目的：构建BMP2/pLEGFP重组逆转录病毒表达载体，并检测其表达的融合蛋白的生物活性。方法：用基因重组技术构建BMP2/pLEGFP重组逆转录病毒表达载体，并通过酶切和PCR鉴定。脂质体法转染COS27细胞，用荧光显微镜检测和Western blotting分析其在COS27细胞表达，细胞活性实验和动物体内异位成骨实验进一步检测融合蛋白的生物活性。结果：经酶切和PCR鉴定重组质粒BMP2/LEGFP构建成功。转染COS27细胞后，荧光显微镜和Western blotting检测均显示融合蛋白在细胞中表达；分泌的产物经细胞活性实验和动物体内异位成骨实验证实具有BMP2和EGFP的双重蛋白活性。结论：基因重组技术成功构建BMP2/pLEGFP重组体，重组体表达的融合蛋白具有GFP和BMP2的双重活性。

目的 探讨利用荧光激活细胞分选技术获得有效重组逆转录病毒包装细胞系的方法。方法 用脂质体介导pLEGFP转染PA317细胞用荧光显微镜观察转染结果：依据EGFP荧光利用流式细胞仪的分选技术获得多克隆和单克隆源性的包装细胞。并利用PCR、RT-PCR对其鉴定；以NIH3T3为靶细胞。对其滴度进行测定。结果 荧光显微镜显示用脂质体介导的方法成功的转染PA317细胞在4次连续流式细胞仪分选后得到了稳定表达的多克隆和单克隆源性的包装细胞。PCR和RT-PCR分别从多克隆和单克隆源性的包装细胞细胞基因组DNA以及多克隆和部分（6/8）单克隆源性的包装细胞培养上清中的重组逆转录病毒RNA中扩增出了插入的EGFP片段。滴度测定显示得到的包装细胞能够产生有效的病毒滴度。结论 荧光激活细胞分选技术是获得有效病毒滴度的包装细胞的有效方法。

目的 探讨大骨节病患者关节软骨细胞凋亡及相关调控因子Bcl-2、Box、Fas及iNos表达分布的特点。方法 收集15例大骨节病儿童和15例正常对照儿童关节软骨。采用脱氧核糖核酸末端转移酶介导的脱氧核糖核酸缺口标记（TUNEL）技术和Bcl-2、Bax、Fas及iNos蛋白免疫组化抗生物素蛋白-生物素-碱性磷酸酶（B-SA）法染色，观察大骨节病儿童和正常对照儿童关节软骨的凋亡细胞和Bcl-2、Bax、Fas及iNos蛋白阳性表达细胞的密度与分布。结果 （1）大骨节病儿童关节软骨中层凋亡阳性细胞数[（33.60±2.71）%]较正常关节软骨[（1.33±0.41）%]显著增多（t=11.59P<001）；（2）大骨节病儿童关节软骨表层和中层Bcl-2、Bax、Fas及iNos的表达显著高于正常关节软骨中的表达（t=11.75～18.65，P<0.01）；大骨节病儿童关节软骨各层Bcl-2、Bax、Fas及iNos表达阳性率有显著性差异（F=73.49～114.42，P<0.01）。以表层最多[分别为（41.93±12.26）%、（45.60±15.78）%、（53.60±16.49）%和（45.47±14.02）%]。其次为中层[分别为（14.93±3.50）%、（13.87±4.32）%、（23.27±4.83）%、（21.67±6.82）%]。结论 大骨节病患者关节软骨细胞凋亡及相关调控因子Bcl-2、Bax、Fas、iNos表达较正常人显著增多。

目的 分析大骨节病患者、病区内对照人群和非病区外对照人群12号染色体上7个短串联重复序列（STR）位点的多态性。方法 采用荧光标记基因扫描方法，对12号染色体上D12S1718、D12S1675、D12S358、D12S367、D12S1638、D12S1646和D12S1682位点在陕西省永寿县、榆林地区大骨节病区患者和病区内对照人群及咸阳地区非病区外对照人群中的多态性进行分析，计算相应人群中7个位点的基因频率、基因型频率，并对各组间基因频率进行x2检验。结果 D12S1718、D12S1675、D12S358、D12S367、D12S1638、D12S1646和D12S1682位点在大骨节病患者中分别检出4、7、7、8、5、5和7个等位基因，5、12、13、11、10、9和13个基因型；在病区内对照人群中分别检出4、9、7、6、6、6和8个等位基因，5、10、12、14、12、9和13个基因型；在非病区外对照人群中分别检出7、9、7、7、5、8和11个等位基因，9、16、17、16、12、15和20个基因型；各组间基因频率进行比较。在D12S367位点和D12S1638位点，患者与病区内对照（D12s367：P=0.034；D12S1638：P=0.041）及非病区外对照间（D12s367：P=0.029；D12S1638：P=0.028）均有显著性差异；在D12S1646位点，患者与病区内对照间无差异（P=0.446），但病区人群与非病区外对照间有差异（患者一非病区外对照：P=0.036；病区内对照。非病区外对照：P=0.039）。结论 大骨节病患者12号染色体的7个STR位点中D12S367和D12S1638等位基因分布显著不同于病区与非病区正常人。

Objective. Kashin-Beck disease (KBD) is a chronic, endemic osteochondropathy principally occurring in children. We investigated apoptotic chondrocyte death and the expression of Bcl-2, Bax, Fas, and inducible nitric oxide synthase (iNOS) in articular cartilage from patients with KBD in order to determine the pathogenesis of chondronecrosis in KBD. Methods. Samples of articular cartilage were divided into 2 groups: control children (15 samples from 15 cases), and children with KBD (15 samples from 15 cases). KBD patients were diagnosed according to "Pathological Criteria to Diagnose KBD in China." Chondrocyte apoptosis was detected by TUNEL staining, and Bcl-2, Bax, Fas, and iNOS-positive articular chondrocytes were stained by immunohistochemistry. Articular cartilage was classified in 3 zones, and positive findings were counted by light microscopy for cytoplasmic staining by polyclonal antibodies of Bcl-2, Bax, Fas, and iNOS and apoptotic chondrocytes by TUNEL. Results. The percentage of positive apoptotic chondrocytes stained by TUNEL in the middle zone of articular cartilage from the KBD patient group (33.60% +/- 2.71%) was higher than that of controls (1.33% +/- 0.41%; p < 0.01). The percentages of chondrocytes staining for Bcl-2, Bax, Fas, and iNOS in KBD patients were significantly higher than in controls (p<0.01); the remarkable difference in Bcl-2, Bax, Fas, and iNOS expression among the upper, middle, and deep cartilage zones was also seen in KBD articular cartilage (p<0.01); and staining for Bcl-2, Bax, Fas, and iNOS in KBD patients was prominent in the upper zone (41.93% +/-12.26%, 45.60% +/-15.78%, 53.60% +/- 16.49%, 45.47% +/- 14.02%, respectively) and the middle zone (14.93% +/- 3.50%, 13.87% +/- 4.32%, 23.27% +/- 4.83%, 21.67% +/-6.82%) of articular cartilage. Conclusion. The apoptotic chondrocytes and Bcl-2, Bax, Fas, and iNOS-positive chondrocytes were significantly more numerous in patients with KBD than in controls.