Apoptotic Effect Ntawm Cistanche Polysaccharides Ntawm Leukemia

Tus sau:

LI Yan,Zhang Jin,ZHANG Jing-Nan,ua al.

Sab hauv Mongolia, Tuam Tshoj

Txhawm rau tshawb xyuas cov nyhuv apoptotic ntawmCistanche polysaccharides( CDP) ntawm tib neegmob leukemiacell kab Jurkat hlwb.Cov txheej txheemMTT kev soj ntsuam tau siv los txheeb xyuas cov txiaj ntsig ntawm CDP ntawm kev loj hlob ntawm tes, Western blotting tau siv los kuaj xyuas qhov ua kom ntawm caspase-9 thiab caspase-3, thiab cov teeb liab apoptotic molecules kuj tau sim.Qhov tshwm simT cell proliferation yog inhibited los ntawm 1 mg / ml thiab 2 mg / ml CDP, T hlwb apoptosis raug ntxias los ntawm 5 mg / ml CDP, thiabcov kab lus ntawm pro-caspase-9 thiab pro-caspase-3 tau txo qis heev. Cov kab lus ntawm cov cell nto receptors CD45 thiab CD71 tau cuam tshuam loj heev los ntawm 5 mg / ml CDP, E K phosphorylation yog induced, thiab JNK phosphorylation tau inhibited.

Cov lus xaus

【Cov lus tseem ceeb】Cistanche polysaccharides; Apoptosis; eceptor; caspase-9; caspase-3

Acute lymphoblastic leukemia (TAG NRHO) yog ib qho mob qog nqaij hlav ntawm cov kab mob lymphatic nrog rau qib siab ntawm kev tiv thaiv kab mob sib kis. , yooj yim rau relapse, thiab lwm yam. Tam sim no, feem ntau siv txoj kev kho mob xws li kev hloov cov pob txha pob txha, induction therapy, immunotherapy, ua ke chemotherapy, thiab lwm yam. Vim tsis muaj peev xwm ntawm feem ntau cov tshuaj chemotherapy rau lub hom phiaj thiab tua cov qog, lawv yuav ua rau mob loj heev rau lub plab zom mov, ntshav system, mob plawv thiab hlab ntsha, thiab lwm yam. Thaum tau txais kev kho mob, nws feem ntau nrog cov kev phiv loj heev, qee qhov ua rau tuag taus. Raws li kev siv tshuaj khomob nce ntxiv, lub cev nyhav dhau los tsim kev kam rau cov tshuaj siv tshuaj khomob, ua rau muaj kev pheej hmoo ntawm tus kabmob.

Yog li ntawd, kev tshawb nrhiav kom tau txais txiaj ntsig zoo, tsis muaj tshuaj lom, thiab kev kho mob leukemia siab heev lossis kev kho mob adjuvant tau dhau los ua qhov tseem ceeb ntawm kev saib xyuas tam sim no.Cistanchehu ua Chagangaoya hauv Mongolian. Nws belongs rau tsev neeg Ledangaceae thiab genus Cistanche. Nws yog kab mob nyob rau hauv cov hauv paus hniav ntawm cov ntoo suab puam thiab muaj lub koob npe nrov ntawm "suab puam ginseng".

Cov khoom xyaw tseem ceeb ntawm Cistanche suav nrog phenylethanoid glycosides, lignans, betaines, sterols, amino acids, thiab polysaccharides. Anti-inflammatory, antiviral, antioxidant, tiv thaiv kab mob thiab lwm yam teebmeem.

Cov kev tshawb fawb tau pom tiasCistanche polysaccharide (CDP),raws li ib qho ntawm nws lub ntsiab active Cheebtsam,muaj anti-tumor thiab immunomodulatory teebmeem.Cov kws tshawb fawb tau pom tias CDP muaj cov txiaj ntsig zoo rau kev tua cov kab mob leukemia K562 thiab THP-1, thiab qhia tias CDP muaj qee lub luag haujlwm hauvkev tiv thaiv lossis kev kho mob leukemia. Tsis tas li ntawd, nyob rau hauv cov tsoos suav tshuaj compound, cistanche, rau kev kho mob ntawm tus mobleukemia, Cistanchekuj plays lub luag hauj lwm raws li ib tug ntawm cov tseem ceeb Cheebtsam. Txawm li cas los xij, tshwj xeeb mechanism ntawm kev txiav txim ntawmCistanchetsis paub. Nyob rau hauv daim ntawv no, kev cais thiab purification ntawmCistanche CDPtau kawm, thiab cov nyhuv tua thiab lwm yam txheej txheem ntawm CDP ntawm tib neeg mob leukemia cell kab Jurkat tau tshawb xyuas.

