Ntu 1: Cistanche Tubulosa Phenylethanoid Glycosides Induce Apoptosis Ntawm Hepatocellular Carcinoma Cells Thiab Txhim Kho Cov nyhuv Antitumor

Hu rau: ali.ma@wecistanche.com

Pengfei Yuan, BSc1, Changshuang Fu, MSc1, Yi Yang, PhD1, Aipire Adila, PhD1, Fangfang Zhou, MSc1, Xianxian Wei, MSc1, Weilan Wang, PhD1, Jie Lv, PhD1, Yijie Li, PhD1, Lijie Xia, PhD1, thiab Jinyao Li, PhD1

Abstract

Cistanche tubulosa yog hom tshuaj suav tshuaj ntsuab thiab siv ntau yam kev ua haujlwm lom neeg. Cov kev tshawb fawb yav dhau los tau pom tiasCistanche tubulosa phenylethanoid glycosides(CTPG) muaj cov tshuaj tiv thaiv kab mob ntawm ntau hom qog hlwb. Txawm li cas los xij, cov teebmeem antitumor ntawm CTPG ntawm HepG2 thiab BEL-7404 hepatocellular carcinoma (HCC) hlwb tseem tsis tau paub. Peb txoj kev tshawb fawb tau pom tias CTPG cuam tshuam qhov kev loj hlob ntawm HepG2 thiab BEL-7404 cov hlwb los ntawm kev cuam tshuam ntawm lub voj voog ntawm tes thiab apoptosis, uas tau cuam tshuam nrog kev ua haujlwm ntawm MAPK txoj hauv kev uas muaj kev tswj hwm phosphorylation ntawm p38, JNK, thiab ERK1/2 thiab mitochondrial-dependent txoj kev uas muaj peev xwm txo tau ntawm mitochondrial membrane. Kev tso tawm ntawm cytochrome c thiab cleavage ntawm caspase-3, -7, -9, thiab PARP tau nce ntxiv los ntawm kev kho CTPG. Tsis tas li ntawd, CTPG cuam tshuam kev tsiv teb tsaws ntawm HepG2 los ntawm kev txo cov qib ntawm matrix metalloproteinase-2 thiab vascular endothelial kev loj hlob yam. Interestingly, CTPG tsis yog tsuas yog txhim kho kev loj hlob ntawm splenocytes tab sis kuj txo cov apoptosis ntawm splenocytes induced los ntawm cisplatin. Hauv H22 qog nas qauv, CTPG ua ke nrog cisplatin ntxiv inhibited kev loj hlob ntawm H22 hlwb thiab txo cov kev mob tshwm sim ntawm cisplatin. Noj ua ke, CTPG inhibited kev loj hlob ntawm HCC los ntawm ncaj qha antitumor nyhuv thiab indirect immunoenhancement nyhuv, thiab txhim kho cov nyhuv antitumor ntawm cisplatin.

Cistanche tubulosa phenylethanoid glycosides, apoptosis, MAPK txoj kev, mitochondria-dependent txoj kev, cisplatin, kev tiv thaiv kab mob

Cistanche muaj cov nyhuv tiv thaiv qog.

Nyem rau Cistanche cov khoom thiab Cistanches

Taw qhia

Mob qog noj ntshav tau kwv yees tias yog thib rau feem ntau kuaj mob qog noj ntshav thiab thib plaub ua rau mob qog noj ntshav thoob ntiaj teb xyoo 2018, nrog rau kwv yees li 841 000 tus neeg mob tshiab thiab 782000 tus neeg tuag txhua xyoo hauv South-Eastern Asia (Mongolia, Cambodia, thiab Nyab Laj) .1 Hauv Suav teb, mob qog noj ntshav yog qhov thib peb ua rau mob qog noj ntshav hauv xyoo 2015.2 Ntau tshaj 90 feem pua ​​​​ntawm cov qog nqaij hlav hauv siab yog hepatocellular carcinoma (HCC) thoob plaws ntiaj teb. Tam sim no, hepatectomy, daim siab hloov pauv, thiab percutaneous ablation yog txoj hauv kev tseem ceeb rau kev kho HCC. Hmoov tsis zoo, cov kev kho mob no muaj txiaj ntsig zoo rau 30 feem pua ​​​​ntawm cov neeg mob tau kuaj pom tias muaj mob qog noj ntshav thaum ntxov, thaum cov neeg mob tau kuaj pom tias muaj mob qog noj ntshav siab (suav txog 40 feem pua) yuav tsum tau cia siab rau kev kho mob palliative kom ntev lawv txoj sia nyob.3,4 Sorafenib ua ke nrog mob siab. Arterial chemoembolism yog ib qho tseem ceeb thiab feem ntau palliative therapy rau cov neeg mob feem ntau nrog HCC siab nyob rau hauv cheeb tsam Asia-Pacific.5 Sorafenib, tau pom zoo los ntawm FDA hauv 2006 rau kev kho mob cancer siab heev, yog ntau yam kinase inhibitor uas inhibits qog cell proliferation thiab vascular. ntau lawm thiab txhawb cov qog cell apoptosis. Sorafenib txuas ntxiv rau tus neeg mob lub sijhawm ciaj sia los ntawm 3 mus rau 5 lub hlis tab sis muaj cov kev mob tshwm sim loj heev thiab muaj feem cuam tshuam nrog qhov tshwm sim ntawm cov tshuaj tiv thaiv.6 Yog li ntawd, nws tseem ceeb heev los tsim cov tshuaj tshiab lossis cov tswv yim rau HCC.

