四氯化碳诱导性肝纤维化大鼠肝组织骨形态发生蛋白-7的表达

目的 探讨四氯化碳(CCl4)诱导性肝纤维化大鼠的肝组织纤维化与骨形态发生蛋白-7(BMP-7)表达的相互关系.方法 将健康雄性 Wistar 大鼠随机分为对照组和肝纤维化组.对照组大鼠每周一、四背部皮下注射橄榄油(0.12ml/100g),肝纤维化组每周一、四背部皮下注射60% CCl4橄榄油(0.3ml/100g),连续注射6、10、16 和21周.实验结束时处死大鼠,取肝中叶,石蜡包埋、切片.行天狼猩红染色,观察肝组织胶原纤维的增生情况;行BMP-7免疫组织化学染色和 Western blotting 检测,观察肝组织.BMP-7的表达.结果 天狼猩红染色显示对照组大鼠仅在肝门静脉及门管区看到少量的胶原纤维,各肝纤维化组大鼠均可见肝组织呈明显的胶原纤维增生,且随着时间的延长,肝纤维化程度逐渐加重;免疫组织化学染色显示,对照组大鼠肝组织中可见极少量的BMP-7表达阳性的细胞,6周肝纤维化组大鼠可见较多的BMP-7阳性表达细胞,但随着实验时间的延长,BMP-7阳性细胞呈递减的趋势,至21周肝纤维化组大鼠肝组织中基本呈阴性表达;Western blotting 法检测大鼠肝组织BMP-7的表达结果与免疫组织化学的结果基本一致.结论 CC14诱导性肝纤维化大鼠肝组织BMP-7的表达与肝纤维化的程度呈负相关的趋势,推测BMP-7可能对肝纤维化具有一定的保护作用.     

人肝干细胞移植增加四氯化碳诱导性肝硬化大鼠白蛋白表达

目的 旨在研究人胚胎肝干细胞移植入四氯化碳(CCl4)诱导性肝硬化大鼠的肝组织后,大鼠血清及肝组织血清白蛋白(ALB)表达的变化情况.方法 将健康雄性Wistar大鼠随机分为对照组和肝硬化组.对照组背部皮下注射橄榄油,CCl4组背部皮下注射CCl4橄榄油.连续注射6周后,肝硬化组再随机分为肝干细胞假移植组和肝干细胞移植组.对照组和假移植组在大鼠肝中叶注射细胞培养液,而移植组则在相应部位注射人胚胎肝干细胞2×106,移植4周后处死所有大鼠.右心室取血离心观察ALB的含量;肝中叶取材,免疫组织化学染色观察人表皮生长因子受体(EGFR)的表达,免疫荧光染色和免疫印迹法(Western blotting)法观察和测定肝组织ALB的表达变化.结果 对照组和肝干细胞假移植组未见人EGFR阳性表达的细胞,肝干细胞移植组则可见大量排列成环状的EGFR阳性表达的细胞.在肝干细胞移植组的血清ALB含量、免疫荧光染色及Western blotting法检测肝组织ALB表达,均明显高于假移植组,而与对照组无明显著异.结论 将人胚胎肝干细胞移植入肝硬化大鼠肝内可以长期存活,并可改善大鼠ALB的表达和分泌.     

人间充质干细胞移植改善四氯化碳诱导性肝硬化大鼠的肝纤维化

目的研究人间充质干细胞（MSCs）移植对四氯化碳诱导性肝硬化大鼠肝纤维化的影响.方法 实验动物随机分为对照组和肝硬化组,肝硬化组采用60%的四氯化碳植物油皮下注射7周制成肝硬化大鼠模型,再随机分成肝硬化7周组、MSC对照组和MSC移植组.在MSC移植组,人胚胎脐带血源的MSCs鉴定后,经肠系膜上静脉注射移植入大鼠体内,3周后处死所有大鼠,行肝组织冰冻切片,抗人表皮生长因子受体（EGF-R）的免疫组织化学显色和天狼猩红染色. 结果肝硬化7周组、MSC对照组和MSC移植组大鼠的血清碱性磷酸酶白蛋白（ALP）、（ALB）和胆固醇（CHO）含量出现了不同程度的异常.肝硬化7周组大鼠肝组织中有大量的胶原纤维增生并形成假小叶;MSC对白蛋白照组与肝硬化7周组相似;仅在MSC移植组的大鼠肝中观察到散在的棕黄色的抗人EGF-R阳性细胞,肝组织中胶原纤维的量明显小于MSC对照组.结论经肠系膜上静脉移植人MSC,可明显改善四氯化碳诱导性肝硬化大鼠的肝纤维化程度,为肝硬化的治疗提供实验依据.     

