Dab tsi yog Cov txiaj ntsig ntawm Cistanche Polysaccharide Ntawm Kev Tiv Thaiv Kev Ua Haujlwm

Hu rau: emily.li@wecistanche.com

Cov tshuaj tiv thaiv kab mob thiab cov txheej txheem ntawm Cistanche Polysaccharide

Zeng Qunli, Zheng Yifan, Lu Zhiliang

[Cov ntsiab lus]Lub Hom Phiaj: Kawm Covtiv thaiv kab mobkev ua ntawmCistanchePolysaccharide(CDPS) thiab cov txheej txheem ntawm nwstiv thaiv kab mobkev cai hauv vitro. Txoj kev: thiazole xiav colorimetric txoj kev (MTT) tau siv los soj ntsuam cov kev cai ntawm CDPS (Cistanche Polysaccharide) ntawm IQS DEX TNF inhibiting kev loj hlob ntawm nas thymic lymphocytes hauv vitro; CDPS (Cistanche Polysaccharide) tau siv los ua lub voj voog ntawm tes thiab ntsuas lub voj voog ntawm tes siv cytometry ntws. Kev txiav txim siab ntawm intracellular Ca2. Cov txiaj ntsig: CDPS (Cistanche Polysaccharide) ntawm qhov siab dua tuaj yeem tiv thaiv cov nyhuv inhibitory ntawm ISQDEX ntawm lymphocyte proliferation thiab inhibitory nyhuv ntawm siab concentration TNF ntawm lymphocyte proliferation, thiab synergize lub proliferation proliferation nyhuv ntawm tsawg concentration TN F ntawm lymphocytes; 100ug / ml 200 ug / ml CDPS (Cistanche Polysaccharide) tuaj yeem ua kom cov hlwb nce ntxiv hauv theem faib; ntau dua concentration (100ug / ml) CDPS (Cistanche Polysaccharide) tuaj yeem txhawb nqa qhov concentration ntawm Ca ion hauv nas thymocytes. Cov ntsiab lus: CDPS (Cistanche Polysaccharide) tuaj yeem txhawb cov hlwb kom nkag mus rau theem faib, thiab nws tau lees paub tias nws cov txiaj ntsig txhawb kev loj hlob ntawm nas thymic lymphocytes cuam tshuam rau nws cov kev txhawb nqa ntawm calcium tso tawm hauv nas thymic lymphocytes.

Cistanche(CistancheDeserticolaYC Ma) yog ib qho khoom siv tsis tshua muaj nqi thiab muaj txiaj ntsig hauv Suav teb hauv kuv lub tebchaws. Nws tau teev npe raws li qib siab hauv "Shen Nong's Materia Medica".Cistanchemuaj lub luag haujlwm ntawm cov ntshav nourishing, nourishing lub raum thiab pab yang, moisturizing cov hnyuv, thiab ncua kev laus. Kev tshawb fawb pharmacological niaj hnub qhia tiasCistanchePolysaccharidetuaj yeem txhim kho lub cevtiv thaiv kab mobmuaj nuj nqi, txhawb DNA synthesis, ntxiv dag zog rau lub cev, txhim kho kev txawj ntse, thiab muaj peev xwm tiv thaiv kev laus [1]. Tus sau tau kawm txog kev cai ntawm CDPS(Cistanche Polysaccharide).), polysaccharide feem ntawmCistanche, ntawmtiv thaiv kab mobmuaj nuj nqintawm nas lymphocytes.

Cistanche Polysaccharidetxhawbkev loj hlob ntawmlymphocytes

1 Cov khoom siv thiab cov txheej txheem

1.1 Reagents thiab tshuaj.

RPM, I 1640 nruab nrab: GIB-CO; HEPES: FARCO; L-glutamine: Shanghai Dongfeng Reagent Factory; Penicillin thiab Streptomycin: North China Pharmaceutical Factory; Concanavalin (ConA), MTT: SIGM A Company; Phytohemagglutinin (PHA): Shanghai Medical Laboratory; Isoproterenol (ISO): Shanghai Tianfeng Pharmaceutical Factory; Mercaptoethanol: Fluka tuam txhab; Cov mis nyuj yug tshiab: Sijiqing Bioengineering Company; Dexamethasone (DEX) tau nthuav tawm los ntawm Dr. Ma Liang, Thawj Tsev Kho Mob ntawm Zhejiang University School of Medicine; Tumor Suppressor Factor (TNF) yog muab los ntawm Yu Yuehan, xibfwb, Department of Cells, School of Life Sciences, Zhejiang University; Tus so ntawm cov reagents yog tag nrho domestically tsim analytical qib.

