Experimental study of neurotrophin-3 overexpression induced pluripotent stem cells in treating rats with diabetes-induced erectile dysfunction
-
摘要:目的
探讨神经营养素-3过表达诱导多能干细胞(iPSC-NT-3)对糖尿病性勃起功能障碍(DIED)大鼠的治疗作用。
方法腹腔注射链脲佐菌素建立糖尿病模型大鼠，皮下注射阿扑吗啡对DIED模型进行验证。选择24只造模成功的DIED大鼠，随机分为模型对照组、诱导多能干细胞(iPSC)治疗组和iPSC-NT-3治疗组; 腹腔注射柠檬酸钠-柠檬酸缓冲液的8只SD大鼠作为正常对照组。各组大鼠腹腔注射戊巴比妥钠进行麻醉, iPSC治疗组和iPSC-NT-3治疗组分别将iPSC和iPSC-NT-3用微量注射针注射入大鼠阴茎海绵体内，正常对照组和模型对照组同法注射同体积的磷酸缓冲液。治疗后第4周，观察各组大鼠的一般情况，评估性功能和阴茎勃起功能。采用定量聚合酶链反应(q-PCR)和Western blot实验检测神经营养素-3(NT-3)、CD31、血管内皮生长因子(VEGF)、内皮型一氧化氮合酶(eNOS)、结蛋白(Desmin)、α-平滑肌肌动蛋白(α-SMA)的mRNA和蛋白的表达。
结果治疗前和治疗后4周，模型对照组、iPSC治疗组和iPSC-NT-3治疗组大鼠体质量和血糖水平的组内、组间差异均无统计学意义(P>0.05)。在大鼠首次爬背时间比较中，模型对照组较正常对照组延长, iPSC治疗组、iPSC-NT-3治疗组较模型对照组缩短, iPSC-NT-3治疗组较iPSC治疗组缩短，差异均有统计学意义(P < 0.05); 在舔嗅次数、骑跨次数和插入次数的比较中，模型对照组均较正常对照组减少, iPSC治疗组、iPSC-NT-3治疗组较模型对照组增加, iPSC-NT-3治疗组较iPSC治疗组增加，差异均有统计学意义(P < 0.05)。在ICP和ICP/MAP的比较中，模型对照组较正常对照组降低, iPSC治疗组、iPSC-NT-3治疗组较模型对照组升高, iPSC-NT-3治疗组较iPSC治疗组升高，差异均有统计学意义(P < 0.05)。在大鼠阴茎海绵体NT-3、CD31、VEGF、eNOS、Desmin和α-SMA的mRNA表达水平比较中，模型对照组较正常对照组降低, iPSC治疗组、iPSC-NT-3治疗组较模型对照组升高, iPSC-NT-3治疗组较iPSC治疗组升高，差异均有统计学意义(P < 0.05)。在大鼠阴茎海绵体NT-3、CD31、VEGF、eNOS、Desmin、α-SMA蛋白水平比较中，模型对照组较正常对照组降低, iPSC治疗组、iPSC-NT-3治疗组较模型对照组升高, iPSC-NT-3治疗组较iPSC治疗组升高，差异有统计学意义(P < 0.05)。
结论iPSC-NT-3可通过促进海绵体组织eNOS、血管内皮及平滑肌的生成而改善DIED大鼠的性功能和勃起功能，是一种潜在的治疗DIED的方法。
Abstract:ObjectiveTo explore the role of neurotrophin-3 overexpression induced pluripotent stem cells (iPSC-NT-3) in treating rats with diabetes-induced erectile dysfunction (DIED).
MethodsThe diabetic model rats were established by intraperitoneal injection of streptozotocin, and the DIED model was verified by subcutaneous injection of apomorphine. Twenty-four rats with successful modeling of DIED were randomly divided into model control group, induced pluripotent stem cell (iPSC) treatment group and IPSC-NT-3 treatment group; eight SD rats with intraperitoneal injection of sodium citrate and citrate buffer were selected as normal control group. Rats in each group were intraperitoneally injected with pentobarbital sodium for anesthesia, the iPSC and the iPSC-NT-3 were respectively injected into the penile sponge of rats through microinjection needle in the iPSC treatment group and the IPSC-NT-3 treatment group, and the equal volume of phosphoric acid buffer was injected by the same method in the normal control group and the model control group. At the 4th week after treatment, the general condition of the rats in each group was observed, and the sexual function and erectile function of the penis were evaluated. Quantitative polymerase chain reaction (q-PCR) and Western blot assay were used to detect the mRNA and protein expressions of neurotrophin-3 (NT-3), CD31, vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), Desmin and α-smooth muscle actin (α-SMA).
