组织工程血管基质的制备与改性

目的：完全脱除猪胸主动脉细胞，对脱细胞血管基质进行改性，增强基质的力学强度，制备组织工程血管支架材料。方法：取家猪的新鲜去除外膜胸主动脉20根，随机分成4组，分别采用胰蛋白酶、TritonX-100及十二烷基硫酸钠（SDS）作为脱细胞试剂对猪胸主动脉进行脱细胞处理，并对脱细胞后的血管基质进行改性，采用HE染色、弹力纤维染色、力学性能测试，以评价脱细胞效果以及材料的力学性能。结果：采用1％TritonX-100单独作用84h既能完全脱除细胞，同时又可完整保留血管基质的三维结构，对胶原纤维、弹力纤维的损伤小，是一种较理想的脱细胞方法。对脱细胞后的基质进行冷冻干燥及真空热交联处理，能有效提高材料的机械强度，冷冻干燥24h后真空120℃下热交联处理12h所获得的材料的机械强度最好，断裂强度可达到1．70MPa。结论：以脱细胞血管基质经冷冻干燥和真空热交联处理后，可以作为血管组织工程的支架材料。     

Estrogen receptor-associated expression of keratinocyte growth factor and its possible role in the inhibition of apoptosis in human breast cancer

Although estrogen is known to play a crucial role in the pathogenesis of breast cancer, the molecular mechanisms underlying the action of estrogen remain elusive. In the present study, we focused on keratinocyte growth factor (KGF) and its receptor (KGFR) in the pathogenesis of breast cancer, as a growth factor mediating estrogen action, since significant roles of KGF were demonstrated in various steroid hormone-dependent tissues. First, using paraffin-embedded specimens from 42 breast cancer patients, we examined expression patterns of KGF and KGFR by both immunohistochemistry using newly generated antibodies and nonradioactive in situ hybridization with T-T dimerized synthetic oligonucleotide probes. We next compared the results with the expression of estrogen receptor (ER) alpha and beta, proliferative activity and apoptotic frequency (TUNEL staining). Also, the similar approaches were taken to analyze the expression and role of KGF in ER-positive (MCF7, ZR-75-1) and ER-negative (SK-BR-3, MDA-MB-231) human breast cancer cell lines in vitro. In the surgical specimens, KGF was expressed in cancer cells as well as stromal cells in 19/42 cases (45%), while KGFR was found in cancer cells in 24/42 cases (57%). The distribution of protein and mRNA in the analysis of both KGF and KGFR expression generally coincided. Moreover, KGF expression was closely associated with the expression of ER alpha, and the coexpression of KGF and KGFR significantly correlated with lower TUNEL index, but not with proliferative activity. In accordance with the in vivo findings, KGF expression was detected only in ER alpha-positive MCF7 and ZR-75-1 cells in vitro. And more importantly, we found the inhibitory effect of KGF upon the induction of apoptosis by anticancer drugs in MCF7 cells. Collectively, our results indicate that ER alpha may be involved in KGF expression, and that KGF may play antiapoptotic roles, rather than mitogenic, in human breast cancer.     

Early rehabilitation program in postmastectomy patients: a prospective clinical trial

The purpose of this study was to determine whether 20 patients who received an early postmastectomy rehabilitation treatment program showed more improvement in range of shoulder motion and functional activities than 13 patients who received instruction for exercise only. Data were obtained at preoperatively, three days after operation, at discharge and at postdischarge one month for each patient on parameters such as range of motion of the ipsilateral shoulder joint, upper extremity circumferential measurements, as well as 10 elements of shoulder function. Postoperatively, both groups showed an increased range of motion of the shoulder joint and improved functional activities, but the group that received postoperative rehabilitation management had a better range of shoulder motion and less difficulty in five items for functional assessment. This study also showed that an early rehabilitation program did not increase postoperative complications. We concluded that an early rehabilitation program or intensive instruction program only by a well-trained physical therapist or physiatrist was beneficial to postmastectomy patients in regaining the function and range of shoulder motion, and significantly better in a rehabilitation group.     

