表达性

Two gene fragments of DEN-1(GenBank accession number DQ886390)and DEN-2(GenBank accession number DQ886391),differentially expressed between the ocular skin tissues of normal and albino turbot,were isolated by mRNA differential display and sequenced.It was confirmed by RT-PCR method that the expression levels of both DEN-1 and DEN-2 were down-regulated in the ocular skin tissue of albino turbot.The sequence homology search of DEN-1 revealed high sequence homologies with the UGGT genes of zebrafish and cattle,and the Ugcgl1 gene of Norway rat.High sequence homologies of DEN-2 were observed with the eya4 gene(the eye absent homolog 4)from red jungle fowl,zebrafish,human,Norway rat,mouse and dog.

应用mRNA差异显示技术，对比研究正常与白化大菱鲆有眼侧皮肤组织，克隆差异表达基因，经测序，RT-PCR 验证，差异表达基因片段DEN-1(GenBank登录号：DQ886390)与DEN-2(GenBank登录号：DQ886391)均在白化大菱鲆有眼侧皮肤组织中下调表达；通过BLAST检索发现，DEN-1与GenBank中的斑马鱼和牛的类似尿苷二磷酸-葡萄糖：糖蛋白葡糖基转移酶基因、与挪威鼠类似尿苷二磷酸-葡萄糖：神经酰氨葡糖基转移酶1(Ugcgl1)基因有较高同源性；DEN-2 与原鸡、斑马鱼、人、挪威鼠、家鼠和狗的眼缺乏同源框4(eya4)基因的同源性均较高。

The positive expression both of p14ARF and mtp53 protein in NSCLC tissues were significantly different from that in nonmal lung tissues and tissues adjacent to the cancer (P.01).The expression of p14ARF protein was correlated with pathological degree（P＜0.01）,but it was uncorrelated with pathological staging,lymphaden metabasis and vessel invasion(P＞0.05).The expression of mtp53 protein was correlated with clinical pathology staging,pathological degree and vessel invasion(P＜0.05 or P＜0.01),but it was uncorrelated with lymphaden metabasis(P＞0.05).(2) The coexpression of p14ARF and mtp53 was 36.7％,their consistency was 38.8％,but the expression of p14ARF protein were negatively correlated with the expression of mtp53 protein(r=-0.3553,P＜0.001).Conclusion The results suggest that the decrease of p14ARF and the increase of mtp53 protein may play an important role in the genesis and development of NSCLC.

结果 (1)在正常肺组织、癌旁组织和NSCLC中p14ARF蛋白的阳性表达率分别为93.3%、83.3%、55.1%，mtp53蛋白的阳性表达率分别为6.7%、44.4%、79.6%；p14ARF蛋白和mtp53蛋白在NSCLC中的表达与正常肺组织和癌旁组织比较均有显著性差异(P＜0.01)；p14ARF蛋白表达与临床病理分期、有无淋巴结转移、有无脉管浸润无关(P＞0.05)，而与病理分化程度有关（P＜0.01）；mtp53蛋白表达与临床病理分期、病理分化程度(P＜0.01)及有无脉管浸润(P＜0.05)有关，而与有无淋巴结转移无关(P＞0.05)；(2)在NSCLC中，pl4ARF与mtp53蛋白共表达为36.7％，一致性为38.8％，二者表达呈负相关(r=-0.3553，P＜0.001)。

Transgenic tobacco plants were obtained through screening with kanamycin. The transgenic tobacco plants could delay TMV infection for about 25 days compared with non-transgenic tobacco plants. Pokeweed antiviral protein Ⅱ is expressed with high level in summer leaves. The expression of PAPⅡ is regulated by season. The total RNA was extracted from pokeweed (Phytolacca americana L.) leaves in summer using the method of TRIzol and used as template to amplify the PAPⅡ gene by RT-PCR and then the gene was cloned into E. coli expression vector and secreted expression pPIC9K vector. The two vectors with PAPⅡ gene were then transferred into E. coli strain BL21 (DE3)-plysS and Pachia pastoris GS115 strain respectively. The specific protein was produced induced by IPTG and methanol.

