Circannual Variation of Cell Proliferation in Lymphoid Organs and Bone Marrow of Dbf1 Male Mice on Three Lighting Regimens

The case of a 40-year-old sighted woman with free-running sleep-wake and melatonin rhythms is presented. The subject was studied for 102 days. During the pre-treatment period, both the sleep-wake and melatonin rhythms had a period of 25.1 hr, similar to the average period of humans living in temporal isolation. Treatment consisted of bright artifical light exposure (2500 lx Vita-Lite) for 2 hr each day upon awakening. Clock time of light exposure was held constant for 6 days and then slowly advanced until the subject was arising at her desired time of day. The subject continued the light treatment at home and was able to live on a 24-hr day for the 30-day follow-up study. While other factors may be operating in this situation, it is possible that the light treatment caused the stabilization of the free-running rhythms, advancement to a normal phase and entrainment to the 24-hr day. We suspect that the tendency to free-run was related to sleep onsets that were abnormally delayed relative to the circadian phase response curve for light. By scheduling a 2-hr pulse of bright light each morning, this tendency to delay would be counteracted by light-induced advances, resulting in normal entrainment.     

Circannual variation of intestinal cell proliferation in BDF1 male mice on three lighting regimens

BDF, male mice were studied over a 24-hr span in winter, spring, summer and fall. For three weeks prior to study, one-third of the animals were kept under a lighting regimen of 8 hr light alternating with 16 hr of darkness (LD 8:16), one-third on a lighting regimen of LD 12:12 and a remainder on a lighting regimen of LD 16:8. During each study, subgroups of animals on all three lighting regimens were killed at 4-hr intervals over a 24-hr span. Twenty minutes prior to being killed, the animals received 5yCi of [³Hthymidine/0.2 ml/20 gm of body weight intraperitoneally. The thymidine uptake in the DNA of the colon and of the small intestine were studied as an index of cell proliferation. A circadian rhythm in [³H]-thymiduie uptake in the colon was found and validated by cosinor analysis. This rhythm was similar in acrophase and amplitude in the animals kept on LD 8:16 and LD 12:12. Also in the mice on LD 16:8, there was a statistically significant circadian rhythm of ('HJ-thymidtne uptake in the DNA of the colon during all four seasons. The acrophases of this rhythm, however, varied widely suggesting free running. A circadian rhythm of pHJ-thymidine uptake in small intestine was less consistent. In animals on all three lighting regimens, however, a circannual variation of f'HJ-thymidine uptake in DNA in colon and small intestine was found with the highest uptake during summer. This study indicates that a lighting regimen of LD 16:8 does not reliably synchronize the circadian rhythm of [³H]-thymidine uptake in the colon. It further shows a circannual rhythm of this function in the colon and in the small intestine which persists under three lighting regimens (LD 8:16, 12:12 and 16:8) maintained for three to four weeks prior to being killed.     

Dna Synthetic Activity in Tumor-Bearing Mice

The rate of DNA synthesis in normal tissues exhibits circadian rhythmicity. However, there have been conflicting reports of the effects of tumor burden on the circadian rhythm of DNA synthesis in non-cancer tissues. We have developed a mouse colon cancer (MC-26) that exhibits different growth under different photoperiods. The purpose of this study was to analyze DNA synthetic activity in tissues removed from tumor-bearing and tumor-free mice maintained under two different photoperiods. Two groups each of approximately 80 male Balb/c mice were acclimated to one of two light-dark cycles, 12L:12D or 6L:18D. Half of each group were injected with 5.0 × 104 MC-26 cells. Twenty-two days later, all mice were killed in subgroups at 4-6 hr intervals over one 24-hr period. Colons and tumors were removed for measurement of DNA synthesis. Results were analyzed by means of one-way analysis of variance (ANO VA) in order to determine whether DNA synthesis varied significantly within groups over the 24-hr period. The DNA synthetic activity, as measured by uptake of tritiated thymidine, exhibited significant temporal variation in the colons of control (tumor-free) mice under both the 12L:12D and 6L:18D photoperiods. The colons of tumor-bearing mice failed to exhibit a fluctuation under a 12L:12D photoperiod but did show a significant 24-hr rhythm under the 6L:18D photoperiod. The subcutaneously growing cancers did not exhibit a circadian variation in DNA synthetic activity under either photoperiod. Both photoperiod and the presence of cancer appear to affect the DNA synthetic activity observed in mice bearing the MC-26 colon cancer.     

