糖尿病基因治疗的实验研究Ⅰ.人胰岛素基因真核细胞表达质粒的构建

为有效开展糖尿病基因治疗，作为第一步，本研究构建了人胰岛素原基因与表达载体PRC／CMV的重组体。首先，从质粒PBCA中酶切回收260bp的人胰岛素原基因片段，利用中间载体PBS．SK构建了含人胰岛素原基困片段的过渡质粒PBS．INS，在此基础上构建了真核细胞表达的人胰岛素基因重组体PRC／CMV．INS。     

Immunoglobulin heavy chain Diversity genes rearrangement pattern indicates that MALT-type gastric lymphoma B cells have undergone an antigen selection process

Gastric MALT lymphoma usually develops from chronic gastritis, the vast majority of which (>90%) is associated with Helicobacter pylori infection. We sequenced the third complementarity determining region (CDR3) of immunoglobulin heavy chain genes in 19 gastric MALT lymphoma clones to determine the pattern of variable (V), diversity (D) and joining (J) gene utilization during immunoglobulin gene rearrangement.
DNA was extracted from paraffin-embedded sections and the rearranged CDR3 regions were amplified using a semi-nested polymerase chain reaction (with primers complementary to the conserved framework-three segment of the variable region [FR3A] and J regions). The DNA used for cloning and sequencing was obtained after purification of monoclonal bands excised from polyacrylamide gels. The N-D-N region specific to each clone was compared with known germline D sequences.
Similarly to that observed in normal and leukaemic B cells, our series of gastric MALT lymphomas showed apparent preferential utilization of genes from the DXP family. In two cases no similarity between the CDR3 nucleotide sequences of the neoplastic clones and the known germline D sequences could be found. In 10/19 analysed alleles the lymphoma B-cell clones appeared to contain two D gene segments (D-D recombination), a rare occurrence in normal individuals but one which has been described as a significant event in the determination of idiotype expression and antigen-binding affinity. Remarkably, despite the use of different D and J segments, the resultant amino acid sequences matched in two patients, suggesting the presence of a common selecting antigen.
The observed pattern of D gene rearrangement suggests that MALT lymphoma B-cell clones have undergone antigen selection, which seems to indicate that the antigen stimulation plays a pivotal role in the development of the lymphoma.     

P53、P16、Bcl-2基因及产物在原发性肺癌中的表达及其临床病理意义

目的 :探讨原发性肺癌 Mtp5 3、p16、Bcl- 2的异常表达与肺癌发生、发展的关系 ,以及它们之间的调控关系。 方法 :用免疫组化技术检测 (L SAB) 114例原发性肺癌组织中 p5 3、p16、Bcl- 2的表达。并用 PCR技术对 6 2例 p16蛋白丢失的肺癌组织中 p16外显子 2 (p16 E2 )的缺失进行分析。 结果:p5 3蛋白异常表达与肺癌组织学类型、分化程度无关 (P >0 .0 5 ) ,但与淋巴结转移情况有关 (P >0 .0 5 )。 p16蛋白阳性表达率与肺癌的组织学类型无关 ,但是其表达水平与非小细胞肺癌 (NSCL C)的细胞分化程度和淋巴结转移密切相关 (P <0 .0 5 )。 NSCL C中 p16E2的缺失率为 45 .2 %,其缺失水平随淋巴结转移和组织学分级的升高而升高 (P <0 .0 5 )。 SCL C(小细胞肺癌 )和正常肺组织中无 p16 E2的缺失。 Bcl- 2在小细胞肺癌中阳性率 6 2 .2 %显著高于 Sq Ca(鳞癌 )的 2 5 %和 Ad Ca(腺癌 )的 9.1%(P <0 .0 5 ) ,与细胞分化程度和淋巴结转移情况无关。另外 ,p5 3与 p16蛋白之间存在调控关系。 结论 :(1) p16、p5 3、Bcl- 2的异常参与肺癌的发生、发展过程。 (2 ) p16基因及产物的异常表达与 NSCL C的组织学分级和淋巴结转移相关 ,可能参与 NSCL C的发生、发展和转移过程。 (3) Bcl- 2反映 SCL C的分之生物学行为和临床     