Cov tshuaj suav tshuaj suav tshuaj (TCM) ib leeg lossis ua ke nrog lwm cov tswv yim tau siv los kho HCC thiab tau pom cov txiaj ntsig kho mob nrog rau lub sijhawm muaj sia nyob ntev, txhim kho lub neej zoo, txo qis kev phiv, thiab lwm yam.7,8Cistancheyog TCM uas muaj phenylethanoid glycosides (PhGs), iridoids, lignin, thiab polysaccharides uas muaj ntau yam kev ua haujlwm lom neeg, xws li tiv thaiv oxidation, tiv thaiv kab mob, tiv thaiv kev laus, txhim kho kev nco, thiab kev tiv thaiv kab mob.9 PhGs tau raug suav tias yog Cov khoom tseem ceeb ntawm Cistanche, thiab muaj ntau lub zog suav nrog kev tiv thaiv oxidation, tiv thaiv kab mob, tiv thaiv apoptosis, hepatoprotection, thiab neuroprotection.10-12 Peb pab pawg tau tshaj tawm tiasCistanchetubulosa phenylethanoid glycosides(CTPG) tuaj yeem cuam tshuam kev loj hlob ntawm melanoma B16-F10 hlwb, esophageal carcinoma Eca-109 hlwb, thiab HCC H22 hlwb los ntawm extrinsic lossis intrinsic signaling txoj hauv vitro lossis hauv vivo; Tsis tas li ntawd, CTPG muaj cov nyhuv immunostimulatory. 13-15

Hauv txoj kev tshawb no, peb tau tshawb xyuas cov nyhuv antitumor thiab cov txheej txheem ntawm CTPG ntawm HepG2 thiab BEL-7404 cov hlwb hauv vitro, tshuaj xyuas cov haujlwm immunomodulatory ntawm CTPG hauv vitro thiab hauv vivo, thiab tau soj ntsuam ntxiv cov txiaj ntsig kho mob ntawm CTPG ua ke nrog cov tshuaj kho mob. tshuaj cisplatin ntawm HCC H22 qog nas hauv vivo. Peb pom tias CTPG tuaj yeem cuam tshuam kev loj hlob ntawm HepG2 thiab BEL-7404 hlwb los ntawm mitochondria-dependent apoptosis thiab MAPK signaling pathway. CTPG cuam tshuam qhov kev tsiv teb tsaws ntawm HepG2 hlwb los ntawm kev txo cov qib ntawm matrix metalloproteinase-2 (MMP-2) ​​thiab vascular endothelial growth factor (VEGF). Tsis tas li ntawd, CTPG txhawb nqa kev loj hlob thiab ua kom lub cev tiv thaiv kab mob thiab txhim kho kev tiv thaiv ntawm nas. Qhov tseem ceeb, CTPG ua ke nrog cisplatin tuaj yeem cuam tshuam ntxiv kev loj hlob ntawm H22 hlwb hauv vivo thiab txo cov kev mob tshwm sim ntawm cisplatin.

Cistanche txhim khu kev tiv thaiv.

