Notch信号通路在宫颈癌患者外周血单个核细胞的表达及其临床意义

目的探讨Notch信号通路在宫颈癌发病机制中的作用。方法采用实时荧光定量PCR和流式细胞术,分别从基因转录和蛋白表达水平检测30例宫颈癌患者和30例健康对照者的外周血单个核细胞（PBMCs）中Notch 1,Notch 2,Notch 3,Notch 4,以及Notch的配体Jagged 1,Jagged 2,Delta 1,Delta 3,Delta 4的表达。结果宫颈癌患者PBMCs中的Notch 1,Notch 2,Jaggcd 1,Jagged 2,Delta 1,Delta 4的mRNA和蛋白的表达均明显高于健康对照组（P〈0．01）,Notch 3,Notch 4,Delta 3的基因和蛋白的表达在两者中差异均无统计学意义（P〉0．05）。结论Notch信号通路的表达在宫颈癌的发病过程中存在一定的变化,可能参与宫颈癌的发生发展,从而为官颈癌的早期诊断和临床治疗提供新线索。     

Notch信号通路在小儿哮喘患者外周血单个核细胞的表达及其临床意义

目的初步探讨Notch信号通路相关分子在小儿哮喘患者中的表达及其临床意义。方法实时荧光定量聚合酶链反应和流式细胞术,分别从基因转录和蛋白表达水平检测20例小儿哮喘患者和20例健康对照者的外周血单个核细胞（PBMC）中Notch1、Notch2、Notch3、Notch4以及Notch的配体Jagged1、Jagged2、DLL1（Delta-like1）、DLL3（Delta-like3）、DLL4（Delta-like4）的表达。结果小儿哮喘患者PBMC中的Notch1、Notch3、Jagged1、Jagged2、Delta1的mRNA和蛋白的表达均明显高于健康对照组（P〈0.01）,Notch2、Notch4、Delta3和Delta4的基因和蛋白的表达在两者中未见明显差异（P〉0.05）。结论 Notch信号通路的表达在小儿哮喘发病过程中有显著改变,可能参与小儿哮喘的发生发展,为深入研究小儿哮喘的发病机制提供思路。     

Notch配体在哮喘小鼠肺中的表达

目的 明确Notch配体（Delta1J、agged1及Jagged2）在小鼠哮喘模型肺中的表达水平，探讨其在哮喘发病机制中的作用。方法 20只BALB/c小鼠随机分为正常对照组和哮喘组（每组10只）。通过卵清蛋白腹腔注射及雾化吸入建立哮喘模型。采用半定量PCR检测Notch配体（Delta1J、agged1及Jagged2）mRNA在哮喘组和正常对照组小鼠肺组织和肺细胞中的表达水平。结果 与正常对照组比较，哮喘小鼠肺组织及肺细胞的Delta1 mRNA表达显著降低（P均〈0.001），Jagged1和Jagged2 mRNA表达显著升高（P〈0.001或0.05）。结论 在哮喘的发病环节中，Notch配体Delta1表现为抑制作用，Jagged1和Jagged2表现为促进作用。     

致敏小鼠树突状细胞和T细胞Notch配体/受体的表达及大剂量过敏原的影响

目的探讨致敏小鼠Notch信号表达缺陷及大剂量过敏原对其影响。方法RT-PCR法分别检测致敏及正常小鼠树突状细胞Notch配体及T细胞Notch受体的mRNA表达情况,并观察大剂量过敏原作用对其表达的影响。结果（1）致敏小鼠树突状细胞Notch配体Jagged1,Jagged2和Delta1的mRNA表达均显著低于正常小鼠,致敏小鼠T细胞Notch1和Notch3的mRNA表达也显著低于正常小鼠（P〈0.05）。（2）10mg/ml的OVA作用下,Jagged1和Notch3的mRNA表达水平较对照组显著升高（P〈0.05）。结论致敏小鼠Notch信号表达缺陷,大剂量过敏原（10mg/ml的OVA）可上调Jagged1和Notch3表达。     

TIF3转基因CHO和COS7细胞株的构建与鉴定

目的：构建TIF3转基因CHO和COS7细胞株并对其构建的细胞株进行鉴定。方法：采用磷酸钙介导转染技术和G418细胞筛选法，以pcDNA3.1/V5-His-TOPO为表达载体，构建TIF3转基因CHO和COS7细胞株；并应用Western Blot技术对转基因表达产物进行分析与鉴定。实验设计分无转染组、载体转染组和目的基因转染组。结果：在7株转染的CHO细胞株中，有3株具有高效稳定表达的TIF3编码蛋白质；在4株转染的COS7细胞株中有2株同样具有高效稳定的TIF3编码蛋白质表达。结论：本研究成功地构建了3株TIF3转基因CHO细胞株和2株TIF3转基因COS7细胞株，它们对今后镉化物相关癌基因的发现及其他生物学功能的研究具有重要的应用价值。     

Notch信号影响耐阿霉素骨肉瘤细胞干细胞特性

目的:探讨耐阿霉素骨肉瘤细胞干细胞特性的改变,并探究Jagged1/Notch1信号通路在其中所起的作用.方法:构建耐阿霉素骨肉瘤细胞,CCK-8实验检测细胞耐药性;Western Blot检测骨肉瘤细胞中干细胞相关基因(Oct4、Sox2、NANOG)蛋白表达水平,及Jagged1、NICD1及Notch信号通路下游...     