酶解乳蛋白对初生仔猪小肠组织学结构及隐窝细胞增殖的影响(英文)

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Circadian and Circannual Variations of Cell Cycle Distribution in the Mouse Bone Marrow

The cell cycle distribution of bone marrow cells from the femurs of female C3H mice has been investigated by flow cytometry according to the time of the day and month of the year. Both circadian and seasonal variations were found for the different cell cycle phases as well as the total cell numbers per femur. Both the mesor, the acrophase and the amplitude of the S, G₂ and (G₁ + G₀) phases varied significantly in some months, while in other months only insignificant rhythms were found. The relative cell cycle distribution only partly reflected variations in the total numbers of proliferating cells, since the total cell number per femur was also variable.

The total numbers of cells in DNA synthesis seem to be higher in the first part of the year, indicating increased cell proliferation during winter and spring. In this period the acrophases of DNA synthesis and G₂ were in the morning, while the second half of the year showed the peak later in the day.

In general, hemopoietic cell proliferation seems to constitute a labile equilibrium with rapidly changing activities.     

Enhanced Gastrointestinal Expression of Cytosolic Malic Enzyme (ME1) Induces Intestinal and Liver Lipogenic Gene Expression and Intestinal Cell Proliferation in Mice

The small intestine participates in lipid digestion, metabolism and transport. Cytosolic malic enzyme 1 (ME1) is an enzyme that generates NADPH used in fatty acid and cholesterol biosynthesis. Previous work has correlated liver and adipose ME1 expression with susceptibility to obesity and diabetes; however, the contributions of intestine-expressed ME1 to these conditions are unknown. We generated transgenic (Tg) mice expressing rat ME1 in the gastrointestinal epithelium under the control of the murine villin1 promoter/enhancer. Levels of intestinal ME1 protein (endogenous plus transgene) were greater in Tg than wildtype (WT) littermates. Effects of elevated intestinal ME1 on body weight, circulating insulin, select adipocytokines, blood glucose, and metabolism-related genes were examined. Male Tg mice fed a high-fat (HF) diet gained significantly more body weight than WT male littermates and had heavier livers. ME1-Tg mice had deeper intestinal and colon crypts, a greater intestinal 5-bromodeoxyuridine labeling index, and increased expression of intestinal lipogenic (Fasn, Srebf1) and cholesterol biosynthetic (Hmgcsr, Hmgcs1), genes. The livers from HF diet-fed Tg mice also exhibited an induction of cholesterol and lipogenic pathway genes and altered measures (Irs1, Irs2, Prkce) of insulin sensitivity. Results indicate that gastrointestinal ME1 via its influence on intestinal epithelial proliferation, and lipogenic and cholesterologenic genes may concomitantly impact signaling in liver to modify this tissue’s metabolic state. Our work highlights a new mouse model to address the role of intestine-expressed ME1 in whole body metabolism, hepatomegaly, and crypt cell proliferation. Intestinal ME1 may thus constitute a therapeutic target to reduce obesity-associated pathologies.     

