CN115433684A - Synchronization method for dictyostelium discodermatum cells - Google Patents
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Abstract
The invention discloses a synchronization method of dictyostelium discodermatum cells, which mainly comprises the following steps: s1: culturing dictyostelium discodermatum cells; s2: starvation treatment of dictyostelium discodermatum cells; s3: collecting cell division information in starvation treatment; s4: providing nutrient substances to the starved dictyostelium discodermatum cells again; after the advantages and the disadvantages of artificial synchronization and natural synchronization are combined, another synchronization method except artificial synchronization and natural synchronization is selected in view of the particularity of the life cycle of the dictyostelium trabeculosum cells, namely, the dictyostelium trabeculosum is starved for a period of time and then the nutrient klebsiella is added, so that the dictyostelium trabeculosum cells can be synchronously split to a certain period relatively quickly in a short time. The research method can shorten the synchronization time and effectively prevent the influence of the interference between the medicines on the experimental result.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to a synchronization method of dictyostelium discodermatum cells.
Background
Dictyostelium discodermatum is a eukaryote, and is often selected as a simple model organism for researching cell development, movement and division due to the characteristics of simple culture condition, short life cycle, easy observation and the like. Meanwhile, dictyostelium trabeculosum cells have many similarities to mammalian cells in terms of behavior, structure and signaling pathways. The strain is an important model organism, plays an important role in researching aspects such as multi-cell origin, development and other human pathogenic mechanisms, and provides help for solving some problems in mammal development. Compared with the expression vector of mammalian cells, the dictyostelium discodermatum has the advantages of low cost, fast cell growth, easy large-scale culture and the like, and a plurality of complex glycoproteins with biological activity are successfully expressed so far. Therefore, dictyostelium discodermatum is likely to become an important expression system for glycoprotein expression systems.
In recent years, tumors, cancers, and the like are diseases that seriously threaten human health. It has been found that the development of these diseases is closely related to the regulation of the cell cycle, and that either the activation of proto-oncogenes or the deregulation of tumor suppressor genes is ultimately linked to the regulation of the cell cycle. Research shows that the sensitivity of a plurality of antitumor drugs for inducing apoptosis or killing tumor cells almost depends on cell cycles, while the cells in a population are in different cell cycle phases under general conditions, and the sensitivity of the cells in different phases to the drugs is different. The cells are synchronously treated to the same period, and anti-tumor and anti-cancer drugs or even radiotherapy are taken as breakthrough or become an effective way for improving the treatment effect of tumors and cancers by selecting proper time points. Therefore, in order to better study the proliferation, metabolism and apoptosis of cells in a certain period, we need to adjust the cells in different phases in a population to be in the same phase by using an experimental method, which is a cell synchronization technology.
Cell synchronization can be classified into natural synchronization and artificial synchronization. The natural synchronization basically has no harm to cells, but the natural synchronization needs long period and has low synchronization efficiency; although the efficiency of artificial synchronization is higher than that of natural synchronization, the artificial synchronization has more or less influence on the cells, and how to reduce the damage to the cells in the synchronization process is an urgent problem to be solved in the current research process.
Disclosure of Invention
In order to solve the technical problems, after the advantages and the disadvantages of artificial synchronization and natural synchronization are combined, another synchronization method except the artificial synchronization and the natural synchronization is selected, namely, the dictyostelium trabeculosum is subjected to starvation treatment for a period of time, and then the nutrient klebsiella is added, so that the synchronization division can be relatively quickly carried out to a certain period in a short time. The research method can effectively prevent the interference between the medicines from influencing the experimental result while shortening the synchronization time;
in order to achieve the technical purpose, the invention is realized by the following technical scheme:
a dictyostelium discodermatum cell synchronization method comprises the following steps:
s1: culturing dictyostelium discodermatum cells;
s2: carrying out cell starvation treatment on dictyostelium discodermatum;
s3: refeeding food KA (klebsiella) to starved dictyostelium cells;
s4: cell division after re-administration of KA was collected at time intervals.
