孫子健,陳 婧,夏衍珩,劉曉燕,蘇海濱,楊昊臻,許 祥,胡瑾華
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粒細(xì)胞集落刺激因子保護(hù)急性損傷人胎肝細(xì)胞的實(shí)驗(yàn)研究
孫子健,陳 婧,夏衍珩,劉曉燕,蘇海濱,楊昊臻,許 祥,胡瑾華
[摘要]目的 建立D-半乳糖胺(D-galactosamine,D-GalN)對(duì)人胎肝細(xì)胞急性損傷的模型,觀察粒細(xì)胞集落刺激因子(Granulocyte-colony stimulating factor, G-CSF)對(duì)人胎肝細(xì)胞損傷的保護(hù)作用。方法 分別用梯度濃度的D-GalN和不同的作用時(shí)間孵育人胎肝細(xì)胞,用四唑鹽比色法(MTT法)檢測(cè)細(xì)胞活性,以確定最佳的人胎肝細(xì)胞急性損傷造模條件。將胎肝細(xì)胞分為4組進(jìn)行不同處理:第1組為空白對(duì)照組,第2組(G組)用G-CSF處理正常細(xì)胞,第3組(ND組)和第4組(GD組)都用D-GalN進(jìn)行損傷造模,但GD組加入G-CSF作為治療,第3組加入等量的0.9%氯化鈉溶液作為實(shí)驗(yàn)對(duì)照。最后用MTT法和乳酸脫氫酶(LDH)釋放量檢測(cè)各組細(xì)胞活性。結(jié)果 當(dāng)D-GalN濃度為10 mg/ml,作用時(shí)間為12 h時(shí),可以殺傷90%以上的人胎肝細(xì)胞,并且可以保證有足夠的藥物反應(yīng)時(shí)間??瞻讓?duì)照組和G組的細(xì)胞活性差異無統(tǒng)計(jì)學(xué)差異,但GD組細(xì)胞活性明顯高于ND組(P<0.05)。結(jié)論 D-GalN對(duì)人胎肝細(xì)胞急性損傷的造模條件為D-GalN 10 mg/ml作用12 h。G-CSF對(duì)D-GalN造成的人胎肝細(xì)胞急性損傷具有保護(hù)作用。
[關(guān)鍵詞]肝功能衰竭; 人胎肝細(xì)胞; 粒細(xì)胞集落刺激因子
[作者單位] 100039 ,北京大學(xué)解放軍第三〇二醫(yī)院教學(xué)醫(yī)院肝衰竭診療與研究中心(孫子健、夏衍珩、許祥、胡瑾華);100039 北京,解放軍第三〇二醫(yī)院肝衰竭診療與研究中心(陳 婧、劉曉燕、蘇海濱、楊昊臻、胡瑾華)
急性肝衰竭病因復(fù)雜,患者肝功能喪失迅速,病情危急,病死率極高,但缺乏有效治療手段[1]。近年來應(yīng)用粒細(xì)胞集落刺激因子(granulocytecolony stimulating factor, G-CSF)治療肝衰竭已在肝衰竭動(dòng)物模型中得到驗(yàn)證,臨床試驗(yàn)也取得了一定效果[2-4]。傳統(tǒng)觀點(diǎn)認(rèn)為G-CSF主要通過促進(jìn)骨髓干細(xì)胞及肝干細(xì)胞(肝卵圓細(xì)胞)在肝臟內(nèi)的增殖以修復(fù)肝臟[5-6],這一過程體現(xiàn)在肝功能或肝組織的恢復(fù)需要一定的時(shí)間。然而不論是臨床觀察還是動(dòng)物實(shí)驗(yàn)中,我們發(fā)現(xiàn)經(jīng)過G-CSF治療的患者或模型小鼠在治療早期均有顯著的肝功能改善,推測(cè)G-CSF可能通過某種機(jī)制直接保護(hù)肝臟細(xì)胞。本研究設(shè)計(jì)體外實(shí)驗(yàn),用D-半乳糖胺(D-galactosamine, D-GalN)對(duì)人胎肝細(xì)胞造成急性損傷,觀察G-CSF是否對(duì)人胎肝細(xì)胞急性損傷具有保護(hù)治療作用。
1.1材料 人胎肝細(xì)胞由上海東方肝膽外科醫(yī)院贈(zèng)予,在配有5%胎牛血清和1%青霉素-鏈霉素的杜爾伯科改良伊格爾培養(yǎng)基(dulbecco modified eagle medium,DMEM)中貼壁生長(zhǎng),置于37 ℃含95%空氣加5%二氧化碳混合氣體的細(xì)胞培養(yǎng)箱中培養(yǎng)。
