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Toxoplasma gondii ni parasite idasanzwe yo mu bwoko bwa protozoan ihindura mikorobe y’ibidukikije byanduye kandi bizwi ko bifitanye isano no gukura kw'ibibyimba byo mu bwonko.Muri ubu bushakashatsi, turakeka ko exosomal miRNA-21 yanduye Toxoplasma itera ikibyimba mu bwonko.Exosomes yavuye muri Toxoplasma yanduye BV2 microglia yaranzwe kandi byemezwa ko selile ya glioma U87 yemejwe.Exosomal microRNA imvugo yerekana isesengura ryakozwe hifashishijwe imirongo ya microRNA na microRNA-21A-5p ifitanye isano na Toxoplasma gondii no gutondeka ibibyimba.Twakoze kandi ubushakashatsi ku nzego za mRNA za gen zifitanye isano na kanseri mu ngirabuzimafatizo za U87 duhindura urwego rwa miR-21 muri exosomes ndetse n'ingaruka za exosomes ku ikwirakwizwa ry'uturemangingo twa glioma U87.Muri exosomes ya U87 selile glioma yanduye Toxoplasma gondii, imvugo ya microRNA-21 iriyongera kandi ibikorwa bya gen antitumor (FoxO1, PTEN, na PDCD4) biragabanuka.BV2 ikomoka kuri exosomes yanduye Toxoplasma itera ikwirakwizwa rya selile U87 glioma.Exosomes itera gukura kwa selile U87 muburyo bwikibyimba cyimbeba.Turasaba ko kwiyongera kwa miR-21 muri microglia ya BV2 yanduye Toxoplasma bishobora kugira uruhare runini nkiterambere ryimikorere ya selile muri selile U87 glioma mukugabanya genes za antitumor.
Biteganijwe ko mu mwaka wa 2018 hamenyekanye abantu barenga miliyoni 18.1 barwaye kanseri yateye imbere ku isi, aho buri mwaka hamenyekana ibibyimba byo mu mitsi yo hagati ya 297.000 (1.6% by'ibibyimba byose) 1.Ubushakashatsi bwakozwe mbere bwerekanye ko ibintu bishobora gutera ibibyimba byo mu bwonko by’umuntu birimo ibicuruzwa bitandukanye bivura imiti, amateka y’umuryango, hamwe n’imirasire ya ionizing biva mu bikoresho byo kuvura no gusuzuma indwara.Ariko, impamvu nyayo itera izo ndwara mbi ntiramenyekana.Hafi ya 20% ya kanseri zose ku isi ziterwa nindwara zanduza, harimo virusi, bagiteri na parasite3,4.Indwara ziterwa na virusi zihungabanya imikorere ya selile yakira, nko gusana ADN no kuzunguruka kwakagari, kandi bishobora gutera uburibwe budakira no kwangiza umubiri.
Indwara zandura zifitanye isano na kanseri y'abantu nizo virusi zikunze kwibasira virusi, harimo papillomavirus ya muntu na virusi ya hepatite B na C.Parasite irashobora kandi kugira uruhare runini mu mikurire ya kanseri y'abantu.Ubwoko butandukanye bwa parasite, aribwo Schistosoma, Opishorchis viverrini, O. felineus, Clonorchis sinensis na Hymenolepis nana, bwagize uruhare mu bwoko butandukanye bwa kanseri y'abantu 6,7,8.
Toxoplasma gondii ni protozoan idasanzwe igenga microen ibidukikije ya selile zanduye.Iyi parasite ngo yandura hafi 30% byabatuye isi, bigatuma abaturage bose bageramiwe 9,10.Toxoplasma gondii irashobora kwanduza ingingo zikomeye, harimo na sisitemu yo hagati (CNS), kandi igatera indwara zikomeye nka meningite yica na encephalite, cyane cyane ku barwayi badafite ubudahangarwa9.Nyamara, Toxoplasma gondii irashobora kandi guhindura ibidukikije byanduye wanduye muguhindura imikurire yingirabuzimafatizo hamwe nubudahangarwa bw'umubiri ku bantu badafite ubudahangarwa, biganisha ku gukomeza kwandura indwara zidakira 9,11.Igishimishije, urebye isano iri hagati ya T. gondii yanduye nubwonko bwibibyimba mu bwonko, raporo zimwe zerekana ko muri vivo yakira impinduka z’ibidukikije bitewe n'indwara idakira ya T. gondii isa na microen ibidukikije.
