HP8：增强子结合蛋白家族的一个新基因的cDNA克隆

以大鼠增强子结合蛋白（Ｃ／ＥＢＰ）ｃＤＮＡ为探针，筛选人胎肝ｃＤＮＡ库，得到一个含有２４８０碱基命名为ＨＰ８的ｃＤＮＡ克隆，其中８４－１１０９位碱基属编码区。经基因数据库（ＥＭＢＬ，９５．６版本）查对未发现同源基因。该基因编码的蛋白Ｃ－末端含有亮氨酸拉链结构，和Ｃ／ＥＢＰ功能区（Ｃ／ＥＢＰ基因家族新成员。基因组Ｓｏｕｔｈｅｒｎ杂交证实该基因为单拷贝基因。在１４种胎儿组织中显示小肠、皮肤高表达，肾     

人原发性肝细胞癌地高辛素标记HBV DNA探针的原位杂交与C－myc和P_（53）癌基因表达相互关系的研究

取自肝癌高发区的４０例人原发性肝细胞癌（ＰＨＣ）标本，应用地高辛素（Ｄｉｇ）标记ＨＢＶＤＮＡ探针作原位杂交，检测ＰＨＣ和癌周肝组织内ＨＢＶＤＮＡ的表达，并用光敏生物素Ｃ－ｍｙｃ癌基因探针原位杂交和用免疫组化检测ＨＢｓＡｇ，ＨＢｃＡｇ及Ｐ５３癌基因在ＰＨＣ和癌周肝组织内的表达。Ｄｉｇ－ＨＢＶＤＮＡ探针肝内总阳性率为８０％，在ＰＨＣ内为６５％，而癌周肝组织中为７３％。ＨＢＶ－ＤＮＡ定位于肝细胞或癌细胞的胞浆和胞核内，而ＨＢＶＤＮＡ阳性颗粒在胞浆内比胞核内多。ＨＢＶＤＮＡ阳性细胞癌周比癌内更多。结果表明，ＰＨＣ病例与ＨＢＶ存在关系密切；癌内ＨＢＶＤＮＡ大都属整合型，而癌周属游离型。研讨ＰＨＣ内ＨＢＶＤＮＡ的表达与ＨＢｓＡｇ，ＨＢｃＡｇ，和Ｐ５３及Ｃ－ｍｙｃ癌基因的表达相互关系及致癌作用。     

黑色素瘤抗原-1基因在肝细胞癌中的表达

目的 检测黑色素瘤抗原－１（ＭＡＧＥ－１）ｍＲＮＡ在肝细胞癌（ＨＣＣ）中的表达,为用ＭＡＧＥ－１基因编码蛋白作为疫苗对ＨＣＣ患者进行免疫治疗提供依据。方法 用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）方法对４５例ＨＣＣ患者组织和相应癌旁肝组织以及１６例肝硬化组织和１２例正常肝组织的ＭＡＧＥ－１ｍＲＮＡ表达情况进行了研究,对６例ＲＴ－ＰＣＲ扩增产物的目的基因片段进行ＤＮＡ序列测定,并以测序证实为ＭＡＧＥ－     

人肝细胞癌及癌旁肝组织HBV-DNA的原位杂交检测

①目的探讨乙型肝炎病毒（ＨＢＶ）ＤＮＡ与增生结节和肝癌的关系。②方法应用原位杂交技术检测５８例肝细胞癌（ＨＣＣ）及癌旁组织中ＨＢＶ－ＤＮＡ的表达。③结果肝细胞癌及癌旁组织中ＨＢＶ－ＤＮＡ阳性率分别为３７．９％（２２／５８）和４４．８％（２６／５８），差异无显著性（χ２＝０．５６，Ｐ＞０．０５）。ＨＢＶ－ＤＮＡ在癌旁组织中主要位于胞浆内，而肝癌组织中以胞核定位为主。２２例ＨＢＶ－ＤＮＡ阳性的ＨＣＣ中，１６例ＨＣＣ及癌旁组织均阳性，６例ＨＣＣ单独阳性，两者差异有显著性（χ２＝９．０９，Ｐ＜０．０１）。６例癌旁增生结节中ＨＢＶ－ＤＮＡ阳性（６／２７），以胞核定位为主。④结论ＨＢＶ感染与ＨＣＣ存在密切关系。癌周组织ＨＢＶ－ＤＮＡ以复制型为主，癌组织内ＨＢＶ－ＤＮＡ以整合型为主；癌旁增生结节为ＨＣＣ的癌前病变。     

