REGENERATIVE PROLIFERATION OF MOUSE EPIDERMAL CELLS FOLLOWING ADHESIVE TAPE STRIPPING

The proliferating cells of mouse epidermis (basal cells) can be separated from the non-proliferating cells (differentiating cells) (Laerum, 1969) and brought into a mono-disperse suspension. This makes it possible to determine the cell cycle distributions (e.g. the relative number of cells in the G^ S and (G₂+ M) phases of the cell cycle) of the basal cell population by means of micro-flow fluorometry. To study the regenerative cell proliferation in epidermis in more detail, changes in cell cycle distributions were observed by means of micro-flow fluorometry during the first 48 hr following adhesive tape stripping. ³H-TdR uptake (LI and grain count distribution) and mitotic rate (colcemid method) were also observed. An initial accumulation of G₂ cells was observed 2 hr after stripping, followed by a subsequent decrease to less than half the control level. This was followed by an increase of cells entering mitosis from an initial depression to a first peak between 5 and 9 hr which could be satisfactorily explained by the changes in the G₂ pool. After an initial depression of the S phase parameters, three peaks with intervals of about 12 hr followed. The cells in these peaks could be followed as cohorts through the G₂ phase and mitosis, indicating a partial synchrony of cell cycle passage, with a shortening of the mean generation time of basal cells from 83-3 hr to about 12 hr. The oscillations of the proportion of cells in G₂ phase indicated a rapid passage through this cell cycle phase. The S phase duration was within the normal range but showed a moderate decrease and the Gj phase duration was decreased to a minimum. In rapidly proliferating epidermis there was a good correlation between change in the number of labelled cells and cells with S phase DNA content. This shows that micro-flow fluorometry is a rapid method for the study of cell kinetics in a perturbed cell system in vivo.     

REGENERATIVE PROLIFERATION OF MOUSE EPIDERMAL CELLS FOLLOWING APPLICATION OF A SKIN IRRITANT (CANTHARIDIN)

The strong skin irritant cantharidin dissolved in benzene was applied to the back of hairless mice. Single cell suspensions of epidermal basal cells were obtained and flow microfluorometric measurements of cellular DNA content were made. Smears were made for autoradiography, and the [³H]TdR labelling index (LI) and mean grain count (MGC) were assessed up to 3 days after cantharidin application. Three successive peaks of cells with S phase DNA content accompanied by three LI peaks were observed. The first two peaks were follwed by peaks of cells in G₂ phase, indicating that after the acute cell injury caused by cantharidin the cells traversed the cell cycle in partial synchrony through two subsequent cell cycles, each of 10–12 hr duration. During this phase of rapid proliferation the LI reached the proportion of cells in S phase, contrary to what is observed in untreated mouse epidermis, where the labelled cells contribute to about half the proportion of cells with S phase DNA content. The first two peaks of cells in S phase and LI coincided with an increased MGC, whereas the third peak was accompanied by a MGC significantly below control values. This indicates that this latter peak is due to a longer DNA synthesis time rather than to a partially synchronized and increased cell proliferation. The duration of the G₁, S and G₂ phases seems to be reduced initially in rapidly proliferating epidermis.     

THE STATHMOKINETIC METHOD IN VIVO TIME-RESPONSE WITH SPECIAL REFERENCE TO CIRCADIAN VARIATIONS IN EPIDERMAL CELL PROLIFERATION IN THE HAIRLESS MOUSE

Groups of hairless mice were injected i.p. with a stathmokinetic dose of 0·15 mg colcemid at seven different times of the day and animals killed 0, 15 and 30 min, 1, 2, 3 and 4 hr after the injection. The proportion of cells in metaphase and ana/telophase was determined in histological sections. The results showed a transient accumulation of metaphases about 30 min after the injection, followed by an increase in metaphases from 1 to 4 hr. Therefore, no value before 1 hr after the colcemid injection should be used in calculations of the mitotic rate. The presence of circadian rhythms with high mitotic activity in the morning and low activity in the evening was confirmed. It is shown by regression analyses that the accumulation period of 4 hr is sufficiently short to reflect circadian variations in epidermal cell proliferation and that the 4-hr accumulation value alone is sufficient to estimate the mitotic rate.     

