DIFFERENTIATION OF RAT LENS EPITHELIAL CELLS IN TISSUE CULTURE (III) FUNCTIONS IN VITRO OF A TRANSFORMED RAT LENS EPITHELIAL CELL LINE

Epithelial cells isolated from one-day-old rat lens were neoplastically transformed using Rous Sarcoma Virus (Schmidt-Ruppin-D strain) and have been in continuous culture for over 30 months. This cell line displays some of the characteristics of lens epithelial cells, but can be shown to represent a permanent cell line in that it has been transferred more than 50 times and is continuing to grow, while primary lens cell cultures have never survived beyond 8 weeks. The infecting viral genome can be rescued from the cell line upon fusion with uninfected chick embryo cells. It is anticipated that this newly established cell line, designated RLE-R, will be useful in studies on cellular differentiation.     

DIFFERENTIATION AND DEDIFFERENTIATION OF RAT LENS EPITHELIAL CELLS IN SHORT- AND LONG-TERM CULTURES

The crystallin synthesis of rat lens cells in cell culture systems was studied in relevance to their terminal differentiation into lens fibers. SDS-gel electrophoresis combined with several immunological techniques showed that γ-crystallin is a fiber-specific lens protein and is not localized in the epithelium of either newborn or adult lenses. When lens epithelial cells of newborn rats were cultured in vitro , α-crystaIlin was detected in many, but not all, of cells cultured for 10 days. Cells with α-crystallin gradually changed their shape into a flattened filmy form and finally differentiated into lentoid bodies. The differentiation of lentoid bodies was also found in cultures of epithelial cells obtained from adult lenses. The molecular constitution of lentoid bodies was the same as that of lens fibers in situ . The differentiation of lentoid bodies occurred successively for 5 months in cultures of lens epithelial cells. Most of the proliferating cells, however, lost α-crystallin during the culture period. Thereafter, they did not show any sign of further differentiation into lens fibers. Four clonal lines were established from these cells. One protein which is specific to the lens epithelium and the neural retina in situ (tentatively named as β_u-crystallin) was maintained in all lines, suggesting that some specific properties of ocular cells remain in the lined cells.     

A SURVEY OF DEHYDROGENASES IN VARIOUS EPITHELIAL CELLS IN THE RAT

Several different epithelial elements that have intense active transport or protein secretory functions were histochemically assayed in several dehydrogenase media by a recently perfected method. The mitochondria represented the only site of activity, not only when tested in the succinate and D-β-hydroxybutyrate media, but also when tested in the lactate, malate, and isocitrate media. The reaction for D-β-hydroxybutyric dehydrogenase in the mouse kidney was curiously limited to the mitochondria of the distal segment of the proximal convoluted tubule, a finding that most convincingly shows that dehydrogenase activity may be differentiated in certain instances from diaphorase activity by the ditetrazole methods and that D-β-hydroxybutyric dehydrogenase is not present in all mitochondria. Tetranitro-BT is favored over nitro-BT in studies conducted on most organs prepared without fixation and on formalin-fixed tissues that consist of lipid-containing or active transport cells.     

大鼠附睾上皮细胞的识别

本文利用免疫荧光技术,用角蛋白和波形蛋白抗体作为组化探针,对不同年龄大鼠的整体和离体培养的附睾上皮细胞进行鉴定,区分出结缔组织成分。并证明用0.05%胰蛋白酶和0.1%胶原酶相继消化附睾管可得到较丰富的上皮细胞,体外培养能维持10d 以上,在培养过程中显示其形态特征。因此本文所用的分离、培养方法所得到的附睾上皮细胞可作为深入研究其功能的一种模型。     

