MORF4基因诱导HeLa细胞衰老

目的建立MORF4基因高表达诱导HeLa细胞衰老模型,探索MORF4基因对HeLa细胞衰老的影响。方法分别将pcDNA3.1(+)/Flag-MORF4和pcDNA3.1(+)空载质粒转染人宫颈癌HeLa细胞株。SA-β-Gal染色检测细胞衰老,MTT法及流式细胞术检测细胞周期变化及细胞的增殖活力,两者共同确定MG-132处理的最适条件。Western印迹检测MORF4、PCNA基因的表达。结果质粒经酶切和测序鉴定,证明pcD-NA3.1(+)/Flag-MORF4质粒中含有目的基因序列;经浓度梯度1.25、2.5、5、10μmol/L MG-132处理转染细胞2、4、24、48 h后,SA-β-Gal染色和MTT检测结果显示10μmol/L MG-132处理24 h的转染细胞明显衰老,细胞活力降低;细胞周期被阻滞在G0/G1期,S期细胞明显减少,增殖受到抑制;Western印迹检测结果显示MORF4蛋白在细胞中的含量明显升高,而与增殖相关基因PCNA表达下调。结论构建的pcDNA3.1(+)/Flag-MORF4质粒在HeLa细胞中表达;并在MG-132作用下,使MORF4基因的表达产物积累,从而影响PCNA蛋白的表达量,抑制HeLa细胞的增殖活性,阻滞细胞周期的进行,导致细胞进入衰老状态。     

Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood.

To study telomere length dynamics in hematopoietic cells with age, we analyzed the average length of telomere repeat sequences in diverse populations of nucleated blood cells. More than 500 individuals ranging in age from 0 to 90 yr, including 36 pairs of monozygous and dizygotic twins, were analyzed using quantitative fluorescence in situ hybridization and flow cytometry. Granulocytes and naive T cells showed a parallel biphasic decline in telomere length with age that most likely reflected accumulated cell divisions in the common precursors of both cell types: hematopoietic stem cells. Telomere loss was very rapid in the first year, and continued for more than eight decades at a 30-fold lower rate. Memory T cells also showed an initial rapid decline in telomere length with age. However, in contrast to naive T cells, this decline continued for several years, and in older individuals lymphocytes typically had shorter telomeres than did granulocytes. Our findings point to a dramatic decline in stem cell turnover in early childhood and support the notion that cell divisions in hematopoietic stem cells and T cells result in loss of telomeric DNA.     

Predicting age at onset of menopause: Testing the “adaptive onset” hypothesis

This study tests the “adaptive onset” hypothesis (AOH) proposed by Kuhle and explores the potential adaptive origins of menopause (Kuhle (2007) [3]). The AOH posits that both menopause and the timing of its onset are adaptive, and that age at onset may be mediated according to the likelihood of successful continued reproduction. Twelve factors were hypothesized to predict whether continued reproduction would be successful. Eight factors were tested using data extracted from the NLS Mature Women cohort. Statistical analyses reveal no support for predictions of the AOH; in fact, a majority of analyses suggest trends opposite from those predicted.     

Impact of inbreeding on fertility in a pre-industrial population

Little is known about the effects of inbreeding on reproduction in modern human societies. It appears indeed that biological effects are hidden by socioeconomic factors, which are the major determinants of fertility. It has been established, in particular, that socially induced reproductive compensation tends to homogenize the number of offspring per family in a given population. Besides, in the field of evolutionary biology, a number of empirical and theoretical studies have shown that the effects of inbreeding are condition dependent. In particular, theoretical developments on the evolution of senescence predict that the deleterious effects of inbreeding should increase with age. We rely on these developments to examine the effects of inbreeding on fertility in a cohort of Canadian women born in the late 19th century. The analysis does not allow for the detection of any effect of inbreeding on the overall number of offspring of women. However, results indicate that high levels of close father inbreeding are associated with a reduction of the productivity of parents during the second half of their reproductive period, as compared with the first half. We suggest that inbreeding depression affects reproduction in modern societies through an interaction with age.     