由于美洲商陆抗病毒蛋白Ⅱ是一个受季节调控表达的蛋白，本实验以美洲商陆夏季叶片为材料，通过对其总RNA的提取、反转录、并用PAPⅡ的特异引物进行PCR扩增，对PAPⅡ进行了基因克隆、序列分析，结果扩增出来的PAPⅡ基因与已经报道的序列同源性是99.9％，然后将该基因构建到原核、真核表达载体上，分别转化了大肠杆菌和毕赤酵母并对它们进行了诱导表达，在两个表达系统中均获得了有活性的PAPⅡ表达蛋白，体外生物测定表明表达的蛋白均具有抑制病毒的活性，PAPⅡ基因在酵母中还没有表达的报道，这为获得具活性PAPⅡ蛋白提供了一种简便可行的方法。

The transgenic tobacco plants could delay TMV infection for about 25 days compared with non-transgenic tobacco plants.Pokeweed antiviral protein II is expressed with high level in summer leaves. The expression of PAPII is regulated by season. The total RNA was extracted from pokeweed (Phytolacca americana L.) leaves in summer using the method of TRIzol and used as template to amplify the PAPII gene by RT-PCR and then the gene was cloned into E.coli expression vector and secreted expression pPIC9K vector. The two vectors with PAPII gene were then transferred into E.coli strain BL21 (DE3)-plysS and Pachia pastor is GS115 strain respectively. The specific protein was produced induced by IPTG and methanol.

由于美洲商陆抗病毒蛋白Ⅱ是一个受季节调控表达的蛋白，本实验以美洲商陆夏季叶片为材料，通过对其总RNA的提取、反转录、并用PAPⅡ的特异引物进行PCR扩增，对PAPⅡ进行了基因克隆、序列分析，结果扩增出来的PAPⅡ基因与已经报道的序列同源性是99.9％，然后将该基因构建到原核、真核表达载体上，分别转化了大肠杆菌和毕赤酵母并对它们进行了诱导表达，在两个表达系统中均获得了有活性的PAPⅡ表达蛋白，体外生物测定表明表达的蛋白均具有抑制病毒的活性，PAPⅡ基因在酵母中还没有表达的报道，这为获得具活性PAPⅡ蛋白提供了一种简便可行的方法。

In order to evaluate the potential of this immunodominant peptide antigen in serodiagnosis of TB, we have undertaken cloning and expression of a large number of esat-6p1 genes in Escherichia coli and purification products.

本实验研究目的是通过构建结核菌ESAT-6蛋白免疫优势段P1的重组多表达载体，在大肠杆菌 BL21（DE3）中进行诱导表达，并对表达产物进行纯化和复性，以获得大量ESAT-6P1抗原，同时对重组多免疫反应性进行鉴定评价，为探索重组多在结核病血清学诊断的应用奠定前期实验基础。

Results (1) The forward and reverse subtracted cDNA libraries of different metastastic potential large cell lung cancer cell lines were successfully constructed;(2) With blue-white colony and dot blot, 307 positive clones in the forward subtracted library and 78 positive clones in the reverse subtracted library were obtained;(3) 55 clones were successfully sequenced in the forward subtracted library. Homolog analysis confirmed 23 differential expression segments, which were similar to the human genes already known, including NME2, NPM1, MT 2A, HSPE1, TAFIA, EPRS, PX19 and EIF3S9 et al;(4) 31 clones were successfully sequenced in the reverse subtracted library. Homolog analysis confirmed 16 differentially expressed segments. 15 of them were similar to the human genes already known, including ANXA2, TUBB, PKN2, GNAS, EEF1A1, SSR2 and RPLPO et al. Only one segment had partial homology to known human genes. This segment was supposed to be the new EST segments which might not have been cloned.

