CN110693908A

CN110693908A - Application of multisystem differentiation sustained stress cells, medicine for treating peripheral nerve injury and preparation method of medicine

Info

Publication number: CN110693908A
Application number: CN201910977582.1A
Authority: CN
Inventors: 陈罡; 王晓冬; 赵雅玉
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2019-10-15
Filing date: 2019-10-15
Publication date: 2020-01-17
Also published as: WO2021073041A1; ZA202006355B

Abstract

The invention provides an application of multisystem differentiation sustained stress cells in preparing a medicine for treating peripheral nerve injury. The invention also provides a medicament for treating peripheral nerve injury, which is characterized by at least comprising cell suspensions of a plurality of groups of differentiated continuously stressed cells. The invention also provides a preparation method of the medicine for treating peripheral nerve injury, which is characterized by comprising the following steps of: (1) sorting to obtain multisystem differentiation continuous stress cells; (2) carrying out suspension culture on the sorted multisystem differentiation continuous stress cells; (3) preparing cell suspension of multi-series differentiation sustained stress cells. The application of the multisystem differentiation sustained stress cells in the preparation of the medicine for treating peripheral nerve injury provides a new choice for clinically treating peripheral nerve injury.

Description

Application of multisystem differentiation sustained stress cells, medicine for treating peripheral nerve injury and preparation method of medicine

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of multi-system differentiated sustained stress cells in preparation of a medicine for treating peripheral nerve injury, a medicine for treating peripheral nerve injury and a preparation method of the medicine for treating peripheral nerve injury.

Background

In recent years, under the influence of factors such as increasing of frequent traffic accidents caused by various natural disasters and unsafe production, about 100 million cases of peripheral nerve injury in China are newly increased every year. The functional recovery after the peripheral nerve injury is a very complex process, relates to the influence of various factors, and is still a difficult problem which besets the medical field up to now. In recent years, researches show that transplantation of mesenchymal stem cells from different sources including bone marrow, umbilical cord and skin can generate better treatment effect on peripheral nerve injury. However, mesenchymal stem cells are a population of cells, the antigenic phenotype is not uniform, and comprises a variety of sub-populations, and no report has been made as to which type or sub-population of cells plays a role in the treatment of peripheral nerve injury. This leads to two consequences: 1) the components of each subtype forming the mesenchymal stem cells are complex, and the proportion of different cell subtypes is changed continuously in the cell culture process, so that the establishment of the quality control standard which accords with the clinically used mesenchymal stem cells is very difficult, and the application of the mesenchymal stem cells in the treatment of peripheral nerve injury is also greatly limited. 2) A great deal of waste of mesenchymal stem cells. Mesenchymal stem cells have a role in the treatment of a variety of diseases, most likely different roles from different subpopulations. Therefore, the search and development of new mesenchymal stem cell therapies to treat peripheral nerve injury has important medical and profound social implications.

Disclosure of Invention

The invention provides application of multisystem differentiation sustained stress cells in preparation of a medicine for treating peripheral nerve injury, a medicine for treating peripheral nerve injury and a preparation method of the medicine for treating peripheral nerve injury, and aims to solve the problems in the background art.

In order to solve the technical problems, the embodiment of the invention firstly provides the application of the multi-differentiated continuously stressed cells in the preparation of the medicine for treating peripheral nerve injury.

Further, the multi-lineage differentiation continuously stressed cell is derived from mesenchymal tissue of bone marrow, fat, umbilical cord or dermis. The multi-lineage differentiation stress cells to be protected in the present invention are not limited to mesenchymal tissues derived from bone marrow, fat, umbilical cord or dermis, but the multi-lineage differentiation stress cells used in the examples of the present invention are derived from bone marrow.

In the present invention, the multi-lineage differentiated continuously stressed cells are suspended in globular or adherent growth. The multisystem differentiation sustained stress cell expresses SSEA-3, Oct3/4, Sox2, CD105 and CD90, and does not express CD34, CD45 and CD11 b.

