人结直肠癌细胞Drosha基因敲除细胞模型的构建

目的 为研究核酸酶Drosha与结直肠癌的关系,利用腺相关病毒（AAV）介导的染色体同源重组技术,构建Drosha基因敲除的HCT116结直肠癌细胞模型.方法 设计引物通过PCR扩增Drosha基因的同源臂,构建Drosha的AAV病毒打靶载体,通过病毒的包装、感染、抗生素新霉素（G418）药物筛选、PCR以及Western blot鉴定获得基因敲除阳性细胞株,通过Cre病毒感染去除抗性基因,最终建立敲除Drosha基因的结直肠癌细胞模型.结果 经过两轮打靶,分别将DNA双链上的Drosha基因去除并经Western blot鉴定,获得Drosha-/-阳性的HCT116细胞株.结论 通过AAV病毒介导的染色体同源重组技术,成功构建Drosha基因敲除的HCT116细胞系.     

表皮葡萄球菌生物被膜形成相关icaC敲除突变株的构建及其表型的改变

目的构建表皮葡萄球菌脚阴性突变株，以期获得仅icaC基因不同而其他遗传背景与表皮葡萄球菌1457株相同的突变株，对脚基因产物在细菌形成生物被膜中的作用进行研究。方法构建同源重组质粒pBT2-△icaC，经金黄色葡萄球菌RN4220株修饰后电转化入表皮葡萄球菌1457株。将含重组质粒的表皮葡萄球菌1457株在40℃多次传代，筛选出icaC敲除突变株（△icaC株）。检测△icaC以株生长曲线，采用微量板半定量法检测细菌生物被膜的形成能力，并采用斑点免疫印迹法检测细菌多糖黏附因子的合成。结果使用同源重组法敲除表皮葡萄球菌1457株基因组中的脚基因，△icaC株的生长曲线与表皮葡萄球菌1457株相似，但生物被膜形成能力及细胞外多糖黏附因子含量显著降低。结论表皮葡萄球菌1457株中脚基因的缺失对细菌生长无明显影响，但显著降低细胞外多糖黏附因子含量及生物被膜形成能力，为进一步研究表皮葡萄球菌脚基因在生物被膜形成中的作用奠定基础。     

硬脑膜额肌悬吊治疗儿童重度先天性上睑下垂

目的 探讨利用异体硬脑膜作为悬吊材料治疗儿童重度先天性上睑下垂的手术效果。方法对27例43眼重度先天性卜睑下垂的儿童行异体硬脑膜额肌悬吊术。结果 术后随访3～32个月，所有患儿外观改善，睑缘弧度美观，符合生理状态。结论 异体硬脑膜额肌悬吊治疗儿童重度先天性上睑下垂，手术方法简单可靠，术后效果良好，值得临床推广。     

Efficient expansion of HIV-1-specific T cell responses by homologous immunization with recombinant Semliki Forest virus particles

Vaccines based on recombinant viruses represent a promising strategy for the development of a prophylactic vaccine against HIV-1. However, despite a proven capacity to stimulate potent HIV-1-specific immune responses, viral systems have limited utility in homologous prime-boost regimens due to the generation of anti-vector immune responses. It is therefore important to develop a diverse set of vaccine candidates that can be combined in different heterologous prime-boost regimens and/or to identify a vaccine candidate that is less sensitive to anti-vector mediated immunity. In this report, we describe the design and pre-clinical immunogenicity of a Semliki Forest virus-based vaccine, VREP-C, encoding Indian origin HIV-1 clade C antigens. We show that a single immunization with VREP-C stimulates HIV-1-specific IFNgamma ELISPOT responses, which were efficiently boosted by a second and a third homologous VREP-C immunization resulting in highly potent cytotoxic T cell responses. These results suggest that VREP-C may be a valuable component of a future prophylactic vaccine against HIV-1.     

Lower level of BRCA2 protein in heterozygous mutation carriers is correlated with an increase in DNA double strand breaks and an impaired DSB repair

The BRCA2 protein is involved in the maintenance of genomic stability through its key role in homologous recombination repair of DNA double strand breaks. Biallelic inactivation of BRCA2 leads to a defect in DNA repair and is associated with a chromosomal instability phenotype. Recent studies on familial breast cancer clusters revealed chromosomal rearrangements and higher rates of sister chromatid exchanges also in heterozygous BRCA2 mutation carriers. In the present study, lymphoblastoid cell lines of heterozygous BRCA2 mutation carriers and of wildtype relatives were compared with regard to BRCA2 mRNA and protein expression and capacity to repair DNA damage induced by gamma-irradiation and mitomycin C. BRCA2+/- cells showed lower amounts of the full-length BRCA2 protein compared to BRCA2+/+ cells. The kinetics of gamma-H2AX protein level revealed distinct defects in DNA double strand break repair in the BRCA2+/- cells. These results are indicative of a haploinsufficiency phenotype in BRCA2+/- cells, suggesting that reduced amounts of functional BRCA2 protein in BRCA2+/- carriers are insufficient for an efficient repair of DNA double strand breaks, a condition that could contribute to the impairment of genomic stability.     

