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ʻO Toxoplasma gondii kahi parasite protozoan intracellular e hoʻololi i ka microenvironment o ka mea hoʻokipa maʻi a ʻike ʻia e pili ana i ka ulu ʻana o ka ulu ʻana o ka lolo.Ma kēia haʻawina, manaʻo mākou he exosomal miRNA-21 mai ka maʻi Toxoplasma e hoʻoikaika i ka ulu ʻana o ka lolo.Ua hōʻike ʻia nā Exosomes mai Toxoplasma-infected BV2 microglia a ua hōʻoia ʻia ka internalization o U87 glioma cell.Ua kālailai ʻia nā ʻōlelo hōʻike exosomal microRNA me ka hoʻohana ʻana i nā arrays o microRNA a me microRNA-21A-5p e pili ana me Toxoplasma gondii a me ka hoʻokaʻawale ʻana i ka tumor.Ua noiʻi pū mākou i nā pae mRNA o nā genes pili i ka maʻi maʻi U87 glioma cell ma o ka hoʻololi ʻana i nā pae miR-21 i nā exosome a me ka hopena o nā exosome ma ke kanaka U87 glioma cell proliferation.I loko o nā exosomes o U87 glioma cell i loaʻa i ka Toxoplasma gondii, ua hoʻonui ʻia ka hōʻike o ka microRNA-21 a ua hoʻemi ʻia ka hana o nā genes antitumor (FoxO1, PTEN, a me PDCD4).ʻO nā exosome i loaʻa mai i ka BV2 i loaʻa i ka maʻi Toxoplasma e hoʻoulu i ka hoʻonui ʻana o nā pūnaewele glioma U87.Hoʻoulu ka Exosome i ka ulu ʻana o nā pūnaewele U87 i loko o kahi kumu hoʻohālike ʻiole.Manaʻo mākou i ka hoʻonui ʻia ʻana o ka exosomal miR-21 i Toxoplasma-infected BV2 microglia hiki ke pāʻani i kahi hana koʻikoʻi ma ke ʻano he mea hoʻoikaika i ka ulu ʻana o ka cell i loko o U87 glioma cell ma ka hoʻohaʻahaʻa ʻana i nā genes antitumor.
Ua manaʻo ʻia ua ʻoi aku ma mua o 18.1 miliona mau hihia o ka maʻi maʻi maʻi maʻi maʻi i ka honua holoʻokoʻa i ka makahiki 2018, me kahi o 297,000 mau maʻi maʻi maʻi ʻaʻai waena i ʻike ʻia i kēlā me kēia makahiki (1.6% o nā maʻi maʻi āpau)1.Ua hōʻike ʻia nā noiʻi mua e pili ana i nā kumu pilikia no ka hoʻomohala ʻana i ka lolo o ke kanaka me nā huahana kemika like ʻole, ka mōʻaukala ʻohana, a me ka radiation ionizing mai ke poʻo therapeutic a me nā mea hana diagnostic.Eia naʻe, ʻaʻole ʻike ʻia ke kumu maoli o kēia mau maʻi ʻino.Ma kahi o 20% o nā maʻi maʻi āpau a puni ka honua i hoʻokumu ʻia e nā maʻi maʻi, me nā maʻi maʻi, bacteria a me nā parasites3,4.Hoʻopilikia nā maʻi maʻi maʻi i nā ʻano genetic o ka cell host, e like me ka hoʻoponopono ʻana i ka DNA a me ka pōʻai cell, a hiki ke alakaʻi i ka mumū mau a me ka pōʻino i ka ʻōnaehana pale5.
ʻO nā maʻi maʻi maʻi e pili ana i ka maʻi kanesa kanaka ka maʻi maʻamau maʻamau, e komo pū me nā maʻi papilloma kanaka a me nā maʻi hepatitis B a me C.Hiki i nā parasites ke pāʻani i kahi kuleana i ka ulu ʻana o ka maʻi maʻi kanaka.ʻO kekahi mau ʻano parasite, ʻo ia hoʻi ʻo Schistosoma, Opishorchis viverrini, O. felineus, Clonorchis sinensis a me Hymenolepis nana, ua hoʻopili ʻia i nā ʻano like ʻole o ke kanesa kanaka 6,7,8.
ʻO Toxoplasma gondii kahi protozoan intracellular e hoʻoponopono ana i ka microenvironment o nā pūnaewele hoʻokipa i hoʻopili ʻia.Ua manaʻo ʻia e hoʻopili kēia parasite ma kahi o 30% o ka heluna kanaka o ka honua, e waiho ana i ka heluna kanaka a pau i ka pilikia9,10.Hiki i ka Toxoplasma gondii ke hoʻopili i nā kino koʻikoʻi, e like me ka pūnaeho puʻupuʻu waena (CNS), a hoʻoulu i nā maʻi koʻikoʻi e like me ka maningitis fatal a me ka encephalitis, ʻoi aku ka nui o nā maʻi immunocompromised9.Eia naʻe, hiki i ka Toxoplasma gondii ke hoʻololi i ke kaiapuni o ka mea maʻi ma o ka hoʻololi ʻana i ka ulu ʻana o ka cell a me nā pane kūlohelohe i nā poʻe immunocompetent, e alakaʻi ana i ka mālama ʻana i kahi maʻi maʻi maʻi asymptomatic9,11.ʻO ka mea hoihoi, ma muli o ka pilina ma waena o ka maʻi maʻi T. gondii a me ka ulu ʻana o ka lolo, ua hōʻike kekahi mau hōʻike i ka hoʻololi ʻana o ke kaiapuni i loko o vivo ma muli o ka maʻi T. gondii mau loa e like me ka microenvironment tumor.
