桔梗皂苷对慢性支气管炎小鼠气道重塑的干预作用研究

探讨桔梗皂苷(kikyosaponin,简称KS)对慢支小鼠气道重塑的干预作用机制.将50只健康小鼠分成正常对照组,模型组和桔梗皂苷低、中、高剂量组,除正常组外,其余4组动物均采用烟熏加浓氨水吸入法建立慢支模型,然后分别用药物进行治疗.实验结束后,取各组小鼠肺组织进行石蜡制片,苏木精-伊红(HE)染色分析气管管壁厚度变化,Masson染色分析肺组织胶原沉积,采用Image-Pro Plus 6.0图像分析软件测量总管壁厚度(WAt/Pbm)、内壁厚度(WAi/Pbm)、平滑肌厚度(WAm/Pbm)以代表气道重塑指标;生化法检测肺组织中羟脯氨酸(Hyp)含量;免疫组织化学染色法观察MMP-9在肺组织中的表达;蛋白印迹术分析MMP-9和TIMP-1在肺组织中的表达水平.HE染色显示,模型组肺组织中支气管明显狭窄,管腔及肺泡中含有大量炎性分泌物,且气道重塑的三项指标均异常增高;免疫组化和三色法显示,模型组小鼠支气管和肺泡细胞中有大量的MMP-9表达,且有胶原蛋白增加明显,生化检测发现模型组小鼠肺组织中Hyp含量亦异常升高,且MMP-9和TIMP-1表达水平升高显著,与正常对照组相比,差异十分显著(P0.01).在连续用药30 d后,与模型组相比,桔梗皂苷各剂量组能明显抑制慢支小鼠支气管胶原纤维增生,改善气道重塑各项指标;减少MMP-9和TIMP-1表达水平,使气管重塑得到显著的减轻(P0.05,P0.01,P0.01).结果显示,桔梗皂苷可明显减轻慢支小鼠气道重塑的病理改变,其干预机制可能是通过清除慢支小鼠肺组织中的炎性因子和自由基的含量,同时又能抑制MMP-9和TIMP-1的表达,从而达到减少胶原蛋白的沉积以减轻气管狭窄而达到逆转气道重塑之目的.     

人脑胶质瘤中MMP-9的表达及意义

目的：探讨MMP-9在人脑胶质瘤中的表达及意义。方法：采用免疫组化S-P法检测45例人脑胶质瘤及32例瘤旁正常脑组织中MMP-9蛋白的表达。结果：MMP-9在正常脑组织、低度恶性及高度恶性脑胶质瘤组织中的阳性表达率分别为15.6%,45.8%,81.0%。结论：MMP-9蛋白表达与胶质瘤的发生和恶性进展有关。     

磷酸钙法将MMP-2，MMP-3双基因干扰载体转入HEK293T细胞的效率观察

检测用磷酸钙法能否将修饰后用于沉默MMP-2,MMP-3基因的慢病毒载体有效地转入到HEK293T细胞。利用磷酸钙法,MMP-2,MMP-3双基因干扰载体（MMP组）与对照质粒（对照组）被分别转染HEK293T细胞,在转染后的第24,48及72h,通过计算绿色荧光蛋白（GFP）阳性细胞数的百分比来判断转染效率。转染24h后,两组细胞均生长状况良好,荧光显微镜下观察,已有大量绿色荧光出现,计算MMP组转染效率为（91．5±6．2）％,对照组转染效率为（80．3±4．7）％;转染后48—72h,两组感染效率均未见明显下降。与对照相比,插入了MMP-2,MMP-3 shRNA模板序列的慢病毒载体没有降低磷酸钙法转染HEK293T细胞效率,反而有所增高。     

IGF-Ⅱ and IGFBP-1 reversely regulate blastocyst implantation in mouse

Insulin-like growth factor (IGF)-Ⅱ and IGF binding protein (IGFBP)-1, members of IGF family, are important in the cyclic development of endometrium and the blastocyst implantation. In the present study, the indirect immunofluorescence showed that IGF-Ⅱ and IGFBP-1 were specifically expressed at the maternal-fetal interface. In a co-culture system, IGF-Ⅱ significantly enhanced the attachment and outgrowth of the blastocyst on monolayer of uterine epithelial cells, while IGFBP-1 did not affect the blastocyst attachment, but markedly inhibited the blastocyst outgrowth. The results of zymography showed that IGF-Ⅱ enhanced the activities of MMP-2 and MMP-9, while IGFBP-1 did not affect the activities of MMP-2 and MMP-9. In conclusion, the equilibrium between the invasion of trophoblast and the inhibition of deciduas may be regulated by the interaction between the IGF-Ⅱ-expressing invading cytotrophoblast and maternal deciduas-derived IGFBP-1.     

