【作者】 刘光伟；

【导师】 龚守良；

【作者基本信息】 吉林大学 ， 放射医学， 2004， 博士

【摘要】 随着核电及核技术的广泛应用，电离辐射对人体健康的影响，尤其是低剂量辐射所致遗传效应问题成为放射医学领域的研究热点。近年来，我们开展了低剂量辐射诱导细胞遗传学适应性反应领域的研究，获得了一些可观的成果。实验中发现，低剂量电离辐射可以诱导生精细胞遗传适应性反应，不仅降低相继大剂量辐射造成的染色体损伤，明显降低子代遗传效应，而且这种效应具有明显的细胞种类规律性，即降低精原细胞和精母细胞的损伤效应，不降低精子细胞和精子的损伤效应。然而，近年来的研究证实，正常的大鼠和小鼠睾丸中存在自发的生精细胞凋亡现象，且随着年龄、生精细胞种类及生精上皮周期不同阶段而发生改变，具有一定规律性。小鼠睾丸生精细胞凋亡主要以有丝分裂前期的精原细胞和精母细胞自发凋亡为主，很少累及精子细胞和精子。已知减数分裂前期时间较长，有许多重要的生物学事件发生，因此这一期间出现自发的细胞凋亡，以淘汰在DNA合成及同源染色体交换遗传物质过程中出现差错的细胞，在调控生精细胞的质量和数量及维持内环境稳定方面具有重要意义。另外，睾丸生精细胞凋亡对电离辐射敏感，很低剂量照射就可引起小鼠生精细胞凋亡增加。并且已经证明，射线作为一种基因毒性因子造成细胞死亡的主要机制是细胞凋亡。由辐射引起的凋亡，不仅呈剂量依赖性特点，还取决于细胞种类和细胞周期。因此，深入探讨低剂量电离辐射对小鼠睾丸生精细胞凋亡的影响及其基因调控，具有重要的意义。本实验主要从整体水平,通过对细胞凋亡及其凋亡相关基因mRNA和其表达蛋白产物的变化分析，研究了低剂量电离辐射对小鼠睾丸生精细胞凋亡的影响及其分子调控机制和低剂量电离辐射诱导生精细胞凋亡适应性反应的规律，并初步探讨其调控机制。1 低剂量电离辐射对小鼠睾丸生精细胞凋亡的影响0.025～0.2 Gy X射线全身照射后0、6、12、18 和24 h分别处死动物，取睾丸，采用常规苏木精-伊红（HE）染色法和原位末端标记（TUNEL）法通过光镜观察小鼠睾丸在生精周期不同阶段各类生精细胞凋亡的剂量及时程效应关系；同时,应用不<WP=127>连续密度梯度离心法分离不同种类生精细胞，流式细胞术检测其细胞凋亡百分率。实验结果表明，0.075 Gy X射线全身照射诱导小鼠生精细胞凋亡具有明显的时程效应关系。照后6 h，精原细胞凋亡率即明显增加，12～18 h达峰值，随后逐渐回降；照后6～12 h精母细胞凋亡率即明显增加，18 h达峰值，随后逐渐回降。照射后各时间点精子细胞和精子凋亡率相对较少，与对照组比较未见显著性差异。提示，0.075 Gy照射诱导生精细胞DNA损伤同时选择性促进精原细胞和精母细胞凋亡增加，随后由于自身修复机制激活及周围组织细胞和巨噬细胞吞噬凋亡小体，异常细胞数明显减少，凋亡百分率逐渐下降。另外，0.025～0.2 Gy X射线全身照射12 h，精原细胞和精母细胞凋亡率变化呈明显的剂量效应关系，但为非线性，而且前者凋亡的峰值明显高于后者。0.025 Gy 照射后，精原细胞凋亡率即明显增高，0.075 Gy其凋亡率增高最为明显，0.2 Gy照射后仍明显高于假照组。0.075和0.1 Gy照射后精母细胞凋亡率明显增高并达峰值。照射后精子细胞和精子凋亡率较低，与对照组比较未见显著性差异。其结果同生精细胞自发凋亡规律即主要以有丝分裂前期精原细胞和精母细胞凋亡为主，很少累及精子细胞和精子相一致。提示，低剂量辐射选择性诱导生精细胞凋亡增加可能具有重要遗传学意义；这也同我们前期工作低剂量辐射诱导细胞遗传损伤适应性反应的结果相符。提示，低剂量辐射选择性诱导生精细胞凋亡可能是低剂量辐射诱导细胞遗传学适应性反应的重要机制之一。2 低剂量电离辐射选择性诱导生精细胞凋亡的基因调控采用免疫组化法分别观察小鼠睾丸发育各阶段各类生精细胞p53、Bcl-2、Bax和Fas蛋白表达和原位杂交法观察其p53 mRNA和Bcl-2 mRNA转录水平的变化。2.1 低剂量X射线照射对生精细胞p53基因表达的影响 野生型p53作为细胞凋亡的诱导基因，在辐射及其他因素诱导细胞凋亡中倍受重视，处于“中心调控”地位，是线粒体和死亡受体信号传导途径的上游“核心”调控分子。本研究表明，0.075 Gy X射线全身照射后12 h，p53蛋白表达阳性的精原细胞明显增多并达峰值，随后逐渐回降；照后6 h，阳性精母细胞开始增多，12 h达峰值，随后逐渐回降；而精子细胞和精子低于精原细胞和精母细胞，与对照组比较未见显著性差异。提示，p53基因蛋白水平调控与低剂量辐射选择性诱导生精细胞凋亡早期反应相关联。同时，其剂量效应关系表明，0.025～0.2 Gy X射线全身照射12 h，p53蛋白表达阳性的精原细胞和精母细胞多呈剂量效应关系，但为非线性，而且前者峰值明显高于后者；精子细胞和精子阳性率较低，与对照组比较未见显著性差异。其结果同低剂量选择性诱导生精细胞凋亡规律相吻合，从而为其提供了分子调控的证据。为进一步探讨其基因调控机制，我们又检测了p53基因转录水平变化。0.075Gy X射线全身照射后3 h，p53表达阳性的精原细胞明显增多，并达峰值，一直持续到6 h；照后3 h，阳性精母细胞开始增多，6 h达峰值，随后逐渐回降，但照后12和18 h仍明显高于对?更多还原