Exosomes izwi nk'itumanaho rinyuranye ritanga ibinyabuzima, harimo proteyine na acide nucleic, biva mu tugari duturanye16,17.Exosomes irashobora kugira ingaruka kumikorere yibinyabuzima nka anti-apoptose, angiogenez, na metastasis muri microc ibidukikije.By'umwihariko, miRNAs (miRNAs), RNAs zidafite kodegisi ya RNA zigera kuri 22 nucleotide mu burebure, ni ingenzi nyuma yo kwanduza gene igenzura ibice birenga 30% bya mRNA byabantu binyuze muri miRNA iterwa no gucecekesha (miRISC).Toxoplasma gondii irashobora guhagarika inzira yibinyabuzima mugucunga imvugo ya miRNA mubakira.MiRNAs yakiriye ikubiyemo ibimenyetso byingenzi byo kugenzura ibinyabuzima byakiriye kugirango bigere ku ngamba zo kubaho kwa parasite.Rero, kwiga impinduka mubakira miRNA umwirondoro wanduye T. gondii birashobora kudufasha kumva neza imikoranire hagati ya nyiricyubahiro na T. gondii neza.Mubyukuri, Thirugnanam n'abandi.15 yatanze igitekerezo ko T. gondii itera kanseri yo mu bwonko ihindura imvugo kuri miRNA yihariye yakira ikura ry’ibibyimba isanga T. gondii ishobora gutera glioma mu nyamaswa zigerageza.
Ubu bushakashatsi bwibanze ku guhindura miR-21 ya exosomal muri microglia yakira yanduye Toxoplasma BV2.Twabonye uruhare rushoboka rwa miR-21 yahinduwe mu mikurire ya selile glioma U87 bitewe no kugumana muri nucleus ya FoxO1 / p27, ikaba ari yo yibasiwe na miR-21 ikabije.
Exosomes yakomotse kuri BV2 yabonetse hakoreshejwe centrifugation itandukanye kandi yemejwe nuburyo butandukanye bwo kwirinda kwanduza ibice bigize selile cyangwa izindi viticles.SDS-polyacrylamide gel electrophorei (SDS-PAGE) yerekanye imiterere itandukanye hagati ya poroteyine zavanywe mu ngirabuzimafatizo za BV2 na exosomes (Igicapo 1A), kandi hasuzumwe ingero zerekana ko Alix yari ihari, yasesenguwe n’iburengerazuba bwahinduye ibimenyetso bya poroteyine za exosomal.Ikimenyetso cya Alix cyabonetse muri poroteyine zidasanzwe ariko ntiziboneka muri poroteyine ya BV2 ya selile (Ishusho 1B).Byongeye kandi, RNA yatunganijwe muri exosomes ikomoka kuri BV2 yasesenguwe hakoreshejwe bioanalyser.18S na 28S ribosomal subunits zagaragaye gake muburyo bwo kwimuka kwa RNA exosomal, byerekana ubuziranenge bwizewe (Ishusho 1C).Hanyuma, microscopi ya elegitoronike yerekanaga ko exosomes yagaragaye yari ifite uburebure bwa 60-150 nm kandi ifite imiterere isa nigikombe isanzwe ya morfologiya ya exosome (Ishusho 1D).
Kuranga exosomes ikomoka muri selile BV2.(A) Urupapuro rwamakuru yumutekano.Poroteyine zitandukanijwe na selile BV2 cyangwa exosomes zikomoka kuri BV2.Imiterere ya poroteyine itandukanye hagati ya selile na exosomes.(B) Isesengura ryiburengerazuba ryibimenyetso bya exosomal (Alix).(C) Isuzuma rya RNA isukuye muri selile BV2 na BV2 ikomoka kuri exosomes ikoresheje bioanalyser.Rero, 18S na 28S ribosomal subunits muri selile BV2 wasangaga gake muri RNA idasanzwe.(D) Ikwirakwizwa rya electron microscopi yerekanaga ko exosomes yitandukanije na selile BV2 yandujwe nabi na acetate ya uranyl 2%.Exosomes igera kuri 60-150 nm mu bunini no mu gikombe (Indirimbo na Jung, amakuru adashyizwe ahagaragara).
Imikorere ya selile ya BV2 ikomoka muri exosomes muri selile ya glioma ya U87 yabonetse hakoreshejwe microscopi conocal.PKH26 yanditseho exosomes iri muri cytoplazme ya selile U87.Nuclei yandujwe na DAPI (Igishusho 2A), byerekana ko exosomes ikomoka kuri BV2 ishobora kwinjizwa mu ngirabuzimafatizo kandi ikagira ingaruka ku bidukikije by'utugingo ngengabuzima.
Kwinjiza imbere ya BV2 ikomoka kuri BV2 muri selile ya glioma U87 na BV2 ikomoka kuri BV2 yanduye Toxoplasma RH yatumye ikwirakwizwa rya selile U87.(A) Exosomes yafashwe na selile U87 yapimwe na microscopi ya conocal.U87 glioma selile yashizwemo na exosomes yanditseho PKH26 (umutuku) cyangwa nta kugenzura amasaha 24.Nuclei yandujwe na DAPI (ubururu) hanyuma ikurikiranwa munsi ya microscope (umurongo wapima: 10 μ m, x 3000).(B) Ikwirakwizwa rya glioma U87 ryagenwe no gukwirakwiza selile.U87 selile glioma yavuwe hamwe na exosomes mugihe cyerekanwe. * P <0.05 yabonetse n'ikizamini cya t. * P <0.05 yabonetse n'ikizamini cya t. * P <0,05 получено по t-критерию Стьюдента. * P <0.05 na t-test yabanyeshuri. * 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 wabonye ukoresheje t-test yabanyeshuri.
