WO2021073041A1

WO2021073041A1 - Application of multi-lineage differentiating stress enduring cells, medicament for treating peripheral nerve injury and preparation method therefor

Info

Publication number: WO2021073041A1
Application number: PCT/CN2020/080794
Authority: WO
Inventors: 陈罡; 王晓冬; 赵雅玉
Original assignee: 南通大学
Priority date: 2019-10-15
Filing date: 2020-03-24
Publication date: 2021-04-22
Also published as: CN110693908A; ZA202006355B

Abstract

Provided is an application of multi-lineage differentiating stress enduring cells in the preparation of a medicament for treating a peripheral nerve injury. Further provided is a medicament for treating a peripheral nerve injury. The medicament at least comprises a cell suspension of multi-lineage differentiating stress enduring cells. Further provided is a preparation method for the medicament for treating a peripheral nerve injury. The method comprises the following steps: (1) sorting to obtain multi-lineage differentiating stress enduring cells; (2) suspension-culturing the sorted multi-lineage differentiating stress enduring cells; (3) preparing a cell suspension of the multi-lineage differentiating stress enduring cells.

Description

Application of multi-lineage differentiation continuous stress cell, medicine for treating peripheral nerve injury and preparation method thereof

Technical field

The invention belongs to the technical field of biomedicine, and specifically relates to the application of multi-lineage differentiated continuous stress cells in the preparation of drugs for treating peripheral nerve injury, the preparation of drugs for treating peripheral nerve damage and the preparation method of drugs for treating peripheral nerve damage.

Background technique

In recent years, affected by factors such as the increase in traffic accidents frequently occurring in various natural disasters, unsafe production and other factors, there are approximately 1 million new cases of peripheral nerve injury in my country each year. Functional recovery after peripheral nerve injury is a very complex process involving many factors, and it is still a problem that plagues the medical community. Recent studies have found that transplantation of mesenchymal stem cells from different sources, including bone marrow, umbilical cord and skin, can produce better therapeutic effects in the treatment of peripheral nerve injury. However, mesenchymal stem cells are a group of cells with heterogeneous antigenic phenotypes and include multiple subtypes. It has not been reported which type or several types of cell subgroups play a role in the treatment of peripheral nerve injury. This leads to two consequences: 1) The composition of the subtypes of mesenchymal stem cells is complex, and the ratio of different cell subtypes is constantly changing during the cell culture process. Therefore, the establishment of mesenchymal stem cells suitable for clinical use It is very difficult to control the standard, which also greatly limits the application of mesenchymal stem cells in the treatment of peripheral nerve injury. 2) A large amount of waste of mesenchymal stem cells. Mesenchymal stem cells have a role in the treatment of many diseases, and it is likely to come from different roles of different subgroups. Therefore, finding and developing new mesenchymal stem cell therapies to treat peripheral nerve injury has important medical significance and far-reaching social significance.

Summary of the invention

The present invention provides the application of multi-lineage differentiated continuous stress cells in the preparation of drugs for treating peripheral nerve injury, a method for treating peripheral nerve injury drugs, and a method for preparing drugs for treating peripheral nerve injury, so as to solve the problems raised in the background art.

In order to solve the above technical problems, the embodiments of the present invention first provide the application of multi-lineage differentiated continuous stress cells in the preparation of drugs for treating peripheral nerve injury.

Further, the multi-lineage differentiated continuous stress cells are derived from the mesenchymal tissue of bone marrow, fat, umbilical cord or dermis. The multi-line differentiated sustained stress cells to be protected in the present invention are not limited to mesenchymal tissue derived from bone marrow, fat, umbilical cord or dermis. The multi-line differentiated sustained stress cells used in the embodiments of the present invention are derived from bone marrow.

In the present invention, the multi-lineage differentiated continuous stress cells grow in suspension into spheres or grow adherently. The multi-lineage differentiated continuous stress cell of the present invention expresses SSEA-3, Oct3/4, Sox2, CD105, and CD90, but does not express CD34, CD45, and CD11b.

