindiki zvakabviswa mukati meawa uye zvakazadzwa neDMEM.Exosomes kubva kuBV2 masero akaparadzaniswa neakagadziridzwa musiyano centrifugation, iyo inonyanya kushandiswa nzira.Dzorera iyo exosome pellet mu300 µl PBS yeRNA kana kuongorora mapuroteni.Iko kusungirirwa kwemaexosomes ega kwakatemwa kushandisa BCA protein assay kit (Pierce, Rockford, IL, USA) uye NanoDrop 2000 spectrophotometer.
Mvura inobva kuBV2 maseru kana exosomes yakabva kuBV2 yakanyungudutswa muPRO-PREP ™ protein extraction solution (iNtRon Biotechnology, Seongnam, Korea) uye mapuroteni akaiswa paCoomassie inopenya yebhuruu yakasvibiswa 10% SDS polyacrylamide gels.Mukuwedzera, mapuroteni akaendeswa kuPVDF membranes kwemaawa maviri.Mabhuroti ekuMadokero akasimbiswa pachishandiswa Alix antibody (Cell Signaling Technology, Beverly, MA, USA) sechiratidzo che exosomal.HRP-conjugated mbudzi anti-mouse IgG (H + L) (Bethyl Laboratories, Montgomery, TX, USA) uye LAS-1000 plus luminescent image analyzer (Fuji Photographic Film, Tokyo, Japan) yakashandiswa sechipiri antibody..Transmission electron microscopy yakaitwa kuti idzidze kukura uye morphology ye exosomes.Exosomes akaparadzaniswa kubva kuBV2 masero (6.40 µg / µl) akagadzirirwa pa carbon-coated meshes uye akasvibiswa zvisina kunaka ne2% uranyl acetate ye1 min.Samples dzakagadzirirwa dzakaonekwa painomhanyisa voltage ye80 kV uchishandisa JEOL 1200-EX II (Tokyo, Japan) ine ES1000W Erlangshen CCD kamera (Gatan, Pleasanton, CA, USA).
BV2-yakagadzirwa exosomes yakasvibiswa uchishandisa PKH26 Red Fluorescent Linker Kit (Sigma-Aldrich, St. Louis, MO, USA) kwemaminitsi gumi nemashanu pakupisa kwekamuri.U87 masero, 2 × 105, ane PKH26-yakanyorwa exosomes (tsvuku) kana pasina exosomes sekutonga kusina kunaka, akaiswa pa 37 ° C kwemaawa makumi maviri nemana mu 5% CO2 incubator.U87 cell nuclei yakasvibiswa neDAPI (blue), masero eU87 akaiswa mu4% paraformaldehyde ye15 min pa4 ° C uye akazoongororwa muLeica TCS SP8 STED CW confocal microscope system (Leica Microsystems, Mannheim, Germany).zvinoonekwa.
cDNA yakagadzirwa kubva kuSiRNA ichishandisa Mir-X siRNA yekutanga strand synthesis uye SYBR qRT-PCR kit (Takara Bio Inc., Shiga, Japan).Real time quantitative PCR yakaitwa pachishandiswa iQ5 real time PCR monitoring system (Bio-Rad, Hercules, CA, USA) uchishandisa maprimers uye matemplate akasanganiswa neSYBR Premix.DNA yakakwidziridzwa kuitira 40 cycles of denaturation pa 95°C for 15 s uye annealing pa60°C kwe60 s.Iyo data kubva kune yega yega PCR maitiro akaongororwa uchishandisa iyo data yekuongorora module yeIQ™5 optical system software (Bio-Rad).Kuchinja kwechikafu mukutaura kwemajini pakati peakasarudzwa chinangwa chemajini uye β-actin/siRNA (uye U6) akaverengerwa pachishandiswa nzira yakajairika.Iyo yekutanga sequences inoshandiswa inoratidzwa muTafura 1.
3 x 104 U87 glioma masero akadyarwa mu96-tsime mahwendefa uye akasanganiswa neToxoplasma-infected exosomes yakabva kuBV2 (50 μg/mL) kana nonpulse exosomes yakabva kuBV2 (50 μg/mL) sekutonga pa12, maawa 18 ne36. .Chiyero chekuwedzera kwesero chakagadziriswa uchishandisa Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) (Supplementary Figures S1-S3) 46.
