NPFFR1基因对鹅卵泡颗粒细胞激素分泌和细胞凋亡的影响研究

神经相关肽受体（RFamide-related peptide receptor,NPFFR1）是促性腺激素抑制激素的主要亲和受体,它在调控动物繁殖方面起着重要作用。为了解NPFFR1对鹅卵巢卵泡发育的作用,本研究以42周龄健康产蛋四川白鹅为试验材料（n=9）,利用RT-qPCR法检测NPFFR1基因在等级前和等级卵泡颗粒细胞中的mRNA表达规律;在颗粒细胞中过表达NPFFR1基因,酶联免疫吸附法检测颗粒细胞上清液（n=9）中雌二醇（estradiol,E2）、孕酮（progesterone,P4）和抗缪勒管激素（anti-Mullerian hormone,AMH）的浓度变化,剩余贴壁细胞作一步法TUNEL检测细胞凋亡情况;转录组测序方法筛选大黄卵泡（8~10 mm）颗粒细胞过表达NPFFR1前后表达差异显著基因,并对差异表达基因进行功能聚类分析。结果显示,除F1等级外,其余等级卵泡颗粒细胞NPFFR1表达量均极显著高于等级前卵泡（P<0.01）;过表达NPFFR1后,等级颗粒细胞上清液中的E2和等级前颗粒细胞上清液AMH的含量显著（P<0.05）降低,但孕酮P4含量变化不显著（P>0.05）;转录组测序共筛选到267个差异表达基因（119个下调,148个上调）,这些基因主要富集在生物节律过程、繁殖进程等生物学过程中;同时,与对照组相比,差异基因AMH显著下调表达（P<0.05）,Clock（clock circadian regulator）、FOS（proto-oncogene,AP-1 trans-cription factor subunit）、Per（period circadian regulator）和ANTXR2（cell adhesion molecule 2）分别极显著（P<0.01）或显著（P<0.05）上调表达。上述试验结果提示,NPFFR1可从激素、细胞凋亡和生物节律等多个环节影响卵泡颗粒细胞,参与调控卵泡的时序等级发育。     

5-Aza-dC对牛成肌细胞MyoD1启动子甲基化及mRNA表达的影响

旨在探究5-氮杂-2-脱氧胞苷（5-Aza-2’-deoxycytidine,5-Aza-dC）对牛成肌细胞的增殖和肌分化因子（myogenic differentiation 1,MyoD1）启动子甲基化以及mRNA表达的影响。本研究复苏了前期冻存的关岭牛成肌细胞,并进行培养,待其生长到对数生长期,再通过不同浓度的5-Aza-dC对牛成肌细胞进行处理,利用流式细胞仪检测细胞凋亡和周期;结合高通量检测方法检测MyoD1启动子甲基化水平,并利用qRT-PCR检测MyoD1及相关基因的表达水平。研究发现,0.1 μmol·L^-15-Aza-dC为最适浓度,该浓度下细胞抗凋亡因子Bcl的表达极显著降低（P<0.01）,促凋亡因子Bax的表达极显著提高（P<0.01）,促凋亡因子Caspase-9的表达显著提高（P<0.05）;周期因子Cyclin A2的表达显著提高（P<0.05）,而细胞因子Cyclin B1、Cyclin D的表达无显著变化;检测空白组和试验组MyoD1启动子甲基化水平发现,试验组甲基化水平极显著低于空白组（P<0.01）;而mRNA的表达水平极显著高于空白组（P<0.01）。5-Aza-dC能够通过改变Caspase-9、Bcl、Bax、Cyclin A2等基因的表达来调节关岭牛成肌细胞的增殖和凋亡,并能够有效降低MyoD1基因启动子的甲基化水平（P<0.01）;极显著提高其mRNA的表达量（P<0.01）。低浓度的5-Aza-dC能通过促进细胞凋亡及调控关岭牛MyoD1的甲基化水平来调控MyoD1的表达;同时推测,MyoD1基因启动子的甲基化水平能够影响关岭牛成肌细胞的生长发育,可为遗传标记辅助关岭牛的改良提供理论参考。     

