KAI1基因对MHCC97-H肝癌细胞生物力学的影响

目的 探讨KAI1基因对MHCC97-H肝癌细胞生物力学的影响.方法 采用微管吸吮技术研究我们前已转染人类KAI1全长正、反义结构基因的人肝癌MHCC97-H细胞粘弹性以及对FN的粘附力等生物力学参数.结果 MHCC97-H肝癌细胞的粘弹性系数K1、K2、μ在转染KAI1正义基因后明显增加(P<0.05),在转染KAI1反义基因后明显降低(P<0.01),而转染空载体后则无明显变化(P>0.1).在FN裱衬表面,各组肝癌细胞的粘附力分别为MHCC97-H-S组685.4±113.6,MHCC97-H-AS组1 124.6±123.5,MHCC97-H-pCI组849.0±126.3及MHCC97-H亲本细胞组824.7±134.1(单位为10-10 N,细胞数为25).与亲本细胞组比较,MHCC97-H细胞对FN的粘附力在转染KAI1正义基因后显著下降(P<0.01),转染KAI1反义基因后显著增加(P<0.01),而转染空载体后无明显变化(P>0.1).结论 从细胞生物力学角度,KAI1基因可能增加肝癌细胞的粘弹性及降低细胞对细胞外基质FN的粘附力,从而抑制转移的初始步骤,进而抑制MHCC97-H肝癌细胞的侵袭和转移.     

不同细胞周期肝癌细胞的粘附特性研究

目的研究大鼠肝实质细胞癌细胞（ＨＴＣ）与不同浓度胶原蛋白Ⅳ裱衬的人工基底膜的粘附特性和Ｇ１、Ｓ期ＨＴＣ细胞与一定浓度胶原蛋白Ⅳ裱衬的人工基底膜的粘附特性。方法同步化Ｇ１和Ｓ期细胞以胸腺嘧啶校苷和秋水仙碱顺序阻断法及胸腺嘧啶核苷双阻断法分别获得,细胞与人工基底膜的粘附特性采用微管吸吮技术测定、结果以胸腺嘧啶核苷和秋水仙碱顺序阻断法及胸腺嘧啶核苷双阻断法可分别获得７２．１０％的Ｇ１期和９８．９４％的Ｓ期ＨＴＣ细胞,ＨＴＣ细胞与胶原蛋白Ⅳ裱衬的人工基底膜的粘附力跟胶原蛋白Ⅳ的浓度成正相关关系,各组之间差异有显著性;Ｇ１期ＨＴＣ细胞的粘附力比Ｓ期显著要大。结论肿瘤早期基底膜含量的增加可能有利于肿瘤细胞的趋化运动和粘附;Ｇ１期肿瘤细胞的粘附力比Ｓ期显著要大,提示其细胞表面粘附分子受体分布的周期性差异以及Ｇ１期肿瘤细胞更易于粘附并穿透基底膜的可能。     

肝苏对人肝细胞和肝星状细胞增殖、氧应激以及细胞外基质表达的影响

背景：有研究显示肝苏对肝细胞具有保护、抗炎、抗氧化和抗肝纤维化的作用,但其作用机制未明。目的：探讨肝苏对人肝细胞和肝星状细胞（HSC）增殖、氧应激以及细胞外基质表达的影响。方法：分别用0．01～1．0mg／ml肝苏培养肝细胞和HSC,以M1丫r法检测肝苏对肝细胞和HSC增殖的影响：用次氮基三乙酸铁（Fe-NTa）和0．05—1．0mg／ml肝苏共同培养肝细胞和HSC,检测超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量;用1．5mg／ml和2．5mg／ml肝苏培养HSC,以酶联免疫吸附测定（ELISA）检测细胞外基质透明质酸（HA）、层黏连蛋白（LN）、I型胶原、Ⅲ型胶原和细胞因子转化生长因子（TGF）-β1含量。结果：在0．05。1．0mg／ml浓度范围内,肝苏可促进肝细胞增殖,但各浓度肝苏对HSC增殖无明显影响。肝苏可增高肝细胞SOD活性,降低肝细胞和HSCMDA含量,但对HSCSOD活性无明显影响。同时肝苏可抑制HSC细胞外基质HA、LN和细胞因子TGF-β1的表达,而I型胶原、Ⅲ型胶原无明显差异。结论：肝苏可促进肝细胞增殖,保护肝细胞和HSC免受氧应激损伤,抑制HSC分泌HA、LN和TGF-β1,提示其具有肝细胞保护、抗氧化和抗肝纤维化作用。     

