基于双荧光和单荧光探针的溶酶体pH值测定方法比较

目的:通过比较基于双荧光探针和单荧光探针的2种溶酶体pH值测定方法,探究一种不依赖四甲基若丹明(TMR)的、基于异硫氰酸荧光素(FITC)单荧光的检测溶酶体pH值方法的可行性。方法:利用人肺癌肺泡基底上皮来源的A549细胞,分别采用FITC/TMR双荧光探针法和FITC单荧光双激发法测定溶酶体pH值;通过计算相对荧光强度的比值FI_(FITC488)/FI_(TMR561)或FI_(FITC488)/FI_(FITC405),绘制pH值标准曲线,用于pH值测定。结果:相对于FITC在Ex488 nm/Em520 nm波长下的荧光强度对pH值极为敏感,其在Ex405 nm/Em422 nm波长下对pH值不敏感,可用于荧光矫正;不同pH值条件下,FI_(FITC488)/FI_(FITC405)比值基本呈线性关系。结论:FITC单荧光双激发法只须使用一种荧光探针即可实现pH值检测,较FITC/TMR双荧光探针法更不易产生系统误差,更适用于溶酶体pH值检测。     

氧化应激对溶酶体储存与转运甲醛的影响

内源甲醛代谢失调被认为是导致阿尔茨海默病的危险因素之一,甲醛蓄积会引起神经细胞的死亡和认知功能的降低.研究表明,细胞内甲醛分布于溶酶体内,而溶酶体功能异常与神经退行性疾病密切相关.本文采用甲醛特异荧光探针,在氧化应激条件下,检测到小鼠脑微血管内皮细胞株bEnd.3和小鼠神经瘤母细胞株N2a溶酶体内甲醛明显升高;在慢性脑低灌注大鼠动物模型中,其脑神经细胞的溶酶体内甲醛也升高(P0.01);LeuLeuOMe处理bEnd.3细胞,使其溶酶体膜通透性增加,导致细胞内甲醛蓄积,而胞外甲醛降低.以上结果证明,溶酶体具有储存和转运甲醛的功能,如果溶酶体出现结构与功能的异常,会导致甲醛代谢失调,造成认知损害.     

分泌型溶酶体与疾病

传统意义的溶酶体被认为是细胞内消化途径的终点站,是含有多种水解酶和脂肪酶的细胞器,可以消化蛋白以及膜结构等.某些特殊类型的细胞中存在分泌型溶酶体,它既有胞内消化的功能,又有调节分泌功能.在调节溶酶体胞吐的蛋白质中,Rab27a蛋白起到了核心作用.相关基因特别是控制胞吐的基因突变,可造成各种免疫缺陷综合症.星型胶质细胞溶...     

睾丸间质细胞——研究自体吞噬的一种正常细胞模型

细胞在生理状态下自体吞噬出现的频率很低,很难用正常细胞来研究自体吞噬活动,一般都通过诱导自体吞噬来获得有关自体吞噬活动的资料。本实验观察了肝、肾、睾丸等组织的32种细胞,发现睾丸间质细胞中自体吞噬出现频率远远高于其他细胞,平均每100个细胞切面中可以看到25个自噬小体,从而为研究自体吞噬的过程和机理提供了一个正常细胞模型。本实验还观察到睾丸间质细胞的自体吞噬活动可分为前自噬小体、早期自噬小体和晚期自噬小体三个阶段,是一个连续的过程。前自噬小体和早期自噬小体不含溶酶体酶,只有在自噬小体与溶酶体接触后,才从后者获取溶酶体酶并将其内容物消化分解,成为晚期自噬小体。由自体吞噬所产生的残余体并不在睾丸间质细胞内积聚,而是通过胞吐作用排出细胞外。     

结核分枝杆菌耐酸机制的研究进展

结核分枝杆菌能在宿主体内长期存活,很大一部分原因是能抵抗吞噬体的酸性环境。细菌一方面能抑制吞噬体与溶酶体融合,干扰吞噬体成熟、酸化过程;另一方面也能通过自身功能抵抗吞噬溶酶体内的酸性杀伤作用。本文主要介绍吞噬体的酸化过程及结核分枝杆菌耐酸机制的最新研究进展。     

