SIMULTANEOUS OVER-EXPRESSION OF INSULIN-LIKE GROWTH FACTOR- Ⅱ (IGF- Ⅱ ) AND IGF- Ⅱ RECEPTOR(IGF- Ⅱ R) GENES IN HUMAN PRIMARY CANCER-IMPLICATION OF AUTOCRINE AND PARACRINE MECHANISM IN AUTONOMOUS GROWTH OF HEPATIC CANCER

This is first report about the simultaneous over-expression of both Insulin-like growth factor (IGF- I ) and its receptor (IGF- I R) at mRNA level in human primary hepatic Cancer (PHC). In 10 PHC samples from China, IGF-I and IGF- I R were both over-expressed, whereas only a background signal was detected in normal liver. In 5 pairs of PHC and its non- tumorous adjacent liver tissues from South Africa, IGF- I and IGF- I R were also over-expressed in PHC. mRNA expression of IGF- I in all 5 cases and IGF- I R in 4 of 5 cases were higher in cancer than non- tumorous adjacent liver tissues. These results strongly implicate that an autocrine and/ or paracrine mechanism might be Involved in formation and progression of PHC.

Correlation of nephroblastoma overexpressed gene (NOV) mRNA levels with cell growth- and migration-related gene expression in osteosarcoma tissue

Objective:To study the correlation of nephroblastoma overexpressed gene (NOV) mRNA levels with cell growth- and migration-related gene expression in osteosarcoma tissue. Methods: A total of 78 patients with osteosarcoma undergoing surgical treatment in our hospital between May 2012 and May 2016 were selected, and the osteosarcoma tissue and paracancerous tissue were collected. Fluorescence quantitative PCR method was used to detect the mRNA expression of NOV as well as pro-proliferation/anti-proliferation genes and invasion genes, and the Pearson test was used to further assess the correlation of NOV mRNA expression with the osteosarcoma cell growth and migration activity.Results: NOV mRNA expression in osteosarcoma tissue was significantly lower than that in paracancerous tissue, FoxM1, STIM1 and Orai1 mRNA expression were significantly higher than those in paracancerous tissue, anti-proliferation genes RanBP9, Tap73, Caspase-3, PTEN and P53 mRNA expression were significantly lower than those in paracancerous tissue, and invasion genes EFEMP1, Notch1, Id1 and Src mRNA expression were significantly higher than those in paracancerous tissue. Pearson test showed that the NOV mRNA expression in osteosarcoma tissue was negatively correlated with pro-proliferation genes FoxM1, STIM1 and Orai1 mRNA expression, positively correlated with anti-proliferation genes RanBP9, Tap73, Caspase-3, PTEN and P53 mRNA expression, and negatively correlated with invasion genes EFEMP1, Notch1, Id1 and Src mRNA expression.Conclusion: NOV expression is relatively low in osteosarcoma tissue, and NOV mRNA expression is negatively correlated with tumor cell proliferation and invasion activity.

WT1、NLR及LMR对儿童急性髓系白血病的诊断价值分析

目的探讨WT1、NLR及LMR对儿童急性髓系白血病的诊断价值。方法选取73例AML初诊患儿作为研究组,另外选取同时期门诊体检的健康儿童作为对照组(50例),比较2组的WT1、NLR及LMR水平等差异。结果研究组患儿的NLR水平高于对照组,而LMR则较对照组低(P<0.05);随着危险度增高,AML患儿的NLR及WT1表达水平逐渐升高,而LMR水平逐渐降低(P<0.05);NLR、LMR及WT1检测鉴别为儿童AML分别为55例、59例及56例,联合检测鉴别儿童AML为69例;各指标联合检测对儿童AML鉴别诊断敏感度、特异度及AUC均明显高于NLR、LMR及WT1单独检测(P<0.05)。结论联合检测WT1及NLR、LMR可显著提高儿童AML的诊断率,但仍需进一步加大样本量行深入验证。

Cyr61/CTGF/Nov family proteins in gastric carcinogenesis

Gastric cancer(GC)is the second leading cause of cancer-related death.The poor survival rate may reflect the relatively aggressive tumor biology of GC.Recently,the importance of the tumor microenvironment in carcinogenesis has emerged.In the tumor microenvironment,tumor cells and the surrounding stromal cells aberrantly secrete matricellular proteins capable of modulating carcinogenesis and regulating metastasis.The Cyr61/CTGF/Nov(CCN)proteins are a family of matricellular proteins with variable roles in many physiological and pathological processes.The evidence suggests that CCN family proteins contribute to GC carcinogenic processes.Here,we briefly review recent research on the effects of CCN family proteins in GC carcinogenesis and the development of new targeted agents in this field.