四妙勇安汤抑制动脉粥样硬化易损斑块炎症反应机制

 通过利用复合因素制备兔动脉粥样硬化（AS）易损斑块模型,研究四妙勇安汤对AS易损斑块的影响及作用机制。随机将40只日本大耳白兔分为正常组10只,实验组30只。正常组予普通饲料,实验组利用复合因素制备兔主AS易损斑块模型,8周时实验组随机分为模型组、辛伐组、四妙组,继续予以高脂饮食;各给药组开始给药至24周取材,观察主动脉的病理形态,检测血清MCP-1、ICAM-1、CRP含量和斑块内HSP60、TNF-α、NF-κB表达。结果显示,模型组内膜明显增厚、纤维帽较薄,斑块内有大量脂质沉积和炎症细胞浸润,血清MCP-1、ICAM-1、CRP随时间延长呈上升趋势,主动脉斑块TNF-α、HSP60、NF-κB表达增加。在给药后各时间点四妙组血清MCP-1、ICAM-1水平均低于模型组（P<0.05或P<0.01）,对CRP水平影响不明显。斑块内TNF-α、HSP60、NF-κB阳性面积百分比均低于模型组（P<0.01）,ICAM-1 MRNA表达低于模型组（P<0.01）。由此得出,四妙勇安汤能够稳定AS易损斑块,其作用可能是通过抑制炎症反应实现的。     

MMP-2 PEX结构域的最佳表达条件

为研究MMP-2 PEX结构域在大肠杆菌BL21(DE3)中的最佳表达条件,直接从构建好的表达载体PET-MMP2 PEX中高效表达MMP-2 PEX结构域蛋白.经SDS-PAGE与软件分析,确定其最佳表达时间为2 h,菌液密度值为0.6时,所表达的蛋白含量最高,而IPTG在一定浓度范围内对表达含量不产生影响.     

MCP-1在子痫前期患者胎盘组织及血清中的表达及意义

通过比较单核细胞趋化蛋白-1（MCP-1）在正常孕妇和子痫前期患者胎盘及血清中的表达,探讨MCP-1在子痫前期发病机制中的作用。选取30例正常晚孕妇女、30例轻度子痫前期患者和30例重度子痫前期患者进行研究。采用免疫组织化学S P法检测MCP-1在胎盘组织中的表达;采用酶联免疫吸附法（ELISA）检测血清MCP-1水平。结果：子痫前期患者胎盘组织和血清中MCP-1的表达均高于正常妊娠组,差异有统计学意义（P〈0.01）,且随着病情加重而递增;子痫前期患者胎盘组织MCP-1表达与血清MCP-1水平呈显著正相关（r=0.725,P〈0.01）。结果表明,MCP-1在子痫前期患者胎盘与血清中的表达均升高,提示MCP-1在子痫前期的发生、发展中可能起着重要的作用。     

Expression of matrix metalloproteinase-28 in human normal cytotrophoblast cells and a choriocarcinoma cell line,JEG-3

There are striking similiarities present between the behavior of invasive placental cells and that of invasive cancer cells. Matrix metalioproteinases (MMPs) are one of the most important mediators. MMP-28, the new member ofMMPs, was sequenced and identified recently. Expression of MMP-28 mRNA and protein in the cytotrophoblast cells anda choriocarcinoma cell line, JEG-3 cell, was conducted by zymography, RT-PCR and Northern blot. There is MMP-28mRNA expression in both the cytotrophoblast cells and JEG-3 cells by RT-PCR. The activity of MMP-28 in cytotrophoblast cells was significantly weaker than that in JEG-3 (P < 0.01) by zymography. Furthermore, mRNA expression of MMP-28 was significantly stronger (P < 0.001) in JEG-3than in human cytotrophoblast cells in a time-dependent way by Northern Blot. Our results suggest that MMP-28 may play a role in some of the tissue-remodeling events associated with normal pregnancy and tumor progression.``     

