快速诱导细胞凋亡及检测方法研究

目的：探讨Ca2＋和Na＋诱导细胞凋亡的最佳浓度及时间,并用甲基绿一派诺宁染色法检测凋亡细胞的形态变化。方法：分别用不同浓度梯度及时间梯度的Ca2＋和Na＋胁迫处理洋葱鳞茎内表皮细胞,得诱导的最佳浓度及时间;用甲基绿一派诺宁染色法检测诱导凋亡的洋葱鳞茎内表皮细胞、大蒜根尖细胞和鸡血红细胞的形态特征变化。结果：诱导处理的最佳Ca2＋和Na＋浓度为0．4mol／L,最适时间约为8h,且CaCl2的诱导效果较NaCl好;经甲基绿一派诺宁染色,洋葱鳞茎内表皮细胞、大蒜根尖细胞、鸡血红细胞凋亡细胞的细胞核均呈蓝紫色,细胞质呈红色。结论：找出了诱导细胞凋亡的最适Ca2＋和Na＋浓度和时间,并检测到细胞凋亡。     

农杆菌介导的MpASR蛋白在洋葱表皮细胞的定位研究

通过比较不同侵染液浓度、侵染时间、取材部位、预处理等条件下的转化效果,优化了农杆菌介导的洋葱鳞茎内表皮细胞转化系统,并且成功检测到大蕉ASR蛋白(MpASR)与GFP的融合蛋白在洋葱表皮细胞中的分布.     

外源NO和H2O2对洋葱鳞片外表皮气孔开度的调控

以洋葱(Allium cepa L.)肉质鳞片外表皮为材料,研究不同浓度及不同处理时间的外源NO和H2O2对洋葱鳞片外表皮上气孔开度的调节作用,并结合NO清除剂血红蛋白(Hb)和H2O2清除剂过氧化氢酶(CAT)研究调控过程中NO和H2O2的相互关系.结果显示:单独施用不同浓度的NO和H2O2均可诱导洋葱鳞片外表皮气孔不同程度关闭,并且浓度越大时间越长,其诱导气孔关闭效应越明显;NO和H2O2共同施用所诱导气孔关闭的效应大于其单独施用效应;Hb和CAT能明显减弱NO和H2O2诱导的气孔关闭.研究表明,NO和H2O2能有效诱导洋葱鳞片上气孔关闭,存在明显的浓度效应和时间效应,且两者可能互相依赖,具有协同效应.     

秋水仙素对洋葱根尖细胞染色体结构变异的影响

用0.05%和0.1%秋水仙素溶液对洋葱Allium cepa根尖进行控时处理,研究不同秋水仙素浓度和不同处理时间对洋葱根尖细胞有丝分裂畸变的影响。结果显示,秋水仙素有促进细胞有丝分裂染色体停滞在中期的能力;不同固定时间对洋葱根尖细胞有丝分裂有影响;秋水仙素对洋葱根尖细胞染色体有畸变效应,诱导产生畸变的能力受溶液浓度和处理时间的影响。     

洋葱鳞茎内表皮细胞中的微管

用超薄切片和原生质体负染的方法,在电镜下观察到洋葱鳞茎内表皮细胞周质微管的存在。其出现时间、分布状况以及排列形态,基本上与考马斯亮蓝染色法观察到的网状结构物相一致。讨论了包被囊泡和微管组织中心可能的作用。     

洋葱内表皮细胞中的网状结构物

使用 Triton 抽提和卡马氏亮蓝染色方法,在洋葱鳞茎内表皮细胞中观察到网状结构物的存在。秋水仙素和细胞松弛素 B 的处理,进一步区分出两种不同的网状结构:近质膜较细的网状结构能被秋水仙素破坏;核周围与核成切线状结合的较粗的网状结构,经细胞松弛素 B 处理后,其形象更为突出。本文讨论了卡马氏亮蓝方法所揭示的带壁细胞的网状结构物。     

纳米银抑制肝癌HepG2细胞活力

目的探讨纳米银(AgNPs)对体外培养的人肝癌细胞株HepG2增殖和凋亡的影响。方法用不同浓度的AgNPs处理肝癌HepG2细胞,采用细胞形态学观察及细胞活力测定(MTT法)来评价AgNPs对HepG2细胞体外增殖的影响,流式细胞仪分析细胞周期分布并检测细胞凋亡率。结果 AgNPs使细胞增殖活性降低,且呈浓度和时间依赖性,细胞呈凋亡形态改变;流式细胞仪检测到细胞凋亡,细胞平均凋亡率呈时间依赖性。AgNPs处理组S期细胞逐渐增多,而G0/G1、G2/M期细胞逐渐减少。结论 AgNPs抑制HepG2细胞生长,使HepG2细胞阻滞于S期,并诱导其凋亡。     