Characterization of NPR1 Genes from Norton and Cabernet Sauvignon Grapevine

Non-expressor of pathogenesis-related genes 1 （NPR1） plays a significant role in the defense responses of plants to pathogens by regulating the expression of defense-related genes. In the present study, we isolated two NPR1 genes from Vitis aestivalis cv. Norton and Vitis vinifera cv. Cabernet Sauvignon, which were referred to as VaNPR1.1 and VvNPR1. 1-CS, respectively. They encode a protein of 584 amino acids with a predicted molecular weight of 64.8 kDa and a theoretical isoelectric point （pI） of 5.74. The predicted amino acid sequences of VaNPR1.1 and VvNPR1.1-CS differ by only one amino acid. Over-expression of VaNPR1.1 gene in Arabidopsis npr1-1 mutant plants restores the transcriptional expression of AtPR-1 gene, though not to the full scale. This result demonstrated that a grapevine VaNPR1.1 possesses a similar function to the Arabidopsis NPR1 in the regulation of defense-related genes. Over-expression of VaNPR1.1 in transgenic Arabidopsis plant increased tolerance to salinity, but had no effect on the drought tolerance. We conclude that VaNPR1.1 is a functional ortholog of AtNPR1 and also involved in grapevine＇s response to the salt stress.

SIRT1 and stem cells: In the forefront with cardiovascular disease, neurodegeneration and cancer

Cardiovascular disease, nervous system disorders, and cancer in association with other diseases such as diabetes mellitus result in greater than sixty percent of the global annual deaths. These noncommunicable diseases also affect at least one-third of the population in low and middle-income countries and lead to hypertension, elevated cholesterol, malignancy, and neurodegenerative disorders such as Alzheimer's disease and stroke. With the climbing lifespan of the world's population, increased prevalence of these disorders is expected requiring the development of new therapeutic strategies against these disabling disease entities. Targeting stem cellproliferation for cardiac disease, vascular disorders, cancer, and neurodegenerative disorders is receiving great enthusiasm, especially those that focus upon SIRT1, a mammalian homologue of the yeast silent information regulator-2. Modulation of the cellular activity of SIRT1 can involve oversight by nicotinamide/nicotinic acid mononucleotide adenylyltransferase, mammalian forkhead transcription factors, mechanistic of rapamycin pathways, and cysteine-rich protein 61, connective tissue growth factor, and nephroblastoma over-expressed gene family members that can impact cytoprotective outcomes. Ultimately, the ability of SIRT1 to control the programmed cell death pathways of apoptosis and autophagy can determine not only cardiac, vascular, and neuronal stem cell development and longevity, but also the onset of tumorigenesis and the resistance against chemotherapy. SIRT1 therefore has a critical role and holds exciting prospects for new therapeutic strategies that can offer reparative processes for cardiac, vascular, and nervous system degenerative disorders as well as targeted control of aberrant cell growth during cancer.

Overexpression of the PdpapERF109 gene enhances resistance of Populus davidiana×P.alba var.pyramidalis to Fusarium oxysporum infection

The key transcription factor gene PdP apE RF109 was cloned from Populus davidiana×P.alba var.pyramidalis(Pdpap),and after overexpression of P dP ap ERF109 in transformants,the gene functions in the resistance response to Fusarium oxysporum infection.Compared with the wild Pdpap,after inoculation with F.oxysporum,the physiological and biochemical characteristics,including relative fresh weight,peroxidase activity,and the percentage of electrolyte leakage showed that,after overexpression of the PdPapERF109 gene,the transformants grew well and displayed significant resistance to F.oxysporum infection.By comparing the reactive oxygen species scavenging capacity of Pdpap plants after pathogen infection,the P dPapERF109-overexpressing plants had significantly better reactive oxygen species scavenging ability than the wild plants.Comprehensive analysis of plant morphology and various physiological and biochemical parameters showed that the overexpression of the P dpapERF109 gene significantly improved the resistance of Pdpap plants to F.oxysporum root rot.Therefore,increasing the expression of the homologous ERF109 gene can be an effective strategy to increase disease resistance in hybrid poplars.

Can overexpression of TGF-β1 gene change the sex ratio in transgenic mice?

