解聚素与金属蛋白酶10功能研究进展

A disintegrin and metalloprotease 10(ADAM10)is an important regulation molecule in the cell membrane. ADAM 10 are often implicated in ectodomain shedding, either to release e. g. growth factors or to initiate further intracellular signaling via regulated intramembrane proteolysis. By this way ADAM10 is involved in the development and growth of the central nervous system (CNS) 、 inflammation and the formation and infestation of cancer. In this paper, the structure and function and four major substrates of the ADAM 10 are briefly introduced.     

血管内皮生长因子受体2的结构功能与信号转导

Angiogenesis, or the formation of new blood vessels out of pre - existing capillaries, is very important in many physiologic and pathologic processes, such as embryonic development, cancer, retinopathies, etc. Vascular endothelial growth factor receptor - 2 (VEGFR-2) plays a key role in angiogenesis. In this review, we discussed the structure, function and signal transduction of vascular endothelial growth factor receptor-2. Understanding these should provide important insights into how new strategies can be devised to interfere in the physiologic and pathologic processes involved in angiogensis.     

Cell mediated gene therapy: A guide for doctors in the clinic

The recent approval of gene therapy products in Europe and Asia and the upsurge of gene therapy products in clinical trials signal the rebound of this technology not only for many orphan diseases but also for non-life threatening diseases. Following the success of induced pluripotent stem(iP S) cells in research, other modified ex vivo gene therapies are also knocking on the door of the clinic. Historically, gene therapy has experienced many ups and downs and still faces many challenges.During the past 10 years, many new ideas have been tried, and the goal of making this technology a more effective treatment modality through greater safety and control is coming within reach. The first clinical trial of i PS cells has begun, and cell mediated gene therapy products have reached phase Ⅲ in some countries. The potential for tumorigenicity and immunogenicity are still concerns with these products, so physicians should understand the biological aspects of engineered cells in the clinic. In this review article, we attempted to provide a summary update of the current state of knowledge regarding this technology: that is, we reviewed products that have finished clinical trials, are still in clinical trials and/or are at the research stage. We also focused on the challenges, future directions, and strategies for making this technology available in the clinic. In addition, the available measures for making gene therapy products safer are within the scope of this article. It is also important to understand the manufacturing process for gene therapy products, because cell characteristics can change during the cell expansion process. When physicians use gene therapy products in the clinic, they should be aware of the viability, temperature sensitivity and stability of these cells because biologic products are different from chemical products. Although we may not be able to answer all possible questions and concerns, we believe that this is the right time for physicians to increase their interest in and understanding of this evolving technology.     

MicroRNA在脑胶质瘤中的作用

icroRNAs (miRNAs) are critical regulators of gene expression. These small, non-coding RNAs are believed to regulate more than one third of protein-coding genes, and have been implicated in the control of many biological processes, including the biology of glioma. The functional significance in some of the miRNAs begins to emerge. This paper reviews the biogenesis of miRNAs, their roles in neuronal development and tumorigenesis of gliomas, and their contribution as tumor biomarkers. Research in this area is quickly gathering pace and is illuminating important aspects of the diseases that may ultimately lead to novel therapeutic interventions, as well as diagnostic and prognostic tools for brain tumors.     

催产素受体与肿瘤

Oxytocin receptor(OTR)is a member of G-protein coupled receptor,which has been found in many tumors and cancer cell lines.Many studies revealed oxytocin(OT)may inhibit the tumor and cancer cell growth and proliferation,but the mechanism of this inhibition is not well known.Some experiments indicated cAMP-PKA system participates in the signal transdution that oxytocin inhibits the cancer cell pro-lifeation .However,other experiments showed the signal transduction for oxytocin in the Hs578T catreinosaco-ma cell is the same as that in the normal celss.In this review,the relationship between OTR and tumors are summarized.     