Diurnal Variation of Cell Proliferation in Three Bacterial Taxa from Coastal North Sea Waters

Pulse-labeling with bromodeoxyuridine (BrdU) in combination with fluorescence in situ hybridization was applied to quantify the percentage of proliferating cells in coastal North Sea waters. In order to assess diurnal variability, we sampled eight or nine times, respectively, within 3 consecutive days at two seasons. Bacteria affiliated with the Roseobacter, SAR86, and NOR5 lineages constituted on average 19% ± 3%, 8% ± 2%, and 6% ± 1% of all cells in May 2002 and 17% ± 3%, 10% ± 2%, and 11% ± 3% in August. The relative abundances of the three populations either remained stable, or they changed very gradually during the observation periods. On average, 38 and 39% of all Bacteria exhibited DNA de novo synthesis in May and August, respectively. The fractions of proliferating cells in bacteria of the SAR86 (May, 59%; August, 72%) and the Roseobacter (48 and 53%) lineages were significantly above the community average. A substantial cell proliferation of population NOR5 (34%) was only encountered in August, concomitant with a dinoflagellate bloom. Significant short-term fluctuations of DNA-synthesizing cells were observed in Roseobacter during May and in NOR5 during August, hinting at a pronounced (temporal or spatial) mesoscale patchiness of growth rates in these populations. Since the BrdU proliferation assay is susceptible to misinterpretation, we also modeled the expected number of labeled cells at increasing BrdU incubation times in a slowly growing bacterial population. We suggest that the absence of visible DNA synthesis in marine bacterioplankton cells after DNA pulse-labeling must not be interpreted as an indication of cell “inactivity.”     

Genomic Basis of Circannual Rhythm in the European Corn Borer Moth

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RTN4对于结肠癌细胞增殖的调控作用

2018年全球癌症统计调查显示,结直肠癌约占患癌新病例的12.1%。因此,寻找新的结肠癌发生有关的基因,发现新的治疗靶点显得尤为迫切。通过数据库分析发现,RTN4基因的表达水平与结肠癌患者生存率的相关性具有统计学意义。针对RTN4基因构建其干扰质粒,将慢病毒作为载体转染结肠癌HCT116细胞中构建敲低RTN4的结肠癌细胞系,最后检测了低表达后RTN4基因的细胞增殖。结果发现,敲低RTN4基因后显著促进了结肠癌细胞HCT116的增殖,研究通过Western blot观察敲低RTN4后HCT116细胞自噬通路相关蛋白p62和LC3的表达情况,发现与对照组相比较,敲低RTN4组LC3转化量（LC3-II/LC3-I）增多,而p62蛋白减少。研究分析了RTN4的潜在抑癌作用,发现敲低RTN4基因会显著增强结肠癌细胞的增殖能力,并且诱导自噬,说明RTN4可能与激活LC3/p62自噬途径有关。     

Supplemental Silk Protein,Sericin, Suppresses Colon Tumorigenesis in 1,2-Dimethylhydrazine-Treated Mice by Reducing Oxidative Stress and Cell Proliferation

This study was done to discover the underlying mechanism of the inhibitory effect of sericin against colon tumorigenesis. Mice were fed a diet with 30 g/kg sericin for 115 d, and given a weekly injection of 1,2-dimethylhydrazine (10 mg/kg body weight) for the initial 10 wk. Dietary supplemental sericin caused a 62% reduction in the incidence of colonic adenoma (P<0.05), but did not affect the incidence of colonic adenocarcinoma. Sericin intake significantly reduced the number of colon adenomas. Consumption of sericin significantly reduced the BrdU labeling index of colonic proliferating cells and the expression of colonic c-myc and c-fos. The levels of colonic 8-hydroxydeoxyguanosine, 4-hydroxynonenal, and inducible nitric oxide synthase protein were significantly suppressed by sericin. The results suggest that dietary sericin suppresses the development of colon tumors by reducing oxidative stress, cell proliferation, and nitric oxide production.     

