细胞衰老的过去、现在和未来:从科学解析到主动干预

高等生物体内的细胞衰老是机体衰老的原始驱动力,通过终止细胞周期循环来应对内外源胁迫性刺激。衰老细胞在整个生命周期中均可出现,但若长期或持续性出现,组织器官的正常功能会因其分泌的大量促炎性蛋白而受到病理性损害。在临床前模型中,针对那些持续存在并导致组织紊乱和器官损伤的衰老细胞进行干预已被证明可以有效延缓衰老、预防或缓解多种增龄相关疾病。尤其重要的,衰老医学界针对选择性清除衰老细胞的小分子药物senolytics的研发为积极预防或干预人类多种增龄相关疾病提供了强大的动力和灿烂的远景。在这篇综述中,笔者将介绍将衰老细胞作为老年疾病治疗靶点的基本原理,讨论近年正在兴起并迅速推广的人类临床试验的技术策略和发展前景,包括那些将小分子化合物senolytics及其相关衰老干预措施转化为临床应用的国际前沿探索。

A Novel Three-parameter Flow Cytometric Analysis for Cell Cycle

To set up a three-parameter method for cell cycle analysis by two-laser flowcy-tometer, which can detect two types of cyclin plus DNA content in one measurement, and thatanalyze unscheduled expression of cyclins. Methods: Three-color fluorescence was used for analysisof two types of cyclins and DNA content simultaneously in individual cells by two-laser flowcytometry. MOLT-4 cells were used to study the expression of major cyclins in mammalian cells. ATriton-X100 permeabilization procedure was optimized for detection of two types of cyclins. Onecyclin was stained directly with a FITC-conjugated monoclonal antibody (mAb), and the other,indirectly with RPE-Cy5-conjugated secondary antibody, while DNA was stained with the fluorochromeDAPI. mAMSA and mimosine treated MOLT-4 cells were used to test this three-parameter method.Results: Permeabilization with 0.5% Triton-XlOO in PBS containing 1% BSA for 5 min on ice providedoptimal conditions for the simultaneous labelling of two cyclins plus DNA in single cells. It wasfound that the emission spectrum of the three dyes (DAPI, FITC and RPE-Cy5) could be measured withno compensation. Based on cyclinA/cyclinE/DNA flow cytometric analysis, asynchronously growingMOLT-4 cells could be divided into 6 compartments (G1o, G1e, G1l, S, G2, and M) simultaneously,allowing for analysis of cell cycle phase specific perturbations without the necessity of cellsynchronization. Unscheduled cyclin B1 expression was observed in G1 cells treated with mimosine andcyclin E in G2 cells treated with mAMSA. We found that unscheduled cyclin expression paralleledexpected cyclin expression. Conclusion: Thus, three-color FCM analysis of cells may not only beapplied to measure unscheduled vs. expected cyclin expression but may also be used to estimate thefraction of cycling cells in up to 6 cell populations.

c-Fos overexpression increases the proliferation of human hepatocytes by stabilizing nuclear Cyclin D1

AIM： To investigate the effect of stable c-Fos over- expression on immortalized human hepatocyte （IHH） proliferation. METHODS： IHHs stably transfected with c-Fos （IHH- Fos） or an empty vector （IHH-C） were grown in me- dium supplemented with 1% serum or stimulated with 10% serum. Cell proliferation was assessed by cell counts, 3H-thymidine uptake and flow cytometry analyses. The levels of cell cycle regulatory proteins （Cyclin DI, E, A） cyclin dependent kinases （cdk） cdk2, cdk4, cdk6, and their inhibitors p15, p16, p21, p27, total and phosphorylated GSK-3β and epidermal growth factor receptor （EGF-R） were assayed by Western blotting. Analysis of O/c/in D1 mRNA levels was performed by reverse transcription-polymerase chain reaction and real-time polymerase chain reaction （PCR） analysis. Stability of Cyclin DI was studied by cycloheximide blockade experiments. RESULTS： Stable c-Fos overexpression increased cell proliferation under low serum conditions and resulted in a two-fold increase in [3H]-thymidine incorpora- tion following serum addition. Cell cycle analysis by flow cytometry showed that c-Fos accelerated the cell cycle kinetics. Following serum stimulation, Cyclin D1 was more abundantly expressed in c-Fos overexpress- ing cells. Cyclin D1 accumulation did not result from increased transcriptional activation, but from nuclear stabilization. Overexpression of c-Fos correlated with higher nuclear levels of inactive phosphorylated GSK- 3β, a kinase involved in Cyclin D1 degradation and higher levels of EGF-R mRNA, and EGF-R protein com- pared to IHH-C both in serum starved, and in serum stimulated cells. Abrogation of EGF-R signalling in IHH- Fos by treatment with AG1478, a specific EGF-R tyro- sine kinase inhibitor, prevented the phosphorylation of GSK-3β induced by serum stimulation and decreased Cyclin D1 stability in the nucleus. CONCLUSION： Our results clearly indicate a positive role for c-Fos in cell cycle regulation in hepatocytes. Importantly, we delineate a new mechanism by which c-Fos could contribute to hepatocarcinogenesis through stabilization of Cyclin D1 within the nucleus, evoking a new feature to c-Fos implication in hepatocellular carcinoma.