1.2人胎肝細(xì)胞急性損傷造模 胎肝細(xì)胞以2×105/孔的密度在12孔板中貼壁穩(wěn)定生長(zhǎng)后,用不含血清的DMEM培養(yǎng)細(xì)胞12 h進(jìn)行饑餓處理。D-GalN用DMEM溶解至濃度為8、9、10和11 mg/ml,以0.4 ml的體積加入各孔,測(cè)試12 h內(nèi)不同濃度藥物對(duì)胎肝細(xì)胞的殺傷力,再選出適宜濃度同法分別孵育細(xì)胞0、4、6、8、10、12和14 h 以確定合適的作用時(shí)間,2種試驗(yàn)均以四唑鹽比色法(MTT法)對(duì)細(xì)胞活性進(jìn)行檢測(cè)。MTT用DMEM溶解后定容至0.5 mg/ml,每孔加入0.2 ml孵育細(xì)胞4 h,吸除MTT液,再每孔加入0.2 ml二甲基亞砜(dimethyl sulphoxide, DMSO)充分溶解細(xì)胞后,從每孔中吸取0.1 ml加入96孔板,用分光光度儀測(cè)A490 nm時(shí)的吸光度。
1.3G-CSF對(duì)胎肝細(xì)胞的保護(hù)作用 如前所述將胎肝細(xì)胞鋪至12孔板,隨機(jī)分為4組:①空白對(duì)照組;② G-CSF組(G組):G-CSF處理正常人胎肝細(xì)胞;③實(shí)驗(yàn)對(duì)照組(ND組):0.9%氯化鈉溶液處理人胎肝細(xì)胞急性損傷模型;④治療組(GD組):G-CSF處理人胎肝細(xì)胞急性損傷模型。對(duì)4組分別做如下處理:首先進(jìn)行饑餓處理,將G-CSF(北京雙鷺?biāo)帢I(yè))用不含血清DMEM配置成10 μg/ml濃度,以0.4 ml體積加入G組和GD組,同時(shí)將相同體積的無血清DMEM加入空白對(duì)照組和ND組,共同孵育12 h。隨后空白對(duì)照組和G組不進(jìn)行處理,而對(duì)ND組及GD組進(jìn)行造模處理(D-GalN濃度由前述實(shí)驗(yàn)獲得),ND組加入0.4 ml溶有濃度為10 mg/ml D-GalN的無血清DMEM(其中混有與GD組內(nèi)G-CSF等體積的0.9%氯化鈉溶液),GD組則加入0.4 ml溶有 D-GalN 和G-CSF的無血清DMEM,其濃度分別為10 mg/ml 和10 μg/ml。以上4組再共同培養(yǎng)12 h,對(duì)細(xì)胞活性進(jìn)行檢測(cè),除了繼續(xù)應(yīng)用上述的MTT法外,同時(shí)抽取細(xì)胞培養(yǎng)液進(jìn)行乳酸脫氫酶(LDH)的濃度測(cè)定。
1.4統(tǒng)計(jì)學(xué)處理 用SPSS19.0軟件進(jìn)行統(tǒng)計(jì)分析。計(jì)量資料呈正態(tài)分布或近似正態(tài)分布,用±s表示。2組間計(jì)量資料比較用成組t檢驗(yàn)(組間方差齊)。P<0.05表示差異有統(tǒng)計(jì)學(xué)意義。
2.1最佳的人胎肝細(xì)胞急性損傷造模條件的確定 分別用梯度濃度的D-GalN孵育人胎肝細(xì)胞12 h,可見當(dāng)濃度達(dá)到10 mg/ml以上時(shí),可殺傷90%以上的胎肝細(xì)胞(表1)。為確定適宜的D-GalN作用時(shí)間,我們又以10 mg/ml的濃度孵育胎肝細(xì)胞,觀察到作用時(shí)間為4 h 時(shí),可殺傷約50%的細(xì)胞,隨著作用時(shí)間的延長(zhǎng),殺傷程度加重,當(dāng)D-GalN孵育12 h,細(xì)胞損傷90%以上(表2)。因此綜合以上情況選擇的造模條件為D-GalN濃度10 mg/ml,作用時(shí)間12 h,這樣既可以劇烈殺傷胎肝細(xì)胞(>90%),又能保證進(jìn)一步檢測(cè)G-CSF保護(hù)作用有足夠的反應(yīng)窗口期。
表1 MTT法檢測(cè)梯度濃度D-GalN對(duì)人胎肝細(xì)胞的殺傷作用(±s)Table 1 Injury effect of D-GalN at gradient concentrations on human fetal hepatocytes detected by MTT method (±s)
表1 MTT法檢測(cè)梯度濃度D-GalN對(duì)人胎肝細(xì)胞的殺傷作用(±s)Table 1 Injury effect of D-GalN at gradient concentrations on human fetal hepatocytes detected by MTT method (±s)
D-GalN 濃度(mg/ml) 0 8 9 10 11 MTT吸光度(標(biāo)準(zhǔn)化OD值) 1.000±0.019 0.299±0.012 0.172±0.037 0.087±0.031 0.073±0.032