Ua ʻike ʻia ʻo Exosome he mau mea kamaʻilio intercellular e hāʻawi ana i ka ʻike olaola, me nā protein a me nā waikawa nucleic, mai nā cell kokoke16,17.Hiki i nā Exosome ke hoʻoikaika i nā kaʻina hana ola e pili ana i ka tumo e like me ka anti-apoptosis, angiogenesis, a me ka metastasis i ka microenvironment tumor.ʻO ka mea nui, ʻo nā miRNA (miRNAs), nā RNA liʻiliʻi ʻole-coding e pili ana i 22 nucleotides i ka lōʻihi, he mea nui ka post-transcriptional gene regulators e hoʻomalu ana ma mua o 30% o ka mRNA kanaka ma o ka miRNA-induced silencing complex (miRISC).Hiki i ka Toxoplasma gondii ke hoʻopau i nā kaʻina hana olaola ma ka hoʻomalu ʻana i ka hōʻike miRNA i nā pūʻali maʻi.Loaʻa i nā Host miRNA nā hōʻailona koʻikoʻi no ka hoʻoponopono ʻana i nā kaʻina hana olaola e hoʻokō ai i ka hoʻolālā ola o ka parasite.No laila, ʻo ke aʻo ʻana i nā loli i ka ʻaoʻao miRNA host ma luna o ka maʻi me T. gondii hiki ke kōkua iā mākou e hoʻomaopopo pono i ka pilina ma waena o ka host a me T. gondii.ʻOiaʻiʻo, ʻo Thirugnanam et al.Ua manaʻo ʻo 15 e hoʻoikaika ʻo T. gondii i ka carcinogenesis lolo ma ka hoʻololi ʻana i kāna hōʻike ma nā miRNA host kikoʻī e pili ana i ka ulu ʻana o ka tumora a ʻike ʻo T. gondii hiki ke hana i nā gliomas i nā holoholona hoʻokolohua.
Ke nānā nei kēia haʻawina i ka hoʻololi ʻana o ka exosomal miR-21 i ka microglia host i loaʻa i ka Toxoplasma BV2.Ua ʻike mākou i kahi hana kūpono o ka exosomal miR-21 i hoʻololi ʻia i ka ulu ʻana o nā cell glioma U87 ma muli o ka paʻa ʻana i ka nucleus o FoxO1 / p27, ʻo ia ka pahuhopu o overexpressed miR-21.
Ua loaʻa nā Exosome i loaʻa mai ka BV2 me ka hoʻohana ʻana i ka centrifugation ʻokoʻa a hōʻoia ʻia e nā ʻano hana like ʻole e pale ai i ka hoʻohaumia ʻana me nā mea cellular a i ʻole nā ​​​​vesicles ʻē aʻe.Ua hōʻike ʻo SDS-polyacrylamide gel electrophoresis (SDS-PAGE) i nā hiʻohiʻona ʻokoʻa ma waena o nā protein i unuhi ʻia mai nā cell BV2 a me nā exosomes (Figure 1A), a ua loiloi ʻia nā laʻana no ka hele ʻana o Alix, i loiloi ʻia e Western blotting o exosomal protein markers i .Loaʻa ka lepili Alix i nā protein exosome akā ʻaʻole i loko o nā protein lysate cell BV2 (Fig. 1B).Eia kekahi, ua kālailai ʻia ka RNA i hoʻomaʻemaʻe ʻia mai nā exosome i loaʻa mai BV2 me ka hoʻohana ʻana i kahi bioanalyzer.18S a me 28S ribosomal subunits i kakaikahi i ikeia ma ka exosomal RNA migration pattern, e hoike ana i ka maemae pono (Figure 1C).ʻO ka hope loa, hōʻike ʻia ka microscopy electron transmission e pili ana nā exosome i ʻike ʻia ma kahi o 60-150 nm ka nui a he ʻano kīʻaha like ʻole o ka exosome morphology (Fig. 1D).
ʻO keʻano o nā exosome i loaʻa mai nā pūnaewele BV2.(A) ʻaoʻao pepa ʻikepili palekana.Ua hoʻokaʻawale ʻia nā protein mai nā cell BV2 a i ʻole exosome i loaʻa mai BV2.ʻOkoʻa nā ʻano protein ma waena o nā cell a me nā exosome.(B) Ka nānā 'ana i nā blot Western o kahi māka exosomal (Alix).(C) Ka loiloi ʻana i ka RNA i hoʻomaʻemaʻe ʻia mai nā cell BV2 a me BV2 i loaʻa i nā exosome me ka hoʻohana ʻana i kahi bioanalyzer.No laila, ʻike ʻole ʻia nā subunits ribosomal 18S a me 28S i nā pūnaewele BV2 i ka RNA exosomal.(D) Ua hōʻike ʻia ka microscopy electron transmission i hoʻokaʻawale ʻia nā exosome i hoʻokaʻawale ʻia mai nā pūnaewele BV2 me ka 2% uranyl acetate.ʻO nā Exosome ma kahi o 60-150 nm ka nui a me ke ʻano kīʻaha (Song and Jung, unpublished data).
ʻO ka internalization pūnaewele o BV2 i loaʻa i nā exosome i loko o U87 kanaka glioma cell i ʻike ʻia me ka microscopy confocal.Hoʻopili ʻia nā exosome i kapa ʻia ʻo PKH26 i loko o ka cytoplasm o nā ʻāpana U87.Ua hoʻopaʻa ʻia ʻo Nuclei me DAPI (Fig. 2A), e hōʻike ana e hiki ke hoʻokomo ʻia nā exosome i loaʻa mai i ka BV2 e nā pūnaewele hoʻokipa a hoʻoikaika i ke kaiapuni o nā cell loaʻa.
ʻO ka hoʻokomo ʻana i nā exosome i loaʻa mai i ka BV2 i loko o nā pūnaewele glioma U87 a me nā exosome i loaʻa mai i ka BV2 i loaʻa i ka Toxoplasma RH i hoʻoulu ʻia i ka hoʻonui ʻana o nā pūnaewele glioma U87.(A) Exosome i hoʻopuni ʻia e nā keena U87 i ana ʻia e ka microscopy confocal.U87 glioma cell i incubated with exosomes labeled with PKH26 (ʻulaʻula) a i ʻole ka mana no nā hola 24.Ua hoʻopaʻa ʻia ka nuclei me DAPI (uliuli) a laila nānā ʻia ma lalo o kahi microscope confocal (paʻa pālākiō: 10 μm, x 3000).(B) U87 glioma cell proliferation ua hoʻoholo ʻia e ka cell proliferation assay.Ua mālama ʻia nā cell glioma U87 me nā exosome no ka manawa i hōʻike ʻia. *P <0.05 i loaʻa ma ka hōʻike t haumāna. *P <0.05 i loaʻa ma ka hōʻike t haumāna. *P < 0,05 получено по t-критерию Стьюдента. *P <0.05 ma ka ho'āʻo-t a ka Haumāna. *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 i loaʻa me ka hoʻohana ʻana i ka hōʻike-t a ka Haumāna.
