β-胡蘿卜素對大白鼠初代肝細胞之生存力,脂質(zhì)過氧化及抗氧化酵

β-胡蘿蔔素對大白鼠初代肝細胞之生存力,脂質(zhì)過氧化及抗氧化酵素活性的影響

許多研究結(jié)果顯示β-胡蘿蔔素具有正面的生物功能，如降低脂質(zhì)過

氧化生成以及降低癌細胞的分裂等，故推論β-胡蘿蔔素具有降低

心血管疾病以及降低癌癥發(fā)生的功效。事實上，曾有研究者發(fā)現(xiàn)

β-胡蘿蔔素不具有抗氧化的效果，並且近年來的人體研究亦發(fā)現(xiàn)

添加β-胡蘿蔔素反而增加心血管疾病與肺癌的發(fā)生，為釐清β-胡蘿蔔素

之抗氧化情形，本研究以體外實驗方式觀察β-胡蘿蔔素對大白鼠

初代肝細胞之細胞生存力、抗氧化酵素活性以及脂質(zhì)過氧化的影響

。採用Wistar品系雄

性大白鼠，以兩階段膠原蛋白酉每的方式分離肝細胞，培養(yǎng)細胞4小

時後進行下列三階段實驗。第一，肝細胞在正常的情況之下持續(xù)培養(yǎng)4

、10、16、22、28以及40小時，並在第16小時更換或不更換培養(yǎng)液

，觀察細胞之生存力以及抗氧化酵素活性的變化以決定後續(xù)實驗的

時間點。由本實驗結(jié)果將後續(xù)實驗的觀察時間點定為12小時；第二

，加入0.05~2mMFeCl3，觀察細胞之生存力以及抗氧化酵素活性的變化

以決定後續(xù)實驗之氧化誘導劑的添加濃度。由本實驗結(jié)果決定以0.1

mM

為添加濃度；第三，將β-胡蘿蔔素添加於培養(yǎng)液中，同時添加或

不添加0.1mMFeCl3，培養(yǎng)12小時之後觀察細胞的生存力、脂質(zhì)過氧

化以及抗氧化酵素活性的改變，實驗中加入α-tocopherol、

retinol、canthaxantnin或α-carotene以作比較。三階段實驗的生化分

析：以lactatedehydrogenaseleakage(LDHleakage)作為細胞

生存力的指標；抗氧化酵素則以分析GSH代謝相關(guān)酵素：麩胱甘月太過

氧化酉每(GSHperoxidase)、麩胱甘月太還原酉每(GSH

reductase)以及麩胱甘月太硫轉(zhuǎn)移酉每(GSHS-transferase)活性

；以thiobarbituricacid-reactivesubstances(TBARS)分

析malondialdehyde(MDA)生成。

結(jié)果顯示：FeCl3降低細胞生存力以及抗氧化酵素活性，並且在0.05~0.1

mM之間隨濃度呈現(xiàn)劑量效應(yīng)。在0.1mMFeCl3的存在下同時加入β-

胡蘿蔔素，發(fā)現(xiàn)抗氧化酵素的活性更顯著降低，且細胞的生存力未獲

得改善，相同的實驗以retinol或α-tocopherol代替β-胡蘿蔔素時

發(fā)現(xiàn)細胞生存力明顯上升。在0.1mMFeCl3的存在下加入10-8~10-5Mβ-

胡蘿蔔素，發(fā)現(xiàn)不同濃度對細胞的影響沒有差異。在不添加0.1mMFeCl3

情形下，β-胡蘿蔔素仍會降低抗氧化酵素活性，相同實驗以

canthaxanthin、retinol或α-carotene代替β-胡蘿蔔素發(fā)現(xiàn)抗氧化

酵素活性不受影響。研究顯示β-胡蘿蔔素對MDA生成沒有明顯的抑制

效果。

研究顯示，β-胡蘿蔔素會降低抗氧化酵素活性，亦發(fā)現(xiàn)β-胡蘿蔔素對脂

質(zhì)過氧化沒有明顯的抑制效果。Theeffectsofβ-caroteneonthecellularviability,lipidperoxidationandactivitiesofantioxidativeenzymesinprimaryrathepatocytes