The late stage of T cell development within mouse thymus

After positive selection and lineage commitment,the TCRαβ~+CD4/CD8 SP medullary thymocytes migrate intoand reside in thymic medulla,where they undergo an ordered program of late stage of T cell functionalmaturation and negative selection to delete self-reactive clones by apoptosis.Accomplishment of this finaldifferentiation pathway,a physiological T cell repertoire is formed:T cells acquire immunocompetence torespond to foreign antigens and tolerance to self-antigens,ready for the emigration to homing to the T cellregions of peripheral lymphoid organs and tissues.In this review,emphases are put on introducing theapproaches applied in this area and our own observations.Basically,we have analyzed the late stage ofmedullary thymocyte phenotypic differentiation pathways of both CD4 SP and CD8 SP medullary thymocytesand the concomitant functional maturation pathway,in particular,of CD4 SP thymocytes.It is to provide astandard to compare the functional capacity of the cells at the developmental stages induced by differentconditions.The cellular and molecular basis of this differentiation process has been partially described.Cellular& Molecular Immunology.2004;1(1):3-11.     

Future directions of clinical laboratory evaluation ol pregnancy

In recent years, our understanding of how the immune system interacts with the developing fetus and placenta has greatly expanded. There are many laboratories that provide tests for diagnosis of pregnancy outcome in women who have recurrent pregnancy loss （RPL） or pre-eclampsia. These tests are based on the premise that immune response to the fetus is equivalent to the adaptive immune response to a transplant. New understanding leads to the concept that the activated innate response is vital for pregnancy and this can result in more effective testing and treatment to prevent an abnormal pregnancy in the future. We describe here only three such areas for future testing： one area involves sperm and semen and factors necessary for successful fertilization; another area would determine conditions for production of growth factors necessary for implantation in the uterus; finally, the last area would be to determine conditions necessary for the vascularization of the placenta and growing fetus by activated natural killer （NK） cells （combinations of killer cell immunoglobulin-like receptor （KIR） family genes with HLA-C haplotypes） that lead to capability of secreting angiogenic growth factors. These areas are novel but understanding their role in pregnancy can lead to insight into how to maintain and treat pregnancies with complicating factors.     

Regulation of antitumor immunity by inflammation-induced epigenetic alterations

Chronic inflammation promotes tumor development,progression,and metastatic dissemination and causes treatment resistance.The accumulation of genetic alterations and loss of normal cellular regulatory processes are not only associated with cancer growth and progression but also result in the expression of tumor-specific and tumor-associated antigens that may activate antitumor immunity.This antagonism between inflammation and immunity and the ability of cancer cells to avoid immune detection affect the course of cancer development and treatment outcomes.While inflammation,particularly acute inflammation,supports T-cell priming,activation,and infiltration into infected tissues,chronic inflammation is mostly immunosuppressive.However,the main mechanisms that dictate the outcome of the inflammation-immunity interplay are not well understood.Recent data suggest that inflammation triggers epigenetic alterations in cancer cells and components of the tumor microenvironment.These alterations can affect and modulate numerous aspects of cancer development,including tumor growth,the metabolic state,metastatic spread,immune escape,and immunosuppressive or immunosupportive leukocyte generation.In this review,we discuss the role of inflammation in initiating epigenetic alterations in immune cells,cancer-associated fibroblasts,and cancer cells and suggest how and when epigenetic interventions can be combined with immunotherapies to improve therapeutic outcomes.     

Recent advances in targeting the autotaxin-lysophosphatidate-lipid phosphate phosphatase axis in vivo

Extracellular lysophosphatidate(LPA) is a potent bioactive lipid that signals through six G-protein-coupled receptors.This signaling is required for embryogenesis,tissue repair and remodeling processes.LPA is produced from circulating lysophosphatidylcholine by autotaxin(ATX),and is degraded outside cells by a family of three enzymes called the lipid phosphate phosphatases(LPPs).In many pathological conditions,particularly in cancers,LPA concentrations are increased due to high ATX expression and low LPP activity.In cancers,LPA signaling drives tumor growth,angiogenesis,metastasis,resistance to chemotherapy and decreased efficacy of radiotherapy.Hence,targeting the ATX-LPA-LPP axis is an attractive strategy for introducing novel adjuvant therapeutic options.In this review,we will summarize current progress in targeting the ATX-LPA-LPP axis with inhibitors of autotaxin activity,LPA receptor antagonists,LPA monoclonal antibodies,and increasing low LPP expression.Some of these agents are already in clinical trials and have applications beyond cancer,including chronic inflammatory diseases.     

