THE DIFFERENTIATING ABILITY OF RAT LENS EPITHELIAL CELLS IN CELL CULTURE

Dissociated cells of lens epithelia of adult rats were monolayerly cultured in vitro. After about 15–20 days' period of active cell growth, such characteristic structures that correspond to "lentoid bodies" described previously in chick cultures were formed. These structures consisted of elongated cells, ultrastructural profile of which was similar with lens fiber. The presence of gamma-crystallin, a marker molecule specific to mature lens fiber, was confirmed in these elongated cells by means of fluorescent antibody technique. The differentiation of lens fiber in vitro was also recognized in clones originating from single lens epithelial cells cultured at very low cell density.     

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.     

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.     

CELL SURFACE CHANGES DURING DEDIFFERENTIATION IN THE METAPLASTIC TRANSFORMATION OF IRIS INTO LENS

Changes at the cell periphery during the dedifferentiative phase of the metaplastic transformation of iris into lens have been studied in Notophthalmus viridescens and Taricha granulosa using cell electrophoresis. Cell surface charge density increases as early as 1–3 days after lens removal. Cells of regenerates at 10–15 days after lentectomy have significantly lower electrophoretic mobilities than those of the irises of nonlentectomized newts. Decrease in surface charge density is due, at least in part, to the loss of ribonuclease- and neuraminidase-sensitive groups from the cell periphery. Loss of negatively charged groups from the cell surface appears to occur as cells go through dedifferentiation. Loss of cell surface components also occurs in the cells of the ventral iris which also undergo dedifFerentiation but do not regenerate a lens.     

家兔晶状体纤维的超微结构:纤维细胞的间隙连接

本文报道晶状体纤维细胞间间隙连接的形态结构。我们利用冰冻断裂技术,在不同部位的球-和-凹连结的头部以及在纤维细胞和纤维细胞之间都观察到间隙连接的存在。通过极其丰富的上述连接,可实现细胞间代谢物和离子的传递。作者认为:对正常晶状体纤维细胞之间的间隙连接的深入了解,将会为晶状体发病机制的研究提供新的线索。     

CHANGES IN CELL FINE STRUCTURE DURING LENS REGENERATION IN XENOPUS LAEVIS

Changes at the level of cell fine structure have been studied during lens regeneration in the toad, Xenopus laevis, where cornea gives rise to the new lens. The transformation of these cells may be divided into three phases. (1) In the cornea, flattened cells become cuboidal and rough endoplasmic reticulum increases in amount. (2) In the new lens vesicle, cisternae of the rough ER break down into vesicles, smooth-walled vesicles and free ribosomes increase in number, and mitochondria can become enlarged and irregular, then centrally attenuated. Rudimentary cilia form. (3) As new lens fibers form, ribosomes become very numerous and low density fibrous elements and dense clumps appear in the cytoplasm. These phases are accompanied by marked nucleolar changes. The changes during the 3rd phase are similar to changes in the lens during normal development. The first two phases show an unexpected morphological complexity.     

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.     

COMPARISON OF THE GROWTH AND DIFFERENTIATION OF EPITHELIAL CELLS FROM NORMAL AND HYPERPLASTIC LENSES OF THE CHICK: STUDIES OF IN VITRO CELL CULTURES

Epithelial cells from hyperplastic lenses of a strain of chicks (Hy-1) selected for high growth rate were dissociated and cultured in vitro and compared with lens epithelial cells from a normal strain (N) in similar conditions. The hyperplastic lens cells showed remarkable motility and adhesiveness after dissociation and formed cell aggregates of various sizes before attaching to the substrate, giving a rather low plating efficiency. The lens structures (lentoid bodies) developed in partially confluent cultures of Hy-1 cells at least three days earlier than those in the cultures from normal control cells, in which the lens structures developed only after the cultures reached confluence. The results of culture at low cell density showed that the Hy-1 cell population consisted of at least two cell types different from each other in growth capacity. These striking differences in in vitro behaviour of dissociated cells from normal and hyperplastic lens epithelia and the results of clonal culture are discussed in relation to the possible mechanisms of abnormal morphogenesis and growth which are likely to be involved in the development of the hyperplastic lens in situ .     

GROWTH OF LENS AND OCULAR ENVIRONMENT: ROLE OF NEURAL RETINA IN THE GROWTH OF MOUSE LENS AS REVEALED BY AN IMPLANTATION EXPERIMENT

The lens of 6-day-old normal mouse was implanted into the lentectomized eye of adult mouse to examine the effect of retina upon the growth of the implanted lens in vivo. The implanted lens grew normally and its transparency was kept for more than 5 months after implantation. The connection between the implanted lens and the ciliary part of the recipient iris was well established with the regeneration of zonular fibers from the recipient. In young lenses implanted reversely into adult eyes, the epithelial cells facing the retina elongated and a new epithelium was formed on the corneal side of the lens within 5 days. Young lenses implanted either in normal or reverse orientation into eyes from which the retina was previously removed did not grow. The cells of the original lens epithelium of these lenses were randomly accumulated beneath the posterior lens capsule, while the anterior portion of the implanted lenses became an epithelial structure without cell elongation. These results suggest that the growth of the implanted lens may be dependent on the retina of the adult eye.     

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.     