肝纤维化大鼠肝细胞内质网形态及GRP78表达的研究

目的: 观察四氯化碳(CCl₄)致大鼠肝纤维化过程中内质网形态及内质网应激标志性蛋白——葡萄糖调节蛋白78(GRP78)的表达变化, 探讨内质网应激在肝纤维化发病机制中可能的作用。方法: 雄性Wistar大鼠皮下注射CCl₄制备肝纤维化模型,分别在4周及8周处死大鼠测定肝脏指数、血清丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)活性, 观察肝组织病理改变和肝细胞内质网形态, 免疫组化和real-time PCR分别检测肝组织GRP78蛋白及mRNA表达变化。结果: 肝纤维化组大鼠肝脏指数、血清ALT和AST活性显著高于正常对照组(P<0.01),肝纤维化明显, 电镜下见肝细胞内质网扩张,数量明显减少; 肝细胞胞浆中GRP78蛋白表达量及mRNA表达量较正常组显著增加(P<0.01)。结论: 在CCl₄诱导的肝纤维化发生过程中肝细胞内质网形态有明显损伤性变化, 内质网应激蛋白GRP78蛋白及基因表达水平明显增加, 提示内质网应激参与肝纤维化发生发展。     

金属硫蛋白降低GRP78和CHOP蛋白表达减轻大鼠砷中毒肝细胞凋亡

目的 研究葡萄糖调节蛋白78(GRP78)、C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白(CHOP)在金属硫蛋白(MT)减轻大鼠砷中毒肝细胞凋亡中的作用.方法 建立MT治疗亚砷酸钠(NaAsO2)诱导的大鼠砷中毒肝损伤模型,用MT治疗2周,以TUNEL法检测肝细胞凋亡,免疫组化法、Western blot检测大鼠肝脏GRP78、CHOP蛋白表达.结果 砷中毒模型组大鼠肝细胞凋亡、肝组织GRP78和CHOP蛋白表达较对照组显著升高(P＜0.05),MT治疗组肝细胞凋亡、肝组织GRP78和CHOP蛋白表达明显回降(P＜0.05),但仍然高于对照组(P＜0.05).结论 MT可通过降低GRP78和CHOP蛋白表达减轻大鼠砷中毒所致的肝细胞凋亡.     

PI3K/Akt信号通路及内质网应激在肝纤维化大鼠肝细胞凋亡中的作用

目的观察磷脂酰肌醇-3激酶/蛋白激酶(PI3K/Akt)信号通路及葡萄糖调节蛋白78(GRP78)、生长停滞及DNA损伤基因(CHOP/GADD153)在四氯化碳(CCl4)诱导的肝纤维化中的表达并探讨其可能的作用。方法将30只SD大鼠随机分为正常组、肝纤维化模型(皮下注射40%CCl4橄榄油溶液)4及8周组。HE染色法观察肝组织病理形态学;用real-time PCR技术检测肝脏内GRP78及CHOP mRNA的表达;用Western blot检测肝脏内PI3K/Akt信号通路中Akt1、磷酸化Akt1及内质网应激相关蛋白GRP78及CHOP的表达;用原位末端转移酶标记(TUNEL)检测细胞凋亡。结果与正常组大鼠比较,肝纤维化模型4及8周组大鼠肝脏内GRP78及CHOP mRNA和蛋白表达均明显升高(P0.05),而肝脏内Akt1和磷酸化Akt1蛋白的表达则较正常大鼠显著降低(P0.05);与正常组大鼠比较,肝纤维化模型4及8周组大鼠肝细胞凋亡显著升高(P0.05)。结论 PI3K/Akt信号通路及内质网应激可能在肝纤维化大鼠肝细胞凋亡中发挥了重要作用。     

