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Toxoplasma gondii bụ parasaiti protozoan intracellular nke na-eme ka microenvironment nke onye ọbịa ahụ butere ya ma mara na ọ na-ejikọta ya na mmụba nke ụbụrụ ụbụrụ.N'ime ọmụmụ ihe a, anyị na-eche na exosomal miRNA-21 sitere na ọrịa Toxoplasma na-akwalite uto ụbụrụ ụbụrụ.E ji mara exosomes sitere na Toxoplasma bu ọrịa BV2 microglia na ntinye nke mkpụrụ ndụ glioma U87.A tụlere profaịlụ okwu microRNA exosomal site na iji usoro microRNA na microRNA-21A-5p jikọtara na Toxoplasma gondii na nhazi etuto ahụ.Anyị nyochakwara ọkwa mRNA nke mkpụrụ ndụ ihe nketa metụtara tumo na mkpụrụ ndụ glioma U87 site n'ịgbanwe ọkwa miR-21 na exosomes yana mmetụta exosomes na mmụba cell U87 glioma mmadụ.N'ime exosomes nke mkpụrụ ndụ glioma U87 nwere Toxoplasma gondii, okwu microRNA-21 na-abawanye ma na-ebelata ọrụ nke mkpụrụ ndụ ihe nketa antitumor (FoxO1, PTEN, na PDCD4).Ezosomes sitere na BV2 butere Toxoplasma na-ebute mmụba nke mkpụrụ ndụ glioma U87.Exosomes na-ebute uto nke mkpụrụ ndụ U87 na ụdị tumor nke òké.Anyị na-atụ aro na ụbara exosomal miR-21 na Toxoplasma bu ọrịa BV2 microglia nwere ike ịrụ ọrụ dị mkpa dị ka onye na-akwalite uto cell na mkpụrụ ndụ U87 glioma site na ibelata mkpụrụ ndụ ihe nketa antitumor.
A na-eme atụmatụ na ihe karịrị nde 18.1 nke ọrịa kansa dị elu ka achọpụtara n'ụwa niile na 2018, yana ihe dị ka 297,000 ụbụrụ ụbụrụ etiti na-achọpụta kwa afọ (1.6% nke etuto ahụ niile) 1.Nnyocha ndị gara aga egosila na ihe ndị dị ize ndụ maka ịmalite ụbụrụ ụbụrụ mmadụ gụnyere ngwaahịa kemịkal dị iche iche, akụkọ ihe mere eme ezinụlọ, na ionizing radieshon sitere na ọgwụgwọ isi na akụrụngwa nyocha.Otú ọ dị, a maghị kpọmkwem ihe kpatara ọrịa ọjọọ ndị a.Ihe dị ka 20% nke ọrịa kansa n'ụwa niile bụ ndị na-efe efe na-ebute, gụnyere nje, nje bacteria na nje nje3,4.Ọrịa nje na-efe efe na-akpaghasị usoro mkpụrụ ndụ ihe nketa nke cell, dị ka nrụzi DNA na okirikiri cell, ma nwee ike ibute mbufụt na-adịghị ala ala na imebi usoro ahụ ji alụso ọrịa ọgụ5.
Ihe ndị na-efe efe metụtara ọrịa kansa mmadụ bụ nje nje virus na-ebutekarị, gụnyere nje papillomavirus mmadụ na nje ịba ọcha n'anya B na C.Nrịanrịa nwere ike ịrụ ọrụ nwere ike ibute ọrịa kansa mmadụ.Ọtụtụ ụdị parasaiti, ya bụ Schistosoma, Opishorchis viverrini, O. felineus, Clonorchis sinensis na Hymenolepis nana, etinyela aka na ụdị ọrịa kansa mmadụ dị iche iche 6,7,8.
Toxoplasma gondii bụ protozoan intracellular nke na-achịkwa microenvironment nke sel ndị nwere nje butere.A na-eche na nje nje a na-ebute ihe dịka 30% nke ndị bi n'ụwa, na-etinye mmadụ niile n'ihe ize ndụ9,10.Toxoplasma gondii nwere ike ibunye akụkụ ahụ dị mkpa, gụnyere sistemu ụjọ nke etiti (CNS), wee bute ọrịa siri ike dịka maningitis na encephalitis na-egbu egbu, ọkachasị n'ime ndị ọrịa na-arịa ọrịa immunocompromised9.Otú ọ dị, Toxoplasma gondii nwekwara ike gbanwee gburugburu ebe onye ọbịa ahụ butere ọrịa site n'ịgbanwe uto cell na nzaghachi mgbochi ọrịa na ndị mmadụ na-adịghị ahụkebe, na-eduga n'ịkwado ọrịa na-adịghị ala ala nke asymptomatic9,11.N'ụzọ na-akpali mmasị, nyere njikọ dị n'etiti T. gondii mgbasa na ụbụrụ ụbụrụ ụbụrụ, akụkọ ụfọdụ na-egosi na na vivo hosta mgbanwe gburugburu ebe obibi n'ihi ọrịa T. gondii na-adịghị ala ala yiri microenvironment tumor.