影响根癌农杆菌介导的香蕉基因转化早期的主要因素

用含质粒载体pCAMBIA2301的根癌农杆菌（Agrobacterium inoculation）转化“641”香蕉（Musa AAA Cavendish subgroup cv.64-1）的薄片外植体，通过测定GUS基因瞬时表达率，对转化早期的主要影响因素进行了研究。结果表明：农杆菌EHA105较适合于介导转化，将薄片置于固体高渗培养基上培养4h后，通过真空减压法使农杆菌接种于其上，获得了较高的瞬时表达率（41.67%），是对照（8.33%）的5倍，农杆菌悬液稀释5倍用于转化较好，共培养3d为宜，薄片外植体越靠近根部，瞬时表达率越高。而外植体预培养会降低瞬时表达率。     

Effect of matrix metallo-proteinase-26 (MMP-26) during embryo implantation in the mouse

Matrix metalloproteinase-26 (MMP-26, endometase and matrilysin-2), a novel member of the MMPs family, is detected not only in the placenta and uterus, but is widely expressed in malignant tumors from different sources as well as in diverse tumor cell lines. However, the function of MMP-26 in the reproductive system has never been reported. Expression of MMP-26 in mouse embryos and the function of the MMP-26 antibody during mouse embryo implantation was examined for the first time by injecting the uterine horn, immunohistochemistry,in situ hybridization, co-culture of mouse blastocysts and uterine monolayer epithelial cells, Western blot, RT-PCR, Northern blot and zymography. Our results show that there is strong expression of MMP-26 mRNA and protein in the mouse embryo. Furthermore, the MMP-26 antibody dramatically inhibited mouse embryo implantation and significantly inhibited adhesion and outgrowth of mouse blastocysts onin vitro uterine monolayer epithelial cells. At the same time, the MMP-26 antibody inhibited the expression of integrin αV mRNA and protein in a dose-dependent manner. These data suggest that MMP-26 may play a role in some of the tissue-remodeling events associated with the invasion of the endometrium by trophoblast cells and facilitate successfully embryo implantation.     

盐酸埃克替尼对ACC-M细胞MMP-2、E-cadherin蛋白表达的影响

盐酸埃克替尼是一种表皮生长因子受体酪氨酸激酶抑制剂,利用免疫细胞化学方法和间接免疫荧光联合流式细胞技术,检测它在体外对人涎腺腺样囊性癌细胞株（ACC-M）MMP-2、E-cadherin 蛋白表达的影响。结果表明:盐酸埃克替尼可降低 MMP-2蛋白表达,增加 E-cadherin 蛋白表达,可抑制人涎腺腺样囊性癌 ACC-M 细胞的浸润、转移。     

不同剂量甾体激素对大鼠动情周期及非动情周期子宫中MMP-2活性的影响

本实验采用酵谱(Zymography)法研究了不同剂量的甾体激素对动情周期及切除双侧卵巢的大鼠子宫中MMP—2活性的影响,结果显示,4种不同剂量的雌二醇(0.05mg,0.1mg,0.3mg0.5mg)对动情后期及切除双侧卵巢后的大鼠子宫中MMP—2均有促进作用,其中67kD,66kD和64kL MMP—2的活性变化与雌二醇剂量变化呈正比关系.动情后期大鼠子宫中MMP—2活性.4种不同剂量(0.2mg,0.4mg,0.6mg和0.8mg)的孕酮对处于动情前期大鼠子宫中MMP—2活性没有显著作用,但是对切除双侧卵巢14天的大鼠子宫中MMP—2活性有明显抑制作用,其活性变化与孕酮剂量成反比.结果揭示:大鼠体内甾体激素分泌量的变化导致子宫MMP—2活性的变化,甾体激素对MMP—2活性的影响与子宫内膜的生理状态密切相关。     