Hepatitis C virus core proteins derived from different quasispecies of genotype 1b inhibit the growth of Chang liver cells

AIM: To investigate the influence of different quasispecies of hepatitis C virus (HCV) genotype 1b core protein on growth of Chang liver cells. METHODS: Three eukaryotic expression plasmids (pEGFP-N1/core) that contained different quasispecies truncated core proteins of HCV genotype 1b were constructed. These were derived from tumor (T) and non- tumor (NT) tissues of a patient infected with HCV and C191 (HCV-J6). The core protein expression plasmids were transiently transfected into Chang liver cells. At different times, the cell cycle and apoptosis was assayed by flow cytometry, and cell proliferation was assayed by methyl thiazolyl tetrazolium (MTT) assay. RESULTS: The proportion of S-phase Chang liver cells transfected with pEGFP-N1/core was significantly lower than that of cells transfected with blank plasmid at three different times after transfection (all P < 0.05). The proliferation ratio of cells transfected with pEGFP-N1/corewas significantly lower than that of cells transfected with blank plasmid. Among three different quasispecies, T, NT and C191 core expression cells, there was no significant difference in the proportion of S- and G0/G1-phase cells. The percentage of apoptotic cells was highest for T (T > NT > C191), and apoptosis was increased in cells transfected with pEGFP-N1/core as the transfection time increased (72 h > 48 h > 24 h). CONCLUSION: These results suggest that HCV genotype 1b core protein induces apoptosis, and inhibits cell- cycle progression and proliferation of Chang liver cells. Different quasispecies core proteins of HCV genotype 1b might have some differences in the pathogenesis of HCV persistent infection and hepatocellular carcinoma.

Expression of positive and negative regulators of cell cycle during wound healing

OBJECTIVE: To detect the expression of cell cycle positive regulators cyclin D(1), cyclin E, CDK(2), CDK(4) and negative regulators p21(cip1), p27(kip1), p16(ink4a) and p15(ink4b) during wound healing in rats. METHODS: Open wounds of full-thickness skin, diameter 1.8 cm, on rat backs were used as the wound model. Wound tissues were harvested on postwounding days 3, 5, 7, 9, 11, 14, 21 and 30. Ki67 expression in granulation tissue was detected by immunohistochemical assay. The patterns of the expression of cyclin D(1), cyclin E, CDK(2), CDK(4) and p21(cip1), p27(kip1), p16(ink4a), p15(ink4b) were detected by Western blot. RESULTS: Cell proliferation in granulation tissue took place predominantly within the first week after injury, with the proliferation peak occurring at postwounding day 5. There were no dramatic variations in the expression of cyclin D(1), CDK(2) and CDK(4) during wound healing. Up-regulated cyclin E was maintained from day 3 to 11 after injury, and then was down-regulated. No expression of p16(ink4a) and p15(ink4b) was found. p21(cip1) was expressed only from day 7 to 14, with peak expression observed on day 9. Constitutive p27(kip1) was expressed throughout wound healing with low levels in the proliferating period of day 3 to 5 and with increased levels in the post-mitotic and remodeling stage. The expression of p21(cip1) and p27(kip1) showed an inverse gradient to that of Ki67. CONCLUSION: p21(cip1) and p27(kip1) play a supervising role in preventing the hyperproliferative tendency in tissue repair.