结果(1)成功构建人大细胞肺癌高低转移株差异表达基因正向消减cDNA文库和反向消减cDNA文库；(2)经蓝白菌落筛选和斑点杂交，正向消减文库获得307个阳性克隆，反向消减文库获得78个阳性克隆；(3)正向消减文库挑选55个克隆成功测序，经同源性分析最终确定差异表达基因片段23个与已知人类全长基因具有很高相似性(95％～100％)，这些基因包括NME2、NPM1、MT2A、HSPE1、TAFlA、EPRS、PX19和EIF3S9等；(4)反向消减文库挑选31个克隆成功测序，经同源性检索和比对最终确定差异表达基因片段16个，其中15个与人类全长基因具有很高相似性(95％～100％)，包括ANXA2、TUBB、PKN2、GNAS、EEF1A1、SSR2和RPLPO等基因。1个片段和己知人类基因仅有部分相似性，表明它可能是未被克隆的人类基因EST片段。

Based on these previous work, in this research, in order to explore the the molecular mechanism of enhanced tumor cell immunogenicity treated by elemene and mytomycin C compositely, we used murine Hca-F ascitic hepatic carcinoma ( high lymphatic metastasis substrain of H22) as an experimental model to compare the immunoprotective effects on Hca-F cells as well as the mechanism of the effects caused by HSP70-peptide complex purified from untreated tumor cells, EC-TCV and BCG; We studied the mechanism of increased tumor cell membrane HSP expression treated with elemene by the techniques of RT-PCR and immunoelectron microscopy; In order to deeply elucidate the antitumor effects and the molecular mechanism of elemene in increasing tumor cell immunogenicity we analyzed and compared the effects of elemene and heat shock on the gene expressions of tumor cell using gene expression profile chip.

本论文在上述研究成果的基础上，选择小鼠肝癌H22的高淋巴道转移亚系Hca-F为模型，进一步比较了未处理肿瘤细胞来源、榄香烯复合瘤苗来源的和卡介苗来源的HSP70-肽复合物对Hca-F的免疫保护效应、效应机制，探讨榄香烯-丝裂霉素复合处理增强肿瘤细胞免疫原性的分子机制；以Hca-F和人肝癌细胞株HepG〓等为模型研究了榄香烯处理增强肿瘤细胞膜热休克蛋白表达的机制；以HepG〓为模型用表达谱基因芯片研究比较了榄香烯与热休克处理对肿瘤细胞基因表达的影响，从而进一步阐明榄香烯抗肿瘤作用及增强肿瘤细胞免疫原性的分子机制。

The results showed that 17 gene fragments were expressed exclusively,26 gene fragments expressed increasingly,and 20 gene fragments expressed decreasingly in incompatible combination compared with either in compatible combination or in control.

结果表明，非亲和性互作中特有的表达片段7条，较亲和性互作及对照表达明显增强的片段21条，表达明显减弱的18条。

Theurinary IL-6 level positively correlated with density of glomerular matrix membrane, global sclerosis, fiber or fibrocellular crescents and interstitial fibrosis (p. 05). According to the degree of density of glomerular matrix membrane and interstitial fibrosis, urinary Col-IV level had better correlation than urinary TGF-betal and IL-6 levels.In IgAN, Col-IV showed increased expression in diseased renal tissue whereas the site of expression of TGF-betal was mainly localized within the cytoplasm of tubular epithelial cells. Interstial expressionwas also present but glomerular TGF-betal expression was found only in patients with heavy mesangial proliferation. There was a significant correlation between glomerular positivity for Col-IV and severity of histological damage. There was also a significant correlation between positivity for TGF-betal and Col-IV in the tubular epithelial and interstitial lesions. In contrast, there was no ralationship between glomerular positivity for TGF-betal and severity of histological damage.The urinary TGF-betal level paralleled tubular TGF-betal expression.

