NCLX gene modified bone marrow mesenchymal stem cell, preparation method and prevention and treatment effect of NCLX gene modified bone marrow mesenchymal stem cell on radiation-induced lung injury

Abstract

The invention relates to the technical field of biology, and provides an NCLX gene modified bone marrow mesenchymal stem cell as well as a preparation method and application thereof, and the bone marrow mesenchymal stem cell contains an NCLX gene or a nucleotide sequence with homology and equal function in the nucleotide sequence of the NCLX gene. The NCLX gene modified bone marrow mesenchymal stem cells have strong tolerance to hypoxia, and have strong resistance to active oxygen. Animal experiments also prove that mice in an experimental group obviously improve lung tissue alveolar collapse and inflammatory cell infiltration conditions and reduce fibrosis of lung tissues, and the NCLX gene modified bone marrow mesenchymal stem cells have the potential of being developed into drugs for treating radiation-induced lung injury. And a new treatment scheme is provided for prevention and treatment application in radiation-induced lung injury.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to NCLX gene-modified bone marrow mesenchymal stem cells, and a preparation method and application thereof. Background Art

[0002] Radiation-induced lung injury (RILI) is an important cause of death in nuclear and radiation accidents, and is also a common side effect of radiotherapy in patients with thoracic tumors. It is reported that 14.6%-37.2% of patients suffer from radiation-induced lung injury during radiotherapy for thoracic tumors, which seriously limits the treatment effect of the tumor and affects the quality of life of patients. RILI has an acute onset and a long course, especially the late fibrosis, which causes serious damage to the health of patients and also brings heavy living and economic burdens to their families. Therefore, the prevention and treatment of radiation lung injury has important practical significance both for medical health and social development.

[0003] Due to the complex mechanism of RILI and the slow and prolonged course of the disease, there has been no effective prevention and treatment method in clinical practice. Existing measures are mainly aimed at the prevention of RILI, including reactive oxygen scavengers, protease inhibitors, cytokine antagonists, etc. These drugs act on a certain link of RILI and can relieve RILI to a certain extent, but they fail to play a good blocking effect on the entire pneumonia-pulmonary fibrosis process. Compared with prevention, clinical treatment measures for RILI are more limited, and glucocorticoids are still the main ones. Although hormones can effectively reduce the early inflammation of RILI, the side effects they bring seriously limit their widespread and long-term application.

[0004] In recent years, the study of mesenchymal stem cells (MSC) has provided new ideas and strategies for the prevention and treatment of RILI. MSC is a type of pluripotent stem cell derived from the early mesoderm with high self-renewal and multidirectional differentiation potential. It is widely present in various tissues such as bone marrow, peripheral blood, umbilical cord blood, and embryonic liver, among which bone marrow is the most common source. MSC has low immunogenicity and can colonize in damaged tissues through "homing" and differentiate into corresponding tissue cells, thereby repairing tissue damage; on the other hand, MSC can also inhibit immune activation and reduce local inflammation through surface molecular contact and exocrine cytokines. MSC is easier to obtain than embryonic stem cells, and is easy to culture and proliferate in vitro. Therefore, the prevention and treatment of diseases with MSC has become a hot topic in medical research in recent years. So far, MSC has shown good therapeutic effects in many diseases, and even in the treatment of some rare diseases such as multiple sclerosis and pulmonary cystic fibrosis, it has also shown great advantages. However, for radiation-induced lung injury, due to the hypoxia and high reactive oxygen environment in the lung after irradiation, the long-term survival of exogenous MSCs in the irradiated lung tissue is limited, weakening the therapeutic effect of MSCs on radiation-induced lung injury. Therefore, how to modify cells at the stem cell level and perform cell therapy for lung injury is a technical problem that needs to be solved urgently by those skilled in the art.

[0005] In 2020, Nature reported that inhibiting mitochondrial Na + / Ca 2+ Activation of the exchange protein (NCLX) can reduce mitochondrial damage. The inventor surprisingly found that the effect of NCLX gene-modified bone marrow mesenchymal stem cells on radiation-induced lung injury is significantly better than that of unmodified mesenchymal stem cells. Summary of the invention

[0006] In view of the shortcomings of the prior art described above, the purpose of the present invention is to provide a NCLX gene-modified bone marrow mesenchymal stem cell and a preparation method thereof and application in the prevention and treatment of radiation lung injury, thereby solving the technical problems that the existing mesenchymal stem cells have a limited survival time in irradiated lung tissue after transplantation in vivo and have a poor effect in treating radiation lung injury.

