Wnt 信号通路与糖尿病性疼痛发生发展机制

WNT SIGNALING IN DIABETICNEUROPATHIC PAIN

张笑丹

11649086

硕士

生物学

宋学军

2018-06-06

2018-07-05

哈尔滨工业大学

深圳

目的：本实验采用腹腔注射链脲佐菌素（Streptozotocin, STZ）构建糖尿病性疼痛（Diabetic neuropathic pain, DNP）模型，通过动物行为学和分子生物化学等手段，探讨 Wnt 信号通路在 DNP 发生发展中的机制和治疗靶点。方法：选取 180 g-220 g 的健康雄性 Sprague Dawley(SD)大鼠，腹腔注射 70mg/kg STZ 诱导糖尿病大鼠模型，腹腔注射等剂量柠檬酸缓冲液作为对照组（Vehicle 组）。制模第 4 周，根据血糖及疼痛行为学将糖尿病大鼠分为高血糖痛敏组（DNP 组）和高血糖不痛敏组（No-pain 组）。另外构建大鼠慢性坐骨神经损伤模型组（CCI 组）做为分子结果对照。制模后 4 周，6 周和 8 周，分别随机选取 Vehicle 组、DNP 组和 No-pain 组的 4 只大鼠脊髓和背根神经节结（Dorsal root ganglia, DRG）的 L4-L5 段，选取 CCI 组大鼠术后 7 天脊髓和 DRG 的 L4- L5 段，采用 Western Blot 的方法测定脊髓中 Wnt 信号通路蛋白 Wnt3a、Wnt5b、Ryk、β-catenin 的表达水平。结果：大鼠腹腔注射 STZ 后，97.6 %出现血糖增高，42.9 %出现持续性疼痛，毛色呈病态，理毛动作减少，瘦弱，尾静脉采血伤口愈合缓慢，容易腹泻，“洗脸”活动增多，垫料潮湿尿多且味道加重。与 Vehicle 组比较，DNP 组和No-pain 组体重明显上升缓慢，制模后第 4 周开始持续缓慢下降（P < 0.01）。 与 Vehicle 组比较，DNP 组和 No-pain 组血糖明显高于 16.6 mmol/L（P < 0.01）， 制模后第 3-4 周达到峰值，之后趋于稳定。DNP 组与 No-pain 组无显著差异（P >0.05）。与 Vehicle 组和 No-pain 组比较，DNP 组机械痛阈值明显下降（P < 0.01）， 制模后第 2 周出现痛敏，在第 5-6 周达到峰值，并持续至第 8 周。Vehicle 组与 No-pain 组无显著差异（P > 0.05）。Western Blot 实验结果显示，在 DNP 组的 第 4、6、8 周脊髓样品中，全细胞 Wnt3a、Wnt5b、Ryk 蛋白水平相较 Control组没有统计学差异（P > 0.05）；核内 β-catenin 蛋白水平相较 Control 组没有统计学差异（P > 0.05）；CCI 组核内 β-catenin 蛋白水平与 Control 组比较明显上调且有统计学差异（P < 0.01）。结论：1. STZ诱导糖尿病大鼠模型，97 %以上大鼠出现血糖增高，42.9 %出现持续性疼痛。2.糖尿病性疼痛在大鼠腹腔注射STZ后第2周发生，第5-6周痛阈达到最低值，并持续低痛阈至第8周。3. Wnt 信号通路在糖尿病性疼痛的发生发展过程中没有明确作用，与其在神经损伤导致疼痛中的机制不同。

Objective: By means of animal behavioral tests and molecular biological meaurements, this study was to explore the possible roles of Wnt signaling in the pathogenesis of diabetic neuropathic pain using rat model of diabetic neuropathic pain(DNP) induced by intraperitoneal injection of streptozotocin (STZ).Method: Healthy male Sprague Dawley (SD) rats weighing 180 g-220 g were selected. STZ (70 mg/kg) was intraperitoneally injected to induce DNP. Four weeksafter STZ injection, the rats were divided into a hyperglycemia group (DNP group) and a hyperglycemic insensitivity group (No-pain group) according to blood glucose and pain behavior. In addition, rats with nerve injury by chronic constriction injury of the sciatic nerve (CCI group) was used as control. The L4-L5 segments of the spinal cord and dorsal root ganglia (DRG) in the groups of Vehicle, DNP and No_x005fpain were collected 4, 6, and 8 weeks after STZ injection. Western Blot was used to determine the expression of Wnt3a, Wnt5b, Ryk and β-catenin in the spinal cord.Results: After intraperitoneal injection of STZ, hyperglycemia appeared in 97.6 % of the rats. 42.9% of the rats showed persisent pain behavors, hair color looked unhealthy, grooming was decreased. The wound in the tail because of vein blood collection recovered slower than usual, more diarrhea and “washing” activities wereobserved. The animals’ body weight in DNP group and No-pain group that increased slower that in control group began to decline from the 4th week after STZ injection(P < 0.01). Blood glucose levels in STZ-rats with pain or without pain increasedsignificantly (>16.6 mmol/L) compared with the control group (<7.0mmol/L) (P <0.01), peaked at 3-4 weeks (24 mmol/L) and then stabilized. There was no significant difference between DNP group and No-pain group (P > 0.05). Compared with Vehicle control and No-pain group, the mechanical pain threshold in DNP rats decreased significantly (P < 0.01). Increased pain-sensitivity occurred in the second week after STZ-injection, peaked in the 5th weeks, and lasted to the 8th weeks observed. There was no significant difference between the Vehicle group and the No-pain group (P > 0.05). Western Blot nanalysis showed that expression of Wnt3a, Wnt5b, and Rykreceptor proteins in the DNP group was not statistically different from those in the Control group (P > 0.05). Further, there was no significant difference in the nuclear expression level of β-catenin protein compared with the Control group (P > 0.05). As a positive control, expression of β-catenin nuclear protein in nerve-injured rats (CCI group) was significantly increased and statistically different from control group (P <0.01).Conclusions: 1. After intraperitoneal injection of STZ, hyperglycemia appeared in 97.6% of the rats, 42.9% of the rats showed persistent pain behaviors. 2. Increased pain-sensitivity occurred in the second week after STZ-injection, peaked in the 5thweeks, and lasted to the 8th weeks observed. 3. Wnt signaling does not seem to beinvolved in the pathogenesis of diabetic neuropathic pain. This is different from that Wnt signaling is critical in the development of pain caused by nerve injury.

糖尿病 糖尿病性疼痛 Wnt 信号通路 β-catenin Ryk

Diabete Diabetic neuropathic pain Wnt signaling β-catenin Ryk receptor

中文

联合培养

南方科技大学

张笑丹. Wnt 信号通路与糖尿病性疼痛发生发展机制[D]. 深圳. 哈尔滨工业大学,2018.