HPM辐照对大鼠生殖系统的影响
摘要
目的：
探讨HPM对大鼠生殖系统的辐照效应与辐射剂量的关系以及HPM辐照引起生殖细胞凋亡的规律。并在蛋白水平探讨HPM辐照引起生殖细胞凋亡的机制。
方法：
1 采用S波段不同平均功率密度(10mW／cm~2、20mW／cm~2、40mW／cm~2、80mW／cm~2)HPM对固定放置的大鼠进行全身照射，照射时间分别为1min、5min、10min和20min，建立动物模型。照射后分别于6h、24h、48h、72h、5d取材，分别经4％多聚甲醛和3％戊二醛固定，制取睾丸／卵巢组织石蜡标本和透射电镜标本。
2 采用光学显微镜和透射电镜分别进行睾丸／卵巢组织形态学和超微结构观察；
3 应用DNA缺口原位末端标记技术(TUNEL法)检测睾丸／卵巢组织凋亡细胞；
4 采用免疫组织化学S-P法检测睾丸／卵巢组织中Bcl-2及C-myc蛋白的表达。
结果：
1 光镜下雄性大鼠受HPM照射后，实验组与对照组相比，
第四军医大学硕士学位论文
20mw/c mZ、smin组开始出现轻度炎细胞浸润、组织充血，水肿;
IOmin组出现曲精小管生精上皮细胞排列紊乱，畸形精子增多。
Zomin组和40 mw/emZ、lmin组可见大鼠梁丸部分曲精小管生精细
胞明显坏死脱落。上述改变随平均功率密度增加，照射时间延长而加
重。40mw/c mZ、smin组部分曲精小管内几乎无细胞，损伤达坏死
极限;透射电镜下翠丸超微结构发生轻度线粒体水肿、内质网扩张
是从Zomw/emZ、smin组开始，lomin组可见精原细胞水肿变性，
发生凋亡，线粒体呈空泡化;20min组毛细血管内皮细胞肿胀，基
底膜分层，断裂。功率增高后的各组均可见部分间质细胞发生变性、
凋亡和坏死崩解。对照组无改变。
Zlomw/emZ、Zomw/emZ辐照雄性大鼠smin，6h组、24h组和
48h组，大鼠肇丸凋亡生精细胞数量明显高于对照组(P<0.01)，以
lomw/emZ、smin、6h组凋亡细胞数量最多，达。61.27士5.9)个/5
个精曲小管。72h组、sd组及1 omw/em，、Zomw/emZ辐照10min
各组与对照组间差别无显著性意义(P>0.05)。
3lomw/emZ、ZomwzemZ辐照雄性大鼠smin、lomin，24h组，
Bel一2和e一my。蛋白染色阳性，表现为(+++)。lomwzem，、lomin，
4sh组，Bel一2蛋白染色阳性为(++)，C一mye蛋白染色(一)，
ZomwzemZ、smin，6h组，Bel一2和e一myc蛋白染色阳性分别为(+
+)和(+);其余实验组Bcl一2蛋白和C一myc蛋白均无表达。
4光镜下雌性大鼠受HPM照射后，实验组与对照组相比，
40mw/c mZ、smin组开始出现组织水肿、充血和炎细胞浸润，
4omw/emZ、10min组开始见卵巢出现损伤，卵泡细胞分离明显，
40mw/emZ、Zomin组及somw/emZ、lmin组、smin组和10min组
出现卵泡内层细胞变性，发育不同阶段的卵泡发生卵泡闭锁。黄体
细胞发生凋亡。20min组则出现组织出血和部分粒层细胞、间质细
胞出现明显坏死崩解。随照射功率密度增加照射时间延长上述病理
变化越明显。透射电镜下超微结构发生轻度线粒体水肿、内质网扩
张，间质内炎细胞浸润是从40mw/c mZ、smin组开始，随照射时间
延长，功率密度增加，各组均可见生殖细胞发生变性、凋亡改变;
第四军医大学硕士学位论文
80mw/c mZ、20min组可见卵母细胞内次级溶酶体增多，微绒毛发
生肿胀。间质细胞凋亡、坏死。Zomw/emZ各组、40mw/emZ、lmin
组及对照组无改变。
s4OmW/emZ的HPM辐照雌性大鼠zomin，6h组、24h组、48h
组sd组凋亡细胞数目明显升高，与对照组相比差别具有非常显著
性意义(P<0.01)。其余实验组与对照组相比差别无意义(P>.05)。
6 HPM辐照大鼠后各24h实验组，Bcl一2和C一my。蛋白染色
呈(++一+++)，4omwzemZ、smin，6h组，Bel一2蛋白染色阳
性为(++)，C一my。蛋白染色(+)，其余实验组二者均无表达。
结论:
1 HPM辐照可导致大鼠皋丸组织和皋丸组织形态发生明显的
病理性改变。对辜丸组织，HPM辐照Zomw/emZ、Zomin或
4omwzemZ、lmin，卵巢组织达到somwzcmZ、lomin，即可对生殖
细胞有致死作用。在相同的辐照条件下，卵巢组织对辐射的耐受能
力强于攀丸组织。
2在一定的辐照剂量与辐照时间范围内，HPM可诱导大鼠辜丸
/卵巢组织发生凋亡，并和辐照后取材时间有关。10mw/c mZ、
20mw/cmZHPM辐照雄鼠smin，40mw/cmZHPM辐照雌鼠10min
即可触发生殖细胞凋亡数目增加，影响大鼠生殖功能。
3不同功率密度的HPM辐照雌鼠和雄鼠，照后24小时，c一myc
及bcl一2蛋白的活化和表达增加，可能是HPM诱导大鼠生殖细胞
凋亡的机制之一。
4在本实验条件下，HPM对大鼠的甚至系统造成损伤存在着
剂量效应关系。HPM的辐照时间和辐照的功率密度存在协同作用
(S”etgism aetion)，导致大鼠生殖细胞的损伤。
本实验的结果为环境和职业中的微波辐射安全防护标准的制
定，有关危害机制的解释提供了生物学研究资料。
Objective:
