【作者】 王志成；

【导师】 龚守良；

【作者基本信息】 吉林大学 ， 放射医学， 2009， 博士

【摘要】 随着核电及核技术的应用,电离辐射越来越多地影响人类生存的环境,对人们健康的影响日益受到关注。电离辐射对机体多系统、多器官、多组织,甚至于细胞、分子水平都能产生一定的作用,特别是大剂量辐射对其产生重要的作用。然而,低剂量辐射对生殖和遗传的作用及其机制还不完全明确,并已成为放射生物学领域的研究热点。睾丸生精细胞对电离辐射极其敏感,本研究的前期工作已证实,低剂量辐射可引起睾丸生精细胞的凋亡增加。射线作为一种物理因子造成细胞死亡的主要机制是细胞凋亡,由辐射引起的生精细胞凋亡不仅具有剂量依赖性特点,还取决于细胞种类和细胞周期;同时,许多基因参与凋亡的调控。线粒体是细胞内重要的细胞器,是氧化磷酸化和形成ATP的主要场所,有细胞“动力工厂(power plant)”之称。另外,线粒体有自身的DNA和遗传体系,因此,只是一种半自主性的细胞器。人类许多疾病,例如肿瘤、糖尿病和聋病等的发生、发展都与线粒体功能缺陷有着密切关系。线粒体为人类机体提供着95%以上的能量,也发挥着包括细胞凋亡、信号转导调控等在内的生化功能。当前,有关线粒体研究,在国内外已深入到生物的生殖、遗传、发育、进化和细胞的代谢、衰老以及疾病等众多重要领域,成为生命科学和分子医学中最活跃的研究前沿课题之一。本文系统地阐述了低剂量电离辐射诱导小鼠睾丸生精细胞凋亡的规律,即0.025～0.2 Gy X射线照射后0～24 h,睾丸生精细胞随剂量增大,时间延长,凋亡逐渐增多;0.075 Gy照射后12 h生精细胞凋亡达到峰值,而后随时间延长、剂量的加大,睾丸生精细胞凋亡逐渐降低到正常水平。同时,阐述了睾丸生精细胞内线粒体的结构和相关生物学功能的剂量-效应、时程-效应规律,并分析了线粒体内相关凋亡蛋白Cyt c、caspase-3、caspase-9和AIF表达及其mRNA水平变化与凋亡发生之间的规律。本研究结果为探讨线粒体在低剂量电离辐射诱导睾丸生精细胞凋亡的调控作用提供了重要的实验证据,为当前辐射生殖和遗传学理论研究提供了新的实验资料,同时也进一步为辐射遗传损伤的防治和辐射防护法规的制订提供了重要的实验依据。更多还原

【Abstract】 With the general application of nuclear power and its technology, the effects of ionizing radiation on the human health, especially on the problems of human reproduction and genetic effect caused by low dose radiation (LDR) have become the research hotspot in radiation biological domain. In recent years, lots of researches have proved that there is spontaneously the apoptosis of spermatogenic cells in rat and mouse testes, which changes with the development time prolongation, spermatogenic cell type and different cycle of seminiferous epithelium, having obvious regularity. Most of spermatogenic cells of spontaneous apoptosis happen in spermatogonia and spermatocytes in mitosis prophase, but it is few in spermatozoa and spermatotids. Spermatogenic cells are very sensitive to ionizing radiation, which is different with the cell type and cell cycle phage. Ionizing radiation with very low dose can enhance the spermatogenic cell apoptosis. Ray is a kind of physics factor. The death way of spermaotgenic cells caused by rays is mainly apoptosis which has dose dependency in the character. It was confirmed that p53 gene and death receptor pathway participate in the regulation of spermatogenic cell apoptosis induced by LDR. Mitochondria participate in the cell apoptosis induced by LDR, which is an important mechanism. At the apoptotic early period, mitochondrial membrane transition pores open widely, mitochondrial potential decreases, endocellular reactive oxygen species enhances, the balance of endocellular Ca2+ homeostasis loses, ATPase activity decreases, Cyt c and AIF proteins in inter mitochondrial membrane release into endochylema, which activate the downstream apoptotic relative factors and cause the cell apoptosis.In the paper, we measured the apoptotic regularity of spermatogenic cells in mouse testes and mitochondrial structure and function changes with the empirical methods on immunology, molecular biology and biochemistry after the mice treated by LDR (0.025– 0.2 Gy), and explored the mechanism that mitochondria participate in the regulation of spermatogenic cell apoptosis. The experiment results showed that spermatogenic cell apoptosis had cell type selectivity and dose- and time-effect regrularities after LDR with 0.025– 0.2 Gy; at the same time, the mitochondrial function changed, mitochondrial membrane potential decreased, the activities of Na+-K+-ATPase and Ca2+-ATPase decreased and the lost balance of Ca2+ homeostasis happened, and there are dose- and time-effect regularities in these results. The mitochondrial structure changed, the mitochondria of spermatogonia, spermatocytes and spermatids swelled and vacuolizated, and their cristae were broken 12 h after whole-body irradiation with 0.075 Gy X-rays; otherwise, the contents of endocellular ROS, NO, NOS and so on enhanced and cellular oxidative damage increased; there were the regularities of dose- and time-effect in mRNA and protein expressions of apoptosis relative gene Cyt c, AIF, caspase-3 and -9, which were similar to the testis spermtogenic cell apoptotic regularity. These results provide the essensial experiment data for exploring the regulation mechanism that the mitochondria participate in the spermatogenic cell apoptosis of mouse testes induced by LDR.1 Effect of LDR on spermatogenic cell apoptosis in mouse testesAfter the mice were sacrificed 3, 6, 12, 18 and 24 h after irradiation with 0.025– 0.2 Gy X-rays, two side testes were got, and the spermatogenic cell apoptosis was detected with TUNEL. The results showed that the spermatogenic cell apoposis displayed