ResultsBefore and 4 weeks after treatment, there were no significant intra-group and between-group differences in body mass and blood glucose level of rats among the model control group, the iPSC treatment group and the iPSC-NT-3 treatment group (P>0.05). In the comparison of primary time of climbing back in rats, the time in the model control group was longer than the normal control group, the time in the iPSC treatment group and the iPSC-NT-3 treatment group was shorter than the model control group, and the time in the iPSC-NT-3 treatment group was shorter than the iPSC treatment group, and all the differences were statistically significant (P < 0.05); in the comparison of licking and smelling times, riding times and inserting times, the times in the model control group were less than the normal control group, the times in the iPSC treatment group and the iPSC-NT-3 treatment group were greater than the model control group, the times in the iPSC-NT-3 treatment group was greater than the iPSC treatment group, and all the differences were statistically significant (P < 0.05). In comparison of ICP and ICP/MAP, the values in the model control group were lower than the normal control group, the values in the iPSC treatment group and the IPSC-NT-3 treatment group were higher than the model control group, and the values in the IPSC-NT-3 treatment group were higher than the iPSC treatment group, and all the differences were statistically significant (P < 0.05). In the comparison of the mRNA expression levels of NT-3, CD31, VEGF, eNOS, Desmin and α-SMA in the cavernous penis of rats, the levels in the model control group were lower than the normal control group, the levels in the iPSC treatment group and the iPSC-NT-3 treatment group were higher than the model control group, the levels in the iPSC-NT-3 treatment group were higher than the iPSC treatment group, and all the differences were statistically significant (P < 0.05). In the comparison of the protein levels of NT-3, CD31, VEGF, eNOS, Desmin and α-SMA in the cavernous penis of rats, the levels in the model control group were lower than the normal control group, the levels in the iPSC treatment group and the IPSC-NT-3 treatment group were higher than the model control group, the levels in the IPSC-NT-3 treatment group were higher than the iPSC treatment group, and all the differences was statistically significant (P < 0.05).
ConclusionThe iPSC-NT-3 can improve the sexual function and erectile function of DIED rats by promoting the production of eNOS, vascular endothelium and smooth muscle in the cavernous tissues, which is a potential treatment for DIED.
-
-
表 1 PCR的引物序列