肝细胞癌组织中P16蛋白表达与丙型肝炎病毒感染的关系

目的：研究肝细胞癌（Ｈｅｐａｔｏｃｅｌｌｕｌａｒ ｃａｒｃｉｎｏｍａ,ＨＣＣ）组织中Ｐ１６蛋白表达与丙型肝炎病毒（Ｈｅｐａｔｉｔｉｓ Ｃｖｉｒｕｓ,ＨＣＶ）感染的关系。方法：用免疫组化ＡＢＣ法检测４６例ＨＣＣ及其癌旁肝组织中Ｐ１６蛋白和ＨＣＶ抗原的表达。结果：Ｐ１６蛋白的阴性表达率在ＨＣＣ癌组织（５８．７％,２７／４６）明显高于其癌旁组织（３６．８％,１４／３８）（Ｐ〈０．０５）,在Ｅｄｍｏｎｄｓ     

鼻咽癌及癌旁粘膜组织中c—erbB—2,EGFR mRNA的表达

应用地高辛配基标记探针和ＤＮＡ－ＲＮＡ原位杂交技术，检测了鼻咽部的癌组织，癌旁粘膜和慢性炎症膜组织中Ｃ－ｅｒｂＢ－２，ＥＧＦＲｍＲＮＡ的表达，结果显示：（１）ＣｅｒｂＢ－２和ＥＧＦＲｍＲＮＡ阳性表达率在癌组织分别为８６．７％（２６／３０）和８３．３％（２５／３０），癌旁粘膜分别为８０．０％（１６／２０）和７０．０％（１４／２０）；慢性炎症粘膜均无表达，（２）二基因同时表达者在癌组织中有２５例（８３     

肝细胞癌c-fos的表达及其与乙型肝炎病毒的关系

目的 研究肝癌组织中ｃ－ｆｏｓ和ｃ－ｃｏｆ ｍＲＮＡ的表达及其与乙型肝炎病毒（ＨＢＶ）的关系。方法 采用免疫组化及原位杂交技术检测５９例肝细胞癌组织及５６例癌旁组织中ＨＢｓＡｇ、ＨＢＶ ＤＮＡ、ｃ－ｆｏｓ蛋白及ｃ－ｆｏｓ ｍＲＡＮ的表达。结果 癌旁肝组织ＨＢｓＡｇ阳性率为８７．５％（４９／５６）。肝癌组织ＨＢＶ ＤＮＡ阳性率为５９．３％（３５／５９）。肝细胞癌组织、癌旁肝组织ｃ－ｆｏｓ蛋白 阳性率     

人染色体8p11.2亚带ANK1位点附近区域表达序列的分离与克隆

目的分离人染色体８ｐ１１．２亚带ＡＮＫ１位点附近区域基因的表达序列。方法采用ｃＤＮＡ选择法从定位于该区域的人工酵母染色体（ＹＡＣ）中分离基因的表达序列，并从分离的表达序列中选取１个序列用于筛选人胎脑ｃＤＮＡ文库。结果获得７个表达序列，其中５个可与目的ＹＡＣ反杂交。所得的表达序列ＢＨＬＣ１５２与鼠的ＣＡｒＧ盒结合因子基因高度同源，ＢＨＬＣ７４与编码心室肌肌球蛋白的碱性轻链１（ＭＬＣ１）基因高度同源，表明ＢＨＬＣ７４可能编码一种ＭＬＣ１的同工蛋白。分离的其它表达序列与数据库内一些位置和功能尚不清楚的表达序列标签（ｅｘｐｒｅｓｅｄｓｅ－ｑｕｅｎｃｅｔａｇ，ＥＳＴ）仅有部分同源性。用表达序列ＢＨＬＣ１１９筛选人胎脑ｃＤＮＡ文库，获得１个插入片段长１９５１ｂｐ的克隆，序列分析表明它编码１７４个氨基酸，该序列与所有已知的蛋白质序列无同源性。Ｎｏｒｔｈｅｒｎ印迹分析结果显示，在人胎脑总ＲＮＡ中呈现１条约３．５ｋｂ带；组织表达分析表明，此系一广泛表达的基因。结论ＢＨＬＣ７４可能作为与８ｐ相关的心脏病候选基因，其它序列至少可以作为定位在ＡＮＫ１位点附近区域的肿瘤抑制基因的初筛基因。     