Preferably, the inoculation method and conditions of the dictyostelium discodermatum cells are that 2mL of HL5 culture medium is added into a 12-well plate, and 10 inoculation is carried out 4 Gently mixing the cells in the exponential growth phase uniformly, and adhering the cells to the wall in the environment of 22 ℃;
preferably, the starvation-treated cells are treated by DB buffer solution, the treatment method is to suck the culture solution in a 12-well plate by using a vacuum pump, and 2ml of newly configured DB buffer solution is gently added along the walls of the wells; removing DB buffer solution by suction, adding 2ml of new DB buffer solution again, and enabling the cells to receive hunger stimulation in an environment at 22 ℃;
preferably, the starvation treatment method is DB buffer, and the food KA is re-administered in the buffer after the cells are starved for 1, 2, 3, 4, 5 and 6 hours respectively;
preferably, before the division condition information is collected, a vacuum pump device is used for sucking DB in a 12-hole plate, a DB buffer solution mixed with KA is added again, a real-time video system is used for recording the cell division conditions of hunger for 1-6h respectively, and a corresponding hunger time range with more cell division is obtained;
preferably, after the starvation treatment range is determined, the nutrient KA is added again in a time area with obvious cell division phenomenon, and the cell division conditions of starvation for 1-6 hours are respectively recorded by a real-time video recording system, so that a corresponding starvation time range with more cell division is obtained;
preferably, the time region in which the splitting phenomenon is more pronounced is after more than 2.5 h.
The invention has the beneficial effects that:
the dictyostelium discodermatum cell synchronization method provided by the invention shortens synchronization time and can effectively prevent interference among medicines from influencing experimental results.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the description below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graph showing the results of cell division in cell starvation treatment for 1-6h;
FIG. 2 is a graph of mean cell division (error fluctuations) for 2-4h of starvation treatment;
FIG. 3 is a graph of mean values of the cleavage rates (error fluctuations) at 2.5h in starvation treatment;
FIG. 4 is a graph of mean division ratio (error fluctuation) for 6h in normal culture;
FIG. 5 is a graph showing the comparison of the division rate between 2.5h of starvation treatment and 6h of normal culture.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A dictyostelium discodermatum cell synchronization method comprises the following steps:
s1: culturing cells of dictyostelium discodermatum;
s2: starvation treatment of dictyostelium discodermatum cells;
s3: collecting cell division information in starvation treatment;
s4: providing nutrient substances to the starved dictyostelium discodermatum cells;
preferably, the inoculation method and conditions of the dictyostelium discodermatum cells are that 2mL of HL5 culture medium is added into a 12-well plate, and 10 inoculation is carried out 4 0.2mL of cells in each exponential growth phase are mixed evenly and gently, and cultured at 22 ℃;
preferably, the starvation-treated cells are treated by DB buffer solution, the treatment method is to suck the culture solution in a 12-well plate by using a vacuum pump, and 2ml of newly configured DB buffer solution is gently added along the walls of the wells; the DB buffer solution is sucked off, and 2ml of new DB buffer solution is added again;
preferably, the starvation treatment method is DB buffer solution, and the cells are respectively starved for 1-6h;
preferably, before the division condition information is collected, a vacuum pump device is used for sucking DB in a 12-hole plate, and a real-time video system is used for respectively recording the division condition of the cells during hunger for 1-6h;
preferably, after the starvation treatment is performed for 1-6h, the nutrient KA is re-added in a time region where the cell synchronization and division phenomenon is obvious.
Preferably, the time zone in which the splitting phenomenon is more pronounced is after more than 2.5 h.
Example 2
Effect of different starvation times on cell synchronization Rate
In order to study the effect of the starvation treatment time on the cell synchronization condition, 6 time periods of starvation treatment, namely 1h, 2h, 3h, 4h, 5h and 6h, were selected in the experiment, and after the corresponding time period of starvation treatment, the KA food was given and the recording of the cell division condition was started. Statistical results are shown in fig. 1, and the number of cell divisions was the greatest after 3h starvation. And the starvation time is less than 2h or more than 5h, the division number of the cells is very small, which is probably related to the multicellular development stage of the cells. Within 2h of starvation, cells are in the initial stage of starting aggregation, and it is likely that intracellular signaling pathways are similar to the vegetative stage, while after 5h of starvation, cells have secreted large amounts of cyclic adenosine monophosphate and aggregation has occurred, and it is difficult to return to the vegetative stage even if food is re-administered.
To further determine the optimal time for starvation treatment, the experimental design continued to narrow the time gradients to 2h, 2.5h, 3h, 3.5h, 4h based on the results of the previous experiments, again after completion of the starvation treatment. Statistical analysis results of cell division are shown in fig. 2, and it can be seen from the results that when cells are given food KA after 2.5h of starvation, a large number of dividing cells can be generated, i.e. a large number of cells have been synchronized.
