LIPOPOLYSACCHARIDE-INDUCEDCYTOTOXICITY AGAINST CULTURED MOUSE HEPATOCYTES AND THE ROLE OF NONPARENCHYMAL LIVER CELLS

Lipopolysaccharide(LPS) was found to induce significant hepatocytotxicity against cultured mouse hepatocytes.Degeneration and necrosis of cultured hepatocytes and decerease of hepatocyte viability were prominent.The asparate transferase level and ^3H-TdR release in the medium were significantly increased after treatment,and the degree of these changes paralleled with LPS concentration.Various other parameters showed no significant difference between the hepatocytes cultured alone and those cocultured with nonparenchymal liver cells.However,if the nonparenchymal liver cells were isolated from mice which had been pretreated with D-galactosamine(GalN)not only was the hepatocyotoxicity induced by LPS enhanced,but the cells also showed certain cytotoxicity against cultured hepatocytes even without LPS,These results suggest that nonparenchymal liver cells might promote LPS induced hepatocyte injury.     

PROTECTIVE EFFECT OF MELATONIN ON NEURAL CELLS AGAINST THE CYTOTOXICITY OF OXYRADICALS

Objective. To investigate the exact mechanism of melatonin to prohibit the apoptosis of neural cells induced by various kinds of cytotoxic agents. Methods. We used the methods of phase contrast microscopy, MTT assay and hoechst dye staining to check this mechanism in SKNSH and U251 cell lines. Results. Both 2mmol/L H2O2 and 0. 5 μmol/L amyloid β- protein (Aβ) induce these two cell lines die via apoptosis. Either melatonin or glutathione can significantly protect both cell lines. The protective effect of 10 μmol/L melatonin is as same as that of 60μmol/L glutathione. Conclusion. Melatonin can partly inhibit the cytotoxicity of H2O2 and Aβ through its role as a free radical scavenger.     

THE MUTUAL BENEFIT ROLE OF LIVER AND PANCREAS IN COMBINED TRANSPLANTATION

The present study observed the mutual benefit role of liver and pancreas in combined hepaticopan-creatic transplantation in rats. The results indicated that pancreas, when transplanted with liver, could survive for a significant long time (13. 4±1.01 days) than it transplanted alone (9. 2±1.14 days) (P< 0. 05, t test). The interstitial rejection was mild and its rejection grade was significantly different from that of pancreas transplanted alone (P<0. 05, X2 test). The liver, when transplanted with pancreas, regenerated with strong competence and contact structure morphologically compared with liver transplanted alone. We think that pancreas could be immunologically protected against rejection and liver can be nutri-tionalized by pancreas in combined liver and pancreas transplantation.     

IDENTIFICATION OF GLUCOSE TRANSPORTER－1 AND ITS FUNCTIONAL ASSAY IN MOUSE GLOMERULAR MESANGIAL CELLS CULTURED IN VITRO

Objective. To evaluate the role of glucose transporter-1 (GLUT1) in the glucose uptake of glomerular mesanglal cells. Methods. Cultured C57/SJL mouse mesangial cells were used in the study. The expression of GLUT1 mRNA was detected by RT-PCR. The expression of GLUT1 protein was detected by immunofluorescence and flow cytometry. The uptake of glucose and its kinetics were determined by 2-deoxy-[3H] -D-glucose uptake. Results. Both GLUT1 mRNA and protein were found in mouse glomerular mesangial cells. 2-deoxy-D-glucose uptake and kinetics assay showed that this glucose transporter had high affinity for glucose and the glucose uptake specificity was further confirmed by phloretin. Conclusion. Functional GLUT1 did present in mouse mesangial cells cultured in vitro and it might be the predominant transporter mediated the uptake of glucose into mesangial cells.     