Autocrine Platelet-derived Growth Factor-Vascular Endothelial Growth Factor Receptor-related (Pvr) Pathway Activity Controls Intestinal Stem Cell Proliferation in the Adult Drosophila Midgut

A dynamic pool of undifferentiated somatic stem cells proliferate and differentiate to replace dead or dying mature cell types and maintain the integrity and function of adult tissues. Intestinal stem cells (ISCs) in the Drosophila posterior midgut are a well established model to study the complex genetic circuitry that governs stem cell homeostasis. Exposure of the intestinal epithelium to environmental toxins results in the expression of cytokines and growth factors that drive the rapid proliferation and differentiation of ISCs. In the absence of stress signals, ISC homeostasis is maintained through intrinsic pathways. In this study, we uncovered the PDGF- and VEGF-receptor related (Pvr) pathway as an essential regulator of ISC homeostasis under unstressed conditions in the posterior midgut. We found that Pvr is coexpressed with its ligand Pvf2 in ISCs and that hyperactivation of the Pvr pathway distorts the ISC developmental program and drives intestinal dysplasia. In contrast, we show that mutant ISCs in the Pvf/Pvr pathway are defective in homeostatic proliferation and differentiation, resulting in a failure to generate mature cell types. Additionally, we determined that extrinsic stress signals generated by enteropathogenic infection are epistatic to the hypoplasia generated in Pvf/Pvr mutants, making the Pvr pathway unique among all previously studied intrinsic pathways. Our findings illuminate an evolutionarily conserved signal transduction pathway with essential roles in metazoan embryonic development and direct involvement in numerous disease states.     

Effect of Chronic Caloric Restriction on the Circadian Regulation of Physiological and Behavioral Variables in Old Male B6C3F1 Mice

The circadian rhythms of food and water consumption, the number of feeding and drinking episodes, oxygen consumption, carbon dioxide production, respiratory quotient, gross motor activity, and body temperature were measured in male B6C3F, mice that were fed ad libitum (AL) or fed a caloric-restricted diet (CR). The CR regimen (60% of the normal AL consumption) was fed to mice during the daytime (5 hr after lights on). CR animals exhibited fewer feeding episodes but consumed more food per feeding bout and spent more total time feeding than AL mice. It appears that CR caused mice to change from their normal “nibbling behavior” to meal feeding. Compared to AL animals, the mean body temperature was reduced in CR animals, while the amplitude of the body temperature rhythm was increased. Spans of reduced activity, metabolism, and body temperature (torpor) occurred in CR mice for several hours immediately before feeding, during times of high fatty acid metabolism (low RQ). The acute availability of exogenous substrates (energy supplies) seemed to modulate metabolism shifting metabolic pathways to promote energy efficiency. CR was also associated with lower DNA damage, higher DNA repair, and decreased proto-oncogene expression. Most of the circadian rhythms studied seemed to be synchronized primarily to the feeding rather than the photoperiod cycle. Night-time CR feeding was found to be better than daytime feeding because the circadian rhythms for AL and CR animals were highly synchronized when this regimen was used.     

Effect of Chronic Caloric Restriction on the Circadian Regulation of Physiological and Behavioral Variables in Old Male B6C3F1 Mice

The circadian rhythms of food and water consumption, the number of feeding and drinking episodes, oxygen consumption, carbon dioxide production, respiratory quotient, gross motor activity, and body temperature were measured in male B6C3F, mice that were fed ad libitum (AL) or fed a caloric-restricted diet (CR). The CR regimen (60% of the normal AL consumption) was fed to mice during the daytime (5 hr after lights on). CR animals exhibited fewer feeding episodes but consumed more food per feeding bout and spent more total time feeding than AL mice. It appears that CR caused mice to change from their normal “nibbling behavior” to meal feeding. Compared to AL animals, the mean body temperature was reduced in CR animals, while the amplitude of the body temperature rhythm was increased. Spans of reduced activity, metabolism, and body temperature (torpor) occurred in CR mice for several hours immediately before feeding, during times of high fatty acid metabolism (low RQ). The acute availability of exogenous substrates (energy supplies) seemed to modulate metabolism shifting metabolic pathways to promote energy efficiency. CR was also associated with lower DNA damage, higher DNA repair, and decreased proto-oncogene expression. Most of the circadian rhythms studied seemed to be synchronized primarily to the feeding rather than the photoperiod cycle. Night-time CR feeding was found to be better than daytime feeding because the circadian rhythms for AL and CR animals were highly synchronized when this regimen was used.     