肝细胞癌患者 Treg 细胞对 CD8＋T 淋巴细胞穿孔素表达的影响

目的：研究肝细胞癌患者免疫抑制性 Treg 细胞对 CD8＋ T 淋巴细胞穿孔素表达的影响。方法采集20例肝细胞癌患者和20名健康人的外周血,用流式分析法检测抗-CD3／CD28刺激48 h 后 CD8＋ T 淋巴细胞的穿孔素表达情况。免疫磁珠分离法分离健康人 CD8＋ T 淋巴细胞和肝细胞癌患者 Treg 细胞,一组 CD8＋ T 淋巴细胞单独培养,另一组CD8＋T 淋巴细胞与 Treg 细胞共同培养,用流式细胞法检测抗-CD3／CD28刺激48 h 后两组 CD8＋ T 淋巴细胞穿孔素表达情况。结果肝细胞癌患者外周血中 CD8＋ T 淋巴细胞穿孔素表达量与健康人相近,分别为（10．74±3．96）％和（12．6±2．48）％,差异无统计学意义（P ＞0．05）。CD8＋ T 淋巴细胞单独培养和与肝癌患者 Treg 细胞共培养后,健康人 CD8＋ T 淋巴细胞穿孔素表达量分别为（34．2±3．65）％和（20．43±4．52）％,差异有统计学意义（t＝11．42,P ＜0．01）。结论肝癌患者免疫抑制性 Treg 细胞可使 CD8＋ T 淋巴细胞穿孔素表达量降低。     

粘附法筛选胃癌细胞亚株

目的：筛选不同表型的胃癌细胞亚株。方法：用体外粘附法筛选对层粘素（ｌａｍｉｎｉｎ，Ｌｍ）有不同粘附力的ＭＫＮ－４５胃癌细胞亚株，并用Ｂｏｙｄｅｎ小室法和裸鼠接种法观察细胞亚株的体内、外侵袭转移能力。结果：经体外反复以层粘素粘附筛选，得到高粘附力的ＭＫＮ－４５Ｌｍ＾＋细胞亚株和低粘附力的ＭＫＮ－４５Ｌｍ＾－细胞亚株。Ｌｍ＾＋细胞体外对人工基膜Ｍａｔｒｉｇｅｌ的侵袭、移行能力及在裸鼠体内的成瘤性和肺转     

狼疮肾炎患者外周血单个核细胞CD134表达的动态变化及意义

目的 探讨CD134共刺激分子的动态变化特点及其存很狼疮肾炎（LN）发病机制中的可能作用。方法 采用流式细胞仪检测活动期和非活动期LN患者外周血单个核细胞（PBMC），在加入抗CD3单抗、白细胞介素（IL）-2刺激培养前、后的不同时段CD134^＋细胞数的动态改变。结果 经抗CD3ε单抗／rIL-2刺激前，活动期LN患者PBMC的CD134^＋细胞百分比已明显增加，主要表现在CD4^＋T细胞亚群上，CD8^＋T细胞则不明显。在刺激活化后，相比于正常对照组，活动期和非活动期LN患者PBMC的CD134^＋细胞百分比12h已明显增加，24h达到高峰，持续到48h仍处于高水平，72～96h后开始下调；至96hi活动期组仍高于正常对照组。结论 不论是活动期或非活动期LN患者，当T细胞受刺激活化后，主要是CD4^＋T细胞表面的CD134分子处于高表达状态和持续时间延长，CD8^＋T细胞只少量且呈一过性地表达：LN患者，特别是活动期LN患者的T细胞处于异常活化状态。     