人胎儿真皮乳头和皮纹发生的研究

本文用扫描电镜法研究了入胎儿的皮纹发生过程,包括初级真皮嵴、次级真皮嵴、真皮乳头和表皮隆线的发生。为研究人皮纹的发生和皮肤的异常提供了皮肤正常发育的形态学依据。共观察111例从第6周到第9个月胎儿的皮纹区皮肤,表明第3个月末胎儿开始形成初级真皮嵴,以后逐渐加深,至第16周嵴的顶端中央产生纵沟形成两条平行的次级真皮嵴;自19周后,次级真皮嵴局部隆起,由波浪形逐渐形成乳头。至30周乳头呈犬牙状。表皮隆线于第4—5月形成,随真皮乳头的增高而渐趋明显。至第6个月,全部皮纹图样已可辨认。本文还讨论了真皮乳头发生的过程。     

CELL POPULATION KINETICS OF PLUCKED AND UNPLUCKED MOUSE SKIN I. UNIRRADIATED SKIN

A detailed study of the cellular proliferation kinetics in interfollicular plucked and unplucked mouse skin has been made in Swiss albino mice, using tritiated thymidine autoradiography. Diurnal variations in mitotic and labelling indices were demonstrated in both systems.
The mean cell cycle times for unplucked and plucked skin were estimated by four different methods and found to be 100 ± 10 and 47 ± 3 hr respectively. Most of the difference was due to the shortening of G₁ phase after plucking. Repeated labelling at intervals shorter than the DNA synthesis times resulted in all the basal layer cells becoming labelled, so that the growth fraction was unity, in unplucked and plucked skin.
A well-defined second wave of labelled mitoses was seen at about 100 hr after labelling the unplucked (i.e. normal) mouse skin.
A double labelling technique using ¹⁴C-TdR and ³H-TdR with a single layer of emulsion gave reasonable values for the duration of the DNA synthesis phase.     

大鼠和小鼠睾丸表皮生长因子表达的免疫组织化学定位观察

为了了解大鼠和小鼠睾丸是否产生ＥＧＦ及其细胞定位,本实验用ＥＧＦ单克隆抗体对大鼠和小鼠睾丸进行了免疫细胞化学定位研究,结果显示：（１）出生后,大鼠和小鼠睾丸即开始产生ＥＧＦ,分泌活动主要位于睾丸间质细胞。（２）至性成熟期,少数精原细胞、精母细胞及个别圆形精子细胞和管周肌样细胞也产生ＥＧＦ,使生精小管尤其是血睾屏障管腔小室侧的ＥＧＦ分泌增加。（３）在本实验中,睾丸支持细胞未见明显ＥＧＦ阳性染色。结果表明,大鼠和小鼠睾丸是可以产生ＥＧＦ的,间质细胞是其主要的ＥＧＦ分泌细胞。进入性成熟期后,少数精原细胞、精母细胞及个别圆形精子细胞和管周肌样细胞也产生ＥＧＦ。大鼠和小鼠睾丸在发育过程中ＥＧＦ分泌量呈上升趋势,至性成熟期达分泌高峰     

PROLIFERATION KINETICS OF THE MOUSE BLADDER AFTER IRRADIATION

The proliferative response of the mouse bladder was investigated, using continuous labelling with tritiated thymidine, at various times after a single dose of radiation. Bladder epithelial and vascular endothelial cells were studied. The cell turnover rate in unirradiated epithelium and endothelium was found to be extremely slow (in excess of 1 year). Irradiation with a single dose of 25 Gy resulted in compensatory proliferation of the epithelium but the response was not initiated for many months. At 3 months after irradiation there was little difference from the control proliferation rate, but from 6 to 22 months after irradiation (the end of the study) there was a period of sustained rapid proliferation with the cell turnover time reduced to approximately 1 week. The increase in proliferative activity observed at 22 months was found to be dose—dependent. Endothelial cells in the blood vessels of the submucosa also showed an increased turnover rate after irradiation and the timing of this reponse was found to be similar to that of the epithelium. The onset of compensatory proliferation in both cell types was found to coincide with marked histological and functional changes in the bladder. In this slowly proliferating tissue, the onset of rapid compensatory proliferation after irradiation is delayed and occurs at the time that functional impairment is observed. This supports the postulate that proliferation is unlikely to contribute much to the sparing effect of prolonged fractionated radiotherapy in slowly dividing tissues.     