A maara exosomes dị ka ndị na-ekwurịta okwu intercellular nke na-ebuga ọdịnaya nke ndu, gụnyere protein na nucleic acid, site na sel ndị agbata obi16,17.Exosomes nwere ike imetụta usoro ndu ndị metụtara etuto dị ka mgbochi apoptosis, angiogenesis, na metastasis na microenvironment tumor.Karịsịa, miRNAs (miRNAs), obere RNA ndị na-abụghị koodu ihe dị ka 22 nucleotides n'ogologo, bụ ihe dị mkpa na-achịkwa usoro ntụgharị okwu transcriptional nke na-achịkwa ihe karịrị 30% nke mRNA mmadụ site na miRNA na-ebute ịgbachi nkịtị (miRISC).Toxoplasma gondii nwere ike imebi usoro ndu site na ịchịkwa okwu miRNA n'ime ndị ọbịa butere ọrịa.MiRNA ndị ọbịa nwere akara dị mkpa maka ịhazi usoro ndu ndị ọbịa iji nweta atụmatụ nlanarị nke nje.Ya mere, ịmụ mgbanwe na profaịlụ miRNA onye ọbịa na ọrịa T. gondii nwere ike inyere anyị aka ịghọta mmekọrịta dị n'etiti onye ọbịa na T. gondii nke ọma.N'ezie, Thirugnanam et al.15 tụrụ aro na T. gondii na-akwalite carcinogenesis ụbụrụ site n'ịgbanwe okwu ya na kpọmkwem miRNA ndị ọbịa jikọtara ya na uto tumor wee chọpụta na T. gondii nwere ike ime ka gliomas na ụmụ anụmanụ na-anwale.
Ọmụmụ ihe a lekwasịrị anya na mgbanwe nke exosomal miR-21 na microglia ndị ọbịa butere Toxoplasma BV2.Anyị hụrụ ọrụ enwere ike nke mgbanwe exosomal miR-21 na uto nke mkpụrụ ndụ U87 glioma n'ihi njide na nucleus nke FoxO1/p27, nke bụ ebumnuche nke miR-21 na-enweghị atụ.
A na-enweta exosomes sitere na BV2 site na iji centrifugation dị iche iche ma kwado ya site na ụzọ dị iche iche iji gbochie mmetọ na ihe ndị dị na cellular ma ọ bụ vesicles ndị ọzọ.SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gosipụtara ụkpụrụ dị iche iche n'etiti protein ndị amịpụtara na sel BV2 na exosomes (Nyocha 1A), na nlele anya maka ọnụnọ Alix, bụ nke nyochara site na Western blotting nke exosomal protein markers na .Achọtara akara Alix na protein exosome mana ọ bụghị na protein lysate cell BV2 (Fig 1B).Na mgbakwunye, a na-enyocha RNA dị ọcha site na exosomes sitere na BV2 site na iji bioanalyzer.18S na 28S ribosomal subunits adịkarịghị ahụ na usoro mbugharị RNA exosomal, na-egosi ịdị ọcha a pụrụ ịdabere na ya (Ọnụ ọgụgụ 1C).N'ikpeazụ, nnyefe eletrọnịkị microscopy gosipụtara na exosomes a hụrụ dị ihe dịka 60-150 nm n'ogo ma nwee ụdị iko dị ka ụdị nke exosome morphology (Fig 1D).
Njirimara nke exosomes ewepụtara na sel BV2.(A) ibe akwụkwọ data nchekwa.E kewapụrụ protein na sel BV2 ma ọ bụ exosomes sitere na BV2.Usoro protein dị iche n'etiti sel na exosomes.(B) Nyocha Western blot nke ihe nrịbama exosomal (Alix).(C) Nlele nke RNA dị ọcha site na sel BV2 na BV2 ewepụtara exosomes site na iji bioanalyzer.Ya mere, 18S na 28S ribosomal subunits na sel BV2 adịghị adịkarị na exosomal RNA.(D) Ntugharị eletrọnịkị microscopy gosipụtara na exosomes dịpụrụ adịpụ na sel BV2 nwere ntụpọ na-adịghị mma na 2% uranyl acetate.Exosomes dị ihe dịka 60-150 nm n'ogo yana ụdị iko (Abụ na Jung, data ebipụtaghị).
A na-ahụ ntinye nke cellular nke exosomes sitere na BV2 n'ime mkpụrụ ndụ glioma mmadụ U87 site na iji microscopy confocal.PKH26 akpọrọ exosomes bụ mpaghara na cytoplasm nke mkpụrụ ndụ U87.Ejiri DAPI mee ihe nuclei (Fig. 2A), na-egosi na exosomes sitere na BV2 nwere ike ịbanye na sel ndị ọbịa ma na-emetụta gburugburu sel ndị nnata.
Ntinye nke exosomes sitere na BV2 n'ime sel U87 glioma na BV2 ewepụtara na exosomes butere Toxoplasma RH butere mmụba nke mkpụrụ ndụ glioma U87.(A) Exosomes nke mkpụrụ ndụ U87 gbachiri site na microscopy confocal tụrụ.Ejikọtara mkpụrụ ndụ glioma U87 na exosomes nke akpọrọ PKH26 (acha uhie uhie) ma ọ bụ enweghị njikwa maka awa 24.Ejiri DAPI (acha anụnụ anụnụ) mee ihe nuclei ahụ wee hụ ya n'okpuru microscope confocal (ogwe ọnụ: 10 μm, x 3000).(B) U87 glioma cell proliferation kpebisiri ike site na nyocha mmụba cell.E jiri exosomes gwọọ mkpụrụ ndụ U87 glioma maka oge egosipụtara. * P <0.05 nwetara site n'ule nwa akwụkwọ. * P <0.05 nwetara site n'ule nwa akwụkwọ. * P <0,05 akwụkwọ ozi maka t-критерию Стьюдента. *P <0.05 site na t-ule nwata akwụkwọ. P <0.05 通过学生t 检验获得。 P <0.05 * P <0,05, ndị na-emepụta ihe na-eme ka t-критерия Стьюдента. * P <0.05 nwetara site na iji t-ule nwata akwụkwọ.