洋葱鳞茎表皮Actin细胞骨架的Rh—Ph荧光和光学显微镜观察

洋葱鳞茎内表皮细胞经Triton X-100处理和多聚甲醛固定之后用Rh-Ph(Rhodamine-Phalloidin)染色,细胞质内可见较丰富的、直径为100—300nm的F-actin束。较粗的F-actin束沿细胞的长轴平行排列,并纵裂成较细的“分枝”,纵裂成的分枝又纵裂成更细的“分枝”。各种大小的F-actin束相互交织在一起构成一个三维的纤丝网络,并且与细胞膜、细胞核和其它细胞器相连。经同样方法处理和固定的细胞用考马斯亮兰R_(250)(Coomassie brilliant blue R_(250))染色之后,细胞质内可见直径为200—300nm的纤丝,形态特征和排列方式和上述在荧光显微镜下看到的F-actin束相同。本研究结果表明洋葱鳞茎内表皮的细胞骨架包含较丰富的F-actin系统;Pena的考马斯亮兰染色法(1980)所显示的结构主要代表F-actin束。     

曲古霉素A诱导人膀胱癌细胞周期阻滞和凋亡的研究

目的:探讨组蛋白去乙酰化酶抑制剂曲古霉素A(TSA)对人膀胱癌T24细胞周期和凋亡的影响。方法:以不同剂量TSA(0.1μM,0.3μM和1μM)处理T24细胞。采用MTT法检测细胞存活率,AnnexinV-PI染色检测细胞凋亡,流式细胞仪检测caspase-3活性,Western blot法检测P21蛋白表达。结果:TSA剂量依赖性降低膀胱癌细胞存活率,促进细胞凋亡,表现为AnnexinV阳性细胞明显增多,同时活化的caspase-3水平增高。TSA还可通过诱导膀胱癌细胞周期阻滞于G2/M期抑制细胞生长,且呈剂量依赖性。结论:TSA通过促进caspase-3激活诱导膀胱癌细胞凋亡,同时诱导细胞阻滞于G2/M期。     

NaCl胁迫对长春花幼苗离子分布和光合作用的影响

以0、50、100、150、200和250mmol·L^-1NaCl的1／2Hoagland营养液处理长春花（Catharanthus roseus）幼苗5d,测定其生物量,根、茎及叶中不同组织细胞中无机离子相对含量,叶绿素含量及光合生理指标。结果表明：NaCl能显著降低长春花幼苗的鲜质量和干质量;对根、茎和叶片横切面X射线徽区分析表明,NaCl胁迫导致长春花体内各组织细胞中Na^＋和Cl^-相对含量显著增加,但在各器官、组织中分布稍有不同：与对照相比,根和茎中都是表皮细胞中增加幅度最大,中柱细胞次之,皮层细胞最低;在叶片中亦是表皮细胞增加幅度最大,依次是皮层细胞、海绵组织细胞及栅栏组织细胞。NaCl胁迫下,K^＋和Ca^2＋相对含量对其的响应特征在不同器官中亦不相同：与对照相比,在根和茎皮层及中柱细胞中的下降幅度低于表皮;而叶中则是栅栏组织细胞最低。盐胁迫能够抑制长春花幼苗生长,打破其体内的离子平衡,但植物为降低盐胁迫的伤害而将过多的Na^＋和Cl^-聚集于表皮细胞。NaCl胁迫下,长春花幼苗叶绿素含量、光合速率（Pn）、细胞间隙CO2浓度（Ci）、气孔导度（Gs）、蒸腾速率（Tr）显著降低,气孔限制值（Ls）升高,说明气孔限制是降低长春花幼苗光合速率的主要因素。     

Salinity induced nuclear and DNA degradation in meristematic cells of soybean (Glycine max (L.)) roots

Changes in the nuclei of meristematic root cells of soybean (Glycine max (L.) Merr. cv. Acme) in response to severe salinity were studied. Root growth was inhibited by 200 mM NaCl, when 1 mM CaCl_2 was present in the culture media. Increasing CaCl_2 up to 5 mM partially prevented this inhibition. However, inhibition also occurred with 100~mM NaCl without CaCl_2. We examined the meristematic cells under a series of NaCl treatments. Nuclear deformation of the cells occurred with 24 h of 150 mM or higher NaCl, and was followed by degradation of nuclei in the apical region of the root. TEM observation and agarose gel electrophoretic analysis confirmed that root tip nuclear DNA deformed or degraded with 150 mM or higher NaCl concentrations.     