To study the relationship between the effect of the radiation of HPM and the time of radiation or average power density of HPM, and the regulation of the apoptosis caused by the HPM radiation. Further more, To discuss the mechanism of the apoptosis of the germ cell induced by the HPM radiation at the protein level.
Methods:
1 The fixed experimental rats were radiated with S wave band 10 mW/cm2, 20mW/cm2, 40mW/cm2, 80mW/cm2 HPM for lminute,5minutes, lOminutes and 20 minutes respectively or sham treatment to build the model of the animal. Testicular or ovarian samples were taken 6h, 24h, 48h, 72h and 5d after radiation , separately fixed with 4% buffered formaldehyde or 3% buffered glutaraldehyde and embedded in paraffin or tetroxide osmate.
2 The morphology and the ultrastructure of the testis or ovary were observed by light microscope or transmission electron microscope respectively.
3 Testicular or ovarian germ cell apoptosis was detected by in situ terminal deoxymudeotityl transferase mediated dUTP nick end labeling
(TUNEL).
4 At the end of the process, immunohistochemical S-P staining was performed to test the expressions of Bcl-2 protein and C-myc protein in germ cell of the testis or ovary in rats. Results:
1 With light microscope, vascular congestion and interstitial edema , slightly inflamatory cell infiltration were observed in the 20 mW/cm2, 5 min group , in addition the germ cells of germinal epithelium irregulation and deformation in the spermatogenesis were also observed in the 20 mW/cm2,10 minutes experimental groups; Partly convoluted seminiferoustub -ules androgone necroses were also observed in 20mW/cm2,20min and 40mW/cm2,lmin group. In 40mW/cm2,5min group, the deadly damage reached the limit, there were nealy no cells in partly convoluted seminiferous tubules. With the transmission electron microscope, slight mitochondrion edema and endoplasm reticulum dilation, spermatogonium degeneration were observed in the 20mW/cm , 5 min group; mitochondrion vacuolation were found in 20mW/cm ,10 minutes experimental groups; a swollen spermato -gonia with rarefaction of nuclear chromosome and obscurity of mitochondria structure with reduplication of splitting of basal membrane were observed in the 20 mW/cm2, 20min, Interstitial cells degeneration, apoptosis and necrosis were also found . But no change was found in contral group.