Mgbe o kwusiri ike na ntinye nke exosomes nke BV2 na-enweta n'ime sel U87 glioma, anyị mere nyocha nke mgbasawanye cell iji nyochaa ọrụ nke BV2 sitere na Toxoplasma na-enweta exosomes na mmepe nke mkpụrụ ndụ glioma mmadụ.Ọgwụgwọ nke mkpụrụ ndụ U87 na exosomes sitere na mkpụrụ ndụ BV2 na-arịa T. gondii gosipụtara na T. gondii na-ebute ọrịa BV2 na-ebute exosomes kpatara mmụba dị elu nke mkpụrụ ndụ U87 ma e jiri ya tụnyere njikwa (Fig 2B).
Tụkwasị na nke ahụ, uto nke mkpụrụ ndụ U118 nwere otu ihe ahụ dị ka U87, dị ka Toxoplasma kpaliri exosomes kpatara ọkwa kachasị elu nke mmụba (data egosighi).Dabere na data ndị a, anyị nwere ike igosi na BV2 sitere na Toxoplasma na-ebute ọrịa exosomes na-arụ ọrụ dị mkpa na mgbasa glioma cell.
Iji nyochaa mmetụta nke Toxoplasma-ọrịa BV2-ebutere exosomes na mmepe tumor, anyị na-agbanye mkpụrụ ndụ U87 glioma n'ime ụmụ oke ọtọ maka ụdị xenograft na ịgbanye BV2-nwetara exosomes ma ọ bụ RH-ebutere BV2-enweta exosomes.Mgbe etuto ahụ pụtachara ka izu 1 gachara, a na-ekewa otu ọ bụla nnwale nke ụmụ oke 5 dabere na nha etuto ahụ iji chọpụta otu ebe mmalite, ma tụọ nha etuto ahụ maka ụbọchị iri abụọ na abụọ.
N'ime ụmụ oke nwere ụdị U87 xenograft, a na-ahụ nha etuto dị ukwuu na ịdị arọ na BV2-ebutere ọrịa exosome nke RH na ụbọchị 22 (Fig. 3A,B).N'aka nke ọzọ, ọ dịghị ihe dị ịrịba ama dị iche iche nke tumor size n'etiti BV2-ewepụtara exosome otu na njikwa otu mgbe exosome ọgwụgwọ.Na mgbakwunye, ụmụ oke etinyere na mkpụrụ ndụ glioma na exosomes n'anya gosipụtara oke etuto ahụ n'ime otu RH-ebutere BV2-exosomes (Fig 3C).Nsonaazụ ndị a na-egosi na exosomes butere Toxoplasma sitere na BV2 na-ebute uto glioma na ụdị etuto òké.
Oncogenesis (AC) nke exosomes sitere na BV2 na ụdị òké U87 xenograft.Nha Tumor (A) na ịdị arọ (B) abawanyela nke ukwuu na ụmụ oke BALB/c gba ọtọ ejiri exosomes butere RH ewepụtara na BV2.A gbanyere ụmụ oke BALB/c gba ọtọ (C) n'okpuru akpụkpọ ahụ yana sel 1 x 107 U87 kwụsịtụrụ na ngwakọta Matrigel.Ụbọchị isii ka ịgbanyechara ya, 100 μg nke exosomes sitere na BV2 ka a na-emeso ya na ụmụ oke.A na-atụle nha tumor na ịdị arọ n'ụbọchị ndị egosipụtara na mgbe a chụrụ àjà, n'otu n'otu. P <0.05. P <0.05. * nkeji <0,05. P <0.05. P <0.05. P <0.05. * nkeji <0,05. P <0.05.
Ihe omuma a gosiputara na 37 miRNA (16 overexpressed na 21 downexpressed) jikọtara ya na mgbochi ma ọ bụ mmepe tumor gbanwere nke ukwuu na microglia mgbe ọrịa na Toxoplasma RH strain (Fig. 4A).Ngosipụta okwu nke miR-21 n'etiti miRNA ndị gbanwere bụ RT-PCR dị adị gosipụtara na exosomes ewepụtara na BV2, exosomes ejiri sel BV2 na U87 gwọọ ya.Nkwupụta nke miR-21 gosipụtara mmụba dị ukwuu na exosomes sitere na mkpụrụ ndụ BV2 butere Toxoplasma gondii (RH strain) (Fig 4B).Ọkwa ngosipụta nke miR-21 dị na sel BV2 na U87 mụbara mgbe ewelitere exosomes gbanwere (Fig 4B).Ọkwa mịR-21 dị n'ụbụrụ n'anụ ahụ nke ndị ọrịa tumor na ụmụ oke nwere Toxoplasma gondii (ụdị ME49) dị elu karịa na njikwa, n'otu n'otu (Fig. 4C).Nsonaazụ ndị a jikọtara ya na ọdịiche dị n'etiti ọkwa ngosipụta nke microRNA buru amụma na enwetara na vitro na in vivo.