1.2 Kev sim tsiaj.

ICR nas, txiv neej, 68 lub lis piam, hnyav 18-20g, 22, tau yuav los ntawm Zhejiang Experimental Animal Center.

1.3 Extraction ntawm Cistanche Polysaccharide [2]:

CistanchePolysaccharideyog muab rho tawm los ntawm peb lub chaw kuaj! Tom qab crushing cov tshuaj crudeCistanche, nws yog immersed nyob rau hauv kub ethanol, muab rho tawm nyob rau hauv boiling dej, thiab precipitated nyob rau hauv ethanol. Qhov nruab nrab enzyme ua ke nrog Sewag txoj kev rau deproteinization, anion DEAE-Sephadex A- 50 pauv chromatography thiab Sephacry S-200 gel filtration chromatography kom tau txais ib feem ntawm CDPS (Cistanche Polysaccharide)). Khaws kom qhuav, siv tsis tiav RPMI1640 los ua kom muaj qhov xav tau ua ntej siv, thiab ua kom tsis muaj menyuam los ntawm ultrafiltration

1.4 Kev Npaj ntawm spleen thiab thymus lymphocytes [3]:

The mice were sacrificed by neck removal. The spleen or thymus was taken aseptically and sieved through a 200-mesh steel mesh. The Hanks solution containing porridge newborn calf serum was used to prepare a single-cell suspension. After centrifugation at 1500 r/min for 10 min, red blood cells were lysed with 0.01 mol/L Tris-NHCl hypotonic solution for 1 min, and then washed with Hanks liquid centrifugation. Finally, the cells were suspended in RPMI1640 containing 10% fresh calf serum for complete culture Solution (containing 2 mmol/LL glutamine, 100 mmol/L sodium pyruvate, 1 mmol/L mercaptoethanol, add penicillin 100 U/ml and streptomycin 100 μg/ml just before use), trypan blue count, live For cells>93, kho qhov concentration ntawm spleen hlwb rau X 10 / ml thiab cov concentration ntawm thymocytes rau × 10 degree / ml

1.5 Lymphocyte proliferation cov tshuaj tiv thaiv:Raws li txoj kev Mosmanmn [4] Kev Txhim Kho: 96-zoo cell kab lis kev cai phaj, ntxiv 10/ml spleen cell suspension los yog dej 10/ml thymocyte ncua kev kawm ntawv 100M1 thiab txawv concentrations ntawm cov tshuaj 10041 nyob rau hauv txhua lub qhov dej. Loj hlob hauv CO2.37C incubator rau 72 teev. 4 teev ua ntej qhov kawg ntawm kab lis kev cai, ntxiv 8μ1 ntawm MTT (5 mg / ml) rau txhua qhov dej. Thaum kawg ntawm kab lis kev cai, aspirate 150"1 ntawm cov supernatant los ntawm txhua qhov dej, thiab ntxiv 0.5N isopropanol hydrochloride 150M1. Maj mam co ntawm lub micro shaker kom yaj tag nrho cov crystals xiav. Ntsuas OD570 ntawm tus nyeem ntawv microplate. Teem txhua qhov kev sim. taw tes ua peb lub qhov dej rov ua dua.

1.6 Kev txiav txim siab ntawm lub zog ntawm lub voj voog ntawm tes [5]:

{{0}}zoo cell kab lis kev cai phaj, ntxiv 4 10 degree /ml thymocyte ncua kev kawm ntawv 1ml rau txhua lub qhov dej, ntxiv ntau yam reagents thiab polysaccharide tov rau 2ml nyob rau hauv muaj zog CO, 37 incubators Tom qab culturing rau 72h, centrifuge ntawm 1500r/min rau 15min, muab pov tseg cov supernatant, ntxiv 7 ethanol los kho cov hlwb precipitated, khaws cia ntawm 4C rau standby test, ntxuav lub hlwb ob zaug nrog PBS, centrifuge ntawm 1500r/min rau 15min, pov tseg cov supernatant, thiab ntxiv Tom qab 0.5ml0.1 RNaseA tau zom ntawm 4C rau 30min, 50ug / ml bromoacetate tau stained rau 30min ntawm 4C, thiab cov fluorescence siv ntawm cov qauv ntawm tes tau ntsuas ntawm FACScan flow cytometer. 10,000 cov ntsiab lus ntsuas ntawm tes tau sau rau txhua tus qauv, thiab cov ntaub ntawv siv Mul tricycle software tsom xam.