2 From 6h experimental groups , the number of apoptosis cells was remarkably larger than the control's till 48h after radiation(P<0.01)in 10 mW/cm2,5min groups and 20 mW/cm2,5min groups. Especially the 10 mW/cm2 ,5min,6 h experimental group reaching the peak (161.27 + 5.9) IS convoluted seminiferous tubules. The change of the other experimental groups was the same as that of the control (P>0.05).
3 The expression of the Bcl-2 protein and C-myc protein were (+ + +)degree in 24 groups after radiation by 10 mW/cm2,20 mW/cm2 HPM for 5min and 10min.In addition, the expression of the Bcl-2 protein was (+ + ) degree and that of the C-myc protein was( - )degree in 10mW/cm2,10min,48h experimental groups; The expression of Bcl-2 protein was ( + + ) degree and
that of C-myc protein was ( + ) degree in 20mW/cm2,5min, 6h experimental groups . No change was found in contral group and the other experimental groups.
4 With light microscope, vascular congestion , interstitial edema and slight inflamatory cell infiltration were observed in the 40 mW/cm2,5 min group, in addition granular cells irregulation were also observed in follicle in the 40mW/cm2,10 minutes experimental groups; granular cells degeneration, lutein cell apoptosis and unilunar follicle, primary follicle and secondary follicle atresia were also observed in 40mW/cm2,20min and 80mW/cm2,lmin group, 5min groups and 10min groups. In 80 mW/cm2,20min group, partly granular cells, Interstitial cells necrosis and tissue haemorrhage were found. With the transmission electron microscope, slight mitochondrion edema and endoplasm reticulum dilation were observed in
探讨HPM对大鼠生殖系统的辐照效应与辐射剂量的关系以及HPM辐照引起生殖细胞凋亡的规律。并在蛋白水平探讨HPM辐照引起生殖细胞凋亡的机制。
方法：
1 采用S波段不同平均功率密度(10mW／cm~2、20mW／cm~2、40mW／cm~2、80mW／cm~2)HPM对固定放置的大鼠进行全身照射，照射时间分别为1min、5min、10min和20min，建立动物模型。照射后分别于6h、24h、48h、72h、5d取材，分别经4％多聚甲醛和3％戊二醛固定，制取睾丸／卵巢组织石蜡标本和透射电镜标本。
2 采用光学显微镜和透射电镜分别进行睾丸／卵巢组织形态学和超微结构观察；
3 应用DNA缺口原位末端标记技术(TUNEL法)检测睾丸／卵巢组织凋亡细胞；
4 采用免疫组织化学S-P法检测睾丸／卵巢组织中Bcl-2及C-myc蛋白的表达。
结果：
1 光镜下雄性大鼠受HPM照射后，实验组与对照组相比，
第四军医大学硕士学位论文
20mw/c mZ、smin组开始出现轻度炎细胞浸润、组织充血，水肿;