Mgbanwe na ngosipụta nke exosomal miP-21a-5p na microglia butere Toxoplasma gondii (RH).(A) Na-egosipụta mgbanwe dị ịrịba ama na siRNA jikọtara ya na mgbochi ma ọ bụ mmepe tumor na-esochi ọrịa T. gondii RH.(B) Achọpụtara ọkwa okwu miR-21 dị n'etiti site na RT-PCR dị adị na exosomes sitere na BV2, exosomes BV2 na-agwọ, na sel U87.(C) Achọpụtara ọkwa okwu miR-21 dị na ụbụrụ ụbụrụ nke ndị ọrịa tumor (N=3) na ụmụ oke nwere Toxoplasma gondii (ME49 strain) (N=3). * P <0.05 nwetara site n'ule nwa akwụkwọ. * P <0.05 nwetara site n'ule nwa akwụkwọ. * P <0,05 было получено с помощью t-критерия Стьюдента. * P <0.05 nwetara site na iji t-ule nke ụmụ akwụkwọ. P <0.05 通过学生t 检验获得。 P <0.05 * P <0,05, ihe ngosi nke t-критерия Стьюдента. * P <0.05 nwetara site na iji t-ule nwata akwụkwọ.
Exosomes sitere na mkpụrụ ndụ BV2 nwere ọrịa RH mere ka uto nke gliomas na vivo na in vitro (Fig 2, 3).Iji chọpụta mRNA ndị dị mkpa, anyị nyochara ọkwa mRNA nke mkpụrụ ndụ ihe nketa antitumor, igbe ndụdụ O1 (FoxO1), PTEN, na ọnwụ cell 4 (PDCD4) akwadoro n'ime mkpụrụ ndụ U87 nwere ọrịa exosomes sitere na BV2 ma ọ bụ RH BV2.Nyocha bioinformatics egosila na ọtụtụ mkpụrụ ndụ ihe nketa metụtara tumor, gụnyere mkpụrụ ndụ ihe nketa FoxO1, PTEN, na PDCD4, nwere saịtị njikọ miR-2121,22.A na-ebelata ọkwa mRNA nke mkpụrụ ndụ ihe nketa antitumor na exosomes sitere na BV2 butere ọrịa RH ma e jiri ya tụnyere exosomes sitere na BV2 (Fig. 5A).FoxO1 gosipụtara ọkwa protein belatara na exosomes sitere na BV2 butere RH ma e jiri ya tụnyere BV2 sitere na exosomes (Nyocha 5B).Dabere na nsonaazụ ndị a, anyị nwere ike ikwenye na exosomes sitere na BV2 nwere ọrịa RH na-agbadata mkpụrụ ndụ ihe mgbochi oncogenic, na-ejigide ọrụ ha na uto etuto.
Toxoplasma RH na-ebute ọrịa BV2 na-ebute exosomes na-ebute nkwụsị nke mkpụrụ ndụ ihe nketa antitumor na mkpụrụ ndụ U87 glioma site na Toxoplasma RH nke BV2 nwetara exosomes.(A) PCR dị adị nke FoxO1, PTEN na PDCD4 okwu na exosomes sitere na T. gondii RH-ọrịa BV2 tụnyere PBS exosomes.Ejiri β-actin mRNA mee ihe dịka njikwa.(B) Ekpebiri okwu FoxO1 site na Western blotting na densitometry data na-enyocha ọnụ ọgụgụ site na iji mmemme ImageJ. * P <0.05 nwetara site n'ule nwa akwụkwọ. * P <0.05 nwetara site n'ule nwa akwụkwọ. * P <0,05 было получено с помощью t-критерия Стьюдента. * P <0.05 nwetara site na iji t-ule nke ụmụ akwụkwọ. P <0.05 通过学生t 检验获得。 P <0.05 * P <0,05, ihe ngosi nke t-критерия Стьюдента. * P <0.05 nwetara site na iji t-ule nwata akwụkwọ.
Iji ghọta mmetụta nke miP-21 na exosomes na usoro mkpụrụ ndụ ihe nketa metụtara tumor, a na-ebugharị mkpụrụ ndụ U87 na onye na-egbochi miP-21 site na iji Lipofectamine 2000 na mkpụrụ ndụ ndị ahụ na-egbute 24 awa mgbe ebufechara ya.FoxO1 na p27 ọkwa ọkwa n'ime sel ndị a na-ebufe na ndị na-emechi miR-21 ka e jiri ya tụnyere mkpụrụ ndụ ejiri BV2 nweta exosomes na-eji qRT-PCR (Fig. 6A,B).Mbufe nke ihe mgbochi miR-21 n'ime sel U87 wedara FoxO1 na okwu p27 nke ukwuu (FIG. 6).
RH nwere ọrịa exosomal BV2 enwetara miP-21 gbanwere okwu FoxO1/p27 na mkpụrụ ndụ glioma U87.Ejiri Lipofectamine 2000 bufee mkpụrụ ndụ U87 site na inhibitor miP-21 na mkpụrụ ndụ ewepụtara ya awa 24 ka ebufechara ya.FoxO1 na p27 ọkwa ọkwa n'ime sel ndị e bufere na ndị na-egbochi miR-21 ka atụnyere ọkwa dị na sel ndị ejiri BV2 nweta exosomes na-eji qRT-PCR (A, B).
