家兔睾丸间质细胞的分离纯化及原代培养

为了研究家兔睾丸间质细胞(Leydigcell,LC)的分离纯化程序及体外生长特点,试验采用4种不同消化程序对家兔睾丸组织进行消化,percoll梯度分离出兔LC,并进行体外培养。结果表明:经4℃保存和34℃孵育,0.5mg/mLⅠ型胶原酶消化40min获得的细胞数最多,经percoll梯度分离的细胞纯度达92%;34℃、5%CO2条件下细胞生长良好,呈现梭形和不规则形2种形状。     

奶牛乳腺上皮细胞和成纤维细胞的体外培养与鉴定

为体外培养纯化出稳定的奶牛乳腺上皮细胞和成纤维细胞,试验通过外科手术的方法取妊娠后期或泌乳期的荷斯坦奶牛乳腺组织,分离乳腺腺泡,用组织块法体外培养奶牛乳腺细胞,应用差时胰酶消化法和差速贴壁法将奶牛乳腺上皮细胞和成纤维细胞分别纯化出来,并用免疫组化的方法对细胞的纯度进行鉴定。结果表明:纯化的奶牛乳腺上皮细胞多为多角形,细胞核呈圆形或椭圆形,核仁清晰可见,多呈鹅卵石样或铺路石样生长,并可分泌乳滴,角蛋白-18反应阳性,波形蛋白反应阴性;纯化的奶牛乳腺成纤维细胞多为长梭形,呈旋涡状或放射状生长,角蛋白-18反应阴性,波形蛋白反应阳性;经纯化后2种细胞的纯度均可达95%以上,可满足后续试验的要求。     

In vitro differentiation of bovine bone marrow‐derived mesenchymal stem cells into male germ cells by exposure to exogenous bioactive factors

Mesenchymal stem cells (MSC) are multipotent progenitor cells defined by their ability to self‐renew and give rise to differentiated progeny. Previous studies have reported that MSC may be induced in vitro to develop into different types of specialized cells including male gametes. In vitro gamete derivation technology has potential applications as an alternative method for dissemination of elite animal genetics, production of transgenic animals and conservation of endangered species. This study aimed at investigating the in vitro effect of BMP4, TGFβ1 and RA on the potential for germ cell (GC) differentiation of bovine foetal MSC (bfMSC) derived from bone marrow (BM). The effect of BMP4, TGFβ1 and RA was analysed on the expression of pluripotent, GC and male GC markers on bfMSC during a 21‐day culture period. bfMSC cultured under in vitro conditions expressed OCT4, NANOG and DAZL, but lacked expression of mRNA of VASA, STELLA, FRAGILIS, STRA8 and PIWIL2. Treatment with exogenous BMP4 and TGFβ1 induced a transient increase (p < .05) in DAZL and NANOG mRNA levels, respectively. However, exposure to RA was more effective in increasing (p < .05) expression of DAZL and regulating expression of OCT4 and mRNA levels of NANOG. These data suggest that bfMSC may possess potential for early GC differentiation, where OCT4, NANOG and specially DAZL may play significant roles in controlling progression along the GC lineage.     

荣昌猪睾丸支持细胞的分离培养

睾丸支持细胞(sertoli cell,SC) 是生精上皮中唯一的体细胞,有着对各级生精细胞及精子提供支持、营养作用;能分泌多种生长因子、营养因子、免疫保护因子等多种生物活性蛋白;     

A long-term in vitro culture of bovine epithelial cells on collagen rafts

In the present work, we established and characterized a 3D functional polarized primary bovine oviduct epithelial cells (BOECs) culture on free-floating type I collagen hydrogels (rafts) at an air-liquid interface (ALI). Intercellular junctions, ultrastructural cellular morphology and the expression of the OVGP1 closely recapitulated those of the in vivo epithelium lining. These morphological and physiological epithelial cell features were maintained under standard DMEM/F12 with 10% foetal bovine serum culture medium for at least 28 days of ALI culture. The versatility of the BOECs raft cultures should allow testing of toxicity compounds, in vitro evaluation of physiological or pathological oviductal states, and the study of epithelial-mesenchymal interactions that are critical for the maintenance of oviductal homeostasis.     

