关键词： LMP-1   椎间盘退变   重组腺病毒   髓核   蛋白多糖   定量分析   BMP   动物模型   基因治疗  

摘要： 本研究构建了LMP-1基因的重组腺病毒载体;成功的进行了人退变髓核细胞的原代培养;首次在人退变椎间盘细胞离体实验和兔退变椎间盘模型组织的活体实验中证实了LMP-1基因可以在退变髓核细胞或组织中持续大量表达,并刺激椎间盘基质蛋白多糖和重要合成代谢细胞因子BMP-2和BMP-7的产生,为基因治疗椎间盘退变开辟了新的路径;首次利用椎间盘退变动物模型定量分析了LMP-1基因在椎间盘退变病程中的变化,为椎间盘退变的发病机制研究提出了新的思考。

关键词： 杆状病毒   GFP   Sox9   椎间盘退变   基因治疗  

摘要： 目的:探讨杆状病毒作为椎间盘退变基因治疗载体的可行性。 方法:本研究从椎间盘发生退变的形态学变化,杆状病毒介导的标志基因GFP转染正常和退变兔椎间盘髓核细胞、介导治疗基因Sox9转染退变的兔椎间盘髓核细胞等方面进行了如下实验:⑴采用共聚焦显微镜和透射电镜,对幼年和成年兔椎间盘髓核组织、细胞以及基质的形态学进行了对比观察与研究。⑵取新西兰大白兔的腰椎间盘进行髓核细胞的分离与培养,分别以6种不同浓度的重组杆状病毒（Ac/CMV/GFP）转染细胞,用荧光显微镜和流式细胞仪检测细胞的转染效率,并检测GFP基因的表达水平。分别将20μL Ac/CMV/GFP注入8只新西兰大白兔的L3-L4 L4-L5、L5-L6椎间盘髓核组织,并注射20μL生理盐水于L1-L2,而L2-L3不作任何处理进行自身对照。观察注射后兔的一般反应,分别于注射后的第7、13、20、28天各处死2只兔,进行髓核组织的切片观察。⑶取新西兰大白兔的髓核细胞进行离体培养,第一组:健康幼兔;第二组:椎间盘退变模型兔;第三组:椎间盘发生自然退变的成年兔。以200MOI的Ac/CMV/GFP转染离体培养的细胞,于第48小时采用流式细胞仪检测转染效率,同时采用HPIAS2000图像分析软件半定量分析外源性GFP基因的表达水平。⑷采用BAC-to-BAC系统构建重组杆状病毒Ac/CMV/Sox9,（Sox9是公认的能够延缓和逆转椎间盘退变的治疗基因）,用它转染离体培养的正常兔椎间盘髓核细胞,并设立Ac/CMV/GFP转染组和空白组作为对照,免疫组化的方法检测Sox9基因在细胞中的表

关键词： 骨形态发生蛋白   基因治疗   髓核细胞   椎间盘退变   细胞培养   基因表达   细胞增殖   细胞外基质  

摘要： 目的 研究整合在腺病毒载体上的人骨形态发生蛋白-7(hBMP-7)基因转染对原代培养的兔髓核细胞生物学活性的影响,探讨其治疗椎间盘退变(IDD)的可行性,为进一步研究提供实验依据。方法将hBMP-7基因整合到腺病毒载体中,构建Ad-hBMP7。然后将其感染体外培养原代兔髓核细胞,采用RT-PCR、Western-blot和免疫细胞化学的方法观察hBMP-7基因在细胞内的表达情况,采用MTT和流式细胞仪的方法观察其对细胞增殖能力的影响,此外还分别采用改进的二甲基亚甲兰比色法和ELISA法观测hBMP-7基因转染对细胞合成蛋白多糖和Ⅱ型胶原能力的影响。结果成功构建了hBMP-7基因的腺病毒载体,将其导入髓核细胞后可以高效表达hBMP-7,并持续3周以上,同时促进了髓核细胞增殖以及合成蛋白多糖和Ⅱ型胶原,较对照组有显著性差异(P<0.05)。结论 腺病毒介导的hBMP-7基因可以在椎间盘细胞内长时间表达,并促进细胞外基质的形成,可以作为目的基因进行IDD的基因治疗体内试验研究。

关键词： Transform Growth Factor Beta   Total Knee Replacement   Anabolic Effect   Aspartic Acid Residue   Total Knee Replacement Patient  

摘要： A recent genetic association study has identified a microsatellite in the coding sequence of the asporin gene as a susceptibility factor for osteoarthritis (OA). Alleles of this microsatellite determine the variable number of aspartic acid residues in the amino-terminal end of the asporin protein. Asporin binds directly to the growth factor transforming growth factor beta and inhibits its anabolic effects in cartilage, which include stimulation of collagen and aggrecan synthesis. The OA-associated allele, with 14 aspartic acid residues, inhibits the anabolic effects of transforming growth factor beta more strongly than other asporin alleles, leading to increased OA liability. We have explored whether the association found in several cohorts of Japanese hip OA and knee OA patients was also present in Spanish Caucasians. We studied patients that had undergone total joint replacement for primary OA in the hip (n = 303) or the knee ( n = 188) and patients with hand OA ( n = 233), and we compared their results with controls ( n = 294) lacking overt OA clinical symptoms. No significant differences were observed in any of the multiple comparisons performed, which included global tests of allele frequency distributions and specific comparisons as well as stratification by affected joint and by sex. Our results, together with reports from the United Kingdom and Greece, indicate that the stretch of aspartic acid residues in asporin is not an important factor in OA susceptibility among European Caucasians. It remains possible that lifestyle, environmental or genetic differences allow for an important effect of asporin variants in other ethnic groups as has been reported in the Japanese, but this should be supported by additional studies.