IOmin组出现曲精小管生精上皮细胞排列紊乱，畸形精子增多。
Zomin组和40 mw/emZ、lmin组可见大鼠梁丸部分曲精小管生精细
胞明显坏死脱落。上述改变随平均功率密度增加，照射时间延长而加
重。40mw/c mZ、smin组部分曲精小管内几乎无细胞，损伤达坏死
极限;透射电镜下翠丸超微结构发生轻度线粒体水肿、内质网扩张
是从Zomw/emZ、smin组开始，lomin组可见精原细胞水肿变性，
发生凋亡，线粒体呈空泡化;20min组毛细血管内皮细胞肿胀，基
底膜分层，断裂。功率增高后的各组均可见部分间质细胞发生变性、
凋亡和坏死崩解。对照组无改变。
Zlomw/emZ、Zomw/emZ辐照雄性大鼠smin，6h组、24h组和
48h组，大鼠肇丸凋亡生精细胞数量明显高于对照组(P<0.01)，以
lomw/emZ、smin、6h组凋亡细胞数量最多，达。61.27士5.9)个/5
个精曲小管。72h组、sd组及1 omw/em，、Zomw/emZ辐照10min
各组与对照组间差别无显著性意义(P>0.05)。
3lomw/emZ、ZomwzemZ辐照雄性大鼠smin、lomin，24h组，
Bel一2和e一my。蛋白染色阳性，表现为(+++)。lomwzem，、lomin，
4sh组，Bel一2蛋白染色阳性为(++)，C一mye蛋白染色(一)，
ZomwzemZ、smin，6h组，Bel一2和e一myc蛋白染色阳性分别为(+
+)和(+);其余实验组Bcl一2蛋白和C一myc蛋白均无表达。
4光镜下雌性大鼠受HPM照射后，实验组与对照组相比，
40mw/c mZ、smin组开始出现组织水肿、充血和炎细胞浸润，
4omw/emZ、10min组开始见卵巢出现损伤，卵泡细胞分离明显，
40mw/emZ、Zomin组及somw/emZ、lmin组、smin组和10min组
出现卵泡内层细胞变性，发育不同阶段的卵泡发生卵泡闭锁。黄体
细胞发生凋亡。20min组则出现组织出血和部分粒层细胞、间质细
胞出现明显坏死崩解。随照射功率密度增加照射时间延长上述病理
变化越明显。透射电镜下超微结构发生轻度线粒体水肿、内质网扩
张，间质内炎细胞浸润是从40mw/c mZ、smin组开始，随照射时间
延长，功率密度增加，各组均可见生殖细胞发生变性、凋亡改变;
第四军医大学硕士学位论文
80mw/c mZ、20min组可见卵母细胞内次级溶酶体增多，微绒毛发
生肿胀。间质细胞凋亡、坏死。Zomw/emZ各组、40mw/emZ、lmin
组及对照组无改变。
s4OmW/emZ的HPM辐照雌性大鼠zomin，6h组、24h组、48h
组sd组凋亡细胞数目明显升高，与对照组相比差别具有非常显著
性意义(P<0.01)。其余实验组与对照组相比差别无意义(P>.05)。
6 HPM辐照大鼠后各24h实验组，Bcl一2和C一my。蛋白染色
呈(++一+++)，4omwzemZ、smin，6h组，Bel一2蛋白染色阳
性为(++)，C一my。蛋白染色(+)，其余实验组二者均无表达。
结论:
1 HPM辐照可导致大鼠皋丸组织和皋丸组织形态发生明显的
病理性改变。对辜丸组织，HPM辐照Zomw/emZ、Zomin或
4omwzemZ、lmin，卵巢组织达到somwzcmZ、lomin，即可对生殖
细胞有致死作用。在相同的辐照条件下，卵巢组织对辐射的耐受能
力强于攀丸组织。
2在一定的辐照剂量与辐照时间范围内，HPM可诱导大鼠辜丸
/卵巢组织发生凋亡，并和辐照后取材时间有关。10mw/c mZ、
20mw/cmZHPM辐照雄鼠smin，40mw/cmZHPM辐照雌鼠10min
即可触发生殖细胞凋亡数目增加，影响大鼠生殖功能。
3不同功率密度的HPM辐照雌鼠和雄鼠，照后24小时，c一myc
及bcl一2蛋白的活化和表达增加，可能是HPM诱导大鼠生殖细胞
凋亡的机制之一。
4在本实验条件下，HPM对大鼠的甚至系统造成损伤存在着
剂量效应关系。HPM的辐照时间和辐照的功率密度存在协同作用
(S”etgism aetion)，导致大鼠生殖细胞的损伤。
本实验的结果为环境和职业中的微波辐射安全防护标准的制
定，有关危害机制的解释提供了生物学研究资料。
Objective:
To study the relationship between the effect of the radiation of HPM and the time of radiation or average power density of HPM, and the regulation of the apoptosis caused by the HPM radiation. Further more, To discuss the mechanism of the apoptosis of the germ cell induced by the HPM radiation at the protein level.
