内质网应激-未折叠蛋白反应在髋臼发育不良软骨可逆性改变中的作用及机制研究

Developmental dysplasia of the hip, or Congenital displasia of the hip, is the most common developmental disorder of the lower extremities in children. The overall frequency of developmental dysplasia of the hip (DDH) is usually reported as approximately 2－3‰. Uncorrected developmental dysplasia of the hip (DDH) is associated with long term morbidity such as gait abnormalities, chronic pain and degenerative arthritis which would cause pain to many patients and force them to receive a complicated total hip replacement surgery. Although the treatment may vary, the goal remains the same; stable positioning of the femoral head in the acetabulum in order for the acetabulum to improve its coverage. Based on the clinic evidence, the earlier stable positioning maintain, the better developmental potiential of the acetabulum cartilage. But it is unkonwn yet what the optimal treatment time point. In our previous study, both behind extremities of newborn SD rats were bound for different time point as DDH animal model.We observed the morphology change of acetabulum in paraffin section with HE staining. It showed that the acetabular shape changed dramatically with the bound duration increased. And acetabular cartilage had the capability to remodeling the shape after the binding released. In histochemistry study, the result showed that type-II pre-collagen accumalation accumulated abnormally in diaplasia chondrocytes. So our hypothesis is that type-II collagen abnormal accumulation might stimulate endroplastic reticulum (ER) stress and its negative feedback signal pathway, unfolding protein reponse(UPR), as one of molecular mechanism in the acetabulum remodeling processure. The ER stress-UPR is activated in response to an accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum. In this scenario, the ER stress-UPR has two primary aims: initially to restore normal function of the cell by halting protein translation and activate the signaling pathways that lead to increasing the production of molecular chaperones involved in protein folding. If these objectives are not achieved within a certain time lapse or the disruption is prolonged, the UPR aims towards apoptosis. In this study, we try to identify the location of abnomal type-II collagen in chondrocytes by co-immunostaining and sub-organ protein extraction assay. The the stimulation of ER stress-UPR signal pathway will be detected by RT-PCR and Western Blotting assay. Finally, the processure of acetabulua chondrocyte development will be represented in Flexer 3D cell culture system to define the stimulation of ER stress-UPR signal pathway. In the mean the hypothesis will be proved other way aroud by siRNA technich. It is important for us to understand the molecular mechanism of DDH chondrocyte reversible development, which will show us the laboratory data and knowledge in cloosing the well treatment time point in clinc.

发育性髋关节发育不良（DDH）是小儿下肢最常见畸形，失去早期诊断及治疗时机的患儿，将最终面临骨关节炎，严重影响生活质量。课题组前期的实验结果发现，II型胶原前蛋白在髋关节发育不良髋臼软骨细胞内异常堆积，提出II型胶原异常堆积并引发内质网－未折叠蛋白反应可能是髋臼发育不良软骨可逆性改变的分子机制的假设。本研究试图：1.利用免疫共荧光及细胞各组分蛋白抽提等技术明确II型胶原前蛋白在细胞内异常堆积的位置；2.通过RT-PCR及Western Blotting等方法检测内质网应激－未折叠蛋白反应通路的激活情况；3. 在Flexer体系中三维培养软骨细胞，模拟髋臼发育不良过程，同时利用siRNA技术逐一阻断内质网应激－未折叠蛋白反应主要信号通路蛋白，正反双向探讨内质网应激－未折叠蛋白反应在髋臼发育不良软骨可逆改变中的作用机制，为临床早期治疗的时间点选择以及愈后评估提供重要的实验数据和理论基础。

发育性髋关节发育不良（DDH）是小儿下肢最常见畸形，失去早期诊断及治疗时机的患儿，将最终面临骨关节炎，严重影响生活质量。DDH的病因尚未明确，治疗方式也存在争议，公认的观点：早期诊断和治疗。关节软骨是由胚胎发育中的间充质细胞分化而来，由软骨细胞和其分泌的细胞间质构成。其中细胞间质起到软骨细胞支架和维持软骨内环境稳定的作用，对于保证软骨细胞的生物合成能力至关重要。课题组前期的实验结果发现，II型胶原前蛋白在髋臼髋关节发育不良软骨细胞内异常堆积，提出II型胶原异常堆积并引发内质网－未折叠蛋白反应可能是髋臼发育不良软骨可逆性改变的分子机制的假设。利用免疫共荧光的方法测定二型胶原及细胞各组分蛋白抽提等技术明确II型胶原前蛋白在细胞内异常堆积的位置为细胞浆内尤以高尔基体内堆积最为显著；提取三维培养系统的支架蛋白并鉴定为II型胶原蛋白，建立了髋关节软骨细胞的体外三维培养系统；通过RT-PCR及Western Blotting等方法检测内质网应激－未折叠蛋白反应通路的激活情况，检测了激活转录因子4（ATF4），激活转录因子6（ATF6）,糖 调节蛋白（GRP78）；等蛋白的表达情况，结果显示随着造模动物双下肢伸髋伸膝位的时间延长，各应激同路蛋白在时间点之间无统计学差异；利用生物可吸收膜作为三维培养的支架，通过骨髓间充质干细胞的分离、鉴定、诱导分化及体外培养，通过观察细胞形态，组织学特性、PCR观察干细胞在三位培养体系的软骨分化特性及与可吸收膜的相容性。通过将三维培养的细胞植入髋关节发育不良的实验动物模型，体内诱导干细胞向髋臼上缘的骨化中心分化，通过微观三维CT断层扫描评估修复髋臼发育不良的有效性。本研究在课题组长期从事髋关节发育不良的基础研究基础上，进一步在分子水平探讨了髋臼发育不良的机制以及自体微创修补髋臼上缘发育不良的可行性方法。为临床和进一步的科学研究提供了必要的理论基础及实验手段。