肝癌微环境中CD4+CD25+调节性T细胞与T细胞免疫的关系

目的 了解肝细胞癌组织CD4 CD25 调节性T细胞(以下简写Treg)与肿瘤微环境T细胞免疫的关系.方法 对52例肝癌组织和癌旁组织用CD4、CD25双重酶标免疫组化染色和用CD8En Vision法染色,对癌组织中Treg细胞和CD4 T、CD8 T、CD4 T/CD8 T比值进行相关性分析.结果 肝癌以及癌旁组织中Treg细胞单个高倍视野平均数分别为7.6308±2.8368、5.1654±1.6718;两组比较有显著差异,P=0.000;肝癌组织中Treg细胞的数量与其浸润性CD4 T淋巴细胞的数量以及CD4 T/CD8 T比值呈显著负相关,r=-0.538,P=0.014;r=-0.545,P=0.000,与浸润性CD8 T淋巴细胞的数量分布无明显相关性,r=-0.403,P=0.078.结论 Treg在肝癌微环境中可能通过细胞接触的方式抑制CD4 T淋巴细胞的增殖来抑制肿瘤局部免疫,使肿瘤细胞逃避免疫监视.因此除去或减少肝癌微环境中的Treg细胞有利于提高肿瘤的免疫治疗效果.     

Ⅳ型和Ⅴ型狼疮性肾炎患者外周血与肾组织淋巴细胞亚群的变化和临床意义

目的:观察Ⅳ型与Ⅴ型狼疮性肾炎(LN)患者外周血淋巴细胞亚群的变化和肾组织淋巴细胞亚群和单核-巨噬细胞的浸润情况,并进一步分析与临床和病理的关系.方法:52例(男性11例,女性41例,年龄18～55岁)初治活动性系统性红斑狼疮,根据2003年ISN/RPS病理分型标准经肾活检确诊为Ⅳ型(n=32)和Ⅴ型(n=20),流式细胞仪测定外周血淋巴细胞亚群(CD3 、CD4 、CD8 、CD20 细胞)和CD4 CD25 Foxp 3 调节性T细胞(Treg细胞)的比例与计数,计算CD4 /CD8 比值.20例年龄、性别匹配的健康志愿者作为对照.同时检测30例Ⅳ型和11例Ⅴ型LN肾组织CD4、CD8、CD20和CD68免疫组化染色,取10例移植肾供肾活检标本作为正常肾组织对照.从中再选取20例(Ⅳ型和Ⅴ型各10例)行CD4和Foxp 3双标免疫组化染色.比较LN患者外周血淋巴细胞亚群和Treg细胞与正常对照的差别,分析Ⅳ型和Ⅴ型之间外周血和淋巴细胞亚群和Treg细胞的变化特点.进一步分析Ⅳ型和Ⅴ型LN肾组织淋巴细胞亚群,特别是Treg以及单核-巨噬细胞浸润的差异和临床病理联系.结果:活动性Ⅳ型、Ⅴ型LN患者与正常人比较外周血CD4 细胞比例、计数和CD4 /CD8 细胞比值及Treg细胞显著降低,CD8 细胞及CD20 细胞明显升高(P＜0.01).Ⅳ型与Ⅴ型相比:(1)外周血CD4 细胞比例[(25.9±6.86)％vs(31.0±7.59)％,P＜0.05]、计数[(288±173)个/μlvs(420±165)个/μl,P＜0.01]和CD4 /CD8 比例降低更明显(0.74±0.31vs1.06±0.57,P＜0.05);(2)外周血CD20 细胞比例无明显差异,但Ⅴ型CD20 细胞计数升高较Ⅳ型明显[(185±136)个/μlvs(268±179)个/μl,P＜0.05];(3)Treg细胞比例[(0.82±0.4)％vs(1.31±0.7)％,P＜0.05]和计数[(8.19±4.26)个 /μlvs(17.5±10.0)个/μl,P＜0.01]Ⅳ型降低更明显.CD4 细胞比例和计数与AI呈负相关(r分别为-0.281和-0.380,P＜0.05),CD4 /CD8 细胞比值与SLEDAI呈负相关(r=-0.307,P＜0.05).Treg细胞比例和计数与SLEDAI呈显著负相关,相关系数分别为-0.411(P＜0.01)和-0.480(P＜0.01),与肾组织AI呈负相关(r为-0.325和-0.473,P＜0.01).肾组织免疫组化见Ⅳ型肾间质浸润的CD4 、CD8 、CD20 和CD68 细胞均高于Ⅴ型病例,其中肾小管浸润的CD20 细胞Ⅳ型明显高于Ⅴ型(119.3±89.7vs36.0±36.0,P＜0.01),肾小球内浸润的CD68 细胞显著高于Ⅴ型(12.4±8.8vs1.7±2.31,P＜0.01).正常肾组织不表达Foxp 3 Treg细胞,绝大多数Foxp 3表达于CD4 细胞,LN中CD4 Foxp 3 Treg细胞主要位于肾间质,Ⅳ型LN CD4 Foxp 3 Treg细胞明显高于Ⅴ型(27.6±18.0 vs2.8±5.0,P＜0.01).结论:Ⅳ型LN患者外周血CD4 细胞数量、CD4 /CD8 细胞比例和Treg细胞数量较Ⅴ型明显降低,Ⅳ型LN肾组织T、B淋巴细胞,特别是调节性T细胞和巨噬细胞浸润较V型明显升高,提示不同病理类型LN的免疫发病机制不同.     