肝细胞癌和癌旁肝组织中c—myc蛋白表达与细胞凋亡的关系

利用原位脱氧核糖核酸末端转移酶标记技术和ＳＰ免疫组化方法检测了肝细胞癌和癌旁肝组织中细胞凋亡和ｃ－ｍｙｃ蛋白的表达。结果显示，癌组织中凋亡细胞密度明显氏于癌旁组织（Ｐ〈０．０１），ｃ－ｍｙｃ蛋白在癌组织和癌旁肝组织中的阳性率分别为４０％（１２／３０）和８６．７％（２６／３０），差异有显著性，其表达强度亦以癌旁肝组织为强（Ｐ〈０．０１）。结果提示，ｃ－ｍｙｃ蛋白的表达既可诱导细胞凋亡又可促进细胞增殖     

胃癌多基因表达的同步检测

胃癌多基因表达的同步检测朱日林，等。中华肿瘤杂志１９９６；１８（３）：１９９应用ＬＳＡＢ和ＡＢＣ法对７５例胃癌及癌旁组织进行了Ｐ５３、ｃ－ｅｒｂＢ－２、ＥＧＦＲ和ｒａｓ表达的研究。结果显示：１。７５例胃癌Ｐ５３、ｃ－ｅｒｂＢ－２、ＥＧＦＲ和ｒａｓ表达...     

HP8: The cDNA clone of a novel member of C/EBP gene family

ＨＰ８：ＴｈｅｃＤＮＡｃｌｏｎｅｏｆａｎｏｖｅｌｍｅｍｂｅｒｏｆＣ／ＥＢＰｇｅｎｅｆａｍｉｌｙ￥（徐砺新）（万大方）（刘彦仿）（李宏年）（张萍萍）（隋延仿）（顾健人）ＸｕＬｉｘｉｎ，ＷａｎＤａｆａｎｇ，ＬｉｕＹａｎｆａｎｇ，ＬｉＨｏｎｇｎｉａｎ，Ｚｈ...     

肝癌内高表达新基因的部分cDNA克隆

目的寻找人肝癌相关的基因。方法应用Ｎｏｒｔｈｅｒｎ杂交技术，对表达序列标记（ＥｘｐｒｅｓｓｅｄＳｅ－ｑｕｅｎｃｅｔａｇｓ，ＥＳＴ）基因克隆进行筛选，以得到的基因片段为探针，筛选人胎肝ｃＤＮＡ文库。结果得到一个在肝癌内高表达的ＥＳＴ基因片段。进一步筛选人胎肝ｃＤＮＡ文库，得到一命名为ＦＬ６的ｃＤ－ＮＡ克隆，核苷酸序列测定表明，ＦＬ６长度为１４６４个碱基，检索最新版本ＧｅｎｅＤａｔａｂａｓｅｓ（ＥＭＢＬ９６．６），未发现同源基因。实验结果还显示该基因在胎儿各组织内有不同的表达特性。结论该基因可能与细胞的增殖、癌变过程相关。     

A novel full-length gene of human ribosomal protein L14.22 related to human glioma

Background This study was undertaken to obtain differentially expressed genes related to human glioma by cDNA microarray and the characterization of a novel full-length gene. Methods Total RNA was extracted from human glioma and normal brain tissues, and mRNA was used as a probe. The results of hybridization procedure were scanned with the computer system. The gene named 507E08 clone was subsequently analyzed by northern blot, bioinformatic approach, and protein expression. Results Fifteen differentially expressed genes were obtained from human glioma by hybridization and scanning for four times. Northern blot analysis confirmed that the 507E08 clone was low expressed in human brain tissue and over expressed in human glioma tissues. The analysis of BLASTn and BLASTx showed that the 507E08 clone was a novel full-length gene, which codes 203 amino acid of protein and is called human ribosomal protein 14.22 gene. The nucleotide sequence had been submitted to the GenBank?with the accession number of AF329277. After expression in E.coli., protein yielded a major band of apparent molecular mass 22 kDa on an SDS-PAGE gel. Conclusions cDNA microarray technology can be successfully used to identify differentially expressed genes. The novel full-length gene of human ribosomal protein 14.22 may be correlated with the development of human glioma.     

