实验性精索静脉曲张大鼠生精状态及IL-1测定与分析
摘要
目的：精索静脉曲张(varicocele,VC)是指精索内蔓状静脉丛的异常伸长、扩张和迂曲,其总发病率为10%～15%；在男性原发性不育病人中40%可见精索静脉曲张。本实验通过建立精索静脉曲张大鼠模型,观察大鼠睾丸重量、睾丸结构变化、外周血性激素水平的变化以及工L-1在大鼠睾丸内的表达变化,观察VC对大鼠下丘脑-垂体-性腺轴的影响及对睾丸内免疫反应的变化,进而为VC导致不育机制研究提供新思路,为VC致男性不育的诊断和治疗提供理论依据。
方法：Wistar大鼠40只,雄性,质量170-200g,购于青岛市药检所,适应性饲养1周,实验饲养条件：室温(20±2)℃,每日光照14h,黑暗10h,饮水、摄食任意。将大鼠随机分为实验性精索静脉曲张(Experimental Varicocele EV)8、12周组(EV8、EV12周组,每组12只)和相应的对照组(每组8只).通过建立精索静脉曲张模型,分别于术后8周、12周分别测量各组大鼠：①左侧精索静脉直径、睾丸重量、睾丸曲细精管生精上皮；②外周血中促卵泡刺激素(FSH)、黄体生成素(LH)和睾酮(T)的水平；③蛋白印迹法检测大鼠双侧睾丸中IL-1蛋白的表达变化。
结果：1睾丸结构改变：光镜下观察,各对照组睾丸曲细精管上皮细胞排列紧密有序,各期生精细胞排列有序,管腔内见大量精子。EV8周组大鼠双侧睾丸均有局灶性损害,呈特殊的“斑点样”表现,即病变的与正常的生精小管交错存在,主要表现为生精上皮退行性变,细胞层数减少,排列疏松、紊乱,精子发生停滞,大量生精细胞脱落于管腔中。EV12周组大鼠双侧睾丸局灶性损害灶增加,睾丸生精上皮内生精细胞层次显著减少,有的仅见1～3层,排列松散、紊乱。
2睾丸质量变化：对照组8周左右侧睾丸质量(1.70±0.06,1.75±0.03),EV8周组左右侧睾丸质量(1.58±0.05,1.68±0.06),对照组12周组左右侧睾丸质量(1.74±0.03,1.77±0.03),EV12周组左右侧睾丸质量(1.50±0.06,1.63±0.04)实验组和对照组右侧睾丸比较差异有统计学意义(P<0.05),实验组和对照组左侧睾丸比较差异有显著统计学意义(P<0.01)EV12周双侧睾丸质量较EV8周组减轻(P<0.01,t=3.73：P＜0.05,t=2.55)。
3血性激素水平的变化：EV 8周组和12周组曲张精索静脉血FSH水平与相应对照组肾静脉血比较明显升高(5.56±0.49,6.46±0.45,4.67±0.56,4.67±0.31)差异有显著统计学意义(P＜0.01)；EV 8周组和12周组曲张精索静脉血LH水平与相应对照组肾静脉血比较明显升高(6.29±0.10,8.12±0.87,5.62±0.42,5.57±0.43)差异有显著统计学意义(P＜0.01)；EV12周组曲张精索静脉血FSH、LH水平较EV8周组升高(P<0.01)；EV8周组和EV12周组精索静脉曲张静脉血中T水平较相应对照组明显降低(3.22±0.2、2,3.03±0.25,2.81±0.40,2.63±0.38)差异有显著统计学意义(P＜0.01)；EV12周组精索静脉曲张静脉血中T水平较EV8周组差异无统计学意义。
4 il-1在大鼠双侧睾丸的表达变化：EV8周组EV12周组较相应对照组双侧睾丸IL-1α和IL-1β蛋白表达量增加,且随精索静脉持续时间的延长逐渐增多,组间比较差异有统计学意义(p＜0.05)
结论：(1)VC对大鼠双侧睾丸曲细精管生精上皮产生明显的损害作用。
(2)VC可引起大鼠外周血中FSH、LH水平的增加和T水平的降低,这可能与VC导致睾丸结构的破坏使睾酮分泌减少反射性的引起FSH、LH水平增加有关。
(3)VC可引起大鼠双侧睾丸IL-1α和IL-1β的表达增加,且随VC持续时间的增加而增加,LI-1表达的增加与VC引起睾丸内免疫反应的变化有关。
Objective
To observe the impact of experimental varicocele (EV) on the bilateral testis and the serum level of follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T)and the level of interleukin-1 (IL-1)in rats.
