病毒胸苷激酶基因治疗人胃癌的实验研究

为观察单纯疱疹病毒脱氧胸苷激酶（ＨＳＶ－ＴＫ）／丙氧鸟苷（ＧＣＶ）基因治疗系统对胃癌细胞的杀伤作用，将ＨＳＶ－ＴＫｃＤＮＡ定向克隆入逆转录病毒载体ｐＤＯＲ－ｎｅｏ中，用ＬＩＰＯＦＥＣＴＡＭＩＮＥ法将该ＨＳＶ－ＴＫ基因转染胃癌细胞系ＳＧＣ－７９０１，测定阳性转染细胞（ＳＧＣ－７９０１／ＴＫ）在体内外对ＧＣＶ的敏感性。结果示ＧＣＶ在体外对ＳＧＣ－７９０１／ＴＫ细胞有明显的杀伤作用，其作用呈现剂量和时间依赖性特点，且同时表现出对周围亲代ＳＧＣ－７９０１细胞的杀伤效应（旁观者效应）。体内实验表明ＧＣＶ可抑制ＨＳＶ－ＴＫ阳性胃癌细胞的生长，使已形成的肿瘤逐渐消失。提示逆转录病毒载体介导的ＨＳＶ－ＴＫ基因转移胃癌细胞，配合以ＧＣＶ治疗，是胃癌基因治疗的又一途径。     

单纯疱疹病毒Ⅰ型胸苷激酶基因转染人肺腺癌细胞A549的体内外实验研究

单纯疱疹病毒Ⅰ型胸苷激酶（ＨＳＶ１－ＴＫ）基因是一个药敏基因，转导该基因的细胞对非毒性抗病毒药更昔洛韦（ＧＣＶ）或阿昔洛韦（ＡＣＶ）敏感，能被其杀灭。通过探讨该基因转染肺癌细胞后对ＧＣＶ或ＡＣＶ的敏感性，为其进入临床治疗肺癌积累实验资料。本文用电穿孔法将含胸苷激酶（ＴＫ）基因的逆转录病毒载体导入人肺腺癌细胞Ａ５４９，在体内外观察转基因细胞对ＧＣＶ的敏感性。材料与方法合ＴＫ基因的逆转录病毒表达载体ＰＬＸＳＮ－ＴＫ参照文献［１］构建，Ａ５４９细胞购自上海细胞所。新霉素（Ｇ４１８）、四甲基偶氮唑盐（Ｍ…     

CD+4自然杀伤T细胞在慢性乙型肝炎病毒感染中的作用

目的 了解ＣＤ４＾＋、ＣＤ８＾＋自然杀伤（ＮＫ）Ｔ细胞在慢性ＨＢＶ感染外周轿中的分布情况,并对其细胞毒性进行分析,阐明其在慢性ＨＢＶ感染中的作用。方法 常规分离外周上核细胞（ＰＢＭＣＳ）,用重组人白细胞介素－１２／２诱导１４ｄ,以鼠抗人ＣＤ４单克隆抗体或抗人ＣｍＡｂ与抗人ＣＤ５６ｍＡｂ分别标记细胞样品,流式细胞术（ＦＣＭ）分析,ＣＤ４＋ＣＤ５６＾＋同时阳性的细胞,即为ＣＤ４＾＋－ＮＫ－Ｔ细胞;Ｃ     