结果 ①IgAN患者尿TGF-β1、IL-6、Col-Ⅳ水平较健康人明显增高(P＜0.01)，该变化与血中的浓度无关(P＞0.05)；②尿TGF-β1水平与小管间质TGF-β1阳性表达呈正相关(P=0.000)，而与小球TGF-β1阳性表达无关(P＞0.05)，尿Col-Ⅳ水平与小球和小管间质Col-Ⅳ阳性表达均呈良好的相关性(P＜0.01)，还与小管间质TGF-β1阳性表达呈正相关(P＜0.05)：③小球Col-Ⅳ阳性表达与肾组织慢性病变密切相关(P＜0.05)，小管间质Col-Ⅳ和TGF-β1阳性表达均与肾小管间质病变呈良好的相关性(P＜0.01)，而小球TGF-β1阳性表达与肾组织损伤无关(P＞0.05)；④尿TGF-β1、Col-Ⅳ水平与肾小球基质基底膜面密度、小管间质病变呈正相关(P＜0.01)，与小球内细胞数呈负相关(P＜0.05)，该结果与其在组织中的表达一致；尿IL-6水平浙江大学硕士学位论文尿TGF一B一、IL一6和Col一IV在IgA肾病中的应用价值与基质基底膜面密度、球性硬化、纤维或细胞纤维新月体所占肾小球百分数及小管间质病变均有显著的相关性(F.05)；在轻度肾病理损伤时，尿'l'G卜pl、I卜6、Col一IV水平即升高，而尿Col一W在反映细胞外基质积聚和间质纤维化程度上比尿TGF一pl和IL一6有更好的相关性。

①Rat MSC and VSMC were cultured and identified, respectively. MSC were labeled with DAPI firstly, and then co-cultivated with VSMC. The changes of morphology and ultrastructure of co-cultured cells were observed. Immunfluorescence analysis was performed by using monoclonal antibodies against specific antigen.②We established the regulatable system in two steps: a stable MSC line expressing rtTA has been constructed and characterized firstly by transfected with pUHD 17-1hyg and then selected by hygromycin B; in a second step, this line was used for trandfer the AT2R gene to MSC to get the well establishing double stable MSC lines;③The expression of AT2R regulated by doxycycline was evaluated by western blot;④The MSCs were transduced into rat carotid arteries with regulatable AT2R gene after the establishment of rat carotid balloon injury restenosis model. The intimal/medial area ratio were measured by digital analysis system.

研究方法：(1)密度梯度离心法及胶原酶消化法分别培养原代大鼠MSC及VSMC，细胞共培养并行免疫荧光化学染色和透射电镜观察超微结构；(2)组成受Dox调控的哺乳动物表达系统的四种成分的转化、扩增及提纯并酶切鉴定；(3)采用常规分子生物学方法连续两个回合转染体外培养的MSC，并分别采用发光计检测不同细胞克隆萤光素酶活性改变以及RT-PCR方法检测AT2R目的基因mRNA表达情况，根据各个细胞克隆受Dox调控表达的程度，选择低背景、高诱导表达AT2R的细胞系，作为双重稳定MSC细胞系；蛋白免疫印迹法观察该细胞系在Dox调控下AT2R表达的时相性、持续性及在不同浓度Dox调控下的表达情况；(4)建立大鼠颈动脉球囊损伤动物模型，将双重稳定MSC在术中导入血管，分别于14 d、28 d进行病理切片，检测可调控AT2R对新生内膜增生的影响；采用RT-PCR免疫组织化学免疫荧光等技术观察AT2R基因在新生内膜中的表达以及细胞外基质成分表达的改变，TUNEL法检测血管组织中细胞凋亡的变化情况。

If you are unfortunate enough to the lovelorn, please tell me, I will help you out, really, please contact me!

如果你不幸失恋了，请告诉我，我会帮助你摆脱困境，真的，请联系我啦！

China's plan to cut energy intensity by 20 percent and pollutant discharges by 10 percent between 2006 and 2010 is a case in point.

中国计划在2006年到2010间降低20%的能源强度和减少10%的主要污染物排放，就是一个这样的例子。