Metrhods
Forty adolescent male rats were involved in this study and were randomly divided into four groups:VC 8-week group (n=12),VC 12-week group (n=12) and the corresponding two sham group (n=8, n=8).The varicocele model was created by partial ligation of the left renal vein.8 or 12 weeks after VC operation,the diameters of left vena spermatica and weight of testis were measured.Observe the seminaferous epithelium of testis under light microscope,and determine the serum levels of FSH, LH and T using radioimmunoassay.The expression of IL-1αand IL-1βin bilateral testis was assessed by Western blot analysis.
Results
The diameters of the left spermatic vein in VC 8-week and VC 12-week group are more broadening than that of control group.Compared with the corresponding control groups, the weight of left testis at VC 8 group and VC12group was significantly lower (P＜0.01;t=11.18、t=4.27 P<0.01,t=3.73)in sequence, so was that of the right testis(P<0.05;t=8.82、t=2.78;t=2.55).The main pathological changes in the testis of the VC rats were degeneration of germinal epithelium,spermatogenesis block,tubular atrophy and androgone desquamation and these lesions were bilatcra land progressive.Compared with control group,the FSH and LH increased, and T decreased significantly (P＜0.01).Compared with control group, the expression of il-1αand il-1βin bilateral testis tissue increased upon progression of varicocele.
Conclusion EV can decrease the weight of the bilateral testis and cause a progressive pathological change of seminiferous tubule;the increased serum level of FSH and LH and decreased T in VC group suggest that EV can induce endocrine disturbance.The increased expression of IL-1αand IL-1βin varicocele shifts the balance in favor of inflammatory and immune responses and causes detrimental effects in testis,which may cause male infertility.
方法：Wistar大鼠40只,雄性,质量170-200g,购于青岛市药检所,适应性饲养1周,实验饲养条件：室温(20±2)℃,每日光照14h,黑暗10h,饮水、摄食任意。将大鼠随机分为实验性精索静脉曲张(Experimental Varicocele EV)8、12周组(EV8、EV12周组,每组12只)和相应的对照组(每组8只).通过建立精索静脉曲张模型,分别于术后8周、12周分别测量各组大鼠：①左侧精索静脉直径、睾丸重量、睾丸曲细精管生精上皮；②外周血中促卵泡刺激素(FSH)、黄体生成素(LH)和睾酮(T)的水平；③蛋白印迹法检测大鼠双侧睾丸中IL-1蛋白的表达变化。
结果：1睾丸结构改变：光镜下观察,各对照组睾丸曲细精管上皮细胞排列紧密有序,各期生精细胞排列有序,管腔内见大量精子。EV8周组大鼠双侧睾丸均有局灶性损害,呈特殊的“斑点样”表现,即病变的与正常的生精小管交错存在,主要表现为生精上皮退行性变,细胞层数减少,排列疏松、紊乱,精子发生停滞,大量生精细胞脱落于管腔中。EV12周组大鼠双侧睾丸局灶性损害灶增加,睾丸生精上皮内生精细胞层次显著减少,有的仅见1～3层,排列松散、紊乱。
2睾丸质量变化：对照组8周左右侧睾丸质量(1.70±0.06,1.75±0.03),EV8周组左右侧睾丸质量(1.58±0.05,1.68±0.06),对照组12周组左右侧睾丸质量(1.74±0.03,1.77±0.03),EV12周组左右侧睾丸质量(1.50±0.06,1.63±0.04)实验组和对照组右侧睾丸比较差异有统计学意义(P<0.05),实验组和对照组左侧睾丸比较差异有显著统计学意义(P<0.01)EV12周双侧睾丸质量较EV8周组减轻(P<0.01,t=3.73：P＜0.05,t=2.55)。