1.1 Main reagents thiab ntsuas

Cistanche hmoov tau yuav los ntawm Sichuan WecistancheGroup Co, Ltd., tib neeg mob leukemia cell kab Jurkat tau yuav los ntawm lub txhab nyiaj ntawm tes ntawm Shanghai Institute of Cell Biology, thiazole xiav (MTT) thiab sodium dodecyl sulfate (SDS) tau yuav los ntawm Beijing Dingguo Changsheng Biotechnology Co., Ltd., fetal bovine serum (FBS) tau yuav los ntawm Hangzhou Sijiqing Bioengineering Materials Co., Ltd., cysteine-muaj caspase -3, caspase-9, CD71, CD45, Phosphorylated c-Jun amino-terminal kinase (p -JNK) thiab phosphorylated extracellular signal-regulated kinase (p-ERK) antibodies tau muas los ntawm Cell Signaling Technology, USA, thiab actin antibodies tau muas los ntawm Shanghai Biyuntian Biotechnology Co., Ltd. Electrochemical Lub luminescence (ECL) substrate tau muas los ntawm Thermo Lub Tuam Txhab Kev Tshawb Fawb ntawm Tebchaws Meskas, thiab lwm cov tshuaj reagents yog qib kev ntsuas hauv tsev. Gel-Saib 6000 ntxiv cov duab ua haujlwm ntse (khoom ntawm Guangzhou Boluteng Biotechnology Co., Ltd.), Revolve FL upside-down fluorescence microscope (khoom ntawm Echo Laboratories, USA), EPOCH microplate nyeem ntawv (khoom ntawm Beijing Bo Teng Instrument Co., Ltd.).

1.2 Sib cais thiab purification ntawm CDP Noj 500 g ntawmCistanche extract hmoov, tsau nws nyob rau hauv 95 feem pua ​​​​ethanol ib hmos, lim nws nrog gauze, sau cov lim residue, boil nws nrog dej kub ntawm 60 degree rau 2 h / lub sij hawm, tag nrho ntawm 3 zaug. Tom qab qhov kawg, cov filtrates tau muab tso ua ke peb zaug, concentrated nyob rau hauv lub siab txo mus rau 2/3 ntawm qhov qub ntim, ntxiv 3 zaug ntawm qhov ntim ntawm 95 feem pua ​​​​ethanol, thiab tso cai rau sawv ntawm 4 degree thaum hmo ntuj. Hnub tom qab, centrifuge ntawm 4 000 r / min rau 10 min, sau cov precipitate, tshem tawm cov protein los ntawm txoj kev Sevag, precipitate nrog kiag li ethanol, khov-qhuav kom tau CDP.

1.3 Cell kab lis kev cai Tib neeg mob leukemia cell kab tau khaws cia hauv cov kua nitrogen hauv Jurkat kuaj. Thaum siv, cov hlwb tau rov zoo sai sai hauv 37 degree dej da dej thiab kab lis kev cai hauv RPMI1640 nruab nrab (muaj 100 U / ml penicillin thiab 100 U / ml streptomycins) nrog 10 feem pua ​​FBS. Cov hlwb raug coj los ntawm 37 degree, 5 feem pua ​​​​CO2 cell incubator, dhau txhua 2 mus rau 3 hnub, thiab cov kev sim tau ua thaum lub hlwb nkag mus rau theem kev loj hlob ntawm logarithmic.

