ABSTRACT
Apoptosis, or programmed cell death, is a common phenomenon which involved in a variety of physiological and pathological conditions in humans, such as neurodegenerative diseases, ischemic injury, autoimmune diseases and cancers. Apoptosis can be detected in vitro by morphology, biochemistry, molecular biology, immunology, and other techniques. Probes for cell apoptosis detection in vivo are still under research and various reagents and methods are constantly emerging. However, none of apoptosis detection methods or reagents are perfect and they all have advantages and disadvantages, as well as suitable scope of application. With the increasing application of apoptosis detection techniques, researchers will be confused about how to choose a suitable method to detect apoptosis and define the application range of each apoptosis detection method. Therefore, it is necessary to compare the benefits and drawbacks of existing apoptosis detection techniques as well as their applicable conditions. This article reviews morphological characteristics, molecular mechanism and specific biochemical changes in apoptotic cells. We summarized various apoptosis-detection methods based on these characteristics that can be used in vitro and in vivo, the advantages and disadvantages of each method and the scope of application. Also, we highlighted the existing tracers that have been used in apoptosis detection in vivo, their potentialities and limitations as well as the clinical applications of apoptosis imaging in multiple disease fields.
ABSTRACT
Human annexin A5 (hAnxA5) as an important functional protein molecule in the human body, widely exists in human cells and body fluids. hAnxA5, a member of annexin group with complex structure, exhibits a variety of biological functions by reversibly and specifically binding phosphatidylserine (PS) in a calciumdependent manner and plays an important role in many human physiological processes. This paper has made induction and summary of the biochemical characteristics, mechanism, biological effects and important biomedical applications of hAnxA5. The hAnxA5 is present in the form of monomer and often exercise biological functions in a polymer. hAnxA5 affects the occurrence and development of pathological phenomena, such as vascular thrombosis disease, autoimmune disease, tumor disease, pulmonary fibrosis and lung injury, nonalcoholic fatty hepatitis, etc. As a biomarker, hAnxA5 has also been adopted in the study of diseases such as tumor, neurodegenerative diseases, heart failure, acute renal injury, asthma and so on. As novel drug candidates, hAnxA5 and its derivatives have been designed and applied to the therapeutic exploration of many kinds of diseases, especially for thrombotic diseases. There are also some gaps and shortcomings for hAnxA5 research. The in-depth of its research will not only expand the understanding of structure and functional relationships, but also promote its application in the field of biomedicine.
ABSTRACT
Annexin is a protein of evolutionarily conserved polygene family that binds to cell membrane phosphatidylserine (PS). PS is closely related to many diseases with a potential as a new drug target. Annexin has a good value in drug discovery and new drug development. Annexin A4 is a member of the annexins family. Annexin A4 involves in a number of cellular functions, such as exocytosis and coagulation. These functions are related to binding of annexin to acidic phospholipids. However, the detail function(s) of annexin A4 has not been fully uncovered. Production of annexin A4 in large quantity is prerequisite for indepth investigation of the structure-function relationship of annexin A4. Human annexin A4 was originally purified from the natural resource at a low yield due to the complex procedure. In the present study, annexin A4 was expressed in a prokaryotic system with a high yield of soluble protein. The plasmid pET28a-annexin A4-EGFP was constructed for the expression. Recombinant annexin A4-EGFP was purified using two methods. Affinity chromatography approach gave a protein yield at purity of 80%. While, the membrane absorption method produced the protein with the purity over 90%. Flow cytometric analysis showed that the annexin A4-EGFP fusion protein could recognize and bind to the apoptotic cells with an affinity PS at 79.58±11.68 nmol·L-1, which is at the same order of magnitude as A5-EGFP. We successfully achieved the efficient expression of annexin A4-EGFP in prokaryotic system, and provided an easy and convenient method for purifying a large amount of annexin A4-EGFP with a high purity. This study has laid a solid foundation for our study of the function of annexin A4 in the future.
ABSTRACT
Objective To introduce a simple and easy method for cell apoptosis analysis as an example with artemisinin to induce apoptosis of human U251 cells. Methods Human U251 cells,cultured in suspension and treated with artemisinin,were freeze-thawed. 30 ?L of 3 mmol/L PI was then added and mixed. After being placed under room temperature for 5 min,the cellular ploidy was analyzed by flow cytometry,and the ratio of cell apoptosis was calculated. Results The ratio of human U251 cell apoptosis induced by artemisinin at a dose of 1?10-4,1?10-5 and 1?10-6mol/L was 33.2%,24.0% and 8.0%,respectively. Conclusion The results showed that artemisinin significantly induced the apoptosis of human U251 cells in a dose-dependent manner. Using this method to analyze cell apoptosis can reduce the procedure of cell treatment and avoid many influential factors. Therefore,it can be regarded as a simple,accurate and rapid method for detecting cell apoptosis.