饰胶蛋白聚糖的抗纤维化和抗肿瘤作用

饰胶蛋白聚糖（Decorin，DCN）属富含亮氨酸小分子蛋白多糖家族成员之一。大量证据表明：DCN通过结合并中和转化生长因子-β（TGF-β），干扰其与受体结合所致的胞外基质过度沉积，以产生抗纤维化和抑制疤痕形成的作用；DCN亦通过激活EGFR／MAPK／p21信号通路和抑制EGF—EGFR介导的促细胞增殖信号通路等机制，抑制肿瘤细胞增殖与转移。基于DCN以上两方面的生物活性，加之源于人体自身产生，其重组产品免疫原性较低，提示DCN对于慢性纤维化和肿瘤等疾病的防治具有潜在的药用开发价值。     

肿瘤坏死因子突变体对胃癌细胞肿瘤坏死因子受体的调节

本文采用放射配基结合分析法和蛋白合成抑制试验研究了肿瘤坏死因子突变体（ＴＮＦ－ｍ）对ＳＧＣ７９０１细胞ＴＮＦ受体的影响。结果表明，ＴＮＦ－ｍ可显著降低ＳＧＣ７９０１细胞表面ＴＮＦ受体数目并呈剂量、时间、温度依赖关系，对受体亲和力无影响，ＴＮＦ－ｍ可使胞浆ＴＮＦ受体数目增加，去除ＴＮＦ－ｍ３ｈ后，膜ＴＮＦ受体大约可恢复６０％，显著高于胰蛋白酶处理组ＴＮＦ受体的恢复率，放线菌素Ｄ对ＴＮＦ－ｍ处理的细胞ＴＮＦ受体的抑制作用显著低于对照组，且ＴＮＦ受体的半衰期约为９０ｍｉｎ．据此认为，ＴＮＦ－ｍ通过介导ＴＮＦ受体的内化从而使膜ＴＮＦ受体数降低。     

鼠表皮生长因子受体胞外段原核表达、纯化与复性

用多聚酶链反应(PCR)方法从既往构建质粒扩增鼠表皮生长因子受体(EGFR)胞外段基因及适宜酶切位点，进而构建原核表达载体。克服质粒丢失的不足，在大肠杆菌中表达目的蛋白，并在变性条件下通过。Ni^2 柱亲和层析纯化表达蛋白，聚丙烯酰胺凝胶电泳(SDS—PAGE)示纯度在95％以上。去除组氨酸标记的纯化蛋白在梯度尿素中透析复性。复性蛋白在表皮生长因子的作用下可以同源二聚化而在非还原SDS—PAGE及蛋白印迹分析中呈二聚体状态，其所产生抗体可以识别复性蛋白及表达于肺癌细胞上的EGFR，提示复性后大肠杆菌表达鼠EGFR胞外段重组蛋白已获得一定空间结构，并具有免疫原性，可以进一步用于EGFR功能及免疫学研究。     

原发性肝癌患者手术治疗前后检测可溶性白细胞介素2受体和肿瘤坏死因子的临床意义

应用双抗体酶联夹心法和放射免疫分析法检测了30名正常人和33例原发性肝癌患者血清中sIL-2R和TNF含量。结果表明：原发性肝癌患者手术前sIL－2R、TNF非常显著地高于正常人（P＜0．001，P＜0．01）；术后2周，血清中sIL－2R与正常人比较有差异（P＜0．01），而TNF则无差异（P＞0．05）。AFP含量高低与sIL－2R和TNF含量无关（r＝0．3826，0．3725；P＞0．05）。     

乳腺癌MDA-231细胞中IL-8与表皮生长因子受体信号通路的关系

目的 探讨IL-8对乳腺癌细胞增殖和侵袭的影响以及IL-8信号通路与表皮生长因子受体(EGFR)信号通路之间的关系.方法 采用ELISA方法检测乳腺癌MDA-231细胞IL-8的分泌及rhEGF、anti-EGFR对IL-8分泌的影响;免疫细胞化学方法检测其膜受体CXCR1和CXCR2的表达;MTT和matrigel invasion(基质凝胶侵袭)方法分析rhIL-8及anti-IL-8对癌细胞增殖和侵袭的影响;Western blot分析rhlL-8及anti-IL-8对EGFR活化的影响.结果 乳腺癌细胞可分泌大量IL-8,其细胞膜表面表达CXCR1和CXCR2两种受体.rhlL-8及其中和抗体对乳腺癌细胞的增殖无明显影响,但可影响其侵袭能力:rhIL-8可提高癌细胞的侵袭活性(P＜0.05),其中和抗体则对癌细胞的侵袭有抑制作用(P＜0.05).rhEGF及anti-EGFR均明显抑制了MDA-231细胞IL-8的分泌(P＜0.05,P＜0.01);未发现rhIL-8对EGFR的酪氨酸磷酸化有任何影响,相反anti-IL-8却诱导了EGFR活化.结论 IL-8不是乳腺癌自分泌的促细胞生长因子,IL-8主要通过促进癌细胞的侵袭而影响肿瘤的发展.乳腺癌中G蛋白耦联受体(GPCR)介导的IL-8信号通路与EGFR信号通路之间并无cross-talk,而是竞争抑制的关系.     