Ma hope o ka hōʻoia ʻana i ka internalization o BV2-derived exosomes i loko o U87 glioma cell, ua hana mākou i ka hoʻonui cell proliferation assays e noiʻi i ke kuleana o BV2-derived Toxoplasma-derived exosomes i ka hoʻomohala ʻana i nā cell glioma kanaka.ʻO ka lapaʻau ʻana i nā pūnaewele U87 me nā exosomes mai nā pūnaewele BV2 T. gondii-infected i hōʻike ʻia e T. gondii-infected BV2-derived exosomes i hoʻonui nui ʻia ka hoʻonui ʻana o nā cell U87 i hoʻohālikelike ʻia me ka mana (Fig. 2B).
Eia kekahi, ʻo ka ulu ʻana o nā cell U118 i loaʻa nā hopena like me U87, ʻoiai ʻo Toxoplasma i hoʻoulu ʻia i nā exosome i hana i nā kiʻekiʻe kiʻekiʻe o ka proliferation (ʻike ʻole i hōʻike ʻia).Ma muli o kēia mau ʻikepili, hiki iā mākou ke hōʻike i ka BV2-derived Toxoplasma-infected exosome i mea koʻikoʻi i ka hoʻonui ʻana o ka cell glioma.
No ka noiʻi ʻana i ka hopena o ka Toxoplasma-infected BV2-derived exosomes i ka ulu ʻana o ka tumo, ua hoʻokomo mākou i nā pūnaewele glioma U87 i loko o nā ʻiole olohelohe no ke kumu hoʻohālike xenograft a hoʻokomo i nā exosomes i loaʻa mai i ka BV2 a i ʻole nā ​​​​exosome i loaʻa mai i ka RH-infected BV2.Ma hope o ka ʻike ʻia ʻana o nā ʻōpū ma hope o 1 pule, ua māhele ʻia kēlā me kēia pūʻulu hoʻokolohua o 5 mau ʻiole e like me ka nui o ka ʻōpū e hoʻoholo ai i kahi hoʻomaka like, a ua ana ʻia ka nui o ka tumora no 22 mau lā.
Ma nāʻiole me ka U87 xenograft kŘkohu, 'oi aku ka nui o ka nui a me ke kaumaha i 'ike 'ia ma ka BV2 i loa'a i ka RH-infected exosome hui ma ka la 22 (Fig. 3A,B).Ma ka ʻaoʻao ʻē aʻe, ʻaʻohe ʻokoʻa koʻikoʻi i ka nui o ka tumora ma waena o ka hui exosome i loaʻa mai i ka BV2 a me ka pūʻulu mana ma hope o ka mālama ʻana exosome.Eia hou, ua hōʻike ʻia nā ʻiole me nā pūnaewele glioma a me nā exosome i ka ʻike maka i ka nui o ka nui o ka tumora i loko o ka hui o RH-infected BV2-derived exosomes (Fig. 3C).Hōʻike kēia mau hopena i ka BV2 i loaʻa i ka Toxoplasma-infected exosome e hoʻoulu i ka ulu ʻana o ka glioma i loko o kahi kumu hoʻohālike ʻiole.
ʻO ka Oncogenesis (AC) o nā exosome i loaʻa mai i ka BV2 i kahi kiʻi ʻiole xenograft U87.Ua hoʻonui nui ʻia ka nui o ka tumor (A) a me ke kaumaha (B) i ka BALB/c nude mice i mālama ʻia me RH-infected exosomes i loaʻa mai BV2.Ua hoʻokuʻu ʻia nā ʻiole ʻole o BALB/c (C) me ka 1 x 107 U87 mau mea i hoʻokuʻu ʻia i ka hui ʻana o Matrigel.ʻEono mau lā ma hope o ka injection, ua mālama ʻia ka 100 μg o BV2-derived exosomes i nā ʻiole.Ua ana ʻia ka nui a me ke kaumaha ma nā lā i hōʻike ʻia a ma hope o ka mōhai ʻana. *P <0.05. *P <0.05. *Р < 0,05. *P <0.05. *P <0.05. *P <0.05. *Р < 0,05. *P <0.05.
Ua hōʻike ka ʻikepili i ka 37 miRNAs (16 overexpressed a me 21 downexpressed) pili i ka immunity a i ʻole ka ulu ʻana o ka tumo i hoʻololi nui ʻia i ka microglia ma hope o ka maʻi me ka Toxoplasma RH strain (Fig. 4A).Ua hōʻoia ʻia nā pae hōʻike pili o miR-21 ma waena o nā miRNA i hoʻololi ʻia e ka RT-PCR manawa maoli i nā exosome i loaʻa mai BV2, nā exosome i mālama ʻia me nā cell BV2 a me U87.Hōʻike ka hōʻike o ka miR-21 i ka piʻi nui o nā exosome mai nā pūnaewele BV2 i loaʻa i ka Toxoplasma gondii (RH strain) (Fig. 4B).Ua hoʻonui ʻia nā pae hōʻike pili o ka miR-21 ma BV2 a me U87 ma hope o ka lawe ʻana i nā exosome i hoʻololi ʻia (Fig. 4B).ʻO nā pae pili o ka hōʻike miR-21 i loko o ka lolo o nā maʻi maʻi a me nā ʻiole i loaʻa i ka Toxoplasma gondii (ME49 strain) ʻoi aku ka kiʻekiʻe ma mua o nā mana, i kēlā me kēia (Fig. 4C).Hoʻopili kēia mau hopena me nā ʻokoʻa ma waena o nā pae hōʻike o nā microRNA i wānana a hoʻopaʻa ʻia i loko o ka vitro a me ka vivo.
