【作者】 李伟；

【导师】 童德文；

【作者基本信息】 西北农林科技大学 ， 细胞生物学， 2013， 博士

【摘要】 苦马豆素（1，2，8-trihyroxyindolizidine，swainsonine，SW）是从豆科黄芪属（Astrgalus）和棘豆属（Oxytropis）等有毒植物中分离得到的一种吲哚兹定生物碱。家畜采食含有SW的植物后不仅能导致中毒死亡，更为严重的是还能引起母畜不孕或流产，给予畜牧业造成巨大的经济损失。到目前为止，关于SW是如何引起妊娠母畜流产的分子机制还不清楚。近年来的研究显示，许多有毒物质是通过诱导黄体细胞凋亡来影响其孕酮分泌进而导致动物不孕或流产。因此，本论文在建立永生化山羊黄体细胞系的基础上，进行SW对山羊黄体细胞孕酮分泌的影响和SW诱导山羊黄体细胞凋亡的信号转导通路研究，以期揭示SW引起妊娠母畜流产的分子机制。研究得到以下结果：1.采取妊娠6～8周的山羊黄体组织，用2mg/mL的胶原酶消化分离黄体细胞，离心收集消化后分散的单个细胞或细胞团，用含有10%胎牛血清的DMEM/DF12培养基悬浮细胞沉淀，置于37oC、5%CO2培养箱中培养。接种培养12h后细胞开始贴壁，呈梭形和多角形，为原代山羊黄体细胞。细胞化学染色显示3β-羟基类固醇脱氢酶（3β-hydroxysteroid dehydrogenase，3β-HSD，类固醇细胞的标志性酶）活性染色为阳性，阳性率达95%以上。通过脂质体2000将带有人端粒酶逆转录酶（human telomerase reversetranscriptase，hTERT）基因的pCI-neo-hTERT真核表达质粒转染进原代山羊黄体细胞，经450μg/mL的G418筛选14d后挑取阳性克隆，传代扩大培养。然后用细胞筛选免疫磁珠进行纯化，转染后第30代和50代黄体细胞与原代细胞相似，呈梭形和多角形、具有单层生长、接触抑制等特点。细胞化学染色显示转染后第30代和50代黄体细胞3β-HSD活性染色鉴定为阳性。RT-PCR和PCR-ELISA检测结果显示，hTERT基因在第30代和50代黄体细胞内稳定表达，并激活了细胞内的端粒酶活性。目前转染的黄体细胞在体外已稳定传至60代。以上试验结果初步表明，在山羊黄体细胞内过表达hTERT基因可激活细胞内的端粒酶活性从而使细胞得到永生化。2. RT-PCR检测结果显示，转染后第30代和50代黄体细胞依然表达原代黄体细胞内合成孕酮的关键基因和受体，如：类固醇敏感调节蛋白（Steroidogenic acute regulatoryprotein，StAR），P450胆固醇侧裂分解酶（P450cholesterol side-chain cleavage enzyme，P450scc），3β-HSD和促黄体激素受体（Luteinizing hormone receptor，LH-R）。此外，放射免疫沉淀（Radioimmunoassay，RIA）检测结果显示，转染后第50代的黄体细胞仍能分泌少量的孕酮，添加8-溴-环化腺苷酸（8-bromo-cAMP）和22-羟胆固醇（22(R)-hydroxycholesterol，22R-HC）后也能显著的刺激转染后黄体细胞孕酮的分泌。细胞生长曲线显示，转染后的黄体细胞比原代黄体细胞具有更快的生长速度，群体倍增时间分别为23.5h和24.2h。核型分析显示，转染后第50代黄体细胞具完整的染色体数（60条）。软琼脂悬浮试验和裸鼠致瘤性试验显示，转染后第50代黄体细胞不具有悬浮生长能力和裸鼠致瘤性。以上试验结果表明，永生化的黄体细胞保留了原代黄体细胞的主要生物学功能和特征，同时不具有肿瘤转化特性。该细胞系为后续研究山羊黄体细胞的功能提供了理想的细胞模型。3．以建立的永生化黄体细胞和原代黄体细胞为受体细胞，研究了SW对黄体细胞孕酮分泌和细胞活性的影响。RIA检测结果显示，低浓度SW（0.4，0.8和1.6μg/mL）处理黄体细胞24h和48h后能显著的提高孕酮的分泌；而高浓度SW（3.2和4.8μg/mL）处理黄体细胞24h和48h后却能显著的降低孕酮的分泌。结果表明，不同浓度SW对黄体细胞孕酮分泌的影响是不同的。进一步用MTT检测细胞活性结果显示，高浓度SW（3.2和4.8μg/mL）处理24h和48h后都能显著的降低细胞活性，而低浓度SW（0.4，0.8和1.6μg/mL）处理24h后对细胞活性没有影响，但是随着处理时间的延长，0.8，1.6μg/mL SW能显著降低细胞活性，这也提示不同浓度SW对黄体细胞活性的影响也不同。4．以建立的黄体细胞系为受体细胞，深入研究不同浓度SW对黄体细胞孕酮分泌和细胞活性影响的分子机制。QRT-PCR和Weseter blot检测结果显示，低浓度SW（0.4，0.8和1.6μg/mL）处理24h后对StAR在mRNA和蛋白水平上都没有显著的影响，但显著增加了P450scc在mRNA和蛋白水平上的表达。只有0.8和1.6μg/mL的SW能显著增加3β-HSD在mRNA和蛋白水平上的表达。表明低浓度SW可能是通过提高P450scc和3β-HSD的表达来增加黄体细胞孕酮的分泌。此外，流式细胞仪（flow cytometry）检测结果表明，3.2和4.8μg/mL SW处理24h和48h后都能显著的抑制细胞周期阻滞在G0/G1期，而0.8和1.6μg/mL的SW处理48h后才能显著的抑制细胞周期阻滞在G0/G1期。Annexin V-FITC/PI双荧光染色流式细胞术检测结果显示，随着SW处理浓度的增加或处理时间的延长，细胞凋亡率逐渐升高，并呈现出剂量和时间依赖性。琼脂糖凝胶电泳显示，4.8μg/mL的SW处理黄体细胞24h，或3.2μg/mL的SW处理48h后，细胞内基因组DNA被切断成180～200bp整数倍大小的片段，表明SW是通过诱导黄体细胞周期阻滞和凋亡来抑制黄体细胞生长和孕酮分泌。5. Western blot检测结果显示，SW是通过下调cyclin E，cyclin D1和CDK2的表达，而上调p21的表达来诱导黄体细胞周期阻滞在G0/G1期。Caspase活性检测显示，SW能激活黄体细胞内Caspase-9和Caspase-3，而对Caspase-8活性没有显著的影响，Caspase-3的底物PARP也随着Caspase-3的活化而发生切割降解。Western blot检测结果显示，SW处理后对黄体细胞内死亡受体通路中的调控分子FasL和Fas的表达没有显著影响，但上调了促凋亡蛋白Bax的表达，下调抑制凋亡分子Bcl-2的表达。QRT-PCR检测结果也显示，SW上调Bax mRNA水平的表达，下调Bcl-2mRNA水平的表达。分离线粒体蛋白和细胞浆蛋白后用Western blot检测显示，促凋亡分子Bax向线粒体转位，而线粒体内凋亡相关的蛋白分子Cyt c释放到细胞浆中，而线粒体第二激活因子（Smac）和凋亡诱导因子（AIF）都没有进而细胞质，表明SW是通过激活Bcl-2家族介导的线粒体通路，进而诱导黄体细胞发生凋亡。此外，特异性的Caspase抑制试验表明，只有特异的Caspase-3，-9和广谱的Caspase抑制剂能显著抑制SW诱导的黄体细胞DNA片段化降解，而Caspase-8抑制剂对SW诱导的细胞凋亡没有显著的影响，表明SW诱导的细胞凋亡依赖于Caspase-9和Caspase-3的活化。以上试验结果表明，SW是通过激活依赖于Casapse级联反应的线粒体通路来诱导黄体细胞凋亡。本论文建立了一株永生化的保持原代黄体细胞主要生物学特征和功能的山羊黄体细胞系，阐明了低浓度SW通过上调P450scc和3β-HSD的表达来增加黄体细胞孕酮分泌，而高浓度SW则通过诱导细胞周期阻滞来抑制细胞增殖，揭示了SW是通过激活依赖于Casapse级联反应的线粒体通路来诱导黄体细胞凋亡的。研究结果不仅提供了为进一步研究山羊黄体细胞生理生化调节机制和各种外源性物质对黄体细胞孕酮分泌影响的永生化黄体细胞系，也为进一步防控严重危害我国西部草原上有毒植物引起的家畜中毒奠定了理论基础。更多还原