Mouse TGF-β1 gene was microinjected into male pronuclei of F2 hybrid fertilized eggs obtained by mating CSJLF1 and C57BL/6J inbred strains to generate transgenic mice with over-expressed TGF-β1 gene. The rate of founder production is 31% and Southern blot analysis of founder mice tail DNAS gave an integration efficiency of 33%. TGF-β1 gene could be stably integrated to the chromosomes of transgenic mice and transmitted to their progeny at a rate of 33% in the second generation. Dot blot analysis of tail RNA of some transgenic mice indicated a moderate expression of the transgene. The most interesting finding of the present work is the striking deviation from the normal male:female sex ratio in transgenic mice,with an average ratio of 6.7:1. The possible nature of the predominance of male sex in transgenic mice overexpressing TGF-β1 is discussed.

Identification of a constitutional mutation in the WT1 gene in Taiwan Residents patients with Wilms tumor

The overall frequency of WT1 gene alterations in Wilms tumor is still unclear in Taiwan. Here we conducted molecular genetic analysis of the WT1 gene in Taiwan Residents patients with Wilms tumor. Polymerase chain reaction and direct sequencing were performed on DNA samples from blood and paraffin-embedded tumor specimens. A constitutional mutation in the WT1 gene was found in one DNA sample from peripheral blood lymphocytes. The remaining DNA samples from peripheral blood lymphocytes and paraffin-embedded tumor specimens were tested negative for both constitutional mutations and somatic mutations. Thus, mutations at other Wilms tumor loci may play an important role in Wilms tumor development.

基因TOPOⅡα、TUBB3、ERCC1在小儿肾母细胞瘤组织中的表达及其临床意义

目的探究基因TOPOⅡα、TUBB3、ERCC1在小儿肾母细胞瘤组织中的表达及其临床意义。方法选择40例肾母细胞瘤患儿的病理组织标本,另同期采集距离肿瘤组织2~3 cm的癌旁组织标本作为对照组。分析两者拓扑异构酶Ⅱα(TOPOⅡα)、3型β微管蛋白编码基因(TUBB3)、核苷酸切除修复交叉互补基因(ERCC1)表达情况;另分析TOPOⅡα、TUBB3、ERCC1与肾母细胞瘤患儿病理特征的相关性。结果癌组织中TOPOⅡα、TUBB3、ERCC1高表达率均高于癌旁组织,差异有统计学意义(P<0.05)。TOPOⅡα高表达者病理分期Ⅲ~Ⅳ占比率高于中低表达者,差异有统计学意义(P<0.05);ERCC1高表达者病理分期Ⅲ~Ⅳ占比率高于中低表达者,差异有统计学意义(P<0.05)。结论TOPOⅡα、TUBB3、ERCC1在小儿肾母细胞瘤中的表达水平均高于癌旁组织,其中TOPOⅡα、ERCC1均与疾病病理分期有关,二者在肾母细胞瘤的发生及发展中具有重要作用。

NOV对大鼠神经干细胞增殖和分化的影响

目的 探讨NOV基因和蛋白对大鼠神经干细胞 (NSCs)增殖和分化的影响。方法 NOV基因重组质粒转染COS 7细胞和NSCs ,收集COS 7细胞和NOV基因修饰COS 7细胞的条件培养液 (简称COS CM和NOV CM ) ,作用于培养的NSCs ,3H TdR掺入检测NSCs的增殖 ,免疫细胞化学和流式细胞仪检测NSCs的分化。结果 ①NOV CM能明显促进NSCs对3H TdR的摄入 ,说明NOV CM能明显促进细胞的增殖 ,另外NOV CM的促细胞增殖作用具有一定的量效关系 ;②免疫细胞化学和流式细胞仪结果显示 ,NOV CM促进NSCs向神经元方向分化 ;③NOV基因修饰NSCs分化时 ,神经元的比例增高。

NOV基因修饰神经干细胞移植对损伤大鼠脊髓功能恢复的影响

目的 观察NOV基因修饰神经干细胞 (NSCs)移植对损伤大鼠脊髓功能的治疗作用。方法 40只Wistar大鼠随机分为对照组 (A组 )、损伤组 (B组 )、NSCs组 (C组 )和NOV NSCs组 (D组 ) ,每组 10只 ;将NSCs悬液注入C组切断脊髓处 ,D组注入等量的NOV基因修饰NSCs悬液 ,B组注射等量的生理盐水。术后 2个月 ,采用BBB评分和皮层体感诱发电位 (CSEP)观察大鼠脊髓功能的恢复程度。结果 ①术后 2月BBB评分B、C、D组大鼠有所恢复 ,但都未达到正常水平 ;C组和D组的大鼠恢复较好 ,评分较高 ,与B组有显著性差异 ;其中D组比C组恢复要好。②脊髓损伤后 ,各组的CSEP波均消失 ,术后 2月除B组外C组和D组的波形均有不同程度的恢复 ,但潜伏期延长 ,其中C组比D组更长 ,两者之间有显著性差异。