PROLIFERATIVE BEHAVIOR OF DIFFERENTIATING CELLS IN THE DEVELOPING RAT PAROTID GLAND

Parotid glands of litters of rats at age intervals from 20 days in utero to 100 days were assayed for DNA content and examined by light- and electron-microscopy. The age differences in total DNA and DNA concentration indicated that there was a rapid rate of proliferation of parenchymal cells until 25 postnatal days, after which the rate declined rapidly, and that there was a rapid increase in cell size between 18 and 25 days. These findings were substantiated by histologic observations, such as the presence of numerous mitotic figures until 25 days of age, and the rapid maturation of the acinar cells between 18 and 25 days. These data suggest that the acinar cells of the rat parotid gland comprise an expanding cell population. Light- and electron-microscopic observations consistently indicated that cells with mitotic figures were about as well differentiated as other parenchymal cells at all stages of gland development, including mature acinar cells observed at ages 23 and 25 days. These observations support the view that the division of cells in advanced stages of differentiation may be important in the growth of certain organs and tissues.     

分步消化法原代培养鼠阴道黏膜上皮细胞的研究

目的探讨大鼠阴道黏膜上皮细胞的体外培养和扩增技术,为构建组织工程化阴道动物模型提供种子细胞。方法取大鼠阴道全层组织,经Dispase酶和胰酶分步消化后,接种于无血清角化细胞培养液中连续培养,观察细胞形态、体外生长特性和超微结构,绘制生长曲线,免疫组化鉴定。结果原代细胞培养24-36 h后开始贴壁,7-10d约80%融合,呈铺路石样外观,可连续传5-6代;扫描电镜下细胞表面可见微绒毛嵴;角蛋白染色阳性,细胞纯度98%;第五代细胞为正常二倍体核型。结论该方法培养的阴道上皮细胞增殖状态良好,细胞纯度高,扩增迅速,可在较短时间内获得大量细胞用于组织工程学研究。     

大鼠前列腺上皮细胞在无血清培养液中增殖的调节

本文报道了未成年大鼠前列腺上皮细胞的无血清培养模型的建立方法,并利用该模型研究了生长因子、催素及性激素对前列腺细胞增殖功能的作用。结果表明：胰岛素、表皮生长因子、转铁蛋白、霍乱霉素和牛垂体提取物对前列腺细胞的增殖有直接的促进作用,糖皮质激素对细胞的增殖无明显影响。催乳素刺激细胞的增值,但与双氢睾酮或雌二醇无协同作用。单独的双氧睾酮或雌二醇对细胞的增殖无显著作用。     

IN VITRO DIFFERENTIATION OF CHICK EMBRYONIC LENS EPITHELIAL CELLS INTO FIBER CELLS

The processes of fiber-cell formation in the lens epithelium of 9-day-old chick embryo in vitro were studied.
Mitotic activity was enhanced during the first 12 hr, but with a drop at the 4th hour of cultivation. After the 24th hour, when the cells began to elongate, almost no mitotic figures or incorporation of ³H-thymidine into the nuclei were observed.
α- and δ-crystallin were contained in and synthesized by the newly isolated lens epithelium. The content and syntheses had diminished by the 12th hour.
In the earlier phase of cultivation, both fiber cell formation and crystallin synthesis were suppressed by treatment with Actinomycin D, but after the 12th hour they were resistant to the antibiotic.
The correlation between cell division and fiber-cell differentiation in the lens epithelium in vitro is discussed and compared with that reported in Wolffian lens regeneration and in developing bovine lens.     

新生大鼠小肠上皮细胞分离培养研究

本实验比较了4种分离大鼠IEC的方法,结果显示联合应用粗胶原酶和中性蛋白酶分离效果最好,细胞贴壁生长能力强。胶原涂膜改善玻璃培养瓶或盖玻片表面的性状有利于细胞贴壁生长。细胞的增殖依赖于培养液的质量、成分及细胞间的相互作用。培养细胞一般1～2天贴壁,7～8天明显增殖,10～14天汇合成片。培养细胞细胞角质蛋白、碱性磷酸酶染色阳性,光镜和电镜检查均显示为IEC。本文所建立的新生大鼠IEC体外培养方法为研究IEC生理和病理提供了一个十分有用的实验模型。     

