Method for enhancing cell membrane permeability

Abstract

The invention discloses a method for enhancing cell membrane permeability, propionibacterium acnes and mouse breast cancer cells are co-cultured, and the membrane permeability of the co-cultured cancer cells is increased. The invention provides an innovative strategy for cancer treatment, namely, by adjusting the permeability of tumor cell membranes, an anti-cancer drug is promoted to enter tumor cells more effectively, the curative effect of the drug is improved, and meanwhile, damage to normal cells is reduced; by inducing the permeability of the tumor cell membrane to be increased, the rate and efficiency of drug molecules passing through the cell membrane can be remarkably improved, and a theoretical basis and technical support are provided for developing a novel drug delivery system.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for enhancing cell membrane permeability. Background Art

[0002] Tumors, a malignant disease that can invade any part of the body, are also called malignant tumors or neoplasms. In recent years, the incidence rate has continued to rise worldwide and has become one of the leading diseases that endanger human health. The current treatment methods for tumors mainly include surgical resection, radiotherapy, chemotherapy, immunotherapy, and targeted therapy. Although the treatment methods are constantly being innovated, some types of tumors are still difficult to cure, which requires more in-depth research and exploration.

[0003] In the biomedical field, the permeability of cell membranes is crucial to the normal physiological function of cells. It is related to the exchange of substances inside and outside the cells and the transmission of signals. When the membrane structure of tumor cells is damaged, its permeability will change significantly, causing a large influx of extracellular calcium ions into the cells. These calcium ions can trigger the programmed cell death mechanism, namely apoptosis, which is an active and orderly form of cell death that helps to remove damaged or excess cells in the body. At the same time, the increase in cell membrane permeability will also cause changes in intracellular osmotic pressure, causing the cells to absorb water and swell and eventually die. This process is called oncosis.

[0004] In addition, destroying the membrane structure of tumor cells can also increase the permeability of chemotherapy drugs, making it easier for drugs to enter the cells, thereby increasing the sensitivity of tumor cells to chemotherapy drugs. This can not only enhance the therapeutic effect of chemotherapy, but also reduce the dosage of drugs and potential side effects. More importantly, the destruction of tumor cell membranes may also make them more vulnerable to attack by the immune system. When the tumor cell membrane is damaged, the antigens on its surface may be more easily recognized by immune cells, thereby stimulating an immune response and further eliminating tumor cells.

[0005] In addition to the impact on tumor cells themselves, the destruction of their membrane structure may also inhibit the metabolic process of the tumor, hinder the proliferation and growth of tumor cells, thereby inhibiting the development of the tumor and reducing its invasion and damage to surrounding tissues. In addition, the destruction of tumor cell membranes may also affect the generation and maintenance of tumor blood vessels. Tumor blood vessels are important channels for tumor cells to obtain nutrients and oxygen. Destroying their membrane structure may make it impossible for tumor blood vessels to continue to provide sufficient nutrients and oxygen to tumor cells, thereby inhibiting the growth and spread of tumors.

[0006] On the other hand, as a common microorganism, the interaction between Propionibacterium acnes and cells may also have a significant impact on the physiological characteristics of cells. Propionibacterium acnes can express a variety of enzymes and antimicrobial peptides, such as lipase, protease, hyaluronan lyase and CAMP. Among them, lipase belongs to the carboxyl ester hydrolase class, which can gradually hydrolyze triglycerides into glycerol and fatty acids. However, excessive intake of unsaturated fatty acids may cause peroxidation reactions due to the unsaturated double bonds in the structure, causing lipid peroxidation in the body. When lipid peroxidation exceeds the physiological balance, it will first destroy the structure and function of the cell membrane, and then cause protein cross-linking, impaired enzyme activity and cell nucleus damage, ultimately affecting the function of the cell.

[0007] In summary, the destruction of tumor cell membrane is of great significance in tumor treatment, and the interaction between Propionibacterium acnes and cell membrane also has a profound impact on the physiological properties of cells, which provides a new perspective for tumor treatment and the interaction between microorganisms and cells. Summary of the invention

[0008] The purpose of the present invention is to use Propionibacterium acnes, a common microorganism, to solve the problem of low drug utilization rate in tumor treatment and provide a method for improving cell membrane permeability.

[0009] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0010] The object of the present invention is to provide a method for improving cell membrane permeability, the specific scheme is:

[0011] A method for enhancing the membrane permeability of mouse breast cancer cells, wherein Propionibacterium acnes is co-cultured with mouse breast cancer cells to increase the membrane permeability of cancer cells.

[0012] The preferred technical solution is: Propionibacterium acnes is activated and cultured and then co-cultured with cells, and the co-culture time of bacteria and cells is 3-12 hours.

[0013] The preferred technical solution is: the concentration of the Propionibacterium acnes suspension is 10 4 CFU / mL-10 6 CFU / mL.