表2 MTT法檢測(cè)在濃度為10 mg/ml 的D-GalN不同時(shí)間對(duì)人胎肝細(xì)胞的殺傷作用(±s)Table 2 Injury effect of D-GalN at the concentration of 10 mg/ml on human fetal hepatocytes detected by MTT method at different time points (±s)
表2 MTT法檢測(cè)在濃度為10 mg/ml 的D-GalN不同時(shí)間對(duì)人胎肝細(xì)胞的殺傷作用(±s)Table 2 Injury effect of D-GalN at the concentration of 10 mg/ml on human fetal hepatocytes detected by MTT method at different time points (±s)
作用時(shí)間(h) 0 4 6 8 10 12 14 MTT吸光度(標(biāo)準(zhǔn)化OD值) 1.000±0.157 0.515±0.032 0.323±0.101 0.274±0.007 0.130±0.027 0.095±0.005 0.015±0.002
2.2G-CSF對(duì)D-GalN造成人胎肝細(xì)胞急性損傷保護(hù)作用測(cè)定 用濃度為10 mg/ml 的D-GalN作用12 h后檢測(cè)人胎肝細(xì)胞活性,結(jié)果如下。
2.2.1MTT法檢測(cè)結(jié)果 MTT法檢測(cè)的是活細(xì)胞與試劑的反應(yīng)能力,以吸光度值表示,值愈大代表細(xì)胞存活數(shù)量愈多,檢測(cè)結(jié)果見表3??瞻讓?duì)照組與G組吸光度差異無統(tǒng)計(jì)學(xué)意義(t=1.425, P=0.227);ND組吸光度明顯低于空白對(duì)照組(t=42.581, P=0.000);GD組吸光度明顯高于ND組(t=3.029,P=0.013)。
2.2.2LDH法檢測(cè)結(jié)果 LDH是在細(xì)胞死亡破裂時(shí)釋放出來,因此LDH的值越高表示細(xì)胞死亡量越大。檢測(cè)結(jié)果見表4??瞻讓?duì)照組與G組LDH濃度差異無統(tǒng)計(jì)學(xué)意義(t=0.316,P=0.768),ND組LDH濃度明顯高于空白對(duì)照組(t=10.301,P=0.000),GD組LDH濃度明顯低于ND組(t=6.779,P=0.000)。
表3 MTT法檢測(cè)4組人胎肝細(xì)胞吸光度( 標(biāo)準(zhǔn)化OD值,±s)Table 3 Absorbance of human fetal hepatocytes of the 4 groups detected by MTT method (standard OD,±s)
表3 MTT法檢測(cè)4組人胎肝細(xì)胞吸光度( 標(biāo)準(zhǔn)化OD值,±s)Table 3 Absorbance of human fetal hepatocytes of the 4 groups detected by MTT method (standard OD,±s)
組別 n 吸光度空白對(duì)照組 6 1.000±0.008 G組 6 0.956±0.011 ND組 6 0.164±0.032 GD組 6 0.221±0.033
表4 LDH法檢測(cè)4組人胎肝細(xì)胞LDH濃度(U/L,±s)Table 4 Concentrations of LDH in fetal hepatocytes of the 4 groups detected by LDH method(U/L,±s)
表4 LDH法檢測(cè)4組人胎肝細(xì)胞LDH濃度(U/L,±s)Table 4 Concentrations of LDH in fetal hepatocytes of the 4 groups detected by LDH method(U/L,±s)
組別 n LDH濃度空白對(duì)照組 6 1.0±0.0 G組 6 1.0±0.0 ND組 6 20.7±4.7 GD組 6 4.0±3.8