Methods:
1 The fixed experimental rats were radiated with S wave band 10 mW/cm2, 20mW/cm2, 40mW/cm2, 80mW/cm2 HPM for lminute,5minutes, lOminutes and 20 minutes respectively or sham treatment to build the model of the animal. Testicular or ovarian samples were taken 6h, 24h, 48h, 72h and 5d after radiation , separately fixed with 4% buffered formaldehyde or 3% buffered glutaraldehyde and embedded in paraffin or tetroxide osmate.
2 The morphology and the ultrastructure of the testis or ovary were observed by light microscope or transmission electron microscope respectively.
3 Testicular or ovarian germ cell apoptosis was detected by in situ terminal deoxymudeotityl transferase mediated dUTP nick end labeling
(TUNEL).
4 At the end of the process, immunohistochemical S-P staining was performed to test the expressions of Bcl-2 protein and C-myc protein in germ cell of the testis or ovary in rats. Results:
1 With light microscope, vascular congestion and interstitial edema , slightly inflamatory cell infiltration were observed in the 20 mW/cm2, 5 min group , in addition the germ cells of germinal epithelium irregulation and deformation in the spermatogenesis were also observed in the 20 mW/cm2,10 minutes experimental groups; Partly convoluted seminiferoustub -ules androgone necroses were also observed in 20mW/cm2,20min and 40mW/cm2,lmin group. In 40mW/cm2,5min group, the deadly damage reached the limit, there were nealy no cells in partly convoluted seminiferous tubules. With the transmission electron microscope, slight mitochondrion edema and endoplasm reticulum dilation, spermatogonium degeneration were observed in the 20mW/cm , 5 min group; mitochondrion vacuolation were found in 20mW/cm ,10 minutes experimental groups; a swollen spermato -gonia with rarefaction of nuclear chromosome and obscurity of mitochondria structure with reduplication of splitting of basal membrane were observed in the 20 mW/cm2, 20min, Interstitial cells degeneration, apoptosis and necrosis were also found . But no change was found in contral group.
2 From 6h experimental groups , the number of apoptosis cells was remarkably larger than the control's till 48h after radiation(P<0.01)in 10 mW/cm2,5min groups and 20 mW/cm2,5min groups. Especially the 10 mW/cm2 ,5min,6 h experimental group reaching the peak (161.27 + 5.9) IS convoluted seminiferous tubules. The change of the other experimental groups was the same as that of the control (P>0.05).
3 The expression of the Bcl-2 protein and C-myc protein were (+ + +)degree in 24 groups after radiation by 10 mW/cm2,20 mW/cm2 HPM for 5min and 10min.In addition, the expression of the Bcl-2 protein was (+ + ) degree and that of the C-myc protein was( - )degree in 10mW/cm2,10min,48h experimental groups; The expression of Bcl-2 protein was ( + + ) degree and
that of C-myc protein was ( + ) degree in 20mW/cm2,5min, 6h experimental groups . No change was found in contral group and the other experimental groups.
4 With light microscope, vascular congestion , interstitial edema and slight inflamatory cell infiltration were observed in the 40 mW/cm2,5 min group, in addition granular cells irregulation were also observed in follicle in the 40mW/cm2,10 minutes experimental groups; granular cells degeneration, lutein cell apoptosis and unilunar follicle, primary follicle and secondary follicle atresia were also observed in 40mW/cm2,20min and 80mW/cm2,lmin group, 5min groups and 10min groups. In 80 mW/cm2,20min group, partly granular cells, Interstitial cells necrosis and tissue haemorrhage were found. With the transmission electron microscope, slight mitochondrion edema and endoplasm reticulum dilation were observed in
引文
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