[0007] In order to solve the above problems, the technical solution provided by the present invention is as follows: a bone marrow mesenchymal stem cell modified with an NCLX gene, wherein the NCLX gene is a nucleotide sequence having at least 90% homology and equivalent function to the NCLX gene nucleotide sequence shown in SEQ ID NO.1.

[0008] The present invention provides a bone marrow mesenchymal stem cell modified with an NCLX gene, wherein the NCLX gene sequence is shown as SEQ ID NO.1.

[0009] The present invention provides a method for preparing bone marrow mesenchymal stem cells modified with NCLX gene, wherein the method comprises the following steps: transfecting NCLX gene or a nucleotide sequence having at least 90% homology and equivalent function with the nucleotide sequence of NCLX gene into bone marrow mesenchymal stem cells to obtain the obtained cells.

[0010] The gene fragment can be transfected into cells by any method in the prior art. Specifically, a recombinant vector containing the NCLX gene or a nucleotide sequence having at least 90% homology and equivalent function to the nucleotide sequence of the NCLX gene can be constructed, and then the recombinant vector can be transfected into stem cells. Various vectors known in the prior art, such as lentivirus, can be used. The bone marrow mesenchymal stem cells are bone marrow mesenchymal stem cells that can be stably passaged.

[0011] Furthermore, the method includes continuing to culture the transfected bone marrow mesenchymal stem cells and selecting target cells that have been successfully transfected.

[0012] Another aspect of the present invention provides the use of the above-mentioned NCLX gene-modified bone marrow mesenchymal stem cells in the preparation of radiation lung injury drugs.

[0013] The radiation lung injury may be an injury from an accident or an injury suffered by a radiotherapy patient. Generally speaking, the injury referred to herein generally refers to a local chest irradiation dose of less than 25 Gy.

[0014] Another aspect of the present invention provides a drug for treating radiation-induced lung injury, wherein the drug contains a therapeutically effective amount of the NCLX gene-modified bone marrow mesenchymal stem cells as described above.

[0015] Furthermore, the medicine also contains excipients acceptable to human body.

[0016] The method of using the drug can be used under the guidance of a doctor. For example, the bone marrow mesenchymal stem cells modified by the NCLX gene can be injected into the patient's body, and the injection dose is determined according to the injury condition.

[0017] Another aspect of the present invention provides a pharmaceutical composition, wherein one of the active ingredients of the pharmaceutical composition is NCLX gene-modified bone marrow mesenchymal stem cells. Other pharmaceutical ingredients may be drugs for treating other symptoms, such as cancer treatment drugs.

[0018] The beneficial effect of the present invention is that the bone marrow mesenchymal stem cells modified by the NCLX gene of the present invention have strong tolerance to hypoxia and strong resistance to reactive oxygen. Animal experiments have also confirmed that the experimental group of mice significantly improved the alveolar collapse and inflammatory cell infiltration of lung tissue, reduced fibrosis of lung tissue, and the bone marrow mesenchymal stem cells modified by the NCLX gene have the potential to be developed into drugs for the treatment of radiation-induced lung injury. A new treatment plan is provided for the prevention and treatment of radiation-induced lung injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shown is the morphology of bone marrow mesenchymal stem cells;

[0020] Figure 2 Shown are fluorescence images of BMSCs transfected with NCLX overexpression and knockdown and their controls;

[0021] Figure 3A The graph shows the EDU fluorescence proliferation results of the experimental group and the control group at 48 hours;

[0022] Figure 3B Shown is the flow cytometry results of cell proliferation in the 48h experimental group and the control group;

[0023] Figure 3C Shown are flow cytometry images of cell apoptosis in the 24h experimental group and control group;

[0024] Figure 3D Shown is the flow cytometry graph of the cell reactive oxygen content in the 1-hour experimental group and the control group;

[0025] Figure 4 Shown is a schematic diagram of local irradiation of lung tissue in mice;

[0026] Figure 5A Shown are H&E, Masson staining, and immunohistochemistry images of CoL-1 and α-SMA in lung tissues of mice at 1 and 8 weeks after irradiation;

[0027] Figure 5B Shown are the fluorescence results of TUNEL detection in mice 1W and 8W after irradiation. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below with reference to the embodiments, but is not limited thereto.