Iji gbanarị nzaghachi mgbochi nke onye ọbịa ahụ, nje parasaiti Toxoplasma na-agbanwe ghọọ cyst anụ ahụ.Ha na-eme ka anụ ahụ dị iche iche, gụnyere ụbụrụ, obi, na akwara skeletal, n'oge ndụ onye ọbịa ahụ ma na-agbanwe nzaghachi mgbochi onye ọbịa ahụ.Tụkwasị na nke ahụ, ha nwere ike ịhazi usoro sel na apoptosis nke sel ndị ọbịa, na-akwalite mmụba ha14,24.Toxoplasma gondii na-ebutekarị mkpụrụ ndụ dendritic, neutrophils, na usoro monocyte/macrophage, gụnyere ụbụrụ microglia.Toxoplasma gondii na-ebute ọdịiche nke macrophages nke M2 phenotype, na-emetụta ọnya na-agwọ ọrịa mgbe ọrịa nje pathogen gasịrị, na-ejikọta ya na hypervascularization na granulomatous fibrosis.Omume omume a nke ọrịa Toxoplasma nwere ike jikọta na akara ndị metụtara mmepe tumor.Gburugburu ebe iro nke Toxoplasma na-achịkwa nwere ike ịdị ka onye na-ebute ọrịa kansa kwekọrọ.Ya mere, enwere ike iche na ọrịa Toxoplasma kwesịrị inye aka na mmepe nke ụbụrụ ụbụrụ.N'ezie, ọnụ ọgụgụ dị elu nke ọrịa Toxoplasma ka akọọrọ na serum nke ndị ọrịa nwere etuto ụbụrụ dị iche iche.Na mgbakwunye, Toxoplasma gondii nwere ike ịbụ ihe ọzọ na-akpata ọrịa carcinogenic ma na-emekọ ihe ọnụ iji nyere carcinogen ndị ọzọ na-efe efe aka ịzụlite etuto ụbụrụ.N'akụkụ a, ọ dị mma ịmara na P. falciparum na Epstein-Barr nje synergistically na-enye aka na nhazi nke lymphoma Burkitt.
Ọrụ nke exosomes dị ka ndị na-achịkwa na ngalaba nyocha ọrịa cancer ka enyochala nke ukwuu.Agbanyeghị, ọrụ exosomes dị n'etiti nje nje na ndị ọbịa butere ọrịa ka aghọtachaghị nke ọma.Ka ọ dị ugbu a, ndị nchịkwa dị iche iche, gụnyere protein ndị zoro ezo, akọwawo usoro ndu nke nje nje protozoan na-eguzogide ọgụ ndị ọbịa ma na-adịgide adịgide.N'oge na-adịbeghị anya, enweela echiche na-eto eto na microvesicles na-ejikọta protozoan na microRNA ha na-emekọrịta ihe na sel ndị ọbịa iji mepụta ebe dị mma maka nlanarị ha.Ya mere, a ga-achọkwu ọmụmụ ihe iji chọpụta njikọ dị n'etiti miRNA exosomal gbanwere na mmụba cell glioma.Mgbanwe MicroRNA (ụyọkọ mkpụrụ ndụ ihe nketa miR-30c-1, miR-125b-2, miR-23b-27b-24-1 na miR-17-92) na-ejikọta na onye na-akwalite STAT3 na macrophages mmadụ butere toxoplasma, na-achịkwa ma na-ebute mgbochi. -apoptosis na nzaghachi nke ọrịa Toxoplasma gondii 29.Ọrịa Toxoplasma na-abawanye nkwupụta nke miR-17-5p na miR-106b-5p, nke jikọtara ya na ọtụtụ ọrịa hyperproliferative 30.Data ndị a na-atụ aro na miRNA ndị ọbịa nke ọrịa Toxoplasma na-achịkwa bụ ụmụ irighiri ihe dị mkpa maka nlanarị nje na pathogenesis na omume ndu ndị ọbịa.
MiRNA ndị gbanwere nwere ike imetụta ụdị omume dị iche iche n'oge mmalite na ọganihu nke mkpụrụ ndụ ọjọọ, gụnyere gliomas: afọ ojuju nke ihe nrịbama uto, enweghị mmetụta na ihe mgbaàmà na-egbochi uto, mgbapụta apoptosis, ikike mmeghari na-akparaghị ókè, angiogenesis, mbuso agha na metastasis, na mbufụt.Na glioma, a chọpụtala miRNA ndị agbanweela n'ọtụtụ ọmụmụ ngosipụta okwu.
N'ime ọmụmụ ihe ugbu a, anyị kwadoro ọkwa dị elu nke okwu miRNA-21 na sel ndị ọbịa butere toxoplasma.Achọpụtala miR-21 dị ka otu n'ime microRNA ndị a na-egosipụtakarị na etuto siri ike, gụnyere gliomas, 33 na okwu ya kwekọrọ na ọkwa glioma.Mgbakọta ihe akaebe na-egosi na miR-21 bụ oncogene ọhụrụ nke na-arụ ọrụ dị ka ihe mgbochi apoptotic na uto glioma ma na-egosipụta nke ukwuu na anụ ahụ na plasma nke ụbụrụ ụbụrụ mmadụ.N'ụzọ na-akpali mmasị, mịR-21 adịghị arụ ọrụ n'ime mkpụrụ ndụ glioma na anụ ahụ na-akpalite mgbochi nke mmụba cell n'ihi apoptosis na-adabere na caspase.Nyocha bioinformatic nke ebumnuche amụma miR-21 kpughere ọtụtụ mkpụrụ ndụ ihe mgbochi tumor jikọtara ya na ụzọ apoptosis, gụnyere ọnwụ cell emepụtara 4 (PDCD4), tropomyosin (TPM1), PTEN, na igbe ndụdụ O1 (FoxO1), nwere saịtị njikọ miR-2121..22.38.
FoxO1, dị ka otu n'ime ihe odide ederede (FoxO), na-etinye aka na mmepe nke ụdị ọrịa cancer mmadụ dị iche iche ma nwee ike ịhazi okwu nke mkpụrụ ndụ ihe nkedo tumor dị ka p21, p27, Bim, na FasL40.FoxO1 nwere ike ijikọ ma mee ka ndị na-emechi okirikiri sel dị ka p27 kwụsịlata uto cell.Ọzọkwa, FoxO1 bụ isi ihe na-eme ka akara ngosi PI3K/Akt ma na-achịkwa ọtụtụ usoro ndu dị ka ọganihu okirikiri cell na ọdịiche sel site na ịgbalite ederede p2742.