肝纤维化大鼠肝细胞生成蛋白与胶原的变化及中药药物的影响

目的：探讨肝纤维化大鼠肝细胞白蛋白及胶原生成的病理变化及扶正化瘀中药药物对其的影响。方法：自CCI4致肝纤维化大鼠肝脏分离肝细胞进行体外培养，检测白蛋白，胶原生成量及组织蛋白酶、胶原酶活性的变化；同时于细胞培养过程中添加扶正化瘀中药体内给药后分离的药物血清。结果：与正常大鼠肝细胞相比，肝纤维化大鼠肝细胞白蛋白生成量下降了14．0％．而细胞外胶原生成量却增加了3．5倍。细胞内的组织蛋白酶及细胞外间质型胶原酶括性分别下降了33．3％与42％。添加中药药物血清后，该肝细胞上述病理变化均获显著改善；而秋水仙碱对细胞外胶原生成率呈现显著抑制作用的同时，亦抑制了细胞白蛋白的生成。结论：肝纤维化大鼠肝细胞生成白蛋白的功能降低，细胞外胶原生成率显著增加，而后者的改变主要与细胞内外的胶原降解括性降低有关。含扶正化瘀中药的血清具有促进肝纤维化大鼠肝细胞向正常肝细胞生理功能转化的作用。     