头帕肿瘤综合征蛋白与肿瘤坏死因子受体介导的坏死样凋亡

头帕肿瘤综合征蛋白(cylindromatosis,CYLD)是一种去泛素化酶,其C-末端USP结构域具有催化功能,可移除受体相互作用蛋白激酶1(receptor interacting protein kinase 1,RIPK1)的K63连接泛素链,调节RIPK1的泛素化水平,从而参与调节肿瘤坏死因子受体1(tumor necrosis factor receptor 1, TNFR1)介导的RIPK依赖的细胞坏死样凋亡等病理生理过程。阐明CYLD对RIPK1去泛素化调节的详细机制,寻找针对CYLD的特异性抑制剂,可为与坏死样凋亡相关的损伤与疾病提供治疗的新策略。     

Detection of gene point mutation in paraffin sections using in situ loop-mediated isothermal amplification

For pathological diagnoses, visualization of genetic status using routine tissue sections is important to determine the relationships between histopathological findings and genetic alterations. Loop-mediated isothermal amplification (LAMP) has been reported to have high levels of specificity and amplification efficiency. An in situ LAMP method was used, along with an amplification refractory mutation system (ARMS) to directly detect a specific point mutation, L858R, which is a mutation of epidermal growth factor receptor (EGFR), useful for the prediction of the effects of the anti-lung cancer drug gefitinib. The investigation was done using two types of cultured cells as well as paraffin-sectioned specimens collected from 26 cases of surgically resected lung cancer. Twelve of the specimens had an L858R mutation and in situ LAMP showed reactions in the nuclei of all cancer cells present in those. Such reactions were also shown on in situ LAMP in three of the remaining 14 cases that were without the L858R mutation. In addition, a few cases showed responses in the nuclei of bronchial epithelium cells located in non-cancerous areas in the vicinity of a positive tumor, which suggested that the mutation had already occurred in the tumorigenic early stage. It is concluded that the present method is useful for pathological and genetic diagnoses.     

Extramammary Paget's disease: analysis of growth signal pathway from the human epidermal growth factor receptor 2 protein

Paget's disease is a skin cancer characterized by characteristic (Paget) cells scattered in the epidermis. Although its prognosis is generally favorable with surgical resection, the clinical outcome turns unfavorable in cases with recurrence and metastasis. Therefore, establishment of effective therapeutic regimens is required for advanced Paget's disease. The human epidermal growth factor receptor 2 (HER2) protein, a transmembrane growth factor receptor, is frequently overexpressed in malignancies, causing activation of the phosphatidylinositol 3 kinase (PI3K) and extracellular signal-regulated kinase (ERK) signal pathways. Recently, HER2-targeting molecular therapy using trastuzumab (Herceptin; Genentech, Inc, South San Francisco, Calif) was revealed to be effective in advanced breast cancers overexpressing HER2 protein. Here, we analyzed the correlation between activation of the HER2 signal pathways and clinicopathologic parameters of 36 extramammary Paget's disease samples from 34 Japanese patients, using immunohistochemical analyses for HER2, phosphorylated HER2, phosphorylated AKT, and phosphorylated ERK proteins. We found overexpression of the HER2 protein in 19.4% (7) of the lesions, 3 of which showed HER2 amplification by chromogenic in situ hybridization. Phosphorylated HER2 protein was detected in 12 lesions (33.3%), including 2 of the 7 HER2-overexpressing lesions. Phosphorylated AKT was detected in approximately 75.0% (27/36) and phosphorylated ERK in 38.9% (14/36). Both HER2 and AKT were simultaneously phosphorylated in 9 cases (25.0%) and HER2 and ERK in 9 cases (25.0%), but all 3 molecules were phosphorylated in only 1 sample. Phosphorylated ERK correlated with the maximum diameter of the tumors (P < .025), but other immunohistochemical parameters failed to show any correlation with clinicopathologic features. These results suggest the contribution of the HER2 signaling pathway to the pathogenesis and progression of some cases of extramammary Paget's disease, for which clinical use of molecular target therapy against the HER2 pathway is warranted.     