Hoʻololi i ka hōʻike o exosomal miP-21a-5p i ka microglia i loaʻa i ka Toxoplasma gondii (RH).(A) Hōʻike i nā loli koʻikoʻi i ka siRNA e pili ana i ka pale ʻana a i ʻole ka ulu ʻana o ka tumo ma hope o ka maʻi T. gondii RH.(B) Ua ʻike ʻia nā pae hōʻike miR-21 pili e ka RT-PCR manawa maoli i nā exosomes i loaʻa mai i ka BV2, nā exosome i mālama ʻia e BV2, a me nā keena U87.(C) Loaʻa nā pae hōʻike miR-21 pili i loko o ka lolo o nā maʻi maʻi maʻi (N = 3) a me nā ʻiole i loaʻa i ka Toxoplasma gondii (ME49 strain) (N = 3). *P <0.05 i loaʻa ma ka hōʻike t haumāna. *P <0.05 i loaʻa ma ka hōʻike t haumāna. *P < 0,05 было получено с помощью t-критерия Стьюдента. *Ua loaʻa ka P <0.05 me ka hoʻohana ʻana i ka ho'āʻo-t a Student. *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 i loaʻa me ka hoʻohana ʻana i ka hōʻike-t a ka Haumāna.
ʻO nā exosome mai nā pūnaewele BV2 i hoʻopiliʻia e RH i alakaʻi i ka uluʻana o nā gliomas i loko o vivo a me in vitro (Fig. 2, 3).No ka ʻike ʻana i nā mRNA kūpono, ua nānā mākou i nā pae mRNA o nā genes target antitumor, pahu forkhead O1 (FoxO1), PTEN, a me ka programmed cell death 4 (PDCD4) i loko o nā keena U87 i loaʻa i nā exosome i loaʻa mai BV2 a i ʻole RH BV2.Ua hōʻike ʻo Bioinformatics analysis i kekahi mau genes pili i ka maʻi tumora, me ka FoxO1, PTEN, a me PDCD4 genes, loaʻa nā pūnaewele paʻa miR-2121,22.Ua hōʻemi ʻia nā pae mRNA o nā genes target antitumor ma RH-infected BV2-derived exosomes i hoʻohālikelike ʻia me BV2-derived exosomes (Fig. 5A).Ua hōʻike ʻo FoxO1 i nā pae protein i hoʻemi ʻia i nā exosomes i loaʻa i ka BV2 i loaʻa i ka RH i hoʻohālikelike ʻia me nā exosomes i loaʻa i ka BV2 (Figure 5B).Ma muli o kēia mau hopena, hiki iā mākou ke hōʻoia i nā exosome i loaʻa mai i ka RH-infected BV2 i hoʻohaʻahaʻa i nā genes anti-oncogenic, e mālama ana i kā lākou kuleana i ka ulu ʻana o ka tumor.
ʻO ka Toxoplasma RH-infected BV2-derived exosomes e hoʻoulu i ka hoʻopau ʻana i nā genes antitumor i loko o U87 glioma cell e Toxoplasma RH-infected BV2-derived exosomes.(A) PCR manawa maoli o FoxO1, PTEN a me PDCD4 hōʻike i nā exosome i loaʻa mai ka T. gondii RH-infected BV2 i hoʻohālikelike ʻia me nā exosomes PBS.Ua hoʻohana ʻia ka β-actin mRNA ma ke ʻano he mana.(B) Ua hoʻoholo ʻia ka ʻōlelo FoxO1 e ka Western blotting a ua loiloi ʻia ka ʻikepili densitometry me ka hoʻohana ʻana i ka papahana ImageJ. *P <0.05 i loaʻa ma ka hōʻike t haumāna. *P <0.05 i loaʻa ma ka hōʻike t haumāna. *P < 0,05 было получено с помощью t-критерия Стьюдента. *Ua loaʻa ka P <0.05 me ka hoʻohana ʻana i ka ho'āʻo-t a Student. *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 i loaʻa me ka hoʻohana ʻana i ka hōʻike-t a ka Haumāna.
No ka hoʻomaopopo ʻana i ka hopena o ka miP-21 i nā exosome ma ka hoʻoponopono gene pili i ka tumo, ua hoʻololi ʻia nā cell U87 me kahi inhibitor o miP-21 me ka hoʻohana ʻana i ka Lipofectamine 2000 a ua ʻohi ʻia nā cell 24 mau hola ma hope o ka lawe ʻana.Ua hoʻohālikelike ʻia nā pae hōʻike FoxO1 a me ka p27 i loko o nā cell i hoʻololi ʻia me nā mea hoʻopiʻi miR-21 me nā cell i mālama ʻia me nā exosomes i loaʻa mai i ka BV2 me ka hoʻohana ʻana i qRT-PCR (Fig. 6A,B).ʻO ka hoʻololi ʻana o ka miR-21 inhibitor i loko o nā keʻena U87 i hoʻohaʻahaʻa nui i ka ʻōlelo FoxO1 a me ka p27 (FIG. 6).
RH-infected exosomal BV2 i loaʻa i ka miP-21 i hoʻololi i ka ʻōlelo FoxO1/p27 i loko o nā pūnaewele glioma U87.Ua hoʻololi ʻia nā pūnaewele U87 me ka inhibitor miP-21 me ka hoʻohana ʻana i ka Lipofectamine 2000 a ua ʻohi ʻia nā cell 24 mau hola ma hope o ka hoʻololi ʻana.Ua hoʻohālikelike ʻia nā pae hōʻike FoxO1 a me p27 i nā cell i hoʻololi ʻia me ka miR-21 inhibitors i nā pae i loko o nā cell i mālama ʻia me BV2-derived exosomes me ka hoʻohana ʻana i qRT-PCR (A, B).