逆转录病毒载体介导的外源性主要组织相容性抗原复合体基因转移及其在T细胞的表达

目的 建立一个在胸腺内表达外源性主要组织相容性抗原复合体(MHC)抗原的实验模型,为下一步研究作准备。方法 应用逆转录病毒载体介导的基因转移技术,首次将外源性MHC基因转移到T淋巴表达并应用聚合酶链反应(PCR)及反转录聚合酶链反应(RT-PCR)检测转染T细胞DNA及mRNA。结果 外源性MHC基因已整合到靶细胞染色体DNA并有效地转录;单克隆抗体免疫荧光染色流式细胞仪检测显示在转染T细胞膜有外源性MHC分子表达;转染效率为46.2％。结论 外源性MHC基因可以转移到T细胞稳定表达,为今后的研究提供了经验。     

转人GM—CSF基因人膀胱癌细胞株的建立及体外生物学特性

经逆转录病毒载体将人GM-CSF基因导入人膀胱癌细胞株BIU-87细胞中,建立了转基因细胞株BIU/GM。经流式细胞仪行细胞DNA周期分析表明GM-CSF基因的导人及表达对BIU-87细胞的增长无影响。免疫荧光测定发现转GM-CSF基因及表达不能促进BIU-87细胞表面HLA-ABC、DR、DQ抗原的表达。转基因瘤细胞株经6000rad X射线照射灭活后,丧失增殖能力,逐步死亡,但能维持一定水平的GM-CSF分泌活性达两周以上。从而为制备灭活的转基因瘤苗提供了初步经验。     

Absent chondrodysplasia punctata in a male with an Xp terminal deletion involving the putative region for CDPX1 locus

This is a follow-up report on a male patient with a 46, Y, r(X) karyotype. Although he had no clinico-radiological features of X-linked recessive chondrodysplasia punctata (CDPX1), molecular studies revealed an Xp terminal deletion involving the putative region for the CDPX1 locus (PABX-DXS31). We suspect that the absence of CDPX1 may be attributable to the nature of the disease and the extreme short stature of the patient (mean – 5.6 S.D.). © 1993 Wiley-Liss, Inc.     

人卵巢癌中肿瘤转移抑制基因nm23-H1编码蛋白的表达

目的：探讨人卵巢癌标本中，肿瘤转移抑制基因ｎｍ２３－Ｈ１编码蛋白ＮＤＰＫ－Ａ表达的临床意义及其与预后的关系。方法：应用免疫组织化学抗生蛋白链菌素－过氧化酶结合（Ｓ－Ｐ）法，检测５８例人卵巢癌标本中ｎｍ２３－Ｈ１蛋白水平。结果：ｎｍ２３－Ｈ１蛋白的表达与患者手术时是否有淋巴结及大网膜转移相关（Ｐ〈０．０５），但原发灶与转移灶间的阳性表达率相差不显著；与患者术后生存时间密切相关（Ｐ〈０．０１），术后生     

Selection of Single Nucleotide Polymorphisms for Association Studies in Candidate Genes

Comparison of polymorphic sites such as single nucleotide polymorphisms (SNPs) within a gene between cases and controls may be useful for establishing a role of this gene in disease susceptibility. The approach includes two steps: the first step is the discovery of the different SNPs within the candidate gene and the second step is the association testing per se that can be done on the entire set of sites discovered or on a subset of these sites only. Selecting a subset of sites may increase the power to detect the association with the candidate gene since a smaller number of tests would then be performed. We proposed a strategy to select sites within a candidate gene and applied it on the Genetic Analysis Workshop 12 candidate gene data. Using these selected sites, we detected an association with candidate genes 1 and 6. © 2001 Wiley‐Liss, Inc.