Embodiments of the present invention further provide a medicament for treating peripheral nerve injury, comprising a cell suspension of at least a plurality of differentiated continuously stressed cells. The medicine for treating peripheral nerve injury can also contain other pharmaceutically acceptable auxiliary materials.

Further, the dosage of the drug of the multisystem differentiation sustained stress cell suspension is 5 ~ 10 × 106 cells/kg body weight counted by the number of multisystem differentiation sustained stress cells, and 1 ~ 5 times of local injection (intrathecal injection, subcutaneous injection, intramuscular injection, etc.), or 5 ~ 10 × 10⁷One/kg body weight, 1 ~ 5 times by intravenous infusion.

The embodiment of the invention also provides a preparation method of the medicine for treating peripheral nerve injury, which is characterized by comprising the following steps: (1) sorting to obtain multisystem differentiation continuous stress cells; (2) carrying out suspension culture on the sorted multisystem differentiation continuous stress cells; (3) preparing cell suspension of multi-series differentiation sustained stress cells.

In the present invention, the method for preparing the multi-lineage differentiation sustained stress cells (comprising the steps of (1) sorting the multi-lineage differentiation sustained stress cells and (2) performing suspension culture on the sorted multi-lineage differentiation sustained stress cells) can be prepared by a method described in the literature (Kuroda, et al, Unit multi-lineage cells in adult human mesenchymal cells, PNAS, 2010, 107: 8639-. The specific preparation method of the multisystem differentiation sustained stress cell comprises the following steps: the method disclosed in the reference (Kuroda, et al., PNAS, 2010, 107: 8639-8643.) prepares the bone marrow cells, uses the characteristic that multi-line differentiation continuous stress cells express SSEA-3+/CD105+/CD 45-to sort the bone marrow cells by using a flow cell sorting technology, performs suspension culture on the sorted multi-line differentiation continuous stress cells, uses a-MEM + 0.9% MethoCult H4100 + 20% FBS culture medium, after 1d, the cells begin to aggregate, the single cells aggregate into cell colonies, then the cell colonies divide and grow again to form stem cell spheres, the number of the 3-5d cells gradually increases, and the cell spheres gradually increase to present a large and transparent shape. The time for balling after subculturing the multisystem differentiation continuous stress cells is obviously shortened compared with that of the cells of the 1 st generation, and the cells can be formed in 2 days from the 2 nd generation. The multi-series differentiation continuous stress cells are identified by adopting the pluripotent stem cell markers SSEA-3, Nanog, Oct4 and Sox 2.

The multisystem differentiation continuous stress cell prepared by the method has good passage stability, and researches show that the cell character is still stable after passage for 60 generations, which is reflected in that cell balls can still form suspension growth; secondly, the cells have the same analgesic effect with the multi-series differentiation sustained stress cells within the passage 5; and thirdly, protein mass spectrometry shows that compared with the multi-series differentiation continuous stress cells of passage 5, the protein expression has no statistical difference in multiple aspects such as cytokines, chemokines, receptors and the like.

Further, the cell suspension of the multi-lineage differentiation continuously stressed cells in the step (3) is prepared according to the following process: digesting the multisystem differentiation continuous stress cells cultured in the step (2) by using 0.25% trypsin-EDTA to prepare single cell suspension, washing by using normal saline, and suspending by using the normal saline to obtain multisystem differentiation continuous stress cell suspension.

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

The technical scheme of the invention has the following beneficial effects:

(1) the application of the multisystem differentiation sustained stress cells in the preparation of the medicine for treating peripheral nerve injury provides a new choice for clinically treating peripheral nerve injury.