共聚焦镜观察凋亡巨噬细胞内pH的变化

用透射电镜观察巨噬细胞的形态学改变,结果显示,地塞米松处理８小时后,大部分巨噬细胞发生凋亡特征变化：胞突缩短、减少,胞膜完整。胞体皱缩,胞质密度增加,其中出现大量空泡。胞核染色质边聚、浓缩。另外用激光扫描共聚焦显微镜（ＡＣＡＳ５７０）和ｐＨ荧光探针ＳＮＡＲＦ┐１／ＡＭ实时检测地塞米松处理巨噬细胞胞浆ｐＨ的动态变化。加入地塞米松,多数巨噬细胞胞浆马上发生快速和短期的碱化。随后,胞浆ｐＨ缓慢降低,胞浆酸化。结果表明,胞浆酸化是细胞凋亡发展的必然过程,胞浆碱化则很可能与细胞凋亡的发生相关,也可能与细胞种类、细胞功能状态相关     

内体—溶酶体运输及其细胞功能

哺乳动物细胞中,内吞作用通过质膜内陷形成囊泡来摄取外界物质,经早内体到达晚内体/溶酶体降解或经再生循环回到质膜。内体运输网络参与细胞一系列重要生命活动,如信号通路调节、细胞器发生以及胞吐作用等。近年来发现Aps、BLOCs、HOPS和ESCRTs等复合体共同参与货物由胞内体到溶酶体或溶酶体相关细胞器的运送。该文主要就这些内体—溶酶体运输系统中重要蛋白复合体的组成和功能进行综述。     

人肝细胞内β-葡萄糖醛酸酶定位定量研究

本实验采用抗β┐葡萄糖醛酸酶（β┐Ｇｌｕｃｕｒｏｎｉｄａｓｅ,简称β┐Ｇ）抗体及胶体金探针技术对人肝细胞超微细胞内β┐Ｇ进行定位及定量研究,结果表明,β┐Ｇ定位于肝细胞中的溶酶体和内质网,并且在溶酶体内分布的量较多,与内质网中分布的量相比有显著性差异（Ｐ＜０．０１）,因此溶酶体可做为研究β┐Ｇ与肝细胞病变关系在细胞超微结构水平的形态学研究的模型     

人膀胱癌细胞纤维肌动蛋白共聚焦激光扫描显微镜观察

探讨膀胱癌细胞纤维肌动蛋白（F－actin)的空间结构。采用共聚焦激光扫描显微镜光学切片技术结合异硫酸氢荧光素－鬼笔环肽（FITC－phalloidin)标记纤维肌动蛋白和碘化丙啶（PI）和标记核酸的荧光探针双重标记技术对膀胱癌细胞纤维肌动蛋白进行形态学观察,结果可见膀胱癌细胞内纤维肌动蛋白微丝形态完整,成细束或细丝状,平行排列地整个细胞或细胞突起,在胞质边缘处较密集。     

溶酶体贮积症的研究进展

溶酶体贮积症是一种罕见的遗传缺陷疾病,溶酶体内未酶解的大分子累积,最终导致细胞功能障碍和临床异常情况。许多溶酶体底物在细胞结构和功能上都有关键的作用,因此溶酶体功能失常的影响非常广泛,如神经受累、间质受累、网状内皮组织受累及胎儿水肿。治疗方法主要有骨髓移植、酶替代疗法、底物减少治疗、基因治疗和分子伴侣治疗。利用转基因及其他一些前沿技术,将有可能彻底根除这些长期困扰人类的溶酶体贮积症。     

受体介导内吞引起的巨噬细胞胞质酸化和胞吐作用

The effects of Con A, WGA, Zymosan A on macrophage cytosolic pH and outflow of lysosomal content through exocytosis were studied with SNAFL-calcein and FITC-Dextran on ACAS570. The results showed all three ligands could induce macrophage cytosolic acidification in about 10 min and kept at the same level hereafter; outflow of lysosomal fluorescent probe through exocytosis appeared in 15-20 min. In resting conditions, macrophage lysosomes mainly distributed in cell center; after stimulated for 15 min by three ligands, the number of lysosomes increased in membrane periphery, in 25-30 min lysosomes moved back toward cell center. We proposed that ligands induced lysosomal pH rises was a basic factor for outflow of lysosomal content through exocytosis, cytosolic acidification inhibited receptor-mediated endocytosis. Cytosolic acidification and outflow of lysosomal content through exocytosis were the results of cellular self-regulation and self-protection during receptor-mediated endocytosis.     