N'ikpeazụ, anyị kwenyere na exosomal miR-21 nke sitere na Toxoplasma-ọrịa microglia nwere ike ịrụ ọrụ dị mkpa dị ka onye na-ahụ maka uto nke mkpụrụ ndụ glioma (Fig 7).Agbanyeghị, a ga-achọkwu ọmụmụ ihe iji chọta njikọ kpọmkwem n'etiti exosomal miR-21, ọrịa Toxoplasma gbanwere, na uto glioma.A na-atụ anya na nsonaazụ ndị a ga-enye mmalite maka ịmụ mmekọrịta dị n'etiti ọrịa Toxoplasma na ihe omume glioma.
A na-atụpụta eserese eserese nke usoro glioma (ụbụrụ) carcinogenesis n'ọmụmụ ihe a.Odee sere na PowerPoint 2019 (Microsoft, Redmond, WA).
Usoro nnwale niile dị na ọmụmụ ihe a, gụnyere iji anụmanụ, dabere na Seoul National University Animal Care na Kọmitii Ọrụ Standard Guidelines wee kwado ya site na Board Review Institutional nke Seoul National University School of Medicine (IRB nọmba SNU- 150715).-2).Emere usoro nnwale niile dịka ntụnye ARRIVE siri dị.
Azụlitere microglia òké BV2 na mkpụrụ ndụ glioma mmadụ U87 na Dulbecco's Modified Eagle's Medium (DMEM; Welgene, Seoul, Korea) na Roswell Park Memorial Institute's Medium (RPMI; Welgene), n'otu n'otu, nke ọ bụla nwere 10% fetal bovine serum, 4 mM l- glutamine, 0.2 mm penicillin na 0.05 mm streptomycin.Azụlitere mkpụrụ ndụ n'ime incubator nwere 5% CO2 na 37 Celsius.Eji eriri cell glioma ọzọ, U118, atụnyere sel U87.
Iji kewapụ exosomes site na T. gondii na-arịa ọrịa RH na ME49, T. gondii tachyzoites (RH strain) na-egbute site na oghere afọ nke 6-izu BALB / c ụmụ oke na-agbanye 3-4 ụbọchị tupu.A na-asacha Tachyzoites ugboro atọ na PBS ma mee ka ọ dị ọcha site na centrifugation na 40% Percoll (Sigma-Aldrich, St. Louis, MO, USA) 43.Iji nweta tachyzoites nke nje ME49, BALB / c ụmụ oke na intraperitoneally 20 anụ ahụ cysts na tachyzoite mgbanwe na cysts anakọtara site na-asacha oghere afọ na 6-8th ụbọchị mgbe ọrịa (PI).Ụmụ oke butere PBS.A na-etolite ME49 tachyzoites n'ime sel agbakwunyere na 100 μg/ml penicillin (Gibco/BRL, Grand Island, NY, USA), 100 μg/ml streptomycin (Gibco/BRL), na 5% nwa ebu n'afọ serum (Lonza, Walkersville, MD) .., USA) na 37 Celsius C na 5% carbon dioxide.Mgbe etolite na mkpụrụ ndụ Vero, ME49 tachyzoites gafere ugboro abụọ site na agịga nlele 25 wee site na nzacha 5 µm iji wepụ irighiri ihe na sel.Mgbe ha sachara, a kwụsịrị tachyzoites na PBS44.A na-echekwa cysts nke Toxoplasma gondii strain ME49 site na intraperitoneal injection nke cysts dịpụrụ adịpụ na ụbụrụ nke ụmụ oke C57BL/6 butere ọrịa (Orient Bio Animal Center, Seongnam, Korea).A na-egbute ụbụrụ ụmụ oke nwere ọrịa ME49 ka ọnwa 3 gachara PI wee gbuo ya n'okpuru microscope iji kewapụ cysts.A na-edobe ụmụ oke ndị butere ọrịa n'okpuru ọnọdụ enweghị nje nje pụrụ iche (SPF) na Ụlọ Akwụkwọ Ọgwụ nke Mahadum Mba Seoul.
E weputara mkpokọta RNA site na exosomes BV2 enwetara, sel BV2 na anụ ahụ na-eji miRNeasy Mini Kit (Qiagen, Hilden, Germany) dịka ntuziaka onye nrụpụta siri dị, gụnyere oge nnabata maka nzọụkwụ elution.Ekpebiri itinye uche RNA na NanoDrop 2000 spectrophotometer.A tụlere ogo microarrays RNA site na iji Agilent 2100 bioanalyzer (Agilent Technologies, Amstelveen, Netherlands).
DMEM nwere 10% exosome-ogbenye FBS kwadebere site na ultracentrifugation na 100,000g maka awa 16 na 4°C wee kpochaa site na nzacha 0.22 µm (Nalgene, Rochester, NY, USA).Azụlitere mkpụrụ ndụ BV2, 5 × 105, na DMEM nwere 10% exosome-depleted FBS na 1% ọgwụ nje na 37°C na 5% CO2.Mgbe awa 24 gachara, a na-agbakwunye tachyzoites nke ụdị RH ma ọ bụ ME49 (MOI = 10) na sel ma wepụ nje ndị na-adịghị emerụ ahụ n'ime otu awa wee jupụta na DMEM.Ekewapụrụ exosomes sitere na sel BV2 site na centrifugation dị iche iche gbanwere, usoro a na-ejikarị eme ihe.Weghachite pellet exosome na 300 µl PBS maka nyocha RNA ma ọ bụ protein.Achọpụtara mkpokọta exosomes dịpụrụ adịpụ site na iji ngwa nyocha protein BCA (Pierce, Rockford, IL, USA) na NanoDrop 2000 spectrophotometer.