RhoC和MMP-9蛋白在结直肠癌中的表达及意义

探讨RhoC和基质金属蛋白酶-9（MMP-9）蛋白在结直肠癌发生发展中的作用。选取新鲜结直肠癌标本作为实验组,正常新鲜结肠粘膜组织作为对照组,应用流式细胞术分别检测RhoC和MMP-9的表达,检测其在不同临床病理特征之间的关系。结果表明,RhoC和MMP-9在结直肠癌组织中高表达（P〈0．05）,且其表达均与Dukes分期、浸润深度及炎细胞浸润相关（P〈0．05）,MMP-9的表达与结直肠癌的淋巴结转移密切相关（P〈0．05）,Rhoc和MMP-9在结直肠癌中的表达呈正相关（r=0．3430,P=0．0420）。流式细胞术联合检测RhoC和MMP-9蛋白的表达有望成为判断预后的重要指标。     

64排CT冠脉序列扩大增强扫描诊断大血管疾病的临床价值

目的:运用64排螺旋CT冠脉序列扩大增强扫描结合回顾性心电门控技术研究在诊断大血管疾病方面的临床价值。方法:选择31例临床怀疑主动脉疾病患者,全部行64排螺旋CT平扫及冠脉序列扩大增强扫描加后续重建,观察主动脉夹层包括范围、破口位置、内膜病变,主动脉瘤及有无合并其他先天性疾病等,总结和归纳病灶的影像学表象及特征。结果:大血管疾病(如主动脉粥样硬化、主动脉夹层及并发的相关疾病、主动脉瘤及先天性疾病等)的影像表现在图像上显示清楚,能够确切显示微小病变结构,克服了搏动伪影等多种干扰因素。结论:运用64排螺旋CT冠脉序列扩大增强扫描结合回顾性心电门控技术可以在很大程度上克服大血管的搏动伪影,清晰显示血管壁、管腔以及病变波及的范围等,从而作出精准的诊断,对临床抢救及治疗发挥重要作用。     

Activation of proteinkinase ERK mediates induction of macrophage MMP-12 by OxLDL

The present study was undertaken to investigate the effect of oxidized low density lipoprotein (oxLDL) on the expression of maerophage matrix metalloproteinase-12 (MMP-12), and the possible mechanisms. Activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was detected by Western blot analysis.Enzymatic activity of MMP-12 was determined by β-casein zymography. RT-PCR analysis was used to measure the mRNA expression level of MMP-12. OxLDL-stimulated macrophages produced increased casein-degrading activities and oxLDL also significantly increased the mRNA level of MMP-12 in a dose-dependent manner.OxLDL stimulated the phosphorylation of ERK1/2 in macrophages. The use of the specific inhibitor indicated that the ERK1/2 signaling pathway was required for the induction of MMP-12.These data demonstrated that oxLDL induced MMP-12 expression in macrophages through an ERK1/2-dependent pathway.     

进展性动脉粥样硬化兔模型的建立方法

目的探究快速、有效地建立进展性动脉粥样硬化兔模型的方法。方法15只新西兰大耳白兔随机分为颈总动脉球囊损伤组（n=8）和假手术组（n=7）,两组均饲喂高脂饮食,14周后结束实验,对颈总动脉和其他血管节段（胸主动脉、主动脉根部和腹主动脉）作病理观察,计算内膜增生面积／内弹力膜围绕面积（％）、平均内膜厚度／内弹力膜围绕面积半径（％）和最大内膜厚度／内弹力膜围绕面积半径（％）,以评价斑块大小;确定病变组织形态学分型（美国心脏协会推荐）以评价斑块病变严重程度。测定实验前后的动物血脂水平。结果高脂喂养14周,在兔主动脉各血管节段中,斑块尺寸由大到小顺序：胸主动脉〉主动脉根部〉腹主动脉。胸主动脉虽在单纯高脂作用的各血管节段中斑块尺寸最大、病变进展最快,但仍主要停留在以内膜细胞外脂质池出现为特点的过渡性病变,较少发展至以脂质核心形成为特征的进展性病变。球囊损伤术使病变最轻微、进展最缓慢的颈总动脉斑块尺寸明显超过胸主动脉（P〈0．01）,并使其发展为进展性动脉粥样硬化病。结论在高脂饮食基础上,球囊损伤法能够快速、有效地建立以脂质核心形成为特征的进展性动脉粥样硬化病变兔模型,为进一步探究不稳定性斑块的形成机制及干预措施提供了适用的动物疾病模型。     