Effect of Changing the Light/Dark Schedule, the Time of Onset of the Light or Dark Period, or the Daylength, on Rhythms of Epidermal Cell Proliferation

Rhythms of labeling and mitotic indices were studied in the hindlimb epidermis of the anuran tadpole Rana pipiens under different light/dark (LD) cycles and daylengths in order to examine the role of the various parameters of the lighting regimen in setting the periods of the rhythms and the timing of the cell proliferation peaks. Altering the time of, or inverting, the 12 h light period on a 24 h day resulted in phase shifting of basically bimodal circadian rhythms with peaks in the light and dark. Thus the cell proliferation rhythms were entrained to the LD cycle. These rhythms also entrained to noncircadian schedules since they lengthened on a 15L : 15D cycle and shortened on a 9L : 9D cycle, although the bimodal characteristic of a peak in the light and a peak in the dark remained. Studies of 18L: 6D and 6L : 18D cycles in which either the time of onset of light or dark was changed relative to the 12L: 12D control indicated that the onset of dark may regulate the timing of the labeling index peaks while the onset of light may determine the time of occurrence of mitotic index peaks. Control of the timing of labeling and mitotic index peaks by different parameters of the LD cycle suggests a mechanism for cell cycle regulation by the environmental lighting schedule. Analysis of the rhythms on all the cycles studied suggested that labeling index rhythms equal the length of, or twice the length of, the dark period. Mitotic index rhythms equal the daylfength or a multiple of the length of the dark period.     

Human Intestinal Circadian Clock: Expression of Clock Genes in Colonocytes Lining the Crypt

Biological clock components have been detected in many epithelial tissues of the digestive tract of mammals (oral mucosa, pancreas, and liver), suggesting the existence of peripheral circadian clocks that may be entrainable by food. Our aim was to investigate the expression of main peripheral clock genes in colonocytes of healthy humans and in human colon carcinoma cell lines. The presence of clock components was investigated in single intact colonic crypts isolated by chelation from the biopsies of 25 patients (free of any sign of colonic lesions) undergoing routine colonoscopy and in cell lines of human colon carcinoma (Caco2 and HT29 clone 19A). Per‐1, per‐2, and clock mRNA were detected by real‐time RT‐PCR. The three‐dimensional distributions of PER‐1, PER‐2, CLOCK, and BMAL1 proteins were recorded along colonic crypts by immunofluorescent confocal imaging. We demonstrate the presence of per‐1, per‐2, and clock mRNA in samples prepared from colonic crypts of 5 patients and in all cell lines. We also demonstrate the presence of two circadian clock proteins, PER‐1 and CLOCK, in human colonocytes on crypts isolated from 20 patients (15 patients for PER‐1 and 6 for CLOCK) and in colon carcinoma cells. Establishing the presence of clock proteins in human colonic crypts is the first step toward the study of the regulation of the intestinal circadian clock by nutrients and feeding rhythms.     