No ka pakele ʻana i ka pane ʻana o ka mea hoʻokipa, ua hoʻololi ʻia ka parasite Toxoplasma i kahi kiko kiko.Hoʻopili lākou i nā ʻiʻo like ʻole, e like me ka lolo, ka puʻuwai, a me ka ʻiʻo iwi, i ke ola o ka mea hoʻokipa a hoʻololi i ka pane kūlohelohe o ka mea hoʻokipa.Eia hou, hiki iā lākou ke hoʻoponopono i ka pōʻaiapili pūnaewele a me ka apoptosis o nā pūnaewele hoʻokipa, e hoʻonui ana i kā lākou proliferation14,24.Hoʻopilikia ka Toxoplasma gondii i nā pūnaewele dendritic host, neutrophils, a me ka laina monocyte/macrophage, me ka microglia lolo.Hoʻokomo ʻo Toxoplasma gondii i ka ʻokoʻa o nā macrophages o ka M2 phenotype, pili i ka ho'ōla ʻana i ka ʻeha ma hope o ka maʻi pathogen, a pili pū me ka hypervascularization a me ka granulomatous fibrosis.Pili paha kēia pathogenesis o ka maʻi Toxoplasma i nā hōʻailona pili i ka ulu ʻana o ka maʻi tumora.ʻO ke ʻano ʻino i hoʻoponopono ʻia e Toxoplasma e like paha me ka precancer pili.No laila, hiki ke manaʻo ʻia ʻo ka maʻi Toxoplasma e kōkua i ka ulu ʻana o ka lolo lolo.ʻO kaʻoiaʻiʻo, ua hōʻike ʻia nā helu kiʻekiʻe o ka maʻi Toxoplasma i ka serum o nā maʻi me nā ʻano lolo lolo.Eia kekahi, ʻo Toxoplasma gondii paha kekahi mea hana kino kino a hana synergistically e kōkua i nā carcinogens infectious e hoʻomohala i ka lolo lolo.Ma kēiaʻano, pono e hoʻomaopopoʻiaʻo P. falciparum a me Epstein-Barr virus synergistically kōkua i ke kūkuluʻana i ka lymphoma o Burkitt.
Ua noiʻi nui ʻia ke kuleana o nā exosome ma ke ʻano he mea hoʻoponopono i ke kahua o ka noiʻi maʻi kanesa.Eia nō naʻe, ʻaʻole i ʻike maikaʻi ʻia ka hana o nā exosome ma waena o nā parasites a me nā mea i hoʻopili ʻia.I kēia manawa, ua wehewehe nā mea hoʻoponopono like ʻole, me nā protein huna, i nā kaʻina hana ola e kū ai nā protozoan parasites i ka hoʻouka kaua a hoʻomau i ka maʻi.I kēia mau lā, ua ulu ka manaʻo e pili ana i nā microvesicles pili protozoan a me kā lākou microRNAs e pili ana me nā cell host e hoʻokumu i kahi wahi kūpono no ko lākou ola.No laila, pono nā noiʻi hou aʻe e ʻike i ka pilina ma waena o nā miRNA exosomal i hoʻololi ʻia a me ka hoʻonui ʻana o ka cell glioma.ʻO ka hoʻololi ʻana o ka MicroRNA (nā ʻano cluster miR-30c-1, miR-125b-2, miR-23b-27b-24-1 a me miR-17-92) e hoʻopaʻa ʻia i ka mea hoʻolaha STAT3 i nā macrophages kanaka i hoʻopili ʻia i ka toxoplasma, ua hoʻoponopono ʻia a hoʻoulu i ka anti -apoptosis i ka pane ʻana i ka maʻi Toxoplasma gondii 29 .Hoʻonui ka maʻi Toxoplasma i ka hōʻike ʻana o ka miR-17-5p a me ka miR-106b-5p, i pili pū me kekahi mau maʻi hyperproliferative 30 .Hōʻike kēia mau ʻikepili i nā miRNA hoʻokipa i hoʻoponopono ʻia e ka maʻi Toxoplasma he mau molekole koʻikoʻi no ke ola parasite a me ka pathogenesis i ka ʻano biological host.
Hiki i nā miRNA hoʻololi ke hoʻololi i nā ʻano ʻano like ʻole i ka wā o ka hoʻomaka ʻana a me ka piʻi ʻana o nā cell malignant, me nā gliomas: pono ponoʻī o nā hōʻailona ulu, insensitivity i nā hōʻailona pale-keʻe, apoptosis evasion, hiki ʻole replicative palena ʻole, angiogenesis, invasion a me metastasis, a me ka mumū.I ka glioma, ua ʻike ʻia nā miRNA i hoʻololi ʻia i loko o nā haʻawina profiling hōʻike.
Ma ka haʻawina i kēia manawa, ua hōʻoia mākou i nā kiʻekiʻe kiʻekiʻe o ka miRNA-21 hōʻike i loko o nā pūnaewele host i hoʻopili ʻia i ka toxoplasma.Ua ʻike ʻia ʻo miR-21 ʻo ia kekahi o nā microRNA i hōʻike pinepine ʻia i nā maʻi maʻi paʻa, me nā gliomas, 33 a me kāna hōʻike e pili ana i ka pae o ka glioma.ʻO ka hōʻiliʻili ʻana i nā hōʻike hōʻike e hōʻike ana ʻo miR-21 he oncogene hou e hana ana ma ke ʻano he mea anti-apoptotic i ka ulu ʻana o ka glioma a ua hoʻohālikelike nui ʻia i nā ʻiʻo a me ka plasma o nā maʻi maʻi lolo kanaka.ʻO ka mea e mahalo ai, ʻo ka miR-21 inactivation i loko o nā glioma cell a me nā ʻiʻo e hoʻoulu i ka pale ʻana i ka hoʻonui ʻana o ka cell ma muli o ka caspase-dependent apoptosis.ʻO ka loiloi bioinformatic o ka miR-21 i wānana i nā pahuhopu i hōʻike ʻia i nā genes suppressor tumor e pili ana i nā ala apoptosis, me ka programmed cell death 4 (PDCD4), tropomyosin (TPM1), PTEN, a me ka pahu forkhead O1 (FoxO1), me ka pūnaewele paʻa miR-2121..22.38.
