大鼠附睾上皮细胞的原代培养及纯化鉴定

目的建立大鼠附睾上皮细胞原代培养及纯化方法。方法利用酶消化法和组织块法对大鼠附睾上皮细胞进行原代培养,然后用胰酶两步消化法进一步纯化附睾上皮细胞,最后分别利用免疫荧光和免疫组织化学染色对原代培养的细胞及相关蛋白表达情况进行鉴定。结果酶消化法较组织块法得到的附睾上皮细胞纯度高,免疫荧光染色结果证明所得附睾上皮细胞主要是主细胞,免疫组织化学结果证明培养的附睾上皮细胞中有雄激素受体和雌激素受体α的表达。结论利用酶消化法对大鼠附睾上皮细胞进行体外培养,方法简单易行,成功率高。     

血-附睾屏障的电生理学及超微结构研究

用玻璃微电极记录大鼠跨附睾上皮细胞电位差。附睾头,附睾体和附睾尾的电位差分别为-5.61±0.42,-4.36±0.38和-4.12±0.23毫伏,腔内为负(均值±S.E.)。用接近生理渗透压(360 mOsm/kg·水),含2%硝酸镧示踪物的灌流液分别从大鼠精索内动脉和附睾管腔内灌流,观察到附睾上皮细胞之间的各种结构形式的连接复合体是血一附睾屏障的超微结构位点。证明小分子的硝酸镧可以通过五合式或纤维式的连接结构,但不能通过附睾上皮细胞间的桥粒复合体和间隙连接。并从生理学和形态学上均证明有血-附睾屏障的存在。棉酚处理后(30毫克/公斤体重/日),附皋的跨上皮电位差的绝对值降低。附睾头,附睾体及附睾尾分别为3.58±0.23,4.13±0.27和3.03±0.18毫伏,腔内为负。其中附睾头与附睾尾的电位变化有明显的统计学意义。附睾上皮细胞对硝酸镧的通透性也稍有增加。     

小鼠近端肾小管上皮细胞原代培养及鉴定

本文旨在建立更有效的小鼠肾小管上皮细胞体外培养及鉴定方法。将小鼠肾脏皮髓质分离,取肾皮质将其充分剪碎,用II型胶原酶消化结合筛网过滤的方法获得小鼠近端肾小管上皮细胞,细胞培养在DMEM中。用倒置显微镜观察细胞形态及生长情况,用流式细胞仪检测细胞增殖能力,用CCK-8检测方法测定活力,用免疫荧光方法鉴定肾小管上皮细胞的纯度。结果显示,免疫荧光鉴定显示95%以上的细胞表达上皮细胞标志蛋白CK18,90%以上的细胞表达近端肾小管上皮细胞标志蛋白Villin、AQP1和SGLT2。细胞可传至第五代,随着传代次数增加,细胞增殖能力逐渐降低。以上结果提示,本研究成功建立了培养高纯度小鼠肾小管上皮细胞的方法,用这一改良方法获得的肾小管上皮细胞可用于后续的离体实验研究。     

Hansson染色法检测小鼠附睾上皮细胞碳酸酐酶的活性

碳酸酐酶（Carbonic Anhydrase,CA）是一种含锌的金属酶,可参与体内CO2和离子转运以及调节酸碱平衡。本文以改良的Hansson法检测CA在雄性小鼠附睾管上皮细胞的分布,为深入研究CA表达及功能提供可行的形态学技术方法。迅速分离ICR雄性小鼠附睾,制成厚度为5μm的冰冻切片。     

胞外ATP在男性生殖道中的作用

胞外ATP除了能广泛作为神经递质外,还被认为是一种旁分泌或自分泌因子。ATP从男性生殖道中的精子或上皮细胞中释放,在调节各种生殖生理功能中起多种作用。本文综述了ATP调节附睾上皮细胞阴离子分泌的信号通路,阐述了ATP对依赖上皮细胞的输精管平滑肌收缩的调节机制,讨论了ATP在男性生殖道中的功能和作用。     