Nyuma yo kwemeza kwinjiza BV2 ikomoka muri BV2 muri selile ya glioma U87, twakoze ubushakashatsi bwo gukwirakwiza selile kugirango dukore iperereza ku ruhare rwa BV2 rukomoka kuri Toxoplasma rukomoka kuri Exosomes mu iterambere ry’uturemangingo twa glioma.Kuvura ingirabuzimafatizo za U87 hamwe na exosomes ziva mu ngirabuzimafatizo za BV2 zanduye T. gondii zerekanye ko T. gondii yanduye BV2 ikomoka kuri BV2 iterwa no kwiyongera cyane mu ngirabuzimafatizo za U87 ugereranije no kugenzura (Ishusho 2B).
Byongeye kandi, gukura kwingirabuzimafatizo za U118 byagize ibisubizo bimwe na U87, nkuko Toxoplasma itera exosomes itera urwego rwo hejuru rwo gukwirakwiza (amakuru aterekanwa).Dufatiye kuri aya makuru, dushobora kwerekana ko BV2 ikomoka kuri Toxoplasma yanduye Exosomes igira uruhare runini mu gukwirakwiza selile glioma.
Kugira ngo dukore iperereza ku ngaruka ziterwa na BV2 ikomoka kuri Toxoplasma yatewe no gukura kw'ibibyimba, twinjije selile U87 glioma mu mbeba zambaye ubusa zerekana urugero rwa xenograft hanyuma dushyiramo exosomes ikomoka kuri BV2 cyangwa RH yanduye BV2 ikomoka kuri RH.Ibibyimba bimaze kugaragara nyuma yicyumweru 1, buri tsinda ryigeragezwa ryimbeba 5 ryagabanijwe ukurikije ubunini bwibibyimba kugirango hamenyekane aho byatangiriye, kandi ubunini bwibibyimba bwapimwe muminsi 22.
Imbeba zifite urugero rwa U87 xenograft, ubunini bwikibyimba nuburemere byagaragaye cyane mumatsinda ya BV2 ikomoka kuri RH yanduye exosome kumunsi wa 22 (Ishusho 3A, B).Ku rundi ruhande, nta tandukaniro rikomeye ryagaragaye mu bunini bw’ibibyimba hagati ya BV2 ikomoka kuri exosome hamwe nitsinda rishinzwe kugenzura nyuma yo kuvura exosome.Byongeye kandi, imbeba zatewe na selile glioma na exosomes zerekanaga muburyo bunini bwibibyimba byinshi mumatsinda ya RH yanduye BV2 ikomoka kuri exosomes (Ishusho 3C).Ibisubizo byerekana ko BV2 ikomoka kuri Toxoplasma yanduye exosomes itera gukura kwa glioma muburyo bwikibyimba cyimbeba.
Oncogenezi (AC) ya BV2 ikomoka kuri exosomes muburyo bwimbeba ya U87 xenograft.Ingano y'ibibyimba (A) n'uburemere (B) yariyongereye cyane muri BALB / c imbeba yambaye ubusa ivurwa na exosomes yanduye RH ikomoka kuri BV2.Imbeba za BALB / c zambaye ubusa (C) zatewe inshinge hamwe na selile 1 x 107 U87 zahagaritswe muri Matrigel ivanze.Iminsi itandatu nyuma yo guterwa, 100 μg za BV2 zikomoka kuri exosomes zavuwe mu mbeba.Ingano n'uburemere byapimwe ku minsi yerekanwe na nyuma yo gutamba. * P <0.05. * P <0.05. * Р <0,05. * P <0.05. * P <0.05。 * P <0.05。 * Р <0,05. * P <0.05.
Amakuru yerekanaga ko miRNA 37 (16 zikabije na 21 zidakabije) zifitanye isano n’ubudahangarwa cyangwa ikibyimba cyahinduwe cyane muri microglia nyuma yo kwandura indwara ya Toxoplasma RH (Ishusho 4A).Urwego rwerekana imvugo ya miR-21 muri miRNA yahinduwe byemejwe nigihe nyacyo RT-PCR muri exosomes ikomoka kuri BV2, exosomes ivurwa na selile BV2 na U87.Kugaragaza miR-21 byagaragaje ubwiyongere bukabije bwa exosomes ziva muri selile BV2 zanduye Toxoplasma gondii (RH strain) (Ishusho 4B).Urwego rwerekana imvugo ya miR-21 muri selile BV2 na U87 rwiyongereye nyuma yo gufata exosomes zahinduwe (Ishusho 4B).Urwego rugereranije rwa miR-21 mumyanya yubwonko bwabarwayi b ibibyimba nimbeba zanduye Toxoplasma gondii (ME49 strain) byari hejuru kuruta kugenzura, (Ishusho 4C).Ibisubizo bifitanye isano no gutandukanya urwego rwimvugo ya microRNAs zahanuwe kandi zemejwe muri vitro no muri vivo.