骨形态发生蛋白9对肝细胞癌肿瘤干细胞干性、增殖和侵袭的影响及机制

目的 探讨骨形态发生蛋白9 (BMP9)对肝细胞癌（HCC）肿瘤干细胞（CSCs）干性、增殖和侵袭的影响及机制。方法 取对数生长期的正常肝细胞、肝癌细胞、肝癌CSCs,采用RT-qPCR法检测BMP9 mRNA表达,采用Western blotting法检测BMP9蛋白表达。采用慢病毒干扰载体转染肝细胞癌肿瘤干细胞（HepG2-CSCs）以敲低BMP9表达,并分成HepG2-CSCs组、HepG2-CSCs-BMP9敲低组。加入MAPK/ERK信号激动剂（DIPQUO）后,将细胞分为三组：HepG2-CSCs组、HepG2-CSCs敲低组及HepG2-CSCs敲低+DIPQUO组。采用RT-qPCR实验检测HepG2-CSCs干性相关分子CD44、SOX2和OCT4表达,采用CCK-8实验检测细胞增殖能力,采用Transwell实验检测细胞侵袭能力,采用蛋白印迹法检测MAPK/ERK通路关键蛋白表达。结果 与正常肝细胞比,肝癌细胞和肝癌肿瘤干细胞（HepG2-CSCs）中BMP9表达上调（P<0.05）。HepG2 CSCs细胞中BMP9 mRNA和蛋白表达高于HepG2细胞（P...     

Mechanical properties of hepatocellular carcinoma cells

AIM: To study the viscoelastic properties of humanhepatocytss and helatocellulsr carcinoma (HCC) cellsunder cytoslelstal perturbation, and to further to study theviscoelastic properties and the adhesive properties of mousehepatorna cells (HTC) in different cell cycls.METHODS: Micropipette aspiration technique was adopted tomeasure viscoelastic coefficients and adhesion force tocollagen coated surface ofthe cells. Three kinds ofcytoskeleton perturbing agents, colchiclnes (Col),cytochalssin D (CD) and vinblastine (VBL), were used totreat HCC cells and hepatocytes and the effects of thesetreatent on cell viscoelastic coefficients were investigated.The experimental results were analyzed with a thres-elsmentstandard linear solid. Further, the viscoelastic properties ofHTC cells and the adhesion force of different cycle HTC cellswere also investigated. The synchronous G1 and S phasecells were achieved through thymine-2-desoryriboside andcolchicines sequential blockage method and thymine-2-desoryriboside blockage method respectively.RESULTS: The elastic coefficients, but not viscouscoefficient of HCC cells (k1 = 103.6± 12.6N.m-2, k2 ＝42.5±10.4N. m-2, μ = 4.5 ± 1.9Pa. s), were significantly higherthan the corresponding value for hepatocytes (K1 = 87.5 ±12.1N.m-2, k2 ＝33.3± 10.3N.m-2, μ=5.9±3.0Pa. s, P＜0.01). Upon treatment with CD, the viscoelastic coefficients ofboth hepotocytes and HCC cells decreased consistently,with magnitudes for the decrease in elastic coefficients ofHCC cells (k1: 68.7 N.m-2 to 81.7N.m-2, 66.3 % to 78.9 %;k2: 34.5 N.m-2 to37.1N.m-2, 81.2% to 87.3 %, P＜0.001)larger than those for normal hepatocytes (k1: 42.6N. m-2 to49.8N.nt-2, 48.7% to56.9 %; k2: 17.2N.m-2 to 20.4N.m-2,51.7 % to 61.3 %, P＜ 0.001). There was a little decrease inthe vlscous coefficient of HCC cells (2.0 to 3.4Pa. s, 44.4 to75.6 %, P＜0.001) than that for hepatocytes (3.0 to 3.gPa.s, 50.8to 66.1% P＜0.001). Upon trastment with Col andVBL, the elastic coefficients of hepatocytes generallyincreased or tended to increase while those of HCC cellsdecreased. HTC cells with 72.1% ofG1 phase and 98. 9 % ofS phase were achieved and high K1, k2 value and low μvalue were the general characlteristics of HTC cells. G1phase cells had higher K1 value and lower tμ value than Sphase cells had, and G1 phase HTC cells had strongeradhesive forces [(275.9±232.8) x 10-10N] than S phase cells[(161.2± 120.4) x l0-10N, P＜0.001).CONCLUSION: The difference in both the pattern and themagnitude of the effect of cytoskeletal perturbing agent onthe viscoelastic properties between HCC cells andhepatocytes may reflect differencss in the state of thecytusieleton structure and function and in the sensitivity toperturbing agent treatment between trinse two types of cells.Change in the viscoelastic properties of cancer cells mayaffect significantly tumor cell invasion and metastasis as wellas interactions between tumor cells and their micro-mechanical environments.     