反义寡脱氧核苷酸在不同作用时点对人胰腺癌细胞系PaTu8988s作用的影响

目的 探讨反义寡脱氧核苷酸（ＡＳＯＤＮ）在不同作用时点对人胰腺癌细胞系ＰａＴｕ８９８８ｓ抑制作用的影响。方法 用人工合成的与Ｋ－ｒａｓ互补的ＡＳＯＤＮ在不同作用时点作用于指数生长期人胰腺癌细胞系ＰａＴｕ８９８８ｓ，并用四甲基偶氮唑盐（ＭＴＴ）法作活细胞测定。结果 人胰腺癌细胞系ＰａＴｕ８９８８ｓ经５０ｕｇ／ｍｌ和１００ｕｇ／ｍｌＡＳＯＤＮ处理后，在１２ｈ、２４ｈ、４８ｈ和７２ｈ的抑制率分别为４２．２５％、６６．２９％、６９．５５％、７４．５８％和６６．２０％、９１．４３％、９８．１８％、９８．３３％。结论 抑制效应从１２ｈ开始出现。４８～７２ｈ最为明显，其后出现时间和效率的饱和现象；抑制率的高峰期随着浓度增大而前移。     

HBV反义全基因真核细胞表达载体的构建及在肝癌细胞内的表达

１．材料与方法：乳哺动物细胞表达载体ｐＢＫ－ＣＭＶ,长约４．５ｋｂ,带有Ｔ_７公用序列。ｐＣＰ１０是ｐＢＲ３２２ＥｃｏＲＩ位点中插入双拷贝头尾相接的ＨＢＶａｙｗ亚型全基因ＨＢＶＤＮＡ重组质粒。ＰＣＲ引物ＸＩ１：１４２８－１４４８ｎｔ５'ＣＧＴ　ＣＣＣＧＴＣ　ＧＧＣＧＣＴＧＡＡＴＣＣ３',ＸＩ_２：１６７２－１６５３ｎｔ５'ＡＧＴＣＣＡＡＧＡＧＴＣＣＴＣＴＴＡＡＧ３'。人肝癌细胞系ＳＭＭＣ－７７２１,出上海细胞所提供。将ＥｃｏＲＩ酶切后回收的全基因ＨＢＶ片段。在Ｔ_４ＤＮＡ连接酶作用下,与ＥｃｏＲＩ酶切…     

逆转录病毒介导HSV-TK基因治疗胰腺癌——实验研究

目的:探讨应用逆转录病毒载体介导HSV-TK基因治疗实验性人胰腺癌细胞系8988的价值。方法:HSV-TK被定向克隆入逆转录病毒载体pMNSM的SV_(40) 下游。重组逆转录病毒载体pMNS-TK-M转染至逆转录病毒包装细胞PA317细胞,产生的重组病毒将HSV-TK转入人胰腺癌细胞系8988细胞内。结果:Southern-blot试验及药敏试验均证实HSV-TK基因已整合至细胞DNA中并完全表达。体外试验证实,HSV-TK阳性8988细胞对ACV的敏感性较母细胞明显为高;裸鼠移植瘤试验证实,腹腔注射ACV有明显阻止移植瘤的形成以及对移植瘤的治疗作用。结论:HSV-TK/ACV有体内治疗胰腺癌的作用,可作为胰腺癌基因治疗的潜在方法之一。     

西藏地区几株虫媒病毒的分离与初步鉴定

对西藏亚西和之那地区采集的蜱，鸟，鼠和家畜血标本用ＢＨＫ１３细胞和１－３日龄乳小白鼠进行了病毒分离，共获得了１７株病毒，对其中两株（Ｔ２０６６，Ｔ２０７６）病毒进行了初步鉴定，包括电镜形态观察，理化，生物学和免疫学性质试验。在９种已知的虫媒病毒及其标准免疫血清（ＳＩＮ，ＣＨＩＫ，ＭＡＫ，ＥＥＥ，ＭＶＥ，ＪＢＥ，ＴＢＥ，ＹＦ－１７Ｄ，ＨＦＲＳ）和分离毒株的交互间接免疫荧光试验中，未发现血清学反应，用     

