缺血再灌注对大鼠颌下腺分泌功能影响的研究

发布时间：2019-05-08 13:28

【摘要】：目的重症角结膜干燥症是由多种原因引起眼表及泪膜异常的疾病,常引起病人眼部不适,视物模糊及泪液不稳定等,严重者会导致眼角膜、结膜病变,甚至引起视力丧失,严重影响患者的生活质量。目前,临床上可以根据患者病情的严重程度采取一些治疗措施,但泪液的分泌的减少严重影响其治疗效果和进一步的眼科治疗。血管化自体颌下腺移植以移植颌下腺分泌的唾液代替泪液,是近来开展的一种治疗角结膜干燥症的新方法,临床研究已证实移植成功的病例,患者眼干症状得到改善,取得了良好的效果。血管化自体移植颌下腺难免要经历失神经和缺血再灌注过程,两者均可能对腺体的分泌功能产生影响。有研究证实神经递质的分泌不足和自主神经受体信号转导通路下调是移植颌下腺分泌功能紊乱的重要机制。移植的腺体除失神经支配外,还经历了缺血再灌注的过程。目前缺血再灌注后颌下腺的损伤—应激反应尚缺乏研究。所以本研究推测缺血再灌注损伤可能是移植腺体早期功能减退或丧失的重要原因。为进一步探究缺血再灌注对移植颌下腺分泌功能状态的影响,本实验建立了大鼠颌下腺原位缺血再灌注模型,在保留大鼠颌下腺分泌神经分支支配下,探讨单纯缺血再灌注后大鼠颌下腺的损伤—应激反应及经历缺血再灌注后大鼠颌下腺分泌功能的变化。材料与方法在保留颌下腺神经分支支配的情况下,建立了大鼠颌下腺原位缺血再灌注模型。颌下腺经历缺血90min后分别再灌注1h、12h、24h、72h,并以此作为观察取样的时间点。本研究于颌下腺导管断端插管,采用Schirmer实验来观察各时间点大鼠颌下腺分泌情况。为进一步观察大鼠颌下腺经历缺血再灌注后的损伤—应激反应,本研究通过HE染色观察颌下腺的组织形态变化；通过透射电镜观察腺体紧密连接超微结构的变化；通过检测组织内活性氧观察腺体的氧应激；通过TUNEL法检测腺体组织细胞的凋亡情况。结果大鼠颌下腺经历缺血再灌注后1h和12h,腺体分泌量显著降低,至再灌注72h腺体的分泌量逐渐恢复正常。组织学观察发现,经历缺血再灌注后,腺体组织开始出现水肿及炎细胞浸润,至再灌注12h最为严重,后逐渐恢复至正常。超微结构进一步观察,紧密连接在再灌注1h和12h变得模糊,电子密度降低。腺体组织活性氧水平及细胞凋亡情况也表现出相同的趋势,表现为在经历再灌注1h和12h后,腺体组织内活性氧信号增多及细胞凋亡水平的显著增高。结论本研究观察缺血再灌注引起的大鼠颌下腺早期的损伤—应激反应,与大鼠颌下腺经历缺血再灌注后早期分泌功能降低的情况相符。本研究证实缺血再灌注损伤后引起的损伤—应激反应是移植腺体早期分泌功能低下的重要原因之一。
[Abstract]:Objective severe keratoconjunctival xerosis is a disease with abnormal surface and lacrimal film caused by a variety of reasons, often causing eye discomfort, blurred vision and unstable tear, etc. Serious cases may lead to corneal and conjunctival lesions. It even causes loss of vision, which seriously affects the quality of life of the patients. At present, some therapeutic measures can be taken according to the severity of the patient's condition, but the decrease of lacrimal secretion seriously affects the therapeutic effect and further ophthalmological treatment. Vascularized submandibular gland transplantation is a new method to treat keratoconjunctival xerosis with saliva secreted from submandibular gland transplantation instead of lacrimal fluid. Clinical studies have confirmed the successful transplantation of the submandibular gland, and the dry eye symptoms of the patients have been improved. Good results have been achieved. Vascularized autotransplantation of submandibular gland will inevitably undergo denervation and ischemia-reperfusion, both of which may affect the secretory function of the gland. It has been proved that insufficient secretion of neurotransmitters and down-regulation of the signal transduction pathway of autonomic nerve receptors are the important mechanisms of the dysfunction of submandibular gland secretion. In addition to denervation, the transplanted glands undergo ischemia-reperfusion. At present, there is still little research on the injury-stress response of submandibular gland after ischemia-reperfusion. Therefore, this study speculated that ischemia-reperfusion injury may be an important cause of early dysfunction or loss of graft glands. In order to further investigate the effect of ischemia-reperfusion on the secretory function of submandibular gland transplantation, a rat model of submandibular gland ischemia-reperfusion in situ was established, which was controlled by preserving the secretory nerve branches of the submandibular gland in rats. To investigate the injury-stress response of submandibular gland in rats after simple ischemia-reperfusion and the changes of submandibular gland secretory function after ischemia-reperfusion. Materials and methods A rat model of in situ ischemia reperfusion of submandibular gland was established by preserving the nerve branches of the submandibular gland. The submandibular gland was reperfused for 1 h, 12 h, 24 h, 72 h after 90min ischemia, and as a time point to observe the sampling. In this study, the submandibular gland secretion was observed by Schirmer test at different time points. In order to observe the injury-stress response of submandibular gland after ischemia-reperfusion, the morphological changes of submandibular gland were observed by HE staining, the ultrastructural changes of tight junctions were observed by transmission electron microscopy (TEM), and the histological changes of submandibular gland were observed by transmission electron microscopy (TEM). Oxygen stress of glands was observed by detecting reactive oxygen species (Ros) and apoptosis of glandular cells was detected by TUNEL method. Results 1 h and 12 h after ischemia-reperfusion, the secretion of submandibular gland decreased significantly, and gradually returned to normal at 72 h after reperfusion. Histological observation showed that edema and inflammatory cell infiltration began to occur in glandular tissue after ischemia-reperfusion, and then returned to normal gradually after 12 hours of reperfusion. Ultrastructural observation showed that the tight junctions became blurred and electron density decreased at 1h and 12h after reperfusion. The levels of reactive oxygen species (Ros) and apoptosis also showed the same trend in glandular tissue. After 1 h and 12 h of reperfusion, the signal of reactive oxygen species (Ros) and the level of apoptosis in glandular tissue increased significantly. Conclusion the early injury-stress response of submandibular gland induced by ischemia-reperfusion is consistent with the decrease of early secretory function of submandibular gland after ischemia-reperfusion in rats. [WT5 "HZ] conclusion [WT5" BZ] [WT5 "BZ] This study confirmed that injury-stress response induced by ischemia-reperfusion injury is one of the important reasons for the early secretory dysfunction of transplanted glands.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R782

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