NOV蛋白免疫反应阳性神经元在鲢、鸡和几种哺乳动物脑的比较发育

目的：研究鲢、鸡、牛、犬和大鼠脑肾母细胞瘤过度表达基因（ｎｏｖ）蛋白免疫反应阳性神经元的比较发育。方法：免疫组织化学技术。结果：低等脊椎动物鲢脑仅有少量ＮＯＶ蛋白免疫反应阳性神经元，分布于延髓的一些核团、丘脑前核、丘脑后核、下丘脑背侧核、下丘脑外侧核。鸡脑ＮＯＶ蛋白免疫反应阳性神经元除广泛分布于脑干外，小脑、间脑和大脑纹状体的多数脑区阳性神经元亦较多。哺乳动物牛、犬和大鼠脑ＮＯＶ蛋白免疫反应阳性神经元广泛分布，脑干、小脑、间脑和大脑都检测到强烈阳性信号。结论：ｎｏｖ基因在从低等脊椎动物到高等脊椎动物的进化过程中非常保守，提示它在脑神经元生长。

NOV对人神经干细胞增殖和分化的影响

目的 探讨NOV蛋白对人神经干细胞 (HNSCs)增殖和分化的影响。 方法 NOV基因重组质粒转染COS 7细胞 ,收集COS 7细胞和NOV基因修饰COS 7细胞的条件培养液 (COS CM和NOV CM ) ,作用于培养的HNSCs,3H TdR掺入液闪检测HNSCs的增殖 ,免疫细胞化学和流式细胞仪检测HNSCs的分化。 结果 (1)NOV CM能明显促进HNSCs对3H TdR的摄入 ,说明NOV CM能明显促进HNSCs的增殖 ,另外NOV CM的促细胞增殖作用具有一定的量效关系 ;(2 )免疫细胞化学和流式细胞仪结果显示 ,NOV CM促进HNSCs向神经元方向分化。结论 NOV蛋白可能具有促进HNSCs增殖和分化的作用。

nm23-H_1mRNA表达与肾母细胞瘤临床行为的关系

应用Northernblot分子杂交技术，检测30例肾母细胞瘤及其对应正常肾组织中nm23－H1mRNA表达。通过nm23－H1mRNA水平的比较及其与临床病理的分析，发现肾母细胞瘤nm23－H1mRNA呈低表达，随着病程的演进和转移的出现，肿瘤细胞中nm23－H1mRNA表达水平呈显著性降低，与肿瘤的病理类型无显著相关性（P＞0．05）。提示nm23－H1RNA低表达可能是肾母细胞瘤演进和转移的重要因素。

大鼠中枢神经系统肾母细胞瘤过度表达基因mRNA神经元的发育——原位杂交与RT-PCR研究

目的研究大鼠ＣＮＳ肾母细胞瘤过度表达基因（ｎｏｖ）ｍＲＮＡ神经元的发育。方法原位杂交组织化学和逆转录ＰＣＲ。结果Ｐ０～Ｐ４：原位杂交方法最早检测到ｎｏｖｍＲＮＡ神经元是Ｐ０，Ｐ０～Ｐ４阳性信号均较弱，分布于脑桥和脊髓等。Ｐ５：丘脑部分核团和海马锥体细胞层观察到阳性神经元。Ｐ８：ｎｏｖｍＲＮＡ神经元数目增多，边缘系统及丘脑明显阳性；大脑皮质和下丘脑阳性较弱。Ｐ１５：丘脑、下丘脑和大脑皮质ｎｏｖｍＲＮＡ神经元增多，其余核团稳定在Ｐ８水平。Ｐ３０：在脑干观察到强烈的阳性信号，大脑皮质强阳性，海马、丘脑和下丘脑标记较强。Ｐ６０：脑桥和延髓的标记仍然很强，大脑皮质、丘脑和下丘脑的阳性信号稳定在Ｐ３０水平。Ｐ３００：ｎｏｖｍＲＮＡ神经元显著减少，阳性信号明显减弱。逆转录ＰＣＲ检测结果与原位杂交所得结果基本相符，在出生前２ｄ检测到ｎｏｖｍＲ－ＮＡ，其表达高峰出现在Ｐ３０～Ｐ６０之间，随之下降，Ｐ１５０和Ｐ３００表达弱。结论ｎｏｖ基因与其他早期反应基因不同，其表达高峰不是在胚胎发育早期，而是在近性成熟时期，提示ｎｏｖ基因主要在神经系统发育、分化及功能活动中起作用。