Gain-of-function polymorphism in mouse and human Ltk: implications for the pathogenesis of systemic lupus erythematosus

Systemic lupus erythematosus (SLE), a complex multigenic disease, is a typical antibody-mediated autoimmune disease characterized by production of autoantibodies against a variety of autoantigens and immune complex-type tissue inflammation, most prominently in the kidney. Evidence suggests that genetic factors predisposing to aberrant proliferation/maturation of self-reactive B cells initiate and propagate the disease. In SLE-prone New Zealand Black (NZB) mice and their F1 cross with New Zealand White (NZW) mice, B cell abnormalities can be ascribed mainly to self-reactive CD5+ B1 cells. Our genome-wide scans to search for susceptibility genes for aberrant activation of B1 cells in these mice showed evidence that the gene, Ltk, encoding leukocyte tyrosine kinase (LTK), is a possible candidate. LTK is a receptor-type protein tyrosine kinase, belonging to the insulin receptor superfamily, and is mainly expressed in B lymphocyte precursors and neuronal tissues. Sequence and functional analyses of the gene revealed that NZB has a gain-of-function polymorphism in the LTK kinase domain near YXXM, a binding motif of the p85 subunit of phosphatidylinositol 3-kinase (PI3K). SLE patients also had this type of Ltk polymorphism with a significantly higher frequency compared with the healthy controls. Our findings suggest that these polymorphic LTKs cause up-regulation of the PI3K pathway and possibly form one genetic component of susceptibility to abnormal proliferation of self-reactive B cells in SLE.     

PCR-RFLP based molecular typing of enteroviruses isolated from patients with aseptic meningitis in Korea

Summary.  We have evaluated PCR–RFLP as a practical method for rapid typing of enteroviruses causing aseptic meningitis in Korea. Through blind examination of 80 clinical isolates from patients with aseptic meningitis, we have compared the results of conventional serotyping with PCR–RFLP based genotyping, which was developed for this study. Among the 80 case isolates, which had been previously typed by routine neutralization test, only 42 cases (52.5%) were matched with typing by PCR–RFLP. The result clearly demonstrated that the enterovirus serotype does not coincide with the genotype. Therefore, the classification of enteroviruses by genotyping with PCR–RFLP, although rapid and simple, may be complicated by regional or seasonal differences. However, the PCR–RFLP method developed in this study is applicable to the epidemiological study of enteroviruses when regional or seasonal differences exist, and is useful in identifying the source of an infection. Received August 6, 2001; accepted April 15, 2002 Published online July 10, 2002     

Fas cDNA克隆、表达及重组蛋白纯化的研究

目的:获得高质量及充足的Fas重组蛋白。方法:采用PCR构建表达重组质粒,亲和层析纯化Fas重组蛋白,ELISA检测其免疫学功能。结果:①PCR扩增获得目的片段约 470 bp。②Sanger双脱氧链终止法测序表明,该 DNA序列仅 174位由A→G突变,属于同义突变,与已知的Fas膜外区氨基酸完全一致。③重组质粒经IPTG诱导有一蛋白得以表达,其目的融合蛋白占细菌总蛋白的17. 4%,分子量约为 34．3 kD。④经 Factor Xa切割 Fas融合蛋白,纯化获得 Fas重组蛋白,Western印迹出现特异性Fas蛋白带,分子量为21．3kD,与预测结果相吻合。⑤ELISA检测 Fas重组蛋白,具有抗体的免疫学活性。结论:成功地表达并纯化了Fas重组蛋白,解决了Fas不易获得的难题,为研究Fas提供了良好的实验材料。     

Rp-cAMP与缺氧神经元Bcl-2表达和细胞凋亡关系的研究

目的探讨培养神经元缺氧后蛋白激酶A(PKA)活性变化及其与神经元缺氧凋亡的关系。方法建立体外培养W istar大鼠胎鼠皮层神经元模型及培养神经元缺氧模型,用不同浓度的PKA抑制剂(Rp-cAMP),在不同的缺氧时相点观察神经元细胞膜和细胞质中PKA的活性、Bc l-2和TUNEL表达的规律。结果随着缺氧时间的延长,培养神经元细胞膜PKA的活性显著增加,Bc l-2表达降低,TUNEL荧光染色阳性率及平均荧光强度显著升高。而应用Rp-cAMP后细胞凋亡情况显著减轻。结论PKA和Bc l-2均参与了缺氧神经元凋亡;PKA抑制剂Rp-cAMP可减轻缺氧神经元凋亡,且该作用与Bc l-2表达降低有关;PKA的激活在缺氧神经元凋亡中起促发作用。     