1株降解黄曲霉毒素B1的解淀粉芽胞杆菌的分离鉴定及其体外脱毒效果

旨在筛选出能够高效降解黄曲霉毒素B₁（AFB₁）的菌株,并对其脱毒活性组分的脱毒效果进行研究,本研究以香豆素为唯一碳源对目标细菌进行初筛,以对AFB₁的降解率为指标进行复筛,并从形态学、生理生化试验和16S rDNA序列分析对筛选菌株进行鉴定;通过测定该菌株不同发酵组分对AFB₁的降解率,来定位其脱毒活性组分,同时采用CCK-8法测定活性组分降解AFB₁后对LMH细胞毒性,并研究了热处理、紫外线照射、强酸、强碱及蛋白酶K处理对活性组分脱毒作用的影响。结果显示,从样品中初筛分离到24株能够在初筛培养基生长良好的菌株,经复筛从中筛选到一株能高效降解AFB₁的菌株Y1-B1,其AFB₁降解率达到73.2%;经形态学、生理生化试验和16S rDNA序列分析确定该菌为解淀粉芽胞杆菌（Bacillus amyloliquefaciens）。对菌株Y1-B1培养物的不同组分进行的脱毒活性研究表明,不同组分对AFB₁的降解率不同,上清液的脱毒能力最强,细菌细胞裂解物的脱毒能力下降明显,菌体悬液和菌体裂解物上清的脱毒能力最差,由此确定解淀粉芽胞杆菌Y1-B1的脱毒能力主要来源于上清液中的某种活性物质;细胞毒性结果显示,与AFB₁对LMH细胞的毒性作用相比,AFB₁被上清液降解后其产物的毒性显著降低。该脱毒活性组分对不同理化因素处理呈现出不同的表现,对高温、紫外照射抵抗力较强,对强酸、强碱抵抗力较差,蛋白酶K仅造成脱毒能力部分减弱。本研究将为解决黄曲霉毒素污染问题提供新的微生物种质资源,同时为后续微生物脱毒制剂的开发奠定基础。     

Mesh-like vascular changes in copper deficiency-induced rat cardiomyopathy

The pathological effects of copper deficiency (COD) are well known. However, the pathogenesis of cardiomyopathy resulting from COD remains unclear. In this study, aimed to elucidate the pathogenesis of COD-induced cardiomyopathy by examining the morphology of the cardiovascular system in copper-deficient rats using histopathology, immunohistochemistry, and scanning and transmission electron microscopy. Changes detected in the myocardium and interstitium were consistent with those reported for COD. Morphological changes included mesh-like changes in the capillary endothelial cells that appear to be a novel finding in COD-induced cardiomyopathy. These changes are hypothesized to result from abnormal vascular remodeling following damage to the basement membrane and due to the mechanical effects of myocardial contractions. Although cardiomyopathy may be associated with microcirculatory disorders arising from these lesions, further investigations are necessary to demonstrate a causal relationship between the pathogenesis of cardiomyopathy and the contribution of these lesions to disease progression.     

AR regulates porcine immature Sertoli cell growth via binding to RNF4 and miR-124a

Sertoli cells are the only somatic cells in the seminiferous epithelium which directly contact with germ cells. Sertoli cells exhibit polarized alignment at the basal membrane of seminiferous tubules to maintain the microenvironment for growth and development of germ cells, and therefore play a crucial role in spermatogenesis. Androgens exert their action through androgen receptor (AR) and AR signalling in the testis is essential for maintenance of spermatogonial numbers, blood–testis barrier integrity, completion of meiosis, adhesion of spermatids and spermiation. In the present study, we demonstrated that AR gene could promote the proliferation of immature porcine Sertoli cells (ST cells) and the cell cycle procession, and accelerate the transition from G1 phase into S phase in ST cells. Meanwhile, miR-124a could affect the proliferation and cell cycle procession of ST cells by targeting 3′-UTR of AR gene. Furthermore, AR bound to the RNF4 via AR DNA-binding domain (DBD) and we verified that RNF4 was necessary for AR to regulate the growth of ST cells. Above all, this study suggests that AR regulates ST cell growth via binding to RNF4 and miR-124a, which may help us to further understand the function of AR in spermatogenesis.     