(2) The multisystem differentiation sustained stress cell adopted in the invention is an adult mesenchymal stem cell newly found in adult bone marrow and dermal tissues in 2010, is a single cell subgroup, and the proportion of the adult mesenchymal stem cell in the adult mesenchymal stem cell is less than 5%. The method has the following characteristics: 1) stage-specific embryonic antigen 3(stage specific embryonic antigen-3, SSEA3) and CDl05 were specifically expressed; 2) stress tolerance exists; 3) the single cells can form cell balls and have strong self-renewal capacity; 4) can differentiate into cells of inner, middle and outer germ layers, but does not form teratoma in nude mice. In the invention, the multisystem differentiation continuous stress cells can promote the function recovery of peripheral nerves after being damaged, and the multisystem differentiation continuous stress cells injected by intravenous injection or under the nerve external mold of the damaged nerves can promote the nerve regeneration of the damaged nerves and promote the recovery of motor and sensory functions, thereby having great clinical application value.

(3) The preparation method of the medicine for treating peripheral nerve injury utilizes the stable passage characteristic of multi-series differentiation continuous stress cells, and is extremely favorable for preparing and treating peripheral nerve injury by providing a large amount of standardized cell products.

Drawings

FIG. 1 is a graph showing the results of the identification of multi-lineage differentiated continuously stressed cells (MUSE cells) according to the present invention; wherein, FIG. 1A is a diagram of the result of collecting human bone marrow cells cultured to the 3 rd generation and sorting by flow cytometry using the characteristics of multisystem differentiation continuous stress cells SSEA-3+/CD105 +; FIG. 1B is a schematic representation of sorted multilineage differentiation sustainably stressed cells in suspension culture from sorted single cells to form cell pellets; FIG. 1C shows the cell morphology after adherent culture after digesting suspension cell beads of multi-lineage differentiation-persistently stressed cells into single cells.

FIG. 2 is a graph showing the functional index of sciatic nerve of mice in the multi-lineage differentiation-sustaining stress cell transplant group and the control group, MUSE (multi-lineage differentiation-sustaining stress cell transplant group) and PBS (control group).

FIG. 3 is a diagram showing the results of detecting the compound muscle action potential of gastrocnemius of mice in a multisystem differentiation continuous stress cell transplantation group and a control group according to the present invention; wherein, FIG. 3A is a diagram of the amplitude of the read action potential of the PowerLab software; fig. 3B is a statistical graph of the mean values.

Detailed Description

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following detailed description is given with reference to specific embodiments.

Example 1

Preparation of multisystem differentiated continuously stressed cells

A bone marrow puncture was performed by a professional doctor above and behind the anterior superior iliac spine of the donor, and 1ml of bone marrow was extracted. Mixing bone marrow and phosphate buffer saline solution at a ratio of 1: 1, adding into upper layer of lymphocyte separation liquid, centrifuging, and separating liquid into four layers, the uppermost layer is 1/2 of liquid column, and is light red plasma layer; the third layer occupies about 1/4 of the liquid column and is a clear lymphocyte separation liquid layer; the boundary of the two layers is a milky cloudy layer with the height of about 5 mm, which is a monocyte layer (the layer is a cell layer to be extracted); the lowest layer is closely attached to the wall of the test tube, is dark red and mainly comprises a red blood cell layer with higher density. The second layer of cells were harvested and cultured in DMEM/F12 complete medium containing 15% FBS at 37 ℃ in 5% CO₂After the cells are cultured in the constant temperature incubator for 24 hours, scattered single cells can adhere to the wall; after 2-3d, the adherent cells begin to divide and proliferate, gradually spread from an initial circle, the two poles of the cells are irregularly oriented, the shapes of the cells are irregular, and mixed cells are arranged around the cells; after 4-7 days the cells grew colony-like, mostly fusiform; after 1w, the cells were flat, multiple-protuberant, and fused with each other. By passage 3, the cells were essentially single in morphology and spindle-shaped, and about 95% of the cells were CD90+/CD45-/CD11 b-as detected by flow cytometry. Collecting cells cultured to the 3 rd generation, sorting the cells by using a flow cytometry sorting technology according to the characteristics of SSEA-3+/CD105+ of Muse cells, carrying out suspension culture on the sorted Muse cells, using a-MEM + 0.9% MethoCult H4100 + 20% FBS culture medium, starting cell aggregation after 1d, enabling single cells to aggregate to form a cell colony, then enabling the cell colony to divide and grow to form stem cell balls, gradually increasing the number of the cells in 3-5d, and gradually enlarging the cell balls to present a large and bright shape. The time for balling after the Muse cell subculture is obviously shorter than that of the 1 st generation cell, and the cell balls can be formed in 2d from the 2 nd generation. By using more than oneMuse cells were identified by the stem cell markers SSEA-3, Nanog, Oct4 and Sox 2.