Cov ntaub ntawv thiab cov txheej txheem

Tsiaj

Kwv yees li 6 mus rau 8 lub lis piam txiv neej BALB / c, Kunming nas, thiab poj niam C57BL / 6 nas tau yuav los ntawm Animal Laboratory Center, Xinjiang Medical University (Urumqi, Xinjiang, Tuam Tshoj) thiab nyob hauv ib qho chaw tsiaj ntawm Xinjiang University nrog chav sov. (RT) ntawm 25 ± 3 degree, thiab 12/12 teev lub teeb-tsaus.

Cell Kab thiab Cell Culture

Lub murine H22 hlwb tau yuav los ntawm Procell Life Science & Technology Co., Ltd. (Wuhan, Hubei, Tuam Tshoj) thiab tib neeg HCC HepG2 thiab BEL-7404 hlwb tau txais los ntawm Xinjiang Key Laboratory of Biological Resources thiab Genetic Engineering, Xinjiang University (Urumqi, Xinjiang, Tuam Tshoj) thiab kab lis kev cai hauv RPMI 1640 nruab nrab (Gibco, USA) los yog Dulbecco's modified Eagle's medium (DMEM) (Gibco) muaj 10 feem pua ​​​​ntawm cov cua sov-inactivated fetal bovine serum (MRC, Tuam Tshoj), 1 feem pua ​​L- glutamine (100mM), 100U/mL penicillin, thiab 100ug/mL streptomycin ntawm 37 degree nyob rau hauv ib tug humidified huab cua ntawm 5 feem pua ​​CO2.

High-Performance Liquid Chromatography (HPLC)

Cov ntsiab lus tseem ceeb ntawm CTPG (Upbio Tech Co., Ltd., Shanghai, Suav teb) tau tsim nyog thiab ntsuas los ntawm HPLC raws li tau hais dhau los.13 A ZORBAX SB-C18 Kem (250 × 4.6mm; 5μm) tau siv thiab theem mobile muaj 0.2 feem pua ​​formic acid tov thiab methanol nrog ib tug gradient los ntawm 23 feem pua ​​mus rau 31 feem pua. Tag nrho cov qauv 10μL tau txhaj tshuaj thiab kuaj pom ntawm 330 nm. Cov qauv echinacoside thiab acteoside (Yuanye, Shanghai, Tuam Tshoj) tau siv los tshuaj xyuas cov khoom ntawm CTPG.

echinacoside cov tshuaj

Kev tsom xam ntawm Cell Viability

Cov teebmeem antitumor ntawm CTPG ntawm HepG2 thiab BEL-7404 hlwb raug soj ntsuam siv MTT (3-(4, 5-dimethyl-2-}thiazolyl)-2, {{7 }}diphenyl-2-H-tetrazolium bromide). HepG2 thiab BEL{11}} hlwb tau plated rau hauv 96- cov phiaj zoo (5 × 104 lub hlwb / lub qhov dej) thiab kho nrog 0, 200, 400, thiab 600 ug / mL CTPG rau 24 thiab 48 teev, feem, tom qab 24 teev ntawm incubation ntawm 37 degree. Txog 35 ug / mL cisplatin (Yuanye) tau siv los ua kev tswj hwm zoo. Tom qab ntawd 100 μL MTT (0.5mg / mL, diluted los ntawm nruab nrab yam tsis muaj FBS nruab nrab) tau ntxiv rau txhua qhov dej thiab kab lis kev cai rau 3 teev ntawm 37 degree thiab 5 feem pua ​​CO2. Tom qab incubation, cov phaj tau centrifuged ntawm 1200 rpm rau 7 feeb, qhov nruab nrab raug tshem tawm thiab 200 ug / mL DMSO tau ntxiv rau txhua qhov dej kom yaj cov formazan crystals. Tus nqi OD490 tau ntsuas los ntawm 96-zoo microplate nyeem ntawv (Bio-Rad Laboratories, CA, USA). Txhawm rau ntsuas qhov cuam tshuam ntawm CTPG ntawm splenocytes, cov hlwb raug cais tawm ntawm C57BL / 6 nas thiab plated rau hauv 96- cov phaj zoo ntawm qhov ntom ntawm 1 × 105 hlwb / zoo. Splenocytes tau kho nrog 0, 200, 400, thiab 600 ug / mL rau 24, 48, thiab 72 teev, feem. Lub cell viability raug xam raws li cov qauv hauv qab no: Cell viability (%)=(ODtreated/ODuntreated) × 100 feem pua ​​.

