AGING OF ENDOCRINE CELLS IN CULTURE

培养中内分泌细胞的老化

基本信息

批准号：
2723890
负责人：
PETER J HORNSBY
金额：
$ 0.5万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-08-01 至 1998-03-31
项目状态：
已结题

项目摘要

The aim of this proposal is to provide insight into cellular aging mechanisms using a model system of bovine and human adrenocortical cells in culture. A hormonally-inducible, -tissue- specific gene, steroid 17a-hydroxylase, will be used as an indicator for general changes in genome in cellular senescence. The mechanism of loss of induction of 17a-hydroxylase in senescence in adrenocortical cells will be investigated. An in vitro transcript elongation assay will be used to measure synthesis rates for 17a-hydroxylase in nuclei from early and late passage cells. The expression of transfected 17a-hydroxylase cDNA in old cells will be examined to investigate the possibility of increased degradation of 17a-hydroxylase mRNA in old cells. Experiments are proposed to identify and characterize a repressor or transcription factor for the 17a-hydroxylase gene which may change in senescence. If evidence for such changes is obtained, the experiments will be extended to investigate whether other steroidogenic enzyme genes are affected, other inducible gene functions, and whether replication is altered by such a factor. Screening for possible repressors or activators will be done by introduction of nuclear extracts by the erythrocyte loading delivery procedure. Expression of 17a-hydroxylase at the individual cell level will be monitored by an in situ hybridization procedure using fluorescence detection, to provide a means for screening nuclear extracts for the presence of regulatory factors. If such factors are detected, they will be used in in vitro experiments using the genomic clone for 17a-hydroxylase. Assays for the presence of DNA-binding proteins in nuclear extracts from senescent or young cells, in conjunction with an in vitro transcription assay, will be used. Senescent adrenocortical cells will be transfected with plasmids comprising 17a-hydroxylase 5' regulatory sequences fused to chloramphenicol acetyltransferase as a reporter gene. Regulation of CAT expression in senescent cells will be investigated using constructs containing various portions of the 5' flanking region of the gene. The nuclease sensitivity of the 17a-hydroxylase gene in senescent cells in comparison to young cells will be studied. Any changes in nuclease sensitivity of the gene in senescence may be followed up by the use of a genomic footprinting procedure. The effects of an immortalizing oncogene will be examined. Adrenocortical cells will be transfected with pLTRmyc, a plasmid containing c-myc under the regulation of the mouse mammary tumor virus long terminal repeat, which provides hormonal control of c-myc expression. Possible immortalization in these transfectants and the effect or myc expression on 17a-hydroxylase induction will be examined.

该提案的目的是深入了解细胞衰老使用牛和人类模型系统的机制培养中的肾上腺皮质细胞。一种激素诱导的，-组织- 特定基因，类固醇17a-羟化酶，将被用作细胞衰老中基因组一般变化的指标。 17a-羟化酶诱导丧失的机制将研究肾上腺皮质细胞的衰老。一个在体外转录延伸测定将用于测量合成早期和晚期传代细胞核中 17a-羟化酶的比率细胞。转染17a-羟化酶cDNA在老年人体内的表达将检查细胞以调查增加的可能性老化细胞中 17a-羟化酶 mRNA 的降解。实验建议鉴定和表征阻遏物或 17a-羟化酶基因的转录因子，可能衰老的变化。如果获得此类变更的证据，实验将进一步扩大，以研究是否有其他类固醇生成酶基因受到影响，其他诱导基因功能，以及复制是否因此类因素而改变。筛选可能的阻遏物或激活物将通过通过红细胞负荷引入核提取物交货程序。 17a-羟化酶的表达单个细胞水平将通过原位杂交监测使用荧光检测的程序，提供了一种手段筛选核提取物是否存在调节因子。如果检测到这些因素，它们将用于体外使用 17a-羟化酶基因组克隆进行的实验。化验核提取物中是否存在 DNA 结合蛋白衰老或年轻细胞，与体外将使用转录测定。衰老的肾上腺皮质细胞将用包含17a-羟化酶5'的质粒转染与氯霉素乙酰转移酶融合的调控序列作为报告基因。衰老细胞中 CAT 表达的调控将使用含有各种成分的构建体来研究细胞基因 5' 侧翼区域的部分。核酸酶衰老细胞中 17a-羟化酶基因的敏感性将研究与年轻细胞的比较。任何变化可以追踪衰老过程中基因的核酸酶敏感性通过使用基因组足迹程序。的影响将检查永生癌基因。肾上腺皮质细胞将用 pLTRmyc（一种含有 c-myc 的质粒）转染在小鼠乳腺肿瘤病毒的调控下长末端重复，提供 c-myc 的激素控制表达。这些转染子可能永生化 myc 表达对 17a-羟化酶诱导的影响将是检查了。