关键词： 椎间盘退变   骨形态发生蛋白-7   体内研究   基因治疗  

摘要： 目的 首先建立椎间盘退变的动物模型,构建含有人类重组BMP-7基因的腺病毒载体,然后将该载体导入椎间盘内,观察其对受损椎间盘的影响。方法 将新西兰大白兔椎间盘用24G针头针刺,观察4周后椎间盘的改变。构建pAd-BMP7重组腺病毒,293细胞包装、扩增和纯化重组腺病毒DNA。将BMP-7基因的腺病毒载体注射入椎间盘内,3天后观察BMP-7的表达情况。将L3/4椎间盘刺伤后注入生理盐水;L4/5椎间盘刺伤后注入Ad-BMP7;L5/6作对照。观察术后2周、4周、8周、12周BMP-7腺病毒载体对新西兰大白兔受损椎间盘的影响。结果 针刺损伤椎间盘4周后,L5/6发生退变。成功构建了腺病毒载体,滴度为1.2×1012PFU/ml。注射BMP-7腺病毒载体,三天后就有BMP-7表达;术后各时间点,L4/5较L3/4都有明显改善。结论 BMP-7基因可以延缓受损椎间盘退变的发生,为进一步实验提供了依据。

关键词： European Population   Japanese Population   Japanese Study   Spanish Population   Repeat Polymorphism  

摘要： Objective. A compelling genetic association with osteoarthritis (OA) of 2 functional alleles in the aspartic acid (D) repeat of the asporin gene was recently reported in a Japanese population. Allele D13 of the repeat encoded OA protection, whereas allele D14 encoded OA susceptibility. The 2 alleles mediate differences in the capacity of asporin to inhibit the cartilage growth factor transforming growth factor 6, with the D14 allele being a particularly potent inhibitor. Our objective was to assess whether the D repeat is associated with OA in UK Caucasians. Methods. The repeat was genotyped in 1,247 patients who had undergone elective joint replacement of the hip or the knee due to end-stage primary OA and in 748 age-matched controls. Results. The D13 allele was more common in controls, and the D14 allele was more common in patients. However, this trend was significant only for men who had undergone hip replacement (P = 0.016, odds ratio 1.48, 95% confidence interval 1.09-2.01). Conclusion. Our data suggest that the asporin polymorphism is not a major influence on OA etiology in Caucasians. The results of our study do not question the veracity of the Japanese report. Instead, our study highlights the complex, heterogeneous nature of OA genetic susceptibility.

关键词： asporin   calmodulin   genetic risk   osteoarthritis   secreted frizzled-related protein 3  

摘要： Purpose of review In the last year there has been considerable success in the identification of genes harbouring susceptibility for primary osteoarthritis. This report brings the reader up-to-date by focusing on three of the more compelling finds. Recent findings A UK group reported an association-of the FRZB gene with,! hip osteoarthritis in females. FRZB codes for secreted frizzled-related protein 3, an antagonist of Writ signalling. The Writ signal transduction pathway is critical for normal development and is also active in adult tissues. Secreted frizzled-related protein 3 helps to maintain articular cartilage and the associated alleles at FRZB reduce the activity of this important protein. A Japanese group has reported an association of the asporin gene ASPN wit h knee and hip osteoarthritis and an association of the calmodulin 1 gene CALM 1 with hip osteoarthritis. Asporin is a cartilage extracellular protein that regulates the activity of transforming growth factor-P. Calmodulin is an intracellular protein that interacts with a number of proteins involved in, signal transduction. The associated alleles at ASPN and CALM1 reduce the ability of chondrocytes to express, the genes encoding aggrecan and-type 11 collagen. Since these are essential structural components of articular cartilage, the ASPN and CALM1 associations are predicted to adversely affect the, maintenance of cartilage. Summary The FRZB, ASPN and CALM1 results are compelling and highlight that polymorphism in signal transduction pathways is a major component, of osteoarthritis susceptibility. This is an exciting observation since signal transduction pathways are malleable and therefore potentially amenable to intervention and modification.

关键词： 椎间盘退变   基因治疗   脊柱退行性疾病   分子生物学   disc   继发病变   手术治疗   分子水平   非手术   遗传学   优越性   免疫学   国内外  

摘要： 椎间盘退变(intervertebral disc degeneration,IDD)引起的一系列脊柱退行性疾病及继发病变在临床上十分多见.非手术和许多手术治疗的效果尚不能令人满意.随着分子生物学、遗传学、免疫学的发展及在医学中的应用,在椎间盘退变的早期针对其分子水平改变的基因治疗逐渐显现出其优越性,国内外学者做了大量的探索.现就其研究近况综述如下.