AN ANALYSIS OF DIFFERENTIATIVE CAPACITY OF PIGMENTED EPITHELIAL CELLS OF ADULT NEWT IRIS IN CLONAL CELL CULTURE

Singly dissociated cells from dorsal and ventral iris epithelia ( iris iridica ) of adult newts were cultured separately at clonal density to analyse their growth and differentiative capacity. Usually some attached cells began to proliferate on 12th day of culture, and grew with loss of melanosomes to form clonal cell colonies. Up to 30 days after inoculation, most of the clonal colonies formed typical epithelial monolayer sheets which consisted mostly of nonpigmented cells. Then, in some of those colonies, cells piled up together and form typical lens structures containing lens antigens. A month and a half after culturing, 30 to 40% of single iris cells, which had been previously marked, grew to form clonal colonies consisting of more than 100 cells. About 30% of these colonies expressed lens specificity and no significant differences in efficiency of colony formation and differentiation were detected between the dorsal cells and the ventral, suggesting that potent cells capable of transdifferentiating into lens cells are evenly distributed in all parts of the newt iris epithelium.     

CELL POPULATION KINETICS OF THE RECOGNIZABLE ERYTHROID CELLS IN THE RAT

The cell population kinetic parameters defining a simple model of the recognizable part of the erythroid system have been determined. Experimental results using tritiated thymidine and radioactive iron autoradiography have provided estimates of the number of cell divisions, transit times and flow rates for all the recognizable stages of the erythroid system. The accuracy of the estimates and the validity of the model employed are discussed.     

兔输卵管上皮细胞解除大鼠早期胚胎发育阻滞的研究

本研究利用兔输卵管上皮细胞(ROEc), 与异种动物大鼠的受精卵共培养,结果大量出现突破2细胞发育阻滞的现象。体外受精卵和体内受精卵的2细胞发育阻滞突破率分别为62%和73%;利用ROEC 条件培养液培养大鼠的体外受精卵,2细胞发育阻滞的突破率达68%,且能顺利发育至桑椹胚和囊胚。将ROEC直接培养在含~(35)S-甲硫氨酸的CZB 培养液中,经SDS-聚丙烯酰胺凝胶电泳及放射自显影,发现在该条件培液中出现了分子量分别为135Kd,68Kd,55Kd,51Kd 和44Kd 由ROEC 分泌的多肽,其中68 Kd 区带最显著,而135 Kd区带非常弱。用(125)~I-酰化剂标记示踪发现,与ROEC 共培养24 h 的大鼠胚胎透明带上含有68Kd 蛋白,并发现55 Kd 蛋白与透明带结合的痕迹。这些蛋白很可能就是胚胎发育调控由母型向合子型过渡并突破早期发育阻滞的关键因子,在功能上似无种属特异性。     

FINE STRUCTURE OF DIFFERENTIATING MOUSE PANCREATIC EXOCRINE CELLS IN TRANSFILTER CULTURE

Fine structural observations have been made in the 11-day embryonic mouse of exocrine cells in pancreatic epithelium developing in tissue culture transfilter from salivary gland mesenchyme of the 13-day embryonic mouse. After 2 days in culture, the exocrine cells show increased cytoplasmic density, abundant ribosomes in aggregate or "rosette" form, and expanded profiles of rough-surfaced endoplasmic reticulum. After 3 and 4 days in culture, the cells exhibit continued expansion of the profiles of endoplasmic reticulum, increased amounts of Golgi membranes, and large areas of light density (prozymogen granules). After 5 days in culture, dense zymogen granules are present in the most highly differentiated cells. In addition, at the filter-epithelial surface, at 2 days, small fibers can be discerned which, after 4 days in culture, show obvious periodicity and are thought to be collagen. The significance of these changes, in relation to the mesenchymal effect, to the onset of specific synthesis and to the stabilization of differentiation is discussed.     