DIFFERENTIATION AND MALIGNANT SUPPRESSION INDUCED BY MOUSE ERYTHROID DIFFERENTIATION AND DENUCLUCLETION FACTOR ON MOUSE ERYTHROLEUKEMIA CELLS

OBJECTIVE: To investigate the roles of mouse erythroid differentiation and denucleation factor (MEDDF), a novel factor cloned in our laboratory recently, in erythroid terminal differentiation. METHODS: Mouse erythroleukemia (MEL) cells were transfected with eukaryotic expression plasmid pcDNA-MEDDF. Then we investigated the changes on characteristics of cell growth by analyzing cells growth rate, mitotic index and colony-forming rate in semi-solid medium. The expressions of c-myc and beta-globin genes were analysed by semi-quantitative RT-PCR. RESULTS: MEL cells transfected with pcDNA-MEDDF showed significant lower growth rate, mitotic index, and colony-forming rate in semi-solid medium (P<0.01). The percentage of benzidine-positive cells was 32.8% after transfection. The expression of beta-globin in cells transfected with pcDNA-MEDDF was 3.43 times higher than that of control (MEL transfected with blank vector, pcDNA3.1), and the expression of c-myc decreased by 66.3%. CONCLUSIONS: MEDDF can induce differentiation of MEL cell and suppress its malignancy.     

DIFFERENTIATION AND MALIGNANT SUPPRESSION INDUCED BY MOUSE ERYTHROID DIFFERENTIATION AND DENUCLEATION FACTOR ON MOUSE ERYTHROLEUKEMIA CELLS

To investigate the roles of mouse erythroid differentiation and denucleation factor (MEDDF), a novel factor cloned in our laboratory recently, in erylhroid terminal differentiation.Methods. Mouse erythroleukemia (MEL) cells were transfected with eukaryotie expression plasmid pcD-NA-MEDDF. Then we investigated the changes on characteristics of cell growth by analyzing cells growth rate, mitotie index and colony-forming rate in semi-solid medium. The expressions of c-mye and β-globin genes were analysed by semi-quantitative RT-PCR.Results. MEL cells transfected with pcDNA-MEDDF showed significant lower growth rate, mitotic index,and colony-forming rate in semi-solid medium ( P<0. 01). The percentage of benzidine-positive cells was 32. 8?ter transfection. The expression of β-globin in cells transfected with pcDNA-MEDDF was 3. 43 times higherthan that of control (MEL transfected with blank vector, pcDNA3. 1), and the expression of c-mye decreased by66. 3% .Conclusions. MEDDF can induce differentiation of     

EFFECTS OF INFLAMMATORY CELLS IN COLD AND WARM ISCHEMIA-REPERFUSION INJURY OF THE GRAFTED LIVER

Objective To investigate whether the impairment of grafted livers after transplantation was induced by the same inflammatory cells both in cold and warm ischemia. Methods Male SD rats were divided into two groups at random,24 donor livers in each group were stored in Ringer's solution at 4℃ for 120min or 240min of transplantation for blood sample and tissue specimen collection. Results Along with the prolongation of cold and warm ischemia time,the serum ALT,AST and LDH level increased gradually after transplantation.Under light microscopy,some hepatocytes presented necrosis after 3h and 6h of transplantation in cold ischemia,and neutrophilic infiltration in sinusoids were evident.Also,a large number of hepatocytes were necrotic 3h or 6h after transplantation in warm ischemia from NHBDs,and lymphocytic infiltration was evident in the sinusoids.The findings in electron microscopy was as the same as those of light microscopy,and the cells which infiltrated the sinusoids in warm ischemia were identified as T lymphocytes. Conclusion The impairment of grafted livers after transplantation appeared to be induced by two different kinds of inflammatory cells in cold and warm ischemia,that is,neutrophils mediated the cold ischemia-reperfusion,and T lymphocytes mediated the warm ischemia-reperfusion from NHBDs,but these findings are to be comfirmed in further investigations.     