引物名称 引物序列 NT-3 forward: 5′- CGTGGCCATTGAACACAGCA - 3′ reverse: 5′- CCCACTGGCGGGTAAGGAAA - 3′ CD31 forward: 5′- CATGGTGGAGCACAGTGGCA - 3′ reverse: 5′- TGGGATGGAGCAGGACAGGTT - 3′ VEGF forward: 5′- CCCACCCACATACATACATT - 3′ reverse: 5′- CTCCCAACTCAAGTCCACA - 3′ eNOS forward: 5′- CTG TGC TGG CAT ACA GAA CC - 3′ reverse: 5′- CTG CCT TGA GTT GGC TCA TC - 3′ Desmin forward: 5′- CTTGATGAGGCAGATGAGGGA - 3′ reverse: 5′- AGCTTCCGGTAGGTGGCAAT - 3′ α-SMA forward: 5′- TTCCCATCCATCGTG - 3′ reverse: 5′- GCCTTAGG GTTCAGC - 3′ GAPDH forward: 5′- GAG AGG GAA ATC GTG CGT GAC-3′ reverse: 5′- CAT CTG CTG GAA GGT GGA CT-3′ 
下载: 导出CSV
表 2 各组大鼠治疗前后血糖和体质量比较(x±s)
组别 治疗前 治疗后 体质量/g 血糖/(mmol/L) 体质量/g 血糖/(mmol/L) 正常对照组(n=8) 424.27±45.28 7.48±1.51 486.82±47.50* 7.63±1.65 模型对照组(n=8) 305.62±37.51# 28.39±4.67# 297.89±41.35# 28.79±4.56# iPSC治疗组(n=8) 298.35±41.15# 29.05±4.25# 291.46±39.71# 29.47±4.47# iPSC-NT-3治疗组(n=8) 302.40±39.67# 28.86±4.49# 295.73±42.55# 29.34±4.38# iPSC: 诱导多能干细胞; iPSC-NT-3: 神经营养素-3基因过表达的iPSC。与治疗前比较, *P < 0.05; 与正常对照组比较, #P < 0.05。
下载: 导出CSV
表 3 各组大鼠治疗后性功能比较(x±s)
组别 首次爬背时间/min 舔嗅次数/次 骑跨次数/次 插入次数/次 正常对照组(n=8) 42.15±4.85 39.72±5.29 15.61±2.72 9.41±1.75 模型对照组(n=8) 114.47±23.57* 13.45±2.17* 4.58±1.21* 0* iPSC治疗组(n=8) 76.21±17.24# 21.86±3.63# 6.62±1.67# 2.79±0.52# iPSC-NT-3治疗组(n=8) 51.39±15.26#△ 27.54±3.75#△ 8.95±2.12#△ 5.79±0.67#△ 与正常对照组比较, *P < 0.05; 与模型对照组比较, #P < 0.05; 与iPSC治疗组比较, △P < 0.05。
下载: 导出CSV
表 4 各组大鼠治疗后勃起功能的比较(x±s)
组别 ICP/mmHg ICP/MAP/% 正常对照组(n=8) 83.15±5.27 75.39±4.75 模型对照组(n=8) 47.38±3.44* 39.50±3.31* iPSC治疗组(n=8) 62.61±4.56# 52.75±4.78# iPSC-NT-3治疗组(n=8) 73.46±5.39#△ 66.32±5.17#△ ICP: 海绵窦内压; ICP/MAP: 海绵窦内压占平均动脉压的
比率。与正常对照组比较, *P < 0.05; 与模型对照组比较,
#P < 0.05; 与iPSC治疗组比较, △P < 0.05。
下载: 导出CSV
表 5 各组大鼠治疗后阴茎海绵体NT-3、CD31、VEGF、eNOS、Desmin和α-SMA的mRNA表达水平比较(x±s)
组别 NT-3 CD31 VEGF eNOS Desmin α-SMA 正常对照组(n=8) 0.87±0.05 1.12±0.07 1.27±0.09 0.78±0.07 1.34±0.12 1.04±0.07 模型对照组(n=8) 0.25±0.03* 0.51±0.04* 0.65±0.05* 0.24±0.03* 0.67±0.05* 0.47±0.04* iPSC治疗组(n=8) 0.39±0.05# 0.65±0.05# 0.78±0.07# 0.49±0.05# 0.85±0.07# 0.69±0.05# iPSC-NT-3治疗组(n=8) 0.64±0.06#△ 0.78±0.06#△ 0.94±0.06#△ 0.62±0.04#△ 1.09±0.06#△ 0.85±0.05#△ NT-3: 神经营养素-3; VEGF: 血管内皮生长因子; eNOS: 内皮型一氧化氮合酶; Desmin: 结蛋白; α-SMA: α-平滑肌肌动蛋白。
与正常对照组比较, *P < 0.05; 与模型对照组比较, #P < 0.05; 与iPSC治疗组比较, △P < 0.05。
下载: 导出CSV
表 6 各组大鼠治疗后阴茎海绵体NT-3、CD31、VEGF、eNOS、Desmin和α-SMA蛋白水平比较(x±s)
组别 NT-3 CD31 VEGF eNOS Desmin α-SMA 正常对照组(n=8) 0.53±0.05 0.74±0.06 0.68±0.06 0.43±0.04 0.81±0.07 0.57±0.05 模型对照组(n=8) 0.16±0.03* 0.24±0.02* 0.31±0.03* 0.13±0.02* 0.34±0.03* 0.21±0.03* iPSC治疗组(n=8) 0.32±0.04# 0.37±0.05# 0.44±0.04# 0.21±0.04# 0.47±0.06# 0.32±0.04# iPSC-NT-3治疗组(n=8) 0.44±0.06#△ 0.51±0.04#△ 0.53±0.05#△ 0.35±0.04#△ 0.63±0.05#△ 0.41±0.04#△ 与正常对照组比较, *P < 0.05; 与模型对照组比较, #P < 0.05; 与iPSC治疗组比较, △P < 0.05。
下载: 导出CSV
-