乙型肝炎病毒-S基因重组逆转录病毒载体诱导小鼠体液和细胞免疫反应

探索逆转录病毒载体在基因治疗乙型肝炎中的应用。方法用ＤＮＡ重组技术构建ＨＢＶ－Ｓ基因重组逆转录病毒载体,电穿孔转染ＰＡ３１７后,用假病毒颗粒感染ＨｅｐＧ２、Ｐ８１５和ＥＬ－４细胞,并进行基因免疫。结果ＨＢＶ－Ｓ基因在上述细胞获得高效表达,在免疫动物体内产生高滴度抗一ＨＢＳ及有效的ＣＴＬ反应。结论该载体肌肉注射后有效激发对ＨＢｓＡｄｅ异性的体液免疫及细胞免疫反应,在细胞内稳定表达,是一种高效的基因转移系统,适用于基因治疗试验。     

重组丙型肝炎病毒基因转染H9细胞模型的建立

目的：建立重组ＨＣＶ基因转染的Ｈ９细胞（人ＣＤ４，Ｔ细胞）模型。方法：以载体ＣＤＺ２（连接有１６９３ｂｐＨＣＶＤＮＡ，包括完整的ＨＣＶ结构区）为模板，通过ＰＣＲ获得高保真的１．７３ｋｂＤＮＡ片段（包括１６９３ｂｐＨＣＶｃＤＮＡ和部分载体ＤＮＡ序列），其特异性被ｓｏｕｔｈｅｒｎｂｌｏｔ证实。将ＨＣＶｃＤＮＡ片段插入载体ｐＣＤ－ＳＲα，经菌落原位杂交以及酶切分斤和ｓｏｕｔｈｅｒｎｂｏＩｔ筛选，获得重组ＨＣＶ（ｐＣＤ－ＨＣＶ），并与ｐＳＶ２－ｇｐｔ载体共转染Ｈ９细胞。结果：从选择性培养基中筛选口阳性克隆细胞，经ＲＴ－ＰＣＲ．ｄｏｔＥＬＩＳＡ和ｗｅｓｔｅｒｎｂｌｏｔ验证表明ｐＣＤ－ＨＣＶ在Ｈ９细胞中可进行复制、转录和表达，表达的约１．３×１０５大小蛋白具有ＨＣＶ抗原性。转染ｐＣＤ－ＨＣＶ的细胞培养至９０天时，仍可检测到ＨＣＶｍＲＮＡ和ＨＣＶ抗原。结论：建立ｐＣＤ－ＨＣＶ转染的Ｈ９细胞模型获得成功。     

逆转录病毒介导的HSV-tk/GCV对肝癌细胞的影响

目的观察单纯疮疹病毒胸苷激酶（ＨＳＶ－ｔｋ）／丙氧鸟苷（ＧＣＶ）自杀基因治疗系统在体内外对人肝癌的杀伤效应。方法构建携带ＨＳＶ－ｔｋ基因的逆转录病毒载体,将该重组逆转录病毒感染人肝癌细胞ＨＣＣ９２０４,筛选稳定表达ｔｋ的细胞克隆ＨＣＣ９２０４／ｔｋ,并测定其在体外对ＧＣＶ的敏感性;ＨＣＣ９２０４／ｔｋ细胞在裸鼠皮下成瘤,用ＧＣＶ治疗并观察疗效。结果ＧＣＶ在体外对ＨＣＣ９２０４／ｔｋ细胞有明显的杀伤作用和旁杀伤效应,裸鼠体内ｅｘｖｉｖｏ实验得到相似结果,在体内外对未转染细胞则无明显毒性．结论在ｉｎｖｉｔｒｏ水平（指完全体外）和ｅｘｖｉｖｏ水平（指一部分体外一部分体内）,表达ＨＳＶ－ｔｋ基因的肿瘤细胞均可被ＧＣＶ有效杀伤,逆转录病毒介导的ＨＳＶ—ｔｋ／ＧＣＶ自杀基因治疗系统有可能成为肝癌的有效治疗方法。     

