人参皂苷Rg1延缓造血干细胞衰老过程中端粒长度和端粒酶活性的变化

目的:探讨端粒长度和端粒酶活性在人参皂苷Rg1延缓造血干细胞(HSC)衰老中的作用.方法:免疫磁性分选法分离纯化小鼠Sca-1+ HSC后分5组:对照组,衰老模型组,Rg1组,Rg1治疗衰老组,Rg1延缓衰老组.衰老相关β-半乳糖苷酶(SA-β-Gal)染色、流式细胞术细胞周期测定和造血祖细胞混合集落(CFU-Mix)培养分析Rg1调控Sca-1+ HSC衰老的生物学作用;Southern blotting与TRAP-PCR SYBR Green法检测端粒长度和端粒酶活性.结果:与衰老模型组相比,Rg1治疗组和Rg1延缓衰老组Sca-1+ HSC的SA-β-Gal染色阳性细胞百分比降低,G1期细胞比例下降,生成CFU-Mix数相对增加,端粒长度缩短明显减少,端粒酶活性增强;Rg1延缓衰老组各检测指标变化均较Rg1治疗组明显.结论:Rg1可能通过激活端粒酶活性和减少端粒长度缩短发挥延缓和治疗Sca-1+ HSC衰老的作用.     

人参皂苷Rg1抗衰老作用可能与改变p16、cyclin D、CDK4的表达有关

目的 :探讨人参皂苷Rg1对抗三丁基过氧化氢 (t BHP)诱导的WI 38细胞衰老作用及其可能细胞周期调控机制。方法 :将WI 38细胞随机分为 4组 ,用不同剂量Rg1预处理。从 30代开始 ,隔代用t BHP作用 ,每次 1h ,共 4次 ,诱导细胞衰老。从光镜、透射电镜观察细胞形态及超微结构 ;流式细胞术分析G1期细胞比例 ;以及SA β 半乳糖苷酶的细胞化学染色 ,确定Rg1的抗衰老作用。并采用免疫印迹技术对CDK4、cyclinD1和p16等表达情况进行检测。结果 :Rg1预处理组与单纯t BHP处理组相比 ,细胞形态体积小、胞体不如后者扁平 ,次级溶酶体减少 ,G1期细胞比例下降 ,SA β 半乳糖苷酶染色阳性细胞百分比下降 ,说明Rg1在t BHP诱导细胞衰老模型中有抗衰老作用。进一步发现用Rg1预处理后 ,p16、cyclinD1表达水平降低、CDK4表达水平增加。结论 :提示Rg1可能通过改变细胞周期调控因子的表达而发挥其抗t BHP诱导的WI 38细胞衰老作用。     

CYP3A4 overexpression enhances the cytotoxicity of the antitumor triazoloacridinone derivative C-1305 in CHO cells

Aim:

To examine how the higher expression level of CYP3A4 isoenzyme influenced the cytotoxicity of the antitumor triazoloacridinone derivative C-1305 in Chinese hamster ovary (CHO) cells.

Methods:

Three CHO cell lines were examined: wild-type CHO cells; CHO-HR cells with overexpression of human cytochrome P450 reductase (CPR); and CHO-HR-3A4 cells with coexpression of human CYP3A4 and CPR. Cellular responses caused by C-1305 were monitored using DAPI staining, cell cycle analysis, phosphatydilserine externalization analysis and SA-β-galactosidase expression analysis. Cell viability was assessed with simultaneous FDA and PI staining.

Results:

Treatment with C-1305 for 72 h exhibited different levels of cytotoxicity in the 3 cell lines, and the values of IC80 in CHO, CHO-HR and CHO-HR-3A4 cells were 0.087±0.005, 0.032±0.0001, and 0.064±0.0095 μmol/L, respectively. The cell cycle analysis revealed that both CHO and CHO-HR cells underwent transient G₂/M arrest, whereas CHO-HR-3A4 cells did not accumulate in this phase. Prolonged exposure up to 120 h caused time-dependent increase in the sub-G₁ fraction in all the 3 cell lines. Treatment with C-1305 caused cell death through apoptosis and necrosis. However, these processes were more pronounced in the transfected CHO cells than in the wild-type cells. The cells surviving after C-1305 exposure underwent senescence.

Conclusion:

CYP3A4 overexpression potently enhances the cellular responses (apoptosis, necrosis and senescence) caused by C-1305 in CHO cells.     