SOME EFFECTS OF BLEOMYCIN ON THE PROLIFERATION, MATURATION TIME and PROTEIN SYNTHESIS OF HAIRLESS MOUSE EPIDERMIS

Hairless mice were given 2 mg Bleomycin i.p. in 1-0 ml saline on two successive days. By a stathmokinetic method, by micro-flow fluorometry and by autoradiography certain kinetic parameters were measured during 10 days after the last injection. Cell counts were made and the turnover time of the differentiating cells estimated. Protein synthesis was estimated by the uptake of radioactive histidine, and dry cell mass measured by weighing. Bleomycin affected cell proliferation in the epidermis by depressing biphasically both the number of cells in, and the passage of cells through, the cell cycle phases: S, G₂ and M, most probably by directly affecting late Gj cells and cells in mitosis. The time between the two minima of depressed DNA synthesis corresponded to the mean generation time of the basal cells. Histidine uptake and dry cell mass were slightly affected, but the turnover time of the differentiating cells was prolonged. Bleomycin thus had a strong long-lasting inhibitory effect on epidermal cell proliferation and a marked inhibitory effect on epidermal cell maturation in mice.     

KINETICS OF CELL RENEWAL, CELL MIGRATION AND CELL LOSS IN THE HAIRLESS MOUSE DORSAL EPIDERMIS

Forty hairless mice were given injections of tritiated thymidine every 4th hour during 10 days. At 24 hr intervals groups of four mice were killed. The numbers of labelled basal and differentiating cells were determined by autoradiography with a stripping film technique. To determine the background activity skin sections from uninjected control mice were subjected to the same stripping film procedure. Another group of hairless mice was given one single pulse labelling with tritiated thymidine. The number of labelled mitoses was scored for 12 hr after the injection. At 10, 12 and 15 hr after the injection, the numbers of labelled basal and differentiating cells were also determined. A mathematical model of cell population kinetics in the epidermis has been suggested. The results of different simulations on this model were compared with the observed results. The curve of mean grain counts under continuous labelling increased from day to day with two well-defined plateaux. The percentage of all labelled cells increased rapidly up to the 3rd day, and thereafter the curves gradually flattened off. When basal cells and differentiated cells were considered separately the labelling index of the basal cells increased rapidly for the first 3 days and then flattened off at the 100% level on the 5th day. The labelling index of the differentiating cells was low during the first 3–4 days. Then a steep increase in the percentage of labelled differentiating cells was seen, but the curve flattened off again close to the 100 % level after the 7th day. The labelled mitosis curve had its maximum 5 hr after the thymidine injection. The curve fell again to almost zero at 12 hr. Ten, 12 and 15 hr after the injection, 6, 7 and 7% respectively of the labelled cells were found in the spinous layer. It was concluded that three grains over each nucleus could be used as lower limit for considering a cell as labelled. On this basis, tritiated thymidine injections every 4th hour can be considered as continuous labelling.     

龙眼剥皮再生的解剖学研究

龙眼(Dimocarpus tongan Lour.)茎干经过大面积环剥,都能再生出新皮。环剥初期,愈伤组织都由近暴露面的射线细胞产生,稍后,其他未成熟木质部细胞也参加愈伤组织的形成,这些愈伤组织一般在靠近表面都可发生木栓形成层,以后迅速形成正常的周皮。在愈伤组织与木质部交界处的未成熟木质部细胞发生维管形成层。新发生的形成层正常地向外分化出次生韧皮部,向内分化出次生木质部。初期有些原来的射线将新形成层带分割成许多小区,二个月后,由于新的形成层不断平周活动,逐渐将形成层连成一圈,以后基本上与正常树皮维管组织的发育一样。     

EFFECTS OF ADENOSINE AND EPIDERMAL GROWTH FACTOR ON THE GROWTH OF MOUSE MAMMARY EPITHELIAL CELLS

The objective of this study was to determine if adenosine alters growth of mammary epithelium. Mouse mammary epithelial cells (NMuMG) were cultured in DMEM supplemented with 10% fetal calf serum. After serum starvation for 24h, EGF (0–100ng/ml) and/or adenosine (0–100μm ) was added. Adenosine at concentrations of 1, 10 or 100μm increased DNA synthesis significantly, when compared to control. Addition of epidermal growth factor (EGF) (10ng/ml) into 1 or 10μm adenosine showed the interaction in DNA synthesis between EGF and adenosine. A similar result was observed when 100μm adenosine added to various concentrations of EGF (0–100ng/ml). In the second mammary gland (thoracic) organ culture studies, mammary development scores were increased by adenosine (100μm ), EGF (100ng/ml) and adenosine plus EGF. These results indicate that the purine nucleoside adenosine stimulates mammary epithelial cell growth and interacts with EGF in DNA synthesis of mouse mammary epithelial cells.     

