1.研究目的

    为了发现新的骨关节炎的生物标记物,我们用iTRAQ方法综合分析了关节软骨。

2.方法

    我们分别收集了OA病人的关节软骨和股骨颈骨折病人的关节软骨,并用iTRAQ的方法发现分子差异,用Western blot进行定量。

3.结果

    我们用iTRAQ方法确定了76OA病人与对照组关节软骨有差异的蛋白。其中,LECT2 (leukocyte cellderived chemotaxin-2), BAALC (brain and acute leukemia, cytoplasmic), and PRDX6(peroxiredoxin-6) 这三个是新的OA生物标记。

4.结论

    用这三个OA生物标记结合其他传统的标记可以更好的反应OA病程和OA预后。

 

5.可借鉴之处(方法)

1)Patients and articular cartilage

Control samples were obtained from 6 patients with femoral neck fracture (4 male and 2 female patients). The samples were collected from femoral head cartilage during bipolar femoral head replacement at Fujita Health University Hospital. OA cartilage samples were obtained from 17 patients with OA (5 male and 12 female patients). These samples were collected from the femoral heads, femoral condyles, and tibial plateaus of patients with primary OA, who underwent total hip or knee replacement surgery at Fujita

Health University Hospital (Table 2).

2)Protein preparation

Samples were snap frozen at ?80°C and cut into 10?m sections by using a cryo-microtome (CM1900; Leica, Grove, IL). The sectioned samples were homogenized by sonication (T10 basic Disperser/Homogenizer; IKA) in RIPA buffer (10mL of 50mM Tris-HCl, pH 8.0, 150mM NaCl, 1% NP40, 0.5% sodium deoxycholate and 0.1% SDS/g cartilage). Then, the samples were centrifuged at 20000×g at 4°C for 10min, and the supernatant was used for analysis. Amicon Ultra Centrifuge Filters 10K (Millipore, Billerica, MA) were used to concentrate proteins by centrifugation at 5000×g. Concentrated samples were stored at ?80°C until use. A random peptide column (Proteominer; BIO-RAD, Hercules, CA) was used to prepare samples for iTRAQ. High-abundance proteins in these samples were excluded, while rare proteins were collected. Eluted samples were concentrated by Amicon Ultra Centrifuge Filters 10K by centrifugation at 14000×g.

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6.评论

    这篇文章主要用到两个技术,iTRAQ和Western都是邦菲可以提供的服务。难点在于关节软骨的处理,包括冷冻切片和超声粉碎。作为1.879分的文章,两方面的技术已经足够,新意在于发现新的biomarkers。借鉴的意义在于,如果样品处理成功,我们可以用大鼠重复这个实验,并增加治疗组,重点Bimakers的发现和治疗的效果。

 

7.参考文献

Daiki Ikeda, Hiroshi Ageta, Kunihiro Tsuchida, et al.(2013). iTRAQ-based proteomics reveals novel biomarkers of osteoarthritis. Biomarkers 18(7): 565-572.

 

2回复在“基于iTRAQ的蛋白质组分析发现新的骨关节炎生物标记

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