Kev txheeb xyuas ntawm Ki-67

Kev kuaj pom ntawm Ki{{0}} tau ua tiav raws li peb txoj kev tshawb fawb yav dhau los.16 Hauv luv luv, BEL-7404 cov hlwb raug kho nrog ntau qhov sib txawv (0, 200, 400, thiab 600ug / mL) ntawm CTPG los yog cisplatin (35 ug / mL). Tom qab 24 teev, cov hlwb raug sau thiab ntxuav nrog PBS, tom qab ntawd kho thiab permeabilized nrog Foxp3 Staining Buffer Set (eBioscience, USA) raws li cov chaw tsim khoom cov lus qhia. Intracellular staining tau ua tiav siv FITC conjugated Ki{10}} antibody (BD Biosciences, San Jose, CA, USA) rau 15 feeb ntawm RT. Cov qauv no tau txheeb xyuas los ntawm kev ntws cytometry (BD FACSCalibur, CA, USA).

Kev soj ntsuam ntawm Cell Apoptosis thiab Cell Cycle

HepG2 thiab BEL-7404 cov noob tau cog ntawm qhov ceev ntawm 2.5 × 105 hlwb / tais thiab incubated ntawm 37 degree thaum hmo ntuj. Cells tau trypsinized thiab sau los ntawm centrifugation tom qab kev kho mob nrog ntau yam ntawm CTPG los yog pretreated nrog caspase inhibitor (Z-VAD-FMK) los yog caspase -3 inhibitor (Ac-DEVD-CHO) (Beyotime, Tuam Tshoj) rau 2 teev ua ntej Kev kho CTPG. Tom qab 24 teev, apoptosis tau kuaj pom los ntawm kev ntws cytometry. Nyob rau hauv luv luv, lub hlwb sau tau ntxuav nrog txias PBS (Gibco) thiab resuspended nyob rau hauv annexin-binding buffer nrog 2.5μL Annexin V-FITC thiab 5μL PI-PE Staining Solution (Solarbio, Beijing, Tuam Tshoj), thiab ces hlwb tau incubated ntawm RT. hauv qhov tsaus ntuj rau 15 feeb. Rau kev tsom xam ntawm lub voj voog ntawm tes, cov hlwb tau sau tom qab kev kho CTPG thiab tsau rau hauv txias 70 feem pua ​​​​ethanol ntawm 4 degree rau 30 feeb. Cells raug stained nrog PI (BD Biosciences) nyob rau hauv qhov tsaus ntuj rau 30 feeb. Cov qauv raug txheeb xyuas los ntawm kev ntsuas cytometer (BD FACSCalibur). Kev nthuav qhia theem ntawm cell apoptosis thiab lub voj voog ntsig txog cov proteins tau kuaj pom los ntawm Western blot.

Western Blot

Western blot tau ua tiav raws li peb txoj kev tshawb fawb dhau los.17HCC tau kho nrog CTPG rau 24 teev. Tom qab ntxuav nrog dej khov-txias PBS ob zaug, tag nrho cov adherent thiab floating hlwb raug sau thiab lysed nyob rau hauv RIPA Lysis Buffer (Beijing ComWin Biotech Co., Ltd) rau 20feeb ntawm dej khov. Tom qab centrifugation ntawm 12000rpm 4 degree rau 10mins, protein concentration tau txiav txim siab siv Bicinchoninic Acid Assay Kit (Thermo Fisher Scientific, USA) raws li cov chaw tsim khoom cov lus qhia. Tib qhov concentration ntawm cov protein tau sib cais los ntawm 12 feem pua ​​SDS-PAGE thiab xa mus rau PVDF daim nyias nyias. Tom qab ntxuav nrog PBST buffer (PBS nrog 0.05 feem pua ​​Tween-20), cov membranes tau thaiv nrog 5 feem pua ​​​​tsis muaj roj mis nyuj ntawm 37 degree rau 1 teev thiab tom qab ntawd incubated nrog cov thawj cov tshuaj tiv thaiv (Cell Signaling Technology, MA, USA) Nyob rau tib lub sij hawm zoo dilutions thaum hmo ntuj ntawm 4 degree. Tom qab ntxuav 3 zaug nrog PBST, daim nyias nyias tau tsim nrog HRP-conjugated Secondary antibodies (eBioscience) rau 2 teev ntawm 37 degree. Cov hom phiaj proteins tau kuaj pom siv ECL cov khoom kuaj (Beyotime). Grayscale scanning cov ntaub ntawv tau txais los ntawm Duab J.

