软骨损伤的基因治疗研究进展

由于软骨组织内无血管,因此其损伤后仅靠其自身来修复是非常有限的.有研究表明,很多细胞因子具有促进软骨形成及软骨样组织修复的功能.由细胞介导的相关基因于损伤部位分泌这些细胞因子促进软骨修复被认为足最理想的治疗方法.基因治疗正是在这一基础上发展起来的一种治疗技术,近年来取得了快速的发展.就近期软骨损伤的基因治疗进展进行综述.     

大鼠小肠缺血再灌注后血中NO,SOD浓度及肺中Bax,Bcl-2表达的改变

目的：研究大鼠小肠缺血再灌注后后血中一氧化氮（NO），超氧化物歧化酶（SOD）的浓度变化以及肺组织中Bax,Bcl-2的表达，探讨小肠缺血再灌注后对肺组织的损伤，方法：建立小肠缺血再灌注模型，分对照 ，再灌注后0，30min，1，2h,1,3,7d共8组，于各时点检测血中Bax,Bcl-2的表达情况。结果：大鼠小肠缺血再灌注后NO浓度0min明显升高，2h时降低，随后升高，7d时达高峰，SOD浓度0min明显下降，2h 时升高，随后下降，7d时达最低，Bax,Bcl-2免疫阳性细胞主要位于肺组织中血管内皮细胞和肺泡上皮细胞，再灌注0min,Bax,Bcl-2阳性细胞率增多，30min时Bax,Bcl-2阳性细胞率均升高分别为17．1％和78．1％，Bcl-2表达高于Bax，两者差别显著（P＜0．01），2h时降低，其后升高，7d时阳性细胞率达高峰分别为94．1％和83．4％，Bax表达明显高于Bcl-2，两者差异显著（P＜0．01）。结论：大鼠小肠缺血再灌注后可引起血中NO，SOD的浓度变化和Bax及Bcl-2阳性细胞在肺组织中的表达改变并可能引起肺组织细胞凋亡和损伤。     

肝细胞癌SCT表现和相关基因表达的初步研究

目的　探讨肝细胞癌细胞中nm2 3 H1基因和C erbB 2基因的表达和其与SCT表现的关系。方法　对 3 6例经手术病理证实且行螺旋CT动、静脉双期增强扫描的肝细胞癌病例 ,观察其SCT表现特征 ,如包膜、子灶、门静脉癌栓、肝门淋巴结转移、肿瘤的大小和强化特征。用免疫组织化学的方法检测癌组织中nm2 3 H1基因和C erbB 2基因的表达情况。结果　肝细胞癌细胞中 ,nm2 3 H1阳性 2 1例 ,阳性率为 5 8.3 % ,转移高危组 (子灶、门脉癌栓、肝门淋巴结转移 )nm2 3 H1基因的表达低于转移低危组 (P <0 .0 5 ) ;C erbB 2基因阳性 2 2例 ,阳性率为 71.1% ,C erbB 2基因的表达在中等大小肝癌组表达高于小肝癌组和大肝癌组 (P <0 .0 5 )。结论　肝细胞癌的SCT表现与nm2 3 H1和C erbB 2基因的表达有一定关系     

白细胞介素-18基因修饰的胎肝细胞经脾移植对小鼠免疫功能的影响

目的：观察表达mIL-18的重组腺病毒基因修饰的胎肝细胞（AdmIL-18/BNL.CL2)经脾移植对正常小鼠免疫功能的影响。方法：实验组小鼠经脾移植AdmIL-18/BNL.CL2，同时设LacZ病毒对照组（Ad-LacZ/BNL.CL2),BNL.CL2细胞对照组及空白对照组。2周后处死，留取血清，制备腹腔巨噬细胞、脾淋巴细胞、肝组织匀浆液，提取肝组织总RNA。采用ELISA法检测各组小鼠血清、腹腔Mφ和脾细胞培养上清、肝匀浆中细胞因子的含量；采用半定量RT－PCR法，检测肝组织细胞因子mRNA相对表达量；以LDH释放法测定腹腔Mφ杀伤活性和脾NK细胞活性，用MTT还原比色法测定脾淋巴细胞的增殖活性。结果：实验组小鼠血清、细胞培养上清及肝匀浆中，IL－18、IL－2、IFN－γ、TNF-α稔均高于其它对照组，而IL－4、IL－10水平则低于对照组；半定量RT－PCR结果与ELISA检测结果一致；同时，实验组腹腔Mφ的杀伤活性和脾NK细胞活性，及脾淋巴细胞增殖活性也明显高于对照组。结论：AdmIL-18能有效转染至胎肝细胞并稳定表达mIL-18;AdmIL-18基因修饰的胎肝细胞经脾移植后，可显著提高肝脏、脾脏免疫细胞活性，活化腹腔Mφ，促进Th1类细胞因子表达，抑制Th2类细胞因子的分泌。     

HLA-DPB1 gene polymorphism and multiple sclerosis: a large case-control study in the southwest of France

The polymorphism at the HLA-DPB1 locus has been characterized in a large number of patients with multiple sclerosis (n = 112) and in healthy controls (n = 115). Both patients and controls lived in the southwest of France (in the Pyrénées Atlantiques) and had similar ethnic background. The typing procedure involved the selective amplification of the second exon of the DPB1 locus by polymerase chain reaction, followed by hybridization of the amplified DNA with 14 sequence-specific oligonucleotide probes. Individual alleles were identified by the pattern of hybridization of the different probes. The distribution of the DPB1 alleles was not significantly different in multiple sclerosis patients and controls (p = 0.11). This does not corroborate the reported association of multiple sclerosis with the primed lymphocyte typing (PLT)-defined DPw4 specificity and is not in favour of a role played by polymorphic residues of the DP molecule in susceptibility to multiple sclerosis.     