[0029] Example 1: Preparation of NCLX gene-modified bone marrow mesenchymal stem cells (BMSC-NCLX)

[0030] 1.1 Preparation method:

[0031] (1) After the bone marrow is taken from male, 4-5 week old, C57BL / 6 mice, it is flushed with F-12 medium and placed in a cell culture incubator (37°C, 5% CO2) for culture. After 48 hours, half of the medium is replaced, and then the full medium is replaced every 2 days. When the cell density in the dish reaches 80-90%, the bone marrow mesenchymal stem cells (BMSC) are stably passaged.

[0032] (2) Construct plasmid vectors for overexpression and knockdown of NCLX and their controls, and package them using lentivirus. The knockdown lentiviral vector is GV493, element sequence: hU6-MCS-CBh-gcGFP-IRES-puromycin, knockdown lentiviral control number: CON313, control insertion sequence: TTCTCCGAACGTGTCACGT; the overexpression lentiviral vector is GV492, element sequence: Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin, overexpression lentiviral control number: CON335; the gene ID of NCLX is 170756, and the sequence is as follows (SEQ ID NO.1).

[0033] (3) The constructed NCLX overexpression and knockdown plasmid vectors and their controls were packaged using 293T cells and viral plasmids, and then transferred into BMSCs that were stably passaged to the third generation. The medium was replaced every 24 hours. After continuous culture for 72 hours, the fluorescence intensity of the BMSCs was observed under a fluorescence microscope to evaluate their construction.

[0034] 1.2 Experimental Results

[0035] (1) On the third day of BMSC culture, some cells were observed to adhere to the wall and were oval or spindle-shaped. On the seventh day, the cell density increased and the cells showed "needle-like" and spindle-shaped changes. Figure 1 ).

[0036] (2) The NCLX overexpression and knockdown plasmid vectors and their control plasmid vectors were transferred into BMSCs stably passaged to the third generation. After 72 hours, GFP fluorescence was visible under a fluorescence microscope ( Figure 2 ), indicating that BMSCs with NCLX overexpression and knockdown as well as their control were successfully constructed.

[0037] Example 2: The proliferation, apoptosis and reactive oxygen resistance of lung parenchymal cells (MLE-12) modified with NCLX gene (i.e., NCLX overexpression) were detected, as well as the changes after irradiation, so as to evaluate the protective effect of BMSC-NCLX on MLE-12 after irradiation.

[0038] 2.1 Main experimental methods

[0039] 2.1.1 Cell proliferation ability: The supernatant of BMSC-NCLX and its control group BMSC-NCLX+OENC cell culture dishes cultured in a normal incubator environment (37°C, 5% CO2) was added to MLE-12 cells, and the MLE-12 cells were placed in a Cs-137 radiation source and irradiated with 12 Gy. 48 hours after irradiation, Edu was used to mark the cells, and the EDU fluorescence intensity was detected by using the DAPI and RHOD channels of a fluorescence microscope and photographed. The experimental group (MLE-12 cells added with BMSC-NCLX supernatant) was compared with the control group (MLE-12 cells added with BMSC-NCLX+OENC supernatant);

[0040] The supernatant of BMSC-NCLX and its control group BMSC-NCLX+OENC cell culture dishes was added to MLE-12, and the MLE-12 cells were irradiated with 12 Gy. 48 hours after irradiation, the proliferation ability of MLE-12 cells was evaluated by flow cytometry using the Edu proliferation kit; the experimental group (MLE-12 cells added with BMSC-NCLX supernatant) and the control group (MLE-12 cells added with BMSC-NCLX+OENC supernatant) were compared.

[0041] 2.1.2 Cell apoptosis ability: The supernatant of BMSC-NCLX and its control group BMSC-NCLX+OENC cell culture dishes was added to MLE-12 cells, and the MLE-12 cells were irradiated with 12 Gy. The apoptosis was detected by flow cytometry using an apoptosis kit 48 hours after irradiation; the experimental group (MLE-12 cells added with BMSC-NCLX supernatant) and the control group (MLE-12 cells added with BMSC-NCLX+OENC supernatant) were compared.

[0042] 2.1.3 Cellular resistance to reactive oxygen species: The supernatant of BMSC-NCLX and its control group BMSC-NCLX+OENC cell culture dishes was added to MLE-12 cells, and the MLE-12 cells were irradiated with 12 Gy. One hour after irradiation, the cells were detected by flow cytometry using a reactive oxygen species kit; the experimental group (MLE-12 cells added with BMSC-NCLX supernatant) and the control group (MLE-12 cells added with BMSC-NCLX+OENC supernatant) were compared.