大鼠附睾钠钾离子主动转运的超微结构研究

在超微结构水平上研究大鼠附睾的离子转运机制,确定离子主动转运相关的钠-钾三磷酸腺苷酶(Na-K-ATPase)的分布。结果表明大鼠附睾上皮细胞具有二类NPP酶活性,即依赖于钾的,对乌巴因敏感的和对Levamisole有抵抗作用的K-NPP酶;不依赖于钾的,对乌巴因不敏感的和对Levamisole有抵抗作用的非K-NPP酶。K-NPP酶只分布在大鼠附睾各段和输出小管近段的主细胞基底膜与侧膜上,非K-NPP酶分布在主细胞的高尔基氏体、内质网膜和致密体上。附睾上皮细胞中的基底细胞、晕细胞和亮细胞均缺少酶的分布。低钾液附睾组织灌流和用棉酚处理动物,均使非钾-NPP酶活性增强,对K-NPP酶活性有抑制作用。     

2种气管黏膜上皮细胞体外培养技术的研究

目的为以猪气管黏膜上皮为细胞模型的研究奠定物质基础,进一步探讨猪气管黏膜上皮细胞的体外传统培养和气液界面培养技术,从而使2种培养技术优势互补。方法对猪气管上皮分离、纯化、培养和传代,并探索上皮细胞最佳冻存复苏条件;复苏后的气管上皮细胞进行气液界面培养,绘制细胞生长曲线和观察细胞纤毛生发情况。利用免疫组化法鉴定上皮细胞。结果4步纯化法可以得到高纯度的气管上皮细胞。使用胎牛血清、DMEM/F12培养液和DMSO的体积分数为50%、40%和10%的冻存体系保存的气管上皮,复苏后细胞存活率平均可达89%。优化后的传统方式培养的上皮细胞可连续传代到第8代,但从第2代开始便观察不到纤毛,转换成气液界面连续培养2代后重新生发纤毛,细胞存活期延长。免疫组化结果显示分离培养细胞为上皮细胞。结论成功建立了2种猪气管上皮细胞培养技术,并找到适宜的气管上皮细胞冻存条件,节省了不断原代取材的成本和时间,并成功实现传统培养细胞冻存复苏后很快适应气液界面的培养并恢复细胞的天然结构,为猪气管黏膜上皮相关研究提供丰富的细胞来源。     

高脂饮食对小鼠附睾内质网应激的影响及褪黑素的干预作用

目的探讨内质网应激在高脂饮食引起的ApoE基因敲除小鼠附睾损伤中的作用及褪黑素（MT）的干预机制。方法将12只ApoE基因敲除的C57BL／6J雄性小鼠随机分为高脂饮食组及MT处理组。高脂饮食组为ApoE基因敲除小鼠,给予高脂饮食;MT处理组给予高脂饲养外,并MT灌胃。以6只野生型C57BL／6J雄性小鼠作为对照组,给予普通饮食。饲养12w后,取附睾组织制片,HE染色观察附睾的病理学形态,免疫组化检测GRP78和CHOP的表达。结果HE染色显示,高脂饮食组小鼠,附睾上皮细胞形态结构不清,细胞萎缩。对照组和褪黑素处理组小鼠附睾上皮细胞形态结构完整,细胞排列整齐。免疫组化显示高脂饮食组小鼠附睾中GRP78、CHOP表达增强（P〈0．01）。MT处理组和高脂饮食组相比,附睾中GRP78、CHOP表达下调（P〈0．01）。结论内质网应激参与高脂饮食导致的附睾损伤;MT可能通过抑制附睾内质网应激,减轻高脂饮食对小鼠附睾的损伤。     

角膜内皮和角膜上皮的细胞培养研究

本文报告了用带塑料环的明胶培养膜培养角膜内皮细胞和角膜上皮细胞的结果。两种细胞在达到生长密集状态后,均具有正常眼内角膜内皮和角膜上皮细胞的形态特征。此方法可用于各种培养细胞的研究,特别是需分隔细胞层顶面和底面培养环境的实验及药物毒性等研究,使实验条件更接近于正常眼内的环境条件。     