吸入高浓度氢气增强四氯化碳损伤性大鼠生精细胞核糖体蛋白S6激酶的表达

目的探讨吸入3%氢气对四氯化碳(CCl4)损伤性大鼠生精细胞核糖体蛋白S6激酶(p70s6k)表达及其增殖功能的影响。方法将18只健康雄性SD大鼠随机分成对照组、CCl4组和氢气治疗组。对照组每周一、四背部皮下注射橄榄油(0.12ml/100g),CCl4组和氢气组每周一、周四皮下注射体积分数60%的CCl4-橄榄油(0.3ml/100g),连续4周。氢气组自实验的第22天吸入3%氢气,1h/d,连续7d。取右侧睾丸和附睾,行石蜡包埋切片,苏木素伊红染色和免疫组织化学染色。取左侧睾丸组织,行Western blotting检测。结果对照组睾丸生精小管内可见多层生精细胞,排列有续,结构完整。CCl4组睾丸生精小管细胞层数减少,结构紊乱,附睾管柱状上皮变薄;氢气组生精细胞数量和附睾管高柱状上皮细胞明显改善。CCl4组大鼠附睾精子密度较对照组降低,氢气组的精子密度明显高于CCl4组(t=4.91,P0.05)。免疫组织化学染色显示,氢气组生精细胞质中p70s6k(t=7.63,P0.05)和PCNA(t=20.08,P0.05)的表达明显强于CCl4组。Western blotting检测显示,氢气组睾丸组织中p70s6k的表达与CCl4组相比明显增强(t=3.64,P0.05)。结论吸入高浓度氢气能明显增强CCl4损伤性大鼠睾丸生精细胞p70s6k的表达,改善CCl4损伤引起的的生精细胞的增殖功能。     

肝细胞生长因子对肝纤维化进程中肝细胞再生及α平滑肌肌动蛋白表达的影响

目的:探讨肝细胞生长因子(hepatocyte growth factor,HGF)在大鼠肝纤维化进程中对肝细胞再生及α平滑肌肌动蛋白(alph smooth mucle actin,α-SMA)表达的影响.方法:将SD大鼠随机分为2组:对照组(n=20)肝70%切除后腹腔注射CCl4和生理盐水8周;实验组(n= 20)肝70%切除后腹腔注射CCl4和HGF 8周.术后8周取残肝行HE、Masson、α-SMA免疫组化染色及电镜观察.结果:HE显示,实验组肝再生指数较对照组明显升高(P<0.05),而Masson和α-SMA免疫组化染色分别显示胶原纤维、α-SMA的表达减少(P<0.05).电镜显示,实验组肝细胞有内大量脂滴,间质胶原纤维减少;肝星状细胞(hepatic stellate cell,HSC)内脂滴较对照组减少.结论:在肝纤维化进程中,HGF可促进肝细胞增殖,抑制HSC活化,减少胶原纤维的形成和α-SMA的表达.     

β-catenin在四氯化碳诱导的小鼠肝纤维化过程中的定位、表达及意义

目的 探讨肝纤维化发生过程中β-连环蛋白(β-catenin)定位、表达及意义.方法 健康雄性昆明小鼠(n=45)随机分为对照组(n=15)与实验组(n=30).实验组小鼠皮下注射50% 四氯化碳(CCl4)-粟米油混合液(6ml/kg),2次∕周,对照组皮下注射同等剂量的粟米油.各组分别于造模1周、4周和8周后取小鼠肝脏,常规制作石蜡切片,Masson染色及Desmin免疫组织化学法观察比较不同组别小鼠肝纤维化病理变化,RT-PCR及免疫组织化学方法检测不同时间点各组小鼠肝组织内β-catenin的表达及定位.结果 Masson染色结果显示,对照组肝汇管区结缔组织内及血管壁有少量细小纤维,实验组小鼠肝脏汇管区和中央静脉及其周围胶原纤维增多;随损伤时间延长纤维增生愈加明显.Desmin免疫组织化学结果显示,各组别均有阳性表达,但损伤组各时间点desmin阳性表达细胞数明显高于对照组(P<0.05 或P<0.001).RT-PCR结果显示,CCl4损伤1周后肝内β-catenin mRNA水平与对照组相比无明显差别(P＞0.05),损伤4周及8周β-catenin mRNA水平则明显下降,与对照组相比差异均有显著性(P<0.01).免疫组织化学结果显示,对照组β-catenin弱表达于肝细胞膜及胆管上皮细胞膜和胞质,而损伤组β-catenin阳性反应主要定位于肝内增生的细胞团及新生胆管上皮细胞质,各组间积分吸光度值差别有显著性(P<0.01).结论 CCl4诱导的肝纤维化过程中β-catenin mRNA表达与蛋白表达不同步,阳性表达细胞主要为新生的细胞团及胆管上皮细胞.     