obvious time course-effect relation after whole-body irradiation with 0.075 Gy X-rays. The spermatogonium apoptotic percentage increased significantly 6 h after irradiation, it reached the peak value at 12– 18 h, then began to cut down; the spermatocyte apoptotic percentage increased obviously 6– 12 h after irradiation, it reached the peak value at 18 h, then began to subdue gradually. These results indicate that 0.075 Gy irradiation promoted selectively the apoptotic increase of spermatogonia and spermatocytes; because the self-repair mechanism was activated and some apoptotic bodies were phagocytized by surrounding histiocyte and macrophage, abnormal cells decreased obviously, and apoptotic percentage cut down gradually. The apoptotic percentage change of spermatogonia and spermatocytes showed a dose-effect relationship 12 h after whole-body irradiation with 0.025– 0.2 Gy, but it was nonlinear, and the apoptotic peak of spermatogonia was higher than that of spermatocytes. After irradiation with 0.025 Gy X-rays, the apoptotic percentage of spermatogonia increased significantly, it was most obvious after irradiation with 0.075 Gy, and it with 0.2 Gy was significantly higher than that with 0 Gy. The apoptotic percentage of spermatocytes increased significantly after irradiation with 0.075 and 0.1 Gy, and reached the peak value. These results indicate that the increase of spermatogenic cell apoptosis induced selectively by LDR might have the important genetics significance.2 Effect of LDR on mitochondrial morphous structure of spermatogenic cellsThe mitochondrial structure changes of spermatogenic cells were measured by transmission electron microscope 12 h after whole-body irradiation with 0.075 Gy X-rays, as compared with those in normal mouse testes. Under common circumstance, the spermatogonium nucleus in mouse testes was big, euchromatin showed a grain shape, mitochondrial appearance was integrity; the spermatocyte heterochromatin was massive, its mitochondrial appearance was integrity; the spermatid nuclear chromatin was dense and uniformity, its mithchondrial appearance was integrity. The mitochondria of spermatogonia and spermatocytes swelled and vacuolizated, and their cristae were broken 12 h after irradiation with 0.075 Gy X-rays; the spermatid was karyopycnosis, the acrosomal vesicle structure was ambiguity, its membrane structure was not clarity, its mitochondrion vacuolizated, the apical body membrane mitochondrial change was not obvious; the distance between spermatogonia and spermatocytes became long, their conjunction disappeared, the spermatocytes necrosed. These results indicate that LDR could induce the changes of spermatogenic cell mitochondrial structure, then induced their function change.3 Effect of LDR on spermatogenic cell function in mouse testesTwo mouse testes were got 0– 24 h after whole-body irradiation with 0.025– 0.2 Gy X-rays. Rhodamine 123 and Fluo-3/AM were used for fluorescent labeling. The mitochondrial membrane potential and Ca2+ homeostasis were measured with FCM; Na+-K+-ATPase and Ca2+-ATPase were measured with biochemical event kit. The results showed that spermatogenic cell mitochondrial membrane potential in mouse testes decreased 3 h after whole-body irradiation with 0.075 Gy X-rays, and reached to the lowest level at 12 h, then began to recover to normal level; [Ca2+]i decreased from 3 h after irradiation, but it increased slightly at 6 h, then it decreased to lowest level at 12 h, and kept at the low level after this; the activities of Na+-K+-ATPase and Ca2+-ATPase decreased from 3 h, and reached to the lowest level at 12 h, and kept at the low level constantly. The mitochondrial membrane potential decreasd 12 h after whole-body irradiation with 0.025– 0.2 Gy X-rays, and reached to lowest level with 0.075 Gy irradiation, then recovered to normal level gradually; [Ca2+]i increased slightly with 0.025 Gy irradiation, then began to decrease and to reached to the lowest level with 0.075 Gy irradiation, and recovered to normal level gradually; the activities of Na+-K+-ATPase and Ca2+-ATPase decreased with 0.025 Gy irradiation and to reached to the lowest level with 0.075 Gy; but the activity of Ca2+-ATPase decreased to lowest level with 0.1 Gy. These results showed LDR could induce spermatogenic cell mitochondrial function in mouse testes changes, and the regularity of dose- and time-effect was similar to apoptotic regularity, it indicated that there was some essential correlation between mitochondrail function and apoptosis.4 Effect of