外源钙降低拟南芥幼苗盐害效应

用5mmol／L CaCl2喷叶后的拟南芥幼苗,在100mmol／L NaCl胁迫下,SOD、CAT和POD的活性以及叶绿素含量降低幅度明显低于对照,MDA含量增加幅度显著低于对照,由此表明钙与拟南芥幼苗盐害效应的降低有关。     

盐胁迫下Ca~(2 )对小麦根无机离子分布和膜脂脂肪酸的影响

用扫描电镜、X—射线能谱仪和等离子耦合吸收光谱(ICP)分析发现,培养在含NaCl培养液中,小麦柜吸收大量Na~ 和Cl~-,K~ 和Ca~(2 )的吸收降低,质膜相对透性提高,K~ 、Na~ 和Cl~-的相对外渗百分率增加。培养在补充CaCl_2含NaCl培养液的,Na~ 和Cl~-含量明显减低,K~ 和Ca~(2 )提高,质膜相对透性下降,K~ 、Na~ 和Cl~-相对外渗百分率减少。由气相色谱分析可知,用含NaCl或补充CaCl_2培养液培养的幼苗,根的膜脂脂肪酸组分没有变化,但培养在补充CaCl_2的培养液中的根,亚麻酸含量和脂肪酸不饱和指数下降。     

钠盐和钙盐胁迫对草莓光合作用的影响

以草莓(Fragaria ananassaDuch.)达塞莱克特品种为试材,采用盆栽法研究了NaCl、CaCl2、Ca(AC)2和Ca(NO3)2胁迫对其生长及光合特性的影响.结果表明,4种盐胁迫下草莓的生物产量、叶绿素含量、净光合速率、蒸腾速率和气孔导度显著降低,且盐浓度越高,下降幅度越大.其中,净光合速率、蒸腾速率和气孔导度的降幅由大到小依次为:Ca(AC)2>Ca(NO3)2>CaCl2>NaCl.与对照相比,NaCl胁迫下草莓根冠比减小,而钙盐胁迫下草莓的根冠比增大,且各盐处理内均随盐浓度增加而增大.钙盐胁迫对草莓光合作用的影响大于钠盐,其中,NaCl胁迫对植株的伤害最小,Ca(AC)2处理的伤害最大.     

Resveratrol Couples Apoptosis with Autophagy in UVB-Irradiated HaCaT Cells

UVB radiation causes about 90% of non-melanoma skin cancers by damaging DNA either directly or indirectly by increasing levels of reactive oxygen species (ROS). Skin, chronically exposed to both endogenous and environmental pro-oxidant agents, contains a well-organised system of chemical and enzymatic antioxidants. However, increased or prolonged free radical action can overwhelm ROS defence mechanisms, contributing to the development of cutaneous diseases. Thus, new strategies for skin protection comprise the use of food antioxidants to counteract oxidative stress. Resveratrol, a phytoalexin from grape, has gained a great interest for its ability to influence several biological mechanisms like redox balance, cell proliferation, signal transduction pathways, immune and inflammatory response. Therefore, the potential of resveratrol to modify skin cell response to UVB exposure could turn out to be a useful option to protect skin from sunlight-induced degenerative diseases. To investigate into this matter, HaCaT cells, a largely used model for human skin keratinocytes, were treated with 25 or 100 µM resveratrol for 2 and 24 hours prior to UVB irradiation (10 to 100 mJ/cm²). Cell viability and molecular markers of proliferation, oxidative stress, apoptosis, and autophagy were analyzed. In HaCaT cells resveratrol pretreatment: reduces UVB-induced ROS formation, enhances the detrimental effect of UVB on HaCaT cell vitality, increases UVB-induced caspase 8, PARP cleavage, and induces autophagy. These findings suggest that resveratrol could exert photochemopreventive effects by enhancing UVB-induced apoptosis and by inducing autophagy, thus reducing the odds that damaged cells could escape programmed cell death and initiate malignant transformation.     

Regulation of intracellular osmotic pressure during the initial stages of salt stress in a salt-tolerant yeast, Zygosaccharomyces rouxii.