ʻO FoxO1, ʻo ia kekahi o nā kumu transcription (FoxO), i ka hoʻomohala ʻana i nā ʻano like ʻole o ke kanesa kanaka a hiki ke hoʻoponopono i ka hōʻike ʻana o nā genes suppressor tumor e like me p21, p27, Bim, a me FasL40.Hiki i ka FoxO1 ke hoʻopaʻa a hoʻōla i nā mea hoʻopaneʻe pōʻaiapili e like me ka p27 e hoʻopau i ka ulu ʻana o ke kelepona.Eia kekahi, ʻo FoxO1 kahi hopena koʻikoʻi o ka hōʻailona PI3K / Akt a hoʻoponopono i nā kaʻina hana ola e like me ka holomua o ka cell cycle a me ka hoʻokaʻawale ʻana o ka cell ma o ka hoʻāla ʻana o ka transcription p2742.
I ka hopena, ke manaʻoʻiʻo nei mākou he mea koʻikoʻi ka exosomal miR-21 i loaʻa mai ka microglia i hoʻopili ʻia e Toxoplasma ma ke ʻano he mea hoʻoponopono ulu o nā cell glioma (Fig. 7).Eia nō naʻe, pono nā noiʻi hou e ʻike i kahi loulou pololei ma waena o ka exosomal miR-21, ka maʻi Toxoplasma i hoʻololi ʻia, a me ka ulu ʻana o ka glioma.Manaʻo ʻia kēia mau hopena e hoʻomaka i ke aʻo ʻana i ka pilina ma waena o ka maʻi Toxoplasma a me ka loaʻa ʻana o ka glioma.
Hoʻopuka ʻia kahi kiʻi schematic o ke ʻano o ka glioma (lolo) carcinogenesis i loko o kēia haʻawina.Kahakiʻi ka mea kākau ma PowerPoint 2019 (Microsoft, Redmond, WA).
ʻO nā protocols hoʻokolohua āpau i kēia noiʻi, me ka hoʻohana ʻana i nā holoholona, ​​ua like ia me ka Seoul National University Animal Care and User Committee Standard Ethical Guidelines a ua ʻae ʻia e ka Institutional Review Board o ka Seoul National University School of Medicine (IRB number SNU- 150715).-2).Ua hoʻokō ʻia nā kaʻina hana hoʻokolohua a pau e like me nā ʻōlelo aʻoaʻo ARRIVE.
BV2 mouse microglia a me U87 kanaka glioma cell i moʻomeheu ʻia ma Dulbecco's Modified Eagle's Medium (DMEM; Welgene, Seoul, Korea) a me Roswell Park Memorial Institute's Medium (RPMI; Welgene), kēlā me kēia, loaʻa kēlā me kēia me 10% fetal bovine serum, 4 mM l- glutamine, 0.2 mM penicillin a me 0.05 mM streptomycin.Hoʻoulu ʻia nā kelepona i loko o kahi incubator me 5% CO2 ma 37 ° C.Ua hoʻohana ʻia kekahi laina glioma cell, U118, no ka hoʻohālikelike ʻana me nā cell U87.
No ka hoʻokaʻawale ʻana i nā exosomes mai nā T. gondii-infected RH a me ME49 strains, ua ʻohi ʻia ʻo T. gondii tachyzoites (RH strain) mai ka ʻōpū o ka ʻōpū o 6-week-old BALB/c mice injected 3-4 mau lā ma mua.Ua holoiʻiaʻo Tachyzoites iʻekolu manawa me ka PBS a hoʻomaʻemaʻeʻia e ka centrifugation ma 40% Percoll (Sigma-Aldrich, St. Louis, MO, USA)43.No ka loaʻaʻana o nā tachyzoites o ke kānana ME49, ua hoʻokomoʻia nāʻiole BALB / c intraperitoneally me ka 20 tissue cysts a me ka hoʻololiʻana o ka tachyzoite i nā cysts i hōʻiliʻiliʻia ma ka holoiʻana i ka'ōpū o ka'ōpū ma ka lā 6-8th ma hope o ka maʻi (PI).ʻO nā ʻiole i loaʻa i ka PBS.Ua ulu ʻia nā tachyzoites ME49 i loko o nā cell i hoʻohui ʻia me 100 μg / ml penicillin (Gibco / BRL, Grand Island, NY, USA), 100 μg / ml streptomycin (Gibco / BRL), a me 5% fetal bovine serum (Lonza, Walkersville, MD) .., USA) ma 37 °C a me 5% carbon dioxide.Ma hope o ka mahi ʻana i loko o nā pūnaewele Vero, ua hoʻoili ʻia ʻo ME49 tachyzoites i ʻelua manawa ma o kahi nila 25 gauge a laila ma o kahi kānana 5 µm e wehe i nā ʻōpala a me nā cell.Ma hope o ka holoi ʻana, ua hoʻokuʻu hou ʻia nā tachyzoites ma PBS44.Ua mālama ʻia nā ʻōpū kiko o Toxoplasma gondii strain ME49 e ka intraperitoneal injection o nā cysts i hoʻokaʻawale ʻia mai ka lolo o nā ʻiole C57BL/6 maʻi (Orient Bio Animal Center, Seongnam, Korea).Ua ʻohi ʻia ka lolo o nā ʻiole ME49-infected ma hope o 3 mau mahina o PI a ʻoki ʻia ma lalo o kahi microscope e hoʻokaʻawale i nā cysts.Ua mālama ʻia nā ʻiole ma lalo o nā kūlana pathogen-free kūikawā (SPF) ma ke kula ʻo Seoul National University School of Medicine.
Ua unuhi ʻia ka huina RNA mai nā exosome i loaʻa mai i ka BV2, nā cell BV2 a me nā ʻiʻo me ka hoʻohana ʻana i ka miRNeasy Mini Kit (Qiagen, Hilden, Kelemānia) e like me nā ʻōlelo a ka mea hana, me ka manawa incubation no ka hana elution.Ua hoʻoholo ʻia ka manaʻo RNA ma kahi NanoDrop 2000 spectrophotometer.Ua loiloi ʻia ka maikaʻi o nā microarrays RNA me ka hoʻohana ʻana i kahi bioanalyzer Agilent 2100 (Agilent Technologies, Amstelveen, Netherlands).