ROLE OF TRANSFORMING GROWTH FACTOR-β IN THE GROWTH INHIBITION OF CULTURED RAT MESANGIAL CELLS IN HIGH GLUCOSE MEDIA

The prenent study was designed to test the hypothesis that the inhibitory effect of elevated glucose levels on mesangial cell growth might be mediated by transforming growth factor β (TGF β). Increased glucose levels inhibited mesangial cell proliferation in a concentration dependent manner. The addition of a rabbit antipncine TGF β1 neutralizing antibody significantly enchanced the cell proliferation in both normal (5.5mM) and high (50mM) glucose media (12% and 30% respectively), and almost completely blocked the growth inhibition induced by high glucose media. TGF β assay demonstrated mesangial cells produced more active than latent TGF β after growth in high glucose media. We conclude that TGF β functions as an autocrine cytokine in mesangial cells growth and the production of TGF β is modulated by high glucose concentration. The growth inhibition induced by high glucose levels may be largely mediated by endogenous TGF β.     

THE EFFECTS OF HSP27 ON THE CYTOTOXICITY OF RAT EMBRYO FIBROBLAST INDUCED BY CISPLATIN

ffeSUnt6 ~h f Etudier l'effet Protecteur de HsP27 contre la cytotoxicitd Preduite per cisplatine surieS fibroblastes ~naires chez ie rat (tranSfoed sons un theoomutant SV40 band antigsne T) (PIHsP27). M6t~ ho effets cytotoriques de cisplatine sur la Proliferation oh cellules PI-HsP27 en PreSenCeon en a~ de HsP27 etaient mesurds per l'emi ~. ~lfots Cisplatine ~trait une cytotoxicitddoc~nte sur ies cellules PI-HsP27 48h aam ie traitement, environ 50% ac cellules etaient mortal permi c…     

THE LOCALIZATION OF ADRENOMEDULLIN IN RAT KIDNEY TISSUE AND ITS INHIBITORY EFFECT ON THE GROWTH OF CULTURED RAT MESANGIAL CELLS

Adrenomedullin（AM），anovelhypotensivepeptideoriginallyisolatedfromhumanpheochromocytoma（１），wassynthesizedinvarioustissues（２，３）．Thecalcitoninreceptor－likereceptor／receptoractivity－modifyingpro－tein－２complexapparentlyservesasafunctionala－     

小鼠自然杀伤细胞杀伤瘤细胞机理的探讨

小鼠脾脏中自然杀伤细胞经4层非连续性 percoll 密度梯度离心法得到分离和纯化.其分离纯度达43%。主要分布于2分层中,形态学特征表现为大颗粒型淋巴细胞的特征,酸性非特异性脂酶呈阳性反应,过氧化物酶反应呈阴性.自然杀伤细胞对 L615白血病细胞具有非抗体介导的杀伤活性即自然杀伤活性.该杀伤活性与自然杀伤细胞释放超氧阴离子自由基有着极其密切的关系,同时此杀伤活性可被内毒素所加强.关于3,4两层细胞,前者表现为非颗粒型大淋巴细胞的形态学特征,后者为小淋巴细胞.它们对瘤细胞皆不表现出杀伤活性,但是当与内毒素作用后,其杀伤活性骤增,可能与受激发后释放超氧阴离子自由基有关.     

大鼠肝细胞与Kupffer细胞共同培养对两者功能和形态影响

目的观察大鼠肝实质细胞与Kupffer细胞体外共同培养时对两者生长、形态及功能的影响。方法采用原位二步Ⅳ型胶原灌注法、Percoll液密度梯度离心法分离Wistar大鼠肝实质细胞与Kupffer细胞;体外将肝实质细胞与Kupffer细胞按6∶1比例共同培养。观察不同情况下肝细胞生存时间和形态,每隔24 h检测培养上清液中清蛋白和谷丙转氨酶(ALT)、谷草转氨酶(AST)的水平,并在培养36 h用放免法检测上清液中白细胞介素1(IL-1)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)含量。结果单独培养组肝细胞的生长、增殖迅速,并向正常肝细胞的形态演变,肝细胞可培养存活至15 d;共同培养组肝细胞生长增殖缓慢,细胞可培养存活至10 d。共同培养组上清液中清蛋白水平在24、36、48、60 h比单独培养组低(t=2.551~3.139,P<0.05);ALT、AST水平在24、36、48、60 h比单独培养组高(t=2.446~3.108,P<0.05);共同培养组Kupffer细胞保持IL-1I、L-6、TNF-α分泌功能,而单独培养组未检测到IL-1I、L-6、TNF-α。结论大鼠Kupffer细胞和肝实质细胞在适宜的培养条件下可进行共同培养,二者可以保持良好的分泌功能,可用于实验研究。     

