CN114404449A

CN114404449A - Application of umbilical cord mesenchymal stem cells in preparation of medicine for repairing acute spinal cord injury

Info

Publication number: CN114404449A
Application number: CN202111683872.9A
Authority: CN
Inventors: 张源; 陈占锋
Original assignee: Zhongsai Stem Cell Genetic Engineering Co ltd
Current assignee: Zhongsai Stem Cell Genetic Engineering Co ltd
Priority date: 2021-12-30
Filing date: 2021-12-30
Publication date: 2022-04-29

Abstract

The invention discloses application of umbilical cord mesenchymal stem cells in preparation of a medicine for repairing acute spinal cord injury, wherein the umbilical cord mesenchymal stem cells have hypoxia tolerance. According to the invention, the umbilical cord mesenchymal stem cell umbilical cord has stronger hypoxia tolerance after gradient hypoxia concentration treatment, and the treatment effect of the umbilical cord mesenchymal stem cell umbilical cord on spinal cord loss repair is effectively exerted after spinal cord transplantation, so that the mesenchymal stem cell transplantation treatment on spinal cord injury becomes a very potential cell treatment mode.

Description

Application of umbilical cord mesenchymal stem cells in preparation of medicine for repairing acute spinal cord injury

Technical Field

The invention relates to application of umbilical cord mesenchymal stem cells, in particular to application of umbilical cord mesenchymal stem cells in preparing a medicine for repairing acute spinal cord injury.

Background

With the rapid development of economy and the popularization of physical exercise, Spinal Cord Injury (SCI) is a serious central nervous system injury, which is a spinal cord site injury caused by various reasons, including primary injury at the initial stage of injury and subsequent secondary injury due to ischemia, hypoxia, inflammation, edema, etc.

Besides causing partial or complete loss of sensory and motor functions below the plane of injury, urinary system complications, respiratory system complications, spasticity, pain, hyperreflexia of vegetative nerves and osteoporosis can also cause serious effects on the quality of life of the patient and even endanger life. The international society for spinal cord (ISCoS) is an international group consisting of professionals managing and studying the progress associated with spinal cord injury. To increase public awareness, the international society for spinal cord sets day 9/5 every year as the day of spinal cord injury. According to incomplete statistics, the number of new spinal cord injury cases worldwide is up to 13.3-22.6 thousands every year, and the treatment and recovery cost of a spinal cord injury patient in a lifetime is up to more than 75 ten thousand dollars on average. The treatment of spinal cord injury patients requires the cooperation of emergency treatment, anesthesia and specialized physicians for critical diseases, surgery, rehabilitation and the like, and each stage is very important for the recovery of nerve functions. About 12000 people suffer from the pain caused by acute SCI each year in the United states, the annual incidence rate of China is over 20/100 ten thousands, primary spinal cord injury and secondary inflammatory reaction thereof can cause irreversible damage to important structures of spinal cords and cause serious obstacle of nerve function, heavy burden is brought to patients, families and society, the cost is over 60 billion dollars each year all over the world, the means for treating SCI is limited at present, and large dose of glucocorticoid medicines such as methylprednisolone and the like are clinically and mostly used in the acute stage, so that the inflammatory response is favorably relieved, but the treatment effect is not obvious, and a plurality of side effects are brought. When the spinal canal is damaged by pressure, the external pressure is relieved by surgical decompression, but the damaged spinal cord is difficult to recover. Edema, reduced blood flow, vasospasm, and oxygen free radicals are all detrimental to the survival of nerve cells during spinal cord injury. Because of the non-regenerability of nerve cells, it is far from enough to relieve the microenvironment which is not beneficial to the survival of the nerve cells, and at the moment, a new idea is provided for treating spinal cord injury by increasing the number of nerve-like cells by utilizing mesenchymal stem cell transplantation.

With the advent of tissue engineering technologies, the integrated use of biological factors, biological materials and cell therapy has brought new eosin for the study and application of SCI repair. The treatment of spinal cord injury mainly aims at improving the microenvironment of inflammation at the injured part and the regeneration and repair of nerve function. The application of stem cells in the development of biomedical technology industry and disease treatment has been greatly advanced, including the source, preparation, quality control, differentiation potential of novel stem cells, curative effect, safety, mechanism and the like for disease treatment, especially the application of technologies such as somatic cell nuclear transplantation, induced pluripotent stem cells, high-throughput accurate analysis and the like greatly promotes the development of stem cell technology, the preclinical key technical problems for disease treatment are gradually solved, stem cell products and technologies are gradually promoted to the clinic, and the stem cell industry has a rapid development trend and is listed in scientific and technological priority development plans and strategic emerging industries by various governments. Umbilical Cord Mesenchymal Stem Cells (UCMSC) are one of mesenchymal adult stem cells derived from neonatal umbilical cord tissues, although research on UCMSC is relatively late, the UCMSC develops rapidly, basic theories and technical researches related to clinical application are relatively systematic and comprehensive, the UCMSC is likely to preferentially become a supporting industry in the development of stem cell technology, and UCMSC technology and products are likely to be widely popularized and applied clinically.