[1] WEN Y, LIU G H, ZHANG Y, et al. MicroRNA-205 is associated with diabetes mellitus-induced erectile dysfunction via down-regulating the androgen receptor[J]. J Cell Mol Med, 2019, 23(5): 3257-3270. doi: 10.1111/jcmm.14212
[2] DING F, SHAN C Y, LI H W, et al. Simvastatin alleviated diabetes mellitus-induced erectile dysfunction in rats by enhancing AMPK pathway-induced autophagy[J]. Andrology, 2020, 8(3): 780-792. doi: 10.1111/andr.12758
[3] 刘安全, 代苗英, 陈挺, 等. 复方玄驹胶囊联合十一酸睾酮治疗糖尿病性勃起功能障碍的疗效分析[J]. 中国性科学, 2019, 28(2): 46-48. doi: 10.3969/j.issn.1672-1993.2019.02.013 [4] 刘贵华, 张弛, 谢云, 等. 尿源干细胞治疗1型糖尿病性勃起功能障碍大鼠的实验研究[J]. 临床泌尿外科杂志, 2018, 33(8): 626-632. doi: 10.13201/j.issn.1001-1420.2018.08.009 [5] LU J, XIN Z X, ZHANG Q, et al. Beneficial effect of PEDF-transfected ADSCs on erectile dysfunction in a streptozotocin-diabetic rat model[J]. Cell Tissue Res, 2016, 366(3): 623-637. doi: 10.1007/s00441-016-2494-7
[6] SONG J Y, SUN T T, TANG Z, et al. Exosomes derived from smooth muscle cells ameliorate diabetes-induced erectile dysfunction by inhibiting fibrosis and modulating the NO/cGMP pathway[J]. J Cell Mol Med, 2020, 24(22): 13289-13302. doi: 10.1111/jcmm.15946
[7] 聂莉, 李琰华, 李俊伟. 2型糖尿病伴勃起功能障碍的研究进展[J]. 中国全科医学, 2020, 23(33): 4267-4273. doi: 10.12114/j.issn.1007-9572.2020.00.499 [8] CASTELA Â, COSTA C. Molecular mechanisms associated with diabetic endothelial-erectile dysfunction[J]. Nat Rev Urol, 2016, 13(5): 266-274. doi: 10.1038/nrurol.2016.23
[9] 燕浩, 肖冬冬, 荣立夺, 等. 脂肪来源干细胞促进糖尿病ED勃起功能恢复的实验研究[J]. 中国男科学杂志, 2019, 33(2): 24-28, 35. https://www.cnki.com.cn/Article/CJFDTOTAL-NXXX201902006.htm [10] ZHOU F, HUI Y, XIN H, et al. Therapeutic effects of adipose-derived stem cells-based microtissues on erectile dysfunction in streptozotocin-induced diabetic rats[J]. Asian J Androl, 2017, 19(1): 91-97. doi: 10.4103/1008-682X.182817
[11] 白刚, 张洪钿, 赖军, 等. NT-3-HUMSCs联合基因沉默SOCS3治疗SD大鼠脊髓损伤后的神经再生修复[J]. 昆明医科大学学报, 2018, 39(3): 31-37. doi: 10.3969/j.issn.1003-4706.2018.03.006 [12] 卢元帅, 徐宁, 杨茂光, 等. 糖尿病性勃起功能障碍发病机制研究进展[J]. 中国实验诊断学, 2019, 23(8): 1476-1478. doi: 10.3969/j.issn.1007-4287.2019.08.065 [13] 胡睿, 张威, 欧宁静, 等. 胰岛素治疗对糖尿病大鼠阴茎组织中miR-126、VEGF、eNOS表达的影响[J]. 天津医科大学学报, 2020, 26(5): 422-428. https://www.cnki.com.cn/Article/CJFDTOTAL-TJYK202005007.htm [14] PANNIER D, PHILIPPIN-LAURIDANT G, BARANZELLI M C, et al. High expression levels of egfl7 correlate with low endothelial cell activation in peritumoral vessels of human breast cancer[J]. Oncol Lett, 2016, 12(2): 1422-1428. doi: 10.3892/ol.2016.4791
[15] 提运荣, 肖冬冬, 卢慕峻. 细胞因子治疗勃起功能障碍的研究进展[J]. 中华男科学杂志, 2019, 25(7): 660-663. https://www.cnki.com.cn/Article/CJFDTOTAL-NKXB201907021.htm -
期刊类型引用(1)
1. 曾海涓，孔佳佳，黄艳秋，靳祥云，李程莉，赖媛婷，刘红美. 1例经皮膀胱穿刺造瘘术后尿液外渗继发蜂窝组织炎患者的护理. 中国老年保健医学. 2023(05): 141-144 . 
百度学术
其他类型引用(0)
计量
- 文章访问数: 143
- HTML全文浏览量: 53
- PDF下载量: 10
- 被引次数: 1
苏公网安备 32100302010246号