1.7 Kev txiav txim siab ntawm intracellular calcium ion concentration [6]

Noj nas thymocytes ib yam li saum toj no. Tshem tawm hauv qhov tsis tiav RPMIl640 kab lis kev cai nruab nrab thiab kho cov cell concentration rau × 10' / ml. Ntxiv 1 ml ntawm thymocyte ncua kev kawm ntawv thiab 1 μM rau txhua lub raj sim Fluo-3AM staining tov 5μ1, 2 feem pua ​​F-275μ1, tom qab incubation ntawm 37 rau 30 min, ntxuav ob zaug nrog tsis tiav RPM1640, 1500r / min, centrifuge rau 10 min, muab pov tseg lub supernatant, thiab ntxiv ntau qhov sib txawv ntawm polysaccharides thiab polysaccharides rau cov hlwb precipitated Tom qab ConA, qhov fluorescence siv tau ntsuas ntawm FACScan flow cytometer. Cov ntaub ntawv ntawm 10,{16}} cov ntsiab lus ntsuas ntawm tes tau sau rau txhua tus qauv thiab tshuaj xyuas los ntawm CELLQUEST software. 1.8 Cov txheej txheem txheeb cais tau siv rau kev txheeb xyuas qhov sib txawv, thiab lub voj voog ntawm tes thiab kev ntsuas calcium ion tau txheeb xyuas los ntawm software ntawm cov flow cytometer.

Cistanche Polysaccharide los ntawm cistanche deserticola

2 Kev tshwm sim

2.1 Cistanche Polysaccharide hauv vitro inhibits kev loj hlob ntawm nas thymic lymphocytes los ntawm ISO DEX TNF

Cov -adrenergic receptor agonist isoproterenol (ISO) thiab chav kawm ntawm adrenal cortex hormone dexamethasone (DEX) inhibit lymphocyte proliferation CDPS (Cistanche Polysaccharide) ntawm qhov siab dua (75 125ug/ml) tuaj yeem tiv thaiv cov nyhuv inhibitory ntawm ISO DEX ntawm lymphocyte proliferation (P<0.01) as="" shown="" in="" figure="" 1.="" tumor="" necrosis="" factor="" (tnf="" β)="" on="" lymphocytes="" at="" high="" concentration="" cdps="">Cistanche Polysaccharide) (125ug / ml) ntawm qhov siab dua tuaj yeem tiv thaiv cov nyhuv inhibitory ntawm TNF, thiab kev sib koom ua ke txhawb kev loj hlob ntawm lymphocytes nrog qis concentration ntawm TNF (Daim duab 2)

2.2 Cistanche Polysaccharide txoj cai ntawm thymus lymphocyte voj voog

CDPS (CistanchePolysaccharide) 25ug/ml 100ug/ml 200#g/ml peb qhov concentrations tau ua rau nas thymic lymphocytes rau 72 teev, cov ntsiab lus DNA hauv cov hlwb raug ntsuas los ntawm FACS, thiab qhov ntsuas kev loj hlob (PI) tau suav raws li tus qauv. Cov txiaj ntsig tau pom tias ob qho tib si 100ug / ml thiab 200ug / ml tuaj yeem ua rau cov hlwb nce ntxiv hauv theem faib, thiab qhov kev loj hlob index (68.0 feem pua, 56.6) ntawm ob qho tib si yog siab dua li pawg tswj hwm (44.6), thiab 200ug / ml. pawg yog me ntsis qis dua 100ug / ml pawg vim hais tias, ntawm qhov concentration ntawm 200ug / ml, nws cov nyhuv ntawm cell proliferation tau ua ib tug downward trend [7]. Tus nqi PI (47.5 feem pua) ntawm 25ug / ml pawg tsis tau siab dua li ntawm pawg tswj hwm.

2.3 Cov nyhuv ntawm Cistanche Polysaccharide ntawm calcium ion concentration hauv nas thymocytes

Cov txiaj ntsig kev sim qhia tau hais tias muaj ntau dua (100 ug / ml) ntawm CDPS (Cistanche Polysaccharide) tuaj yeem txhawb nqa qhov nce hauv calcium ion concentration hauv nas thymocytes Piv nrog rau pawg tswj hwm, muaj qhov sib txawv tseem ceeb (P<0.01), and="" there="" is="" a="" certain="" effect="" relationship="" with="" the="" action="" time="" (figure="">

3 Kev sib tham

Cistanche, feem ntau hu ua"dej ginseng", yog ib tug qhuav, scaly fleshy qia ntawmCistanche.Cistanchemuaj lub luag haujlwm ntawm nourishing raum yang, nourishing essence, ntshav thiab hnyuv. Nws yog ib hom kev zoo siab ntawm pituitary-adrenal cortex los yog muaj cov tshuaj hormones zoo ib yam li adrenal cortex. Cov tshuaj suav muaj txiaj ntsig uas tuaj yeem tswj tau covtiv thaiv kab mobmuaj nuj nqintawm lub cev. Peb cov kev tshawb fawb yav dhau los qhia tias CDPS (Cistanche Polysaccharide) hauv vitro tuaj yeem txhawb nqa kev loj hlob ntawm thymic T lymphocytes uas tsis tau qhib lossis qhib los ntawm ConA PHA. Ua ke nrog ConA PHA, muaj ob hom T cell stimulator, peb ntseeg tias CDPS (Cistanche Polysaccharide) yog Badjuvant rau T hlwb, thiab peb kuj pom tias CDPS (Cistanche Polysaccharide) tuaj yeem ua rau muaj qhov tso tawm ntawm nas splenic lymphocytes ntawm ntau dua [8]

