【作者】 王雷；

【导师】 林德贵； 蒋金书； 吕俊宣；

【作者基本信息】 中国农业大学 ， 预防兽医学， 2005， 博士

【摘要】 硒化合物在抗肿瘤药物中已显示出广阔开发应用前景,硒抗肿瘤作用机制的研究及硒化合物与其它抗肿瘤药物的联合使用已成为目前研究热点,本实验利用辐照氧化性损伤小鼠模型、鸡胚绒毛尿囊膜模型、荷艾氏腹水癌ICR小鼠模型和前列腺癌（LNCaP）细胞研究了含硒化合物（Selenite、MSeA和SeMC）的抗肿瘤作用和机制,观察了硒与VCR联合使用对艾氏腹水癌和LNCaP肿瘤细胞的作用。并利用联合严重免疫缺陷型小鼠（SCID）成功建立了荷人源性LNCaP皮下肿瘤、荷LNCaP原位前列腺肿瘤、荷LNCaP皮下肿瘤去势三个SCID小鼠模型。其结果如下: 1) Na₂SeO₃和SeMC对受辐照损伤的小鼠有保护作用。4.5G剂量的辐射线全身照射小鼠会引起小鼠身体强烈的氧自由基损伤,服用了含硒化合物Na₂SeO₃和SeMC后,能显著提高机体含硒酶GSH-Px的水平;延长小鼠的生存期;升高血液中白细胞含量;使睾丸精子的畸形率降低;血液中SOD、GSH和GSH-Px的含量较辐照组明显升高。这与它们均及时清除辐照损伤在体内产生的氧自由基,并能及时为含硒酶补充充足的硒,从而减轻自由基对机体的损伤有关。 2) Na₂SeO₃和SeMC在药物—甲基纤维素膜（20μg/鸡胚的剂量）对于鸡胚尿囊膜局部血管的形成有抑制作用,较对照组差异显著PM<0.05。但是作用机制仍需进一步研究。 3) 4μg/gbw剂量的Na₂SeO₃和SeMC单独作用于ICR小鼠腹腔艾氏腹水癌时,小鼠的生存周期没有明显的延长。但是它们能有效的抑制肿瘤细胞的增殖,使G1期细胞比率升高,血清中的SOD、GSH和GSH-Px的水平升高而MDA含量下降,提示机体抗氧化水平的提升。SeMC与VCR连用时,对EAC肿瘤细胞的作用效果更强,能明显的抑制肿瘤细胞的增殖。2μg/gbw SeMC和0.4μg/gbw VCR联用,可以明显的升高血清中SOD和GSH的含量,降低MDA的水平,表明机体的抗氧化性损伤的水平升高。流式细胞术检测SeMC与VCR联用能促进肿瘤细胞的凋亡,延长小鼠的生存期,说明SeMC与VCR在抗EAC肿瘤方面具有良好的协同作用。 4) VCR具有抗LNCaP细胞的作用,它可以诱导LNCaP细胞凋亡,并且作用的细胞周期点是在G2/M期,会引起G2期肿瘤细胞比例的升高。Na₂SeO₃+VCR组和MSeA+VCR的联合用药对LNCaP肿瘤细胞的作用更强,其诱导细胞凋亡的比例显著升高（P<0.05）,增殖期（S期）细胞比例减少,所以硒与VCR联合使用具有抗LNCaP肿瘤细胞的协同作用。 5) 摘除双侧睾丸成功建立了荷LNCaP肿瘤去势小鼠模型。摘除双侧睾丸及其附睾后,LNCaP实体瘤的生长速度减慢甚至停滞;肿瘤细胞分泌到血清中的前列腺特异性抗原（PSA）蛋白浓度迅速下降;处于增殖状态的肿瘤细胞数量急剧减少,流式细胞术检测增殖细胞比例明显下降;肿瘤细胞的凋亡数量增加,TUNEL法检测显示肿瘤细胞中阳性细胞数与未去势小鼠相比明显增多,存在显著性差异（P<0.05）;电镜观察可以发现大量肿瘤细胞处于凋亡状态,核膜周围出现大量深染染色质,胞内存在大量凋亡小体。结果表明雄激素对于LNCaP肿瘤的生长具有明显的支持作用。在实验中同时观察到了在长期处于无雄激素或者极低量雄激素的环境下,对于雄激素更多还原