AGE-DEPENDENT CHANGE IN THE TRANSDIFFERENTIATION ABILITY OF CHICK NEURAL RETINA IN CELL CULTURE*

We examined how the transdifferentiation ability of neural retinal cells into lens and/or pigment cells in call culture is changed with the development of the donor. Cells dissociated from neural retinas of chick embryos ranging from 3-day-old to the stage immediately before hatching and of 3-day-old chicks were cultured for about 60 days. The results clearly indicated that the transdifferentiation ability decreased with age. The latest developmental stage at which the differentiation of lens cells took place was in 18-day-old embryos. A gradual decrease in this ability was shown by the comparison of crystallin content in cultures prepared from embryos at different stages. The differentiation of pigment cells was recognized in cultures of neural retinas earlier than in 15-day-old embryos. Such loss of the ability of neural retinal cells to transdifferentiate into pigment cells earlier than into lens cells can be partially attributed to inhibitory factors accumulated in medium conditioned with many neuronal cells present in cultures.     

A DEMONSTRATION OF LENS FORMING-CELLS IN NEURAL RETINA IN CLONAL CELL CULTURE

Clonal cultures with 1,000–3,000 cells were prepared from cells harvested from high density cultures of neural retina of 8-day-old chick embryos. About 1.14% and 0.31% of inoculated cells developed into recogniziable colonies in Eagle's MEM and in Ham's F-12 supplemented with fetal calf serum respectively. Of these colonies, lentoid bodies of authentic lens nature were differentiated in 10% and 33.52% in MEM and F-12 respectively. Cells harvested from high density cultures of the anterior and posterior portions of the neural retina were clonally cultured. Plating efficiency was much higher in the anterior cells than in the posterior ones and clonies with lentoid differentiation were developed only in clonal cultures of the anterior cells.     

小鼠颌下腺上皮细胞的培养及表皮生长因子的鉴定

体外培养小鼠颌下腺细胞,形态学观察可见有上皮样细胞生长。免疫细胞化学及蛋白质印迹转移分析结果表明,体外培养的颌下腺上皮样细胞可合成并分泌表皮生长因子。     

EPITHELIAL CELL MIGRATION IN THE INTESTINE

Despite significant advances in our understanding of the roles of the cytoskeleton and matrix receptors in cell locomotion, derived largely fromin vitrostudies on the movement of epithelial cell sheets and isolated cells, the mechanism of epithelial cell migration in the adult intestine remains an enigma. The primary function of the epithelial cell cytoskeleton seems to be in the maintenance of the apical region of the epithelium facing the gut lumen. There we find the brush border, with its associated enzymes, and the intercellular adhesion complexes that give the epithelium its cohesiveness and its barrier function. Curiously, there is little in the way of an organized cytoskeleton in the basal region of the epithelium adjacent to the basement membrane on which the epithelium is presumed to migrate. In this short review, I focus on what is known about epithelial migration from our understanding of the structure of the epithelium and from studies on wound healing, and indicate some avenues for future study.     

EFFECTS OF PUROMYCIN ON THE STRUCTURE OF RAT INTESTINAL EPITHELIAL CELLS DURING FAT ABSORPTION

This report provides information on the morphology of rat intestinal epithelial cells during fat absorption. In addition, the role of protein metabolism in this process has been evaluated by blocking its synthesis with puromycin and studying the fine structure of mucosal cells from rats at various times after fat intubation. The results indicate that SER-derived vesicles, containing fat droplets, migrate from the apical cytoplasm of the absorptive cell and fuse with saccules or vacuoles of the Golgi complex. Arguments are made that the Golgi complex is important in completing chylomicron formation and in providing appropriate enveloping membranes for the chylomicron. Such membranes may be necessary for Golgi vacuoles to fuse with the lateral cell membranes and release chylomicra. Puromycin treatment causes the absorptive cell to accumulate increased quantities of lipid that are devoid of membrane during fat absorption. In addition, puromycin-treated cells contain much less RER and Golgi membranes are strikingly decreased in number. In this paper we discuss the consequences of these abnormalities and suggest that continued protein synthesis by the RER is required in order to generate Golgi membranes. If such membranes are absent the cell's ability to discarge chylomicra is impaired and lipid accumulates.