Ua hoʻomākaukau ʻia ʻo DMEM me 10% exosome-poor FBS e ultracentrifugation ma 100,000g no 16 mau hola ma 4 ° C a kānana ʻia ma kahi kānana 0.22 µm (Nalgene, Rochester, NY, USA).ʻO nā pūnaewele BV2, 5 × 105, i hoʻouluʻia i DMEM i loaʻa ka 10% exosome-depleted FBS a me ka 1% antibiotic ma 37 ° C a me 5% CO2.Ma hope o 24 mau hola o ka incubation, ua hoʻohui ʻia nā tachyzoites o ka strain RH a i ʻole ME49 (MOI = 10) i nā cell a ua wehe ʻia nā parasite non-invading i loko o hoʻokahi hola a hoʻopiha hou ʻia me DMEM.Ua hoʻokaʻawale ʻia nā Exosome mai nā pūnaewele BV2 e ka centrifugation ʻokoʻa i hoʻololi ʻia, ke ʻano i hoʻohana nui ʻia.Hoʻopau hou i ka pellet exosome i 300 µl PBS no ka RNA a i ʻole ka nānā ʻana i ka protein.Ua hoʻoholo ʻia ka neʻe ʻana o nā exosomes kaʻawale me ka BCA protein assay kit (Pierce, Rockford, IL, USA) a me kahi NanoDrop 2000 spectrophotometer.
Ua hoʻoheheʻe ʻia nā wai mai nā cell BV2 a i ʻole exosomes i loaʻa mai ka BV2 i loko o ka PRO-PREP™ protein extraction solution (iNtRon Biotechnology, Seongnam, Korea) a ua hoʻouka ʻia nā protein ma luna o Coomassie brilliant blue stained 10% SDS polyacrylamide gels.Eia kekahi, ua hoʻololi ʻia nā protein i nā membrane PVDF no 2 mau hola.Ua hōʻoia ʻia nā blots Western me ka hoʻohana ʻana i ka antibody Alix (Cell Signaling Technology, Beverly, MA, USA) ma ke ʻano he hōʻailona exosomal.HRP-conjugated goat anti-mouse IgG (H + L) (Bethyl Laboratories, Montgomery, TX, USA) a me ka LAS-1000 plus luminescent image analyzer (Fuji Photographic Film, Tokyo, Iapana) ua hoʻohana ʻia ma ke ʻano he antibody lua..Ua hana ʻia ka microscopy electron e aʻo i ka nui a me ka morphology o exosomes.Ua hoʻomākaukau ʻia nā exosome i hoʻokaʻawale ʻia mai nā pūnaewele BV2 (6.40 µg/µl) ma luna o nā meshes carbon-coated a ʻino ʻia me ka 2% uranyl acetate no 1 min.Ua ʻike ʻia nā mea hoʻohālike i hoʻomākaukau ʻia ma ka volta wikiwiki o 80 kV me ka hoʻohana ʻana i kahi JEOL 1200-EX II (Tokyo, Iapana) i hoʻolako ʻia me kahi kāmela ES1000W Erlangshen CCD (Gatan, Pleasanton, CA, USA).
Hoʻopiliʻia nā exosome i loaʻa mai i ka BV2 me ka PKH26 Red Fluorescent Linker Kit (Sigma-Aldrich, St. Louis, MO, USA) no nā minuke 15 i ka mahana wela.ʻO nā pūnaewele U87, 2 × 105, me ka PKH26-labeled exosomes (ʻulaʻula) a i ʻole exosome ma ke ʻano he mana maikaʻi ʻole, ua hoʻopili ʻia ma 37 ° C no nā hola 24 i kahi incubator 5% CO2.Ua hoʻopaʻa ʻia ka nuclei cell U87 me DAPI (uliuli), ua hoʻopaʻa ʻia nā cell U87 i 4% paraformaldehyde no 15 min ma 4 ° C a laila nānā ʻia i kahi ʻōnaehana microscope confocal Leica TCS SP8 STED CW (Leica Microsystems, Mannheim, Kelemānia).ʻike ʻia.
Ua hana ʻia ka cDNA mai ka siRNA me ka hoʻohana ʻana iā Mir-X siRNA first strand synthesis a me SYBR qRT-PCR kit (Takara Bio Inc., Shiga, Iapana).Hana ʻia ka PCR quantitative maoli me ka ʻōnaehana ʻike PCR iQ5 maoli (Bio-Rad, Hercules, CA, USA) me ka hoʻohana ʻana i nā primers a me nā templates i hui pū ʻia me SYBR Premix.Ua hoʻonui ʻia ʻo DNA no 40 mau pōʻai o ka denaturation ma 95 ° C no 15 s a me ka annealing ma 60 ° C no 60 s.Ua kālailai ʻia ka ʻikepili mai kēlā me kēia pane PCR me ka hoʻohana ʻana i ka module analysis data o ka iQ™5 optical system software (Bio-Rad).Ua helu ʻia nā hoʻololi pili i ka hōʻike gene ma waena o nā genes i koho ʻia a me β-actin/siRNA (a me U6) me ka hoʻohana ʻana i ke ʻano kuʻekuʻe maʻamau.Hōʻike ʻia nā ʻōkuhi mua i hoʻohana ʻia ma ka Papa 1.
3 x 104 U87 glioma cell i kanu ʻia i loko o nā pā 96-well a hui pū ʻia me Toxoplasma-infected exosomes i loaʻa mai BV2 (50 μg / mL) a i ʻole nonpulse exosomes i loaʻa mai BV2 (50 μg / mL) e like me nā mana ma 12, 18 a me 36 mau hola. .Ua hoʻoholo ʻia ka nui o ka hoʻonui ʻana o ka cell me ka hoʻohana ʻana i ka Cell Counting Kit-8 (Dojindo, Kumamoto, Iapana) (Nā Kiʻi Hoʻohui S1-S3) 46.