LDR on reactive oxygen species of spermatogenic cells in mouse testesTwo mouse testes were got 0– 24 h after whole-body irradiation with 0.025– 0.2 Gy X-rays. DCFH-DA is a fluorescent labelled compound. ROS contens were measured with FCM. NO content and NOS activity were measured with biochemical event kit. The results showed that ROS content increased with time prolongation 3– 24 h after 0.075 Gy irradiation, it reached the most value at 12 h, then kept at the high level at 18– 24 h; NO content decreased slightly 3 and 6 h after irradiation, then began to increase, reached the peak value and kept at high level; NOS activity increased with time prolongation, and reached to the highest at 12 h, then kept at the high level. ROS content increased gradually 12 h after whole-body irradiation with 0.025– 0.2 Gy X-rays, it increased to maximum, then decreased a little and kept at high level; NO contents began to increase after 0.025 Gy irradiation, but decreased slightly after 0.075 Gy irradiation, then increased again and to the maximum after 0.2 Gy irradiation; NOS activity increased to maximum after 0.075 Gy irradiation, then recovered gradually to normal level with the dose enhancing. These results indicate that LDR could induce ROS increase of spermatogenic cells in mouse testes, there was a causal relation between LDR and mitochondrial damage, which related closely to apoptosis.5 Effect of LDR on expressions of relative gene mRNA and proteins in mouse testesTwo mouse testes were got 0– 24 h after 0.025– 0.2 Gy X-rays irradiation. The changes of Cyt c, caspase-3 and AIF mRNA in mouse testes were measured with real time PCR; the of expressions of Cyt c and caspase-3 protein were measured with immunohistochemistry and FCM, respectively; the expressions of Cyt c, caspase-9, -3 and AIF proteins were measured with Western blot.5.1 Effect LDR on expressions of Cyt c gene mRNA and protein in mouse testesCyt c locates center posision in the cell apoptosis regulated by mitochondrial pathway. It activated caspase families in down stream, then the spermatogenic cell apoptosis happened 3– 24 h after 0.075 Gy irradiation. Cyt c mRNA expression decreased slightly 6 h after irradiation, it increased at 12 h and reached to the maximum at 24 h. The results in immunohistochemistry showed that Cyt c positive spermatogonia and spermatocytes tended to increase, and reached to peak value 12 h after irradiation, then cut down; Cyt c positive spermatids and spermatozoa were less in the initial stage after irradiation, and increased significantly 12 h after irradiation, but were less than that positive spermatogonia and spermatocytes. The results in FCM and Western blot showed that Cyt c expression increased significantly with time prolongation, and reached to the maximum value at 12 h, then began to decrease. Cyt c mRNA increased with the increase of doses 12 h after irradiation with 0.025– 0.2 Gy X-rays, and reached to the maximum value after 0.1 Gy irradiation, then cut down. The results in immunohistochemistry showed that Cyt c positive spermatogonia and spermatocytes were more 12 h after irradiation with 0.025～0.2 Gy X-rays, and increased more obvious after 0.075 Gy irradiation; but that spermatids and spermatozoa were less. The results in FCM and Western blot showed that Cyt c protein expression increased with the increase of doses, and reached to the peak value after 0.075 Gy irradiation, then decreased. The expressions of Cyt c mRNA and protein had dose- and time-effect regularities, and as the regularity of apoptosis, these provide the evidence for the spermatogenic cell apoptosis regulated by Cyt c.5.2 Effect of LDR on spermatogenic cell caspase-3 and -9 in mouse testesThe caspase families were the important transfered chain and the executor of apoptotic pathway. Caspase-3 mRNA increased with time prolongation 3– 24 h after 0.075 Gy irradiation, and reached the maximum at 24 h. The results in immunohistochemistry showed that caspase-3 positive spermatogonia increased significantly 3 h after irradiation, but spermatocytes did significantly 6 h after irradiation. Caspase-3 positive spermatogonia and spermatocytes reached the perk value 12 h after irradiation, then began to decrease; caspase-3 positive spermatids and spermatozoa were less than that positive spermatogonia and spermatocytes; the results in FCM and Western blot showed that caspase-3 protein expressed most 12 h after irradiation. Caspase-3 mRNA increased with the increase of doses 12 h after the irradiation with 0.025– 0.2 Gy