受体介导内吞对巨噬细胞膜电位、胞浆和溶酶体pH的影响

本文利用荧光标记方法测定了刀豆素Ａ、麦芽凝集素、酵母多糖刺激引起的巨噬细胞膜电位、胞浆ｐＨ溶酶体ｐＨ的变化。结果显示三种配体均导致细胞膜电位超极化,胞浆ｐＨ降低、溶酶体ｐＨ或高,三个生理参量趋于稳定时间稍有不同。胞浆ｐＨ的降低可能有抑制内吞的作用,溶酶体ｐＨ上升是触发溶酶体内容物外排的基本因素。内吞引起的这些变化是细胞代谢过程中自我调节和保护的表现。     

Tilorone acts as a lysosomotropic agent in fibroblasts

Tilorone, an amphiphilic cationic compound with antiviral activity perturbed the lysosomal system. In cultured fibroblasts tilorone induced storage of sulfated glycosaminoglycans, enhanced secretion of precursor forms of lysosomal enzymes, inhibited intracellular proteolytic maturation of lysosomal enzymes, and inhibited receptor-mediated endocytosis of lysosomal enzymes. In isolated lysosomes tilorone was found to increase pH and to abolish the ATP-dependent acidification. These effects suggest that tilorone acts like a weak base that accumulates in acid compartments of the cells, raises the pH therein and interferes with lysosomal catabolic activity and with receptor-mediated transport of lysosomal enzymes.     

Macrophages function as a ferritin iron source for cultured human erythroid precursors

Iron is essential for the survival as well as the proliferation and maturation of developing erythroid precursors (EP) into hemoglobin-containing red blood cells. The transferrin-transferrin receptor pathway is the main route for erythroid iron uptake. Using a two-phase culture system, we have previously shown that placental ferritin as well as macrophages derived from peripheral blood monocytes could partially replace transferrin and support EP growth in a transferrin-free medium. We now demonstrate that in the absence of transferrin, ferritin synthesized and secreted by macrophages can serve as an iron source for EP. Macrophages trigger an increase in both the cytosolic and the mitochondrial labile iron pools, in heme and in hemoglobin synthesis, along with a decrease in surface transferrin receptors. Inhibiting macrophage exocytosis, binding extracellular ferritin with specific antibodies, inhibiting EP receptor-mediated endocytosis or acidification of EP lysosomes, all resulted in a decreased EP growth when co-cultured with macrophages under transferrin-free conditions. The results suggest that iron taken up by macrophages is incorporated mainly into their ferritin, which is subsequently secreted by exocytosis. Nearby EP are able to take up this ferritin probably through clathrin-dependent, receptor-mediated endocytosis into endosomes, which following acidification and proteolysis release the iron from the ferritin, making it available for regulatory and synthetic purposes. Thus, macrophages support EP development under transferrin-free conditions by delivering essential iron in the form of metabolizable ferritin.     

Endosome pH measured in single cells by dual fluorescence flow cytometry: rapid acidification of insulin to pH 6

The acidification of various ligands was measured on a cell by cell basis for cell suspensions by correlated dual fluorescence flow cytometry. Mouse 3T3 cells were incubated with a mixture of fluorescein- and rhodamine-conjugated ligands, and the ratio of fluorescein and rhodamine fluorescence was used as a measure of endosome pH. The calibration of this ratio by both fluorometry and flow cytometry is described. Dual parameter histograms of average endosome pH per cell versus amount of internalization were calculated from this data, for samples in the absence and presence of chloroquine added to neutralize acidic cellular vesicles. The kinetics of acidification of insulin were measured and compared with previous results obtained with the chloroquine ratio technique. Rapid acidification of internalized ligand was observed both for insulin, which was mostly internalized via nonspecific pathways, and for alpha 2-macroglobulin, which was mainly internalized by specific receptor-mediated endocytosis. The average pH observed for internalized insulin was less than pH 6 within 10 min after addition of insulin. At 30 min, the average pH began to decrease to approximately pH 5, presumably because of fusion of endosomes with lysosomes.     