Therearemanyresearchesindicatethatβ-carotenehas

manybiologicalfunctions,suchasreductinglipid

peroxidationanddecreasingmitogenesisofcancer

cells.Becauseoftheseobservations,ithasbeensuggestedthat

peoplewhoconsumemorefruitsandvegetablescontainingβ-

carotenehavesomewhatlowerriskofcancerand

cardiovasculardisease.However,ithasalsobeen

foundthatβ-carotenehasnoeffectonlipidperoxidationand

mayhasadverseeffectontheincidenceoflungcancer

andontheriskofdeathresultingfromlungcancer

andcariovasculardiseases.Inthispresentstudy,I

examinedtheeffectofβ-caroteneonthecellularactivitiesof

anti-oxidativeenzymes,onthecellularviability

andonthelipidperoxidationinprimaryrat

hepatocytes.

Primaryrathepatocyteswerepreparedfrommale,8-week-old

Wistarstrainratsbytwo-stepcollagenase

perfusion.Theisolatedcellswereincubatedin

platingmedium.Afterfour-hourplating,themediumwasremoved

andreplacedbythesamemediumcontainingnofetalbovineserum

(FBS).Thenthreeexperimentalstepswere

followed.Inthefirststep,thecellswere

incubatedinnormalconditionfor4,10,16,22,28and40hours

withorwithoutchangingtheculturemediumat16

hours.Changesofactivitiesoftheantioxidative

enzymesandcellularviabilityweremeasuredtodescribe

timecourseofthecells.12-hourplatingwasseclectedas

appropriateincubationtimeforsubsequent

experiments.Inthesecondstep,cellswereincubated

Theeffectsofβ-caroteneonthecellularviability,lipidperoxidationandactivitiesofantioxidativeenzymesinprimaryrathepatocyteswith0.05~2mMFeCl3for12hours.Changesofactivitiesofthe

antioxidativeenzymes,cellularviabilityandlipidperoxidation

weremeasuredtodescribedoseeffectofFeCl3.

0.1mMwasselectedassuitableconcentrationfor

subsequentexperiments.Inthethirdstep,cellswere

incubatedwithβ-caroteneandwithorwithout0.1mMFeCl3for

12hours.Theeffectofβ-carotenewereobservedby

measuringtheactivitiesoftheantioxidative

enzymes,cellularviabilibtyandthelipidperoxidation.

α-tocopherol,retinol,canthaxanthin

orα-carotenewereadded

inthemediumbythesameprotocolincontrast

withβ-carotene.Lactatede-hydrogense(LDH

leakage)wasanalysedasindexofcellularviability.GSH

peroxidase,GSHreductaseandGSHS-transferaseweremeasuredas

indicesofantioxidativeenzymes.Theanalysisof

thiobarbituricacidreactivesub-stances(TBARS)

isindexofmalondialdehyde(MDA)production.

Theresultsindicatethatcellsincubatedwith0.1mMFeCl3for

12hoursexhibitedreducedthecellularviabilityand

reducedtheactivitiesofanti-oxidativeenzymes.

Addingβ-caroteneinto0.1mMFeCl3treatedcellssigni-

ficantlyreducedtheactivitiesofantioxidativeenzymes.The

effectofvariousconcentrationofβ-caroteneon

cellswasnotsignificantlydifferentfromeachothers

anditisindicatedthattheadditionofβ-carotenedid

notimprovethecellularviability.However,Addingα-tocopherol

orretinolinto0.1mMFeCl3treatedcells

significantlyincreasedcellularviability.

Incorporatingβ-caroteneintononFeCl3treatedcells,the

decreaseofactivitiesofantioxidativeenzymes

wasrevealed.Whencanthaxanthin,retinolorα-carotene

wereappliedinsteadofβ-carotene,theactivitiesofanti-

oxidativeenzymeswerenotdifferentfromcontrolgroup.The

resultindicatesthattheadditionofβ-carotenehadno

eff