A forest-based approach to identifying gene and gene gene interactions

Multiple genes, gene-by-gene interactions, and gene-by-environment interactions are believed to underlie most complex diseases. However, such interactions are difficult to identify. Although there have been recent successes in identifying genetic variants for complex diseases, it still remains difficult to identify gene-gene and gene-environment interactions. To overcome this difficulty, we propose a forest-based approach and a concept of variable importance. The proposed approach is demonstrated by simulation study for its validity and illustrated by a real data analysis for its use. Analyses of both real data and simulated data based on published genetic models show the effectiveness of our approach. For example, our analysis of a published data set on age-related macular degeneration (AMD) not only confirmed a known genetic variant (P value = 2E-6) for AMD, but also revealed an unreported haplotype surrounding single-nucleotide polymorphism (SNP) rs10272438 on chromosome 7 that was significantly associated with AMD (P value = 0.0024). These significance levels are obtained after the consideration for a large number of SNPs. Thus, the importance of this work is twofold: it proposes a powerful and flexible method to identify high-risk haplotypes and their interactions and reveals a potentially protective variant for AMD.     

HLA基因、CC10基因和结节病

结节病是一种多器官系统受累的肉芽肿性疾病,常侵犯肺部、双肺门淋巴结。其病因目前尚不清楚,本文仅就有关结节病相关基因研究中的两个基因HLA基因、CC10基因和结节病的关系作一简要综述。     

RFamide-related peptide gene is a melatonin-driven photoperiodic gene

In seasonal species, various physiological processes including reproduction are organized by photoperiod via melatonin, but the mechanisms of melatonin action are still unknown. In birds, the peptide gonadotropin-inhibiting hormone (GnIH) has been shown to have inhibitory effects on reproductive activity and displays seasonal changes of expression. Here we present evidence in mammals that the gene orthologous to GnIH, the RFamide-related peptide (RFRP) gene, expressed in the mediobasal hypothalamus, is strongly regulated by the length of the photoperiod, via melatonin. The level of RFRP mRNA and the number of RFRP-immunoreactive cell bodies were reduced in sexually quiescent Syrian and Siberian hamsters acclimated to short-day photoperiod (SD) compared with sexually active animals maintained under long-day photoperiod (LD). This was contrasted in the laboratory Wistar rat, a non-photoperiodic breeder, in which no evidence for RFRP photoperiodic modulation was seen. In Syrian hamsters, the reduction of RFRP expression in SD was independent from secondary changes in gonadal steroids. By contrast, the photoperiodic variation of RFRP expression was abolished in pinealectomized hamsters, and injections of LD hamsters with melatonin for 60 d provoked inhibition of RFRP expression down to SD levels, indicating that the regulation is dependent on melatonin. Altogether, these results demonstrate that in these hamster species, the RFRP neurons are photoperiodically modulated via a melatonin-dependent process. These observations raise questions on the role of RFRP as a general inhibitor of reproduction and evoke new perspectives for understanding how melatonin controls seasonal processes via hypothalamic targets.     

Evidence for gene recombination in FCGR3 gene variants

Background and Objectives  The genes encoding the Fcγ receptors (FcγR) IIIa and IIIb ( FCGR3A and FCGR3B ) are clustered on chromosome 1 band q23–24 and exhibit allelic polymorphism. We investigated the molecular basis of additional new FCGR3 genomic variation.
Materials and Methods  A segment shared by FCGR3A and FCGR3B containing the polymorphic nucleotide positions 141, 147, 227, 266, and 277 in exon 3 was cloned and sequenced from genomic DNA of 30 donors and 3 bacterial artificial chromosome (BAC) clones. A mixture consisting of isolated FCGR3B * 2 - and FCGR3A - plasmids was cloned and sequenced as well. Additionally, nucleotide databases were screened for clones with variant FCGR3 sequences.
Results  A total of 12 FCGR3 variants defined by the polymorphic positions were detected in whole blood genomic DNA from 23 of 24 FCGR3B * 2 and/or FCGR3B * 3 positive donors, the DNA from two of three BAC clones and in the DNA mixture of isolated FCGR3B * 2 - and FCGR3A - plasmids.
Conclusion  Nucleotide exchanges of the variants were non-random and resulted from two alternative nucleotides present in one of the polymorphic position of the basic FCGR3 forms. Polymerase chain reaction (PCR) artefacts cannot be excluded as origin of new variants, but there is strong evidence that at least two variants are the result of a somatic recombination.     