Laminin deposition to type IV collagen enhances haptotaxis, chemokinesis, and adhesion of hepatoma cells through beta1-integrins

BACKGROUND/AIMS: In hepatocellular carcinoma, laminin deposition to type IV collagen along the sinusoids is observed with the development of arterial network, coinciding with intrahepatic metastasis. We investigated the influence of laminin deposition to type IV collagen on hepatoma cell adhesion, motility and secretion of matrix metalloproteinases (MMPs), which are indispensable behaviors for tumor metastasis. METHODS: Hepatoma cell lines (KYN-1, -2 and -3) were used. The expression of integrin subunit mRNAs in hepatoma cells was confirmed by RT-PCR. The influence of laminin addition to type IV collagen on the adhesion, chemokinesis, and migration of KYN-1, -2 and -3 was evaluated by the haptotactic migration, phagokinetic track motility, and cell adhesion assays. The effects of integrin subunits on these activities were evaluated using the function-blocking antibodies for integrins. Phosphorylation of MEK1/2 and secretion of MMPs were investigated by Western blotting and gelatin zymography. RESULTS: Integrin alpha1, alpha2, alpha3, alpha6 and beta1 subunit mRNAs were detected. The combination of type IV collagen and laminin enhanced the migration, chemokinesis, and adhesion of hepatoma cells compared to that of type IV collagen when used alone. The enhanced activity was significantly suppressed by function-blocking antibodies for integrin alpha1, alpha2, alpha3, alpha6 and beta1 subunits. Hepatoma cells cultured on the combination of type IV collagen and laminin showed phosphorylation of MEK1/2 and increased secretion of MMPs. CONCLUSIONS: The addition of laminin to type IV collagen enhances hepatoma cell adhesion and motility through beta1-integrins.     

Changes in the Distribution of Laminin,Fibronectin, Type IV Collagen and Heparan Sulfate Proteoglycan during Colony Formation by Chick Retinal Pigment Epithelial Cells In Vitro

Retinal pigment epithelial cells isolated by non-enzymatic means from 8 day old chick embryos were grown as explants on glass covers lips in culture. Retinal pigment epithelial cells grown in this way form colonies in which three zones, each containing cells with distinctly different morphology, pigmentation, adhesion pattern and cytoskeletal organization, can be distinguished (Turksen et al., 1983). Using specific antisera against laminin, fibronectin, type IV collagen, and heparan sulfate proteoglycan, we have found differences in the distribution of these basement membrane components in the three zones of each colony. When cells were stained with laminin, type IV collagen and fibronectin antibodies, extensive filamentous arrays were observed on the substratum side of the cells. In contrast to type IV collagen which was deposited to a similar degree in all three zones of each colony, laminin and fibronectin were most prominent in the central zone in which the packed cuboidal differentiated cells are located. In contrast to the other components, the heparan sulfate proteogylcan appeared to be associated primarily with the cell surface. Our results support the general view that basement membrane components could influence cell shape through an effect on the cytoskeleton, and playa role in the maintenance and expression of the differentiated state. Thus retinal pigment epithelial cells might provide a very useful model system for studying the interactions between the cytoskeleton and extracellular matrices and the biosynthesis of the BM components in epithelial cells.     

