【摘要】目的探讨雷公藤及铅、镉等重金属元素对睾丸间质细胞一氧化氮合酶(NOS)活性的影响。方法用NADPH-d黄递酶组织化学方法，观察给予氯化镉、醋酸铅、雷公藤1、2、3、4周后，小鼠睾丸间质NOS活性的变化。结果正常组间质细胞呈NOS强阳性反应，而曲精小管生精细胞均呈NOS阴性反应。给予雷公藤2周内NOS反应无变化，第3和第4周后，NOS阳性的睾丸间质细胞数量无明显变化，但NOS活性稍降低。给予醋酸铅和氯化镉1周后，NOS阳性的睾丸间质细胞数量明显减少，并随给药时间延长而加重，NOS活性逐渐降低，给药4周后NOS阳性的睾丸间质细胞几乎不着色。结论雷公藤虽然具有明显抗生育作用，但对NOS阳性的睾丸间质细胞数量及NOS活性无明显影响；而铅、镉等对睾丸有毒理作用的重金属元素，不但可使NOS阳性的睾丸间质细胞减少，NOS活性也明显降低。探讨雷公藤及铅、镉等重金属元素对睾丸间质细胞一氧化氮合酶(NOS)活性的影响。方法用NADPH-d黄递酶组织化学方法，观察给予氯化镉、醋酸铅、雷公藤1、2、3、4周后，小鼠睾丸间质NOS活性的变化。结果正常组间质细胞呈NOS强阳性反应，而曲精小管生精细胞均呈NOS阴性反应。给予雷公藤2周内NOS反应无变化，第3和第4周后，NOS阳性的睾丸间质细胞数量无明显变化，但NOS活性稍降低。给予醋酸铅和氯化镉1周后，NOS阳性的睾丸间质细胞数量明显减少，并随给药时间延长而加重，NOS活性逐渐降低，给药4周后NOS阳性的睾丸间质细胞几乎不着色。结论雷公藤虽然具有明显抗生育作用，但对NOS阳性的睾丸间质细胞数量及NOS活性无明显影响；而铅、镉等对睾丸有毒理作用的重金属元素，不但可使NOS阳性的睾丸间质细胞减少，NOS活性也明显降低。

【中图分类号】R329.4R322.6⁺4【文献标识码】A

【文章编号】0529-1356(2000)02-183

EFFECTS OF TRIPTERYGIUM WILFORDII F, LEAD AND CADMIUM ON NOS ACTIVITY OF MICE TESTIS INTERSTITIAL CELLS

MA Wen-ling,ZHANG Feng,ZHANG Chao-xin,ZHANG Xing-he,FAN Yu-bing

（Department of Anatomy, Bethune Military Medical College, Shijiazhuang 050081,China;）

LIU Wei，SHAO Xin-xi

（Institute of Military Medicine of Beijing Military Area,Shijiazhuang 050081,China）

【Abstract】Objective To explain the effects of nitric oxide (NO) on testis interstitial cells (IC) after administration of tripterygium wilfordii f.(TWF), lead acetate and cadmium chloride.Methods Using NADPH-d histochemistry to investigate activity change of NOS in the testis of mice, which were survived for 1,2,3,4 weeks after administration of TWF, lead acetate and cadmium chloride.Results The interstitial cells (IC) of the testis were strong NOS-activity in the normal group, spermatogenic cells were all NOS-negative. Two weeks after administration of TWF, The NOS-activity had no change, during 3rd and 4th weeks NOS-positive IC had not obvious changes in number, but NOS activity was a bit lower. One week after administration of lead acetate and cadmium chloride, the numbers of NOS-positive IC reduced significantly. With prolonging administration of such metal salts NOS activity gradually reduced. In 4 weeks after administration of the such metal salts, there were rarely seen NOS-positive IC, and they were almost not stained.Conclusions TWF may have remarkable antifertility whereas the numbers and the activity of NOS of IC are not affected by TWF. In contrast, cadmium and lead were harmful to the testis. They not only reduce the numbers of IC, but also interfere NOS activity significantly.