REGENERATION OF THE SUNFLOWER CAPITULUM AFTER CYLINDRICAL WOUNDING OF THE RECEPTACLE

Using the young capitulum of Helianthus annuus L., a cylindrical plug of undifferentiated receptacle tissue, 1 mm in diameter, was isolated from lateral communication with the rest of the receptacle surface by a vertical circular wound cut, while retaining continuity with the subapical meristem. Within 24 hr, active cell division was induced at the inner and outer surfaces of the wound and in the receptacle epidermis bordering the wound edges, creating a rounded rim at the top of the wound. Within 3–6 days, floral initials, spaced 133–166 μm apart appeared on the flanks of both rims and later on the top of the plug and surrounding receptacle surface. The first formed initials developed into involucral bracts or ray florets and the later ones into disc florets which were organized into contact parastichies, the number of which did not conform with the Fibonacci series. The base of the plug developed into a stem-like structure completing the regeneration of a fully formed functional capitulum. This operation was demonstrated for two sunflower cultivars and occurred in both long and short daylengths.     

REGENERATION OF UTERINE EPITHELIUM AFTER EXPERIMENTAL ABLATION IN THE RAT

Regeneration of the uterine luminal epithelium was studied after its mechanical removal in progesterone-primed rats, leaving one control horn intact. Pulse labelling with [³H]TdR during regeneration, showed a rapid peak of labelling index in remaining glands. A differentiated and highly labelled luminal epithelium reappeared at 34 hr, thereafter showing a rapidly declining LI. After initial depletion, the glandular cell population size was restored within 64 hr, whereas luminal epithelium cell numbers became stabilized at about half normal level. Grain counts after prelabelling showed more rapid dilution in gland cells of stripped uterine horns, indicating accelerated cycling of previously dividing cells. Thymidine labelling indices also showed that, after removal of the epithelium, almost all gland cells became rapidly committed to divide. On average, less than two cell cycles were necessary to restore stable glandular and epithelial population sizes. Numbers of labelled cells were also drastically increased in myometrium and serosa of treated horns. This suggests a non-specific mechanism for stimulation of mitotic activity after ablation of epithelium.     

小鼠卵母细胞的冷冻—解冻及其体外发育的研究

试验比较四种防冻剂对小鼠核泡期(germinal vesicle stage)无卵丘细胞的卵母细胞(下称裸卵),在各种不同条件下冷冻-解冻的影响,并用乙二醇为防冻剂,冷冻-解冻后,形态正常的裸卵1137枚,经体外培养排出第一极体、发育达中期Ⅱ的成熟率为43.2％(491/1137)。用体外获能的小鼠附睾尾精子进行体外受精的受精率为25.4％(31/122)。继续培养进一步发育至2-细胞期的发育率为17.3％(55/318)。对照组(新鲜裸卵)的成熟率为48.6％(158/325),受精率为26.4％(388/144)。两者无显著差异(P<0.05)。     

大鼠脊髓损伤后表皮生长因子受体在脊髓的表达特点

目的研究大鼠脊髓损伤(spinal cord injury,SCI)后表皮生长因子受体(epidermal growth factor re-ceptor,EGFR)在脊髓的表达特点及意义。方法健康成年雄性SD大鼠,随机分为4组(每组10只):假手术组,SCI术后3 d、7 d和14 d组。应用Basso Beattie Bresnahan(BBB)评分观察大鼠行为学改变;逆转录-聚合酶链反应(RT-PCR)检测损伤段脊髓组织中EGFR mRNA表达水平;免疫组织化学方法观察损伤段脊髓灰质中EGFR蛋白表达情况;并对EGFRmRNA及蛋白表达情况与BBB评分进行相关性分析。结果行为学观察发现大鼠脊髓损伤后下肢神经功能逐步恢复;RT-PCR结果显示EGFR mRNA在假手术组大鼠脊髓中微量表达,SCI术后3 d表达显著升高,随后趋于下降,14 d时仍高于假手术组(P<0.01);免疫组织化学染色显示损伤段脊髓灰质中EGFR阳性细胞数在损伤后3 d显著高于假手术组(P<0.01),随后趋于下降,但14 d时仍高于假手术组(P<0.01);EGFR mRNA及蛋白的表达均与BBB评分呈显著负相关(r=-0.956,P<0.05;r=-0.966,P<0.05)。结论EGFR在大鼠脊髓损伤后具有时相分布特点,且与动物行为呈负相关,提示其表达可能阻碍损伤后的神经功能恢复。     