人nov基因全长cDNA克隆、测序及其组织表达谱分析

采用特异性mRNA富集技术,从人早期胚胎中得到人nov基因(novH)的cDNA克隆,序列分析表明,该基因cDNA序列由2389个碱基对组成,包含一段长1071bp的开放阅读框(ORF),3′端含有加尾信号AATAAA和ply(A)尾巴.Northern杂交显示,在Hela细胞和肾上腺组织中有表达的novHmRNA,大小约为2.5kb;多组织RNA点杂交分析结果进一步表明,novH基因在肾上腺和主动脉中的表达水平相对较高,在成人肾、肝、肺和小肠组织中亦有少量的表达.

NOV蛋白在切割海马伞大鼠海马中表达的变化

通过切割右侧海马伞制备大鼠海马伞损伤模型,应用Westernblotting、免疫组织化学技术,观察切割海马伞后不同时程海马中肾母细胞瘤过度表达基因(NOV)蛋白表达的变化,并对结果进行图像处理和统计学分析。Westernblotting结果显示,NOV蛋白在切割海马伞后3d表达开始上升,14d达高峰后缓慢下降。切割海马伞后14d切割侧和正常侧相比较,海马CA1-CA3区的锥体细胞层及齿状回颗粒层NOV阳性细胞数目无显著性差异(P>0.05),但切割侧NOV阳性细胞明显比正常侧深染,两侧比较平均灰度值有显著性差异(P<0.01)。结合本课题组以往的工作,本研究结果提示,切割海马伞后海马中高表达的NOV蛋白可能参与了诱导神经干细胞迁移和向神经元分化的过程。

RGS4蛋白在小儿肾母细胞瘤组织中的表达及临床意义

目的分析G蛋白信号调节蛋白4(RGS4)在小儿肾母细胞瘤组织和癌旁组织中的表达差异,探讨RGS4与小儿肾母细胞瘤发生发展的关系。方法收集37例小儿肾母细胞瘤患者术后肾癌组织及8例癌旁组织,采用免疫组织化学法检测RGS4蛋白的表达水平;另收集患者手术切除新鲜癌组织及对应癌旁组织标本各8例,采用q RT-PCR和Western blot分别检测RGS4 m RNA和蛋白的表达水平。结果免疫组织化学染色结果显示,RGS4蛋白在小儿肾母细胞瘤组织及癌旁组织中均存在阳性表达,其在小儿肾母细胞瘤组织中的高表达率低于癌旁组织[37.83%(14/37)vs.87.5%(7/8),χ2=4.675,P<0.05];RGS4 m RNA在小儿肾母细胞瘤组织中的表达水平显著低于癌旁组织(1.064±0.549 vs.5.374±0.735,t=13.290,n=8,P<0.01);Western blot结果显示,RGS4蛋白在小儿肾母细胞瘤组织中的表达量低于癌旁组织(0.301±0.092 vs.0.779±0.041,t=13.424,n=8,P<0.01)。结论 RGS4在小儿肾母细胞瘤组织中表达下调,可能与小儿肾癌的发生发展有关。

nov基因在不同种属动物脊髓中的表达

用地高辛标记的cRNA探针原位杂交方法研究了鲢、青蛙、蛇、鸡、牛、犬和猫脊髓中肾母细胞瘤过度表达基因（nov）mRNA阳性神经元的种系发育。结果显示，低等脊椎动物鲢、青蛙和蛇脊髓中仅有少量novmRNA阳性神经元，分布于灰质腹角。鸡脊髓中阳性神经元除主要分布于脊髓腹角外，中央灰质也有少量分布。哺乳动物牛、大和猫脊髓灰质中novmRNA阳性神经元分布广泛，背腹角、中央灰质及中央核区都检测到很强的杂交信号。以上结果表明，nov基因在从低等脊椎动物到高等脊椎动物的进化过程中非常保守，这种保守性提示nov基因在脊髓神经元发育、分化及正常生理功能中可能具有重要作用。