小鼠和豚鼠精子在附睾成熟过程中Ca~(2 )分布变化规律的研究

小鼠附睾头精子,其头部Ca~(2 )在顶体前区顶体外膜内侧结合最多,Ca~(2 )沉淀反应颗粒于该处呈连续层状。附睾头豚鼠精子其头部结合Ca~(2 )含量很少,且主要结合于顶体前区腹面顶体外膜内侧。小鼠附睾体和附睾尾精子Ca~(2 )的分布特征基本上和附睾头精子相同。但豚鼠附睾尾精子顶体外膜内侧无Ca~(2 )结合。和附睾头、附睾尾的附睾液相比,附睾体附睾液基质内具有大量Ca~(2 )存在。附睾体柱状上皮细胞的微绒毛切面上也具有Ca~(2 )沉淀反应颗粒,微绒毛可能与附睾液Ca~(2 )含量的调节有关。精子尾部Ca~(2 )主要分布于线粒体内,在质膜内、外两侧和线粒体外膜外侧也结合有少量的Ca~(2 )。和小鼠精子相比,豚鼠精子尾部线粒体内具有大量的Ca~(2 )。     

Isolation, culture and characteristics of epididymal epithelial cells from adult cats

A tissue-culture system in which cells retain defined ultrastructural and functional characteristics was established to provide a basis for functional investigations of the epididymal duct in the cat. A widely used culture protocol for rat epididymal epithelium was used as a starting point and subsequently modified. The cellular population of the cat's epididymal epithelium was isolated by successive collagenase and trypsin digestion. A high yield of isolated cells obtained with good viability, were cultured in DMEM/F12 medium supplemented with foetal bovine serum, in absence or in presence of additional dihydrotestosterone (1 nM). The plated primary cultures reached confluence within 5-8 days, producing a monolayer of cohesive cells. Samples taken after 6 days in culture were processed for transmission and scanning electron microscopies. Immunocytochemical staining was used to estimate the purity of the epithelial cell population in the monolayers. The cell cultures displayed several functional traits of in vivo epithelia, including [35S] hypotaurine and [35S] taurine production. These results demonstrate that primary cultures of epididymal epithelial cells isolated from sexually mature cats maintain several differentiated characteristics of the intact organ and therefore provide a valuable system for the study of epididymal epithelial cell functions, metabolic activities and their regulation in cats.     

Structural and functional aspects of cultured epididymal epithelial cells isolated from pubertal rats

Acidic epididymal glycoprotein (AEG) and androgen-binding protein (ABP) antisera were used to study functional activities of primary cell cultures of the epididymal epithelium of 20--23-day-old rats. Extensive AEG immunoreactivity was associated with almost all epithelial cells of the distal caput, corpus and cauda epididymidis. ABP immunoreactivity was solely confined to some epithelial cells of the caput epididymidis. AEG and ABP immunoreactive cells were identified as principal cells. Morphological studies of enzymically dispersed aggregates of the epididymal epithelial cells showed that stromal cells were satisfactorily removed and that cell aggregates consisted of a predominant population for cells displaying the morphological characteristics of principal cells. Scanning and transmission electron microscopic studies of cultured epididymal epithelial cells in monolayers demonstrated that microvilli and pit-like invaginations of the cell surface were preserved during the first 7--10 days of culture and then gradually disappeared. Other characteristic subcellular structures such as Golgi apparatus and rough endoplasmic reticulum cisterna were preserved. Electrophoretic analysis of [35S]methionine-labelled secretory polypeptides released by epididymal epithelial cells into the culture medium demonstrated a distinct protein band pattern which differed from that observed in the medium of cultured rat Sertoli cells. These results demonstrate that primary cultures of epididymal epithelial cells isolated from sexually immature rats maintain several differentiated characteristics of the intact organ and therefore provide a valuable system for the study of epididymal epithelial cell function.     