氟化钠对L02人正常肝细胞衰老及相关蛋白表达影响的实验研究

目的 研究氟化钠（NaF）是否会诱导人肝细胞 L02衰老及衰老相关标志蛋白水平的改变。方法 不同浓度 (0、1、2、3、4、5、6、7和8mmol/L) NaF 处理 L02人正常肝细胞,CCK-8 法检测细胞存活率的变化以筛选 NaF 的作用浓度;衰老标志物 β-半乳糖苷酶 (β-galactosidase, β-Gal) 染色检测衰老阳性细胞;实时荧光定量 PCR (Real-time PCR) 和蛋白免疫印迹 (Western Blot) 测定细胞衰老标志蛋白 p16,p21 mRNA 和蛋白的表达水平;流式细胞术分析细胞周期的变化情况。结果 CCK-8 结果显示,NaF 浓度为 1、4、7mmol/L时 L02细胞的存活率（87.38±0.93、66.72±2.81、52.17±2.04）均低于对照组［(100.00±0.00),均 P<0.01］,且每组之间的细胞存活率差异存在统计学意义（均 P<0.01）,因此 NaF 选用1、4、7 mmol/L 处理L02细胞作为低氟组、中氟组、高氟组开展研究;β-半乳糖苷酶染色结果显示,低、中、高氟组中细胞衰老率 (23.15±0.23、36.06±0.40、65.36±0.82)均高于正常组［(3.27±0. 27),P<0.01］;Real-time PCR 结果显示,p16、p21 mRNA在低、中、高氟组中表达(2.29±0.19、3.44±0.30、5.25±0.32;1.88±0.22、3.22±0.24、7.33±0.30)表达均高于正常对照组［(1.00±0.00;1.00±0.00),均P<0.01］。低、中、高氟组中p16蛋白水平(93.68±3.40、116.25±7.30、122.31±3.00)均高于对照组［(78.07±4.17),P<0.05,P<0.01,P<0.01),低、中、高氟组中p21蛋白水平(87.61±5.22、121.62±4.27、147.95±7.81)均高于对照组［(61.32±4.56),均P<0.01)］;流式结果显示,低、中、高氟组 L02 细胞周期均停滞在 G2 期,与对照组相比,差异具有统计学意义 (P<0.01)。结论 NaF 处理的人肝细胞 L02 中细胞衰老率及衰老标志蛋白 p16、p21 的 mRNA 和蛋白表达升高,这可能提示了 NaF 可导致 L02 细胞衰老,进一步说明细胞衰老在氟化物诱导的肝损伤中可能具有重要作用。     

马齿苋多糖对衰老小鼠免疫调节作用的研究

目的：本研究旨在探讨中药马齿苋多糖对衰老小鼠免疫调节作用。方法：皮下注射D-半乳糖建立衰老小鼠模型,用马齿苋多糖进行治疗,同时设正常对照组、阳性对照组和阴性对照组,用药30d后处死小鼠,进行免疫指标检测。结果：用D-半乳糖造模以后,小鼠胸腺指数、脾脏指数和T淋巴细胞转化值都降低（P〈0.05）。用马齿苋治疗后,小鼠胸腺指数、脾脏指数和T淋巴细胞转化值都增生（P〈0.05）。结论：马齿苋多糖增强机体免疫力,马齿苋多糖对衰老小鼠具有免疫调节作用。     

淫羊藿苷对快速老化小鼠SAMP8学习记忆功能影响的研究

目的:研究淫羊藿苷(Icaritin,ICA)对快速老化小鼠SAMP8学习记忆功能的影响。方法:按体重随机挑选10只8月龄抗快速老化小鼠SAMR1和50只同月龄快速老化小鼠SAMP8,并将SAMP8随机分为5组(模型组、多奈哌齐组和ICA50mg/kg、100mg/kg、200mg/kg组),灌胃给药1个月,在给药前后分别用Morris水迷宫、SMG-2迷宫和跳台方法检测SAMR1和SAMP8的学习记忆功能能力。结果:与给药前相比,给药后的快速老化小鼠SAMP8定位航行和空间搜索能力以及被动反应能力得到提高。结论:ICA可以改善SAMP8的学习记忆能力。     

HDAC4 stabilizes SIRT1 via sumoylation SIRT1 to delay cellular senescence

The nicotinamide adenine dinucleotide‐dependent protein deacetylase silent information regulator 2 (Sir2) regulates cellular lifespan in several organisms. Histone deacetylase 4 (HDAC4) belongs to the class IIa group of HDACs; this class of HDACs is composed of proteins that are important regulators of gene expression that control pleiotropic cellular functions. However, the role of HDAC4 in cellular senescence is still unknown. This study shows that the expression patterns of HDAC4 and Sirtuin 1 (SIRT1; the mammalian homolog of Sir2) are positively correlated during cellular senescence. Moreover, the overexpression of HDAC4 delays senescence, whereas the knockdown of HDAC4 leads to premature senescence in human fibroblasts. Furthermore, it is demonstrated that HDAC4 increases endogenous SIRT1 expression by enhancing its sumoylation modification levels, thereby stabilizing its protein levels. This study, therefore, provides a new molecular mechanism for the regulation of cellular senescence.     