The embodiment of the present invention additionally provides a medicament for treating peripheral nerve injury, which is characterized in that it includes at least a cell suspension of multi-lineage differentiated continuous stress cells. The medicine for treating peripheral nerve injury of the present invention may also contain other pharmaceutically acceptable auxiliary materials.

Further, the drug dosage of the multi-lineage differentiation continuous stress cell suspension is that the number of multi-lineage differentiation continuous stress cells is 5-10×106/kg body weight, local injection (intrathecal injection, subcutaneous injection, intramuscular injection) Injection, etc.) 1 to 5 times; or 5 to 10×10 ⁷ pieces/kg body weight, intravenous infusion 1 to 5 times.

The embodiment of the present invention additionally provides a preparation method for the treatment of peripheral nerve injury, which is characterized in that it comprises the following steps: (1) sorting to obtain multi-lineage differentiated continuous stress cells; (2) pair sorting Suspension culture of the multi-line differentiated continuous stress cells; (3) Preparation of cell suspension of multi-line differentiated continuous stress cells.

In the present invention, the preparation method of multi-lineage differentiated sustained-stress cells (including step (1) sorting to obtain multi-lineage differentiated sustained-stress cells; step (2) suspension of the sorted multi-lineage differentiated sustained-stress cells Culture;), which can be prepared by the method described in the literature (Kuroda, et al., Unique multipotent cells in adult human mesenchymalcell populations. PNAS, 2010, 107: 8639-8643.). The specific method for preparing multi-lineage differentiated continuous stress cells is as follows: Reference (Kuroda, et al., PNAS, 2010, 107: 8639-8643.) discloses the method for preparing bone marrow cells, and expresses them using multi-lineage differentiated continuous stress cells. The characteristics of SSEA-3+/CD105+/CD45- Use flow cytometric sorting technology to sort bone marrow cells, and carry out suspension culture of sorted multi-lineage differentiated continuous stress cells, using a-MEM+0.9% MethoCult H4100 +20% FBS medium, after 1 day of culture, the cells begin to accumulate, and single cells aggregate to form cell colonies, and then the cell colonies divide and grow to form stem cell spheres. The number of cells gradually increases after 3-5 days, and the cell spheres gradually increase and appear large. And the translucent form. The spheroidization time of multi-lineage differentiated continuous stress cells after subculture is significantly shorter than that of the first generation cells. Starting from the second generation, the spheroids can generally be formed within 2 days. The pluripotent stem cell markers SSEA-3, Nanog, Oct4 and Sox2 were used to identify the multi-lineage differentiated continuous stress cells.

The multi-lineage differentiated continuous stress cells prepared by the method of the present invention have good passage stability. Studies have shown that the cell characteristics remain stable after 60 generations, which is reflected in the following: 1. Cell spheres can still grow in suspension; 2. And passage 5. The multi-lineage differentiated continuous stress cells within one generation have the same analgesic effect; 3. The protein profile analysis shows that compared with the multi-lineage differentiated continuous stress cells of the 5th generation, the cytokines, chemokines, receptors, etc. There are no statistical differences in protein expression in many aspects.

Further, the cell suspension of the multi-lineage differentiated sustained-stress cells in the step (3) is prepared according to the following process: the multi-lineage differentiated sustained-stress cells cultured in step (2) are digested with 0.25% trypsin-EDTA A single cell suspension is prepared, washed with physiological saline, and then suspended in physiological saline to obtain a multi-lineage differentiation continuous stress cell suspension.

Further, the cell concentration of the multi-lineage differentiation sustained-stress cell suspension in the multi-lineage differentiation sustained-stress cell suspension is 1-10×107 cells/ml.

The beneficial effects of the above technical solutions of the present invention are as follows:

(1) The application of the multi-lineage differentiated continuous stress cells of the present invention in the preparation of drugs for treating peripheral nerve injury provides a new option for clinical treatment of peripheral nerve injury.