LncRNA RP1调控周期蛋白质促进结肠癌细胞增殖

长链非编码RNAs（long non-coding RNAs, lncRNAs）是一类无蛋白质编码功能,长度大于 200 nt的RNAs。qRT-PCR实验证实,lncRNA RP1-506.5（命名为RP1）在人结肠癌细胞株中的表达量明显高于人正常结肠上皮细胞（P<0.01）。RP1在结肠癌组织中的表达量为癌旁组织中表达量的8.5倍。在HCT116中,上调RP1的表达,同时在HCT8中沉默RP1的表达,探讨RP1对结肠癌细胞生物学特性的影响。MTS实验、活细胞工作站增殖实验,结合平板克隆实验发现,过表达RP1能明显促进结肠癌细胞HCT116的增殖能力。而在HCT8细胞中沉默RP1表达后,该细胞的增殖能力明显减弱。流式细胞周期实验结果表明,RP1能促进细胞周期快速通过G1/S检测点,并能加速S期进程。荧光定量PCR、Western印迹实验发现,在HCT116中细胞中,上调RP1的表达后,P21的表达水平下调,细胞周期蛋白D1（cyclinD1）、依赖细胞周期蛋白激酶6(CDK6)表达水平上调;当沉默LncRNA RP1的表达后,能上调P21的表达水平,下调cyclinD1、CDK6的表达水平。这些结果表明,LncRNA RP1可通过调控周期相关蛋白质的表达促进结肠癌细胞增殖。     

TERT对小鼠EL-4淋巴瘤细胞增殖调控的初步研究

为了研究端粒酶催化亚基TERT与细胞周期相关基因的调控关系及其在肿瘤细胞增殖中的调控作用,应用基因芯片及RT-PCR等技术,对靶向mTERT的RNA干涉后的小鼠EL-4淋巴瘤细胞进行细胞周期相关基因的表达谱分析,筛选到43个基因在TERT受抑制前后存在表达差异,并且全部为下调基因.表明在小鼠EL-4淋巴瘤细胞中内源性TERT的表达抑制,导致了调控G1期和S期进程的相关细胞周期相关基因的表达变化,并可能通过此途径影响肿瘤细胞的生长和增殖.     

RNA干扰敲减PIWIL1表达对人结肠癌细胞SW620增殖，粘附及侵袭能力影响

PIWIL1为AGO蛋白(Argonaute proteins )PIWI亚家族的成员之一,在睾丸中特异表达. 其在干细胞自我更新、RNA干扰和翻译调节中起着重要的作用.本文采用实时PCR方法检测PIWI基因家族中PIWIL1、PIWIL2、PIWIL3、PIWIL4在人结肠癌SW620细胞中mRNA表达水平,首次证实了在SW620细胞中,PIWIL1相对PIWIL2、PIWIL3、PIWIL4,其表达水平最高（P﹤0.05）.构建表达针对PIWIL1的小发卡结构干扰RNA的重组质粒,并用Western 印迹验证其达到较高的干扰效率.用脂质体将重组质粒转染入SW620细胞中,通过MTT实验、集落形成实验、细胞聚集实验及细胞侵袭转移实验,分别观察到其可抑制SW620细胞的生长、增殖、侵袭转移能力,增强了SW620细胞的粘附能力.提示PIWIL1可能在结肠癌发生、发展过程中发挥作用.     

靶向XBP1S的RNA干扰质粒对细胞增殖的影响

目的 构建靶向人XBP1S的siRNA真核表达载体（pSUPER-XBP1S）并观察其对人HeLa细胞和HepG2细胞增殖能力的影响.方法 设计并合成针对XBP1S基因的siRNA,退火成互补双链后克隆至真核表达载体pSUPER构建重组质粒,并将其转染入HeLa细胞和HepG2细胞中.采用RT-PCR检测转染前后XBP1S在HeLa细胞和HepG2细胞中的转录,Western印迹检测转染前后XBP1S蛋白的表达;MTT法、细胞计数检测重组质粒对HeLa细胞和HepG2细胞增殖能力的影响.结果 重组质粒能有效地抑制HeLa细胞和HepG2细胞中XBP1S基因的转录和表达;转染HeLa细胞和HepG2细胞后,细胞增殖抑制率及细胞增殖数与对照组比较,差异有统计学意义（P〈0.05）.结论 成功构建了靶向人XBP1S的siRNA表达载体pSUPER-XBP1S,并且有效的抑制了HeLa细胞和HepG2细胞中XBP1S的转录和表达,有效抑制了细胞的增殖能力.