【Abstract】 Swainsonine (1,2,8-trihyroxyindolizidine, SW), a natural toxin, is an indolizidinealkaloid isolated from locoweed a number of poisonous plants including Astragalus andOxytropis species. Ingestion of SW-containing plants not only cause livestock death, but alsoresult in infertility or abortion in the livestock, which causes tremendous loss annually to thelivestock industry. Up to now, the molecular mechanisms for SW causes female abortion isstill unknown. Recent years, many researchers have demonstrated that some toxicologicalcompounds can induce luteal cell apopotsis and cause abnormal luteal function throughinhibition of progesterone production, resulting in reproductive failure. Therefore, in thisstudy, we firstly established a transfected steroidogenic caprine luteal cell line, which wasused to investigate the effects of SW on progesterone secretion and cell viability and themechanisms involved in these processes in vitro. Finally, the exact molecular pathways ofSW-induced apoptosis were studied scientificly. The results were as follows:1. Caprine corpora lutea (CL) judged to be in early (6to8weeks) stages of pregnancy werecollected. Luteal cells were isolated from CL by2mg/mL collagenase digestion. Luteal cellsobtained was collected by centrifugation, cultured in DMEM/DF12supplemented with10%fetal calf serum at37oC and5%CO2. The cells became adherent after12h culture. Twoshapes cells were found, spindle and polygonal. Cytochemistry staining showed that this2shapes cells were positive for3β-hydroxysteroid dehydrogenase (3β-HSD) activity, a markerfor steroidogenic cells, with95%of positive rate. Primary luteal cells were transfected with aplasmid pCI-neo-hTERT containing the human telomerase reverse transcriptase (hTERT)gene by Lipofectamin2000. Clones were selected after450μg/mL G418resistance for14d,and positive clones were amplified. After purifying by magnetic cell separation kit, thetransduced luteal cells at passage30and50grew as confluent monolayers with the typicalspindle and spherical shaped morphology, which were similar to primary luteal cells atpassage. Cytochemistry staining showed that the transfected luteal cells at passage30and50were positive for3β-HSD activity. Results from RT-PCR and TRAP-ELISA assay confirmed that transduced luteal cells steadily expressed hTERT gene and exhibited higher telomeraseactivity at passage30and50. Up to now, the cells have been cultured for60passages in vitro.Taken together, our results demonstrate that over-expression of hTERT in caprine luteal cellscan reactivate telomerase and immortalize luteal cells.2. RT-PCR