The ovalbumin gene: Cloning of the natural gene

The structural ovalbumin DNA sequences are not contiguous and are separated by multiple "intervening regions" in native chicken DNA. EcoRI, a restriction endonuclease that does not cleave the structural ovalbumin DNA sequences, digests the natural ovalbumin gene into three distinct fragments of 2.4, 1.8, and 9.5 kilobase pairs in length by cleaving within these "intervening regions." The 2.4-kilobase pair fragment contains only about 450 nucleotide pairs of coding sequence, with the rest being intervening sequences. This DNA fragment was cloned in bacteria by using the certified EK2 vector lambdagtWES.lambdaB after enrichment from total EcoRI-digested chicken DNA by a combination of RPC-5 column chromatography and preparative agarose gel electrophoresis. Five out of approximately 20,000 recombinant phage plaques were capable of hybridizing with a (32)P-labeled Hha I fragment of a recombinant plasmid pOV230 containing the entire structural ovalbumin gene. DNA amplified in these recombinant phages, lambdagtWES.OV2.4, was shown to contain the same restriction endonuclease cleavage sites as in the 2.4-kilobase pair EcoRI fragment previously determined by restriction mapping of total genomic chicken DNA. The intervening sequences were allowed to hybridize with excess total chicken DNA and oviduct nuclear RNA after nick-translation. They were found to be unique chicken DNA sequences, and appeared to be transcribed in their entireties during gene expression. Like the structural gene sequences, the expression of the intervening sequences is also inducible by steroid hormones.     

Antiangiogenic gene therapy for hepatocellular carcinoma using angiostatin gene

Recent studies have reported that antiangiogenic gene delivery into cancer cells inhibits growth of certain tumors in vivo. Hepatocellular carcinoma (HCC) is a hypervascular cancer, and antiangiogenic gene therapy might be suitable for HCC. In the present study, we investigated the antiangiogenic effects of angiostatin gene transduction into HCC both in vitro and in vivo. Angiostatin gene was cloned into a pSecTag2B mammalian expression vector to construct pSecTag2B-ANG. pSecTag2B or pSecTag2B-ANG were transfected into an HCC cell line, PLC/PRF/5, and then stable transfectants were obtained by Zeocin selection. pSecTag2B or pSecTag2B-ANG transfection did not alter the expression of vascular endothelial growth factor (VEGF), a potent angiogenic stimulator, or pigment epithelium-derived factor (PEDF), an angiogenic inhibitor, in PLC/PRF/5 cells. However, conditioned media (CM) derived from pSecTag2B-ANG-transfected PLC/PRF/5 cells (CM-ANG) suppressed the proliferation and migration of human umbilical vein endothelial cells (HUVEC) by 35% and 50%, respectively, relative to their effects on nontransfected cells. In in vivo experiments, pSecTag2B-ANG stable transfected (CM-Mock) and nontransfected cells (CM-N) were mixed at various proportions and the mixed cells were subcutaneously implanted into athymic mice. Suppression of tumor growth was noted in mice implanted with angiostatin gene-transfected cells, and such suppression was proportional with the percentage of transfected cells. Analysis of the vascular density in these tumors showed that the tumor growth suppression effect of angiostatin gene correlated with suppression of tumor vascularity. In conclusion, antiangiogenic gene therapy using angiostatin gene is potentially suitable for the treatment of patients with HCC.     

Sex-based differences in gene transmission and gene expression.

The higher prevalence of certain diseases among women suggests involvement of genetic mechanisms linked to the sex chromosomes or of sex-limited gene expression that may be developmentally or hormonally regulated. Analysis of genetic markers and gene expression patterns provides the means for testing hypotheses related to these mechanisms.