1.4 MTT sim Jurkat hlwb nyob rau theem kev loj hlob ntawm logarithmic, centrifuged kom tau cell pellet, thiab suav ntawm 5 × 105 hlwb / ml. Siv ib daim 96-zoo cell kab lis kev cai phaj, inoculate 100 ul hlwb nyob rau hauv txhua lub qhov dej, thiab tom qab ntawd ntxiv 100 ul RP-MI1640 ua tiav nruab nrab raws li pab pawg tswj; pawg sim tau muab faib ua 3 pawg, ua ntej siv RPMI1640 nruab nrab los npaj cov kev sib txawv ntawm CDP, thiab tom qab ntawd ntxiv 100 ul ntawm RP-MI1640 nruab nrab rau txhua qhov dej. ul Jurkat hlwb thiab 100 ul CDP tov tau siv los ua qhov kawg ntawm CDP ntawm 1, 2, thiab 5 mg / ml, raws li. Txhua pab pawg tau teeb tsa nrog 5 qhov sib npaug thiab incubated hauv 37 degree cell incubator rau 48 teev. Tom qab incubation, ntxiv 20 ul ntawm MTT (qhov concentration ntawm cov tshuaj Tshuag yog 5 mg / ml) rau hauv txhua lub qhov dej, muab nws rov qab rau hauv lub cell incubator, thiab txuas ntxiv incubate rau 4 h, thiab thaum kawg ntxiv 100 ul ntawm 20 feem pua. SDS kom yaj formazan ntawm 37 degree thaum hmo ntuj, thiab siv nws rau hnub tom qab. Lub absorbance ntawm 570 nm ntawm txhua pab pawg tau kuaj pom los ntawm tus nyeem ntawv microplate, thiab qhov inhibition tus nqi sib txawv ntawm CDP ntawm Jurkat cell proliferation yog xam los ntawm cov mis.

1.5 Western blotting Jurkat hlwb nyob rau hauv lub logarithmic kev loj hlob theem tau coj thiab noob nyob rau hauv ib tug 6- zoo phaj ntawm ib tug ceev ntawm 2 × 105 hlwb / ml, 500 ul / well, thiab 500 ul RPMI1640 tiav nruab nrab tau ntxiv rau kev tswj. pab pawg. Teem 3 pawg sim, 1, 2, thiab 5 mg / ml pab pawg kho mob polysaccharide, CDP

Dissolved hauv RPMI1640 ua tiav qhov nruab nrab ua ntej, 500 ul ntawm cov hlwb thiab 500 ul ntawm CDP cov kev daws teeb meem sib txawv tau ntxiv rau 6- zoo phaj thiab incubated ntawm 37 degree rau 48 h. Tom qab kab lis kev cai, cov xov tooj ntawm tes raug tshem tawm ntawm txhua pawg tau sau, centrifuged ntawm 1 500 r / min rau 5 min kom tau txais cov cell pellets, thiab ntxuav ob zaug nrog pre-cooled phosphate-buffered saline (PBS). Tom qab ntxuav, 20 ul ntawm cell lysis tsis tau ntxiv rau txhua pawg, thiab cov hlwb raug lysed rau dej khov rau 30 feeb. Tag nrho cov protein tau muab rho tawm thiab cov cell concentration tau hloov kho siv Bradford txoj kev. 20 ug ntawm cov protein tau muab los ntawm txhua pawg rau SDS-polyacrylamide gel electrophoresis (PAGE), thiab cov protein tau raug xa mus rau polyvinylidene fluoride (PVDF) membrane los ntawm electroporation. Thaiv nrog nonfat mis hmoov nyob rau hauv chav tsev kub rau 1 h, ntxiv luav los tiv thaiv tib neeg caspase -3 thiab caspase -9 tshuaj tiv thaiv ntawm ib tug dilution ratio ntawm 1:1 000, incubated nyob rau hauv chav tsev kub rau 1 h, yaug 3 zaug nrog phosphate Tween buffer (PBST) shaker (10 min/time), ntxiv horseradish peroxidase (HRP)-labeled secondary antibody (1:1 000), incubate rau 1 h ntawm chav tsev kub, yaug 3 zaug nrog PBST, yaug 1 zaug nrog PBS, ntxiv ECL substrate, thiab siv Lub chaw ua haujlwm ntse duab tau siv rau kev thaij duab, thiab kev hloov pauv ntawm txhua pab pawg tau muab piv rau kev kawm apoptosis ntawm Jurkat hlwb induced los ntawm CDP. Rau kev kuaj pom ntawm apoptosis txoj kev muaj feem cuam tshuam nrog cov proteins, kab lis kev cai ntawm tes thiab CDP kev kho mob tau zoo ib yam li saum toj no. Tom qab cov tshuaj tiv thaiv, cov hlwb tau lysed rau cov protein ntau. Cov protein tau pauv mus rau PVDF membrane los ntawm SDS-PAGE thiab electroporation. Tom qab thaiv nrog skimmed mis hmoov rau 1 h, anti-CD71, CD45, p-JNK thiab p-ERK cov tshuaj tiv thaiv (1:1 000) ntxiv, thiab incubated ntawm chav tsev kub rau 1 h. HRP-labeled Secondary antibody (1:1 000) tau incubated rau 1 h, thiab thaum kawg, lub ntse imaging workstation tau siv rau imaging los kawm txog cov teeb liab molecules ntawm CDP-induced Jurkat cell apoptosis. Cov txiaj ntsig kev sim bands tau grey-scaled nrog Image J (1.8.0).

