Objective: To investigate the protective effect of human urine-derived stem cells (USCs) on erectile function and cavernous structure in rats with cavernous nerve injury (CNI).
Methods: Sixty adult male SD rats with normal sexual function were randomly divided into four groups of equal number: sham operation, bilateral CNI (BCNI) model control, phosphate buffered saline (PBS), and USC. The BCNI model was established in the latter three groups of rats by clamping the bilateral cavernous nerves. After modeling, the rats in the PBS and USC groups were treated by intracavernous injection of PBS at 200 μl and USCs at 1×106/200 μl PBS respectively for 28 days. Then, the maximum intracavernous pressure (mICP) and the ratio of mICP to mean arterial pressure (mICP/MAP) of the rats were calculated by electrical stimulation of the major pelvic ganglions, the proportion of nNOS- or NF200-positive nerve fibers in the total area of penile dorsal nerves determined by immunohistochemical staining, the levels of endothelial cell marker eNOS, smooth muscle marker α-SMA and collagen I detected by Western blot, and the smooth muscle to collagen ratio and the cell apoptosis rate in the corpus cavernosum measured by Masson staining and TUNEL, respectively.
Results: After 28 days of treatment, the rats in the USC group, as compared with those in the PBS and BCNI model control groups, showed significant increases in the mICP (［81 ± 9.9］ vs ［31 ± 8.3］ and ［33 ± 4.2］ mmHg, P <0.05), mICP/MAP ratio (0.72 ± 0.05 vs 0.36 ± 0.03 and 0.35 ± 0.04, P <0.05), the proportions of nNOS-positive nerve fibers (［11.31 ± 4.22］% vs ［6.86 ± 3.08］% and ［7.29 ± 4.84］% , P <0.05) and NF200-positive nerve fibers in the total area of penile dorsal nerves (［27.31 ± 3.12］% vs ［17.38 ± 2.87］% and ［19.49 ± 4.92］%, P <0.05), the eNOS/GAPDH ratio (0.52 ± 0.08 vs 0.31 ± 0.06 and 0.33 ± 0.07, P <0.05), and the α-SMA/GAPDH ratio (1.01 ± 0.09 vs 0.36 ± 0.05 and 0.38 ± 0.04, P <0.05), but a remarkable decrease in the collagen I/GAPDH ratio (0.28 ± 0.06 vs 0.68 ± 0.04 and 0.70 ± 0.10, P <0.05). The ratio of smooth muscle to collagen in the corpus cavernosum was significantly higher in the USC than in the PBS and BCNI model control groups (17.91 ± 2.86 vs 7.70 ± 3.12 and 8.21 ± 3.83, P <0.05) while the rate of cell apoptosis markedly lower in the former than in the latter two (3.31 ± 0.83 vs 9.82 ± 0.76, P <0.01; 3.31 ± 0.83 vs 9.75 ± 0.91, P <0.05).
Conclusions: Intracavernous injection of USCs can protect the erectile function of the rat with cavernous nerve injury by protecting the nerves, improving the endothelial function, alleviating fibrosis and inhibiting cell apoptosis in the cavernous tissue.
目的： 探讨尿源干细胞（USC）对海绵体神经损伤性勃起功能障碍（CNIED）大鼠勃起功能和阴茎海绵体组织结构的保护作用。方法： 60只成年雄性SD大鼠随机平均分为4组（n=15只/组）：假手术组、双侧海绵体神经钳夹损伤组（BCNI组）、PBS组、USC组。假手术组予暴露双侧海绵体神经后直接关闭手术切口，其余3组均予血予血管钳钳夹双侧海绵体神经1 min，建立CNIED模型；PBS组和USC组分别予阴茎海绵体注射 PBS（200 μl）或USC（1×106 细胞/ 200 μl PBS）。治疗28 d后测定大鼠的最大海绵体内压（mICP）和mICP/平均动脉压（mICP/MAP），并通过Western印迹检测海绵窦内皮细胞标志物NOS，平滑肌标志物α-SMA，以及Collagen I，通过免疫组化检测海绵体阴茎背神经内的神经标志物（nNOS、NF-200），Masson染色检测海绵体平滑肌/胶原比值，以及TUNEL染色检测海绵窦内细胞凋亡水平。 结果： 治疗28 d后，USC组大鼠的mICP及mICP/MAP均较PBS组［（81±9.9) mmHg vs (31±8.3) mmHg，0.72±0.05 vs 0.36±0.03，P<0.05］和BCNI组 ［(81±9.9) mmHg vs (33±4.2) mmHg，0.72±0.05 vs 0.35±0.04，P<0.05］ 显著升高。免疫组化结果显示：USC组大鼠背神经内nNOS、NF-200阳性神经纤维面积的比例(%)均较PBS组(11.31±4.22 vs 6.86±3.08，27.31±3.12 vs 17.38±2.87，P<0.05)和BCNI组 (11.31±4.22 vs 7.29±4.84，27.31±3.12 vs 19.49±4.92，P<0.05) 显著增加；Western印迹检测结果显 示：USC组大鼠eNOS/GAPDH比值较PBS组（0.52±0.08 vs 0.31±0.06，P<0.05）和BCNI组（0.52±0.08 vs 0.33±0.07，P<0.05）均显著提高，α-SMA含量亦较PBS组（1.01±0.09 vs 0.36±0.05，P<0.05）和BCNI组（1.01±0.09 vs 0.38±0.04，P<0.05）显著提高，而Collagen I含量较PBS组（0.28±0.06 vs 0.68±0.04，P<0.05）和BCNI组（0.28±0.06 vs 0.70±0.10，P<0.05）显著降低;且Masson染色结果示USC组大鼠阴茎平滑肌/胶原比值(%)亦较PBS组（17.91±2.86 vs 7.70±3.12，P<0.05）和BCNI组（17.91±2.86 vs 8.21±3.83，P<0.05）显著升高。TUNEL染色显示USC组大鼠海绵窦内细胞的凋亡指数（%）较PBS组（3.31±0.83 vs 9.82±0.76，P<0.01）和BCNI组（3.31±0.83 vs 9.75±0.91，P<0.05）显著降低。 结论： USC可以通过保护神经、改善海绵窦内皮功能和海绵体纤维化，抑制细胞凋亡，显著保护CNIED大鼠的勃起功能。.
Keywords: cavernous nerve injury; erectile dysfunction; rat model; urine-derived stem cell.