(2) The multi-lineage differentiated continuous stress cell used in the present invention is a newly discovered adult mesenchymal stem cell in adult bone marrow and dermal tissue in 2010. It is a single cell subgroup, among which The ratio is less than 5%. It has the following characteristics: 1) Specific expression of stage-specific embryonic antigen-3 (SSEA3) and CD105; 2) Stress tolerance; 3) Single cells can form cell spheres with strong Self-renewal ability; 4) It can differentiate into cells of the inner, middle and outer germ layers, but does not form teratomas in nude mice. In the present invention, multi-lineage differentiated continuous stress cells can promote functional recovery after peripheral nerve injury, intravenous injection or injection of multi-lineage differentiated continuous stress cells under the nerve outer mold of the injured nerve can promote the nerve regeneration of the injured nerve, and It can promote the recovery of motor and sensory functions, and has great clinical application value.

(3) The preparation method of the medicine for treating peripheral nerve injury of the present invention utilizes the stable passage characteristics of multi-lineage differentiated continuous stress cells, which is extremely beneficial to the requirement to provide a large number of standardized cell products to prepare and treat peripheral nerve injury.

Description of the drawings

Figure 1 is a diagram of the identification results of multi-lineage differentiated continuous stress cells (MUSE cells) in the present invention; among them, Figure 1A is the collection of human bone marrow cells cultured to the third generation, using multi-lineage differentiated continuous stress cells SSEA-3+/ The characteristics of CD105+ were sorted by flow cytometry; Fig. 1B is the sorted multi-lineage differentiated continuous stress cells that can form cell spheres from the sorted single cells in suspension culture; Fig. 1C is The cell morphology of the suspension cell spheres of multi-lineage differentiated continuous stress cells after digestion into single cells and adherent culture.

Fig. 2 is a diagram showing the detection of mouse sciatic nerve function index in the multi-line differentiated continuous stress cell transplantation group and the control group of the present invention, MUSE (multi-line differentiated continuous stress cell transplantation group), PBS (control group).

Fig. 3 is a graph showing the detection results of the gastrocnemius compound muscle action potential of mice in the multi-lineage differentiation continuous stress cell transplantation group and the control group of the present invention; among them, Fig. 3A is a graph of the action potential amplitude read by the PowerLab software; Fig. 3B is the average statistics Figure.

Detailed ways

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will describe in detail in conjunction with specific embodiments.

Example 1

Preparation of Multi-lineage Differentiated Persistent Stress Cells

A professional doctor will perform bone marrow aspiration on the donor’s anterior superior iliac spine, and extract 1ml of bone marrow. Add bone marrow and phosphate buffered saline solution to the upper layer of the lymphocyte separation solution at a ratio of 1:1. After centrifugation, the liquid is divided into four layers. The uppermost layer occupies about 1/2 of the liquid column and is a light red plasma layer; the third layer is about 1/4 of the liquid column is the clear lymphocyte separation layer; at the junction of the two layers is a milky white cloud-like layer about 5mm high, which is the mononuclear cell layer (this layer is the cell layer to be extracted); The bottom layer is closely attached to the test tube wall and is dark red, mainly composed of a dense red blood cell layer. Take the second layer of cells and use DMEM/F12 complete medium containing 15% FBS. After culturing in _{a constant temperature incubator at 37°C and 5% CO 2} for 24 hours, scattered single cells can be seen to adhere to the wall; The parietal cells began to divide and proliferate, gradually spreading from the initial round shape, the cell poles face irregularly, the shape is irregular, and there are mixed cells around; after 4-7 days, the cells grow in colonies, mostly in the shape of a spindle; after 1w, the cells are flat. Many protrusions, cell bodies fused with each other. After the third generation, the cell morphology was basically single and spindle-shaped. About 95% of the cells detected by flow cytometry were CD90+/CD45-/CD11b-. Collect the cells cultured to the third passage, use the characteristics of Muse cells SSEA-3+/CD105+ to sort by flow cytometry, and carry out suspension culture on the sorted Muse cells, using a-MEM+0.9% MethoCult In H4100+20% FBS medium, the cells began to aggregate after 1 day, and single cells aggregated into clusters to form cell colonies, and then the cell colonies divided and grew to form stem cell spheres. The number of cells gradually increased after 3-5 days, and the cell spheres gradually increased and appeared large. And the translucent form. The spheroidization time of Muse cells after subculture is significantly shorter than that of the first generation of cells. Starting from the second generation, the spheroids can generally be formed within 2 days. The pluripotent stem cell markers SSEA-3, Nanog, Oct4 and Sox2 were used to identify Muse cells.