Nyem qhov no kom paub Cistanche cov nyhuv ntawm kev tiv thaiv qog thiab Anti-cancer Effect

1.6 Kev txheeb xyuas txheeb cais SPSS21.

2 cov txiaj ntsig ntawm Cistanche muaj nuj nqi rauleukemia

2.1 Cell proliferation theem

CDP tau raug cais tawm kom tiav, nrog ntau dua 9{{20}} feem pua ​​​​qab zib thiab tsawg dua 5 feem pua ​​​​cov ntsiab lus protein. Tus nqi inhibition ntawm cell proliferation nyob rau hauv 1 mg / ml CDP pawg yog (19. 1 ± 2. 1) feem pua, thaum uas nyob rau hauv 2 mg / ml thiab 5 mg / ml CDP pawg yog (44. 2 ± 5. 2. ) ) feem pua ​​​​thiab (47. 8 ± 4. 4) feem pua, qhia tias CDP tuaj yeem cuam tshuam qhov kev loj hlob ntawm Jurkat hlwb nyob rau hauv koob tshuaj. 2.2 Piv nrog rau pawg tswj hwm (0.62±{26}}. }}7), theem ntawm apoptosis hauv Jurkat hlwb kho nrog 5 mg / ml CDP ua rau cov enzymes ntawm caspase-9 thiab caspase{{30}}. Pro-form pro-caspase-9 ( 0. 09 ± 0. 02) thiab pro-caspase-3 ( {{ 63}}. 12 ± 0. 01) tau txo qis, thiab 1 mg/ml thiab 2 mg/ml CDP pawg tau txo qis rau pro-caspase-3 hauv hlwb. Caspase-9 (0.58±0.04, 0.51±0.01) thiab pro-caspase-3 cov ntsiab lus (0.48±0.04, 0.44±0.05) tsis muaj qhov cuam tshuam tseem ceeb, uas yog, qis Cov concentration ntawm CDP tsis ua rau apoptosis ntawm Jurkat hlwb, tab sis feem ntau inhibited cell proliferation, raws li qhia nyob rau hauv daim duab 1.

1-4: pab pawg neeg,

1 mg / ml CDP pawg, 2 mg / ml CDP pawg, 5 mg / ml CDP pawg; tib yam li daim duab hauv qab no

Daim duab 1 Cov nyhuv ntawm CDP ntawm caspase-9 thiab caspase-3 hauv Jurkat hlwb

Kev cuam tshuam ntawm Cistanche polysaccharides cuam tshuam rau leukemia

2.3 Cov txheeb ze qhia qib ntawm CD71 thiab CD45 hauv pawg tswj hwm apoptosis receptor yog 1.00±0.04, {{10}}.54± 0 {20}}2. 5 mg/ml CDP pawg tuaj yeem cuam tshuam CD71 ({27}} 05±0 02). 01) thiab CD45 ({38}}. 13 ± {{40}}. 03); 1 mg / ml thiab 2 mg / ml CDP pawg tuaj yeem cuam tshuam qhov kev qhia ntawm CD71 (0. 52 ± 0. 06, 0. 43 ± 0. 07) rau qee qhov Tsis muaj qhov cuam tshuam loj rau kev qhia ntawm CD45 (0.51). ± 0.01, 0.48 ± 0.02). Saib daim duab 2.