血管生成中DLL4/Notch信号通路的作用及其与VEGF、NO的关系

Notch信号通路在生物进化过程中高度保守,广泛存在于各种生物体中.哺乳动物的Notch信号通路由4种Notch受体(Notch1～4)和5种配体(Jagged1、Jagged2、 DLL1、DLL3和DLL4)组成,与血管发生及功能形态关系密切.     

Notch信号和毛细胞发生

细胞分化的最终方向依赖于各种因素的相互作用。包括细胞增殖、迁移、生长、分化。在多细胞生物发育中，需要通过邻近细胞间的相互作用，对各谱系细胞分化进行精细的调控。研究表明，Notch信号存在于脊椎动物前庭及耳蜗系统。被认为是与听毛细胞终极分化有关的细胞内部信号途径。Notch信号途径有3种作用方式：(1)旁侧抑制(2)谱系决定(3)边界形成。多细胞器官中细胞终极分化的机制之一就是由Notch信号及其配体Delta。serrate或Jagged介导的。     

CHO细胞中VMAT2的鉴定及应用

目的鉴定转染囊泡单胺转运体(vesicular monoamine transporter-2,VMAT2)基因在中国仓鼠卵巢细胞(Chinese hamster ovary cell,CHO)细胞中VMAT2的表达情况。方法采用细胞免疫荧光染色、RT-PCR、免疫电镜的方法,分别从形态学、分子生物学和微观亚细胞结构角度来确定VMAT2的表达。结果免疫荧光染色显示VMAT2-CHO表达明亮的绿色荧光;逆转录聚合酶链反应电泳显示VMAT2-CHO出现180 bp的阳性条带;VMAT2-CHO细胞中免疫胶体金颗粒分布在细胞核周膜性结构上。结论转染VMAT2基因的CHO细胞可以稳定且大量表达VMAT2,有望成为研究VMAT2功能与帕金森病关系的单一的细胞模型。     

Immunolocalization of notch signaling protein molecules in a maxillary chondrosarcoma and its recurrent tumor

Background

Notch receptors are critical determinants of cell fate in a variety of organisms. Notch signaling is involved in the chondrogenic specification of neural crest cells. Aberrant Notch activity has been implicated in numerous human diseases including cancers; however its role in chondrogenic tumors has not been clarified.

Method

Tissue samples from a case of primary chondrosarcoma of the maxilla and its recurrent tumor were examined immunohistochemically for Notch1-4 and their ligands (Jagged1, Jagged2 and Delta1) expression.

Results

Both primary and recurrent tumors were histopathologically diagnosed as conventional hyaline chondrosarcoma (WHO Grade I). Hypercellular tumor areas strongly expressed Notch3 and Jagged1 in spindle and pleomorphic cells suggesting up-regulation of these protein molecules at sites of tumor proliferation. Expression patterns were distinct with some overlap. Differentiated malignant and atypical chondrocytes demonstrated variable expression levels of Jagged1, and weak to absent staining for Notch1, 4 and Delta1. Protein immunolocalization was largely membranous and cytoplasmic, sometimes outlining the lacunae of malignant chondrocytes. Hyaline cartilage demonstrated a diffuse or granular precipitation of Jagged1 suggesting presence of soluble Jagged1 activity at sites of abnormal chondrogenesis. No immunoreactivity for the other Notch members was observed. Calcified cartilage was consistently Notch-negative indicating down-regulation of Notch with cartilage maturation. Stromal components namely endothelial cells and fibroblasts variably expressed Notch1, 3 and Jagged1 but were mildly or non-reactive for the other members.

Conclusions

Results indicate that Notch signaling pathway may participate in cellular differentiation and proliferation in chondrosarcoma. Findings implicate Notch3 and Jagged1 as key molecules that influence the differentiation and maturation of cells of chondrogenic lineage.     