Example 3
Comparison of cell synchronized division at 2.5h starvation and normal culture
After 2.5h of starvation was determined as the optimal synchronized split time, the synchronized split results of 2.5h of starvation cells were used as the experimental group. Continuously researching the synchronization result after 2.5h of starvation treatment, adding the nutrient KA after 2.5h of starvation treatment, continuously photographing for 3h after 0.5h of starvation treatment, and obviously observing that the cell division rate is highest when 2h of photographing is observed (as shown in figure 3), wherein the whole experiment lasts for 6h. Within the time range of 6h, the synchronous division phenomenon of the cells is obvious, the synchronous division rate in the whole experimental process is counted, and the synchronous division rate of the cells after 2.5h of starvation treatment reaches 59.51 percent. The results of synchronized division of starvation-treated 2.5h cells were used as experimental groups.
The division results of the dictyostelium basilicum cultured normally for 6h were arranged as a control group. It can be found that the cells under normal culture have no synchronization phenomenon in the first three hours, and the synchronization phenomenon, namely cell division, gradually appears by 4-6 hours. But the synchronous division rate of the cells is very low, the synchronization phenomenon is not obvious, and the observation shows that the total synchronous division rate of the cells under normal culture for 6 hours only reaches 7.04 percent.
In order to more intuitively sense the difference of synchronous division rate of the cells of the dictyostelium gracilis under 2.5h of hunger treatment and 6h of normal culture, the experiment and the statistical data are repeated, and a comparison graph is made to show that: after 2.5h of starvation treatment, the division rate of the cells reaches 59.51 percent, while after 6h of normal culture, the division rate of the cells is only 7.04 percent and is far less than the synchronization efficiency of the cells after 2.5h of starvation treatment. That is, the starvation treatment can effectively increase the synchronization efficiency of the cells, and efficiently synchronize the cells to a specific period in a short time.
From the above results analysis we can clearly see that: under the normal culture condition, the time for synchronously splitting the dictyostelium discodermatum cells is long, and the phenomenon of synchronously splitting the cells is not obvious. The food KA is added after the dictyostelium trabeculosum is subjected to starvation treatment, the dictyostelium trabeculosum can be induced to be synchronously split, the nutrient KA is added after the dictyostelium trabeculosum is subjected to starvation treatment for 2.5 hours, the cell synchronization effect is most obvious, and the synchronization degree can reach 59.51% on average in a short time. Compared with the cell synchronization effect of dictyostelium discodermatum under normal culture, the starvation treatment not only can greatly improve the cell synchronization division rate of dictyostelium discodermatum, but also can shorten the cell synchronization time to a certain extent, and provides a novel idea for the research of medical services and the like.
Claims (7)
1. A dictyostelium discodermatum cell synchronization method is characterized by comprising the following steps:
s1: culturing cells of dictyostelium discodermatum;
s2: carrying out cell starvation treatment on dictyostelium discodermatum;
s3: refeeding food KA (klebsiella) to starved dictyostelium cells;
s4: cell division after re-administration of KA was collected at time intervals.
2. The method for synchronizing dictyostelium discodermatum cells according to claim 1, wherein said method for inoculating dictyostelium discodermatum cells comprises adding 2mL of HL5 medium to 12-well plate, and inoculating 10 4 And (3) slightly and uniformly mixing the cells in the exponential growth phase, and enabling the cells to adhere to the wall in an environment at 22 ℃.
3. The dictyostelium discodermatum cell synchronization method according to claim 1, wherein the starvation-treated cells are treated with DB buffer by aspirating culture medium from 12-well plates with a vacuum pump, and gently adding 2ml of newly configured DB buffer along the walls of the wells; the DB buffer was aspirated, 2ml of fresh DB buffer was added again, and the cells were subjected to starvation stimulation at 22 ℃.
4. The method for synchronizing dictyostelium discodermatum cells according to claim 1, wherein the starvation treatment is DB buffer, and the food KA is re-administered in the buffer after the cells are starved for 1, 2, 3, 4, 5 and 6 hours respectively.
5. The method for synchronizing dictyostelium trabeculosum cells according to claim 1, wherein before the division information is collected, DB in a 12-well plate is sucked by a vacuum pump device, DB buffer mixed with KA is added again, and the cell division conditions of starvation for 1-6h are recorded by a real-time video recording system, so as to obtain a corresponding starvation time range with more cell division.
6. The method for synchronizing dictyostelium discodermatum cells as claimed in claim 1, wherein after said starvation treatment range is determined, the nutrient KA is added again in a time zone where the cell division phenomenon is more obvious, and the cell division conditions of starvation for 1-6h are recorded by a real-time video recording system, so as to obtain a corresponding starvation time range with more cell divisions.
7. The method for synchronizing dictyostelium discodermatum cells according to claim 6, wherein said time zone in which said division phenomenon is more pronounced is after more than 2.5 hours.
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