Daim duab 2 Cov nyhuv ntawm CDP ntawm kev qhia ntawm receptors ntawm lub cell membrane nto

2.4 Apoptosis-txog txoj hauv kev

Cov kev qhia txheeb ze ntawm p-JNK thiab p-ERK hauv pawg neeg siv yog 1.20±0.08 thiab 0.21±0 .03, raws. 5 mg / ml CDP pawg induced apoptosis thiab txo cov phosphorylation theem ntawm JNK. ( 0. 32 ± 0. 04), nce qib phosphorylation ntawm ERK (1. 13 ± 0. 05); 1 mg / ml, 2 mg / ml CDP nce qib phosphorylation ntawm JNK (1. 12 ± 0. 06 , 1. 06 ± 0. 06) tsis muaj txiaj ntsig zoo, tab sis tseem nce qib phosphorylation ntawm ERK. (0. 54 ± 0. 04, 0. 63 ± 0. 02). Saib daim duab 3.

Daim duab 3 Cov nyhuv ntawm CDP ntawm phosphorylation ntawm JNK thiab ERK

3 Kev sib tham

Nyob rau hauv xyoo tas los no, kev tshawb fawb tau pom tias CDP muaj qee yam cuam tshuam rau cov kab mob leukemia, tab sis tseem tsis tau paub txog cov txheej txheem tshwj xeeb.

Nyob rau hauv tus txheej txheem ntawm apoptosis, caspase-9 yog ib qho ntawm cov khoom siv hluav taws xob xaim hluav taws xob hauv cov txheej txheem caspase-dependent apoptosis. Tom qab lub teeb liab stimulation, pro-caspase-9 yog hydrolyzed loj heev los tsim ib daim ntawv activated ntawm cleaved-caspase-9, uas txuas ntxiv xa cov cim apoptotic downward los txhawb cov txheej txheem ntawm apoptosis. Caspase-3 yog ib qho effector-type caspase molecule thiab yog ib qho tseem ceeb ntawm cov teeb liab molecule hauv cov txheej txheem ntawm cellular caspase-dependent apoptosis. Nws kuj muaj nyob rau hauv daim ntawv ntawm pro-enzyme (pro-caspase-3) nyob rau hauv ib txwm mob. Thaum qhib los ntawm hydrolysis, ib tug loj npaum li cas ntawm cleaved-caspase yog tsim. -3, nws yuav lyse ntau yam ntawm cov protein molecules tseem ceeb hauv cov hlwb thiab nws thiaj li ua rau apoptosis. CDP belongs rau lub active polysaccharides ntawm lom macromolecules thiab tsis tuaj yeem nkag mus rau hauv cov hlwb los ntawm cov cell membrane. Yog li ntawd, CDP induces apoptosis los ntawm kev cuam tshuam rau kev qhia thiab kev faib tawm ntawm receptors nyob rau saum npoo ntawm Jurkat cell membranes, yog li xa cov teeb liab mus rau sab hauv ntawm cov hlwb, thiab thaum kawg ua rau cov tsev neeg caspase. protein, ua rau cell apoptosis. Txoj kev tshawb no qhia tau hais tias tsawg concentration ntawm CDP tsis induce apoptosis ntawm Jurkat hlwb, tab sis tsuas yog inhibits cell proliferation. CD71, tseem hu ua transferrin receptor, yog hom II transmembrane glycoprotein receptor koom nrog hauv kev nqus hlau thiab tswj kev loj hlob ntawm tes thiab kev loj hlob. CD71 tau qhia ntau heev rau cov neeg mob uas mob ntshav qab zib mellitus thiab cuam tshuam nrog cov tshuaj tiv thaiv thawj zaug, uas yog, kev qhia ntawm CD71. Tus nqi siab dua, qhov ua rau cov txiaj ntsig ntawm thawj chemotherapy. Yog li, CD71 yog ib qho ntawm cov cim rau kev kuaj mob. CD45 yog qhia nyob rau saum npoo ntawm txhua hom leukocytes, tseem hu ua leukocyte common antigen. Nws belongs rau hom I transmembrane glycoprotein thiab zoo sib xws rau kev loj hlob thiab kev sib txawv ntawm T hlwb. CD45 muaj ntau hom kev qhia sib txawv thiab hom allosteric hauv ntau hom kab mob leukemia, yog li nws tuaj yeem siv los ua ib qho ntawm cov cim rau kev kuaj mob sib txawv thiab kev tiv thaiv kab mob ntawm cov kab mob leukemia. CD45 monoclonal antibody tau siv dav hauv kev kho mob leukemia, tshwj xeeb tshaj yog cov tshuaj tiv thaiv kab mob leukemia. Friesen et al. pom tias radionuclides