Ua kūʻai ʻia mai Orient Bio (Seongnam-si, South Korea) wahine 5-week old BALB/c mau ʻiole a mālama ʻia i loko o nā hīnaʻi sterile ma ke ana wela (22±2°C) a me ka haʻahaʻa (45±15°C).%) ma ka lumi wela (22±2°C) a me ka haʻahaʻa (45±15%).Ua hana ʻia kahi pōʻai kukui 12-hola a me kahi pōʻeleʻele 12-hola ma lalo o SPF (Seoul National University School of Medicine Animal Center).Ua hoʻokaʻawale ʻia nā ʻiole i ʻekolu mau pūʻulu o 5 mau ʻiole i kēlā me kēia a ua hoʻopili ʻia nā hui āpau i lalo me ka 400 ml o PBS i loaʻa nā glioma 1 x 107 U87 a me ka mea ulu i hoʻemi ʻia ʻo BD Matrigel ™ (BD Science, Miami, FL, USA).ʻEono lā ma hope o ka hoʻokomo ʻia ʻana o ka maʻi tumora, ua hoʻokomo ʻia ka 200 mg o nā exosome i loaʻa mai nā cell BV2 (me/me ka ʻole o Toxoplasma maʻi) i loko o ka pūnaewele tumor.He iwakāluakūmālua mau lā ma hope o ka loaʻa ʻana o ka maʻi tumora, ua ana ʻia ka nui o nā ʻiole i kēlā me kēia hui me kahi caliper ʻekolu manawa i ka pule, a ua helu ʻia ka nui o ka tumor e ka ʻano: 0.5 × (ākea) × 2 × lōʻihi.
ʻO ka hōʻike hōʻike MicroRNA me ka hoʻohana ʻana i ka miRCURYTM LNA miRNA array, 7th hanauna he, mmu a me rno arrays (EXIQON, Vedbaek, Denmark) e uhi ana i 1119 mau ʻiole maikaʻi ʻia ma waena o 3100 kanaka, ʻiole a me ka ʻiole hopu miRNA.I loko o kēia kaʻina hana, ua hoʻoneʻe ʻia ka 250 a i ka 1000 ng o ka RNA a pau mai ka 5′-phosphate ma ka mālama ʻana me ka phosphatase alkaline intestinal bipi a ukali ʻia me ka lepili me ka Hy3 green fluorescent dye.A laila, hoʻohui ʻia nā laʻana i hōʻailona ʻia e ka hoʻouka ʻana i nā paheʻe microarray me ka hoʻohana ʻana i kahi pahu keʻena hybridization (Agilent Technologies, Santa Clara, CA, USA) a me kahi kit hybridization slide (Agilent Technologies).Hana ʻia ka Hybridization no 16 mau hola ma 56 ° C, a laila holoi ʻia nā microarrays e like me nā ʻōlelo a ka mea hana.Ua nānā ʻia nā kiʻi paheʻe microarray i hoʻohana ʻia me kahi ʻōnaehana microarray scanner Agilent G2565CA (Agilent Technologies).Hoʻokomo ʻia nā kiʻi i kālai ʻia me ka hoʻohana ʻana i ka polokalamu Agilent Feature Extraction software version 10.7.3.1 (Agilent Technologies) a ua helu ʻia ka ikaika fluorescence o kēlā me kēia kiʻi me ka faila GAL pili o ka protocol Exiqon i hoʻololi ʻia.Waiho ʻia ka ʻikepili Microarray no ka haʻawina o kēia manawa ma ka waihona GEO ma lalo o ka helu komo GPL32397.
Hoʻopili ʻia nā ʻōlelo hōʻike o nā miRNA exosomal makua i ka microglia o RH a i ʻole ME49 i hoʻopili ʻia me Toxoplasma me ka hoʻohana ʻana i nā mea hana pūnaewele.Ua ʻike ʻia nā miRNA e pili ana me ka hoʻomohala ʻana i ka ʻōpū me ka hoʻohana ʻana i ka miRWalk2.0 (http://mirwalk.umm.uni-heidelberg.de) a kānana ʻia me ka ikaika o ka hōʻailona maʻamau (log2) ʻoi aku ma mua o 8.0.Ma waena o nā miRNA, ua ʻike ʻia nā miRNA i hōʻike ʻokoʻa ʻia ma mua o 1.5-fold i hoʻololi ʻia e ka kānana kānana o nā miRNA i hoʻololi ʻia e RH a i ʻole ME49 strains i loaʻa i ka T. gondii.
Ua kanu ʻia nā cell i loko o nā papa ʻeono (3 x 105 cell / well) ma opti-MEM (Gibco, Carlsbad, CA, USA) me ka hoʻohana ʻana iā Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).Hoʻoulu ʻia nā cell i hoʻololi ʻia no 6 mau hola a laila hoʻololi ʻia ke ʻano i ke ʻano hou.Ua ʻohi ʻia nā kelepona ma hope o 24 mau hola ma hope o ka lawe ʻana.
Hana ʻia ka ʻikepili helu me ka hoʻohana ʻana i ka t-test a Student me Excel (Microsoft, Washington, DC, USA).No ka hoʻāʻo ʻana i nā holoholona holoholona, ​​​​ua hana ʻia kahi ANOVA ʻelua me ka hoʻohana ʻana i ka polokalamu Prism 3.0 (GraphPad Software, La Jolla, CA, USA). Ua manaʻo ʻia nā waiwai P <0.05 he koʻikoʻi nui. Ua manaʻo ʻia nā waiwai P <0.05 he koʻikoʻi koʻikoʻi. Значения P <0,05 считались статистически значимыми. Ua manaʻo nui ʻia nā helu P <0.05. P 值< 0.05 被认为具有统计学意义。 P ‼< 0.05 Значения P <0,05 считались статистически значимыми. Ua manaʻo nui ʻia nā helu P <0.05.
Ua ʻae ʻia nā protocols hoʻokolohua āpau i hoʻohana ʻia i kēia noiʻi e ka Institutional Review Board o ke Kula Nui o Seoul National University School of Medicine (IRB number 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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