Isolation and characterization of epididymal epithelial cell plasma membranes

Sperm maturation and storage occur in a unique milieu created in large part by the epididymal epithelium. To learn more about the interaction of the epididymal epithelial cell with both luminal and systemic environments, we now report on the preparation and characterization of epididymal epithelial cell plasma membranes. A preparation enriched for epididymal epithelial cell plasma membranes was isolated from collagenase-digested epididymal tubule fragments by hand-Dounce homogenization, differential centrifugation, and sucrose gradient centrifugation. The final membrane fraction was enriched 11-fold for the plasma membrane marker 5'-nucleotidase; 2.6-fold for the lysosomal marker acid phosphatase, and 3-fold for the Golgi marker thiamine pyrophosphatase. No enrichment was observed for mitochondrial or endoplasmic reticulum enzyme markers. Specific and saturable transferrin-binding activity was also detected in the final preparation. Electron microscopy revealed the presence of vesicles and sheets of membranes as well as an occasional Golgi apparatus. The plasma membrane fraction was used to generate monoclonal antibodies. Of 102 wells exhibiting growth, 12 were positive by immunofluorescent staining of frozen sections. Ten of these recognized determinants in epithelial cells, and 2 stained peritubular smooth muscle cells. Most of the epithelial cell-specific antibodies stained brush border alone or in combination with the basolateral plasma membrane. Three antibodies stained the Golgi apparatus. Most antibodies were specific for particular epididymal regions, 3 also recognized determinants in the kidney, and 1 stained residual bodies in the testis.     

Rat epididymal epithelial cells and 17beta-estradiol synthesis under hCG stimulation in vitro

Epithelial cells of human and animal epididymis display features of steroidogenic cells. Rat epididymal epithelial cells in vitro produce androgens which are converted to 17beta-estradiol, and released into the medium. The regulation of the epididymal steroidogenesis is not fully understood but it could be expected that it remains under LH influence. In previous study we observed that the morphology of rat epididymal epithelial cells in vitro was affected by hCG and the increase of amount of lipid droplets, glycogen and PAS-positive substances was observed. The present studies show the organelles which take part in synthesis of steroids in rat epididymal epithelial cells in vitro and the effect of hCG on E2 synthesis. The cells were cultured in the medium with/without DHT and without DHT in supplementation with hCG. After hCG stimulation the amount of an active mitochondria were increased when compared to the amount of mitochondria in the epididymal epithelial cells cultured without DHT. Ultrastructure of the cells was similar to the cells cultured with DHT, while the cytoplasm of the cells cultured without DHT was disorganized. The synthesis of 17beta-estradiol was stimulated by hCG, that exerted its effect through LH/hCG receptors, localized in the epididymal epithelial cells.     

Distribution of a proteinase inhibitor of epididymal origin in the tissues and secretions of the male reproductive tract of mice

Monoclonal antibodies to a low molecular weight, acid-stable acrosin-trypsin inhibitor isolated from epididymal homogenates were used to localize the inhibitor in tissues and secretions of the male reproductive tract of mice. The inhibitor, identified by indirect immunofluorescence, is present in the testes and in the apical portion of the epithelial cells from the caput region of the epididymis of both intact and efferentiectomized animals. Sperm isolated from the testes and caput epididymal region show inhibitor-positive fluorescence on the anterior acrosomal region. The inhibitor could not be localized on ductus or electroejaculated sperm or ductus sperm previously incubated in a purified inhibitor solution. Furthermore, the inhibitor was not visible in the copulatory plug of recently inseminated animals or on sperm recovered from the uterus or from an artificial capacitating medium. The inhibitor could be detected by an enzyme-linked immunosorbent assay in the supernatants of detergent treated or frozen-thawed caput sperm but not of ductus sperm. The data suggest that the inhibitor, made in both the testes and caput epididymis where it associates with the sperm, is lost from the sperm or irreversibly masked during the epididymal sojourn.     

Possible involvement of microtubules and microfilaments of the epididymal epithelial cells in 17beta-estradiol synthesis

The rat epididymal epithelial cells revealed features of steroidogenic cells and released 17beta-estradiol (E2) into the culture medium. In steroidogenic cells, elements of the cytoskeleton due to their influence on organelle distribution are implicated in the regulation of steroidogenesis. In the present study, the morphology of cultured epididymal epithelial cells in light, scanning and transmission electron microscopes was evaluated. The organization of microtubules and microfilaments revealed by fluorescence microscopy, and the concentration of E2 in cultured medium were also studied. The epididymal epithelial cells were cultured in different conditions: in the medium with or without exogenous testosterone (T) and in the co-culture with Leydig cells as a source of androgens. The cells in co-culture located close to Leydig cells were rich in glycogen, PAS-positive substances and lipid droplets, in higher amount than the cells cultured with addition of exogenous testosterone. Stress fibers and microtubules of epididymal epithelial cells cultured with exogenous T and in co-culture with Leydig cells presented typical structure, and numerous granular protrusions appeared on the surface of the cells. Disorganization of microtubules and shortening of stress fibers as well as the smooth cell surface deprived of granular protrusions were observed in the epididymal epithelial cells cultured without T. Change of the cytoskeleton organization caused by the absence of androgen in culture medium resulted in an increased E2 secretion.     