The identification results are shown in Figure 1. Figure 1A is the collection of human bone marrow cells cultured to the third generation, using the characteristics of the multi-lineage differentiation continuous stress cell SSEA-3+/CD105+ for sorting using flow cytometry sorting technology the result of. This result indicates that approximately 0.89% of the cultured bone marrow cells are multi-lineage differentiated continuous stress cells. Figure 1B shows that the sorted multi-lineage differentiated continuous stress cells can form cell spheres from the sorted single cells in suspension culture. This result indicates that the continuous stress cells in multi-lineage differentiation can be expanded in vitro in a large amount. Figure 1C shows the cell morphology when the suspension cell pellets of multi-lineage differentiated continuous stress cells are digested into single cells and then subjected to adherent culture. This result shows that the multi-lineage differentiated continuous stress cells can also be cultured adherently, and the cell morphology is consistent. Can be used for further experiments.

Example 2

Multi-lineage differentiated continuous stress cells for the treatment of mice with sciatic nerve crush

1) Mouse sciatic nerve clamp injury model

The mouse was continuously anesthetized with isoflurane gas on the operating platform, a 1 cm incision was made on the left buttocks, and a special forceps 0.3 cm from the lower edge of the piriformis muscle was used to clamp the sciatic nerve 3 times, 10 seconds each time, 10 seconds interval, squeeze The width of the compression injury is 2 mm. The distal end of the injury was marked on the nerve outer mold with 8-0 micro sutures, and the wound was sutured.

2) Multi-lineage differentiation and continuous stress cell transplantation.

(1) Intravenous injection: Multi-lineage differentiated continuous stress cells were injected through the tail vein at a dose of ^{1-10×10 7 cells/kg body weight.}

(2) Local injection: Multi-lineage differentiated continuous stress cells were injected under the nerve outer mold at the clamp injury at a dose of ^{1-10×10 6 cells/kg body weight.}

3) Detection of mouse sciatic nerve function index

Make a self-made wooden trough with a length of 40 cm, a width of 8 cm, and a height of 10 cm. Cut 70 g of white paper into the same length and width as the wooden trough and spread it on the bottom of the trough. After the mouse's hind feet are colored with paint, the mouse is placed on one end of the trough and allowed to walk to the other side of the trough by itself, leaving 5-6 foot prints on each hind limb. Select the clearly imprinted foot print to measure the three indexes of the normal foot (N) and the injured foot (E): A, PL (footprint length); B, TS (toe width); C, IT (middle toe) width). Substituting the above index into the Bain formula, calculate the sciatic nerve function index. Bain formula: SFI=109.5(ETS-NTS)/NTS-38.3(EPL-NPL)/NPL+13.3(EIT-NIT)/NIT-8.8. Sciatic nerve function index SFI=0 is normal, -100 is complete injury.

The mouse sciatic nerve function index was tested, and the results are shown in Figure 2: The sciatic nerve function index of the mice in the multi-lineage differentiated continuous stress cell transplantation group was better than that of the control group, suggesting that the multi-lineage differentiated continuous stress cell can promote the recovery of nerve injury.