IFN-γ在沙眼衣原体呼吸道感染中免疫防御机制的探讨

目的:检测与IFN-γ作用相关的酶吲哚胺2,3二氧化酶(IDO)、诱导性一氧化氮合成酶(iNOS)和NADPH氧化酶(ox)gp91在沙眼衣原体呼吸道感染中的表达及与机体防御的关系,探讨衣原体感染中IFN-γ免疫防御作用的机制.方法:用沙眼衣原体小鼠肺炎株(MoPn)通过鼻腔感染C57BL/6(H-2b)小鼠,用过氧化物酶连接的鼠抗衣原体脂多糖单抗染色HeLa 229细胞,检测衣原体在肺组织的生长;用RT-PCR检测衣原体感染后第7及14天小鼠肺组织IFN-γ、IDO、iNOS和gp91NADPH ox mRNA表达.结果:MoPn呼吸道感染后小鼠肺组织匀浆衣原体活性测定,于感染后第2天,HeLa 229细胞内可见有衣原体包涵体生长,IFU值增高,于感染后第7天IFU达最高水平,以后逐渐下降,至感染后21天基本恢复到基线水平.与未感染的对照组比较,Th1细胞因子IFN-γ于感染后第7天表达显著增高,感染后14天有所降低,但仍维持较高水平;同时衣原体感染可显著诱导与IFN-γ作用相关的三种酶IDO、iNOS和gp91 NADPH ox在小鼠肺组织的表达,感染后第7及14天,IDO,iNOS及gp91 NADPH ox的表达与对照组比较均有显著差异,其中IDO和gp91 NADPH ox于感染后第7天mRNA表达增高显著(P＜0.01),14天略有下降(P＜0.05).结论:衣原体呼吸道感染诱导Th1细胞因子IFN-γ mRNA高表达,参与宿主对衣原体的清除及机体免疫防御,此作用可能与其相应的酶IDO、iNOS和gp91 NADPH ox表达增高有关.     

TCRVβ7.1基因修饰T细胞对乳腺癌细胞杀伤作用的研究

目的：观察TCPVβ7．1基因转染前后正常人外周血淋巴细胞对乳腺癌细胞株杀伤活性的影响。方法：脂质体包裹PdWA3.1vβ7.1后转染健康人PBMC，流式细胞仪检测PdWA3.1Vβ7.1基因表达，改良MIT法检测TCRVβ7．1基因转染前后正常人外周血淋巴细胞对乳腺癌细胞株杀伤活性。结果：TCRVβ7．1基因转染可显著增加正常人PBMC该基因表达，转染前后正常人外周血淋巴细胞对乳腺癌细胞株杀伤活性有显著性差异。结论：用TCR基因修饰可明显提高正常人PBMC对乳腺癌细胞杀伤作用。     

抗SARS-CoV抗原的人源Fab段噬菌体抗体库的构建

目的 :利用抗SARS冠状病毒IgG抗体阳性的SARS康复患者外周血淋巴细胞 ,构建人源Fab段抗体文库。方法 :制备外周血淋巴细胞总RNA ,逆转录成cDNA。以其为模板 ,利用针对家族特异性Ig基因的引物扩增重链Fd段和轻链基因 ,并重组到噬菌粒载体pComb3中 ,将重组噬菌粒载体电转化大肠杆菌XL 1Blue,酶切鉴定抗体库的重组率 ,并测定噬菌体抗体库的库容量。结果 :构建了源于SARS康复患者血清中抗Fab段的抗体文库 ,轻链、重链Fd段基因的重组率分别为91%和 75 % ,库容量为 7.2 3× 10 7。结论 :成功地构建了抗SARS CoV抗原的人源Fab段噬菌体抗体库