楊麗萍,張廣州,袁菊芳,趙 爽,白杰英,曾 林
(軍事醫(yī)學(xué)科學(xué)院實(shí)驗(yàn)動(dòng)物中心,北京 100071)
研究報(bào)告
D-gal致大鼠急性肝損傷過程中肝卵圓細(xì)胞增殖及遷延
楊麗萍,張廣州,袁菊芳,趙 爽,白杰英,曾 林
(軍事醫(yī)學(xué)科學(xué)院實(shí)驗(yàn)動(dòng)物中心,北京 100071)
目的用D-gal建立大鼠急性肝損傷模型,觀察肝損傷后再生過程中肝卵圓細(xì)胞的增殖和遷延。方法建立大鼠急性肝損傷模型,于第1、3、7和14天分別取肝組織,分別行病理、免疫組織化學(xué),觀察卵圓細(xì)胞的分布遷移情況,并取第7天肝組織進(jìn)行組織電鏡觀察匯管區(qū)新增生細(xì)胞超微結(jié)構(gòu)。結(jié)果病理切片顯示肝細(xì)胞變性壞死程度以第7天和第14天為主,出現(xiàn)新增生的細(xì)胞。免疫組化示隨時(shí)間陽性細(xì)胞明顯增多,分布于匯管區(qū),并向小葉中心遷移,形成大量的膽小管,并有部分向壞死區(qū)遷移。透射電鏡有新生內(nèi)源性細(xì)胞,小于成熟的肝細(xì)胞,細(xì)胞器較少,有細(xì)胞緊密連接,以數(shù)個(gè)細(xì)胞排列成小膽管,與免疫組化一致。結(jié)論在大鼠急性肝損傷時(shí)HOC被活化、增殖,并向肝小葉中心遷移,全程參與了肝再生過程。
急性肝損傷;肝卵圓細(xì)胞;增殖;遷延
干細(xì)胞是一類無限期在體內(nèi)整個(gè)生命活動(dòng)中有自我更新能力的細(xì)胞。在適當(dāng)條件下或給予適當(dāng)信號(hào),干細(xì)胞可分化為具體不同來源的細(xì)胞[1-6]。其中,肝臟干細(xì)胞是近幾年研究的熱點(diǎn),由于其來源于自體而沒有移植后的免疫排斥反應(yīng),是合適的肝臟損傷后肝衰竭的移植物并有望成為一種新的臨床方法。肝臟內(nèi)的成體干細(xì)胞就是肝卵圓細(xì)胞,本實(shí)驗(yàn)探討大鼠急性肝損傷模型中,肝損傷后再生過程中肝卵圓細(xì)胞的增殖和遷延,為肝臟修復(fù)機(jī)制提供可能的機(jī)制。
1.1 材料
Wistar大鼠10只購于軍事醫(yī)院科學(xué)院動(dòng)物所,體重120±20 g。D-胺基半乳糖(D-Gal)購買于重慶醫(yī)科大學(xué),批號(hào):951124。小鼠抗大鼠OV6單克隆抗體(北京中杉金橋生物技術(shù)公司);免疫組化試劑盒(福州邁新生物技術(shù)公司);DMEM/F12(1∶1)、H-DMEM(美國(guó)Gibco公司)。
1.2 方法
Wistar大鼠10只,雌雄不限,用D-gal腹腔注射,調(diào)整文獻(xiàn)中D-gal劑量,每只大鼠腹腔注射400 mg/kg。分別于注射D-Gal后第l、3、7、14天,將大鼠處死,行病理、免疫組織化學(xué),觀察卵圓細(xì)胞的分布遷移情況,并取第7天肝組織進(jìn)行組織電鏡觀察匯管區(qū)新增生細(xì)胞超微結(jié)構(gòu)。
2.1 病理變化
D-gal腹腔注射24 h常規(guī)病理切片見肝細(xì)胞變性、糖原消失及局灶性炎性浸潤(rùn),部分肝細(xì)胞破裂,門脈區(qū)出現(xiàn)少量炎性細(xì)胞浸潤(rùn)(圖1)。3 d時(shí)肝細(xì)胞變性壞死,炎性細(xì)胞浸潤(rùn),以門管區(qū)顯著。7 d時(shí)匯管區(qū)卵圓形細(xì)胞增多,并出現(xiàn)少量新生的小膽管及肝細(xì)胞(圖2)。14 d時(shí)匯管區(qū)出現(xiàn)大量增生的卵圓形細(xì)胞,并形成較多新生膽管,向肝小葉遷移(圖3)。
2.2 組織免疫組化OV6的表達(dá)
組織免疫組化觀察OV6表達(dá),發(fā)現(xiàn)第1天在匯管區(qū)僅有少量表達(dá)(圖4),第7天在匯管區(qū)陽性細(xì)胞大幅增加,并新生的小膽管明顯較對(duì)照組增多,并開始向肝實(shí)質(zhì)內(nèi)遷移(圖5),第14天上述陽性細(xì)胞廣泛分布于肝小葉中,參與了肝臟的修復(fù)(圖6)。
2.3 肝組織透射電鏡觀察
取第7天大鼠肝組織,透射電鏡結(jié)果發(fā)現(xiàn)匯管區(qū)有較多的新生細(xì)胞,大小約7~15 μm,胞核大,核圓形或卵圓形,胞質(zhì)相對(duì)較少,胞質(zhì)內(nèi)較少細(xì)胞器(圖7)。