Notch signaling and ghost cell fate in the calcifying cystig odontogenic tumor

Notch signaling is an evolutionarily conserved mechanism that enables adjacent cells to adopt different fates. Ghost cells (GCs) are anucleate cells with homogeneous pale eosinophilic cytoplasm and very pale to clear central areas (previous nucleus sites). Although GCs are present in a variety of odontogenic lesions notably the calcifying cystic odontogenic tumor (CCOT), their nature and process of formation remains elusive. The aim of this study was to investigate the role of Notch signaling in the cell fate specification of GCs in CCOT. Immunohistochemical staining for four Notch receptors (Notch1, Notch2, Notch3 and Notch4) and three ligands (Jagged1, Jagged2 and Delta1) was performed on archival tissues of five CCOT cases. Level of positivity was quantified as negative (0), mild (+), moderate (2+) and strong (3+). Results revealed that GCs demonstrated overexpression for Notch1 and Jagged1 suggesting that Notch1-Jagged1 signaling might serve as the main transduction mechanism in cell fate decision for GCs in CCOT. Protein localizations were largely membranous and/or cytoplasmic. Mineralized GCs also stained positive implicating that the calcification process might be associated with upregulation of these molecules. The other Notch receptors and ligands were weak to absent in GCs and tumoral epithelium. Stromal endothelium and fibroblasts were stained variably positive.     

Effects of Xinfeng capsule on pulmonary function based on treg-mediated notch pathway in a rat model of adjuvant arthritis

OBJECTIVE:To observe the impact of xinfeng xapsule(XFC) on pulmonary function in a rat model of adjuvant arthritis(AA) and to investigate the mechanism of action.METHODS:Forty rats were randomly divided into four groups of ten:normal control(NC);model control(MC);tripterygium glycosides tablet(TPT);and xinfeng capsule(XFC).Except for the NC group,AA was induced in all rats by intracutaneous injection of 0.1 mL Freund’s complete adjuvant in the right paw on the 19th day.NC and MC groups were given(0.9%) physiological saline.The TPT and XFC groups were given TPT(10 mg/kg) and XFC(1.2 g/kg),respectively.Thirty days after administration,changes in paw edema(E),the arthritis index(AI),pulmonary function,levels of regulatory T-cells(Treg),ultrastructure of lung tissue,and expression of Notch receptors and ligands in lung tissue were observed.RESULTS:In the MC group,E and the AI were increased and pulmonary function significantly decreased;the structure of alveolar type-II cells was damaged;ratios of Treg in peripheral blood were reduced;and expression of Notch receptors such as Notch3 and Notch4 and ligands such as Delta1 in lung tissue were significantly increased whereas expression of Notch1,Jagged1 and Jagged2 were significantly decreased.After intervention with XFC,E and the AI were decreased;pulmonary function was enhanced;the structure of alveolar type-II cells was improved;and expression of Treg,Notch1,Jagged1,Jagged2 was elevated,whereas that of Notch3,Notch4 and Delta1 was reduced.CONCLUSION:XFC can not only inhibit E and the AI and improve joint symptoms,it can also improve pulmonary function and reduce inflammation in lung tissue.These actions could be carried out through increases in the expression of Treg,Notch receptors(Notch1) and ligands(Jagged1,Jagged2),and reductions in the expression of Notch3,Notch4 and Delta1.These phenomena would reduce the deposition of immune complexes and the inflammatory response in lung tissue,thereby improving joint symptoms and pulmonary function.     

重组CHO细胞中不同启动子对含MAR表达载体转基因表达的影响

目的 分析在重组CHO细胞中不同启动子对含核基质结合区(MAR)表达载体转基因表达的影响.方法 PCR扩增CMV启动子及β-珠蛋白MAR,构建含β-珠蛋白MAR表达载体pCAT1,随后将CMV启动子替代pCAT1上SV40启动子构建CMV启动子驱动的表达载体pCAT2.pCAT1、pCAT2不含MAR的对照载体同时转染CHO细胞,G418筛选稳定转化的细胞株,酶联免疫吸附试验(ELISA)分析氯霉素乙酰转移酶(CAT)基因的表达水平.结果 含MAR表达载体转染的细胞CAT酶表达量比不合MAR的pCATG和pCAT3载体转染的细胞高,分别提高2.14倍和1.25倍(P＜0.05);而由SV40启动子驱动含MAR表达载体pCAT1转染的细胞CAT酶表达水平明显比由CMV启动子驱动的pCAT2载体高3.26倍(P＜0.05).结论 在稳定重组CHO细胞中MAR能够提高转基因的表达水平,SV40启动子与MAR组合其启动效率优于CMV启动子与MAR组合.     