GRP78在胃癌生长中的作用*

 目的： 探讨内质网分子伴侣葡萄糖调节蛋白78（GRP78）参与胃癌细胞生长的作用。方法: 回顾性分析34例胃癌及34例癌旁组织,利用组织微阵列技术,构建组织阵列,采用免疫组织化学技术SP法检测该阵列中GRP78蛋白在胃癌及其癌旁组织中表达情况。利用Western blotting检测人体胃癌组织、癌旁组织（肿瘤旁1 cm处）和正常组织（远离肿瘤≥10 cm处）中GRP78蛋白的表达情况。利用Western blotting检测人胃癌细胞SGC7901和过表达GRP78的SGC7901-H78细胞（稳定转染GRP78）中GRP78和生长相关蛋白cyclin D1的表达情况。结果: (1) 免疫组织化学检测发现：人体胃癌组织中GRP78表达水平明显高于癌旁组织和正常组织,且与性别、分化程度相关（P<0.05）; (2) Western blotting检测发现：胃癌组织中GRP78蛋白的表达明显高于癌旁组织和正常组织;(3)  Western blotting检测发现： 相对于SGC7901细胞,SGC7901-H78细胞中GRP78蛋白的表达明显升高,同时检测生长相关蛋白cyclin D1的表达发现,随着GRP78表达的上调,cyclin D1蛋白表达增加。结论: GRP78蛋白的高表达可能参与了胃癌细胞的生长。     

Glucose regulated protein 78 (GRP78) is overexpressed in colorectal carcinoma and regulates colorectal carcinoma cell growth and apoptosis

Glucose regulated protein 78 (GRP78) plays an important role in the development and progression of cancer. However, the role of GRP78 in colorectal carcinoma still remains unclear. In this study, using immunohistochemistry and RT-PCR, we explored the expression patterns of GRP78 in colorectal carcinoma. We knocked down GRP78 expression in RKO cells using shRNA-GRP78. Apoptosis and proliferation assay were performed. We found increased GRP78 expression with progression along the normal tissue–adenoma–carcinoma sequence, while there was no difference in the relative mRNA expression of GRP78 among normal, adenoma and colorectal carcinoma. The shRNA-GRP78 plasmid caused a significant reduction of GRP78 expression at both mRNA and protein levels. Furthermore, knockdown of GRP78 not only efficiently suppressed proliferation of RKO cells, but also induced early apoptosis of cells. In conclusion, our study demonstrated that GRP78 may play some important roles in the development and progression of colorectal carcinomas. The expression of GRP78 is associated with the enhanced proliferation of colorectal carcinoma cells and with their resistance to apoptosis.     

Two polymorphisms in the epithelial cell-derived neutrophil-activating peptide (ENA-78) gene

Increased expression of epithelial cell-derived neutrophil-activating peptide (ENA-78) has been reported in several immune and inflammatory conditions suggesting its role in inflammatory response. We have identified two single nucleotide polymorphisms in the promoter and exon 2 of the ENA-78 gene by scanning the full length gene using DHPLC DNA fragment analysis and DNA sequencing. The polymorphism at position +398 (A/G from the first ATG codon) in exon 2 results in a synonymous substitution not resulting in an amino acid change. The promoter polymorphism was found at position -156 (C/G from the first ATG codon). An assay was designed for the detection of the polymorphisms using SNapshot ddNTP primer extension, followed by capillary electrophoresis (ABI 3100). Allele and genotype frequencies for the promoter -156 polymorphism are presented for 107 healthy Spanish and 54 UK Caucasians. Frequencies for the exon 2 polymorphism are also presented for 63 UK Caucasians.     

Expressions and significance of CXC chemotactic factors about GROα, ENA-78 and NAP-2 at rat asthma

Objective To observe the expressions of grouth-related oncogen (GRO)α, epithelial neutrophil activating protein-78 (ENA-78) and neutrophil-activating peptide-2 (NAP-2) of rat asthma. And to investigate the role of neutrophil in the pathogenesis of asthma exacerbation. Methods In this experi-ment, the rat model of asthma were randomly divided into two groups on average, including asthma group and control group. Levels of ENA-78 at blood neutrophil were detected by flow cytometry method. The ex-pressions of GROα protein at bronchial wall and NAP-2 protein at blood neutrophil were detected by immuno-histochemieal method. Results Levels of GROα, ENA-78 and NAP-2 proteins in asthma group [0.138 ±0.009(A value), 97.65±13.99(MFI), 0.198±0.016(A value), respectively]were significantly higher than those in control group[0.077±0.010(A value), 50.79±8.66(MFI), 0.079±0.015(A value), re-spectively], all P < 0.01. Conclusion Levels of GROα, ENA-78 and NAP-2 were increased at rat asth-ma. They may be participate in inflammation of asthma exacerbation. Neutrophil may promote inflammatory cells influxing into airway wall via increasing synthesis of CXC chemotactic factors.     