showed that the transfeceted luteal cells at passage30and50expressed genesencoding key proteins, enzymes and receptors inherent to normal luteal cells, such assteroidogenic acute regulatory protein (StAR), cytochrome P450cholesterol side-chaincleavage enzyme (P450scc),3β-HSD and luteinizing hormone receptor (LH-R). In addition,radioimmunoassay (RIA) showed that the transfeceted luteal cells produced detectablequantities of progesterone upon8-bromo-cAMP (8-Br-cAMP) or22(R)-hydroxycholesterol(22R-HC) stimulation. The growth curve showed that the transfected luteal cells grow morerapidly than primary luteal cells (Population Doubling Time (PDT):23.5:24.2h). Karyotypeanalysis showed that the transfected luteal cells have normal chromosome complement with amodal chromosome number of60. Furthermore, Soft agar assay and the xenograft in nudemice showed that the transfected luteal cells have no neoplastic transformation in vitro and invivo. Thes results demonstrated that immortalized luteal cells by hTERT retain their originalcharacteristics without neoplastic transformation, and may provide a useful model for studiesof luteal cell functions.3. Transfected and primary luteal cells were used to investigate the effects of SW onprogesterone secretion and cell viability. RIA showed that higher concentrations of SW (3.2and4.8μg/mL) treatments significantly inhibited progesterone production in the presence orabsence of22R-HC and pregnenolone in both transfected and primary luteal cells at24and48h when compared to the control group. However, SW at lower concentrations (0.4,0.8and1.6μg/mL) significantly stimulated the basal progesterone production and enhanced22R-HC-stimulated progesterone production when compared to the control group, while0.8and1.6μg/mL of SW significantly promoted pregnenolone-stimulated progesteroneproduction when compared to the control group in both transfected luteal cells and primaryluteal cells. MTT assay showed SW concentrations were lower than1.6μg/mL within24h or0.8μg/mL within48h. However, with the increase of treatment time and SW concentrations,cell viability significantly decreased when compared to the cells without SW treatment.4. Results from QRT-PCR and Weseter blot assay showed that StAR did not showsignificant changes at both mRNA and protein levels in different concentrations of SW-treatedcells compared to control cells. However, the expression of P450scc significantly increased atboth mRNA and protein levels in the luteal cells treated with0.4,0.8and1.6μg/mL of SWfor24h, compared to the control. The expression of3β-HSD significantly increased at both mRNA and protein levels in the luteal cells treated with0.8and1.6μg/mL of SW for24h. Inaddition, flow cytometry showed that after24h treatment, the percentage of cells in G0/G1phase increased from51%in the control group cells to63%and68%in the cells treated with3.2and4.8μg/mL of SW, respectively. After48h treatment, SW at0.8,1.6,3.2and4.8μg/mL significantly altered the cell