Immunohistochemical expression of HER - 2 and Ki - 67 in granulosa cell tumor in bitches

Granulosa cell tumour, an ovarian neoplasm of stromal origin, is an important tumour related to oestrogenic dominance syndrome and cystic endometrial hyperplasia–pyometra complex. In order to analyse ovarian tumour´s malignant potential, immunohistochemical markers can be used, such as anti-HER2 and anti-Ki-67. The aim of this study was to evaluate the expression of immunohistochemical markers HER-2 and Ki-67 in granulosa cell tumour from bitches´ ovaries. In HER-2 immunomarker analysis using the HercepTest^® method, most tumours were classified as 2+ (moderate labelling). Concerning Ki-67 immunomarker, only one case was described as having a high proliferative index. An association was found between immunostained cell percentage by anti-HER-2 antibodies and high pleomorphism, represented by the pattern of follicular/trabecular tumour arrangement. There was no correlation between anti-Ki-67 and anti-HER-2 antibody immunostaining intensities, probably due to only one case with a high Ki-67 index. With an effective protocol for HER-2 and Ki-67 immunohistochemical identification in granulosa cell tumours in bitches, it was possible to characterize this neoplasm proliferation profile.     

The influence of progesterone and prostaglandin F2α on decorin and the adhesion of caruncular epithelial cells of bovine placenta at early-mid pregnancy—Part II

One of the most important processes determining the proper course of gestation and its physiological termination in cows is the adhesion of epithelial cells allowing for direct contact of maternal and foetal parts of the placenta. Throughout pregnancy, placental cells are under strict hormonal control, which among others regulates the concentration and activity of specific proteins participating in the extracellular matrix remodelling of foetal membranes. The aim of the study was to evaluate the influence of progesterone and prostaglandin F_2α on the adhesion of epithelial cells at early-mid pregnancy in cows. Additionally, the impact of selected hormones on anti-adhesive properties of decorin was evaluated. Caruncular epithelial cells were isolated from healthy cows during pregnancy, immediately after slaughter. Primary cell cultures derived from the 2nd and 4th month of gestation were used in the experiments. The viability of cells was assessed by MTT assay. The adhesion of cells to fibronectin was measured spectrophotometrically. The activity of metalloproteinases was confirmed by the metalloproteinase assay. Progesterone (10^–5 and 10^–7 mol/L) and prostaglandin F_2α (10^–4_, 10^–5 and 10^–7 mol/L) increased the viability of bovine caruncular epithelial cells in the 2nd month of pregnancy. The treatment with prostaglandin F_2α significantly reduced the number of adherent cells from the 2nd month of gestation at the doses of 10^–4 and 10^–5 mol/L. Both progesterone and prostaglandin F_2α were shown to have an effect of decorin resulting in both a decrease in metalloproteinase activity and an increase in adhesion of cells to fibronectin.     

甘肃多儿国家级自然保护区维管植物区系分析

基于野外实地考察、植物标本的采集与鉴定以及结合文献研究的方法,分析了多儿保护区维管植物区系的物种组成和分布区类型。结果表明,该区维管植物共115科469属1323种,包括蕨类植物14科25属61种,裸子植物4科8属25种,被子植物97科436属1237种。多儿保护区维管植物区系优势现象明显,≥20种的优势科共879种,占该区维管植物总种数的66.44%;小属和单种属成分比例较大,占该区维管植物总属数的95.31%,总种数的75.51%。科级水平上,热带科（30.43%）稍多于温带科（26.96%）;属级水平上,温带科（72.49%）明显高于热带科（13.01%）;种级水平上,中国特有种有545种,其中538种与周边省份所共有,体现了该区系明显的过渡性和交汇性。该保护区维管植物区系地理成分复杂多样,属的分布有15个分布型和5个变型,温带地理成分占绝对优势,尤其以北温带分布为主。该区珍稀濒危野生植物较多,被列入保护名录有37种;一些古老、孑遗植物在该保护区具有良好的原始类群分布。本研究进一步完善了多儿保护区维管植物区系特征,为以后了解该保护区生物多样性提供可靠依据。