A na-agbanye mmiri sitere na mkpụrụ ndụ BV2 ma ọ bụ exosomes sitere na BV2 na ngwọta mmịpụta protein PRO-PREP ™ (iNtRon Biotechnology, Seongnam, Korea) na protein eburula na Coomassie na-acha anụnụ anụnụ nwere ntụpọ 10% SDS polyacrylamide gels.Na mgbakwunye, a na-ebufe protein na akpụkpọ anụ PVDF maka awa 2.A kwadoro mgbochi ọdịda anyanwụ site na iji ọgwụ mgbochi Alix (Technology Signaling Cell, Beverly, MA, USA) dị ka ihe nrịbama exosomal.HRP-conjugated ewu anti-mouse IgG (H + L) (Bethyl Laboratories, Montgomery, TX, USA) na a LAS-1000 gbakwunyere luminescent image analyzer (Fuji Photographic Film, Tokyo, Japan) ji mee ihe dị ka ihe mgbochi nke abụọ..A na-eme microscopy eletrọn nnyefe iji mụọ nha na ọdịdị nke exosomes.A na-akwado Exosomes dịpụrụ adịpụ na sel BV2 (6.40 µg / µl) na meshes mkpuchi carbon na ntụpọ na-adịghị mma na 2% uranyl acetate maka 1 min.A hụrụ ihe atụ ndị a kwadebere na voltaji na-agba ọsọ nke 80 kV site na iji JEOL 1200-EX II (Tokyo, Japan) nke nwere igwefoto ES1000W Erlangshen CCD (Gatan, Pleasanton, CA, USA).
Ezosomes sitere na BV2 sitere na iji PKH26 Red Fluorescent Linker Kit (Sigma-Aldrich, St. Louis, MO, USA) maka nkeji iri na ise na okpomọkụ.U87 mkpụrụ ndụ, 2 × 105, na PKH26-labeled exosomes (red) ma ọ bụ enweghị exosomes dị ka njikwa na-adịghị mma, na-agbanye na 37 ° C maka awa 24 na 5% CO2 incubator.U87 cell nuklei nwere ntụpọ na DAPI (acha anụnụ anụnụ), sel U87 na-edozi na 4% paraformaldehyde maka 15 min na 4 ° C wee nyochaa na Leica TCS SP8 STED CW confocal microscope system (Leica Microsystems, Mannheim, Germany).hụrụ.
Emepụtara cDNA site na siRNA site na iji Mir-X siRNA mbụ strand synthesis na SYBR qRT-PCR kit (Takara Bio Inc., Shiga, Japan).Emere PCR ezigbo oge site na iji sistemu nchọpụta PCR ozugbo (Bio-Rad, Hercules, CA, USA) site na iji primers na ndebiri agwakọta na SYBR Premix.A kwalitere DNA maka okirikiri 40 nke denaturation na 95ºC maka 15 s na mgbake na 60°C maka 60 s.A tụlere data sitere na mmeghachi omume PCR ọ bụla site na iji modul nyocha data nke iQ™5 ngwa ngwa ngwa anya (Bio-Rad).Mgbanwe ndị emetụtara na mkpụrụ ndụ ihe nketa n'etiti mkpụrụ ndụ ihe mgbaru ọsọ ahọpụtara na β-actin/siRNA (na U6) gbakọrọ site na iji usoro mgbanaka ọkọlọtọ.E gosipụtara usoro izizi eji eme ihe na Tebụl 1.
A na-akụ mkpụrụ ndụ 3 x 104 U87 glioma n'ime efere 96 nke ọma ma jikọta ya na exosomes nke Toxoplasma na-ebute site na BV2 (50 μg / mL) ma ọ bụ exosomes na-abụghị nke sitere na BV2 (50 μg / ml) dị ka njikwa na 12, 18 na 36 awa. .A na-ekpebi ọnụọgụ cell site na iji Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) (Mgbakwunye S1-S3) 46 .
A zụtara ụmụ nwanyị BALB/c gba ọtọ dị izu 5 n'aka Orient Bio (Seongnam-si, South Korea) wee debe ya n'otu n'otu n'ime oghere na-adịghị ọcha na ọnụ ụlọ (22± 2°C) na iru mmiri (45± 15°C).%) na ụlọ okpomọkụ (22± 2°C) na iru mmiri (45± 15%).Emere okirikiri ọkụ elekere 12 na okirikiri gbara ọchịchịrị nke awa iri na abụọ n'okpuru SPF (Seoul National University School of Medicine Animal Center).E kewara oke oke n'ime otu atọ nke ụmụ oke 5 nke ọ bụla na otu niile ka etinyere subcutaneously na 400 ml nke PBS nwere sel glioma 1 x 107 U87 yana ihe na-eto eto belatara BD Matrigel ™ (BD Science, Miami, FL, USA).Ụbọchị isii ka ịgba ọgwụ mgbochi tumor gasịrị, 200 mg nke exosomes sitere na mkpụrụ ndụ BV2 (na / na-enweghị ọrịa Toxoplasma) na-agbanye n'ime ebe akpụ ahụ.Ụbọchị iri abụọ na abụọ mgbe ọrịa tumor gasịrị, a na-eji caliper tụọ ụbụrụ ụbụrụ nke ụmụ oke na otu ọ bụla ugboro atọ n'izu, wee gbakọọ olu tumor site na usoro: 0.5 × (obosara) × 2 × ogologo.