子宫颈癌中MHC-Ⅰ类分子抗原呈递相关蛋白GRP78、CRT、ERP57表达及意义

目的 探讨子宫颈炎组织和子宫颈癌组织中GRP78、CRT、ERP57蛋白与HPV16型感染在维吾尔族和汉族妇女中的表达及相关关系.方法 采用免疫组化SP法和PCR技术检测子宫颈炎组织69例(汉族38例,维吾尔族31例)以及子宫颈癌组织124例(汉族36例,维吾尔族88例)中GRP78、CRT、ERP57蛋白的表达和HPV16的感染情况.结果 HPV16型感染在子宫颈癌中的阳性表达率(31.45%)高于宫颈炎组(11.59%),差异有统计学意义(P<0.05).GRP78、CRT、ERP57 3种蛋白在宫颈癌中的表达(95.16%、90.32%、90.32%)均高于与其各自在宫颈炎中的表达(89.96%、50.72%、49.28%),差异均有统计学意义(P<0.05).GRP78和CRT在汉族妇女宫颈癌中的表达(100.00%、97.22%)均高于各自在汉族妇女宫颈炎中的表达(81.58%、39.47%),并且差异均有统计学意义(P<0.05).CRT和ERP57在维吾尔族妇女宫颈癌中的表达(87.50%、94.32%)均高于各自在维吾尔族妇女宫颈炎中的表达(64.51%、35.48%),并且差异均有统计学意义(P<0.05).除此之外,仅ERP57在维吾尔族妇女宫颈癌中的表达(94.32%)高于其在汉族宫颈癌中的表达(80.56%),差异有统计学意义(P<0.05).而通过对GRP78、CRT、ERP57蛋白与HPV16的相关性分析发现:GRP78、CRT、ERP57蛋白在维吾尔族妇女宫颈癌中的阳性表达率与HPV16型感染均存在正相关关系(r=0.278,P<0.05;r=0.429,P<0.05;r=0.222,P<0.05).结论 通过对GRP78、CRT、ERP57 3种蛋白和HPV16联合检测,可能作为检查子宫颈癌的标记物,提高宫颈癌的检出率;ERP57蛋白在宫颈癌中的表达存在民族间差异.     

GRP78 inhibits macrophage adhesion via SR-A

Class A scavenger receptor （SR-A） plays an important role in macrophage adhesion. However, the underlying mechanism remains unclear. We previously found that 78 kDa glucose-regulated protein （GRP78） inhibited SR- A-mediated ligand internalization into macrophage by binding to SR-A. The aim of the study was to investigate whether GRP78 could regulate SR-A-mediated cell adhesion. We demonstrated that GRP78 bound directly to SR-A by fluorescence resonance energy transfer （FRET） assay. Overexpression of GRP78 inhibited macrophage adhesion via SR-A. These results suggest that GRP78 may act as an inhibitor of macrophage adhesion via SR-A.     

Expression of autocrine motility factor receptor (AMFR) in human breast and lung invasive micropapillary carcinomas

The aim of this study was to evaluate the clinicopathological significance of autocrine motility factor receptor (AMFR) expression in a variety of human invasive micropapillary carcinomas (IMPC). AMFR expression was compared in 111 samples of a variety of human IMPCs which had intrinsic non-micropapillary components and with 26 cases of control pulmonary adenocarcinoma (CPA, carcinoma without an IMPC component) by immunohistochemistry (IHC). In the 137 cases analysed, AMFR expression was significantly elevated in the IMPC components compared to the non-IMPC components (p = .005) and normal tissues (p < .001). AMFR expression was also higher in the IMPC samples compared to their intrinsic non-IMPC components (p = .0234). Between the 69 cases of lung IMPC and 26 cases of CPA, AMFR expression was notably higher in the IMPC components than in the CPA components (p = .0455). However, there was no significant difference between the non-IMPC components in the lung and the CPA components (p = .4584). Moreover, in breast cancer, elevated AMFR expression was not significantly correlated with mixed type or pure type IMPC (p = .5969) or with age, gender, T stage, or lymph node metastasis (LNM). Between IMPC and CPA of the lung, there was no statistical significance in age, T stage, and LNM, where AMFR expression was higher in IMPC (p = .0071). Thus this study demonstrated that AMFR was overexpressed in a variety of human IMPC components compared with non-micropapillary components. This suggests that AMFR expression is a potential new prognostic indicator for different types of human IMPC, which might thus be a new therapeutic target.