CHANGES IN THE SKIN FLORA OF COD AFTER WASHING AND ICING

SUMMARY: Bacterial counts done on the skin of North Sea cod show that while washing with running sea water greatly reduced the numbers on newly caught fish, contact with ice from a trawler's ice bunker resulted in an immediate large increase on the washed fish.
Washing did not materially alter the percentage representation of the various genera present, but icing with trawler bunker ice could cause major alterations and often added types of bacteria similar to those present on newly caught fish, indicating contamination of the ice on board the trawler.     

大鼠皮肤切伤后成纤维细胞EGFR胶原基因表达的影响

４５只大鼠,分成７个生前损伤组,１个死后损伤组和１个正常对照组。每组５只,背部皮肤切口造创,生前伤组按伤后０、５、１０、１５、３０、６０、９０、１２０ｍｉｎ不同时间和死后在伤口缘取皮,进行切片和免疫组化实验,观察ＥＧＦＲ的基因表达。结果表明,正常和死后组不表达,生前伤组表达区沿表皮基底层和皮下组织分布,表达随时间而增强。表达率Ｐ与时间Ｔ的对数呈高度正相关（ｒ＝０．９８５）。随建立了回归方程。用回归方程计算出的表达率和时间,接近实测值。根据回归方程推导,时间是以２为底表达率为参数的指数函数。它可能提示,时间Ｔ与成纤维细胞的分裂增殖周期和数量有关。表达率作为特征参数,标志着细胞膜ＥＧＦＲ传递信息的特性。说明细胞膜ＥＧＦＲ在相互传递信息中,相互影响表达结果     

A FINE STRUCTURE STUDY OF LIPID IN MOUSE LIVER REGENERATING AFTER PARTIAL HEPATECTOMY

The fine structure of liver 3½ to 72 hours after partial hepatectomy has been compared with that of liver from sham-operated animals; all animals were 60- to 90-day old male mice of the C3H strain. Numerous small bodies with diameters ranging from 300 to 1,000 A have been observed distributed randomly throughout the cytoplasm of the hepatic parenchymal cells at early intervals after partial hepatectomy. In material fixed in osmium tetroxide and embedded in methacrylate, they appear as uniformly electron-opaque bodies, but in permanganate-fixed liver, they display only a peripheral rim of electron-opaque material surrounding a clear core. Each of these cytoplasmic bodies appears to be located within a vesicle. A few of the opaque bodies are also present in sinusoids and in the spaces of Disse; these bodies are not located within vesicular structures. Fat droplets of various sizes are easily distinguished in regenerating liver; with the increase in number of these fat droplets at later postoperative intervals, there occurs a concomitant decrease in the number of cytoplasmic bodies. It is suggested that the cytoplasmic bodies contain some lipid component. Possible explanations of the origin, nature, and fate of the cytoplasmic bodies are discussed.     

长江三峡地区典型灌丛的生物量及其再生能力

 灌丛是三峡地区典型的退化生态系统类型。本文采用收获法和模拟砍伐实验研究了三峡地区铁仔灌丛、椎木灌丛、荆条灌丛和黄栌灌丛的生物量及黄栌灌丛、椎木灌丛地上部分砍伐后的再生能力。研究结果表明,这4种类型的灌丛总生物量分别为22.5±5.1、21.0±3.7、16.9±7.5和13.6±2.4t·hm-2,相当于同纬度地带性生态系统常绿阔叶林(30年林龄)的10％一25％ 4种灌丛灌木层占总生物量、地上部分生物量和地下部分生物量的90％以上。在生物量—物种序列中,前5种植物占总生物量的84％ 以上。不同地点灌丛生物量的比较表明,同一种类型灌丛,亚热带和暖温带地区总生物量没有明显差异。通过模拟砍伐实验,黄栌灌丛、被木灌丛地上部分全部砍伐后1年地上部分生物量就可以恢复到对照的42.7％和62.0％,说明这些灌丛类型具较高的生长速度和很大的恢复潜力。