反复冻融及超深低温处理的异体肌腱移植实验研究

为比较有创与无创冷冻方法处理的肌腱异体移植的效果，探索不同冷冻处理技术对肌腱免疫源性的影响及获得成功的异体移植的条件，采用有创反复冻融及无创超深低温（－１９６℃）冻存技术分别处理肌腱，液氮下保存３个月后异体移植，以自体肌腱移植作对照。腱细胞活力测定显示，反复冻融处理的肌腱细胞全部失活，而超深低温处理的肌腱细胞活力为（９２．５±３．４）％。组织学观察显示，反复冻融及超深低温处理的异体移植肌腱均产生了不同程度炎性细胞浸润，且腱周粘连均较自体移植肌腱重。主动屈曲功能测定、羟脯氨酸含量测定及生物力学性能测定显示，反复冻融与超深低温组无显著性差异（Ｐ＞０．０５），且后两项指标显著较自体移植肌腱差。认为：①反复冻融及超深低温处理的肌腱异体移植获得一定成功，两者无显著性意义。②既要保留肌腱细胞的活性，又要去除肌腱中的抗原呈递细胞，可能是冷冻处理的肌腱异体移植获得成功的关键。③损伤了肌腱细胞成分，降低了肌腱抗原性，不等于能获得成功的异体移植     

Receptors for vasoactive intestinal peptide on murine lymphocytes turn over rapidly

The interaction of the neuropeptide vasoactive intestinal peptide (VIP) with its receptors on murine mesenteric lymph node lymphocytes (MLN) has been re-examined in detail. Intact MLN actively internalize surface bound VIP. The rate constants associated with the insertion of receptors at the MLN surface, the internalization of VIP occupied and unoccupied receptors and the elimination of the peptide were determined. At 37 degrees C, MLN insert approximately 140 VIP receptors cell-1 min-1 at their surface, and the rate of internalization of occupied receptors (0.23 min-1) was much greater than that of the unoccupied receptors (0.02 min-1). Exposure of MLN to non-saturating concentrations of VIP markedly altered the expression of VIP receptors at the lymphocyte surface. The rapid turnover of VIP receptors combined with the differential clearance of occupied and unoccupied receptors from the cell surface provides a mechanism by which homologous regulation of VIP receptor expression can occur on these lymphocytes.     

应用同源基因定量PCR方法快速检测Down综合征

本文设计一对引物,用聚合酶链反应同时扩增位于21号染色体的人肝型磷酸果糖激酶基因(PFKL-CH21)以及与其高度同源的位于1号染色体上人肌型磷酸果糖激酶基因(PFKM-CH1),然后同光密度扫描进行定量比较分析,结果显示,在31例正常人这两个同源基因的扩增产量比值0.939±0.161;而16例Down综合征患者的比值约为1.589±0.164,两组相比有高度显著性差异(P＜0.001),正常组与异常组的比值分布不存在重叠区,阴阳性结果易于判断,因此这种同源基因定量聚合酶联反应有可能发展成一种新的快速诊断Down综合征的方法.     

同种异体软骨细胞修复耳廓缺损的动物实验研究

目的探讨兔同种异体软骨细胞和自行制备生物材料高孔隙率聚乳酸(poly—DL-lactide,PDLLA)支架复合物修复兔耳廓软骨缺损的可行性。方法 18只大白兔分为软骨细胞/PDLLA复合物移植组、单纯PDLLA对照组和空白对照组,将体外培养的兔同种异体软骨细胞和自行制备的PDLLA支架形成复合物,修复兔耳廓软骨缺损。分别于植入后6周、12周、18周取材,HE和甲苯胺蓝染色了解兔耳廓缺损修复情况。结果术后18周,软骨细胞/PDLLA复合物移植组软骨缺损区为软骨组织修复,新生软骨与正常软骨问连接好,单纯PDLLA对照组和空白对照组为纤维软骨或纤维组织修复。结论同种异体软骨细胞/自制PDLLA复合物移植可较好地修复兔耳廓软骨缺损。     

介导野生型p53基因转移的重组腺病毒的构建研究

应用同源重组方法成功地构建了携带野生型ｐ５３基因的复制缺陷型重组腺病毒，通过光镜、电镜、酶切、ＰＣＲ共扩增等方法证实了构建载体的正确性，并使常规的同源重组方法进一步改进，简化了载体构建的操作程序，结果准确，成功率高。本结果可用于以腺病毒为载体进行肿瘤、心血管疾病等基因治疗的研究。