The identification results are shown in FIG. 1, wherein FIG. 1A shows the results of collecting human bone marrow cells cultured to the 3 rd generation and sorting by flow cytometry using the characteristics of multi-lineage differentiation continuously stressed cells SSEA-3+/CD105 +. This result indicates that approximately 0.89% of the cells in cultured bone marrow cells are multisystem differentiation-sustaining stress cells. FIG. 1B is a schematic representation of sorted multilineage differentiation sustainably stressed cells in suspension culture from sorted single cells to form cell pellets. This result indicates that cells with continued stress in multisystem differentiation can be expanded in vitro in large numbers. FIG. 1C shows the cell morphology after adherent culture after digesting suspension cell beads of multi-lineage differentiation-persistently stressed cells into single cells. The result shows that the multisystem differentiation continuous stress cells can also be cultured in an adherent way and have consistent cell morphology. Can be used for further experiments.

Example 2

Multisystem differentiated sustained stress cells for treating sciatic nerve clamp injury mice

1) Mouse sciatic nerve clamping injury model

Mice were continuously anesthetized with isoflurane gas on the surgical platform, a 1 cm incision was made in the left hip, sciatic nerve was clamped 3 times with a specially made forceps 0.3 cm from the lower edge of piriformis, 10 seconds each time, 10 seconds intermittently, and the width of the crush injury was 2 mm. The far end of the injury is marked on the nerve external mold by 8-0 micro suture, and the wound is sutured.

2) Multisystem differentiation sustained stress cell transplantation.

(1) Intravenous injection of multi-differentiated continuously stressed cells according to 1 ~ 10X 10⁷The dose per kg body weight was injected via tail vein.

(2) Local injection of multisystem differentiation sustained stress cells according to the formula of 1 ~ 10X 10⁶The dose per kg body weight was injected under the extraneural model at the site of the injury.

3) Mouse sciatic nerve function index detection

A wooden groove with the length of 40cm, the width of 8cm and the height of 10cm and two open ends is manufactured, 70g of white paper is cut into the same length and width as the wooden groove, and then the white paper is laid at the bottom of the groove. After the hind legs on both sides of the mouse are colored with pigment, the mouse is placed at one end of the trough and is made to walk to the other side of the trough by itself, and 5-6 footprints are left on each hind limb. The footprints with clear footprints were selected to measure 3 indices of normal (N) and injured lateral (E) feet, respectively: A. PL (footprint length); B. TS (toe width); C. IT (medial toe width). Substituting the indexes into Bain formula to calculate 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 sciatic nerve function index of the mice is detected, and the result is shown in figure 2: the mouse sciatic nerve function index of the multisystem differentiation continuous stress cell transplantation group is better than that of the control group, and the fact that the multisystem differentiation continuous stress cells can promote recovery of nerve injury is suggested.