Impinduka mu mvugo ya exosomal miP-21a-5p muri microglia yanduye Toxoplasma gondii (RH).(A) Yerekana impinduka zikomeye muri siRNA zijyanye n'ubudahangarwa cyangwa ikibyimba nyuma yo kwandura T. gondii RH... * P <0.05 yabonetse n'ikizamini cya t. * P <0.05 yabonetse n'ikizamini cya t. * P <0,05 было получено с помощью t-критерия Стьюдента. * P <0.05 yabonetse ukoresheje t-test yabanyeshuri. * 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 wabonye ukoresheje t-test yabanyeshuri.
Exosomes ituruka muri selile BV2 yanduye RH yatumye imikurire ya gliomasi muri vivo no muri vitro (Ishusho 2, 3).Kugirango tumenye mRNAs zifite akamaro, twasuzumye urwego rwa mRNA rwimikorere ya antitumor, agasanduku ka forkhead O1 (FoxO1), PTEN, hamwe nurupfu rwa selile 4 (PDCD4) muri selile U87 zanduye exosomes zikomoka kuri BV2 cyangwa RH BV2.Isesengura rya bioinformatics ryerekanye ko ingirabuzimafatizo nyinshi zifitanye isano n’ibibyimba, harimo na FoxO1, PTEN, na PDCD4, zifite miR-2121,22.mRNA urwego rwa antitumor genes yagabanutse muri RH yanduye BV2 ikomoka kuri exosomes ugereranije na BV2 ikomoka kuri BV2 (Ishusho 5A).FoxO1 yerekanye igabanuka rya poroteyine muri RH yanduye BV2 ikomoka kuri exosomes ugereranije na BV2 ikomoka kuri Exosomes (Ishusho 5B).Dufatiye kuri ibyo bisubizo, twakwemeza ko exosomes ikomoka kuri BV2 yanduye RH igabanya ingirabuzimafatizo zirwanya oncogeneque, zigakomeza uruhare rwazo mu mikurire y’ibibyimba.
Toxoplasma RH yanduye BV2 ikomoka kuri exosomes itera guhagarika ingirabuzima fatizo za selile ya glioma U87 na Toxoplasma RH yanduye BV2 ikomoka kuri exosomes.(A) Igihe nyacyo PCR ya FoxO1, PTEN na PDCD4 imvugo muri exosomes ikomoka kuri T. gondii RH yanduye BV2 ugereranije na PBS exosomes.act-actin mRNA yakoreshejwe nkigenzura.. * P <0.05 yabonetse n'ikizamini cya t. * P <0.05 yabonetse n'ikizamini cya t. * P <0,05 было получено с помощью t-критерия Стьюдента. * P <0.05 yabonetse ukoresheje t-test yabanyeshuri. * 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 wabonye ukoresheje t-test yabanyeshuri.
Kugira ngo dusobanukirwe n'ingaruka za miP-21 muri exosomes ku kugenzura ibibyimba biterwa na gene, selile U87 zandujwe hamwe na inhibitor ya miP-21 ikoresheje Lipofectamine 2000 hanyuma selile zisarurwa nyuma y'amasaha 24 zanduye.Urwego rwa FoxO1 na p27 murwego rwoherejwe na selile zandujwe na miR-21 inhibitor zagereranijwe na selile zavuwe na BV2 zikomoka kuri exosomes ukoresheje qRT-PCR (Ishusho 6A, B).Ihererekanyabubasha rya miR-21 muri selile U87 ryagabanije cyane imvugo ya FoxO1 na p27 (FIG. 6).
RH yanduye exosomal BV2 ikomoka kuri miP-21 yahinduye imvugo ya FoxO1 / p27 muri selile U87 glioma.U87 selile zandujwe na miP-21 inhibitor ikoresheje Lipofectamine 2000 hanyuma selile zisarurwa nyuma yamasaha 24 zanduye.Urwego rwa FoxO1 na p27 murwego rwoherejwe na selile zandujwe na miR-21 inhibitor zagereranijwe nurwego muri selile zavuwe na BV2 zikomoka kuri exosomes ukoresheje qRT-PCR (A, B).
Kugirango uhunge ubudahangarwa bw'uwakiriye, parasite ya Toxoplasma ihinduka cyst tissue.Zirandura uduce twinshi, harimo ubwonko, umutima, n'imitsi ya skeletale, mubuzima bwa nyirubwite kandi bigahindura ubudahangarwa bw'umubiri.Byongeye kandi, barashobora kugenzura ingirabuzimafatizo hamwe na apoptose ya selile yakira, bigatuma ikwirakwizwa ryayo 14,24.Toxoplasma gondii yiganjemo kwanduza selile dendritic selile, neutrophile, hamwe na monocyte / macrophage, harimo na microglia yo mu bwonko.Toxoplasma gondii itera itandukaniro rya macrophage ya M2 phenotype, igira ingaruka ku gukira ibikomere nyuma yo kwandura virusi, kandi ikanajyana na hypervascularization na fibrosis granulomatous.Iyi myitwarire itera indwara ya Toxoplasma irashobora kuba ifitanye isano nibimenyetso bifitanye isano no gukura kw'ibibyimba.Ibidukikije byangwa na Toxoplasma birashobora kumera nkibisanzwe.Kubwibyo, dushobora gutekereza ko kwandura Toxoplasma bigomba kugira uruhare mu mikurire yibibyimba byo mu bwonko.Mubyukuri, umubare munini w’ubwandu bwa Toxoplasma wagaragaye muri serumu y’abarwayi bafite ibibyimba bitandukanye mu bwonko.Byongeye kandi, Toxoplasma gondii irashobora kuba indi kanseri itera kanseri kandi igakorana kugirango ifashe izindi kanseri zanduza kwandura ibibyimba mu bwonko.Ni muri urwo rwego, birakwiye ko tumenya ko virusi ya P. falciparum na Epstein-Barr igira uruhare mu mikorere ya lymphoma ya Burkitt.
