压力性尿失禁小鼠模型的建立及效果评价

Establishment and evaluation of mouse model of stress urinary incontinence

目的探讨阴道扩张(VD)法构建压力性尿失禁(SUI)小鼠模型的可行性。方法于2015年10~12月,选择135只无特定病原体(SPF)级雌性未育C57BL/6小鼠为研究对象,按照VD法构建SUI模型的时间,将其随机分为VD 1、3、7、14、28d组(各组为15只小鼠)和对照组(60只小鼠)。分别于VD法进行SUI建模后的第1、3、7、14及28天,检测小鼠膀胱最大容量(MBC)、膀胱漏尿点压(BLPP)及腹压漏尿点压(ALPP)等尿动力学指标。对照组小鼠未进行处理,直接检测MBC、BLPP及ALPP。以±1.96 s估计对照组小鼠MBC、BLPP及ALPP的正常参考值范围,分析VD各组小鼠SUI建模成功率和SUI模型耐久度。使用明胶酶谱法检测各组小鼠阴道前壁组织基质金属蛋白酶(MMP)-2与-9表达。对VD各组与对照组MBC、BLPP、ALPP、MMP-2与-9进行统计学分析。实验过程中对动物的处置符合动物伦理学要求。各组小鼠周龄及体重等比较,差异均无统计学意义(P>0.05)。结果 1VD 1、3、7、14、28d组MBC、BLPP、ALPP分别与对照组比较,差异均有统计学意义(F=11.060,P=0.000;F=71.670,P=0.000;F=61.120,P=0.000)。VD 1、3及7d组MBC、BLPP和ALPP,均分别较对照组显著降低,差异均有统计学意义(MBC:tD=7.115、4.528、3.909,均为P=0.000;BLPP:tD=18.461、15.949、14.294,均为P=0.000;ALPP:tD=16.198、12.895、12.700,均为P=0.000)。VD 14和28d组BLPP、ALPP分别与对照组比较,均显著降低,差异均有统计学意义(BLPP:tD=7.494、3.104,P=0.000、0.003;ALPP:tD=6.356、2.031,P=0.000、0.046),但VD 14和28d组MBC分别与对照组比较,差异均无统计学意义(tD=1.086、0.469,P=0.293、0.640)。2对照组C57BL/6小鼠MBC、BLPP及ALPP的正常参考值范围分别为0.06~0.15mL、34.55~53.81cm H_2O(1cm H_2O=0.098kPa)及33.88~58.96cmH_2O。3VD 1、3d组SUI建模成功率均>93%,VD 7d组约为90%,VD 14d组约为60%,VD 28d组仅约为30%。C57BL/6小鼠SUI模型耐久度为2~4周。4VD 1、3、7、14、28d组小鼠阴道前壁组织MMP-2与-9表达水平分别与对照组比较,差异均有统计学意义(F=55.130,P=0.000;F=18.850,P=0.000);而VD 3、7和14d组分别较对照组显著增加,并且差异均有统计学意义(MMP-2:t=5.257、17.280、11.620,均为P=0.000;MMP-9:t=2.953、9.538、5.450,P=0.042、0.001、0.006);VD 1、28d组分别与对照组比较,差异均无统计学意义(MMP-2:t=0.004、2.360,P=0.997、0.078;MMP-9:t=0.245、2.156,P=0.818、0.097);VD 7d组二者均最高,与VD 3和14d组分别比较,差异均有统计学意义(MMP-2:t=7.733、6.526,P=0.002、0.003。MMP-9:t=5.468、6.257,P=0.005、0.003)。结论 VD法构建的SUI小鼠模型耐久度为2~4周,VD法建模后第3~7天的SUI小鼠模型最为稳定,最适宜用于SUI发病机制及其治疗等研究。综合比较尿动力学指标和SUI建模成功率,对SUI小鼠模型建模效果进行分析,较单纯比较尿动力学指标更为可靠。由于本研究纳入样本量较小,以及膀胱置管时不同操作者技术水平不同等因素的影响,小鼠尿动力学指标的正常参考值范围及VD法构建的SUI小鼠模型耐久度,仍有待进一步研究证实。

