跟腱断裂病因学关键靶标信号传导通路研究

The degenerative changes of Achilles tendon caused by over-used injury are the basic pathological change of Achilles tendon rupture. The Achilles tendon tissues obtained from the patients of Achilles tendon rupture and some others of normal Achilles tendon will be histopathologically analyzed firstly. In addition, for the result of qualitative and functional analysis on some key protein, the differential proteomics technique will be used to compare the tissue of Achilles tendon, the serum and the urine. Thereby to analysis the influence of the differential protein expression profiling to Achilles tendon rupture. The immunohistochemistry will be employed to detect stabilities of the some key proteins on Achilles tendon tissues and serum, then the mRNA transcription level on these key proteins will be confirmed by using the RT-PCR technology. Based on the Achilles tendon tissue, serum and urine which obtained from the patients on our previous studies, the localized and systematic differential protein expression profile will be compared, and some related key targets will be identified and verified. The influences of ruptured tissue on differential protein expression profiling will be observed and compared with the normal tissue to clarify the relationship from differential protein expression maps on how the disease was developed. This will facilitate to precisely interpret the basic pathological change of ruptured tendon and to investigate the effected mechanism of etiology on this disease caused by some key targets. Simultaneously, a new approaches and technique for early warning, diagnosis and prognosis for Achilles tendon rupture would be explored.

跟腱过度使用性损伤导致跟腱退行性改变是发生跟腱断裂伤的基础病理。因此，首先将跟腱断裂患者与正常跟腱者跟腱组织进行组织病理学分析，再分析两者跟腱组织、血清、尿液差异蛋白质谱，并获得某些关键蛋白的定性和功能分析，探讨断裂跟腱对差异蛋白质谱的影响。然后对跟腱组织、血清运用免疫组织化学技术检测在先前组织中某些差异蛋白存在的稳定性，再进行关键靶标信号传导通路探索。本研究根据我们前期获得的断裂跟腱与正常跟腱、血清等组织，运用差异蛋白质组学技术，对局部与全身差异蛋白质谱进行比较，并对某些关键靶标进行鉴定与验证以及信号传导通路探索。与正常组织对比，观察断裂伤者组织对差异蛋白质谱的影响，阐明差异蛋白质谱与本病发生之间的联系，从而进一步对本病病理基础给以精确的阐释，探讨本病病因关键靶标的作用机理，同时为本病提供早期预警、诊断以及预后的新方法、新技术。

目的：经皮重建缝合方法与开放微创手术治疗急性跟腱断裂伤（本病），同时完善生物医学标本；将本病与正常人跟腱组织进行组织病理学分析；对跟腱组织、血清、尿液进行差异蛋白质谱比较，从而获得某些关键蛋白的定性和功能分析；细胞培养、慢病毒转染，从细胞水平等方面对靶标进行验证；探索关键靶标对信号传导通路的影响；探讨CRMP-2在跟腱愈合过程中的作用。方法：本病患者分为经皮重建入路手术治疗和开放微创切口进行评价疗效；对跟腱形态学、组织学、免疫组化与超微结构进行对照研究；应用蛋白质组学技术对本病组织、血清、尿液进行研究，通过细胞培养、慢病毒转染对关键靶标进行验证；选取关键靶标进行信号传导通路影响的研究；建立大鼠跟腱断裂修复模型，组织学观察固定组与运动治疗组的差异，再通过免疫组化研究CRMP-2意义。结果：1）经皮重建治疗具有手术时间短、术中出血量少、住院时间短、切口感染率低优点。2)组织病理学分析提示本病组跟腱排列紊乱，肌腱围肥厚充血，正常细胞结构消失、呈玻璃样变性，脂肪组织浸润较多见，毛细血管增生明显。3)本病组与正常组比较，血清特有的差异蛋白37个，尿液163个，跟腱373个。确定APOA4，PK，PRDX1，PGK1，PKM等为关键靶标。4）随着细胞培养时间的延长，两组细胞有不同程度的形态和数量的改变，本病组细胞数量少，体积小、形状不规则，排列比较紊乱。5) 两组细胞培养、慢病毒转染获得成功，正在完善信号通路研究。6)制动组胶原纤维排列紊乱、胶原纤维损伤，Western证实CRMP-2的印迹升高。结论：经皮重建法更具有推广性。Ⅰ型与III型胶原纤维在跟腱组织中的比例及形态学改变对本病的发生和发展起着重要作用，APOA4，PK，PRDX1，PGK1，PKM等一系列关键靶标对跟腱基础疾病信号传导通路起着重要作用，须进一步研究。正常跟腱及本病组跟腱在相同条件下的细胞生长速度及数量有差别，本病组细胞增殖不明显、细胞间隙变大、细胞数目明显减少、细胞体积较小。运动治疗可能通过上调CRMP-2蛋白表达促进愈合。