Uruhare rwa exosomes nk'ubugenzuzi mu bijyanye n'ubushakashatsi bwa kanseri rwakorewe ubushakashatsi bwimbitse.Nyamara, uruhare rwa exosomes hagati ya parasite na hostes zanduye ntirurasobanuka neza.Kugeza ubu, abagenzuzi batandukanye, harimo na poroteyine zasohotse, basobanuye inzira y’ibinyabuzima uburyo parasite ya protozoan irwanya ibitero byabakiriye kandi ikomeza kwandura.Vuba aha, habaye imyumvire ikura ivuga ko mikorobe ziterwa na protozoan hamwe na microRNA zabo zikorana na selile zakira kugirango habeho ibidukikije byiza kugirango babeho.Kubwibyo, ubushakashatsi buracyakenewe kugirango umenye isano iri hagati ya miRNAs yahinduwe na selile ya glioma.Guhindura MicroRNA (genes genes miR-30c-1, miR-125b-2, miR-23b-27b-24-1 na miR-17-92) ihuza porotokoro ya STAT3 muri macrophage yanduye toxoplasma, irategurwa kandi itera anti -apoptose mu gusubiza indwara ya Toxoplasma gondii 29.Indwara ya Toxoplasma yongera imvugo ya miR-17-5p na miR-106b-5p, ifitanye isano n'indwara nyinshi ziterwa na hyperproliferative 30.Aya makuru yerekana ko miRNAs yakira igengwa nubwandu bwa Toxoplasma ari molekile zingenzi zo kubaho kwa parasite no gutera indwara mu myitwarire y’ibinyabuzima.
Ihinduka rya miRNAs irashobora guhindura ubwoko bwimyitwarire itandukanye mugihe cyo gutangiza no gutera imbere kwingirangingo mbi, harimo na glioma: kwihaza kubimenyetso byikura, kutumva ibimenyetso bibuza gukura, kwirinda apoptose, ubushobozi bwo kwigana butagira imipaka, angiogenezi, gutera na metastasis, no gutwika.Muri glioma, miRNAs yahinduwe yagaragaye mubushakashatsi bwinshi bwerekana imiterere.
Muri ubu bushakashatsi, twemeje urwego rwo hejuru rwa miRNA-21 mu ngirabuzimafatizo zanduye toxoplasma.miR-21 yamenyekanye nka imwe muri microRNA ikunze gukabya cyane mubyimba bikomeye, harimo glioma, 33 kandi imvugo yayo ifitanye isano na glioma.Ibimenyetso bifatika byerekana ko miR-21 ari igitabo cyitwa oncogene gikora nk'ikintu kirwanya antoptotique mu mikurire ya glioma kandi kikaba gikabije cyane mu ngingo na plasma z'ubwonko bw'ubwonko bw'abantu.Igishimishije, miR-21 idakora muri selile na glioma itera kubuza ikwirakwizwa ry'uturemangingo bitewe na apoptose iterwa na caspase.Isesengura rya bioinformatike ya miR-21 yahanuwe ryagaragaje ingirabuzimafatizo nyinshi zifata inzira zifata inzira ya apoptose, harimo urupfu rwa selile 4 (PDCD4), tropomyosine (TPM1), PTEN, hamwe nagasanduku ka O1 (FoxO1), hamwe na miR-2121..22.38.
FoxO1, kimwe mu bintu byandikirwa abantu (FoxO), igira uruhare mu iterambere rya kanseri zitandukanye z’abantu kandi irashobora kugenga imiterere ya genes suppressor genes nka p21, p27, Bim, na FasL40.FoxO1 irashobora guhuza no gukora ingirabuzimafatizo ya selile nka p27 kugirango ikure imikurire ya selile.Byongeye kandi, FoxO1 nigikorwa cyingenzi cyerekana ibimenyetso bya PI3K / Akt kandi ikagenga inzira nyinshi yibinyabuzima nko gutera imbere kwingirabuzimafatizo no gutandukanya selile binyuze mugukoresha inyandiko ya p2742.
Mu gusoza, twizera ko exosomal miR-21 ikomoka kuri microglia yanduye Toxoplasma ishobora kugira uruhare runini mugukuza imikurire ya selile glioma (Ishusho 7).Nyamara, ubushakashatsi buracyakenewe kugirango tubone isano itaziguye hagati ya exosomal miR-21, kwandura Toxoplasma, no gukura kwa glioma.Ibisubizo byitezwe gutanga intangiriro yo kwiga isano iri hagati yanduye Toxoplasma nindwara ya glioma.