肝卵圓細(xì)胞被認(rèn)為是肝臟的干細(xì)胞,在肝臟中數(shù)量極少,但是,當(dāng)肝臟受到損傷時(shí),可以大量的增殖,并能夠修復(fù)部分損傷的肝細(xì)胞,暫時(shí)替代肝臟的功能[7-12]。本實(shí)驗(yàn)對(duì)D-gal造成的急性肝臟損傷時(shí)肝卵圓細(xì)胞的活化增殖進(jìn)行研究,探討肝臟損傷時(shí)可能的干細(xì)胞替代治療途徑。
3.1 肝損傷模型制備
目前,有以下幾類肝損傷模型的建立方法[13-15]:(1)化學(xué)性肝損傷動(dòng)物模型,如四氯化碳性肝損傷;D-氨基半乳糖性肝損傷;醋氨酚性肝損傷。(2)免疫性肝損傷動(dòng)物模型,如卡介菌加脂多糖誘導(dǎo)法;丙酸桿菌或痤瘡丙酸桿菌加脂多糖誘導(dǎo);刀豆蛋白A誘導(dǎo)法。(3)酒精性肝損傷動(dòng)物模型,如急性酒精性肝損傷;慢性酒精性肝損傷。(4)外科手術(shù)動(dòng)物模型,主要有三種:肝缺血模型、部分肝切除模型、全肝切除模型等。在本實(shí)驗(yàn)中,我們選擇了D-gal來作為大鼠肝臟損傷模型,D-gal用法是用生理鹽水配制成100 g/L,并用1 mol/L氫氧化鈉(NaOH)調(diào)節(jié)其pH值至7.10。但造成肝損傷的劑量,國(guó)內(nèi)、外報(bào)告差別較大(100 mg/kg~2300 mg/kg)[16-20],大鼠或小鼠1次性腹腔注射600~900 mg/kg,也有人連續(xù)兩次,間隔24 h,造成急性肝損傷模型,染毒24~48 h后處死動(dòng)物,檢查肝功、病理及脂質(zhì)過氧化等有關(guān)指標(biāo)[21-23]。在繼Dixit、Blitzer等成功建立實(shí)驗(yàn)動(dòng)物鼠和兔的D-gal誘導(dǎo)的肝損傷模型后,有學(xué)者成功制備大動(dòng)物犬的D-gal模型[24-26]。
本實(shí)驗(yàn)在預(yù)實(shí)驗(yàn)的基礎(chǔ)上,發(fā)現(xiàn)給大鼠400 mg/kg的D-gal即可使大鼠的肝臟受到中等程度的損傷,且不會(huì)導(dǎo)致大鼠的死亡,其操作可靠,重復(fù)性較強(qiáng),肝損傷一致性較好。
3.2 肝卵圓細(xì)胞增殖和遷延過程
有研究者為誘導(dǎo)大鼠肝臟卵圓細(xì)胞,連續(xù)給予低濃度的2-乙酰氨基芴(2-AAF)抑制肝細(xì)胞的增生,在處理中期(7~10 d),實(shí)施肝2/3切除術(shù),由于肝細(xì)胞大量丟失,肝細(xì)胞生長(zhǎng)反饋信息缺失導(dǎo)致卵圓細(xì)胞的快速生長(zhǎng)。卵圓細(xì)胞先出現(xiàn)在膽管周圍,然后有些遷移入肝實(shí)質(zhì)。通過病理切片發(fā)現(xiàn),匯管區(qū)有卵圓形細(xì)胞的增殖,隨著實(shí)驗(yàn)的進(jìn)展,卵圓形細(xì)胞向肝小葉內(nèi)遷移,在第14天時(shí),肝卵原細(xì)胞已經(jīng)散布在肝實(shí)質(zhì)內(nèi),代替損傷的肝細(xì)胞修復(fù)肝臟。而免疫組化OV6也證實(shí)了增殖的體積稍小的細(xì)胞是卵圓細(xì)胞。透射電鏡發(fā)現(xiàn)發(fā)現(xiàn)匯管區(qū)有較多的新生細(xì)胞,大小約7~15 μm,胞核大,核圓形或卵圓形,胞質(zhì)相對(duì)較少,胞質(zhì)內(nèi)較少細(xì)胞器。我們的實(shí)驗(yàn)結(jié)果證實(shí),肝卵圓細(xì)胞始終貫穿于肝臟修復(fù)的全過程,可以為急性肝衰竭進(jìn)行暫時(shí)的支持,對(duì)臨床有積極的意義。
肝干細(xì)胞的分裂增生方式被認(rèn)為是一種不對(duì)稱增生,在子代2個(gè)細(xì)胞中,一個(gè)保持干細(xì)胞的特性,另一個(gè)分化成組織細(xì)胞,參與組織損傷修復(fù).在正常肝臟,肝干細(xì)胞處于休眠期,不分裂增生。當(dāng)肝干細(xì)胞被激活后,必須在適宜的微環(huán)境中才能分化成熟。故我們認(rèn)為[27-29],肝臟損傷后修復(fù)的方式可能如下:大鼠損傷肝臟早期就有小部分卵圓細(xì)胞被激活,初步參與了肝臟的修復(fù),但是由于數(shù)量太少,無法完全修復(fù)損傷肝臟,因而,病理表現(xiàn)肝細(xì)胞壞死、炎性細(xì)胞浸潤(rùn)等改變,隨著時(shí)間,激活的卵圓細(xì)胞越來越多,在肝臟微環(huán)境刺激下,開始沿膽小管向肝實(shí)質(zhì)內(nèi)遷移,在遷移過程中得到成熟,分化為成熟的肝細(xì)胞,發(fā)揮了肝細(xì)胞的功能,對(duì)肝臟衰竭提供了暫時(shí)的修復(fù)。為此,有必要進(jìn)一步研究肝卵圓細(xì)胞具體的分化調(diào)控機(jī)制。