[0014] Beneficial effects:

[0015] 1. Innovative treatment strategy: The present invention provides an innovative strategy for cancer treatment, which is to promote anticancer drugs to enter tumor cells more effectively by regulating the permeability of tumor cell membranes, thereby improving drug efficacy and reducing damage to normal cells.

[0016] 2. Enhance drug delivery efficiency: By inducing increased permeability of tumor cell membranes, the rate and efficiency of drug molecules passing through cell membranes can be significantly improved, providing a theoretical basis and technical support for the development of new drug delivery systems.

[0017] 3. Promote cell biology research: This method can not only be used for cancer treatment research, but also as a powerful tool for studying cell biology issues such as cell membrane structure and function, cell-cell interactions, and cell signal transduction, thereby promoting in-depth development of research in related fields.

[0018] 4. High biosafety: As one of the normal flora of human skin, Propionibacterium acnes is relatively safe to use, and the biosafety of experiments and treatments can be further ensured by precisely controlling the co-incubation conditions and lipase activity.

[0019] 5. Potential clinical application prospects: This method has shown great potential in the treatment of malignant tumors such as breast cancer. In the future, it is expected to be transformed into a clinically applicable treatment method through further optimization and clinical trials, bringing good news to patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram comparing the lactate dehydrogenase content in the control group (a) and the Propionibacterium acnes treatment group (b) under the same treatment conditions.

[0021] Figure 2 Schematic diagram comparing the cell membrane permeability of the control group (a) and the Propionibacterium acnes treated group (b).

[0022] Figure 3 Schematic diagram of the MoS2 group (a) and the group treated with Propionibacterium acnes and MoS2 together (b) MoS2 entering the cells.

[0023] Figure 4 Schematic diagram of tumor cell survival rate in the control group (a), Propionibacterium acnes group (b), MoS2 group (c), and Propionibacterium acnes and MoS2 co-treated group (d). DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0025] Example 1

[0026] A method for enhancing the membrane permeability of mouse breast cancer cells comprises the following steps:

[0027] (1) Strain activation: A ring of Propionibacterium acnes was selected from the frozen glycerol tube and inoculated into liquid thioglycollate medium and cultured at 37°C for 36 h.

[0028] (2) Preparation of Propionibacterium acnes suspension: Use fresh liquid thioglycollate medium to dilute the mature Propionibacterium acnes in step 1 to obtain a Propionibacterium acnes suspension with a concentration of 106 CFU / mL.

[0029] (3) Co-culture: Mouse breast cancer cells (4T1) were placed in the lower chamber of Transwell for culture, and RPMI-1640 medium containing 10% fetal bovine serum and 1% double antibody (100U / mL) was used for culture at 37°C and 5% CO2. When the cells grew to 70% to 80%, 1.5mL RPMI-1640 medium and a 106CFU / mL Propionibacterium acnes suspension were added to the Transwell chamber, respectively, and the control group and Propionibacterium acnes group were set. After the Transwell chamber was placed in the lower chamber of Transwell for co-incubation for 3, 6, 9, and 12 hours, the Transwell chamber was taken out, the cell supernatant in the lower chamber of Transwell was aspirated, and the leakage of intracellular macromolecular substance lactate dehydrogenase (LDH) was detected.

[0030] The results are as follows Figure 1 As shown in the figure, within 12 hours of treatment, compared with the slight change in the control group, the Propionibacterium acnes group had obvious lactate dehydrogenase leakage under the same observation conditions, reaching a peak at the sixth hour. Therefore, it was determined that Propionibacterium acnes could cause changes in cell membrane permeability.

[0031] Example 2

[0032] A method for enhancing the membrane permeability of mouse breast cancer cells

[0033] (1) Strain activation: Same as Example 1

[0034] (2) Preparation of Propionibacterium acnes suspension: Same as Example 1

[0035] (3) Co-culture: Mouse breast cancer cells (4T1) were cultured in the lower chamber of the Transwell using RPMI-1640 medium containing 10% fetal bovine serum and 1% double antibody (100 U / mL) at 37°C and 5% CO2. When the cells grew to 70% to 80%, 1.5 mL of RPMI-1640 medium and a 106 CFU / mL Propionibacterium acnes suspension were added to the Transwell chamber, respectively, to set up a control group and a Propionibacterium acnes group. After the Transwell chamber was placed in the Transwell lower chamber and incubated for 6 hours, the Transwell chamber was taken out, the old culture medium was removed, and the above cells were collected, digested with trypsin, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, washed with pre-cooled PBS, and centrifuged again at 1000 rpm into a 1.5 mL conical bottom centrifuge tube, centrifuged into a pellet, and electron microscopy fixative was slowly added to fill the centrifuge tube with fixative to completely immerse the sample in the fixative. After being fixed at 4°C for 12 hours, the morphology of the cell membrane was observed using a biological transmission electron microscope.

[0036] The results are as follows Figure 2 As shown in the figure, when cells are co-incubated with Propionibacterium acnes, obvious holes can be observed on the cell membrane. The experimental results show that Propionibacterium acnes can destroy the structure of the cell membrane and change the permeability of the cell membrane.