Igishushanyo mbonera cyerekana uburyo bwa glioma (ubwonko) kanseri itera kanseri muri ubu bushakashatsi.Umwanditsi ashushanya muri PowerPoint 2019 (Microsoft, Redmond, WA).
Porotokole zose zigeragezwa muri ubu bushakashatsi, harimo no gukoresha inyamaswa, zari zikurikije amabwiriza ngenderwaho y’imyitwarire y’imyitwarire ya kaminuza ya Seoul y’igihugu cya Seoul kandi yemejwe n’inama ishinzwe isuzuma ry’ikigo cy’ishuri ry’ubuvuzi rya kaminuza nkuru ya Seoul (IRB nimero SNU- 150715).-2).Inzira zose zigeragezwa zakozwe hakurikijwe ibyifuzo bya ARRIVE.
Imbeba ya BV2 ya microglia na U87 ingirabuzimafatizo ya glioma yabantu yarezwe muri Dulbecco's Modified Eagle's Medium (DMEM; Welgene, Seoul, Koreya) hamwe na Roswell Park Memorial Institute's Medium (RPMI; Welgene), buri kimwe kirimo 10% ya serumu y'inda, 4 mM l- glutamine, 0.2 mM penisiline na 0,05 mM streptomycine.Ingirabuzimafatizo zarezwe muri incubator hamwe na 5% CO2 kuri 37 ° C.Undi murongo wa selile glioma, U118, wakoreshejwe kugereranya na selile U87.
Kugira ngo exosomes itandukane na T. gondii yanduye RH na ME49, T. gondii tachyzoites (RH strain) yasaruwe mu cyuho cy’inda cy’imbeba BALB / c zimaze ibyumweru 6 zatewe inshinge iminsi 3-4 mbere.Tachyzoites yogejwe inshuro eshatu hamwe na PBS hanyuma isukurwa na centrifugation muri 40% Percoll (Sigma-Aldrich, St. Louis, MO, USA) 43.Kugirango ubone tachyzoite yumuvuduko wa ME49, imbeba za BALB / c zatewe inshinge zidafite intanga 20 hamwe na tachyzoite ihinduka muri cysts yakusanyijwe no koza umwobo winda kumunsi wa 6-8 nyuma yo kwandura (PI).Imbeba zanduye PBS.ME49 tachyzoite yakuze mu ngirabuzimafatizo zongerwamo 100 μg / ml penisiline (Gibco / BRL, Ikirwa Kinini, NY, Amerika), 100 μg / ml streptomycine (Gibco / BRL), na serumu y'inda 5% (Lonza, Walkersville, MD) .., Amerika) kuri 37 ° C na 5% ya dioxyde de carbone.Nyuma yo guhinga mu ngirabuzimafatizo za Vero, ME49 tachyzoite yanyujijwe kabiri binyuze mu rushinge rwa 25, hanyuma ikanyura muyungurura 5 µm kugirango ikureho imyanda na selile.Nyuma yo gukaraba, tachyzoite yahagaritswe muri PBS44.Tissue cysts ya Toxoplasma gondii strain ME49 yabungabunzwe no guterwa intraperitoneal yatewe na cysts itandukanijwe nubwonko bwimbeba C57BL / 6 zanduye (Orient Bio Animal Centre, Seongnam, Koreya).Ubwonko bwimbeba zanduye ME49 bwasaruwe nyuma y amezi 3 ya PI hanyuma bucukurwa munsi ya microscope kugirango itandukanya cysts.Imbeba zanduye zabitswe mu bihe bidasanzwe bitarwaye indwara (SPF) mu Ishuri Rikuru ry’Ubuvuzi rya kaminuza ya Seoul.
RNA yose yakuwe muri BV2 ikomoka kuri exosomes, selile BV2 na tissue ikoresheje miRNeasy Mini Kit (Qiagen, Hilden, Ubudage) ukurikije amabwiriza yabakozwe, harimo nigihe cyo gukuramo intambwe yo gukuraho.Imyitozo ya RNA yagenwe kuri NanoDrop 2000 yerekanwe.Ubwiza bwa microarray ya RNA bwasuzumwe hifashishijwe bioanalyser ya Agilent 2100 (Agilent Technologies, Amstelveen, Ubuholandi).