【Abstract】 This experiment investigated the effect and mechanism of selenium compound by using radiation oxidational trauma mice model, chorioallantoic membrane chicken model and ICR mice modle bearing EAC and cells with prostate cancer, and observed the effect of the use of selenium and VCR at the same time to ICR and LNCaP cancer cells. Three SCID mice models—hominine LNCaP subcutaneous tumour, LNCaP prostate tumour and LNCaP subcutaneous tumour castrated are successfully built by using sever combined immunal deficince mice. The following are the results:1. Na2SeO3 and SeMC have the function of protecting the mice suffering radiation oxidational trauma. When mice are radiated with 4.5G radiant ray, their body will be severely damaged by active oxygen radical. After taking the Selenium compound of Na2SeO3 and SeMC, the mice’s level of GSH-Px is highly improved, their survival period is prolonged, the quantity of leucocyte in blood is increased, the rate of deformed testicle sperm is lowered, the content of SOD, GSH and GSH-Px is obviously increased compared with the radiant group. This may be because selenium compounds can help clean the active oxygen radical produced after suffering radiant trauma in time, and supply GSH-Px enzyme with enough selenium in time, thus the trauma of organism is decreased.2. Na2SeO3 and SeMC has inhibiting effect to the forming of local angiopoiesis in chicken embryo when the dosage of 20μg/chicken embryo drugs-mythylfibrin membrane is taken. The difference is obvious compared with the control group(P<0.05). But function mechanism needs further investigation.3. When only the dosage of 4μg/gbw Na2SeO3 and SeMC has effect on EAC in ICR mice, the mice’s living period is not obviously prolonged. But they can effectively inhibit the growth of cancer cells, make the rate of G1 period cells and the level of SOD, GSH and GSH-PX in serum increase ,and the MDA in serum decrease , hint at the increase of the organism’s antioxidant level. When SeMC and VCR are both used, their effects to EAC cells are stronger, and the growth of cancer cells can be obviously inhibited. When 2μg/gbw MSeC and 0.4μg/gbw VCR are both used, the content of SOD and GSH in serum is obviously increased and MDA level is decreased. It represents that the organism’s level of antioxidant trauma is increased. Flow Cytometry(FCM) finds out that the use of SeMC and VCR at the same time facilitates the death of cancer cells and prolongs mice’s living period. It proves that SeMC and VCR have good coordinated roles in the aspect of resisting EAC tumour.4. VCR has the effect of anti-LNCaP cell. It can induce the LNCaP cell to die. The G2/M is the central cell cycle point effected by VCR and it causes the rate of G2 tumour cells to increase. The combination of Na2SeO3+VCR and MSeA+VCR hasstronger effect on LNCaP tumour cells. The rate of inducing cells to die is greatly increased (P<0.05) and the rate of S-period cells during growing period is decreased . So the combining use of selenium and VCR has coordinated role in resisting LNCaP cancer cells.5. The mice castrated model bearing LNCaP is successfully built by extirpating the double spermaries . After the double spermaries are extirpated, the growth of LNCaP tumour is lowered, even stopped. The concentration of prostate special antigen albumen is rapidly decreased, the quantity of tumour cells in growing condition is sharply reduced. Flow Cytometry(FCM) shows the rate of growing cells is obviously decreased. The cancer cells’ mortality is increased. TUNEL detect shows that the quantity of positive cells in the cancer cells is obviously augmented compared with uncastrated mcie and the difference is significantly ( P<0.05 ) . Observation by electrical microscope finds that a great many of cancer cells are in the apoptosis state. A great quantity of dark chromatin appears around the karyotheca, lots of apoptosis body are in the tumor cells. The above results show that female hormone has obvious supporting role to the growth of LNCaP tumour. It is also observed that when LNCaP tumour cells dep更多还原