[0043] 2.2 Main experimental results

[0044] 2.2.1 Cell proliferation ability: (1) After Edu labeling, the cell proliferation ability was detected by using the DAPI and RHOD channels of a fluorescence microscope to detect the EDU fluorescence intensity. The results showed that the fluorescence intensity of the experimental group was significantly increased compared with the control group, that is, there was a higher proportion of red fluorescence (594fluorescence) positive cells, indicating that the proliferation ability of MLE-12 cells added with BMSC-NCLX supernatant was improved. In addition, after 12Gy irradiation treatment, the fluorescence intensity of the experimental group decreased less than that of the control group, indicating that irradiation had little effect on the proliferation ability of MLE-12 cells added with BMSC-NCLX supernatant ( Figure 3A );

[0045] (2) Flow cytometry results showed that the cell proliferation rate of the experimental group increased compared with that of the control group, that is, EDU-labeled cells had a higher proportion of red fluorescence-positive cells, showing two peaks of red fluorescence negative weak staining (left peak) and positive strong staining (right peak), corresponding to non-proliferating cells and proliferating cells, respectively. In addition, the proliferation capacity of the experimental group after 12 Gy irradiation was significantly increased ( Figure 3B ); These results indicate that the addition of BMSC-NCLX supernatant has a good protective effect on irradiated MLE-12.

[0046] 2.2.2 Cell apoptosis ability: Flow cytometry results showed that the apoptosis rate of the control group was 5.38±2.4%, while that of the experimental group was 4.08±1.3%; 48h after irradiation, the apoptosis rate of the control group was 26.00±3.4%, while that of the experimental group was 8.74±2.3% ( Figure 3C ). The above results show that adding BMSC-NCLX supernatant can reduce the apoptosis rate of MLE-12 cells and has a good protective effect on irradiated MLE-12 cells.

[0047] 2.2.3 Cellular resistance to reactive oxygen species: (1) After loading the experimental and control group cells with reactive oxygen species probes, it can be seen that under normal circumstances, the fluorescence intensity of the control group is higher than that of the experimental group, that is, the positive peak of the control group (the range of the reactive oxygen species probe fluorescence ratio P2 in the flow cytometry diagram) is significantly shifted to the right compared with the experimental group (with a fluorescence ratio of 10 4 As the benchmark), it shows that adding BMSC-NCLX supernatant can reduce the content of reactive oxygen species in MLE-12 cells; (2) MLE-12 cells were further irradiated with 12 Gy, and the content of reactive oxygen species was detected by flow cytometry 1 hour after irradiation. It was found that the experimental group could more significantly reduce the content of reactive oxygen species in MLE-12 cells after irradiation than the control group ( Figure 3D ); In summary, it was shown that the addition of BMSC-NCLX supernatant could have a strong resistance to reactive oxygen species in MLE-12 cells, and had a more significant protective effect on irradiated MLE-12 cells.

[0048] Example 3: Therapeutic effect of bone marrow mesenchymal stem cells with NCLX gene knockdown (BMSC-NCLX+KD) on radiation-induced lung injury in mice

[0049] 3.1 Main experimental methods

[0050] (1) The constructed experimental group BMSC-NCLX+KD cells and the control group BMSC-NCLX+KDNC cells were cultured at 1×10 5 Number / mouse, injected into C57BL / 6 mice via tail vein injection;

[0051] (2) 2 hours after cell infusion, the mice were subjected to local irradiation of lung tissue. The specific method is as follows: male, 6-8 weeks old, C57BL / 6 mice were anesthetized and placed in a mouse box for irradiation, and their limbs were fixed. The small box for irradiation is a transparent plastic box. After the mice are fixed, 60 The irradiation was performed with a Co-γ-ray source. During the irradiation, only the chest cavity was exposed, and other parts were shielded with lead blocks. The total irradiation dose was 25 Gy, and the dose rate was 1 Gy / min ( Figure 4 ).

[0052] (3) After local thoracic irradiation of mice, lung tissues were collected at 1 week (1w) and 8 weeks (8w) after irradiation for H&E staining and Masson staining, as well as immunohistochemical detection of CoL-1, α-SMA, and TUNEL to evaluate lung tissue damage and the degree of fibrosis ( Figure 5A \B).