DMEM hamwe na 10% exosome-ikennye FBS yateguwe na ultracentrifugation kuri 100.000g mumasaha 16 kuri 4 ° C hanyuma iyungurura binyuze muri 0.22 µm muyunguruzi (Nalgene, Rochester, NY, USA).Ingirabuzimafatizo za BV2, 5 × 105, zarezwe muri DMEM zirimo 10% FBS ya exosome-yagabanutse na antibiotike 1% kuri 37 ° C na 5% CO2.Nyuma yamasaha 24 yubushakashatsi, tachyzoite yumuvuduko wa RH cyangwa ME49 (MOI = 10) yongewemo muri selile hanyuma parasite idatera yakuweho mugihe cyisaha imwe yuzuzwa DMEM.Exosomes yavuye muri selile BV2 yatandukanijwe na centrifugation itandukanye, uburyo bukoreshwa cyane.Hagarika pellet ya exosome muri 300 µl PBS kugirango RNA cyangwa isesengura rya poroteyine.Ubwinshi bwa exosomes yihariye bwagenwe hakoreshejwe ibikoresho bya poroteyine ya BCA (Pierce, Rockford, IL, USA) hamwe na NanoDrop 2000 yerekanwe.
Ibicuruzwa biva mu ngirabuzimafatizo za BV2 cyangwa exosomes zikomoka kuri BV2 byatewe muri PRO-PREP solution igisubizo cyo gukuramo poroteyine (iNtRon Biotechnology, Seongnam, Koreya) kandi poroteyine zashyizwe kuri Coomassie ubururu bwiza cyane bwanditseho 10% ya SDS polyacrylamide.Byongeye kandi, poroteyine zoherejwe muri membrane ya PVDF amasaha 2.Blots zo muburengerazuba zemejwe hakoreshejwe antibody ya Alix (Ikimenyetso cya selile yerekana ibimenyetso, Beverly, MA, USA) nk'ikimenyetso cya exosomal.HRP-conjugated ihene irwanya imbeba IgG (H + L) (Laboratoire ya Bethyl, Montgomery, TX, Amerika) hamwe na LAS-1000 hiyongereyeho isesengura ry'amashusho ya Luminescent (Filime y'amafoto ya Fuji, Tokiyo, Ubuyapani) yakoreshejwe nka antibody ya kabiri..Ikwirakwizwa rya electron microscopi ryakozwe kugirango ryige ingano na morphologie ya exosomes.Exosomes yitandukanije na selile BV2 (6.40 µg / µl) yateguwe kuri meshes isize karubone kandi yandujwe nabi na acetate ya uranyl 2% kuminota 1.Ingero zateguwe zagaragaye kuri voltage yihuta ya 80 kV ukoresheje JEOL 1200-EX II (Tokiyo, Ubuyapani) ifite kamera ya ES1000W Erlangshen CCD (Gatan, Pleasanton, CA, USA).
BV2 ikomoka kuri BV2 yandujwe hakoreshejwe PKH26 Red Fluorescent Linker Kit (Sigma-Aldrich, St. Louis, MO, USA) muminota 15 mubushyuhe bwicyumba.U87 selile, 2 × 105, hamwe na PKH26 yanditseho exosomes (umutuku) cyangwa nta exosomes nkigenzura ribi, yashyizwe kuri 37 ° C mumasaha 24 muri incubator ya 5% ya CO2.Nuclei selile U87 yandujwe na DAPI (ubururu), selile U87 zashyizwe muri 4% paraformaldehyde muminota 15 kuri 4 ° C hanyuma zisesengurwa muri sisitemu ya microscope ya Leica TCS SP8 STED CW (Leica Microsystems, Mannheim, Ubudage).biragaragara.
cDNA yashizwemo kuva siRNA ikoresheje Mir-X siRNA synthesis ya mbere yumurongo hamwe na SYBR qRT-PCR kit (Takara Bio Inc., Shiga, Ubuyapani).Igihe nyacyo PCR cyakozwe hakoreshejwe iQ5 igihe nyacyo sisitemu yo kumenya PCR (Bio-Rad, Hercules, CA, USA) ikoresheje primers na templates bivanze na SYBR Premix.ADN yongerewe imbaraga kuri cycle 40 yo gutandukana kuri 95 ° C kuri s 15 na anne kuri 60 ° C kuri 60 s.Ibyatanzwe muri buri reaction ya PCR byasesenguwe hifashishijwe isesengura ryamakuru ya software ya iQ ™ 5 ya sisitemu ya optique (Bio-Rad).Impinduka zijyanye nimvugo ya gene hagati yintego zatoranijwe hamwe na act-actin / siRNA (na U6) zabazwe hakoreshejwe uburyo busanzwe bwo gutonda.Urutonde rwa primer rukoreshwa rwerekanwe kumeza 1.
3 x 104 U87 selile glioma yabibwe mumasahani 96-yivanze hanyuma ivangwa na exosomes yanduye Toxoplasma ikomoka kuri BV2 (50 μg / mL) cyangwa exosomes idasanzwe ikomoka kuri BV2 (50 μg / mL) nkigenzura kumasaha 12, 18 na 36. .Igipimo cyo gukwirakwiza selile cyagenwe hakoreshejwe Kit Kubara Akagari-8 (Dojindo, Kumamoto, Ubuyapani) (Imibare yinyongera S1-S3) 46.