解聚素与金属蛋白酶10功能研究进展

解聚素与金属蛋白酶10(ADAM10)是细胞膜上一种重要的调节分子.它通过调节性膜内蛋白水解(regulated intramembrane proteolysis),引起底物分子的胞外功能区的脱落,释放各种生长因子,或者调节细胞内的蛋白水解,进一步启动细胞内的信号转导途径.ADAM10通过这种方式参与了中枢神经系统的发育和生长、炎症反应以及肿瘤的形成和侵袭.本文仅就ADAM10的分子结构,4类主要的作用底物和发挥的功能作一简要介绍.     

结肠癌组织中MICA和ADAM10的表达及其相关性

目的 研究MHC I类相关分子A(MHC class Ⅰ chain-related A,MICA)及ADAM10在结肠癌中的表达及其与临床病理学参数之间的关系,并探讨两者的相关性,为结肠癌细胞表面MICA蛋白脱落机制的研究提供组织学依据.方法 应用免疫组织化学方法,采用SP法检测有完整资料的90例结肠癌组织及30例癌旁组织中MICA和ADAM10的表达差异.结果 在癌组织和癌旁组织中,MICA蛋白表达阳性率分别为67.8%和7.1%,ADAM10蛋白表达阳性率分别为82.2%和20%,差异均具有显著性(P<0.01),并且与淋巴结转移有关(P<0.05),MICA与ADAM10表达呈负相关性(r=-0.258,P<0.05).结论 MICA的表达降低与ADAM10的表达升高,可能共同参与了肿瘤的免疫逃逸及肿瘤的转移;且ADAM10在膜型MICA的脱落机制中发挥重要作用.     

早孕滋养细胞膜型及可溶性趋化因子CXCL16表达调控

目的 探讨趋化因子CXCL16在人早孕滋养细胞表达和释放的调控机制.方法 原代培养人早孕滋养细胞,实时定量RT-PCR、免疫化学和ELISA方法分析CXCL16的表达和分泌;ELISA方法分析细胞因子IFN-γ、TNF-α、IL-4刺激前后滋养细胞CXCL16的表达和分泌水平;ELISA方法分析ADAM10治疗前后滋养细胞CXCL16的表达及分泌水平.结果 滋养细胞表达并分泌趋化因子CXCL16;IFN-γ治疗后滋养细胞表达和分泌CXCL16水平均显著上升(P<0.01),但TNF-α和IL-4并不影响CXCL16的表达或分泌;ADAM10可以加速CXCL16自滋养细胞的脱落,但并不上调CXCL16的合成.结论 IFN-γ和ADAM10参与调控母胎界面滋养细胞趋化因子CXCL16的合成和分泌.     

小鼠ADAM10条件性基因打靶载体的构建与鉴定

目的构建小鼠ADAM10条件性基因打靶载体,为建立ADAM10条件性敲除小鼠模型奠定基础。方法以正常小鼠（129/Svj）基因组DNA为模板,采用长片段PCR方法,扩增小鼠ADAM10基因第2外显子及其侧翼序列。通过引入LoxP位点和TK基因等步骤,获得ADAM10条件性基因打靶载体。结果经限制性内切酶酶切鉴定和测序证实,构建的小鼠ADAM10基因条件性打靶载体符合设计要求。结沦成功构建了小鼠ADAM10条件性基因打靶载体,为建立ADAM10条件性基因敲除小鼠打下了基础。     

靶向沉默ADAM10基因对心肌细胞N-cadherin加工的影响

目的:研究心肌细胞中解整合素金属蛋白酶家族(ADAMs)成员ADAM10在神经型钙黏蛋白(N-cadherin)底物加工过程中的重要性,为探求该加工途径在维持心肌组织结构形态和完整性中的作用打下基础。方法:将质粒sc-270165 ADAM10 siRNA(r)转染入大鼠心肌细胞(H9c2),建立ADAM10稳定沉默的心肌细胞株。通过Western blotting检测心肌细胞N-cadherin及其C末端片段(CTF)的蛋白表达,流式细胞术检测心肌细胞表面N-cadherin的表达。利用黏附实验检测其对细胞黏附能力的影响。结果:与阴性对照组相比,ADAM10基因沉默组心肌细胞N-cadherin蛋白表达提高,CTF蛋白表达减少;细胞表面N-cadherin的表达增多,心肌细胞黏附能力提高。结论:心肌细胞中ADAM10在N-cadherin的加工水解过程中可能发挥了作用。     