Regulation of neural development by glial cell line-derived neurotrophic factor family ligands

Glial cell line-derived neurotrophic factor (GDNF) and its three relatives constitute a novel family of neurotrophic factors, the GDNF family ligands. These factors signal through a multicomponent receptor complex comprising a glycosylphosphatidylinositol-anchored cell surface molecule (GDNF family receptor (GFR) alpha) and RET tyrosine kinase, triggering the activation of multiple signaling pathways in responsive cells. Recent gene-targeting studies have demonstrated that GDNF family ligands are essential for the development of a diverse set of neuronal populations and we have now started to understand how these ligands uniquely regulate the formation and sculpting of the nervous system. Recent studies have also revealed interactions by multiple extracellular signals during neural development. The deciphering of GDNF family ligand signaling in neural cells promises to provide vital new insights into the development and pathology of the nervous system.     

In vivo fluorescence imaging for cancer diagnosis using receptor-targeted epidermal growth factor-based nanoprobe

Receptor-targeted imaging is emerging as a promising strategy for diagnosis of human cancer. Herein, we developed an epidermal growth factor-based nanoprobe (EGF-NP) for in vivo optical imaging of epidermal growth factor receptor (EGFR), an important target for cancer imaging. The self-quenched EGF-NP is fabricated by sequentially conjugating a near-infrared (NIR) fluorophore (Cy5.5) and a quencher (BHQ-3) to EGF, a low-molecular weight polypeptide (6.2 kDa), compared to EGFR antibody (150 kDa). The self-quenched EGF-NP presented great specificity to EGFR, and rapidly internalized into the cells, as monitored by time-lapse imaging. Importantly, the self-quenched EGF-NP boosted strong fluorescence signals upon EGFR-targeted uptake into EGFR-expressing cells, followed by lysosomal degradation, as confirmed by lysosomal marker cell imaging. Consistent with cellular results, intravenous injection of EGF-NP into tumor-bearing mice induced strong NIR fluorescence intensity in the target tumor tissue with high specificity against EGFR-expressing cancer cells. Signal accumulation of EGF-NP in tumor was much faster than that of EGFR monoclonal antibody (Cetuximab)-Cy5.5 conjugates due to the rapid clearance from the body and tissue permeability of low-molecular weight EGF. This self-quenched, EGF-based imaging probe can be applied for diagnosis of various cancers.     

Association of tumor necrosis factor receptor 2 (TNFR2) polymorphism with susceptibility to systemic lupus erythematosus

Multiple genetic as well as environmental factors are considered to be involved in the development of systemic lupus erythematosus (SLE). A number of previous studies have suggested a possible role for tumor necrosis factor (TNF) in the pathogenesis of SLE. In addition, one of the candidate loci suggested by the genome-wide linkage analysis corresponds to the chromosomal position encompassing the TNF receptor 2 gene (TNFR2). The purpose of this study was to analyze the polymorphism of TNFR2 and its possible association with the susceptibility to SLE, using the case-control association analysis. Polymorphism screening of the exons containing previously reported nonsynonymous base substitutions was carried out by the polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) method, using genomic DNA from 81 Japanese patients with SLE and 207 healthy individuals. Two alleles were present in exon 6, coding for methionine (196M) and arginine (196R) at position 196. 30 of 81 patients (37.0%) with SLE were positive for the 196R allele, which was significantly more frequent compared with 39 of 207 healthy individuals (18.8%) (chi2=10.6, df=l, P=0.001, odds ratio=2.53, 95% CI: 1.45-4.43). Genotype analysis revealed that the presence of one 196R allele was sufficient for rendering susceptibility. The association of 196R allele with SLE was independent from that of HLA-DRB1*1501. In conclusion, the TNFR2 196R allele was found to be significantly associated with the susceptibility to SLE in the Japanese population. Further population and functional studies will be of particular importance to establish TNFR2 as one of the susceptibility genes to SLE.     