Induction of senescence in cancer cells by the G-quadruplex stabilizer, BMVC4, is independent of its telomerase inhibitory activity

BACKGROUND AND PURPOSE Telomerase is the enzyme responsible for extending G-strand telomeric DNA and represents a promising target for treatment of neoplasia. Inhibition of telomerase can be achieved by stabilization of G-quadruplex DNA structures. Here, we characterize the cellular effects of a novel G-quadruplex stabilizing compound, 3,6-bis(4-methyl-2-vinylpyrazinium iodine) carbazole (BMVC4). EXPERIMENTAL APPROACH The cellular effects of BMVC4 were characterized in both telomerase-positive and alternative lengthening of telomeres (ALT) cancer cells. The molecular mechanism of how BMVC4 induced senescence is also addressed. KEY RESULTS BMVC4-treated cancer cells showed typical senescence phenotypes. BMVC4 induced senescence in both ALT and telomerase-overexpressing cells, suggesting that telomere shortening through telomerase inhibition might not be the cause for senescence. A large fraction of DNA damage foci was not localized to telomeres in BMVC4-treated cells and BMVC4 suppressed c-myc expression through stabilizing the G-quadruplex structure located at its promoter. These results indicated that the cellular targets of BMVC4 were not limited to telomeres. Further analyses showed that BMVC4 induced DNA breaks and activation of ataxia telangiectasia-mutated mediated DNA damage response pathway. CONCLUSIONS AND IMPLICATIONS BMVC4, a G-quadruplex stabilizer, induced senescence by activation of pathways of response to DNA damage that was independent of its telomerase inhibitory activity. Thus, BMVC4 has the potential to be developed as a chemotherapeutic agent against both telomerase positive and ALT cancer cells.     

补益营卫方对衰老表皮结构蛋白K17的影响

目的:观察补益营卫方对衰老表皮结构蛋白K17的基因和蛋白水平的影响。方法:将3个月龄小鼠(年轻组)与14个月龄小鼠(老年组)表皮细胞消化分离出来,然后进行传代培养,其中14个月龄小鼠(老年组)的表皮细胞分为2组,一组用常规方法培养细胞,另一组用含2. 5%的补益营卫方培养基进行培养,采用荧光定量RT-PCR法和Western Blotting法分别检测细胞K17mRNA表达和细胞K17蛋白表达。结果:与年轻组比较,老年组的K17mRNA和蛋白表达水平均升高(P <0. 05);经药物干预后小鼠表皮细胞中K17mRNA和蛋白水平较老年组明显降低(P <0. 05)。结论:补益营卫方可通过抑制衰老表皮结构蛋白K17的表达,起到延缓皮肤衰老作用。     

人胚肺成纤维细胞衰老过程中胰岛素样生长因子2表观遗传活化作用

目的观察体外培养人胚肺成纤维细胞复制性衰老过程中及过氧化氢诱导早衰持续阶段胰岛素样生长因子2(IGF2)表观遗传学修饰。方法荧光定量PCR检测IGF2的mRNA表达,甲基化特异PCR检测其启动子区甲基化改变,染色质免疫沉淀结合定量PCR检测组蛋白修饰,包括组蛋白H3、H4乙酰化,H3(Lys4)及H4(Lys20)甲基化修饰。结果细胞复制性衰老过程中,中年细胞组及复制性衰老细胞组IGF2的mRNA表达升高,早衰持续组升高显著;细胞衰老过程中,仅复制性衰老细胞组在启动子区-658～-456bp具有一定的甲基化水平;复制性衰老细胞组的组蛋白修饰在启动子区-856～-634bp以H4乙酰化,H3甲基化修饰为主;+9～+145bp的修饰,复制性衰老细胞组受组蛋白H3及H4甲基化联合修饰,早衰持续组以H3甲基化修饰为主。结论细胞衰老过程中,IGF2启动子区组蛋白修饰联合调控其mRNA表达,复制性衰老与早衰的调控机制存在差异。