WTX基因：第一个位于X染色体上的肾母细胞瘤抑癌基因

肾母细胞瘤又称肾胚胎瘤,德国外科医生Max Wilms于1899年曾对该瘤的特性发表了详细的描 述,故又称为Wilms瘤[1].     

增强DNA免疫效应的研究进展

新近迅速起来的ＤＮＡ疫苗由于兼有重组亚单位疫苗的安全性和减毒活疫苗的有效性，因而受到人们的高度重视。ＤＮＡ疫苗虽具有多得优点，但在诱导的免疫效应上还未尽如入意。研究者们尝试着采取各种措施以增强ＤＮＡ疫苗的免疫效应，本文对近年来几种比较有效的增强措施作一综述。     

多CTL表位DNA疫苗诱导特异性CTL应答的研究

目的:构建多CTL表位DNA疫苗诱导特异性CTL应答。方法:将编码两个HCVCTL表位(H-2d)的基因序列插入真核表达载体pcDNA3.1中,构建多CTL表位DNA疫苗。用其免疫BALB/c小鼠后,以经不同CTL抗原肽冲击的P815细胞(H-2d)作为靶细胞,进行CTL杀伤试验。结果:多CTL表位DNA疫苗可诱导机体产生针对其编码的两种HCVCTL表位的特异性CTL免疫应答,且总的特异性CTL杀伤效应增强。结论:通过天然侧翼氨基酸相连的多个CTL表位为编码序列构建的多CTL表位DNA疫苗,不但能诱导机体产生针对各个CTL表位的特异性CTL应答,而且各特异性CTL应答的联合作用可显著增强总的CTL杀伤效应。     

肾母细胞瘤介入治疗后的病理学研究

目的探讨介入治疗对肾母细胞瘤病理学的影响。方法对30例术前介入治疗后的肾母细胞瘤与16例术前化疗、17例单纯手术组的肿瘤和20例曾作芯针活检的组织进行病理观察对照研究。对三组各15例组织作TUNEL染色与SP法免疫组化染色检测肿瘤凋亡和PCNA表达。结果介入组肿瘤纤维性假包膜厚度平均2,4±0．7mm,平均坏死面积占肿瘤77．5％,肿瘤内中型血管坏死闭锁率达93．3％,瘤体内均见淡黄色碘油残留及大片泡沫状组织细胞浸润;化疗组纤维性假包膜厚度平均2．1±0．9mm,平均坏死面积占肿瘤51．2％。中型血管坏死闭锁率达43．8％。单纯手术组纤维性假包膜厚度平均1．1±0．4mm,平均坏死面积占肿瘤14％,瘤体内未见中型血管坏死及闭锁。介入组术前芯针活检结果与术后瘤体病理比较发现间叶型,术后瘤体坏死相对较少,其余类型坏死均较明显,尤以胚芽型为甚。介入组与单纯手术组相比肿瘤细胞增殖活性明显降低,凋亡细胞显著增多,但与化疗组相比无差异。结论术前介入治疗,能使瘤体内血管迅速坏死、闭锁、肿瘤大片坏死,同时伴有纤维组织增生、纤维性假包膜形成;肿瘤内碘油长期滞留及泡沫状组织细胞浸润,有利于肿瘤细胞杀灭作用;组织类型与治疗敏感性有关;介入治疗也可通过抑制细胞增殖,诱导凋亡达到控制肿瘤的目的。     

增强DNA免疫效应的研究进展

新近迅速发展起来的ＤＮＡ疫苗由于兼有重组亚单位疫苗的安全性和减毒活疫苗的有效性，因而受到人们的高度重视。ＤＮＡ疫苗虽具有多种优点，但在诱导的免疫效应上还未尽如人意。研究者们尝试着采取各种措施以增强ＤＮＡ疫苗的免疫效应，本文对近年来几种比较有效的增强措施作一综述。     