高血压大鼠血管平滑肌细胞GRP94和CHOP的表达及替米沙坦的影响

观察替米沙坦对高血压大鼠动脉血管平滑肌细胞内质网应激相关因子葡萄糖调节蛋白94(GRP94)和C/EBP同源蛋白(CHOP)的表达,以及血管中层厚度的变化的影响。将48只成年雄性SD大鼠随机等分为假手术对照组(对照组)、手术模型组和替米沙坦组。对照组分离腹主动脉,但不行狭窄术;模型组行腹主动脉狭窄术(TAC);替米沙坦组行腹主动脉狭窄术并给予每天替米沙坦1.5 mg/kg。喂养6周用颈动脉插管法测定各组大鼠的动脉压(MAP);利用图像分析软件测量血管中层的厚度。用免疫组织化学法和western-blot法检测GRP94和CHOP的表达水平。结果:①手术模型组大鼠的MAP显著高于相应的对照组和替米沙坦组,有统计学意义(P<0.05)。②手术模型组的GRP94表达量高于对照组和替米沙坦组,有统计学意义(P<0.05)。③手术模型组的CHOP蛋白表达量高于对照组和替米沙坦组,有统计学意义(P<0.05)。④手术模型组大鼠血管平滑肌中层厚度厚于对照组和替米沙坦组,有统计学意义(P<0.05)。说明腹主动脉狭窄致高血压可引起血管平滑肌细胞内质网应激反应,导致GRP94表达及CHOP表达的增加。替米沙坦可能通过减轻内质网应激,逆转高血压所致的血管平滑肌细胞的损伤作用,对血管平滑肌细胞有保护作用。     

大蒜素对肺腺癌细胞侵袭生物学行为影响的初步研究

通过MTT法、细胞粘附试验、Transwell细胞迁移和侵袭试验检测不同浓度的大蒜素对肺腺癌细胞的活性、粘附、迁移与侵袭能力的变化;(RT-qPCR)逆转录-定量聚合酶链反应检测不同浓度的大蒜素对TIMP/MMP平衡的影响.本研究中发现大蒜素可呈剂量依赖性抑制肺腺癌细胞的活性、粘附、迁移和侵袭能力.大蒜素主要降低基质金...     

Correlation of matrix metalloproteinase－2, －9, tissue inhibitor－1 of matrix metalloproteinase and CD44 variant 6 in head and neck cancer metastasis

This study aimed to explore the molecular mechanism in tumor invasion and metastasis. The expression of matrix metalloproteinase-2, -9 (MMP-2, MMP-9), tissue inhibitor-1 of matrix metalloproteinase (TIMP-1), cell adhesion molecule 44 variant 6 (CD44v6), HER2/neu and p53 was investigated in 154 patients with head and neck squamous cell carcinoma (SCC) by ABC and ImmunoMax immunohistochemical method. Their clinical relevance and correlation were analysed. The expression of MMP-2, MMP-9, TIMP-1, CD44v6, HER2/neu and p53 was found in cancer cells in 87.01%, 85.71%, 68.18%, 98.05%, 55.19% and 50.65% cases respectively. Linear regression and correlation analysis revealed that there was close positive relationship (P<0.05) between the expression of MMP-2 and MMP-9, TIMP-1 and CD44v6, HER2/neu and MMP-9, MMP-2 and p53. Up-regulation of MMP-2 was accompanied by advanced T stage (P<0.01). There was also a trend of MMP-2 expression being related with tumor metastasis. Increased expression of HER2/neu was found in patients with tumor recurrence(P<0.05). The expression of TIMP-1 was higher in laryngeal cancer than that in pharyngeal cancer, and higher in keratinizing and non-keratinizing SCC than that in basaloid SCC(P<0.05). These findings suggested that MMP-2 and MMP-9, HER2/neu and MMP-9, MMP-2 and p53 had a coordinate function in aggression of tumor; that MMP-2 had a more important function than MMP-9 in tumor invasion and metastasis; and that HER2/neu might serve as a biomarker for poor prognosis in HNSCC.