纳米粒介导ROCK-Ⅱ-siRNA基因治疗阿尔茨海默病的体内研究

Intrahippocampal injection of PEG-PEI/ROCK-Ⅱ-siRNA improves learning and memory deficits in a mouse model of Alzheimer's disease

目的探讨海马内注射PEG-PEI/ROCK-Ⅱ-siRNA纳米复合物对阿尔茨海默病模型小鼠(SAMP8)学习、记忆、认知及海马组织胆碱乙酰转移酶(ChAT)活性的影响。方法 6月龄SAMP815只小鼠分为治疗组(8只)及阴性对照组(7只),另选5只SAMPR1鼠作为正常对照组。治疗组给予PEG-PEI/ROCK-Ⅱ-siRNA,N/P=50、阴性对照组给予PEG-PEI/ROCK-Ⅱ-Scr,正常对照组给予生理盐水,给药均为海马注射,3 d给药1次,给药5次。30 d后各组进行Morris水迷宫检测行为学改变,免疫组织化学检测海马ChAT的表达。结果 PEG-PEI/ROCK-Ⅱ-siRNA治疗组与阴性对照组相比,定位航行试验潜伏期显著缩短[第1天:(37.02±3.30)s vs.(40.68±3.59)s,t=2.77,P<0.01;第3天(23.00±2.70)s vs.(31.35±1.80)s,t=3.35,P<0.01],穿越目的象限区域次数明显增加(4.50±1.68 vs.2.41±0.76,t=1.97,P=0.096),目的象限停留距离比值(0.66±0.25 vs.0.34±0.05,t=2.49,P<0.05)及时间比值(0.57±0.13 vs.0.30±0.04,t=3.95,P<0.01)都较阴性对照组提高,海马组织ChAT活性也增加(CA1区阳性率0.32±0.08 vs.0.17±0.01,t=25.21,P<0.01;CA3区阳性率0.32±0.02 vs.0.16±0.01,t=17.91,P<0.01),差异具有统计学意义。治疗组与正常对照组相比,Morris水迷宫结果及海马组织ChAT活性无统计学差异。结论脑内注射PEG-PEI/ROCK-Ⅱ-siRNA纳米复合物能提高SAMP8鼠的学习和记忆能力,这种作用可能通过增加海马组织ChAT含量而得以实现。

Objective To evaluate whether intrahippocampal injection of PEG-PEI/ROCK-I1- siRNA improves spatial and learning deficits, and enhances activity of choline acetyl transferase （CHAT）in SAM mice. Methods Fifteen SAMP8 male mice and 5 SAMR1 male mice were assigned to PEG-PEI/ ROCK- 11 -siRNA group （ 8 SAMP8 ）, PEG-PEI/ROCK- II -Scr group （ 7 SAMP8 ） and normal group （ 5 SAMR1 ）. We injected every 3 days and lasting 14 days. A total volume of 2. 86 μl noncomplex or saline was intrahippocampally injected according to the stereotaxic apparatus. One month after injection, Morris water maze（MWM） was applied to evaluate the spatial and leaning function of mice in each group and the activity of ChAT was detected by immunohistochemistry. Results Mice in PEG-PElf ROCK- lI -siRNA group showed decreased escape latency in navigation test from the first trial [ （ 37. 02 ± 3.30 ） s vs. （ 40. 68 ± 3. 59 ） s, t = 2. 77, P 〈 0.01 ] to the third trial [ （23.00 ±2. 70 ） s vs. （ 31.35 ±1. 80 ） s, t = 3.35, P 〈 0. 01 ], increased times crossing the destination quadrant in the spatial probe test than that in the PEG-PEI/ROCK-II-scr group（ times crossing the destination quadrant ： 4. 50 ±1.68 vs. 2.41 ± 0.76, t = 1.97, P = 0. 096 ; distance percentage ：0. 66 ± 0. 25 vs. 0. 34 ±0. 05, t = 2. 49, P 〈 0. 05 ; time percentage ： 0. 57 ± 0. 13 vs. 0. 30 ± 0. 04,t = 3.95, P 〈 0. 01 ）, as well the ChAT activity in the hippocampus （ percentage of positive cells in CA1 region ：0. 32±0. 08 vs. 0. 17 ± 0. 01 ,t = 25.21, P 〈 0.01 ;and 0. 32 ± 0. 02 vs. 0. 16± 0.01, t = 17.91, P 〈 0. 01 in CA3 region）. Compared with normal group, mice in PEG-PElf ROCK-II-siRNA group showed no significant difference io the MWT, as well as in ChAT activity. Conclusion We conclude that intrahippocampal injection of PEI-PEG/ ROCK-II-siRNA improves spatial and learning deficits in SAM mice, and it may be achieved through enhancing the activity of CHAT.