Nyocha okwu MicroRNA site na iji miRCURYTM LNA miRNA array, ọgbọ nke asaa nwere, mmu na rno arrays (EXIQON, Vedbaek, Denmark) na-ekpuchi ụmụ oke mara mma nke 1119 n'etiti mmadụ 3100, òké na oke miRNA jidere nyocha.N'oge usoro a, e wepụrụ 250 ruo 1000 ng nke ngụkọta RNA na 5'-phosphate site na ọgwụgwọ nke nwa ehi alkaline phosphatase na-esote ya na ịdebanye aha na Hy3 green fluorescent dye.A na-ejikọta ihe atụ ndị a kpọrọ aha site na ị na-ebu ihe mmịfe microarray site na iji ngwa ngwa ngwakọ (Agilent Technologies, Santa Clara, CA, USA) na ngwa mmịfe ngwakọ (Agilent Teknụzụ).A na-eme hybridization maka awa 16 na 56 Celsius C, mgbe ahụ, a na-asacha microarrays dịka ndụmọdụ ndị nrụpụta si dị.A na-enyocha ihe mmịfe microarray a haziri site na iji sistemụ nyocha microarray Agilent G2565CA (Agilent Technologies).Ebubata onyonyo enyochagoro site na iji ụdị sọftụwia Agilent Feature Extraction 10.7.3.1 (Agilent Teknụzụ) wee gbakọọ ike ọkụ nke onyonyo ọ bụla site na iji faịlụ GAL kwekọrọ nke protocol Exiqon gbanwetụrụ.A na-edobe data microarray maka ọmụmụ ihe ugbu a na nchekwa data GEO n'okpuru nọmba ntinye GPL32397.
A na-enyocha profaịlụ nkọwa nke miRNA exosomal tozuru oke na microglia nke ụdị RH ma ọ bụ ME49 butere Toxoplasma site na iji ngwaọrụ netwọk dị iche iche.Achọpụtara miRNA ndị metụtara mmepe etuto site na iji miRWalk2.0 (http://mirwalk.umm.uni-heidelberg.de) wee wepụta ya na ike mgbama ahaziri ahazi (log2) karịa 8.0.N'ime miRNA, ahụrụ miRNA ndị egosipụtara n'ụzọ dị iche ka achọpụtara ka ọ karịrị okpukpu 1.5 gbanwere site na nyocha nzacha nke miRNAs nke ụdị RH ma ọ bụ ME49 butere T. gondii gbanwere.
A na-amịpụta mkpụrụ ndụ na efere isii nke ọma (3 x 105 cell / well) na opti-MEM (Gibco, Carlsbad, CA, USA) na-eji Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).A na-emepe mkpụrụ ndụ ndị a gbanwere maka awa 6 wee gbanwee usoro ahụ ka ọ bụrụ nke ọhụrụ zuru oke.A na-egbute sel 24 awa mgbe ebufechara ya.
A na-eme nyocha ọnụọgụgụ nke ọma site na iji t-ule nwata akwụkwọ nwere ngwanrọ Excel (Microsoft, Washington, DC, USA).Maka nyocha anụmanụ nnwale, a na-eme ANOVA ụzọ abụọ site na iji sọftụwia Prism 3.0 (GraphPad Software, La Jolla, CA, USA). A na-ahụta ụkpụrụ P- <0.05 dị ka ọnụ ọgụgụ dị mkpa. A na-ahụta ụkpụrụ P- <0.05 dị ka ọnụ ọgụgụ dị mkpa. Значения P <0,05 считались статистически значимыми. A tụlere ụkpụrụ P <0.05 dị ka ọnụ ọgụgụ dị mkpa. P 值< 0.05 被认为具有统计学意义。 P na <0.05 Значения P <0,05 считались статистически значимыми. A tụlere ụkpụrụ P <0.05 dị ka ọnụ ọgụgụ dị mkpa.
Usoro nnwale niile ejiri mee ihe n'ọmụmụ ihe a bụ ndị ụlọ ọrụ nyocha ụlọ ọrụ nke Seoul National University School of Medicine (IRB nọmba 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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Rasheed, S., Rehman, K. & Akash, MS Nghọta banyere ihe ize ndụ nke etuto ụbụrụ na usoro ọgwụgwọ ha. Rasheed, S., Rehman, K. & Akash, MS Nghọta banyere ihe ize ndụ nke etuto ụbụrụ na usoro ọgwụgwọ ha.Rashid, S., Rehman, K. na Akash, MS Nyochaa ihe ize ndụ maka ụbụrụ ụbụrụ na nnukwu ọgwụgwọ ọgwụgwọ. Rasheed, S., Rehman, K. & Akash, MS 深入了解脑肿瘤的危险因素及其治疗干预措施。 Rasheed, S., Rehman, K. & Akash, MS Nghọta miri emi nke ihe ize ndụ ụbụrụ ụbụrụ na ntinye ọgwụgwọ.Rashid, S., Rehman, K. na Akash, MS Nyochaa ihe ize ndụ maka ụbụrụ ụbụrụ na nnukwu ọgwụgwọ ọgwụgwọ.Sayensị biomedical.Onye na-ere ọgwụ.143, 112119 (2021).
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