本文選題：豬 + 顆粒細(xì)胞��； 參考：《西北農(nóng)林科技大學(xué)》2015年碩士論文

【摘要】：砷元素是動(dòng)物體存在的一種微量元素,其有益性和毒性并存,嚴(yán)重影響著家畜的生活環(huán)境和健康。目前,砷對(duì)家畜生殖方面的研究主要集中在雄性生殖毒性及機(jī)理方面,對(duì)雌性生殖毒性方面的研究較少,顆粒細(xì)胞是卵巢中重要的功能細(xì)胞群,在雌性動(dòng)物生殖活動(dòng)中扮演重要角色。本試驗(yàn)以體外培養(yǎng)的豬卵巢顆粒細(xì)胞(pGCs)為研究模型,為后續(xù)繁殖生物學(xué)研究選擇適合的卵泡來源,并探究亞砷酸鈉(NaAsO2)對(duì)pGCs增殖凋亡的影響。本試驗(yàn)采用機(jī)械法剖剪取得豬卵泡并進(jìn)行分級(jí),機(jī)械十字剪法獲得pGCs,臺(tái)盼藍(lán)染色方法計(jì)算細(xì)胞活率,倒置顯微鏡下觀察細(xì)胞形態(tài),四甲基偶氮唑藍(lán)法(MTT)測定體外培養(yǎng)顆粒細(xì)胞生長曲線,實(shí)時(shí)熒光定量方法(qRT-PCR)檢測促卵泡刺激素受體基因(FSHR)及促黃體生成素受體基因(LHR)的轉(zhuǎn)錄水平表達(dá)情況,進(jìn)一步鑒定顆粒細(xì)胞;以合適直徑卵泡范圍內(nèi)的卵巢顆粒細(xì)胞作為試驗(yàn)對(duì)象,人膽囊收縮素-8方法(CCK-8)檢測NaAsO2對(duì)細(xì)胞存活的影響,qRT-PCR方法檢測FSHR、LHR、CYP-19、ER1和ER2的轉(zhuǎn)錄水平的表達(dá)情況;FITC-AnnexinV/PI雙染法檢測細(xì)胞凋亡情況,酶聯(lián)免疫吸附試驗(yàn)法(ELISA)檢測激素分泌情況。主要得到了如下研究結(jié)果:1.確定了最適的體外培養(yǎng)pGCs培養(yǎng)體系為:基礎(chǔ)培養(yǎng)基DMEM/F12、胎牛血清(FBS)3%、牛血清白蛋白(BSA)0.3%、胰島素50ng/mL、促卵泡生成素(FSH)0.1 IU/mL、青霉素-鏈霉素1%。2.豬卵巢中,大型(≥5mm)、中型(2~5mm)、小型(2mm)3種不同直徑卵泡中獲得顆粒細(xì)胞的數(shù)量分別為1.71×108、4.01×108、4.19×108,顆粒細(xì)胞活率分別為68%、75%、72%,純度分別為97%、95%、90%,體外培養(yǎng)生長增殖狀態(tài)方面,中型卵泡中獲得的顆粒細(xì)胞生長與增殖狀態(tài)最佳、FSHR基因表達(dá)情況為小型表達(dá)量最高、大型和中型無顯著性差異(P0.05),LHR表達(dá)量情況為大型最高且顯著高于中型和小型卵泡(P0.05)。綜合以上結(jié)果分析后,我們將中型卵泡作為本試驗(yàn)后續(xù)研究的卵巢顆粒細(xì)胞的最適卵泡來源。3.CCK-8法生長曲線檢測結(jié)果顯示,NaAsO2作用影響pGCs的存活率及生長狀態(tài):高濃度NaAsO2(50μM)組pGCs出現(xiàn)死亡現(xiàn)象,低濃度(0.08μM)幾乎沒有影響,而中間濃度(0.4μM、2μM、10μM)組間沒有顯著差異(P0.05),且10μM NaAsO2作用24h后出現(xiàn)活性增強(qiáng)現(xiàn)象。4.FITC-AnnexinV/PI雙染法檢測結(jié)果表明,NaAsO2(0、0.08μM、0.4μM、2μM、10μM、50μM)處理pGCs,除了10μM NaAsO2處理組細(xì)胞凋亡情況出現(xiàn)了緩解現(xiàn)象外,其他各組均隨著NaAsO2濃度的升高,顆粒細(xì)胞凋亡率隨之升高,呈現(xiàn)劑量依賴性。10μM NaAsO2作用的pGCs中凋亡相關(guān)基因qRT-PCR檢測結(jié)果顯示,在作用24h時(shí)顯著上調(diào)Bcl-2基因,并下調(diào)Caspase-3基因表達(dá),與FITC-AnnexinV/PI雙染法檢測結(jié)果一致。5.10μM NaAsO2作用不同時(shí)間(4h,8h,12h,24h),qRT-PCR結(jié)果顯示,在作用4h,8h,12h時(shí),FSHR、LHR、CYP19、ER1和ER2的基因表達(dá)量整體上有下調(diào)趨勢,而在藥物作用24h后出現(xiàn)上調(diào)趨勢。同一作用時(shí)間不同濃度NaAs O2處理組,qRT-PCR結(jié)果顯示,FSHR、LHR、CYP19、ER1和ER2的表達(dá)量都隨NaAsO2濃度的升高而呈現(xiàn)降低趨勢,具劑量依賴型。6.ELISA測定結(jié)果,NaAsO2處理組與對(duì)照組,以及不同濃度NaAsO2處理組組間孕酮和雌二醇含量差異均不顯著(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).

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S814

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本文編號(hào)：1858628