LH release from dispersed bovine pituitary cells in culture: in vitro effects of estradiol and procedural variables

Procedures for cell dissociation and in vitro culture were validated to investigate secretion of luteinizing hormone (LH) from bovine anterior lobe (AL) pituitary cells. The concentration of trypsin used for dissociation affected cell yield, cell loss during preincubation, LH secretion, and response to gonadotropin-releasing hormone (GnRH). Optimum results were obtained with trypsin concentrations of 8-16 micrograms/mg fresh tissue. Duration of preincubation and of experimental culture markedly affected LH secretion and response to GnRH. Immediately after dissociation, cells contained relatively low quantities of LH, but they were able to release a substantial proportion of this LH. Basal release of LH and GnRH-induced release of LH were highly correlated with total quantities of LH, and all three parameters increased with time of preincubation until 24 hr. Experimental treatments of 2 hr duration were optimal for investigating GnRH stimulation of LH release, whereas longer treatments may be required to investigate effects of agents that inhibit the release of LH. Preincubation of dissociated AL cells with physiological concentrations of estradiol increased all three LH parameters. Progesterone had no effect either alone or in combination with estradiol. In conclusion, the procedures described for cell dissociation and culture of suspended cells provide a useful tool for studying release of LH from the bovine AL cell.     

昆明白小鼠胚胎生殖细胞的分离与培养

以昆明白品系小鼠胎儿生殖嵴为材料，以不同的培养液分离培养胚胎生殖细胞（EG cells），发现小鼠胎儿肝细胞条件培养液的效果好于未添加白血病抑制因子（LIF）的基础培养液，而差于添加了1000IU／mL LIF的基础培养液。同时，试验对比了从不同胎龄胎儿分离培养原始生殖细胞（PGCs）的情况，鉴定了EG细胞的生物学特性；EG细胞经体外培养，可以分化为神经样细胞、上皮样细胞和简单类胚体。     

Bovine ovarian stem cells differentiate into germ cells and oocyte‐like structures after culture in vitro

Stem cells have been isolated from ovaries, and their ability to differentiate into oocytes in vitro has been demonstrated for mice and human, but not for bovine species. The aims of this study were to isolate germline stem cells from bovine ovaries and to evaluate the effects of bone morphogenetic proteins (BMPs) 2 and 4, and follicular fluid on the differentiation of these stem cells into oocyte‐like structures. The ovarian stem cells were isolated and cultured in α‐MEM⁺ supplemented with BMP2, BMP4 or follicular fluid. On days 0 and 14, cells were evaluated for their morphological appearance, viability, expression of alkaline phosphatase and for markers of germ cell formation (VASA and DAZL) and oocyte development (GDF9, ZPA and SCP3) by qPCR. Levels of mRNA were analysed using ANOVA and Bonferroni test (p < .05). The results showed that at day 0, ovarian stem cells expressed specific markers of pluripotency (OCT4, SOX). In addition, these cells were positive for alkaline phosphatase, which is a marker commonly used to identify primordial germ cells (PGCs). After the period of differentiation, cells had morphological features that resemble PGCs and oocyte‐like cells (OLCs). An increase, ranging from five to 14 times, in the expression of VASA was observed in cells cultured in medium supplemented with BMPs and follicular fluid, while the increase in DAZL expression ranged from four to six times. In addition, OLCs had an increase in expression of mRNAs for GDF9, ZPA and SCP3 that ranged from two to eight times. In conclusion, OLCs can be differentiated in vitro from ovarian stem cells and BMPs and follicular fluid are effective in stimulating the expression of mRNAs for germ cell and oocyte markers.     

