Fangfa Zeng

Bio: Fangfa Zeng is an academic researcher from Nanchang University. The author has contributed to research in topics: Cell migration & Glutaraldehyde. The author has an hindex of 2, co-authored 5 publications receiving 49 citations.

Effects of long-term serial cell passaging on cell spreading, migration, and cell-surface ultrastructures of cultured vascular endothelial cells

Abstract: The effects of serial cell passaging on cell spreading, migration, and cell-surface ultrastructures have been less investigated directly. This study evaluated the effects of long-term serial cell passaging (totally 35 passages) on cultured human umbilical vein endothelial cells which were pre-stored at −80 °C as usual. Percentage- and spread area-based spreading assays, measurements of fluorescently labeled actin filaments, migration assay, and measurements of cell-surface roughness were performed and quantitatively analyzed by confocal microscopy or atomic force microscopy. We found that the abilities of cell spreading and migration first increased at early passages and then decreased after passage 15, in agreement with the changes in average length of actin filaments. Recovery from cold storage and effects of cell passaging were potentially responsible for the increases and decreases of the values, respectively. In contrast, the average roughness of cell surfaces (particularly the nucleus-surrounding region) first dropped at early passages and then rose after passage 15, which might be caused by cold storage- and cell passaging-induced endothelial microparticles. Our data will provide important information for understanding serial cell passaging and implies that for pre-stored adherent cells at −80 °C cell passages 5–10 are optimal for in vitro studies.

Determination of the lowest concentrations of aldehyde fixatives for completely fixing various cellular structures by real-time imaging and quantification

Abstract: The effectiveness of fixatives for fixing biological specimens has long been widely investigated. However, the lowest concentrations of fixatives needed to completely fix whole cells or various cellular structures remain unclear. Using real-time imaging and quantification, we determined the lowest concentrations of glutaraldehyde (0.001–0.005, ~0.005, 0.01–005, 0.01–005, and 0.01–0.1 %) and formaldehyde/paraformaldehyde (0.01–0.05, ~0.05, 0.5–1, 1–1.5, and 0.5–1 %) required to completely fix focal adhesions, cell-surface particles, stress fibers, the cell cortex, and the inner structures of human umbilical vein endothelial cells within 20 min. With prolonged fixation times (>20 min), the concentration of fixative required to completely fix these structures will shift to even lower values. These data may help us understand and optimize fixation protocols and understand the potential effects of the small quantities of endogenously generated aldehydes in human cells. We also determined the lowest concentration of glutaraldehyde (0.5 %) and formaldehyde/paraformaldehyde (2 %) required to induce cell blebbing. We found that the average number and size of the fixation-induced blebs per cell were dependent on both fixative concentration and cell spread area, but were independent of temperature. These data provide important information for understanding cell blebbing, and may help optimize the vesiculation-based technique used to isolate plasma membrane by suggesting ways of controlling the number or size of fixation-induced cell blebs.

Method of low temperature induction off-spreading of cell for dynamic measurement of medicament influence on cell spreading

Abstract: A method of low temperature induction off-spreading of cell for dynamic measurement of medicament influence on cell spreading The method is characterized by comprising the following steps: planting adherent cells in a suspension state on a culture aperture or plate and incubating in an incubator to complete spread the cells; adding medicament and incubating the cells and the medicament together for 20 min-1 h and removing the medicament; placing the cells treated by the medicament under a microscope and shooting a morphology image before cell spreading; absorbing the aqueous solution and adding 4 DEG C cell nutrient solution or buffer; dynamically observing off-spreading state of the cells by a microscope immediately, continuously obtaining morphology images of the cells every few minutes for 30 min-1 h; measuring adherence area of each cell or a mean value of adherence areas of all the cells at a certain time point by a piece of software; and comparing the obtained data with blank data and analyzing to obtain data of medicament influence on cell spreading. The invention has advantages of simple process, no damage on cells, rapid detection, dynamic observation or measurement and good effect.