5-vhiki-ekura mbeva dzevakadzi BALB/c dzakashama dzakatengwa kubva kuOrient Bio (Seongnam-si, South Korea) uye dzakachengetwa imwe neimwe mumakeji asina utachiona patembiricha yekamuri (22 ± 2 ° C) uye humidity (45±15°C).%) pane tembiricha yekamuri (22±2°C) uye mwando (45±15%).A 12-awa chiedza kutenderera uye 12-awa yakasviba kutenderera kwakaitwa pasi peSPF (Seoul National University School of Medicine Animal Center).Mbeva dzakapatsanurwa mumapoka matatu emakonzo e5 rimwe nerimwe uye mapoka ese akaiswa jekiseni pasi ne 400 ml yePBS ine 1 x 107 U87 glioma masero uye kukura chinhu chakaderedzwa BD Matrigel ™ (BD Science, Miami, FL, USA).Mazuva matanhatu mushure mejekiseni rebundu, 200 mg ye exosomes yakatorwa kubva kuBV2 masero (ane / asina Toxoplasma utachiona) akaiswa panzvimbo yebundu.Mazuva makumi maviri nemaviri mushure mekutapukirwa nebundu, ukuru hwebundu rembeva muboka rega rega hwakayerwa necaliper katatu pasvondo, uye bundu vhoriyamu yakaverengerwa neformula: 0.5 × (hupamhi) × 2 × kureba.
MicroRNA kutaura kwekuongorora uchishandisa miRCURYTM LNA miRNA array, 7th generation ine, mmu uye rno arrays (EXIQON, Vedbaek, Denmark) inovhara 1119 mbeva dzinonyatsozivikanwa pakati pe3100 vanhu, mbeva uye rat miRNA kutora probes.Mukati mekuita uku, 250 kusvika 1000 ng yeRNA yakazara yakabviswa kubva ku5'-phosphate nekurapwa nemhuru intestinal alkaline phosphatase ichiteverwa nekunyora neHy3 green fluorescent dhayi.Mienzaniso yakanyorwa yakazosanganiswa nekurodha masiraidhi emicroarray pachishandiswa hybridization chamber kit (Agilent Technologies, Santa Clara, CA, USA) uye masiraidhi ekubatanidza masiraidhi (Agilent Technologies).Hybridization yakaitwa kwemaawa gumi nematanhatu pa56 ° C, ipapo ma microarrays akashambidzwa maererano nemazano emugadziri.Iwo akagadziridzwa microarray masiraidhi akabva aongororwa uchishandisa Agilent G2565CA microarray scanner system (Agilent Technologies).Mifananidzo yakaongororwa yakaunzwa kunze kwenyika pachishandiswa Agilent Feature Extraction software version 10.7.3.1 (Agilent Technologies) uye fluorescence intensity yemufananidzo wega wega yakagadziriswa pachishandiswa GAL faira inoenderana nemodified Exiqon protocol.Microarray data yechidzidzo chazvino inoiswa mudhatabhesi yeGEO pasi penhamba yekupinda GPL32397.
Mataurirwo ezvinyorwa zveexosomal miRNAs yakakura mu microglia yeRH kana ME49 strains yakabatwa neToxoplasma yakaongororwa pachishandiswa maturusi akasiyana etiweki.miRNAs ine chekuita nekukura kwebundu yakaonekwa pachishandiswa miRWalk2.0 (http://mirwalk.umm.uni-heidelberg.de) uye yakasefa neyakajairwa chiratidzo chesimba (log2) yakakura kupfuura 8.0.Pakati pemiRNAs, miRNA yakaratidzwa zvakasiyana-siyana yakawanikwa inopfuura 1.5-yakashandurwa nekuongororwa kwesefa ye miRNAs yakashandurwa neRH kana ME49 mashizha ane T. gondii.
Masero akadyarwa mumahwendefa matanhatu-tsime (3 x 105 masero / zvakanaka) mune opti-MEM (Gibco, Carlsbad, CA, USA) achishandisa Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).Masero akatapurirwa akagadzirwa kwemaawa matanhatu uye ipapo yepakati yakashandurwa kuita nyowani yakazara svikiro.Masero akakohwewa maawa makumi maviri nemana mushure mekutapurirana.
Ongororo yenhamba yainyanya kuitwa pachishandiswa t-test yeMudzidzi neExcel software (Microsoft, Washington, DC, USA).Pakuongorora mhuka dzekuyedza, nzira mbiri ANOVA yakaitwa pachishandiswa Prism 3.0 software (GraphPad Software, La Jolla, CA, USA). P-values ​​<0.05 yakaonekwa seyakakosha. P-tsika <0.05 yaionekwa seyakakosha. Значения P <0,05 считались статистически значимыми. P maitiro <0.05 aionekwa seakakosha. P 值< 0.05 被认为具有统计学意义。 P <0.05 Значения P <0,05 считались статистически значимыми. P maitiro <0.05 aionekwa seakakosha.
Zvose zvibvumirano zvekuedza zvakashandiswa muchidzidzo ichi zvakagamuchirwa neInstitutional Review Board yeSeoul National University School of Medicine (IRB nhamba 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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