空白對(duì)照組與G組人胎肝細(xì)胞存活數(shù)量差異無統(tǒng)計(jì)學(xué)意義,提示G-CSF對(duì)人胎肝細(xì)胞無損傷作用;ND組明顯低于空白對(duì)照組,提示造模成功;GD組明顯高于ND組,提示提示G-CSF對(duì)D-GalN所致的人胎肝細(xì)胞損傷具有直接保護(hù)作用。
D-GalN有特異的肝毒性,能夠耗盡肝細(xì)胞內(nèi)的三磷酸尿苷,從而阻斷小分子化合物的合成,繼而引起肝細(xì)胞的凋亡。它的這一特性已廣泛應(yīng)用于肝損傷的動(dòng)物實(shí)驗(yàn)?zāi)P蚚7],我們也選擇D-GalN進(jìn)行造模。人類原代肝細(xì)胞被認(rèn)為是體外研究肝病最理想的實(shí)驗(yàn)對(duì)象,但獲取效率極低且實(shí)驗(yàn)難度高,而人胎肝細(xì)胞因其仍可增殖而易于培養(yǎng),并且已具備一系列肝細(xì)胞基因的表達(dá),已較多地應(yīng)用于肝臟疾病的體外研究[8]。我們首次成功在體外應(yīng)用D-GalN對(duì)人胎肝細(xì)胞制作急性損傷模型,10 mg/ml D-GalN作用于人胎肝細(xì)胞12 h后即可造成90%以上的細(xì)胞損傷,同時(shí)也保證進(jìn)一步驗(yàn)證G-CSF保護(hù)作用所需的反應(yīng)窗口期。這一模型的構(gòu)建,為探究G-CSF對(duì)胎肝細(xì)胞的直接作用提供了便利。MTT法可用于檢測(cè)細(xì)胞活性和藥物毒性,而利用死亡細(xì)胞釋放LDH的量來評(píng)定細(xì)胞存活程度是更為精準(zhǔn)的方法[9]。在判定G-CSF對(duì)胎肝細(xì)胞保護(hù)作用時(shí),我們同時(shí)應(yīng)用了這2種檢測(cè)方法,以提高實(shí)驗(yàn)結(jié)果的準(zhǔn)確性。我們的實(shí)驗(yàn)發(fā)現(xiàn)ND組與GD組相比,無論從MTT法還是LDH法的測(cè)定結(jié)果來看,GD組的細(xì)胞都顯示了更高的細(xì)胞活性,證實(shí)G-CSF在體外可以直接保護(hù)人胎肝細(xì)胞減輕D-GalN的毒害作用,并且此保護(hù)作用相當(dāng)顯著。
關(guān)于G-CSF保護(hù)人胎肝細(xì)胞的機(jī)制,我們考慮一是由于G-CSF可能會(huì)幫助胎肝細(xì)胞增殖來維持較高的細(xì)胞活性,二是G-CSF可能直接保護(hù)胎肝細(xì)胞抵御凋亡壞死。對(duì)于第一種可能,從表3 和4的空白對(duì)照組和G組結(jié)果來看,G-CSF對(duì)胎肝細(xì)胞并無增殖作用。而對(duì)第二種可能,已有研究者在許多腦缺血和心肌梗死的實(shí)驗(yàn)中發(fā)現(xiàn)G-CSF能通過抵抗凋亡來保護(hù)神經(jīng)元和心肌細(xì)胞[10-11]。因此我們?cè)O(shè)想G-CSF對(duì)于人胎肝細(xì)胞的保護(hù)也可能是通過抗凋亡作用實(shí)現(xiàn)的,對(duì)于其具體的分子機(jī)制,以及是否適用于原代肝細(xì)胞和體內(nèi)實(shí)驗(yàn),還需后續(xù)的實(shí)驗(yàn)驗(yàn)證。
【參考文獻(xiàn)】
[1] 聶青和,朱婷. 肝衰竭并發(fā)癥治療新進(jìn)展[J]. 傳染病信息,2014,27(4) : 204-208.
[2] Theocharis SE, Papadimitriou LJ, Retsou ZP, et al. Granulocytecolony stimulating factor administration ameliorates liver regeneration in animal model of fulminant hepatic failure and encephalopathy[J]. Dig Dis Sci, 2003, 48(9):1797-1803.
[3] Duan XZ, Liu FF, Hu JH, et al. Granulocyte-colony stimulating factor therapy improves survival in patients with hepatitis B virus-associated acute-on-chronic liver failure[J]. World J Gastroenterol, 2013, 19(7):1104-1110.