4) Mouse gastrocnemius compound muscle action potential detection

Mice were continuously anesthetized with isoflurane gas on the surgical platform, dissociating both sciatic nerves. The stimulation electrode is a double-hook electrode and is arranged below a nerve trunk at the position of the proximal end of the sciatic nerve clamp injury, which is 0.3 cm away from the lower edge of the piriformis, and the normal lateral positions are the same. The recording electrode is a bipolar needle electrode, is inserted into the middle of the abdominal part of the gastrocnemius muscle, the arrangement direction of the two electrodes is vertical to the fiber direction of the gastrocnemius muscle, the distance between the two-stage tips is 2 mm, the stimulation intensity is 5 milliamperes, the stimulation interval is 0.25 milliseconds, each animal is measured for 3 times, the average value is taken for statistics, and the action potential amplitude is read through PowerLab software.

The results of the mouse gastrocnemius compound muscle action potential detection are shown in fig. 3: the action potential amplitude of the gastrocnemius compound muscle of the mice in the multisystem differentiation continuous stress cell transplantation group is higher than that of the control group, and the multisystem differentiation continuous stress cell is prompted to promote the recovery of nerve injury.

The invention utilizes a mouse model of sciatic nerve clamping injury, and proves that the nerve regeneration of the injured nerve can be promoted and the recovery of motor and sensory functions can be promoted by injecting multi-system differentiated continuously stressed cells under intravenous injection or nerve external models of the injured nerve for the first time.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. Application of multisystem differentiation sustained stress cells in preparing a medicine for treating peripheral nerve injury.

2. The use of multi-lineage differentiated continuously stressed cells according to claim 1, in the preparation of a medicament for the treatment of peripheral nerve injury, wherein the multi-lineage differentiated continuously stressed cells are derived from mesenchymal tissue of bone marrow, fat, umbilical cord or dermis.

3. A medicament for the treatment of peripheral nerve injury comprising a cell suspension of at least a plurality of differentiated continuously stressed cells.

4. The drug for the treatment of peripheral nerve injury of claim 3, wherein the amount of said suspension of multisystem differentiation-sustaining stress cells is 5 ~ 10X 10 as counted as the number of multisystem differentiation-sustaining stress cells⁶Injecting the mixture 1 ~ 5 times or 5 ~ 10X 10 times per kg body weight⁷One/kg body weight, 1 ~ 5 times by intravenous infusion.

5. A method for preparing a medicament for treating peripheral nerve injury according to claim 3, comprising the steps of: (1) sorting to obtain multisystem differentiation continuous stress cells; (2) carrying out suspension culture on the sorted multisystem differentiation continuous stress cells; (3) preparing cell suspension of multi-series differentiation sustained stress cells.

6. The method for preparing a medicine for treating peripheral nerve injury according to claim 5, wherein the suspension culture of the multi-lineage differentiation stress-sustaining cells in the step (2) comprises the following processes: using a-MEM + 0.9% MethoCult H4100 + 20% FBS culture medium, after 1 day of culture, starting cell aggregation, aggregating single cells to form cell colonies, then dividing and growing the cell colonies again to form stem cell balls, gradually increasing the cell number in 3-5 days, gradually increasing the cell balls, and presenting a large and transparent shape; the balling time of the multi-series differentiation continuous stress cells after subculture is obviously shortened compared with that of the cells of the 1 st generation, and the cell balls can be formed in the next day from the 2 nd generation; the multi-series differentiation continuous stress cells are identified by adopting the pluripotent stem cell markers SSEA-3, Nanog, Oct4 and Sox 2.

7. The method for preparing a drug for treating peripheral nerve injury according to claim 5, wherein the cell suspension of the multi-lineage differentiation stress-sustaining cells in step (3) is prepared by the following process: digesting the multisystem differentiation continuous stress cells cultured in the step (2) by using 0.25% trypsin-EDTA to prepare single cell suspension, washing by using normal saline, and suspending by using the normal saline to obtain multisystem differentiation continuous stress cell suspension.

8. The method of claim 5, wherein the cell concentration of the multi-lineage differentiation stress cells in the multi-lineage differentiation stress-sustaining cell suspension is 1 ~ 10 x 10⁷One per ml.