cDNA microarray in isolation of novel differentially expressed genes related to human glioma and clone of a novel full-length gene

Background This investigation was undertaken to obtain differentially expressed genes related to human glioma using cDNA microarray and the characterization of one novel full-length gene. Methods Total RNA was extracted from human glioma tissues and normal brain tissues, and mRNA was used to make probes. After hybridization and washing, the results were scanned using a computer system. The gene named 681F05 clone was an expressed gene to human glioma through four-time hybridization and scanning. Subsequently northern blot analysis was performed by northern blot, 5‘RACE and bioinformatics. Results Fifteen differentially expressed genes to human glioma were obtained through four-time hybridization and scanning. Northern blot analysis confirmed that 681F05 clone was low-expressed in human brain tissues and over-expressed in human glioma tissues. The analysis of BLASTn and BLASTx showed that 681F05 clone is two cDNA clones encoding two novel proteins that are highly identified to the cyclophilin isoform 10 of C. Elgans, respectively. Sequence analysis revealed the two cDNA clones are two different splicing variants of a novel cycophilin-like gene (PPIL3a and PPIL3b). Conclusions cDNA microarray technology can be successfully used to identify differentially expressed genes. The novel full-length gene of human PPIL3 may be correlated with the formation of human glioma.     

小鼠衰老相关新基因Arzc的克隆

目的鉴别和克隆与小鼠衰老相关的新基因.方法采用改进的差异显示反转录聚合酶链反应(DDRT-PCR)技术分析正常老化和年轻BALB/C小鼠大脑皮层mRNA的差异表达,差异显示的新表达序列标签(EST)经Northern杂交验证后,作为出发序列,利用生物信息学方法设计引物,以小鼠大脑皮层总RNA的反转录产物为模板通过PCR克隆全长cDNA.结果差异显示分析共获得表达差异明显的EST 42条,序列同源性分析显示其中29条为已知基因cDNA片段,13条可能为新EST.对新EST进行Northern杂交验证后,对确证在正常老化鼠皮层表达明显下调的1个EST,通过生物信息学方法克隆全长cDNA,得到1个新的1 382 bp的cDNA序列,命名为Arzc,获得GenBank注册号AY344585.该序列含有1个261 bp的开放阅读框,推测的编码多肽包含86个氨基酸残基,与噬菌体编码的重组酶/整合酶的一段氨基酸序列有较高同源性.结论鉴别并克隆到一个可能与小鼠衰老相关的新基因Arzc.     

mRNA差异显示技术在紫杉醇诱导的表达基因克隆中的应用

作者应用mRNA差异显示、DNA序列分析和Northern杂交技术，对紫杉醇处理人乳癌细胞诱导的表达基困进行cDNA克隆研究，12个克隆有明确的mRNA水平表达改变，其中6个cDNA克隆核旮酸序列与已知的cDNA序列有较高的同源性，其余6个尚无同源性基因。克隆C3P3与人腺苷基蛋氨酸合成酶基因序列同源性达99％，紫杉醇诱导该酶基因转录活动和基因产物活性增高。提示mRNA差异显示技术在筛选mRNA水平表达差异的基因cDNA克隆研究中是可行的和有效的。     

Biological function of a novel gene overexpressed in human hepatocellular carcinoma