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Proliferation and Protraction of Hepatic Oval Cell during Acute Liver Injury Induced by D-gal in Rat
YANG Li-ping,ZHANG Guang-zhou,YUAN Ju-fang,ZHAO Shuang,BAI Jie-ying,ZENG Lin
(Laboratory Animal Center of the Academy of Milifary Medical Science,Beijing 100071,China)
ObjectiveTo establish a rat model with the combination of D-galactosamine(D-GalN),and to observe the protraction and proliferation of oval cells during liver regeneration following hepatic damage。MethodsD-galactosamine was injected to abdominal cavity in the rat so as to duplicate liver injury model.After rats were put to death on 1,3,7 and 14 days respectively,distribution and migration of OV6 positive cells were tested by tissue immunohistochemistry and finally ultrastructure of new proliferating cells was observed by transmission electron microscope in the converge duct。ResultsPathological section and immunohistochemistry results showed that HOCs gradually migrated from hepatic lobule in the portal vein to liver and confluent with normal liver cells board.OV6 positive cells were moderately increased in number by the tissue immunohistochemistry and even developed into a number of gallbladder tubules,some of which crawled into necrosis areas.Moreover,the new cells with less organells,smaller than the mature liver cells in size,lined up to form small bile duct under thetransmission electron microscope,which displayed similarities to the findings by immunohistochemistry。ConclusionHOC can be activated and proliferated following liver damage and it is involved in liver regeneration.
Acute liver injury;Hepatic oval cell;Proliferation;Protraction
R329.25
A
1671-7856(2010)10-0006-04
10.3969/j.issn.1671.7856.2010.10.003
2010-06-12
楊麗萍(1972-),女,助理研究員,主要從事干細(xì)胞與信號(hào)轉(zhuǎn)導(dǎo)基礎(chǔ)研究。E-mail:majxmajc@sohu.com
曾林(1965-),男,研究員,博士,研究方向:實(shí)驗(yàn)動(dòng)物病理學(xué)。E-mail:Zenglin1965@126.com