肾母细胞瘤微卫星不稳定性与临床病理的关系

目的探讨微卫星不稳定性（ｍｉｃｒｏｓａｔｅｌｉｔｅｉｎｓｔａｂｉｌｉｔｙ,ＭＳＩ）与肾母细胞瘤临床病理的关系。方法采用ＰＣＲ扩增,变性聚丙烯酰胺凝胶电泳等技术,选用分别定位于Ｘ及２１号染色体的ＡＲ和ＵＴ７６２微卫星标记位点,对５０例肾母细胞瘤标本作ＭＳＩ测定。按ＮＷＴＳ－Ⅱ病理分类法,将５０例分成间变型和无间变型,以及按ＮＷＴＳ－Ⅲ病期分类法可分为四期。结果ＡＲ位点ＭＳＩ阳性１４例（２８％）,ＵＴ７６２位点阳性１０例（２０％）,同一病例两个位点均阳性２例（４％）,总阳性病例２２例（４４％）。早期肿瘤（Ⅰ,Ⅱ期３８例）中１６例阳性（４２．１％）,晚期肿瘤（Ⅲ,Ⅳ期１２例）６例阳性（５０％）,（Ｐ＞０．０５）。间变型（１４例）１０例阳性,阳性率７１％,显著高于无间变型（３６例中１２例阳性）的阳性率３３％（Ｐ＜０．０５）。并发现手术边缘组织２例阳性。结论微卫星不稳定性对区分肾母细胞瘤的恶性程度有一定意义。     

树突状细胞靶向性DNA疫苗的抗肿瘤免疫效应

目的:构建含OVA-Fc融合基因并对树突状细胞具有靶向性的DNA疫苗,评价其在肿瘤治疗中的作用。方法:构建真核表达载体OVA-Fc-pcDNA3.1,以脂质体转染法将其导入CHO细胞,用流式细胞术和ELISA法检测融合蛋白OVA-Fc的表达。建立E.G7-OVA荷瘤小鼠模型,用51Cr释放实验测定免疫小鼠脾脏细胞毒性T淋巴细胞(CTL)的抗肿瘤活性。通过观察荷瘤小鼠肿瘤的体积和生存期评价该肿瘤疫苗的疗效。结果:酶切鉴定和序列测定证明,真核表达载体OVA-Fc-pcDNA3.1构建正确。用流式细胞术和ELISA法均表明,转染的CHO细胞能表达OVA-Fc融合蛋白。OVA-Fc能激发CTL的杀伤活性,发挥抗肿瘤作用,从而减缓肿瘤的生长,延长荷瘤小鼠的生存期。结论:含OVA-Fc-pcDNA3.1的树突状细胞靶向性DNA疫苗能在体内有效地激发抗瘤免疫应答,为进一步开展临床实验奠定了基础。     

CSE1L在神经母细胞瘤化疗DNA损伤修复过程中的表达

目的 研究核转运因子CSE1L在化疗后神经母细胞瘤DNA损伤修复过程中的表达变化并初步探索其作用机制,为神经母细胞瘤诊疗提供线索和理论依据.方法 通过免疫组化染色方法对19例未化疗和28例经化疗的神经母细胞瘤组织中CSE1L表达水平进行分析.阿霉素处理SH-SY5Y细胞6小时和12小时建立DNA损伤模型,并通过免疫荧光方法检测DNA损伤修复标志物g-H2AX和53BP1在DNA损伤位点的聚集情况;免疫印迹检测阿霉素诱导DNA损伤后CSE1L以及53BP1、FEN-1和EXO-1等DNA损伤相关蛋白的变化水平.慢病毒敲减CSE1L后,免疫印迹检测CSE1L、53BP1、FEN-1和EXO-1蛋白水平变化.结果 未化疗组CSE1L阳性率(73.7％)高于化疗组(42.9％)并具有显著差异(卡方检验P=0.037);阿霉素可诱导DNA损伤修复标志物g-H2AX和53BP1在DNA损伤位点的聚集;并诱导53BP1、CSE 1L、FEN-1的表达水平在处理6小时先增高、在12小时降低,EXO-1表达持续增高;敲减CSE1L可降低53BP1和FEN-1表达,但促进EXO-1表达.结论 CSE1L在化疗后神经母细胞瘤样本中表达降低,并参与DNA损伤修复过程.     