PTHrP和Notch双信号通路调控骨骺干细胞的增殖

目的 研究PTHrP、Notch双信号系统对骨骺干细胞增殖的调控作用.方法 体内实验:取24 h内新生大鼠股骨体外器官培养,分别用PTHrP信号系统激活剂PTHrP(1～34)和PTHrP受体竞争抑制物PTHrP(7～34),及Notch信号系统激活剂Jagged1/Fc和抑制剂DAPT处理,空白对照加PBS缓冲液,培养72 h后收集标本行HE、Brdu及免疫组化染色方法检测.体外实验:构建PTHrP重组质粒和RNAi慢病毒表达载体转染体外培养的骨骺干细胞,收集转染后细胞用免疫印迹检测.结果 与对照组相比,PTHrP(1～34)、Jagged1/Fc处理组静止区骨骺干细胞层长度在生长板全长中比值及Brdu阳性细胞率明显增高,促细胞增殖作用明显,而PTHrP(7～34)、DAPT处理组则抑制骨骺干细胞增殖.且PTHrP明显促进Notch信号通路配体Jagged1与受体NICD蛋白表达.结论 PTHrP、Notch信号通路均可促进骨骺干细胞增殖,且PTHrP可上调Notch表达进而促进骨骺干细胞的增殖.

Abstract：

Objective To study the regulation of the proliferation of epiphysis stem cells by the PTHrP (parathyroidhormone related peptide) and Notch signaling systems. Methods An organ culture system of femurs of SD rat in 24 h after birth was employed. PTHrP ( 1 - 34) was used as the activator of the PTHrP signaling pathway and PTHrP (7 - 34) as the antagonist of PTH (parathyroidhormone) -receptor. For Notch signaling system, Jaggedl/Fc was used as the activator and DAPT as its inhibitor. The femurs were cultured in DMEM ( Dulbecco's modified Eagle's medium)/F12 medium while phosphate buffered saline was used for the control groups. Hhematoxylin and eosin staining and bromodeoxyuridine analysis were used to analyze the length of the epiphysis stem cells zone and the proliferation of epiphysis stem cells. The expression of NICD (Notch intra-cellular domain) and Jaggedl were analyzed by immunohistochemistry. The epiphysis stem cells were transfected with the lentiviral vectors with rat PTHrP gene overexpression or inhibition properties, the cells transfected with the PGC-GFP-lentivirus or NC-GFP-lentivirus were used as control. Western blot was employed to detect the expression of NICD and Jaggedl genes. Results PTHrP (1 -34) and Jagged1/Fc could dramatically elevate the rate of epiphysis stem cells zone by the whole growth plate length measurement while PTHrP (7 - 34 ) and DAPT could decrease the rate. Brdu analysis also showed that the number of proliferative epiphysis stem cells could be up-regulated by the PTHrP (1 -34 ) or Jagged1/Fc signaling. By contrast, the treatment with PTHrP (7 -34) or DAPT reduced the number of proliferative epiphysis stem cells. Immunohistochemistry and Western blot showed a significantly elevated expression of NICD and Jagged1 when PTHrP signaling was activated while a reductive expression of NICD and Jagged1 when PTHrP signaling was inactivated. Conclusion Both of PTHrP and Notch signaling system could promote the proliferation of epiphysis stem cells. And the PTHrP signaling can stimulate Notch signaling to promote the proliferation of epiphysis stem cells.     

Notch-Jagged/Delta信号通路在佐剂关节炎大鼠肺功能 降低中的作用

目的：观察佐剂关节炎(AA)大鼠肺功能、肺组织Notch信号通路的变化，探讨AA大鼠肺功能降低的机制。 方法：将30只大鼠随机分为正常组和模型组，每组15只，向模型组大鼠右后足跖皮内注射弗氏完全佐剂0.1 mL致 炎，复制成AA模型。致炎30 d后，观察两组大鼠足跖肿胀度、关节炎指数、肺功能、肺病理形态学及肺组织Notch受 体/配体表达的变化。结果：与正常组相比，模型组大鼠足跖肿胀度、关节炎指数、肺功能参数0.3 s内平均呼气流量 (FEV0.3/FVC)、肺泡炎程度、肺组织Notch3和Notch4及Jagged2的表达升高(P<0.05或P<0.01)；50%肺活量的最大呼气流 量(FEF50)、75%肺活量的最大呼气流量(FEF75)、用力最大呼气流量(PEF)降低，肺组织Notch1，Jagged1，Delta1的表达 降低(P<0.01)。相关分析显示：大鼠肺功能参数FEV0.3/FVC与Notch4呈正相关，FEV0.3/FVC，FEF25，FEF50分别与足跖 肿胀度，Notch3，Delta1的表达呈负相关(P<0.05或P<0.01)。结论：AA大鼠在发生足跖肿胀、关节炎症同时出现肺功 能降低，且肺功能参数与Notch受体/配体呈相关性，提示致炎后免疫复合物沉积于组织局部，激活Notch信号通路， 通过级联放大效应协同参与肺组织损伤，导致肺功能降低。