Qee cov kev tshawb fawb tau pom tias recombinant TNF tuaj yeem ua rau apoptosis ntawm ib txwm nas thiab tib neeg T lymphocyte pawg hauv vitro, thiab cov nyhuv no tuaj yeem cuam tshuam los ntawm IIL-2 IL-1 IFN thiab IIL-4 nrog dexamethasone (DEX) Nws tuaj yeem inhibit lymphocyte proliferation los ntawm inhibiting lymphocyte qhia thiab tso tawm ntawm IL-2 thiab down-regulating zus tau tej cov high-affinity IL-2R. Qhov kev sim no pom tias muaj ntau dua ntawm CDPS (CistanchePolysaccharide) tuaj yeem tiv thaiv cov teebmeem ntawm qhov siab ntawm TN F ntawm lymphocyte proliferation. Inhibition, synergistic nrog qis concentration ntawm TNF los txhawb kev loj hlob ntawm lymphocytes, thiab tuaj yeem tiv thaiv cov nyhuv inhibitory ntawm ISQ DEX ntawm lymphocyte proliferation. Cov teebmeem no yog los ntawm CDPS (CistanchePolysaccharide) los txhawb kev loj hlob ntawm T lymphocytes thiab yog autocrine los ntawm T cells IL-2 thiab lwm yam cytokines kom tiav.

Nyob rau hauv xyoo tas los no, muaj ntau txoj kev tshawb fawb txog kev siv tshuaj tiv thaiv kab mob ntawm cov nroj tsuag polysaccharides, tab sis ob peb txoj kev tshawb fawb ntawmtiv thaiv kab mobkev tswj mechanism ntawm polysaccharides, tshwj xeeb tshaj yog cov teeb liab transduction txoj kev ntawm polysaccharides ua rau lymphocytes nrog Ca2 raws li tus thib ob tub txib. Tsis muaj ntawv ceeb toom. Qhov kev sim no tau tshawb fawb ua ntej hauv cheeb tsam no. Intracellular signaling molecules xws li cAM R Ca", IP3 DG cGMP, thiab lwm yam, ntawm cov Ca" muaj qhov tseem ceeb ntawm lub cev muaj zog ntawm cov hlwb. Qhov nce hauv intracellular Ca² concentration yog ib qho tseem ceeb tshwm sim nyob rau hauv thaum ntxov theem ntawm lymphocyte activation thiab proliferation. Cov txheej txheem no feem ntau tshwm sim nyob rau hauv thawj kaum tawm vib nas this mus rau feeb thaum lub hlwb tau qhib los ntawm stimulators. Txoj kev classic ntawm T cell activation yog phosphatidylinositol (PIB) los metabolize phosphatidyl leeg. Cov dej cawv metabolite 1,4.5-inositol triphosphate (IP3) tuaj yeem txhawb kev tso tawm Ca" khaws cia rau hauv cov mesh sab hauv los ntawm kev khi rau cov khoom mesh sab hauv, nce qib ntawm intracellular Ca2, thiab cov concentration ntawm intracellular Ca. * nce ntxiv. Siab tuaj yeem txhawb kev tsim cov NF-AT (nudeic yam tseem ceeb ntawm activated T cell), tom kawg muaj qhov cuam tshuam los txhawb kev qhia ntawm IIL-2 noob. ) ntawm CDPS (CistanchePolysaccharide) muaj txiaj ntsig zoo rau 5 10 min Lub Sijhawm muaj qhov cuam tshuam loj rau kev nce siab ntawm Ca* hauv nas thymocytes, thiab muaj kev sib koom ua ke nrog ConA. Yog li ntawd, peb xav tias CDPS (Cistanche Polysaccharide) txhawb kev tso tawm calcium hauv nas thymic lymphocytes kom nce cov calcium ion concentration hauv cov cell. Qhov kev nce ntxiv txhawb qhov kev qhia ntawm IL-2 noob thiab ua rau kev loj hlob ntawm T hlwb, uas yog ib qho ntawm cov theoretical foundations ntawmCistanchePolysaccharidestswj cov nyhuv ntawm lub cev

Cistanche Polysaccharide

Cov ntaub ntawv

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