增敏化合物丁胱亚磺酰亚胺对Acc—2细胞的药物毒性

目的研究新型增敏化合物丁俄亚磺筑亚胶（buthioninesulfoximine，BSO）对ACC-2细胞药物毒性。方法采用克隆形成法研究在有氧状态和手氧状态下，BSO对ACC-2细胞的药物毒性，在实验中BSO的药物剂量分别是200、100、50、40、20μmol／L。结果在有氧和乏氧状态下，BSO对Acc-2细胞的半数抑制剂量分别为12μmol／L和80μmol／L。结论BSO的药物毒性较小，BSO在乏氧状态对Acc-2细胞的药物毒性强于有氧状态下对Acc-2细胞的药物毒性，     

大鼠肝脏细胞超微结构的立体形态观察

肝脏是冷冻割断法较难制做的器官之一。本文显示肌细胞、枯氏细胞内膜结构的立体构象,结构完整清晰,立体感强。肝细胞内膜结构极为丰富致密,主要以线粒体,粗、滑面内质网构成。线粒体为小卵圆形,外表光滑。其内线粒体嵴丰富,为圆柱形;粗面内质网,如透射电镜下所见,浅层紧密排列,未见明显囊腔;滑面内质网呈短分枝管状与粗面内质网连续,构成胞内膜结构的大部。肝细胞脂滴为圆球形,表面光滑,无包膜,直接与周围膜结构相接触。枯氏细胞内线粒体为长圆杆状;吞噬体表现为膜分隔的无定形结构,内含部分残质。本结果揭示了肝细胞、枯氏细胞内透射电镜下难以全面观察的膜结构的全貌。     

IL-2激活的TIL对自体肿瘤细胞的杀伤活性及其与LAK细胞活性的比较

肿瘤浸润性淋巴细胞(TIL)经白细胞介素2(IL-2)体外培养后具有很强的体内外抗肿瘤作用,且有一定的靶细胞特异性,其抗肿瘤效果强于淋巴因子激活的杀伤细胞即LAK细胞(P<0.01)。从瘤体中新鲜分离到的TIL对自体肿瘤细胞的杀伤活性极低,经IL-2体外培养后,其杀伤活性逐渐增高,以培养至7～25d的杀伤活性最强,这与IL-2使TIL分泌3种抗癌淋巴因子包括IL-2、IFN-γ、淋巴毒素(LT)增加有关。体外培养25d后,TIL的抗肿瘤活性下降,实验表明这与培养过程中TIL的Lyt-2~+细胞(Tc)减少而L3T4~+细胞(T_H)增多有关。TIL经冻存复苏和IL-2体外培养后仍保持很强的抗肿瘤活性,冻存前后比较未见显著差异(P>0.05),这为间断地运用TIL治疗复发性、晚期肿瘤提供了一条可行的途径。     

黄芩有效成分对四氯化碳致伤的原代培养大鼠肝细胞的作用

作者采用原代培养大鼠肝细胞研究黄芩有效成分的护肝作用，并对其中含量较多的结晶单体进行结构鉴定。结果表明：黄芩的乙酸乙酯萃取物和正丁醇萃取物（１．０～２．５ｍｇ／ｍｌ）均可使四氯化碳致伤的肝细胞培养液中ＡＬＴ活性显著降低（Ｐ＜０．００１）。从黄芩乙酸乙酯萃取物分离出来的晶Ⅱ、Ⅲ、Ⅳ在所试浓度下均有显著护肝作用；晶Ⅰ则主要为直接抑制酶活性作用。经理化鉴别和光谱分析，晶Ⅰ、Ⅱ分别为汉黄芩素和黄芩素。     