X-rays, and reached to the maximum after 0.075 Gy irradiation, then decreased; the results in immumohistochemistry showed that caspase-3 positive spermatogonia and spermatocytes were more, but that positive spermatids and spermatozoa were less, that positive spermatogonia and spermatocytes were most after 0.075 Gy irradiation. At the same time, caspase-9 expression had dose- and time-effect relation after irradiation. The results in immunohistochemistry showed caspase-9 positive spermatogonia began to increase 3 h after 0.075 Gy irradiation, and reached to the peak value 12 h after irradiation, then decreased; that positive spermatocytes were more than that positive spermatogonia, increased at 6 h, and reached to the peak value 12 h after irradiation, and kept at an the high level; otherwise, their not were the regularities in that positive spermatids and spermatozoa after irradiation unlike that positive spermatogonia and spermatocytes. The results in Western blot showed that caspase-9 expressed less 6 h than that in the normal after irradiation, but began to increase at 12 h and reached to the peak value 24 h after irradiation. The results in immunohistochemistry showed that caspase-9 positive spermatogonia and spermatocytes in the mice were mostly 12 h after whole-body irradiation with 0.025– 0.2 Gy X rays, but that positive spermatids and spermatozoa were less, and were maximum with 0.075 irraidation. The results in Western blot showed that caspase-9 expression decreased slightly after 0.05 Gy irradiation, and expressed most with 0.075 and 0.1 Gy irradiation, then cut down after 0.2 Gy irradiation. When the apoptosis of spermatogenic cells happened, especially Cyt c activated the apoptotic pathway, Cyt c, Apaf-1 and dATP linked to complex activated caspase-9 and caspase-3 which is apoptotic executeor. These results indicate that caspase-3 and -9 mRNA and protein level changes were the same to the dose- and time- effect of spermatogenic cell apoptosis, which show that both caspase-3 and -9 participate in apoptotic regulation at gene transcription and translation levels.5.3 Effect of LDR on AIF gene expression of apermatogenic cells in mouse testesThat AIF incuced apoptosis is not depended on caspase families. SIF releases from mitochondria into cytoplasm, locates at nucleus, and caused chromosome clumping to perimeter of nucleus and DNA Klenow fragment breakage. Finally, the apoptosis of cells happen. AIF mRNA changed not obvious 6 and 12 h after whole-body irradiation with 0.075 Gy X-rays, as compared with that at 0 h, and increased to the maximum at 24 h. The results in immunohistochemistry showed that AIF positive spermatogonia and spermatocytes increased 3 h after irradiation, continued to increased with time prolongation, and reached to maximum 12 h after irradiation, then decreased; that positive spermatids and spermatozoa were less, and these changes had not regularity with the time prolongation. The results in Western blot showed that AIF protein expressed greatly 12 h after irradiation, then decreased. AIF mRNA increased with the increase of doses 12 h after irradiation with 0.025– 0.2 Gy X-rays, and reached to the maximum after irradiation with 0.1 Gy. The results in immunohistochemistry showed that AIF positive spermatogonia, spermatocytes, spermatids and spermatozoa increased gradually with the increase of dosed, that positive spermatogonia, spermatids and spermatozoa were less than that positive spermatocytes. The results in Western blot showed that AIF protein expressed greatly after irradiation with 0.075 Gy, but the expression was less after irradiation with 0.1 and 0.2 Gy. The dose- and time-effect regularities of AIF mRNA and protein expressions were consistent to the apoptotic regularity of spermatogenic cells in mouse testes. This proved thoroughly that AIF took part in apoptosis regulation. In one word, LDR (0.025– 0.2 Gy)could change spermatogenic cell mitochondrial fuction in mouse testes, decreasing mitochondrial membrane potential and ATPase activity and disturbing Ca2+ homeostasis; at same time, LDR might induce the increase of intracellular reactive oxygen species and the enhancement of oxidative damage. Cyt c and AIF releases between mitochondrial membranes increased, and activated capsase pathway and switched on apoptosis. We see from these results that Cyt c, AIF, caspase-3, and -9 genes regulated apoptosis from transcription and translation, and provided the evidences for exploring mitochondria regulated spermatogenic cell apoptotic mechanisms.更多还原