Background
Exosomes are a type of cytology-to-outside cysts that were first found and named by pan and johnstone in a sheep web weave. As research progresses, almost all cells, including blood cells, immunocellular cells, cancer cells, stem cells, etc., are found to produce excreta, not only in cell culture fluids, but also in blood, urine, breast milk, saliva, chest cavity fluids, etc. The excreta can carry some of the substances in the feed cell and is considered to be an important vehicle for inter-cell communication and material exchange, which can directly or indirectly regulate cell functions and biological behaviour. Research has shown that the participation of the excrete in the development of tumour diseases can be an important marker in the incubation of tumours. The application of the excreta is mainly in the three main areas of disease treatment, biomarkers, and drug delivery。
I. Excretion and purity of the body
The efficient and highly purified accumulation of the excreta is key to achieving its organizational analysis and functional research. However, the small size and density of the excretion, combined with the heterogeneity of the particle size and composition, may vary in the form of the excretion even from the same cell, causing enormous difficulties in extracting the excrete. A variety of extraction methods based on the size, density and immunometric pattern of the excreta have been developed, including hypervelocity centrifugation, density gradient centrifugality, polymer deposition, ultrafiltration, size detoxification chromatography, immunokinism, etc。
Figure 1 common excretion separation methods
1. Hypervelocity centrifugal method: first to remove cells, cell fragments and large cytological bubbles in samples from 300, 2000 and 10,000 g centrifugal forces, then to collect the excretion under 110 000 g conditions, and finally to wash sediments with phosphate buffer fluids (pbs, abs962) to remove solublely disturbed proteins (figure 1a)
2. The density gradient centrifugal method: it is a more stringent hypervelocity centrifugal method capable of further purification of the cystics based on sample density to overcome contamination caused by protein co-deposition. Common separated media include iodine gsalol and sugar cane. In the case of sugar, for example, the pre-positioning of the sugar/d2o solution at different gradients is required, followed by the addition of the substance to be separated. Under hypervelocity centrifugal forces, the excretion will be present in the range (1. 13 ~1. 19 g/ml) and the collection of the fractions will be pure (figure 1b)
3 the size-deductive chromatography method: separation of the excrete by a different particle size, using polymer gel filling as a separate medium (figure 1c). When the chromatography column is filled with stable, porous gel particles, and the solution containing the excreta is added to the chromatography column, the smaller particles will be caught in the hole, while the larger molecule will not enter the hole and be washed off first. Thus, at different stages, the desorption component will consist of molecules of different sizes, starting with larger excretion particles, followed by smaller proteins and lipid particles
4. Superfiltration: the rationale is the same as traditional membrane separation. This method usually uses a superfilter with a relative molecular retention of 100 kda and allows for the concentration of samples while extracting the excretion, especially for mass samples such as cell culture fluids and urine (figure 1d)
5 prosthetic method: the prosthesis and extraction method is currently the best excretion method and the only method to identify a specific cell source. Relatives and extractions of the excrete are mainly achieved through the specific interactions between antibodies and the protein of the excrete (figure 1e). The antibodies used are mono- or multi-resistant, and the target proteins can be trans-film proteins such as cd63, cd9 and cd81, which are commonly present on the surface of the endocrine body, and membrane proteins and upper skin cell slime. Accompanying molecules (epcam, etc.) may also be marked proteins unique to the surface of the carcinogenic cell excretion
6 co-deposition based on polymers: the polymer deposition method is a common strategy for commercial excretion reagents, of which polyethanol is currently the most commonly used deposition agent. It is generally accepted that the rationale for polymer deposition is that polymers reduce the resolution of the excretion via "jacking" of water molecules, leading to deposition under low-speed centrifuge conditions (figure 1f)。
Ii. Expressions of the excreta
At present, external genre bodies such as electroscopes, dynamic light dispersion, nanoparticle tracker, protein immunosuppression, enzymes immunosuppression and fluid cell meters can be shown。
Information on the morphology of the excreta can be obtained through mirrors, which vary according to the environment in which they are operated. For example, in scanning mirrors, trans-radioscopes and atomic microscopes, the excreta displays a cylindrical tea tort form (figure 2a), which may be caused by the extreme dehydration of the samples during fixed or dyed processes, resulting in the fall of the excrete. On the contrary, the excretion is rounded in the refrigerated mirrors and, because it does not dehydrate, the resulting excretion form is likely to be closer to the actual cystic state (figure 2b). In addition, information on excretion phosphorus bi-molecular thickness, particle distribution, etc., can also be provided because of the higher resolution of the lenses and atomic microscopes (figure 2c)。
Figure 2a the sub-genocrine of scanning mirrors presents a cylindrical teatoll。
Fig. 2b refrigeration mirrors present a circular shape of the excretion, possibly closer to the true condition of the cystic。
Figure 2c double-molecular layers of excrete phosphorus can be observed under a penetrator/atomic electroscope。
Figure 2
While several commonly used forms of representation are still inadequate, a combination of methods can improve the accuracy of the representation of the excreta. As technologies develop, accurate, rapid and reliable analysis of the excreta is expected to be achieved in the future。
Iii. Tracement techniques for the excreta
In vitro tagging or in vitro tracers of isolated excrete may facilitate further study of the functions of the excrete. There are a number of methods of marking the excreta, such as fluorescent tags, bioton tags, quantum point tags, immunomarks, isotope tags, genetic markers, etc. Of these, fluorescent tags, because of their ease of operation, are the most common method of marking the excreta of scientists. The fluorescent material marked with the excreta includes dir, dii, dio, did, among others, where the near infrared fluorescent of dir can penetrate cells and tissues and is often used to trace in small animals. In addition, pkh26 and pkh67 reagent boxes marked with cell membranes were used to mark the excreta。
The following is an example of the literature detailing the specific steps that dir has taken to mark the excretion and use it for internal tracking。
1. Ready to separate purified excreta
2. Mark 1h of the excreta at 37°c with 2 μm dir, then purge with superev 0. 5。
3. Injecting different levels of the peritoneal cavity into mice. After 24h injection, mice are anaesthetized and made using the nir-ii imaging system h imaging system。
In order to further analyse the location of the excreta in the tissue, tissues are screened, fixed and sliced, and dir signals are tested using the niconti2-e+a1r co-focused microscope (ex/em = 748/780nm)。
Figure 3 after evs treated 24h, most of the fluorescent signals remained at the injection level (a) in the normal mice. On the contrary, in ap mice treated by evs, fluorescent signals spread to the abdomen and pancreas showed greater fluorescent signals than ap mice treated with f4/80-evs。