Isolation and culture of rabbit primordial germ cells

Primordial germ cells (PGCs) are embryonic precursors of the gametes of adult animals and are considered stem cells of the germline. Since their proliferation in vitro correlates well with the schedule of developmental changes in vivo, they might be interesting research tools for genomic imprinting, germ-cell tumors and fertility. Furthermore, once primordial germ cells are separated and placed on a feeder layer with cytokines, they become cultured pluripotent cell lines called embryonic germ (EG) cells. EG cells share several important characteristics with embryonic stem (ES) cells as they can also contribute to the germ line of chimeras. To investigate the characteristics of PGCs and establish rabbit EG (rEG) cells, we cultured rabbit PGCs (rPGCs) in vitro with various combinations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and forskolin on inactivated mouse embryonic fibroblast (MEF) feeder layers. The present study found PGC proliferation in early cultures and induction of rEG-like colonies. These cells expressed pluripotent markers, such as alkaline phosphatase activity, OCT-4, Sox-2 and SSEA-1, in the undifferentiated state; however, the cells did not develop into a teratoma when injected into the kidney capsules of SCID mice, although the restricted differentiation potentials to neural cells were determined via embryoid body formation. From these characteristics and further characterization of the germ stem cell markers Vasa, SCP-1 and SCP-3, we suggested that these were hybrid cells with characteristics somewhere between PGC and EG cells.     

贵州香猪睾丸发育中支持细胞和生精细胞数量变化观察

为了解香猪睾丸发育过程中生殖细胞和支持细胞数变化规律,用手术取出30,40,50,70,90和110日龄(每个年龄组n=3～4)香猪右侧睾丸,经中性多聚甲醛固定,石蜡包埋,组织切片采用免疫组化SP法,用单克隆抗体GATA-4检测睾丸支持细胞的特异生长转录因子-4,经DAB显色、苏木素复染。光镜下核呈棕色者为支持细胞,核呈蓝色者则为生殖细胞;经显微照相并用Scion image软件测量生精小管及管壁面积。结果:30～110日龄睾丸支持细胞数维持在稳定水平(P>0.05),而生殖细胞数随日龄增加而增多,70日龄生殖细胞数快速增多(P<0.05),持续到110日龄。同样,从70日龄开始睾丸生精小管和管壁面积显著性增大(P<0.05)。香猪睾丸支持细胞快速增殖发生在30日龄前,而生殖细胞数随着日龄的增长而增多。     

Generation of multinucleated giant cells in vitro from bovine monocytes and macrophages

The generation of multinucleated giant cells (MGC) from cells of the bovine monocyte-macrophage lineage was investigated. Freshly isolated monocytes were incubated with the conditioned medium (CM) of peripheral blood mononuclear cell cultures treated with Concanavalin A for 1-4 days (CM1 to CM4). Only CM1 generated MGC despite similar concentrations of IFNgamma in all CMs. Nevertheless, MGC formation from monocytes was enhanced by adding either macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF), MGC formations from macrophages were observed only when macrophages were cultured with GM-CSF plus CM. These results indicate that several mechanisms to generate MGC from bovine monocytes-macrophage lineage cells exist, and that GM-CSF is a major mediator of MGC formation in cattle.     

胰岛素、胰高血糖素对体外培养肝细胞DGAT2mRNA的影响

为阐明神经内分泌因子胰岛素、胰高血糖素在奶牛脂肪代谢过程中的调控作用,用荧光定量PCR方法检测胰岛素、胰高血糖素对肝细胞二酰基甘油酰基转移酶（DGAT2）mRNA丰度的影响。结果表明低浓度的胰岛素能促进脂肝细胞内DGAT2mRNA的表达;胰高血糖素抑制脂肝细胞内DGAT2mRNA表达。由此证明：胰岛素和胰高血糖素能直接调控肝细胞中的DGAT2基因mRNA的表达。     

山羊雄性胎儿生殖细胞建立干细胞系初探

体外培养山羊50~68日龄雄性胎儿生殖细胞,并检测它们的碱性磷酸酶(AP)活性和Oct-4蛋白,探讨性别分化后的生殖细胞用于建立干细胞系的可行性及检测指标。当山羊胎儿睾丸细胞体外培养时,生殖细胞及其来源的细胞克隆均呈AP阴性和Oct-4蛋白阴性,其中有部分细胞克隆表现为AP假阳性。山羊胎儿生殖细胞克隆呈隆突状生长,多为圆形,与周围细胞界限分明,但克隆内细胞间界限不清。细胞克隆至少可以培养3代以上。研究结果显示,山羊雄性胎儿生殖细胞可以用于建立生殖系来源的干细胞系;AP和Oct-4蛋白不适宜用来检测体外培养的山羊胎儿生殖细胞及其来源的细胞系。     