Objective To explore the feasibility of building a stable mouse model of stress urinary incontinence （SUI） by vaginal dilation （VD） method. Methods From October to December 2015, a total of 135 cases of 8-week-old female C57BL/6 mice were selected as research subjects. They were randomly divided into VD 1, 3, 7, 14 and 28 d group （15 cases of mice in each group） and control group （60 cases of mice）. The mouse model of SUI in VD 1, 3, 7, 14 and 28 d group was built by VD method, and the maximum bladder capacity （MBC）, bladder leak point pressure （BLPP） and abdomen leak point pressure （ALPP） were detected in 1, 3, 7, 14 and 28 d after VD, respectively. The mice in control group didnrt receive any treatment and their MBC, BLPP and ALPP were detected directly. The normal reference ranges of MBC, BLPP and ALPP in control group were estimated by x：J= 1. 96 s, and they were used to analyzed the successful modeling rate and the SUI model durability of all VD groups. The expression of matrix metalloproteinases （MMP）-2 and -9 in paries anterior vagina of mice in every group was detected by gelatin zymograpby method. MBC, BLPP, ALPP, MMP-2 and -9 in all VD groups and control group were statistically analyzed. All experimental protocols were approved by the Institutional Animal Care and Use Committee. There were no statistical differences in weight and week-old between all groups of mice （P = 0. 05）. Results （DSingle factor analysis of variance results showed that there were significant differences between all VD groups and control group in MBC, BLPP and ALPP （F= 11. 060, P=0. 000~ F= 71. 670, P=0. 000 F=61. 120, P=0. 000）. The MBC, BLPP and ALPP in VD l, 3 and 7 d group were significantly decreased compared with those in control group, and all the differences were statistically significant （MBC： tD^7. 115, 4. 528, 3. 909~ all P=0. 000. BLPP.. tD= 18. 461, 15. 949, 14.294 allP=0.000% ALPP： to=16. 198, 12. 895, 12.700 all P=0.000）. Compared with control group, the BLPP and ALPP were significantly decreased in VD 14 and 28 d group, and all the differences were statistically significant （BLPP： tD = 7. 494, 3. 104 P= 0. 000, 0. 003. ALPP： tD = 6. 356, 2. 031 P= 0. 000, 0. 046）, however, there were no significant differences in MBC （tD = 1. 086, 0. 469 P=0. 293, 0. 640）. （1）The normal reference range of C57BL/6 mice＇s MBC was 0.06- 0.15 mL, BLPP was 34. 55-53. 81 cmH2O （1 cmH2O = 0. 098 kPa） and ALPP was 33. 88- 58.96 cmH2O. （2）The successful modeling rates in VD 1 and 3 d group both were 93%, and about 90%, 60% and 30% in VD 7, 14, 28 d group, respectively. The SUI model durability was 2 to 4 weeks. QSingle factor analysis of variance results showed that there were significant differences between all VD groups and control group in MPP-2 and -9 （F=55. 130, P=0. 000） F=18. 850, P= 0. 000）. Compared with control group, the expression of MMP-2 and -9 in paries anterior vagina of VD 3, 7 and 14 d group were significantly increased （MMP-2： t= 5. 257, 17. 280, 11. 620 P= 0.006, 0.000, 0.000. MMP-9.. t=2.953, 9.538, 5. 450 P=0. 042, 0.001, 0.006）. However, there were no differences in VD 1 and 28 d group compared with control group （MMP-2.. t= 0. 004, 2.360 P=0.997, 0.078. MMP-9： t=0. 245, 2. 156, P=0. 818, 0. 097）. The expressions of MMP-2 and -9 in VD 7 d group both were the highest, and there were significant differences compared withVD3 and 14 d group （MMP-2.. t=7.733, 6. 526 P=0.002, 0.003. MMP-9： t=5.468, 6. 257, P=0. 005, 0. 003）. Conclusions The durability of VD mouse model of SUI is 2 to 4 weeks, the SUI mouse model within 3 to 7 days after its building by VD method is the most stable and reliable subject to research the pathogenesis and therapy of SUI. Comprehensively analyzing the parameters of urinary dynamics and the effectiveness of SUI model are better and more reliable than just analyzing the parameters of urinary dynamics. Because of the small sample size in this study and the influence of different operational capabilities of different operators and many other uncontrollable factors, the normal reference ranges of MBC, BLPP and ALPP and the effectiveness of SUI model should be confirmed by further researches.