3血性激素水平的变化：EV 8周组和12周组曲张精索静脉血FSH水平与相应对照组肾静脉血比较明显升高(5.56±0.49,6.46±0.45,4.67±0.56,4.67±0.31)差异有显著统计学意义(P＜0.01)；EV 8周组和12周组曲张精索静脉血LH水平与相应对照组肾静脉血比较明显升高(6.29±0.10,8.12±0.87,5.62±0.42,5.57±0.43)差异有显著统计学意义(P＜0.01)；EV12周组曲张精索静脉血FSH、LH水平较EV8周组升高(P<0.01)；EV8周组和EV12周组精索静脉曲张静脉血中T水平较相应对照组明显降低(3.22±0.2、2,3.03±0.25,2.81±0.40,2.63±0.38)差异有显著统计学意义(P＜0.01)；EV12周组精索静脉曲张静脉血中T水平较EV8周组差异无统计学意义。
4 il-1在大鼠双侧睾丸的表达变化：EV8周组EV12周组较相应对照组双侧睾丸IL-1α和IL-1β蛋白表达量增加,且随精索静脉持续时间的延长逐渐增多,组间比较差异有统计学意义(p＜0.05)
结论：(1)VC对大鼠双侧睾丸曲细精管生精上皮产生明显的损害作用。
(2)VC可引起大鼠外周血中FSH、LH水平的增加和T水平的降低,这可能与VC导致睾丸结构的破坏使睾酮分泌减少反射性的引起FSH、LH水平增加有关。
(3)VC可引起大鼠双侧睾丸IL-1α和IL-1β的表达增加,且随VC持续时间的增加而增加,LI-1表达的增加与VC引起睾丸内免疫反应的变化有关。
Objective
To observe the impact of experimental varicocele (EV) on the bilateral testis and the serum level of follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T)and the level of interleukin-1 (IL-1)in rats.
Metrhods
Forty adolescent male rats were involved in this study and were randomly divided into four groups:VC 8-week group (n=12),VC 12-week group (n=12) and the corresponding two sham group (n=8, n=8).The varicocele model was created by partial ligation of the left renal vein.8 or 12 weeks after VC operation,the diameters of left vena spermatica and weight of testis were measured.Observe the seminaferous epithelium of testis under light microscope,and determine the serum levels of FSH, LH and T using radioimmunoassay.The expression of IL-1αand IL-1βin bilateral testis was assessed by Western blot analysis.
Results
The diameters of the left spermatic vein in VC 8-week and VC 12-week group are more broadening than that of control group.Compared with the corresponding control groups, the weight of left testis at VC 8 group and VC12group was significantly lower (P＜0.01;t=11.18、t=4.27 P<0.01,t=3.73)in sequence, so was that of the right testis(P<0.05;t=8.82、t=2.78;t=2.55).The main pathological changes in the testis of the VC rats were degeneration of germinal epithelium,spermatogenesis block,tubular atrophy and androgone desquamation and these lesions were bilatcra land progressive.Compared with control group,the FSH and LH increased, and T decreased significantly (P＜0.01).Compared with control group, the expression of il-1αand il-1βin bilateral testis tissue increased upon progression of varicocele.
Conclusion EV can decrease the weight of the bilateral testis and cause a progressive pathological change of seminiferous tubule;the increased serum level of FSH and LH and decreased T in VC group suggest that EV can induce endocrine disturbance.The increased expression of IL-1αand IL-1βin varicocele shifts the balance in favor of inflammatory and immune responses and causes detrimental effects in testis,which may cause male infertility.
引文
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