The accumulation of glycerol and inorganic ions as it related to osmotic pressure, and the regulation of intracellular osmotic pressure in a salt-tolerant yeast, Zygosaccharomyces rouxii, were examined for several hours after salt stress. Intracellular contents of glycerol increased for up to 6 h in media supplemented with 1 M and 2 M NaCl and did not increase in medium containing 3 M NaCl. Intracellular contents of Na+ and Cl- reached a maximum value within 1 and 3 h, respectively, in all NaCl-containing media and increases were proportional to the concentration of NaCl in the medium. As glycerol was accumulated in cells, the intracellular contents of Na+ and Cl- gradually decreased in media containing 1 M and 2 M NaCl. After salt stress, cell volume decreased within 1 h and the original volume was re-established for 3 to 6 h in media with 1 M and 2 M NaCl but not in medium with 3 M NaCl. Intracellular concentrations of solutes, which were calculated from the total contents of glycerol and inorganic ions and the cell volume, became almost equivalent to the external osmotic pressure within 1 h after salt stress. Experiments using various inhibitors showed that a large amount of ATP was required not only for the synthesis and accumulation of glycerol but also for the exclusion of Na+ and Cl- from cells under salt-stressed conditions.     

Effects of abscisic acid and proline on adaptation of tobacco callus cultures to salinity and osmotic shock

Tobacco callus ( Nicotina tabacum cv. Badischer Geudertheimer) took up sorbitol rapidly and without a lag period from media with up to 0.7 M of the polyol. Accumulation of proline was greatly enhanced under these conditions and was proportional to the absorbed sorbitol, while the viability of the callus cultures was quite low after a few hours of incubation. Under moderate conditions (0.1 M sorbitol) as well as under severe osmotic shock (0.7 M sorbitol), the cells adapted by adjusting the sorbitol/proline ratio to ca 3. NaCl (0.1 M ) had the same effect as sorbitol (0.7 M ) on the survival rate, but only slightly affected proline synthesis in the first hours of incubation. Addition of 10⁷ or 10 ⁵ M abscisic acid (ABA) did not increase the proline content, but 10 ⁷ M ABA delayed the deleterious effect of NaCl and improved the state of the cells. No influence of abscisic acid during the incubation with sorbitol could be detected. Two different strategies for the adjustment of tobacco callus to salinity and sorbitol are suggested: Non-ionic stress is controlled by the accumulation of proline, whereas ABA could be involved in the adaptation to ionic stress.     

Salinity–induced modulations in the protective defense system and programmed cell death in <Emphasis Type="Italic">Nostoc muscorum</Emphasis>

To study the biochemical adaptive responses of the blue green algae Nostoc muscorum to the salinity- induced stress they were exposed to various concentrations (5, 10, 15, 20 or 200 mM) of sodium chloride (NaCl). A dose-dependent inhibition of total protein content showed an adverse effect of NaCl on the growth of N. muscorum. Four-day treatment of NaCl (5–20 mM) progressively increased the content of the total peroxide with subsequent increase of the superoxide dismutase (SOD) activity, proline and total phenol content only up to 10 mM NaCl. Higher concentrations of NaCl caused significant decrease in both the enzymatic and non-enzymatic antioxidants. Induction of two polypeptides of ~29.10 and 40.15 kD as well as upregulation of many polypeptides as compared to control indicates the induction of SOD and dehydrin-like proteins, which supports the theory of adaptation against the salt stress. Furthermore, adaptation of N. muscorum to lower concentrations (5–20 mM) of NaCl was also confirmed by no fragmentation of DNA while DNA fragmentation indicating programmed cell death (PCD) could only be seen at 200 mM NaCl for 12 hours. We hypothesized that proline may confer a positive role to combat salinity stress and the same was confirmed by treatment of the test blue green algae with exogenous proline (1 and 10 μM). The results exhibited 16% reduction in the level of total peroxides, which is a well known oxidative stress marker in the 10 μM proline-treated NaCl group as compared to direct exposure to NaCl.     

Na+、K+、Mg2+、Ca2+和葡萄糖溶液作为授精-激活介质对中华鲟精子受精率的影响

以不同浓度的NaCl、KCl、MgCl2、CaCl2溶液和葡萄糖溶液作为授精介质,研究了中华鲟(Acipenser sinensis)的受精效果.结果显示,适量的阳离子和葡萄糖作为激活授精介质时中华鲟卵受精率都有所提高.在实验设置浓度范围内25 mmol/L NaCI溶液、0.1 mmol/L KCl溶液、1 mmol/L MgCl2溶液、1 mmol/LCaCh溶液和50 mmol/L葡萄糖溶液浓度下,受精率分别可达到最高值,依次为87.72%、86.82%、82.24%、89.76%、80.92%.随着实验浓度继续增加,受精率反而呈下降趋势.结果显示,作为人工配制的中华鲟精子授精一激活介质,最适NaCI溶液浓度在25 mmol/L附近,最适葡萄糖溶液浓度在25 mmol/L附近,最适KCI溶液浓度≤0.1 mmol/L,最适MgCl2溶液浓度≤1 mol/L,最适CaCh溶液浓度≤1 mmol/L.