[4] Garg V, Garg H, Khan A, et al. Granulocyte colony-stimulating factor mobilizes CD34+cells and improves survival of patients with acute-on-chronic liver failure[J]. Gastroenterology , 2012, 142(3):505-512 .e501.
[5] Lei Y, Liu Z, Han Q, et al. G-CSF enhanced SDF-1 gradient between bone marrow and liver associated with mobilization of peripheral blood CD34+cells in rats with acute liver failure[J]. Dig Dis Sci, 2010, 55(2):285-291.
[6] Piscaglia AC, Shupe TD, Oh SH, et al. Granulocyte-colony stimulating factor promotes liver repair and induces oval cell migration and proliferation in rats[J]. Gastroenterology, 2007, 133(2):619-631.
[7] Lin X, Zhang S, Huang R, et al. Protective effect of tormentic acid from Potentilla chinensis against lipopolysaccharide/ D-galactosamine induced fulminant hepatic failure in mice[J]. Int Immunopharmacol, 2014, 19(2):365-372.
[8] Zhou M, Huang Y, Cheng Z, et al. Revival, characterization, and hepatitis B virus infection of cryopreserved human fetal hepatocytes [J]. J Virol Methods, 2014, 207:29-37.
[9] Smith SM, Wunder MB, Norris DA, et al. A simple protocol for using a LDH-based cytotoxicity assay to assess the effects of death and growth inhibition at the same time[J]. PLoS One, 2011, 6(11):e26908.
[10] Schneider A, Krüger C, Steigleder T, et al. The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogenesis[J]. J Clin Invest, 2005, 115(8):2083-2098.
[11] Harada M, Qin Y, Takano H, et al. G-CSF prevents cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes[J]. Nat Med, 2005, 11(3):305-311.
(2015-12-30 收稿 2016-02-20 修回)
(責(zé)任編委 王永怡 本文編輯 張?jiān)戚x)
Protective effect of granulocyte-colony stimulating factor on acute injury of human fetal hepatocytes
SUN Zi-jian, CHEN Jing, XIA Yan-heng, LIU Xiao-yan, SU Hai-bin, YANG Hao-zhen, XU Xiang, HU Jin-hua*
Liver Failure Treatment and Research Center, 302 Military Hospital of China-Peking University Teaching Hospital, Beijing 100039, China
*Corresponding author, E-mail: hjh@medmail.com.cn
[Abstract]Objective To establish the model of acute injury of human fetal hepatocytes induced by D-galactosamine (D-GalN) and observe the protective effect of granulocyte-colony stimulating factor (G-CSF) on the injury of fetal hepatocytes. Methods The human fetal hepatocytes were hatched by gradient concentrations of D-GalN at different action time, and their activities were detected by MTT assay, in order to determine the optimal conditions for establishing the model of acute injury of human fetal hepatocytes. The human fetal hepatocytes were divided into 4 groups, which received different treatments: the first group (control group) was the blank control group; normal fetal hepatocytes of the second group (group G) were treated with G-CSF; the injury was induced by D-GalN in both the third group (group ND) and the fourth group (group GD), but the human fetal hepatocytes of group GD were treated with G-CSF, and those of group ND with the same amount of normal saline instead of G-CSF. The activities of the hepatocytes of each group were detected by both MTT assay and LDH release assay. Results More than 90% of human fetal hepatocytes could be killed by D-GalN at the concentration of 10 mg/ml for 12 hours. The medicine had enough time to play its protective effect. The activities of the hepatocytes were not significantly different between the control group and group G, while that of group GD was significantly higher than that of group ND (P<0.05). Conclusions The model of acute injury of human fetal hepatocytes can be established by D-GalN at the concentration of 10 mg/ml for 12 hours. G-CSF has a protective effect on the acute injury of human fetal hepatocytes induced by D-GalN.
[Key words]liver failure; human fetal hepatocytes; granulocyte-colony stimulating factor
[通訊作者]胡瑾華,E-mail: hjh@medmail.com.cn
[基金項(xiàng)目]國(guó)家自然基金面上項(xiàng)目(81171641);北京市科技計(jì)劃 首都特色臨床應(yīng)用研究項(xiàng)目(Z131107002213157)
DOI:10.3969/j.issn.1007-8134.2016.02.007
[文獻(xiàn)標(biāo)志碼][中國(guó)圖書資料分類號(hào)] R512.62 A
[文章編號(hào)]1007-8134(2016)02-0089-03