COMPARISON OF NEURONAL AND LENS PHENOTYPE EXPRESSION IN THE TRANSDIFFERENTIATING CULTURES OF NUERAL RETINA WITH DIFFERENT CULTURE MEDIA*

The effects of three different culture media (Eagle's MEM, F-12 and L-15) on the transdifferentiation of 8-day chick embryonic neural retina into lens cells, were examined with respect to the expression of two phenotypes. One type referred to neuronal specificity (as represented by the level of cholineacetyl-transferase, CAT, activity) and the other to lens specificity (as represented by content of α-and δ-crystallin). In 7-day cell cultures before the visible differentiation of lentoid bodies, CAT activity was detected in all media. But, its level was about 9 times higher in cultures with L-15 than in those with MEM and 3 times higher than in F-12. In 26-day cultures, CAT activity was practically undetectable. The production of α-and δ-crystallin was detected in cultures at 26 days. There were quantitative differences in the crystallin content with different media, and it was highest in cultures with L-15. The results indicate that conditions most favourable to the maintenance of the neuronal specificity in cell cultures of neural retina, can also support the most extensive transdifferentiation. The possibility of direct transdifferentiation of once neuronally specified cells into lens cells in cultures with L-15 has been suggested to explain the present results.     

EYE LENS WEIGHT AND AGE IN AFRICAN ELEPHANTS

Eye lens dry-weights have been determined for 543 African elephants from three populations in East Africa. When plotted against estimated ages based on tooth replacement and wear criteria they indicate growth curves with rapid initial growth in lens weight, succeeded by a phase of rectilinear growth which apparently persists throughout life.
Parameters for the regressions of lens dry weight on age have been calculated by sex and locality. Confidence limits are fitted and no significant difference in the growth rates can be demonstrated, except for a sex difference in the values for the α intercept.
Variability at age is slightly greater in males than females, but is little greater than i3 indicated by studies on other species using known-age animals. This is taken to confirm the accuracy of the age criteria adopted and leads to conclusions on their precision.
It is suggested that this method might provide an objective check on the accuracy and precision of age estimates in other species.     

INDIVIDUAL VARIATION IN THE DISTRIBUTION OF LENS POTENCY IN WOLFFIAN LENS REGENERATION

After lentectomy in newts, lens regeneration originates from the iris. The regenerant was externally observed with a stereomicroscope as a depigmented area (DA) of the iris, and the extent of DA up to 15 days after lentectomy was measured. The extent of DA was found to differ among individuals, whereas it was the same in both eyes of each animal. In a number of animals one eye was used for lentectomy. After measuring the DA, two groups of animals were selected; a "W-group" with an extremely wide DA that deviated from the standard value, and "N-group", with an extremely narrow DA. Six iris sectors obtained from the animals of the W-group or N-group were implanted into lentectomized eyes of other animals to investigate the difference in the distribution of lens potency in these two groups. Animals of the W-group possessed a wider distribution of lens potency than animals of the N-group. Pulse-labelling with ³H-thymidine on lentectomized eyes of both groups was done 0, 3, 5, 7 and 12 days after lentectomy. DNA-synthesis began earlier and continued longer in the dorsal part of the iris of the W-group than in that of the N-group. The distribution of lens potency in the iris is discussed on the basis of these findings.     

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。     

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.     

大鼠脑神经干细胞系(RNSC-FMU 1)的建立和鉴定

采用无血清培养液分离和培养新生SD 大鼠脑的神经干细胞,以机械分散的方法传代,成功地建立了大鼠脑神经干细胞系(RSNC-FMU 1)。该细胞系可在体外长期传代,至今已在体外连续生长超过21个月(>100代),保持了神经干细胞的特性和正常的核型,经诱导可分化成为神经元、星形胶质细胞和少突胶质细胞,具有较旺盛的自新能力,倍增时间约为20h,并可冷冻保存,裸鼠体内移植证实其不具有成瘤性。该细胞系为神经干细胞研究提供了一个良好的工具。     

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。     

AN EVALUATION OF ERYTHROPOIESIS IN THE YOUNG RAT

Estimates of the cell population kinetic parameters have been obtained for the erythroid cells of the young growing rat using the technique of labelled mitoses and these results have been analysed by a computer programme. The phases of the cell cycle for the proliferating cells have been shown to be of shorter duration than generally reported. Together with the differential cell count and initial labelling index these data have enabled estimates of the growth fraction, birthrate, flow rate, number of divisions and transit time to be determined for each compartment.     

DIFFERENTIATION AND TRANSDIFFERENTIATION IN VITRO OF NEURAL RETINA FROM MUTANT CHICKENS WITH HYPERPLASIC LENS EPITHELIUM1)

Mutant chickens, Hy-1 and Hy-2, show abnormalities in growth and differentiation of the lens epithelium. In this study, neural retinal cells (NR cells) from 3.5-day-old embryos of these mutants were cultured, and the differentiation in vitro was compared with the cells of the normal strain. Hy-1 cells in vitro were characterized by a delay in the first appearance of neuronal cells (N-cells) and by excessive production of this cell type at later stages. By contrast, the Hy-2 cells were indistinguishable from the normal cells in the early phase of culturing. In spite of the marked difference of Hy-1 NR cells in neuronal differentiation up to about 7 days in culture, the transdifferentiation of lens and pigmented cells occurred to a similar extent and with the same time schedule as cultures of normal cells. A number of lentoid bodies were formed by about 10 days. The relative composition of the three major classes of crystallins in transdifferentiated lens cells was almost identical between normal and Hy-1 strains. The results were discussed in comparison with the previous results of cell culture of NR of 8-day embryonic mutant chickens, and it was concluded that the process of transdifferentiation in cell culture is different between NR from 3.5-day-old and 8-day-old embryos.