Longevity in vitro of human CD4+ T helper cell clonesderived from young donors and elderly donors, or fromprogenitor cells: age-associated differences in cellsurface molecule expression and cytokine secretion

The effectiveness of the adaptive immune system reliesupon extensive proliferation of an initially smallnumber of antigen-specific T cells. At the end of asuccessful response, the majority die by apoptosis anda small minority joins the memory cell pool. Uponre-challenge with antigen, these memory cells mustagain undergo clonal expansion in order to mediate aneffective response. Thus, T cells are subjected tomarked proliferative stress which may result in clonalexhaustion due to replicative senescence. In othersystems made up of rapidly proliferating cells (e.g. inthe gut) individual clones are identical and arereplaced at the end of their lifespan bydifferentiation from a stem cell reservoir. However,because of the unique clonal distribution of antigenreceptors on T cells, mere replacement with other Tcells is not sufficient to maintain the integrity ofthe system. Moreover, the very source of new T cellsdecreases with age (due to thymic involution).Therefore, the adaptive immune system may be uniquelysusceptible to the deleterious effects of replicativesenescence. Particularly in humans, in vivo studies ofthe behaviour of individual T-cell clones in the bodyis difficult. However, T-cell longevity, measured asproliferative capacity in terms of populationdoublings, can be usefully modelled at the clonallevel in vitro. This paper discusses the surprisinglylittle that is known about the average longevity,variation between clones, and the maximal longevity ofhuman T cells under clonal culture conditions invitro. From our own studies, we show that averagelifespan of human T cells is as little as 17 PD;however, established clones reach 35 PD on average,with maximum longevity generally in the region of 60–80 PD, regardless of the source of the cloned cells.Expression of surface molecules in general did notdiffer strikingly between young and old donors, butthe frequency of clones secreting IL-10, and theamount secreted per clone was higher in the elderlythan in the young. Conversely, the frequency of clonessecreting IL-6 and the amount secreted per clone washigher in the young.     

Telomerase and cancer: time to move from a promising target to a clinical reality.

The past 25 years have seen unparalleled advances in our understanding of the molecular basis of cancer. As a result, novel molecular targets have been identified that provide great potential for the development of new cancer diagnostics and therapies. Four key features of cancer cells distinguish them from their normal counterparts: loss of cell-cycle regulation, loss of control over invasion and metastasis, failure of apoptotic mechanisms, and bypass of senescence. This review examines our understanding of the bypass of senescence and the process of immortalization during carcinogenesis. In addition, the realistic opportunities for telomerase in cancer diagnostics and the challenges faced in clinical trial design for telomerase therapeutics are discussed.     

Activation of fibroblast growth factor receptor 3 and oncogene-induced senescence in skin tumours

Background  The activation of oncogenes is an important step in tumorigenesis, and recently, oncogene-induced senescence (OIS) was proposed as a critical barrier against malignant transformation in normal primary cells.
Objectives  The aim of this study was to examine the activation of fibroblast growth factor receptor 3 (FGFR3) as an oncogene product and OIS in human skin tumours.
Methods  We investigated the activation of FGFR3 and OIS by mutation and immunohistochemical analysis in skin tumours, including seborrhoeic keratosis, actinic keratosis (AK), Bowen's disease (BD), basal cell carcinoma (BCC) and squamous cell carcinoma (SCC).
Results  Activated point mutations of FGFR3 were identified in four of 22 cases (18%) of seborrhoeic keratosis, but no mutation was detected in the other skin tumours. Twenty-seven of 31 cases (87%) of seborrhoeic keratosis showed moderately to strongly positive expression of the FGFR3 protein, but almost all the other skin tumours were negative. On the other hand, almost all the seborrhoeic keratoses showed negative immunoreactivity for antiphoshohistone H2AX (γ-H2AX) as a marker of OIS, but 17 of 22 cases (77%) of AK were moderately to strongly positive. Immunoreactivity for γ-H2AX was significantly greater in AK than in seborrhoeic keratosis, BD, BCC and SCC.
Conclusions  The activation of FGFR3 might be a common feature in the tumorigenesis in seborrhoeic keratosis, although the activation does not induce a typical oncogenic signal in keratinocytes. In addition, OIS due to some oncogenic signals rather than activation of FGFR3 might be involved in the early skin carcinogenesis related to chronic ultraviolet radiation exposure.     

