亚砷酸钠对体外培养的猪卵巢颗粒细胞增殖活性的影响

发布时间：2018-05-07 22:02

本文选题：猪 + 颗粒细胞　；参考：《西北农林科技大学》2015年硕士论文

【摘要】：砷元素是动物体存在的一种微量元素,其有益性和毒性并存,严重影响着家畜的生活环境和健康。目前,砷对家畜生殖方面的研究主要集中在雄性生殖毒性及机理方面,对雌性生殖毒性方面的研究较少,颗粒细胞是卵巢中重要的功能细胞群,在雌性动物生殖活动中扮演重要角色。本试验以体外培养的猪卵巢颗粒细胞(pGCs)为研究模型,为后续繁殖生物学研究选择适合的卵泡来源,并探究亚砷酸钠(NaAsO2)对pGCs增殖凋亡的影响。本试验采用机械法剖剪取得猪卵泡并进行分级,机械十字剪法获得pGCs,台盼蓝染色方法计算细胞活率,倒置显微镜下观察细胞形态,四甲基偶氮唑蓝法(MTT)测定体外培养颗粒细胞生长曲线,实时荧光定量方法(qRT-PCR)检测促卵泡刺激素受体基因(FSHR)及促黄体生成素受体基因(LHR)的转录水平表达情况,进一步鉴定颗粒细胞;以合适直径卵泡范围内的卵巢颗粒细胞作为试验对象,人胆囊收缩素-8方法(CCK-8)检测NaAsO2对细胞存活的影响,qRT-PCR方法检测FSHR、LHR、CYP-19、ER1和ER2的转录水平的表达情况;FITC-AnnexinV/PI双染法检测细胞凋亡情况,酶联免疫吸附试验法(ELISA)检测激素分泌情况。主要得到了如下研究结果:1.确定了最适的体外培养pGCs培养体系为:基础培养基DMEM/F12、胎牛血清(FBS)3%、牛血清白蛋白(BSA)0.3%、胰岛素50ng/mL、促卵泡生成素(FSH)0.1 IU/mL、青霉素-链霉素1%。2.猪卵巢中,大型(≥5mm)、中型(2~5mm)、小型(2mm)3种不同直径卵泡中获得颗粒细胞的数量分别为1.71×108、4.01×108、4.19×108,颗粒细胞活率分别为68%、75%、72%,纯度分别为97%、95%、90%,体外培养生长增殖状态方面,中型卵泡中获得的颗粒细胞生长与增殖状态最佳、FSHR基因表达情况为小型表达量最高、大型和中型无显著性差异(P0.05),LHR表达量情况为大型最高且显著高于中型和小型卵泡(P0.05)。综合以上结果分析后,我们将中型卵泡作为本试验后续研究的卵巢颗粒细胞的最适卵泡来源。3.CCK-8法生长曲线检测结果显示,NaAsO2作用影响pGCs的存活率及生长状态:高浓度NaAsO2(50μM)组pGCs出现死亡现象,低浓度(0.08μM)几乎没有影响,而中间浓度(0.4μM、2μM、10μM)组间没有显著差异(P0.05),且10μM NaAsO2作用24h后出现活性增强现象。4.FITC-AnnexinV/PI双染法检测结果表明,NaAsO2(0、0.08μM、0.4μM、2μM、10μM、50μM)处理pGCs,除了10μM NaAsO2处理组细胞凋亡情况出现了缓解现象外,其他各组均随着NaAsO2浓度的升高,颗粒细胞凋亡率随之升高,呈现剂量依赖性。10μM NaAsO2作用的pGCs中凋亡相关基因qRT-PCR检测结果显示,在作用24h时显著上调Bcl-2基因,并下调Caspase-3基因表达,与FITC-AnnexinV/PI双染法检测结果一致。5.10μM NaAsO2作用不同时间(4h,8h,12h,24h),qRT-PCR结果显示,在作用4h,8h,12h时,FSHR、LHR、CYP19、ER1和ER2的基因表达量整体上有下调趋势,而在药物作用24h后出现上调趋势。同一作用时间不同浓度NaAs O2处理组,qRT-PCR结果显示,FSHR、LHR、CYP19、ER1和ER2的表达量都随NaAsO2浓度的升高而呈现降低趋势,具剂量依赖型。6.ELISA测定结果,NaAsO2处理组与对照组,以及不同浓度NaAsO2处理组组间孕酮和雌二醇含量差异均不显著(P0.05)。
[Abstract]:Arsenic is a trace element in animal body. Its benefit and toxicity coexist, which seriously affect the living environment and health of domestic animals. At present, the research of arsenic on domestic animal reproduction is mainly focused on the toxicity and mechanism of male reproduction, and the research on female reproductive toxicity is less. Granulosa cells are important functions in the ovary. The cell group, which plays an important role in the reproductive activities of female animals. This experiment uses the porcine ovarian granulosa cells (pGCs) in vitro as the research model, chooses the suitable follicle source for subsequent reproductive biology, and explores the effect of sodium arsenite (NaAsO2) on the proliferation and apoptosis of pGCs. Grade, mechanical cross shear method was used to obtain pGCs, trypan blue staining method was used to calculate cell viability, cell morphology was observed under inverted microscope, four methyl azazolium method (MTT) was used to determine the growth curve of granulosa cells in vitro, and real-time fluorescence quantitative method (qRT-PCR) was used to detect the follicle stimulating hormone receptor gene (FSHR) and luteinizing