4) Detection of compound muscle action potential of mouse gastrocnemius muscle

The mice were continuously anesthetized with isoflurane gas on the operating platform to free the sciatic nerves on both sides. Using the PowerLab data acquisition system, the stimulating electrode is a double hook-shaped electrode placed under the nerve trunk 0.3 cm from the lower edge of the piriformis muscle at the proximal end of the sciatic nerve clamp, and the normal side position is the same. The recording electrode adopts a bipolar needle-shaped electrode inserted into the middle of the gastrocnemius muscle. The two poles are arranged perpendicular to the gastrocnemius fiber direction. The two-stage tips are separated by 2 mm. The stimulation intensity is 5 mA. The stimulation interval is 0.25 milliseconds. Each animal is measured 3 times. Take the average value for statistics, and read the action potential amplitude through PowerLab software.

The mouse gastrocnemius compound muscle action potentials were tested, and the results are shown in Figure 3: The action potential amplitude of the mouse gastrocnemius compound muscle in the multi-lineage differentiated continuous stress cell transplantation group was higher than that of the control group, suggesting that multi-lineage differentiated continuous stress cells can promote Recovery of nerve damage.

The present invention uses the mouse model of sciatic nerve clamp injury to prove for the first time that intravenous injection or injection of multi-lineage differentiated continuous stress cells under the nerve outer mold of the injured nerve can promote the nerve regeneration of the injured nerve, and can promote the recovery of motor and sensory functions.

The above are the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications are also It should be regarded as the protection scope of the present invention.

Claims

Application of multi-lineage differentiated continuous stress cells in preparing medicine for treating peripheral nerve injury.
The application of the multi-line differentiated persistent stress cell in the preparation of a medicine for the treatment of peripheral nerve injury according to claim 1, wherein the multi-line differentiated persistent stress cell is derived from the mesenchyme of bone marrow, fat, umbilical cord or dermis. Qualitative organization.
A medicament for treating peripheral nerve injury, which is characterized in that it comprises at least a cell suspension of multi-lineage differentiated continuous stress cells.
The drug for the treatment of peripheral nerve injury according to claim 3, wherein the drug dosage of the multi-lineage differentiation continuous stress cell suspension is 5-10. ×10 6 /kg body weight, local injection 1-5 times; or 5-10×10 7 /kg body weight, intravenous infusion 1-5 times.
A preparation method of a medicine for the treatment of peripheral nerve injury according to claim 3, characterized in that it comprises the following steps: (1) sorting to obtain multi-lineage differentiated sustained stress cells; (2) pairing the sorted Suspension culture of multi-line differentiated and sustained stress cells; (3) Preparation of cell suspension of multi-line differentiated and sustained stress cells.
The method for preparing a medicine for treating peripheral nerve injury according to claim 5, wherein the suspension culture of multi-lineage differentiated continuous stress cells in the step (2) comprises the following process: using a-MEM+ In 0.9% MethoCult H4100+20% FBS medium, the cells begin to aggregate after 1 day of culture, and single cells aggregate to form cell colonies, and then the cell colonies divide and grow to form stem cell spheres. The number of cells gradually increases after 3-5 days, and the cell spheres gradually The spheroidization time after subculture of multi-lineage differentiated continuous stress cells is significantly shorter than that of the first generation of cells. Starting from the second generation, the spheroids can be formed on the next day; Competent stem cell markers SSEA-3, Nanog, Oct4 and Sox2 were used to identify multi-lineage differentiation and continuous stress cells.
The preparation method of a medicine for treating peripheral nerve injury according to claim 5, wherein the cell suspension of multi-lineage differentiated continuous stress cells in the step (3) is prepared according to the following process: Step (2) The cultured multi-lineage differentiation sustained stress cells were digested with 0.25% trypsin-EDTA to prepare a single cell suspension, washed with normal saline, and then suspended in normal saline to obtain a multi-lineage differentiation sustained stress cell suspension.
The preparation method of a medicine for the treatment of peripheral nerve injury according to claim 5, wherein the cell concentration of the multi-lineage differentiated and sustained-stress cells in the multi-lineage differentiation and sustained-stress cell suspension is 1-10 ×10 7 pieces/ml.