Influence of the hypogonadism (hgn) locus on female reproduction and ovarian development in an altered genetic background

Background and Aims:   The hypogonadic rat ( hgn/hgn ) shows male sterility controlled by a single recessive gene hgn . The hgn/hgn females detected by the presence of renal hypoplasia in the HGN inbred strain show a reduced fertility. Recently, the gene responsible for male hypogonadism was mapped on chromosome 10 by a linkage analysis using only male backcross progeny. Because backcross females could not be categorized as affected or normal because of variations in their renal sizes, we could not examine female fertility in the backcross progeny. In the present experiment, we examined whether the gene mapped on rat chromosome 10 has any influences on female reproduction and ovarian development.
Methods:  The assumptive hgn/hgn females were identified in the backcross progeny by microsatellite markers closely linked to the hgn locus. Postnatal body growth, the weights of reproductive organs, estrus cycle and ovarian histology were examined. In addition, backcross embryos were genotyped, and their body growth and ovarian development was examined.
Results:  The hgn/hgn females showed growth retardation, a short reproductive life and an abnormal ovarian histology as adults. Regarding embryonic development, hgn/hgn females showed body growth retardation and ovarian hypoplasia.
Conclusion:  The mutation of the hgn mapped on chromosome 10 causes not only male sterility but also female reduced fertility related to ovarian hypoplasia beyond the altered genetic background. (Reprod Med Biol 2006; 5 : 227–234)     

自拟方参黄冲剂延缓衰老临床浅识

在临床实践的基础上总结了自拟方参黄冲剂延缓衰老的临床体会,提出了针对不同类型患者灵活变化辨证施治的原则,以期为临床延缓衰老提供新的理论和方法。     

Cellular senescence occurred widespread to multiple selective sites in the fetal tissues and organs of mice

Cellular senescence protects multicellular organisms from tissue overgrowth including cancer, and contributes to tissue ageing. With stable cell cycle arrests, cellular senescence has been mostly studied in the adult tissues of mammals. In the present study, we report widespread cellular senescence within certain time windows of late‐phase normal development of mouse embryos. Using in situ senescence‐associated β‐galactosidase (SA‐β‐gal) staining, we showed SA‐β‐gal activity in selected cell populations of the brain, stomach, interdigital webs, tail, ear, limbs and nasal mouth area on gestation day 14.5 of the mouse embryos. On day 18.5 of gestation, selected cells in the intestines and bone developmental areas showed SA‐β‐gal activity. The chondrocytes in ossification zones were significantly marked by the activities of SA‐β‐gal, p21, p15 and Hp1Y, suggesting activation of the cell cycle checkpoint by the p53 and Rb pathways, and development of senescence‐associated heterochromatic foci. Throughout gestation days 14.5–18.5, the trophoblast cells in the labyrinth layer of the placentas also showed strong activities of SA‐β‐gal, p53 and p21. Increased expressions of p19, p16 and Rb of the p16/Rb pathway, and reduced expressions of Ki67 were also observed in the placentas. Taken together, the present findings suggest that cellular senescence represents an essential mechanism at multiple sites including the fetal bone forming zones and placenta during mammalian embryonic development, playing potential roles in the full embryonic development of tissue growth and organ formation.