Tagged CD45 mAb tuaj yeem ua rau apoptosis nyob rau hauv cov tshuaj tiv thaiv kab mob leukemia cell kab los ntawm kev ua kom lub tsev neeg cov proteins (xws li caspase-3, caspase-8, thiab caspase-9). CDP induces apoptosis ntawm Jurkat hlwb, ua ntej los ntawm kev ua ntawm lub cell membrane nto receptors CD45 thiab CD71, cuam tshuam rau kev qhia thiab kev faib tawm ntawm ob, xa cov cim apoptosis mus rau hauv lub hlwb, thiab thaum kawg ua rau caspase-9 thiab caspase{{8} }}, ua rau apoptosis. Txawm li cas los xij, ob qho tib si caspase-9 thiab caspase-3 yog nyob rau hauv qab ntawm txoj kev apoptotic, thiab cov kab hluav taws xob sab saud yuav tsum tau tshawb nrhiav ntxiv. Nyob rau hauv txoj kev tshawb no, CDP ntawm ib tug concentration ntawm 5 mg / ml induced apoptosis ntawm Jurkat hlwb thaum inhibiting kev qhia ntawm CD45, uas zoo ib yam li cov CD45 monoclonal antibody tau piav saum toj no. Yog li, los ntawm kev xav ntawm apoptosis-txog receptors CD45 thiab CD71,Cistanchemuaj qee yam peev xwm rau kev kho mob los yog kev kho mob ntawm leukemia.Mitogen-activated protein kinase systems (MAPKs) tau nthuav tawm hauv txhua lub hlwb eukaryotic.

Cistanche ntsiav tshuaj rau kev txhim kho kev tiv thaiv kab mob ntawm Leukemia kho

Nws yog ib qho tseem ceeb heev cov ntaub ntawv kis tau tus mob txoj kev uas ua extracellular stimulation teeb liab kis mus rau lub intracellular thiab yog tsim los ntawm serine/threonine protein kinases, xws li ERK, JNK, thiab p38MAPK. Nws cov phosphorylation thiab dephosphorylation yog cov hloov pauv ntawm cov haujlwm ntawm tes thiab koom nrog thiab tswj cov txheej txheem ntawm kev loj hlob ntawm tes, kev loj hlob, kev sib txawv, kev ua kom thiab apoptosis. Txoj kev tshawb no qhia tias JNK dephosphorylation koom nrog CDP-induced T cell apoptosis, thaum ERK phosphorylation koom nrog ob txheej txheem hauv CDP inhibits cell proliferation thiab induces apoptosis.

Hauv kev xaus,Cistanche CDPmuaj cov nyhuv cytotoxic ntawm T-lymphocytic leukemia cell kab Jurkat, uas qhia tias cov concentrations qis (qis dua 2 mg / ml) inhibit cell proliferation, thaum siab concentrations (5 mg / ml) induce apoptosis.Cistanche CDPinduces apoptosis hauv Jurkat hlwb feem ntau los ntawm kev cuam tshuam thiab hloov cov hlwb.

Kev nthuav qhia ntawm apoptosis-txog receptors CD71 thiab CD45 ntawm daim nyias nyias xaim xaim mus rau sab hauv ntawm cov hlwb, ntxiv rau ERK phosphorylation thiab JNK dephosphorylation, thiab thaum kawg ua kom cov caspase, uas nyob rau hauv lem activates caspase {{4} } los ua kom apoptosis.