cycle distribution, compared to the control group. Thepercentage of cells at G0/G1phase increased from52%to81%. Results from Western blotassay further confirmed that higher concentrations of SW induced luteal cell cycle arrest inG0/G1through down-regulating the expression of Cyclin E, Cyclin D1, CDK2, andup-regulating the p21protein levels. Annexin V-FITC/PI staining assay showed that over1.6μg/ml of SW induced cell apoptosis after48h treatment, and over3.2μg/ml of SW inducedcell apoptosis after24h treatment. DNA fragmentation assay showed that the cells treatedwith4.8μg/mL SW for48h showed a typical DNA ladder pattern.5. Caspase activity analysis showed that SW treatment increased the activities of Caspase-9and Caspase-3without affecting that of Caspase-8. After Caspase-3activation, the full-length(116kDa) poly ADP ribose polymerase (PARP) were cleaved to active form of85kDa, ahallmark of apoptosis. z-VAD-fmk (pan caspase inhibitor), z-LEHD-fmk(caspase-9specificinhibitor), or z-DEVD-fmk (caspase-3specific inhibitor) significantly inhibited SW-inducedapoptosis, whereas z-IETD-fmk (caspase-8inhibitor)did not, suggested SW-induced apoptosisdependent on Caspase-9and Caspase-3activation. However, the expression of Fas, Fas ligand(FasL) or caspase-8activity did not appear significant changes in the process of SW-inducedapoptosis. Both QRT-PCR and Western blot assay showed that SW treatment up-regulatedBax, down-regulated Bcl-2expression, promoted Bax translocation to mitochondria, activatedmitochondria mediated apoptotic pathway, which in turn caused the release of cytochrome c,the activation of caspase-9and caspase-3. In addition, SW treatment did not affect theexpression levels of Smac and AIF in the mitochondrial fractions.Taken together, the present study we established and evaluated a stable steroidogeniccaprine luteal cell line through transfection of a plasmid containing the hTERT gene. Theimmortalized luteal cells still retained their original characteristics, including steroidbiosynthesis capability, and the expression of key steroidogenic genes that are modulated bydifferent physiologic inducers, and will provide an important tool for the study of corpusluteum function. In addition, this study also illustrated that lower concentrations of SWinduced progesterone production through up-regulation of P450scc and3β-HSD withoutaffecting cell viability, whereas higher concentrations of SW induced cell cycle arrest andapoptosis throuth activation of mitochondrial pathway. The results may be a potentiallyvaluable and reliable cell model to be used to study function and regulation of normal caprine luteal cells. Furthermore, it could be used as an in vitro model to assess the effects of manyphysiological, biological, pharmacological and toxicological events and compounds on CLfunction. The results may also provide theoretical basis for prevention and control livestockpoisoning caused by toxic plants that serious damage to the grassland of west China.更多还原