Imbeba z’ibyumweru 5 BALB / c imbeba zambaye ubusa zaguzwe muri Orient Bio (Seongnam-si, Koreya yepfo) kandi zigashyirwa ku giti cyazo mu kasho kitagira ubushyuhe ku cyumba cy’ubushyuhe (22 ± 2 ° C) n’ubushuhe (45 ± 15 ° C).%) ku bushyuhe bwicyumba (22 ± 2 ° C) nubushuhe (45 ± 15%).Umucyo w'amasaha 12 n'umuzenguruko w'amasaha 12 byakozwe munsi ya SPF (Seoul National University of Medicine Centre Animal Centre).Imbeba zagabanyijwemo amatsinda atatu yimbeba 5 buri tsinda kandi inshinge zose zatewe inshinge 400 ml ya PBS irimo selile 1 x 107 U87 glioma kandi ibintu bikura byagabanije BD Matrigel ™ (BD Science, Miami, FL, USA).Iminsi itandatu nyuma yo guterwa ibibyimba, mg 200 za exosomes zikomoka muri selile BV2 (zanduye / zidafite Toxoplasma) zatewe mu kibyimba.Iminsi 22 nyuma yo kwandura ikibyimba, ubunini bwikibyimba cyimbeba muri buri tsinda bwapimwe na caliper inshuro eshatu mucyumweru, kandi ingano yikibyimba yabazwe na formula: 0.5 × (ubugari) × 2 × uburebure.
Isesengura rya MicroRNA ukoresheje miRCURYTM LNA miRNA array, igisekuru cya 7 gifite, mmu na rno array (EXIQON, Vedbaek, Danimarike) ikubiyemo imbeba 1119 ziranga neza mubantu 3100 bafata abantu, imbeba nimbeba miRNA.Muri ubu buryo, 250 kugeza 1000 ng ya RNA yose yakuwe kuri 5′-fosifate hifashishijwe inyana zo mu nda ya alkaline fosifata ikurikirwa no gushyiramo irangi ryatsi rya Hy3.Ibyitegererezo byanditseho byahujwe no gupakira amashusho ya microarray ukoresheje ibikoresho byo kuvanga ibyumba (Agilent Technologies, Santa Clara, CA, USA) hamwe nigikoresho cyo kuvanga (Agilent Technologies).Hybridisation yakozwe mu masaha 16 kuri 56 ° C, hanyuma microarray yozwa hakurikijwe ibyifuzo byabayikoze.Amashusho yatunganijwe ya microarray yahise asuzumwa hifashishijwe sisitemu ya micranray ya Agilent G2565CA (Agilent Technologies).Amashusho yabikijwe yatumijwe mu mahanga hifashishijwe porogaramu ya Agilent Feature Extraction software 10.7.3.1 (Agilent Technologies) kandi ubukana bwa fluorescence bwa buri shusho bwagereranijwe hifashishijwe dosiye ya GAL ijyanye na protocole ya Exiqon yahinduwe.Amakuru ya Microarray kubushakashatsi burimo ashyirwa mububiko bwa GEO munsi ya numero GPL32397.
Imyirondoro yerekana imiterere ya miRNA ikuze muri microglia ya RH cyangwa ME49 yanduye Toxoplasma yasesenguwe hakoreshejwe ibikoresho bitandukanye byurusobe.miRNAs ifitanye isano niterambere ryibibyimba yamenyekanye ikoresheje miRWalk2.0 (http://mirwalk.umm.uni-heidelberg.de) hanyuma ikayungururwa hamwe nuburemere bwibimenyetso bisanzwe (log2) burenze 8.0.Muri miRNAs, miRNAs zitandukanye zitandukanye wasangaga zirenga inshuro 1.5 zahinduwe nisesengura rya filteri ya miRNAs yahinduwe na RH cyangwa ME49 yanduye T. gondii.
Ingirabuzimafatizo zabibwe mu masahani atandatu (3 x 105 selile / iriba) muri opti-MEM (Gibco, Carlsbad, CA, USA) ukoresheje Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).Ingirabuzimafatizo zahinduwe zimaze amasaha 6 hanyuma uburyo bwo guhinduka buhinduka uburyo bushya bwuzuye.Ingirabuzimafatizo zasaruwe nyuma yamasaha 24 nyuma yo kwimurwa.
Isesengura mibare ryakozwe ahanini hifashishijwe t-test yabanyeshuri hamwe na software ya Excel (Microsoft, Washington, DC, USA).Kubushakashatsi bwinyamaswa zigeragezwa, inzira ebyiri ANOVA yakozwe hakoreshejwe software ya Prism 3.0 (GraphPad Software, La Jolla, CA, USA). P-gaciro <0.05 byafatwaga nkibisobanuro bifatika. P-gaciro <0.05 byafatwaga nkibisobanuro bifatika. Значения П <0,05 считались статистически значимыми. P agaciro <0.05 byafatwaga nkibyingenzi. P 值 <0.05 被 认为 具有 统计学 意义。 P 值 <0.05 Значения П <0,05 считались статистически значимыми. P agaciro <0.05 byafatwaga nkibyingenzi.
Porotokole zose zigeragezwa zikoreshwa muri ubu bushakashatsi zemejwe n’ikigo gishinzwe isuzuma ry’ikigo cy’ubuvuzi cya kaminuza nkuru ya Seoul (IRB nimero 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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