Kev soj ntsuam ntawm Mitochondrial Membrane Potential (Δψm)

Δψm tau txiav txim los ntawm membrane-permeable JC-1 dye (Beyotime). Luv luv, HepG2 thiab BEL-7404 hlwb tau kho nrog ntau qhov sib txawv ntawm CTPG (0, 200, 400, thiab 600ug/mL) rau 24 teev. Txhua lub hlwb tau sau thiab ntxuav nrog JC-1 ntxuav tsis. Cells raug stained nrog JC-1 fluorescent sojntsuam raws li cov chaw tsim khoom cov lus qhia rau 20 feeb ntawm RT. Tom qab ntxuav nrog PBS ob zaug, tag nrho cov qauv tau soj ntsuam los ntawm cov cytometry ntws (BD FACSCalibur).

Hoechst 33342 Staining

Hoechst 33342 staining tau ua tiav raws li peb txoj kev tshawb fawb yav dhau los.16 Cov kev hloov morphological ntawm nuclei raug tshuaj xyuas siv cov membrane-permeable DNA-binding dye Hoechst 33342 (Beyotime). Luv luv, cov hlwb raug inoculated nyob rau hauv ib tug 6-zoo phaj ntawm qhov concentration ntawm 1 × 105 hlwb/zoo nyob rau hauv 2mL nruab nrab. Thaum nce mus txog 70 feem pua ​​​​rau 80 feem pua ​​​​ntawm qhov sib xyaw, cov hlwb tau kho nrog 200, 400, thiab 600ug / mL ntawm CTPG lossis cisplatin rau 24 teev. Cov hlwb raug ntxuav nrog PBS thiab kho nrog 4 feem pua ​​​​cov dej khov-txias paraformaldehyde ntawm 4 degree rau 10 feeb. Tom qab ntxuav nrog PBS, cov hlwb tau stained nrog Hoechst 33342 ntawm 4 degree rau 10 feeb. Cov qauv tau pom los ntawm fluorescence inverted microscope (Nikon Eclipse Ti-E, Nyiv).

cistanche extract

Migration Assay

HCC cell migration tau kuaj pom los ntawm kev tshuaj xyuas qhov txhab. Nyob rau hauv luv luv, HepG2 hlwb (2 × 104/well) tau noob nyob rau hauv ib tug 24- zoo phaj. Tom qab ncav cuag 8{16}} feem pua ​​​​ntawm qhov sib txuam, qhov nruab nrab ntawm txhua qhov dej tau khawb ib zaug nrog 20μL pipette taub. Tom qab ntxuav nrog PBS, cov hlwb raug kho nrog cisplatin (35ug / mL) lossis ntau qhov sib txawv (0, 200, 400, thiab 600ug / mL) ntawm CTPG ntawm 37 degree. Tom qab 24 teev, cov duab ntawm txhua tus qauv raug coj mus rau hauv lub tshuab tsom iav (Nikon Eclipse Ti-E). Qhov nruab nrab ntawm kev tsiv teb tsaws ntawm tes tau txheeb xyuas los ntawm Duab J. Qhov feem pua ​​​​ntawm qhov txhab kho tau raug xam los ntawm qhov sib npaug: qhov txhab kho (%)=(1-kos thaj tsam ntawm lub sijhawm taw tes / kos thaj tsam ntawm 0hours) × 100 feem pua. Kev nthuav qhia theem ntawm cell migration-related proteins MMP-2 thiab VEGF tau kuaj pom los ntawm Western blot.

Proliferation thiab Apoptosis ntawm Splenocytes

Rau kev soj ntsuam kev loj hlob, splenocytes raug cais tawm ntawm 3 C57BL / 6 nas thiab stained nrog CFSE (eBioscience). CFSE cov ntawv sau npe tau raug inoculated rau hauv 24-cov phiaj zoo ntawm qhov ntom ntawm 2 × 106 hlwb hauv 1 mL nruab nrab ntawm ib qhov dej thiab kho nrog ntau qhov sib txawv ntawm CTPG (200, 400, thiab 600 ug / mL) lossis ua ke nrog 35 ug. /ml cisplatin rau 72 teev. Flow cytometry tsom xam tau ua tom qab staining nrog CD3-APC thiab CD19-PE (BD Biosciences). Rau kev soj ntsuam apoptosis, splenocytes tau inoculated rau hauv 24- cov phiaj zoo ntawm qhov ntom ntawm 2 × 106 hlwb hauv 1 mL nruab nrab ntawm ib qhov dej thiab kho nrog ntau qhov sib txawv ntawm CTPG (200, 400, thiab 600 ug / mL) lossis ua ke nrog cisplatin rau 24 teev. Flow cytometry tsom xam tau ua tom qab staining nrog 2.5 μL Annexin V-FITC thiab 5 μL PI-PE Solution.