Phospholipid content and lamellar structures in the epididymal epithelial cells of rats treated chronically with lead acetate [Pb(II)].

The electron-microscopic observations accomplished covered epididymal epithelial cells of rats receiving lead acetate for five times longer than the duration of one spermatogenesis. These cells were found to possess a large number of vacuoles and conglomerates containing plicated membranes or tightly packed myelin-like lamellar formations. Further observations also revealed the formation of lamellar structures in mitochondria, dilatation of cisternae in the Golgi apparatus, and increased phagocytosis of spermatozoa by epithelial cells. The presence of a large amount of membranous material correlated with the increased content of phospholipids in epididymal epithelial cells. It may be suggested that the presence of such a great quantity of lamellar structures in epididymal epithelial cells of rats treated chronically with lead is the result of several processes, including the augmented synthesis of membranes associated with encircling the deposits of lead, autophagy in the cells, as well as intensified phagocytosis of spermatozoa.     

In vitro culture of epithelial cells from the caput, corpus, and cauda epididymis of Sus domesticus

This work describes a protocol to culture epididymal epithelial cells from the caput, corpus, and cauda regions of Sus domesticus. Epididymal epithelial fragments were obtained by dissection and enzymatic digestion with collagenase. About 30 epididymal fragments from each epididymal region were cultured in 24-well culture plates with supplemented RPMI-1640 medium at 37 degrees C, 5% CO2 in air, and 100% humidity. A confluent monolayer of polygonal and tightly packed epithelioid cells from the three epididymal regions was obtained after 12-16 days in culture and maintained in vitro for more than 60 days. The proportion of epididymal epithelial cells in these cultures was assessed by immunofluorescent staining for cytokeratins. Throughout the 2 months of culture, about 80% of the cells were cytokeratin-positive. Electron microscopy observations indicated that cultured cells from caput, corpus, and cauda epididymal regions were tightly adhered to each other by junctional complexes and that stereocilia were present in their apical membranes. Moreover, the presence of an extensive rough endoplasmic reticulum, Golgi apparatus and numerous vesicles in the cytoplasm suggested that cultured cells maintained secretory and absorptive activities. These results show that the epididymal epithelial cells in culture from S. domesticus retain some fundamental features that characterize the epididymal epithelium in the intact organ. This system might be a valuable tool for studying the mechanism of sperm maturation in vitro, including epididymal cell secretions and the analysis of regional differences.     

Steroidogenic characteristics of in vitro cultured epididymal epithelial cells of the rat

The paper presents the steroidogenic features of cultured epithelial cells of rat epididymis and their ability to synthesize steroid hormones. The cytoplasm of epididymal epithelial cells accumulated lipid droplets and contained active enzymes of steroidogenesis. Numerous mitochondria with lamellar cristae occurred near lipid droplets. Frequently, mitochondria formed a direct contact with lipid droplets and smooth endoplasmic reticulum. The hormone assay showed that the epididymal epithelial cells cultured without dihydrotestosterone synthesized and released the following steroids: dehydroepiandrosterone (DHEA), testosterone (T) and 17beta-estradiol (E). The levels of DHEA and T were very low. The concentration of E detected in media of cultured epididymal epithelial cells exceeded many times the concentration of E in control media. The cytoplasmic presence of organelles and enzymes that participated in the steroid synthesis indicated their similarity to steroidogenic cells. Epididymal epithelial cells were capable of moderate in vitro synthesis of androgens. It cannot be excluded that steroidogenesis in the cultured epididymal epithelial cells is maintained to sustain 17beta-estradiol synthesis pathways.