缺氧、再给氧对培养大鼠心肌细胞的损伤和尼可地尔的保护作用

本实验观察缺氧、再给氧对培养大鼠心肌细胞的损伤和尼可地尔(Nicorandil)的保护作用。缺氧组和缺氧加Nicorandil组给予充氮生长液;正常组和正常加Nicorandil组给予正常生长液。各组在实验9h时均用正常生长液置换原生长液。缺氧组心肌细胞在3h后搏幅减弱,频率变慢,节律失常。9h后约有1/3细胞停搏,细胞形态改变,上清液内乳酸脱氢酶(LDH)和肌酸激酶(CK)明显增高。更换正常生长液后15h内细胞病变和酶谱变化更趋严重,停搏细胞持续增加。缺氧加Nicorandil组内在Nicorandil剂量为0.5～2mg/ml时,细胞形态基本正常,很少节律失常,上清液内LDH和CK明显低于缺氧组。正常组和正常加Nicorandil组细胞搏动频率、节律和上清液内LDH、CK均无明显改变。     

乙双吗啉和γ射线联合应用对小鼠小肠上皮细胞的影响

本文研究乙双吗啉(AT1727)和γ射线联合应用对动物小肠上皮细胞的作用。腹腔注射AT1727 160mg/kg,在放射前30min给药时,其斜率和单纯放射组相仿,DMF值为1.09;放射后30min给药时,D_o值增加1.3倍,DMF值为1.05。在使用相等分割剂量做分割放射时,显示出他们之间明显的相互作用。结果显示:联合应用增加了辐射生物效应,且和所用药物浓度有关,和二者之间应用次序和相隔时间也有关。     

体外诱导小鼠胚芽干细胞分化为肝细胞的实验研究

目的寻找一种诱导小鼠胚芽干细胞向肝细胞定向分化的最佳诱导剂。方法分离提取孕11 d小鼠胚胎的原始生殖细胞,体外培养扩增后,一部分胚芽干细胞接种到6孔培养板中。向不同孔中分别加入不同浓度的碱性成纤维细胞生长因子（bFGF）、表皮生长因子（EGF）、β-神经生长因子（β-NGF）、维甲酸（RA）、肝细胞提取液进行诱导分化。另一部分胚芽干细胞与胎肝组织共培养。将培养12 d的细胞采用靛青绿摄入试验和白蛋白（ALB）免疫细胞化学染色来鉴定原始生殖细胞向肝细胞分化的情况。结果与胎肝组织共培养和添加了β-NGF、肝细胞提取液的胚芽干细胞能够诱导成肝样细胞,这些肝样细胞能够摄取ICG并表达ALB,上述3组细胞ALB阳性细胞数较对照组明显增多（P〈0.05）。bFGF、EGF、RA并无诱导分化作用。结论β-NGF、胎肝组织产生的细胞因子及肝细胞提取液中的细胞因子可诱导小鼠胚芽干细胞向肝细胞分化。     

卵圆细胞双向分化与实验性肝硬变组织发生关系的探讨

作者动态观察了 CCl_4诱发大鼠肝硬变中卵圆细胞(OC)的变化。用药后4周肝汇管区 OC 始增生,8～12周汇管区及肝小叶内出现大量团状或条索状排列的 OC,其周围常伴 desmin 阳性细胞存在。OC 聚集处,体积缩小,嗜碱性增强,趋向围成管腔;肝细胞大片坏死区,OC 分散,体变大,胞浆丰富,颇与再生肝细胞相近。18周后肝硬变形成时,管状排列的 OC 演变为新生胆管。结果显示OC 可能向肝细胞和胆管上皮细胞双向分化,作为一种“干细胞”参与肝硬变发生。