牛磺酸对大鼠缺血心肌Bcl-2和Bax蛋白表达的影响

目的：探讨大鼠心肌缺血损伤与凋亡相关基因bcl-2和bax表达的关系及牛磺酸的影响。方法：结扎大鼠冠状动脉左前降支建立心肌缺血模型。检测心肌线粒体中超氧化物歧化酶（SOD）、Ca^2 -ATPase活性及MDA（丙二醛）含量，用免疫组化法检测Bcl-2和Bax蛋白在心肌中的表达量。结果：结扎冠脉左前降支可致心肌线粒体MDA含量升高，SOD和Ca^2 －ATPase活性下降；缺血心肌Bax蛋白表达呈显著升高；牛磺酸能明显减少缺血心肌线粒体MDA的生成，降低心肌Bax蛋白的表达，增加Bcl-2蛋白的表达。结论：结扎大鼠冠状动脉左前降支导致的心肌缺血损伤与Bcl-2和Bax蛋白的表达有关。     

p21WAF1与p27KIP1真核双表达载体的构建鉴定及在胃癌细胞中的表达

目的 构建p21WAF1与p27KIP1真核双表达载体,体外转染胃癌7901细胞,观察其在胃癌细胞中的表达及相关特性.方法 提取人全血总RNA,利用逆转录-聚合酶链反应(RT-PCR)扩增p21WAF1和p27WAF1基因并将其分别克隆到真核双表达载体pIRES中构建重组质粒pIPES-p27KIP1-p21WAF1,经酶切和测序鉴定重组质粒.脂质体介导重组质粒plRES-p27KIP1.p21WAF1"转染胃癌7901细胞,收集并提取转染后12、24、48、72 h和5 d各细胞的总RNA,逆转录成cDNA,荧光定量聚合酶链反应(FQ-PCR)检测各细胞中p21WAF1和p27KIP1在RNA水平的表达.带有绿色荧光蛋白的pIRES-EGFP作为对照.结果 RT-PCR扩增分别获得约500和600 bp大小的产物,重组质粒pIRES-p27KIP1.p21WAF1经酶切和测序鉴定证实为p21WAF1和p27KIP1基因.转染胃癌7901细胞48 h时转染率为68%.与空质粒对照组比较,重组质粒转染胃癌7901细胞12、24、48、72 h和5 d后FQ-PCR证实p27KIP1的Ct值(cycle threshold,Ct)分别减少8.2、10、10、7.4和6.7,p21WAF1的Ct值分别减少7.4、8.2、8.2、6.3和5.70其中24 h和48 h的Ct值减少最大.结论 真核双表达载体pIRES-p27KIP1.p21WAF1构建成功并能在胃癌7901细胞内高效表达.     

Differential Regulation of RGS-2 by Constant and Oscillating PTH Concentrations

PTH has diverse effects on bone metabolism: anabolic when given intermittently, catabolic when given continuously. The cellular mechanisms underlying the varying target cell response are not clear yet. PTH induces RGS-2, a member of the Regulator of G-protein Signaling protein family, via cAMP/PKA, and inactivates PKC-mediated signaling. To investigate intracellular signaling pathways with different PTH concentration-time patterns, we treated UMR 106-01 osteoblast-like cells in a perfusion system. PTH was administered intermittently (4 min/h, 10⁻⁷ M) or continuously at an equivalent cumulative dose (6.6 × 10⁻⁹ M). cAMP was measured using radioimmunoassay, mRNA levels using real-time rtPCR and ribonuclease protection assay, and protein levels using Western immunoblotting. A single PTH pulse transiently increased cAMP levels by 2000% ± 1200%. In contrast to continuous PTH exposure, cAMP induction remained unchanged with intermittent PTH, ruling out desensitization of the PTH receptor. In continuously perfused cells, RGS-2 abundance was three to five times higher than in cells intermittently exposed to PTH for up to 12 h. MKP-1 and -3 were significantly less induced with pulsatile PTH; exposure-mode-dependent differences in MMP-13 and IGFBP-5 were small. Pulsatile but not continuous PTH administration prevents PTHrP receptor desensitization and accumulation of RGS-2 in osteoblasts, which should preserve PKC-dependent signaling.     

Gene expression profiling of human lymph node metastases and matched primary breast carcinomas: Clinical implications

The genetic program that drives tumor metastasis and the mode and timing of its initiation are of great practical significance to clinical management. Modern technical advances open new opportunities for gaining useful relevant information. Gene expression profiles of histologically‐verified viable tissue from lymph node metastases were compared with those of matched primary breast cancers from 10 different patients, among samples from over 400 cases, using high‐throughput oligonucleotide arrays comprising probes for 22,000 genes. It was observed that metastases have very similar expression signatures to their parent tumors. However, detailed computational analysis revealed that a small number of genes were consistently differentially expressed between 100% of tumors and metastases, suggesting that these are mechanistically important. Lists of such candidate genes, of potential clinical interest, are provided. We interpret these results in the framework of a meta‐analysis of previous investigations by others and ourselves and of existing clinical knowledge on the behavior of human tumors. The collective data show that metastases resemble their primary tumors but the signatures obtained in different studies are not sufficiently reproducible or informative to be prognostically useful, although they do give valuable insights into the pathogenesis and biology of human tumor metastasis. The findings indicate that the genetic program encoding metastasis is implemented progressively over time although, occasionally, this evolution can occur rapidly, early in the life of the neoplasm. The important clinical significance of this deduction is that, in most patients, early detection provides time for appropriate therapeutic intervention to be effective in obstructing metastasis.