[0037] Example 3

[0038] Propionibacterium acnes promotes MoS2 uptake by improving cell membrane permeability

[0039] In order to verify the conclusion of "Propionibacterium acnes can improve the permeability of cell membranes" proposed in Example 2, the anti-tumor drug MoS2 was selected, and the effect of Propionibacterium acnes assisted MoS2 cell delivery was investigated at the cellular level. The experiment set up a MoS2 incubation group alone and a MoS2 administration group after pre-incubation of Propionibacterium acnes for 6 hours.

[0040] For specific cell culture conditions, see Example 1. 4T1 cells were cultured in the lower chamber of Transwell. When the cells grew to about 70%, 1.5 mL of RPMI-1640 medium and a 106 CFU / mL Propionibacterium acnes suspension were added to the Transwell chamber respectively. After incubation for 6 hours, the chamber was taken out, and MoS2 with a final concentration of 500 μg / mL was added to the cells. After incubation for 3 hours, a biological transmission electron microscope was used to observe the entry of MoS2 into the cells.

[0041] The results are as follows Figure 3As shown, compared with the MoS2 group, the amount of MoS2 in the cells was obviously increased in the group that was pre-incubated with Propionibacterium acnes for 6 hours and then added with MoS2, indicating that after the cells were pretreated with Propionibacterium acnes, the permeability of the cell membrane could be increased, thereby increasing the delivery efficiency of MoS2 into the cells.

[0042] Example 4

[0043] Propionibacterium acnes can effectively enhance the anti-tumor effect of MoS2

[0044] In order to verify the effect of Propionibacterium acnes assisted MoS2 cell delivery mentioned in Example 3 on improving drug efficacy, 4T1 cells were cultured in the lower chamber of Transwell. When the cells grew to about 70%, 1.5 mL of RPMI-1640 medium and a 106 CFU / mL Propionibacterium acnes suspension were added to the Transwell chamber respectively. After incubation for 6 h, the chamber was taken out and MoS2 with a final concentration of 500 μg / mL was added to the cells. 2， After culturing for 6 hours, the cytotoxic effect of each group on 4T1 cells was detected by CCK-8 detection kit using an enzyme marker at 450 nm.

[0045] The results are as follows Figure 4 As shown in the figure, compared with the blank control group, Propionibacterium acnes itself had no significant inhibitory effect on the proliferation of 4T1 cells, while the MoS2 alone group and the group pre-incubated with Propionibacterium acnes and then administered with MoS2 could significantly kill tumor cells. Compared with the MoS2 alone group, the pre-incubation with Propionibacterium acnes could significantly increase the killing effect of MoS2 on cells and improve the anti-tumor effect of MoS2.

[0046] In summary, the present invention provides a new method for inducing cancer cell membrane permeability to assist anti-tumor drugs in killing cancer cells. The method has the advantages of simple operation, low cost, significant effect, etc., and has broad application prospects.

[0047] The above-mentioned embodiments only express the implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A method for enhancing the permeability of mouse breast cancer cell membranes, characterized in that Propionibacterium acnes is co-cultured with mouse breast cancer cells to increase the permeability of cancer cell membranes. After activation and culture, the Propionibacterium acnes forms a Propionibacterium acnes suspension which is then co-cultured with the cells. The co-culture time of the bacteria and cells is 3-12 hours.

2. The method according to claim 1, characterized in that The concentration of the Propionibacterium acnes suspension was 10 4 -10 6 CFU / mL.

3. The method according to claim 1, characterized in that The following steps are involved: (1) Strain activation: A ring of Propionibacterium acnes was picked from the frozen glycerol tube and inoculated into liquid thioglycollate medium and cultured at 37°C for 36 h; (2) Preparation of Propionibacterium acnes suspension: Use fresh liquid thioglycollate medium to dilute the mature Propionibacterium acnes in step 1 to a concentration of 10 6 CFU / mL of Propionibacterium acnes bacterial suspension; (3) Co-culture: Mouse breast cancer cells (4T1) were placed in the lower chamber of the Transwell and cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% double antibody (100 U / mL) at 37°C and 5% CO2. When the cells grew to 70% to 80%, 1.5 mL of RPMI-1640 culture medium and 106 CFU / mL Propionibacterium acnes suspension were set as control group and Propionibacterium acnes group. After the Transwell chamber was placed in the Transwell lower chamber for incubation for 6 hours, the Transwell chamber was taken out, and the old culture medium was removed. The above cells were collected, digested with trypsin, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, washed with pre-cooled PBS, and centrifuged again at 1000 rpm into a 1.5 mL conical bottom centrifuge tube. Centrifuged into a pellet, electron microscopy fixative was slowly added, and the fixative filled the centrifuge tube so that the sample was completely immersed in the fixative. After being fixed at 4°C for 12 hours, the morphology of the cell membrane was observed using a biological transmission electron microscope.

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