【Abstract】 With the development of nuclear power and nuclear technology, the effect of ionizing radiation on human health, especially genetic effect problems caused by low dose radiation (LDR) have become a research hotspot in radiomedical domain. In recent years, we have obtained some considerable results on the adaptive response induced by LDR in cell genetics. The adaptive response not only reduces the chromosome damage to subsequent larger dose irradiation, showing a better filial generation genetic effect, but also has significant regularity in cell types. Namely LDR can reduce the damage effect of spermatogonia and spermatocytes and not that of spermatids and spermatozoa. However, it was confirmed that there is a kind of spontaneous spermatogenic cell apoptotic phenomena in normal rat and mouse testis, and it can vary with ages, cell types and different cycles of seminiferous epithelium. The spontaneous apoptosis of spermatogonia and spermatocytes were taken as the main during mitosis prophase in mouse testis. We have known many important biological events taken place in mitosis prophase, so this kind of spontaneous apoptosis had an important significance in eliminating abnormal cells during DNA synthesis and homologous chromosome exchanging genetic materials, controling the quality and quantity of spermatogenic cells and maintaining the homeostasis. Moreover, spermatogenic cell apoptosis is very sensitive to ionizing radiation and very low dose irradiation can induce it to increase. The main mechanisms of rays as a kind of genetic toxicity factor causing cell death was regarded as apoptosis, which not only had dose-dependent characters, but it also depended on cell types and cell cycle. Therefore, it is very important to further explore the effect of LDR on mouse spermatogenic cell apoptosis and its molecular mechanisms. In this dissertation through analyzing the apoptosis and its related gene mRNA and protein expressions, we studied the effect of LDR on mouse spermatogenic cell apoptosis and its molecular mechanisms and the apoptotic adaptive response induced by LDR and its rudiment mechanisms. <WP=132>Effects of LDR on mouse spermatogenic cell apoptosisThe Kunming mice were sacrified and testis was got 0, 6, 12, 18 and 24 h after 0.025～0.2 Gy irradiation. The apoptotic dose and time course effect relationships during the different stages of seminous epithelium cycles were observed using hematoxylin- eosin (HE) staining and TdT-mediated dUTP nick end labeling (TUNEL) methods; discontinuity density gradient centrifugation to separate different types spermatogenic cells and flow cytometry method (FCM) to detect its apoptotic percentage.The results showed that the apoptosis of spermatogenic cells had a significant time-course relationship after 0.075 Gy X-ray whole body irradiation (WBI) in mice. Spermatogone cell apoptosis increase 6 h after irradiation with 0.075 Gy X-rays, reached its peak 12～18 h and subsequently decrease; spermatocyte cell apoptosis began to increase 6～12 h after irradiation with the same dose irradiation, reached its peak 18 h, and subsequently began to decrease. The apoptotic percentages of spermatids and spermatozoa at every time point after irradiation were relatively lower than those of spermatogonia and spermatocytes, and the apoptotic percentages didn’t have significant difference between every time point and 0 h point. The results indicate that 0.075 Gy irradiation could cause the DNA damage of spermatogenic cells, selectively promote the spermatogone and spermatocyte apoptosis, and subsequently significantly decrease the abnormal cell number because of the activation of self-repair mechanisms and swallowing effects of macrophages and nearby cell.In addition, the apoptotic percentages of spermatogonia and spermatocytes had a significant dose-effect relationship 12 h after LDR WBI, but nonlinear, and the former apoptotic peak obviously higher than that of the latter. Spermatogone cell apoptotic percentages significantly increased after 0.025 Gy irradiation. The apoptotic percentage更多还原