Low cytoplasmic pH inhibits endocytosis and transport from the trans- Golgi network to the cell surface

A fibroblast mutant cell line lacking the Na+/H+ antiporter was used to study the influence of low cytoplasmic pH on membrane transport in the endocytic and exocytic pathways. After being loaded with protons, the mutant cells were acidified at pH 6.2 to 6.8 for 20 min while the parent cells regulated their pH within 1 min. Cytoplasmic acidification did not affect the level of intracellular ATP or the number of clathrin-coated pits at the cell surface. However, cytosolic acidification below pH 6.8 blocked the uptake of two fluid phase markers, Lucifer Yellow and horseradish peroxidase, as well as the internalization and the recycling of transferrin. When the cytoplasmic pH was reversed to physiological values, both fluid phase endocytosis and receptor-mediated endocytosis resumed with identical kinetics. Low cytoplasmic pH also inhibited the rate of intracellular transport from the Golgi complex to the plasma membrane. This was shown in cells infected by the temperature-sensitive mutant ts 045 of the vesicular stomatitis virus (VSV) using as a marker of transport the mutated viral membrane glycoprotein (VSV-G protein). The VSV-G protein was accumulated in the trans-Golgi network (TGN) by an incubation at 19.5 degrees C and was transported to the cell surface upon shifting the temperature to 31 degrees C. This transport was arrested in acidified cells maintained at low cytosolic pH and resumed during the recovery phase of the cytosolic pH. Electron microscopy performed on epon and cryo-sections of mutant cells acidified below pH 6.8 showed that the VSV-G protein was present in the TGN. These results indicate that acidification of the cytosol to a pH less than 6.8 inhibits reversibly membrane transport in both endocytic and exocytic pathways. In all likelihood, the clathrin and nonclathrin coated vesicles that are involved in endo- and exocytosis cannot pinch off from the cell surface or from the TGN below this critical value of internal pH.     

Autophagy of metabolically inert substances injected into fibroblasts in culture

We have examined the morphologic characteristics of fibroblasts cultured from the beige mouse, a genetic variant phenotypically similar to human Chediak-Higashi syndrome (CHS). These cultured fibroblasts are characterized by large, amorphous dense body inclusions in their cytoplasm, often as large as the cell nucleus. Using time-lapse video phase-contrast microscopy, we have observed the formation of these large dense bodies through fusion of relatively normal-appearing lysosomes in the beige mouse fibroblast. After formation of these large inclusions, cells occasionally extruded the contents of these structures through apparent fusion with the plasma membrane and rapid exocytosis. Fibroblasts cultured from normal black mice showed no evidence of fusion between lysosomes or exocytosis of lysosomes. However, the uptake of extracellular medium through macropinocytosis, subsequent actions of lysosomes on these macropinosomes through saltatory motion, cellular migration and ruffling activity appeared normal in beige mouse fibroblasts. Immunocytochemical localization of α₂-macroglobulin, a normal serum protein commonly incorporated into lysosomes in cultured fibroblasts by receptor-mediated endocytosis, showed that the large dense bodies contained α₂-macroglobulin, in keeping with their lysosomal origin. This suggested further that receptor-mediated endocytosis in these cells was relatively normal. In addition, light and electron microscopic cytochemistry showed these large inclusions to be acid-phosphatase positive, further characterizing them as lysosomal. The electron microscopic appearance of these dense inclusions was consistent with their origin through repeated fusion of lysosomes. It is suggested that a primary defect in this disease may be the ability of mature lysosomal membranes to fuse, unlike normal lysosomal membranes, indicating perhaps an alteration in some specific component of the lysosomal membranes in CHS.     