封闭N-cadherin解整合素金属蛋白酶水解位点的单链抗体的制备及鉴定

目的 制备封闭神经型钙黏素(N-cadherin)解整合素金属蛋白酶水解位点(ADAM)的单链抗体(scFv),并进行鉴定.方法 首先利用RT-PCR技术从分泌抗N-cadherin的ADAM水解位点单克隆抗体(mAb)细胞株中扩增出重链(VH)和轻链(VL)可变区基因片段,然后通过重叠延伸PCR法(SOE-PCR),构建成scFv基因片段.再将其克隆入原核表达载体pET-28a中,在大肠杆菌中诱导表达,通过镍柱纯化和复性后,用SDS-PAGE、ELISA和Western blot法等测定重组蛋白的生物学活性.结果 PCR、酶切和测序表明scFv片段长744 bp,编码248个氨基酸.scFv基因表达载体转化E.coli BL21(DE3),经IPTG诱导表达出相对分子质量(Mr)约29 000的目的蛋白,主要为包涵体形式,经变性,纯化和复性后,获得纯度达90％以上的scFv蛋白,ELISA和Western blot法检测表明可溶性scFv可以与N-cadherin的ADAM水解位点序列多抗原短肽和全长N-cadherin结合.结论 成功构建并表达封闭N-cadherin的ADAM加工位点单链抗体.     

Ectodomain shedding of Fcα receptor is mediated by ADAM10 and ADAM17

FcαR (CD89) plays important roles in immunoglobulin A (IgA)‐mediated immune responses. Soluble forms of FcαR (sFcαR) are found in the culture supernatants of FcαR‐expressing cells, in human serum and in the serum of FcαR transgenic mice, and have been suggested to be produced through a proteolytic process. However, little is known about the mechanism involved in the proteolytic release of sFcαR. In this study, we investigated the shedding mechanism of FcαR and determined the nature of the proteinase involved in FcαR shedding. In chemical inhibitor assays, shedding of FcαR was dramatically inhibited by EDTA, EGTA and a broad‐spectrum metalloproteinase inhibitor, GM6001, suggesting that a metalloproteinase was responsible for FcαR shedding. Overexpression of dominant‐negative mutants of ADAM (a disintegrin and metalloproteinase) 10 and ADAM17 markedly inhibited the production of sFcαR. Finally, knockdown of both endogenous ADAM10 and endogenous ADAM17 inhibited FcαR shedding, demonstrating that ADAM10 and ADAM17 were involved in the shedding of FcαR. The characterization of ADAM10 and ADAM17 as sFcαR‐releasing enzymes provides a novel insight into the molecular mechanism of sFcαR production and will help in further elucidation of the physiological and pathological roles of sFcαR.     

Contribution of putative genetic factors and candidate gene variants to inter-individual variation of circulating fractalkine (CX3CL1) levels in a large UK twins’ sample

Objective

Soluble fractalkine (sFRACT) is involved in the pathogenesis of several clinical diseases. Our major objective was to determine to what extent its variation is governed by genetic factors and whether this genetic variation could be attributable to SNPs in five candidate genes: CX3CL1, CX3CR1, ADAM10, ADAM17 and AREG.

Methods

Plasma levels of sFRACT and 38 SNPs, with minor allele frequency >0.1 were examined in a large twin sample drawn from the general UK population. The discovery sample included 3306 middle-aged females: 1172 MZ twins and 2134 DZ twins. A replication sample of 1675 twins was used to validate the major association results obtained in genetic association analysis in the discovery sample. We implemented variance component analysis to estimate contribution of putative genetic, (including above SNPs) and environmental factors to sFRACT variation.

Results

sFRACT was found not to vary with either age or BMI. Putative genetic factors (heritability) explained 43.6 ± 3% of the total variation of plasma sFRACT levels. However, we found no evidence of association between sFRACT and any of the examined SNPs, despite having >85% power to detect an association of just 1% of the variance explained. The results in the discovery and replication samples were in good agreement suggesting these findings are real.

Conclusion

Our results suggest involvement of genetic factors to inter-individual variation of sFRACT levels in a general human population. However, further studies are required to determine genetic polymorphisms affecting sFRACT variation.