Method for quantitatively measuring cell spreading rate based on idealized cell radius

Abstract: The invention relates to a method for quantitatively measuring cell spreading rate based on idealized cell radius. The method is characterized by comprising the steps of: planting adherent cells in a suspended state on culture holes or plates, capturing the morphology of the cells at different time points by using imaging equipment during the cell adhering and spreading process, measuring the adhering area of each cell, idealizing the cells before and after spreading to be round, and calculating the cell spreading rate (growth rate of the cell radius within unit time). According to the invention, the idealized cell radius is used as a standard, the spreading rate is calculated according to the cell spreading time and the change of the cell radius, and the defects of the traditional method are solved; and compared with the traditional cell spreading measuring method, the method has the advantages that the method is more accurate, the required measuring equipment is simple, and the calculation method is simple and feasible, and can be integrated to measuring equipment.

Method for quantitatively measuring cell spreading rates

Abstract: The invention provides a method for quantitatively measuring the cell spreading rates. The method is characterized by planting adherent cells in the state of suspension in culture holes or plates, using imaging equipment to capture the morphologies of the cells at different time points in the processes of cell adherence and spreading, measuring the adherence area of each cell and computing the cell spreading rates according to the formula Rs=(An+1-An)/(Tn+1-Tn). The method has the following characteristics: the spreading rates are computed by taking the adherence areas of the cells as the standard according to the time spent on cell spreading and variation of the adherence areas; the defects of the traditional methods are overcome; the method provided by the invention is more accurate compared with the traditional cell spreading measurement methods; the needed measurement equipment is simple; and the computation method is also simple and feasible and is easy to integrate in the measurement equipment.

Extracellular vesicles of multiple myeloma cells utilize the proteasome inhibitor mechanism to moderate endothelial angiogenesis

Abstract: Bone marrow microenvironment is known to support angiogenesis, thus contributing to progression of multiple myeloma (MM). Bortezomib, a proteasome inhibitor (PI) widely used in MM treatment, has anti-angiogenic activity. Extracellular vesicles (EVs), shedding from cell surface, serve as mediators in cell-to-cell communication. We have hypothesized that MM cells (MMCs) treated with bortezomib generate EVs that could diminish angiogenesis, thus limiting MM progression. In the present study, EVs were obtained from MMCs (RPMI-8226), untreated (naive) or pre-treated with bortezomib. EVs were outlined using NanoSight, FACS, protein arrays and proteasome activity assays. The impact of MMC-EVs on endothelial cell (EC) functions was assessed, employing XTT assay, Boyden chamber and Western blot. A high apoptosis level (annexin V binding 70.25 ± 16.37%) was observed in MMCs following exposure to bortezomib. Compared to naive EVs, a large proportion of bortezomib-induced EVs (Bi-EVs) were bigger in size (> 300 nm), with higher levels of annexin V binding (p = 0.0043).They also differed in content, presenting with increased levels of pro-inflammatory proteins, reduced levels of pro-angiogenic growth factors (VEGFA, PDGF-BB, angiogenin), and displayed lower proteasome activity. Naive EVs were found to promote EC migration and proliferation via ERK1/2 and JNK1/2/3 phosphorylation, whereas Bi-EVs inhibited these functions. Moreover, Bi-EVs appeared to reduce EC proteasome activity. EVs released from apoptotic MMCs following treatment with bortezomib can promote angiogenesis suppression by decreasing proliferation and migration of EC. These activities are found to be mediated by specific signal transduction pathways.

Vimentin Plays a Crucial Role in Fibroblast Ageing by Regulating Biophysical Properties and Cell Migration.

Abstract: Ageing is the result of changes in biochemical and biophysical processes at the cellular level that lead to progressive organ decline. Here we focus on the biophysical changes that impair cellular function of human dermal fibroblasts using donors of increasing age. We find that cell motility is impaired in cells from older donors, which is associated with increased Young's modulus, viscosity, and adhesion. Cellular morphology also displays parallel increases in spread area and cytoskeletal assembly, with a threefold increase in vimentin filaments alongside a decrease in its remodelling rate. Treatments with withaferin A or acrylamide show that cell motility can be modulated by regulating vimentin assembly. Crucially, decreasing vimentin amount in cells from older individuals to levels displayed by the neonatal donor rescues their motility. Our results suggest that increased vimentin assembly may underlay the aberrant biophysical properties progressively observed at the cellular level in the course of human ageing and propose vimentin as a potential therapeutic target for ageing-related diseases.