Proton-translocating ATPase and lysosomal cystine transport

A proton-translocating ATPase was identified in highly purified lysosomes from Epstein-Barr virus-transformed human lymphoblasts. Activity of this ATPase caused acidification of highly purified, fluorescein isothiocyanate dextran-loaded lysosomes and correlated with the ATP-dependent efflux of lysosomal cystine. The lysosomal ATPase was distinct from mitochondrial F1-ATPase in its responses to a variety of inhibitors. Although ATP-dependent lysosomal cystine efflux is not demonstrable in cultured lymphoblasts from individuals with nephropathic cystinosis, ATPase activity and acidification in lysosomes from these cells is comparable to that in noncystinotic lysosomes. ATPase activity in lymphoblasts from normal individuals was 543 +/- 79 nmol/mg/min while in lymphoblasts from cystinotic individuals this activity was 541 +/- 25 nmol/mg/min. ATP-dependent acidification of lysosomes from normals was -0.5 +/- 0.1 pH units compared to -0.5 +/- 0.1 pH units in cystinotic lysosomes. Activity of the lysosomal proton-translocating ATPase is a necessary, but not sufficient, condition for lysosomal cystine efflux.     

Acidification of endocytic compartments and the intracellular pathways of ligands and receptors

Several hormones, serum proteins, toxins, and viruses are brought into the cell by receptor-mediated endocytosis. Initially, many of these molecules and particles are internalized into a common endocytic compartment via the clathrin-coated pit pathway. Subsequently, the ligands and receptors are routed to several destinations, including lysosomes, the cytosol, or the plasma membrane. We have examined the mechanism by which sorting of internalized molecules occurs. A key step in the process is the rapid acidification of endocytic vesicles to a pH of 5.0-5.5 This acidification allows dissociation of several ligands from their receptors, the release of iron from transferrin, and the penetration of diphtheria toxin and some viral nucleocapsids into the cytoplasm. Transferrin, a ligand that cycles through the cell with its receptor, has been used as a marker for the recycling receptor pathway. We have found that in Chinese hamster ovary (CHO) cells transferrin is rapidly segregated from other ligands and is routed to a complex of small vesicles and/or tubules near the Golgi apparatus. The pH of the transferrin-containing compartment is approximately 6.4, indicating that it is not in continuity with the more acidic endocytic vesicles which contain ligands destined to be degraded in lysosomes.     

Haptoglobin Genotype-dependent Differences in Macrophage Lysosomal Oxidative Injury

The major function of the Haptoglobin (Hp) protein is to control trafficking of extracorpuscular hemoglobin (Hb) thru the macrophage CD163 receptor with degradation of the Hb in the lysosome. There is a common copy number polymorphism in the Hp gene (Hp 2 allele) that has been associated with a severalfold increased incidence of atherothrombosis in multiple longitudinal studies. Increased plaque oxidation and apoptotic markers have been observed in Hp 2-2 atherosclerotic plaques, but the mechanism responsible for this finding has not been determined. We proposed that the increased oxidative injury in Hp 2-2 plaques is due to an impaired processing of Hp 2-2-Hb complexes within macrophage lysosomes, thereby resulting in redox active iron accumulation, lysosomal membrane oxidative injury, and macrophage apoptosis. We sought to test this hypothesis in vitro using purified Hp-Hb complex and cells genetically manipulated to express CD163. CD163-mediated endocytosis and lysosomal degradation of Hp-Hb were decreased for Hp 2-2-Hb complexes. Confocal microscopy using lysotropic pH indicator dyes demonstrated that uptake of Hp 2-2-Hb complexes disrupted the lysosomal pH gradient. Cellular fractionation studies of lysosomes isolated from macrophages incubated with Hp 2-2-Hb complexes demonstrated increased lysosomal membrane oxidation and a loss of lysosomal membrane integrity leading to lysosomal enzyme leakage into the cytoplasm. Additionally, markers of apoptosis, DNA fragmentation, and active caspase 3 were increased in macrophages that had endocytosed Hp 2-2-Hb complexes. These data provide novel mechanistic insights into how the Hp genotype regulates lysosomal oxidative stress within macrophages after receptor-mediated endocytosis of Hb.