DNA疫苗与抗肿瘤免疫

DNA疫苗是近年发展起来的新一代疫苗 ,能够诱导宿主体内细胞毒T细胞反应和抗体反应。大量实验研究证明 ,注射抗肿瘤DNA疫苗能获得肿瘤保护性免疫效应 ,在肿瘤免疫治疗中应用前景看好。本文对DNA疫苗抗肿瘤免疫的机制、影响因素及应用作一综述     

DNA介导免疫的研究进展

DNA介导免疫是指将质粒DNA导入动物组织后，质粒DNA原位表达并产生特异的体液和细胞免疫应答。目前主要应用的基因导入方法是质粒DNA直接注射骨骼肌，并可在质粒DNA注射前应用再生诱导剂提高导入基因的蛋白表达水平以增强免疫效果。DNA介导免疫多被应用于抗感染免疫研制成核酸疫苗。     

Cytotoxic T-Lymphocyte Responses Elicited to Wilms' Tumor Gene WT1 Product by DNA Vaccination

We recently have reported that Wilms' tumor gene WT1 is highly expressed not only in leukemias but also in various types of solid tumors and that WT1 protein is a novel tumor antigen against which cytotoxic T lymphocytes (CTLs) can be elicited by immunization with 9-mer WT1 peptides capable of binding to major histocompatibility complex (MHC) class I molecules. In the present study, plasmid DNA encoding murine full-length WT1 protein was injected intramuscularly into C57BL/6 mice. The mice vaccinated with the WT1 plasmid DNA elicited CTLs against the WT1 protein, and the CTLs specifically killed WT1-expressing tumor cells in a MHC class I-restricted manner. Furthermore, the vaccinated mice rejected the challenges of WT1-expressing tumor cells and survived with no signs of autoimmunity caused by the CTLs. These results demonstrated that vaccination with the WT1 plasmid DNA can elicit CTL responses specific for the WT1 protein, resulting in the acquisition of rejection activity against challenges of WT1-expressing tumor cells. This WT1 DNA vaccination may find clinical application for various types of solid tumors as well as leukemias.     

新型颗粒性肽-DNA复合疫苗有效激发肿瘤抗原特异性CTL反应

目的设计新型抗肿瘤治疗性疫苗。方法以多聚赖氨酸为桥梁，将抗原肽与其编码基因并置于同—疫苗颗粒，进而设计出颗粒性肽-DNA复合疫苗。结果该疫苗可激发体内肿瘤特异性CTL反应，并对P815肿瘤具有特异性杀伤活性。结论该颗粒性肽-DNA复合疫苗有希望成为一种有效的肿瘤疫苗形式。     

Ⅰ型人免疫缺陷病毒gag-gp120嵌合基因核酸疫苗的构建与鉴定

目的：构建含Ⅰ型人免疫缺陷病毒(HIV-1)gag—gp120嵌合基因核酸疫苗的表达质粒。方法：将gag和gp120连接后的嵌合基因插入到真核表达载体pVAX1中，构建真核表达质粒pVAXGE。用脂质体法将构建的重组质粒转染Hela细胞72h后，取转染的Hela细胞进行RT—PCR检测和和Dot—ELISA分析。结果：重组质粒转染细胞的总RNA中，可扩增出目的基因的转录产物。Dot-ELISA的结果显示，目的基因在Hela细胞内得到表达。结论：成功地构建了表达gag—gp120嵌合基因的核酸疫苗质粒，为HIV—1核酸疫苗的制备奠定基础。     

TLR9-/- and TLR9+/+ mice display similar immune responses to a DNA vaccine

Plasmid DNA continues to attract interest as a potential vaccine-delivery vehicle. However, the mechanisms whereby immune responses are elicited by plasmids are not fully understood. Although there have been suggestions regarding the importance of CpG motifs in plasmid immunogenicity, the molecular mechanisms by which CpG motifs enhance immune responses to DNA vaccines are not well understood. As Toll-like receptor 9-deficient (TLR9-/-) mice fail to respond to the adjuvant effects of CpG oligonucleotides, we used these mice to determine the effect of CpG motifs in plasmids used for DNA immunization. In the study described below, we report that DNA immunization was as effective in eliciting antigen-specific antibody and at stimulating antigen-specific interferon-gamma (IFN-gamma)-secreting cells in TLR9-/- mice as in TLR9+/+ mice. This study illustrates that DNA vaccines elicit immune responses by multiple mechanisms and demonstrates that TLR9 is not essential for the induction of immune responses following DNA immunization.     