Cell Adhesive,Protein Binding,and Antigenic Properties of Laminin

The cell adhesive, protein-binding and immunological properties of laminin were studied. When present on a solid-phase, laminin promoted to some degree the adhesion of various types of cells including fibroblasts, but it was less active than insolubilized fibronectin, type I collagen, or type IV collagen. When laminin was present in solution, it promoted the adhesion of cells to surfaces that would otherwise be nonadhesive. No significant binding of laminin to type IV collagen could be demonstrated by affinity assays employing radioactively labeled ligands or enzyme-labeled antibodies. Binding of laminin to anti-laminin and to polyornithine could be readily demonstrated under the same conditions. The possible autoantigenicity of laminin was studied by immunizing mice with rat and mouse laminin. Rat laminin induced antibodies that reacted strongly with rat laminin and weakly with mouse laminin in enzyme immunoassay. Mouse laminin did not induce any detectable antibodies. These results confirm earlier work on cell adhesive properties of laminin but show that laminin is different from fibronectin in that laminin promotes adhesion of cells better when presented to the cells in a soluble form. They do not support the contention that laminin would bind directly to type IV collagen or that it would be autoantigenic.     

Integrin receptors on aortic smooth muscle cells mediate adhesion to fibronectin, laminin, and collagen.

Extracellular matrix receptors on vascular smooth muscle cells help in anchoring the cells during contraction and in promoting cellular migration after vessel injury. We found that rat aortic smooth muscle cells attach to surfaces coated with fibronectin, laminin, and collagen types I and IV. Cell attachment to these substrates appears to be mediated by members of the beta 1 integrin family of extracellular matrix receptors. Antibodies to the beta 1 subunit not only demonstrated the presence of integrin complexes in focal adhesion plaques but also blocked cell adhesion to the different substrates. Ligand-affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis isolated a series of receptor complexes that were recognized by antisera to beta 1 integrin receptors. Each of the receptors appeared to be a heterodimer in which one of several alpha subunits shared a common 120-kDa (nonreduced) beta 1 subunit protein. The rat aortic smooth muscle cells had one alpha subunit (150 kDa nonreduced, 140 kDa reduced) that bound exclusively to fibronectin. There was a second alpha subunit (150 kDa nonreduced, 160 kDa reduced) that bound exclusively to collagen type I. In addition, there was a third alpha subunit (185 kDa nonreduced, 200 kDa reduced) that was promiscuous and bound to collagen types I and IV as well as to laminin; the 185-kDa alpha subunit appeared to bind to collagen more efficiently than it did to laminin. Thus, smooth muscle cells express multiple integrin receptors with different ligand specificities that appear to mediate cell interactions with the extracellular matrix.     

The integrin, alpha6beta1, is necessary for the matrix-dependent activation of FAK and MAP kinase and the migration of human hepatocarcinoma cells.

Expression of the integrin, alpha6beta1, a receptor for laminins, is associated with the progression of hepatocellular carcinoma (HCC). The approach to investigating the alpha6beta1 integrin signaling in HCC cells was to express a deletion mutant of the beta4 integrin cytoplasmic domain (beta4-Deltacyt) in 2 HCC cell lines, HepG2 and Huh7. Expression of this mutant prevents formation of the alpha6beta1 heterodimer. As expected, adhesion of both the HepG2/beta4-Deltacyt and Huh7/beta4-Deltacyt transfectants to laminin, but not to collagen, was reduced compared with the mock transfectants. However, migration of the beta4-Deltacyt transfectants toward both collagen and laminin was inhibited, suggesting a role for alpha6beta1 in the signaling of migration. Migration of HCC cells requires mitogen-activated protein (MAP) kinase. The adhesion of the beta4-Deltacyt transfectants to collagen resulted in a substantial reduction in MAP kinase activation in comparison with the mock transfectants, although their ability to activate MAP kinase in response to epidermal growth factor (EGF) stimulation was not impaired. In addition, matrix adhesion of the beta4-Deltacyt transfectants did not stimulate the tyrosine phosphorylation of focal adhesion kinase (FAK), and this defect correlated with reduced binding of adaptor protein Grb2 to FAK. These results suggest that FAK tyrosine phosphorylation is dependent on alpha6beta1 expression, and that FAK-Grb2 association plays a central role in alpha6beta1-mediated activation of MAP kinase. Moreover, the expression of alpha6beta1 in HCC cells is necessary for FAK/MAP kinase-dependent migration.     