磺胺喹噁啉对小鼠睾丸的病理损害及生精细胞凋亡作用

按毒理学方法给雄性小鼠灌喂不同剂量磺胺喹噁啉,检测对小鼠睾丸的病理学损害,探讨其对雄性小鼠生殖器官产生的可能危害,采用DNA Ladder条带和TUNEL法检测了磺胺喹噁啉对生精细胞凋亡的影响。结果表明：1／30LD50组和1／20LD50组精原细胞、初级精母细胞、次级精母细胞和精子细胞基本正常,仅少部分核出现形状不规则的现象。而1／10LD50组和1／10LD50组则病理变化明显,表现为部分曲精小管上皮细胞减少、分层不明显;各级生精细胞细胞膜部分破损、有细胞质溶解现象,核体积皱缩或轮廓不清,核膜有溶解,细胞内线粒体嵴减少或外膜破损;成熟的精子细胞数量减少或从支持细胞上脱落。小鼠睾丸DNA Ladder条带检测结果表明磺胺喹噁啉对诱导生精细胞凋亡没有明显的作用,原位末端标记法检测磺胺喹噁啉各用药组小鼠生精细胞凋亡指数均有所增加,但较对照组差异不显著（P〉0．05）,也没有表现出明显的正相关变化。说明大剂量使用磺胺喹噁啉会对小鼠睾丸产生器质性的损害,但对睾丸生精细胞凋亡没有明显的诱导或抑制作用。     

牛乳腺上皮细胞的分离培养

为了对牛乳腺上皮细胞(MECs)进行分离、培养和鉴定,并研究细胞分泌功能,试验通过胶原酶消化法分离得到了牛乳腺上皮细胞,采用传代法对细胞进行纯化,对细胞标志蛋白进行免疫荧光染色鉴定,通过体外诱导和RT-PCR分析鉴定细胞的分泌功能。结果表明:分离到的牛乳腺上皮细胞具有典型乳腺上皮细胞的形态特征,表达广谱角蛋白,经诱导后可分泌β-酪蛋白。     

牛体外受精胚胎培养技术研究

在COCs体外培养过程中添加共培养物质和采用不同体积的液滴进行培养,以探讨牛卵母细胞的体外培养效果。结果表明,卵丘细胞、卵丘细胞+bFF共培养系统对胚胎发育均有促进作用,其中以卵丘细胞+bFF共培养系统对桑椹胚和囊胚的发育最好,其囊胚率为10.63%,显著高于对照组的7.23%(P<0.05),高于卵丘细胞组的8.50%(P>0.05)。用培养皿微滴培养(50μL)和用4孔板大体积培养(500μL)的试验表明,大体积培养比微滴培养有优势,桑椹胚和囊胚率均较高,其中囊胚率为10.23%,显著高于培养皿微滴培养的9.07%(P<0.01)。     

Characterization of the bovine secondary in vitro antibody response

In the present report, the bovine secondary in vitro antibody response to keyhole limpet hemocyanin is described. The induction of anti-KLH antibody was not dependent upon the presence of mitogen but was antigen specific (KLH vs ovalbumin). Furthermore, this response was dependent upon cell density (10(6) per well), antigen dose (1 to 10(-5) ug/culture) and time in culture (5 days). The antibody produced was specific for KLH as measured in several binding assays. An unresponsive state was detected with high concentrations of KLH (more than 10 ug per culture) which was not due to the formation of immune complexes but to the inactivation of B and/or T cells. The induction of the antibody response was dependent on the presence of macrophages (syngeneic or allogeneic) and their presence could not be replaced with 2-mercaptoethanol.     

Induction of bovine plaque-forming cells in vitro

A culture system was developed for the in vitro inoculation of bovine splenic lymphocytes with sheep RBC and trinitrophenylated proteins. The addition of pokeweed mitogen to the cultures was necessary for the induction of plaque-forming cells to the antigen. This culture system required the use of autologous serum and the addition of mitogen.