Objective To clone the full-length of a differentially expressed cDNA fragment, LC27, and study its biological function tentatively. Methods Northern blot was used to analyze the expression pattern of LC27 in hepatocellular carcinoma, matched nontumor liver tissues, fetal liver and normal adult liver tissues, as well as BEL-7402 hepatocellular carcinoma cell line ESTs splicing and 5’ rapid amplification of cDNA ends (5’ RACE) were used to clone the full-length of LC27 cDNA.An antisense oligodeoxynucleotide approach was used to investigate the biological role of the gene in the proliferation of BEL-7402 cells. Results A 2186 bp novel cDNA with an open reading frame encoding a 283 amino acid protein was cloned.Analysis of the deduced amino acid sequence indicated that it is 38% (88/229) identical to human Golgi 4-transmembrane spanning transporter MTP.The gene and the encoded protein was termed hepatocellular carcinoma overexpressed transmembrane protein (hotp) and HOTP, respectively.Hotp mRNA was almost undetectable in normal adult liver and fetal liver tissues.However, it was significantly up-regulated in hepatocellular carcinoma and some matched nontumor liver tissues, as well as BEL-7402 cells.The proliferation of BEL-7402 cells was suppressed by an antisense oligodeoxynucleotide against hotp mRNA at a concentration of 50 μg/ml. Conclusion HOTP may be an integral membrane transporter protein.The overexpression of the gene in hepatocellular carcinoma may play an important role in hepatocarcinogenesis and disease progression.     

人肿瘤坏死因子样蛋白全长cDNA的克隆及原核表达

目的分离、克隆编码人肿瘤坏死因子样蛋白(TNFLP)的全长cDNA,并对其功能进行初步研究。方法从本室构建的λZAP表达胎脑文库中分离到编码TNFLP的全长cDNA。应用多种软件对该基因及其编码蛋白进行分析,寻找其同源蛋白,分析其亲、疏水性及其存在的结构域和基序。以Northern印迹分析检测TNFLP的mRNA的组织分布,并用谷胱苷肽-S-转移酶(GST)原核表达系统分段表达TNFLP的N端和C端蛋白。结果TNFLP全长共2112bp,编码一208个氨基酸的蛋白。亲、疏水性分析显示,TNFLP有两个疏水区,且C端95个氨基酸与小鼠的TNF-α一致性为42%。基序分析显示,TNFLP的C端有一内质网膜定位信号-TYKRL,与TNF-α完全一致。人的TNFLP位于人的16号染色体上。Northern杂交显示,TNFLP在心、脑和胰腺中呈高表达,可见一个转录本。用GST原核表达系统,分段表达了TNFLP的N端和C端。结论初步分析了TNFLP的组织表达谱,并分段表达了其N端和C端,为进一步研究其功能提供了条件。     

应用抑制性差减杂交技术克隆小鼠胸腺细胞发育相关新基因

目的:克隆胸腺细胞发育相关新基因.方法:应用抑制性差减杂交技术(suppression subtractive hybridization, SSH),构建了胸腺基质细胞系cDNA差减杂交文库,结合RACE及RT-PCR进一步克隆并验证新基因的cDNA全长序列.结果:筛选两个不同胸腺基质细胞系的差异表达基因,获得了差异表达的cDNA片段,克隆后挑选阳性克隆进行测序,成功克隆8个新基因片段.对其中两个新基因克隆C55和C91进行Northern杂交分析,mRNA转录体长度分别为1.4 kb及1.5 kb,C55的表达在两种胸腺基质细胞系中存在显著差异.进一步克隆了C55和C91两个新基因的cDNA全长序列,已被GenBank所接受.结论:抑制性差减杂交技术是克隆新基因片段的有效方法;成功克隆8个新基因片段及两个新基因的cDNA全长序列.     

一种新型单羧酸转运体基因的分离与克隆

目的 分离、克隆编码肾脏甲酸盐（formate)和（或）草酸盐（oxalate)转运体的cDNA,以研究Cl^-在近端肾小管转运的机制。方法 根据细菌Oxlt(formate-oxalate交换体）的基因序列在哺乳动物表达序列标签（express sequence tag,EST)数据库中查找与其相似的cDNA。按筛选到的cDNA设计探针,应用小鼠多组织杂交膜分析该基因的组织表达谱。结果 在小鼠肾cDNA文库中有一个与Oxlt极其相似的EST,序列分析显示了其有一编码429个氨基酸蛋白质的开放阅读框。从编码的顺序中可发现该序列与单羧酸转运家族（monocarboxylate transporter,MCT)极为相近,与小鼠MCT1的氨基酸一致性为30％,与小鼠MCT2为33％,与大鼠MCT329％。其中第14－158个氨基酸中有25％与OxlT相同。Northern印迹发现,新的基因在小鼠肾脏中表达最多,肝脏和心脏中极少表达。结论 该基因是一新型的MCT家族成员,主要在肾脏中表达,其功能可能是介导肾小管的formate转运。