【Key words】 NO; Testis; Interstitial cell; Tripterygium wilfordii f; Infertility; NADPH-diaphorase histochemistry; Mouse

一氧化氮(nitric oxide, NO)广泛存在于人体的心血管系统、神经系统、消化系统和免疫系统，既兼有第二信使和神经递质的性质，又是效应分子，具有复杂的生物学效应。近年来证明雄性生殖管道^［1］和生殖腺^［2］均存在一氧化氮合酶(nitric oxide synthase, NOS)纤维和细胞，并证明雄性生殖系统中的NO与阴茎勃起、睾丸微循环的调节、雄激素的分泌、精子的成熟、运动及获能均有密切关系^［3］。睾丸内NOS存在于间质细胞内^［4］，但有关睾丸间质细胞所产生的NO作用尚不十分明确，也未见到有关雄性不育模型睾丸内NOS阳性细胞变化的报道。本研究用新型抗雄性生育药物雷公藤(tripterygium wilfordii f, TWF)制备雄性不育模型，与铅和镉所造成的睾丸损伤模型相比较，观察了雄性小鼠睾丸内NOS阳性的间质细胞数量和NOS活性变化，以探讨NO在雷公藤抗生育和铅、镉对睾丸毒理损伤中所发挥的作用。

材料和方法

1.动物分组及模型的制备

取雄性昆明种小鼠48只，分为对照组、雷公藤组、醋酸铅组和氯化镉组，每组各12只。分别用双蒸水0.25ml/d、雷公藤多甙片40mg/(kg·d)(湖南株洲市制药三厂出品，批号970601)，醋酸铅1000mg/(kg.d)(A.R,温洲市化学用料厂，批号950803)和氯化镉7mg/(kg.d)(A.R，亭新化工试剂厂，批号970201)于早9时灌胃，连用7d，从第2周开始醋酸铅用量减半，其余各组维持原剂量不变，连用6d，停药1d，共给药4周。于1～4周末每组各灌注固定3只小鼠。

2.组织切片制备及NADPH-d组织化学反应

每周末每组动物各取3只，用4%水合氯醛(60mg/kg)腹膜腔麻醉，仰卧位开胸，经左心室推注0.9%氯化钠10ml，缓慢推注4%多聚甲醛50ml，立即取双侧睾丸，固定于上述固定液8h后(4℃)，移入15%蔗糖1h，30%蔗糖过夜(4℃)，取睾丸经液氮速冻后冰冻冠状切片，片厚30μm，裱于涂铬矾明胶载片上，应用改进NADPH-d黄递酶组织化学方法进行NOS组织化学反应。步骤如下：用含1%Triton X-100 0.1mol/L磷酸缓冲液孵育30min，用含0.25%NBT和0.5%NADPH、0.1mol/L磷酸缓冲液37℃反应3h，室温条件下反应2h，1%Triton X-100 0.1mol/L磷酸缓冲液稳定30min，用0.1mol/L磷酸缓冲液冲洗终止反应，常规脱水、透明、封片、光镜观察。

结果和讨论

对照组小鼠睾丸内NOS阳性细胞密集分布于曲精小管之间的间质内。而曲精小管壁的生精细胞均呈NOS阴性反应，阳性细胞为卵圆型，呈强阳性反应，胞浆被染成深蓝色，直径约18μm(图1)。雷公藤组用药1周后，NOS阳性睾丸间质细胞的数量和着色程度与对照组相比无明显变化，用药2周后NOS着色稍变淡，用药第3、4周末，染色变淡的同时，细胞数量略有减少(图2)。醋酸铅组在用药1周后NOS细胞活性无变化，但NOS阳性细胞的数量减少，2周末数量减少且染色变淡，第3周末和第4周末NOS细胞数量明显减少，曲精小管之间仅留数个，甚至完全消失，细胞着色变淡(图3)。氯化镉组在用药1周后NOS阳性的睾丸间质细胞数量减少，染色无明显变化，2周后NOS细胞明显减少，染色变淡，第3、4周后染色很淡，数量很少，相邻曲精小管间仅存数个细胞(图4)。

1NOS阳性细胞密集分布于睾丸间质，呈卵圆形，直径约18μm，胞核较大，曲精小管壁细胞均呈NOS阴性反应×100

2应用雷公藤4周后，NOS阳性睾丸间质细胞数量略减少，染色稍变淡×100

3应用醋酸铅4周后，NOS阳性睾丸间质细胞数量明显减少，染色很淡×100

4应用氯化镉4周后，NOS阳性睾丸间质细胞数量明显减少，染色很淡×100

Fig.1 NOS-positive cells distributed in the interstitium of the testis, this cells were oval in shape, the diameter of them were averaged about 18μm and their nuclei were large. But the cells of seminiferous tubule were NOS-negative. ×100

Fig.2 The numbers of NOS-positive interstitial cells decreased slightly and they were stained faintly 4 week after administration of TWF. ×100

Fig.3 The numbers of NOS-positive interstitial cells decreased significantly and the stained were much light 4 week after administration of lead acetate. ×100

Fig.4 The numbers of NOS-positive interstit