血小板源生长因子在低氧性肺动脉平滑肌细胞增殖中的作用

运用流式细胞仪及细胞原位杂交方法测定低氧对培养的新生牛肺动脉平滑肌细胞（ＰＡＳＭＣ）生长的影响，并探讨血小板源生长因子（ＰＤＧＦ）在其中的作用，结果表明：培养的ＰＡＳＭＣ的低氧１２ｈ时，由Ｇ０／Ｇ１期进入Ｓ斯的细胞数明显增多，为对照组的１５７．７％（Ｐ〈０．０５）；ＰＤＧＦ可进一步促进低氧状态下ＰＡＳＭＣ的增殖，Ｓ期细胞为对照组的２４２．３％（Ｐ〈０．０１），原位杂交结果显示：低氧（１２ｈ）条件下     

人HER2胞外区基因重组及重组蛋白的纯化

为在体外纯化可溶性HER2胞外区蛋白并提高其原核表达产量，将多聚酶链式反应（PCR）方法获得的HER2基因胞外段编码区重组至pGEX-6P-1质粒，转化大肠杆菌BL21，采用不同的诱导条件分析融合蛋白的表达及其溶解性，不溶的包涵体经变性复性，与裂解液上清分别经Olutathione Sepharose4B亲合纯化融合蛋白。结果发现，重组蛋白在原核细胞中以可溶和不可溶两种表达形式存在，但以不溶的包涵体形式为主。低温（30℃）、较高的细菌密度（A600—1．8）和适当的培养基添加剂能有利于可溶性融合蛋白的表达。Olutathione Sepharose4B亲合纯化细菌裂解液上清和复性后的融合蛋白，其产量为1．23mg／L菌液，最大限度地获得了可溶性的HER2蛋白胞外段，为HER2特异性抗体的制备及其功能研究打下了良好的基础。     

肺癌患者化疗前后SIL-2R和TNF检测的临床意义

目的 :探讨了 4 0例肺癌患者化疗前后血清中SIL - 2R和TNF含量的变化。方法 :应用放免法和酶免法检测 4 0例肺癌患者血清中SIL - 2R和TNF含量并与 35名正常人作比较。结果 :肺癌患者在化疗前血清中SIL - 2R和TNF含量显著地高于正常人组 (p <0 0 1 ) ,化疗后明显下降 (p <0 0 5 )。 结论 :血清中SIL- 2R和TNF水平可作为肺癌患者免疫功能监测指标之一 ,并可作为疗效判断参考     

Intratumoral heterogeneity of HER2 protein and amplification of HER2 gene in salivary duct carcinoma

Salivary duct carcinoma (SDC) is an aggressive adenocarcinoma of the salivary glands, and accounts for 1–3% of all malignant salivary gland tumors, resembling morphologically invasive ductal carcinoma (IDC) of the breast. In contrast to IDC of the breast and gastric carcinoma (GC), the study of human epidermal growth factor receptor 2 (HER2) in SDC has not progressed. Therefore, we investigated the relationship between HER2 protein expression and amplification of the HER2 gene, and compared them in terms of intratumoral heterogeneity (ITH) in 13 cases of SDC using immunohistochemistry and dual color in situ hybridization. We found seven cases with protein overexpression (53.8%) and five cases with gene amplification (38.5%) in accordance with ASCO/CAP guidelines. ITH of HER2 protein expression was seen in seven cases (53.8%). Interestingly, the ratio of the HER2 gene showed homogenous distribution with or without the presence of ITH of HER2 protein expression. SDC tends to have more ITH of HER2 protein similarly to GC, in contrast to IDC of the breast. ITH of HER2 protein in SDC has no heterogeneity of the HER2 gene amplification. The mechanism of HER2 protein expression in SDC might proceed through a more complex pathway relative to that of IDC of the breast.     

外阴营养不良患者病变组织中性激素受体的检测

目的：研究外阴营养不良与性激素受体的关系。方法：采用直接荧光法，测定了１１例患者病变组织的性激素受体（ＳＨＲ）。其中增生型４例，硬化苔藓型４例，混合型３例。并对其中８例进行了外阴未发病组织ＳＨＲ对照。结果：（１）患者病变组织及未发病组织表皮各层及真皮层内均有不同程度的雌激素受体（ＥＲ）、孕激素受体（ＰＲ）、雄激素受体（ＡＲ）阳性率；（２）患者病变组织及未发病组织基底层ＥＲ、ＡＲ无或少于表皮其它各层     

肺癌患者化疗前后血清TSGF、SIL-2R和血浆VEGF检测的临床意义

目的:探讨了肺癌患者化疗前后血清TSGF、SIL-2R和血浆VEGF水平的变化及临床意义.方法:应用ELISA测定32例肺癌患者血浆VEGF含量和血清SIL-2R含量,光电比色法测定血清TSGF含量,并与30名正常健康人作比较.结果:肺癌患者在化疗前血清TSGF、SIL-2R和血浆VEGF水平非常显著地高于正常人组(P＜0.01),经化疗后6个月与正常人比较仍有显著差异(P＜0.05).结论:肺癌患者的发生和发展与血清TSGF、SIL-2R和血浆VEGF水平密切相关.