HIV-1 B''亚型gag-env融合基因的DNA疫苗构建和免疫原性分析

目的 构建HIV-1 B′亚型中国流行株gag和env融合基因的DNA疫苗，对其免疫原性进行研究。方法 根据已报道的HIV-1 B′亚型RIA2分离株gag和env基因的氨基酸序列按哺乳动物密码子使用频率进行优化并人工合成基因，插入真核表达载体pDRVISV1．0中，构建表达RL42 gag-env，融合蛋白的DNA疫苗，pSVRL／GE。用Western blot和抗gag p55抗体细胞内染色的方法体外检测pSVRUGE的表达效率。DNA疫苗pSVRL/GE免疫BALB／c小鼠后，用ELISPOT检测小鼠的细胞免疫反应。结果 限制性内切酶鉴定表明融合基因已成功插入pDRVISV1．0载体中，Western blot证实融合基因可有效表达融合蛋白；细胞内染色结果表明，pSVRL/GE转染的293T细胞中49．8％表达gag p55，荧光强度均值为924；而空载体pDRVISV1．0转染的293T细胞中非特异背景染色只有0．5％。经免疫的小鼠脾细胞体外用H-2^d限制性表位肽AMQMIKET刺激后，ELISPOT检测显示，pSVRUGE免疫小鼠每10^6脾细胞形成226个斑点（SD=140），而空载对照组每1驴脾细胞形成29个斑点（SD=16）（P〈0．05）。结论 所构建的DNA疫苗pSVRL/GE可高效表达相应抗原蛋白，并可有效激活机体的细胞免疫反应。     

中国株HIV-1核心蛋白Gag核酸疫苗的构建与表达

目的 构建含HIV－1中国流行株B亚型核心蛋白Gag基因的真核表达质粒，并在体外进行表达和鉴定。方法 将Gag基因插入到核酸疫苗载体质粒pVAX1中，构建真核表达质粒pVAXGAG。用脂质体法。将重组质粒转染人Hela细胞后进行表达产物的检测。结果 间接免疫荧光检测显示，转染重组质粒的细胞表面有绿色荧光。Western blot和Dot ELISA分析显示，重组质粒转染细胞的裂解物中存在表达的Gag蛋白。结论 构建的核酸苗可在体外进行表达，且表达的蛋白具有特异性。     

MUC1基因疫苗诱导小鼠特异性CTL和体液免疫应答

目的 :观察MUC1基因疫苗诱导小鼠特异性杀伤性T细胞及体液免疫应答的作用。方法 :采用股四头肌肌肉注射 ,将构建的MUC1基因疫苗pcDNA3.1 MUC1免疫雌性BALB/c小鼠 ,每次间隔 3wk ,共 3次。最后 1次免疫后第 3周 ,接种表达MUC1的EMT6乳腺癌细胞进行免疫保护实验。用 4h51Cr释放法检测小鼠脾细胞特异性CTL杀伤活性 ;免疫组化染色法检测小鼠血清特异性抗体的水平。结果 :在效靶比为 10 0∶1、5 0∶1、2 5∶1、12 .5∶1时 ,MUC1基因疫苗免疫组特异性CTL对EMT6靶细胞杀伤活性分别为 5 4 .1%、39.8%、2 6 .4 %和2 0 .1% ,对照组分别为 13.2 %、10 .0 %、8.2 %、7.2 %和 11.7%、9.8%、7.7%、7.0 % ,前者与后二者差异显著 (P <0 .0 1)。免疫组化染色检测显示 ,人乳腺癌组织MUC1呈染色阳性 ;MUC1基因疫苗免疫组仅见 4 0 % (4/ 10 )的小鼠有肿瘤形成 ,而 pcDNA3.1对照组和生理盐水阴性对照组 10 0 %可见肿瘤形成、生长 ,表明MUC1基因疫苗免疫组小鼠具有一定的免疫保护作用。结论 :MUC1基因疫苗可诱导小鼠产生特异性CTL及体液免疫应答 ,对小鼠体内荷瘤可能具有一定的预防作用     