Kev Ntsuam Xyuas Kev Nyab Xeeb thiab Kev Ua Haujlwm Immunostimulatory ntawm CTPG Hauv Vivo

Rau kev ntsuam xyuas hauv vivo immunostimulatory kev ua ub no ntawm CTPG, 6 mus rau 8 lub lis piam txiv neej Kunming nas tau muab faib ua 7 pawg (5 nas / pawg). Kev txhaj tshuaj subcutaneous (sc, 200, 400 mg / kg), txhaj tshuaj intraperitoneal (ip, 200, 400 mg / kg), thiab kev tswj hwm intragastric (ig, 200, 400 mg / kg) tau siv txhua 2 hnub rau tag nrho 7 zaug. , thaum pawg tswj tsis tau txais kev kho mob. Cov nas tau hnyav txhua hnub thiab cov xwm txheej ntawm cov nas tau pom txhua hnub. Tom qab kev kho CTPG, cov kab mob ntawm cov nas tau sib cais thiab hnyav. Cov khoom nruab nrog cev indexes raug xam raws li cov mis: organ index=cev hnyav (mg)/lub cev hnyav (g). Cov splenocytes tau sau, suav, thiab stained nrog anti-CD3-APC, anti-CD19-PE, anti-CD49b FITC lossis anti-CD4-APC, anti-CD{{ 22}}PE, anti-CD8-FITC (BD Biosciences). Cov qauv raug kuaj pom los ntawm kev ntws cytometry (BD FACSCalibur).

Antitumor Efficacy of CTPG Combined with Cisplatin in H22 Tumor-Bearing MiceAbout 6 mus rau 8 lub lis piam txiv neej cov nas BALB/c tau txhaj subcutaneously nrog H22 hlwb (1.0 × 106 ib nas nyob rau hauv 100 μL PBS). Thaum cov qog hlav mus txog kwv yees li 60mm3, cov qog nqaij hlav nas tau muab faib ua 4 pawg (6 nas / pawg) thiab kho nrog cisplatin (4mg / kg), CTPG (400mg / kg), cisplatin (4mg / kg) ntxiv rau CTPG ( 400mg / kg), lossis tsis muaj kev kho mob (tswj pab pawg), feem. Cov nas mob qog tau txhaj tshuaj CTPG nyob rau hnub 5, 7, 9, 11, thiab 13th hnub, feem. Cisplatin tau txhaj tshuaj rau hnub 7 thiab 11th. Cov qog ntim thiab lub cev hnyav tau ntsuas txhua hnub. Cov qog ntim tau suav raws li hauv qab no: Cov qog ntim (V)=a × b2/2, uas a thiab b sawv cev rau txoj kab uas hla ntev tshaj plaws ntawm cov qog ntsuas los ntawm vernier caliper, feem. Hnub tim 20, lub cev thiab cov qog tau raug cais tawm thiab hnyav. Cov splenocytes tau sau, suav, thiab stained nrog anti-CD3-APC thiab anti-CD19-PE lossis anti-CD4-FITC thiab anti-CD8-APC lossis anti-CD11b-PE thiab anti-Gr-1-APC lossis anti-CD4-FITC, anti-CD25-APC, thiab anti-Foxp3-PE (BD Biosciences) . Tom qab ntawd, cov zaus thiab cov naj npawb ntawm cov hlwb tau txheeb xyuas los ntawm kev ntws cytometry (BD FACSCalibur).

Cistanche txhim kho cov nyhuv tiv thaiv qog thiab txhim kho kev tiv thaiv.

Yog xav paub ntxiv, thov nias daim duab.

Kev txheeb cais

Tag nrho cov ntaub ntawv tau nthuav tawm raws li qhov ntsuas ± tus qauv yuam kev ntawm qhov nruab nrab (SEM). Kev txheeb xyuas txheeb cais tau ua los ntawm kev tshuaj xyuas ib / ob txoj kev ntawm kev sib txawv (ANOVA) siv Prism5.0 software. P<.05 was="" considered="" statistically="">

NYEEM NO PART 2