hormone receptor gene (LHR). Transcriptional level expression, further identification of granulosa cells; ovarian granulosa cells in a suitable diameter follicle as experimental object, human cholecystokinin -8 method (CCK-8) to detect the effect of NaAsO2 on cell survival, qRT-PCR method to detect the expression of FSHR, LHR, CYP-19, ER1 and ER2; FITC-AnnexinV/PI double staining detection Apoptosis, enzyme linked immunosorbent assay (ELISA) detection of hormone secretion. The main results are as follows: 1. the optimum culture pGCs culture system in vitro is the basic culture medium DMEM/F12, fetal bovine serum (FBS), bovine serum albumin (BSA) 0.3%, insulin 50ng/mL, follicle stimulating hormone (FSH) 0.1 IU/mL, penicillin chain In the ovary of mycophenin 1%.2., large (> 5mm), medium (2~5mm), and small (2mm) 3 different diameter follicles, the number of granulosa cells is 1.71 * 108,4.01 * 108,4.19 x 108 respectively, and the living rates of granulosa cells are 68%, 75%, 72% respectively, and the purity is 97%, 95%, 90% respectively. The expression of FSHR gene expression was the highest, and there was no significant difference between large and medium size (P0.05). The expression of LHR was the highest and significantly higher than that of medium and small follicles (P0.05). The result of.3.CCK-8 growth curve detection showed that the effect of NaAsO2 on the survival rate and growth state of pGCs: high concentration NaAsO2 (50 mu M) group pGCs died, low concentration (0.08 mu M) had almost no effect, but the intermediate concentration (0.4 mu M, 2 u M, 10 micron M) had no significant difference (P0.05), and the activity increased after 10 micron M. The results of.4.FITC-AnnexinV/PI double staining showed that NaAsO2 (0,0.08 mu M, 0.4 mu M, 2 mu M, 10 mu M, 50 mu M) treated pGCs. Except for the apoptosis of 10 mu M NaAsO2 treatment group, the death rate of granular cells increased with the increase of the concentration of NaAsO2. The results of apoptosis related gene qRT-PCR showed that the gene expression was significantly up-regulated and the expression of Caspase-3 gene was down regulated at the time of action of 24h, and the expression of Caspase-3 gene was consistent with the results of FITC-AnnexinV/PI double staining. There was a downward trend in the body and up trend after the drug action of 24h. The same action time was different in the NaAs O2 treatment group. The qRT-PCR results showed that the expression of FSHR, LHR, CYP19, ER1 and ER2 decreased with the increase of NaAsO2 concentration, with dose-dependent.6.ELISA determination, NaAsO2 treatment group and control group, and different concentration. There was no significant difference in progesterone and estradiol levels between the NaAsO2 treatment group and the control group (P0.05).

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S814

【参考文献】