A DNA vaccine encoding a chimeric allergen derived from major group 1 allergens of dust mite can be used for specific immunotherapy

Immunization with DNA-based constructs has been shown to be against the antigen and the response is skewed in such a way as to ameliorate the symptoms of allergic disease. This approach is particularly useful in the treatment of allergic inflammatory diseases, such as asthma. The major group 1 allergen from house dust mites is one of the triggers of allergic asthma. This study explores whether a chimeric gene R8, derived from the major group 1 allergen of house dust mite species (Dermatophagoides farinae and Dermatophagoides pteronyssinus), can be expressed in Human Embryonic Kidney 293 cells (HEK 293T) and whether such a construct can be used as a DNA vaccine in asthma therapy. The eukaryotic expression vector pcDNA3.1 was used to express the R8 molecule in HEK 293T cells and successful expression of R8 was confirmed using a fluorescence microscope and western blot analysis. The efficacy of R8 as DNA vaccine was also assessed in a mouse asthma model. The in vivo data showed that R8 rectified the TH1/TH2 imbalance typical of allergic inflammation and stimulated the proliferation of regulatory T (Treg) cells. Immunization with the R8 construct also decreased serum allergen-specific IgE production in this mouse asthma model. Our findings suggest that R8 may be a feasible potential DNA vaccine for specific immunotherapy (SIT) in the treatment of allergic asthma.     

Infrequent mutation of the WT1 gene in 77 Wilms' tumors

Homozygous deletions in Wilms' tumor DNA have been a key step in the identification and isolation of the WT1 gene. Several additional loci are also postulated to contribute to Wilms' tumor formation. To assess the frequency of WT1 alterations we have analyzed the WT1 locus in a panel of 77 Wilms' tumors. Eight tumors showed evidence for large deletions of several hundred or thousand kilobasepairs of DNA, some of which were also cytogenetically detected. Additional intragenic mutations were detected using more sensitive SSCP analyses to scan all 10 WTl exons. Most of these result in premature stop codons or missense mutations that inactivate the remaining WTl allele. The overall frequency of WTl alterations detected with these methods is less than 15%. While some mutations may not be detectable with the methods employed, our results suggest that direct alterations of the WTl gene are present in only a small fraction of Wilms' tumors. Thus, mutations at other Wilms' tumor loci or disturbance of interactions between these genes likely play an important role in Wilms' tumor development. © 1994 Wiley-Liss, Inc.     

HBV与HCV融合DNA疫苗的构建及其体液免疫应答

目的 构建含乙型肝炎病毒（HBV）表面抗原基因（S区基因）与丙型肝炎病毒（HCV）核心抗原基因（C区基因）的嵌合真核表达载体,观察preS1和preS2基因对HBV表面抗原及HCV核心抗原体液免疫的影响。方法 用PCR方法,分别扩增HBV S区基因和HCV C区基因。将S区基因克隆入真核表达载体pcDNA3.1,酶切鉴定后,大量提取质粒并免疫Balb/c小鼠,用ELISA法检测抗HBs和抗HCV抗体。结果 成功地扩增出目的基因片段,克隆后酶切鉴定结果正确,序列分析与文献报告相一致。免疫后检测到抗HBs和抗HCV抗体。preS1与preS2基因对构建的融合DNA疫苗的体液免疫应答有一定的抑制作用。抗HBs抗体的产生低于只含S基因的真核表达载体;preS1基因对抗HCV抗体的产生具有抑制作用,而preS2无影响。结论 不同长度的HBV S区基因可影响抗HBs和抗HCV抗体的产生。