Effects of vitamin A on collagen metabolism by cultured rat liver cells

Conflicting results have been reported concerning the phenotypes of collagen produced by cultured Ito cells. These variations may be attributed to differences in pretreatment, i.e., with or without vitamin A to facilitate the separation of Ito cells. In the present study, the effects of vitamin A on collagen metabolism by Ito cells and hepatocytes of rats were analyzed. In cultured Ito cells, staining reactions to type I collagen increased, and those to type IV collagen and laminin decreased after pretreatment with vitamin A. The rate of collagen synthesis by Ito cells decreases significantly by treatment with vitamin A. The decrease was clearer in degraded collagen than in intact collagen. The synthesis of type I collagen increased and that of type IV collagen significantly decreased in Ito cells by treatment with vitamin A. In the hepatocytes, the staining reaction to type I collagen increased with vitamin A pretreatment. The net collagen and type III collagen synthesis in hepatocytes decreased by treatment with vitamin A. These results indicate that vitamin A modifies collagen metabolism in different cell types in different ways.     

Effects of vitamin A on collagen metabolism by cultured rat liver cells.

Conflicting results have been reported concerning the phenotypes of collagen produced by cultured Ito cells. These variations may be attributed to differences in pretreatment, i.e., with or without vitamin A to facilitate the separation of Ito cells. In the present study, the effects of vitamin A on collagen metabolism by Ito cells and hepatocytes of rats were analyzed. In cultured Ito cells, staining reactions to type I collagen increased, and those to type IV collagen and laminin decreased after pretreatment with vitamin A. The rate of collagen synthesis by Ito cells decreases significantly by treatment with vitamin A. The decrease was clearer in degraded collagen than in intact collagen. The synthesis of type I collagen increased and that of type IV collagen significantly decreased in Ito cells by treatment with vitamin A. In the hepatocytes, the staining reaction to type I collagen increased with vitamin A pretreatment. The net collagen and type III collagen synthesis in hepatocytes decreased by treatment with vitamin A. These results indicate that vitamin A modifies collagen metabolism in different cell types in different ways.     

Hepatocyte survival depends on beta1-integrin-mediated attachment of hepatocytes to hepatic extracellular matrix.

BACKGROUND: A major drawback of allogeneic hepatocyte transplantation is the lack of sustained survival of the transplanted cells in the recipient liver parenchyma. The purpose of this study was to determine the effect of the presence or absence of hepatic extracellular matrix (ECM) molecules on hepatocyte survival and function following hepatocyte isolation for transplantation purposes, and the role of beta1-integrin molecules therein. METHODS: Hepatocytes, either untreated or treated with anti-beta1 integrin antibodies or RGD peptides, were seeded on wells precoated with collagen type I, type IV, laminin, fibronectin or polyhydroxyethylmehacrylate. The extent of attachment and apoptosis was evaluated. RESULTS: When hepatocytes were added into wells precoated with either fibronectin, or collagen type IV, rapid spreading and prolonged survival occurred, in contrast to hepatocytes that were seeded in wells precoated with collagen type I or polyhydroxyethylmehacrylate. Pretreatment of the cells with anti-beta1-integrin antibodies resulted in reduction of cell attachment to laminin, fibronectin, collagen I, and collagen IV. Synthetic RGD (arginine-glycine-aspartate)-peptides and anti-beta1 antibodies inhibited apoptosis of cultured hepatocytes. CONCLUSIONS: Our findings indicate that embedding of hepatocytes within their normal liver ECM surroundings maintains their survival. When detached from their natural surrounding hepatocytes enter into apoptosis, unless treated with anti-beta1-integrin antibodies or RGD peptides. This knowledge will allow improvement of hepatocyte transplantation efficiency.