[0053] 3.2 Experimental results:

[0054] (1) The lung tissue pathological results 1 week after irradiation showed that the therapeutic effect of BMSC after NCLX knockdown was significantly lower than that of normal BMSC cells, indicating that the NCLX gene affects the therapeutic effect of BMSC cells in improving acute lung injury caused by irradiation in mice. That is, the H&E staining results showed that after irradiation, the alveoli of the lung tissue of the control group mice infused with BMSC-NCLX+KDNC cells (abbreviated as CON in the result figure) collapsed, there was obvious inflammatory cell infiltration in the bronchi, and the airway smooth muscle septa thickened; while the mice infused with BM In the experimental group of SC-NCLX+KD cells (abbreviated as KD in the result figure), the degree of lung tissue lesions in mice was aggravated, bronchial stenosis, alveolar collapse were more severe, and more obvious inflammatory cell infiltration was seen around the airways and blood vessels, and the difference was statistically significant (P < 0.05); the results of immunohistochemistry (α-SMA and COL-1) showed that the expression levels of α-SMA and CoL-1 in the experimental group of mice infused with BMSC-NCLX+KD cells were significantly increased compared with those in the control group, and the difference was statistically significant (P < 0.05) ( Figure 5A );

[0055] (2) The lung tissue pathological results at 8 weeks after irradiation showed that knockdown of NCLX reduced the inhibitory effect of BMSC on pulmonary fibrosis in irradiated mice. Masson staining results showed that a small amount of blue collagen (positive area) was visible in the lung tissue of the control group mice infused with BMSC-NCLX+KDNC cells (abbreviated as CON in the results), and the subbronchial collagen deposition in the experimental group mice infused with BMSC-NCLX+KD cells (abbreviated as KD in the results) was significantly aggravated compared with the control group mice (P < 0.05); the immunohistochemical results showed the same trend as the expression level in the lung tissue of 1 week mice ( Figure 5A );

[0056] (3) TUNEL staining of lung tissues at 1 and 8 weeks after irradiation showed that NCLX knockdown BMSCs significantly increased irradiation-induced apoptosis of lung parenchymal cells (including alveolar epithelium and vascular endothelium) compared with normal BMSCs, i.e., an increase in red fluorescence (594fluorescence) positive cells ( Figure 5B ). The above results fully demonstrate that BMSC-NCLX can significantly improve the lung injury of mice caused by irradiation, and the protective effect of BMSC on lung tissue is significantly reduced after knocking down NCLX.

[0057] The above is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, which all belong to the protection scope of the present invention.

[0058] Sequence Listing

[0059] SEQ ID NO.1

[0060]

Claims

1. A bone marrow mesenchymal stem cell modified by NCLX gene, characterized in that: The NCLX gene is a nucleotide sequence having at least 90% homology and equivalent function to the NCLX gene nucleotide sequence shown in SEQ ID NO.

1.

2. The NCLX gene-modified bone marrow mesenchymal stem cells according to claim 1, characterized in that: The NCLX gene sequence is shown in SEQ ID NO.

1.

3. The method for preparing NCLX gene-modified bone marrow mesenchymal stem cells according to claim 1, characterized in that: The method comprises the steps of transfecting the NCLX gene or a nucleotide sequence having at least 90% homology and equivalent function with the nucleotide sequence of the NCLX gene into bone marrow mesenchymal stem cells to obtain the product.

4. The method according to claim 3, characterized in that The method comprises constructing a recombinant vector containing the NCLX gene or a nucleotide sequence having at least 90% homology and equivalent function to the nucleotide sequence of the NCLX gene, and then transfecting the recombinant vector into stem cells.

5. The method according to claim 4, characterized in that The vector is a lentivirus, and the bone marrow mesenchymal stem cells are bone marrow mesenchymal stem cells that can be stably passaged.

6. Use of the bone marrow mesenchymal stem cells modified with the NCLX gene as claimed in claim 1 or 2 in the preparation of a drug for radiation-induced lung injury.

7. A drug for treating radiation-induced lung injury, characterized in that: The drug contains a therapeutically effective amount of the NCLX gene-modified bone marrow mesenchymal stem cells according to claim 1 or 2, wherein the bone marrow mesenchymal stem cells contain the NCLX gene.

8. The drug according to claim 7, characterized in that The medicine also contains auxiliary materials acceptable to human body.

9. A pharmaceutical composition, characterized in that One of the active ingredients of the pharmaceutical composition is the NCLX gene-modified bone marrow mesenchymal stem cell drug according to claim 7 or 8, and also includes drugs for treating other symptoms.

10. The pharmaceutical composition according to claim 9, characterized in that The medicine for treating other symptoms is a cancer treatment medicine.

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