Disclosure of Invention

The invention aims to provide a new application of umbilical cord mesenchymal stem cells.

Specifically, the invention relates to application of umbilical cord mesenchymal stem cells in preparation of a medicine for repairing acute spinal cord injury.

In the invention, the umbilical cord mesenchymal stem cells have hypoxia tolerance capability, can adapt to the hypoxic environment of the spinal cord part after spinal cord injury, can play a role in improving the treatment effect.

The common mesenchymal stem cell culture is carried out in CO₂The concentration of 5% is approximately 21% and is closer to the oxygen content of air. The oxygen concentration in human healthy body is generally 1% -12%, and when pathological changes occur, the oxygen concentration at pathological tissue positions is lower (even below 1%) due to insufficient blood supply. The inventor finds that after the spinal cord injury, edema, blood flow reduction, vasospasm, oxygen free radical generation and injury partThe serious shortage of oxygen content is not good for the survival of nerve cells. Because of the non-regenerability of nerve cells, it is far from sufficient to relieve the microenvironment which is not beneficial to the survival of nerve cells, and the survival rate of MSC directly returned to the traditional culture system is low, so that the aim of curing diseases is difficult to achieve. Because the oxygen concentration at the spinal cord is low, when mesenchymal stem cell transplantation treatment is carried out, the transplanted mesenchymal stem cells need to adapt to the change of the gas concentration from 21% of oxygen to low oxygen, and the change can cause the death of a large amount of mesenchymal stem cells, so that the treatment effect cannot be achieved at all. Therefore, the mesenchymal stem cells are used for repairing acute spinal cord injury, and the mesenchymal stem cells are required to have hypoxia tolerance capability so as to greatly improve the action capability and the treatment effect of the cells.

However, the direct hypoxia treatment of mesenchymal stem cells can improve the survival rate of mesenchymal stem cells, but the effect is not obvious, and the method is not suitable for clinical application. Therefore, the method performs gradient hypoxia treatment on the umbilical cord mesenchymal stem cells, increases the hypoxia tolerance capacity of the umbilical cord mesenchymal stem cells, has higher survival rate when being used for spinal cord part transplantation, and effectively improves the action capacity and treatment effect of the cells.

In the invention, the gradient hypoxia treatment is specifically that umbilical cord mesenchymal stem cells are placed in a cell culture box, the initial concentration of oxygen is 20%, and then the gradient decrease of the oxygen concentration is performed every 2 hours until the final concentration of oxygen is below 2%. The gradient decreases the oxygen concentration within 5%.

The invention has the following advantages:

1. the Umbilical Cord Mesenchymal Stem Cells (UCMSC) have rich material sources, and experiments prove that the UCMSC has a repairing effect on spinal cord injury, so that the transplantation of the UCMSC on the spinal cord injury becomes a cell treatment mode with great potential.

2. The survival rate of the umbilical cord mesenchymal stem cells is low in a hypoxia environment, and even if the umbilical cord mesenchymal stem cells are subjected to direct hypoxia treatment, the transplantation of a spinal cord part cannot achieve an effective treatment effect. The method is characterized in that the conventional culture system is changed, the condition of proliferation and growth of cells is changed in vitro, the gradient low-oxygen concentration treatment is carried out on the umbilical cord mesenchymal stem cells, the cells are in a low-oxygen environment in advance through the gradient decreasing oxygen concentration treatment, the oxygen resistance mechanism is changed, the low-oxygen tolerance capacity of the umbilical cord mesenchymal stem cells is enhanced, the survival capacity of the cells in vivo is finally improved, and the treatment effect of the umbilical cord mesenchymal stem cells on the loss of spinal cords is effectively exerted after the spinal cords are transplanted.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.

First, extract mesenchymal stem cells from umbilical cord of newborn and expand to 3 generation cells

1. The umbilical cord specimen is taken from the umbilical cord of a newborn delivered by a healthy pregnant woman in term of the month of full term, and the umbilical cord collection has the informed consent of the pregnant woman. After sterile collection, the cells are placed into a special sample collection box, transported at 4-8 ℃, and sent to a laboratory within 6 hours and sent into a stem cell separation laboratory.

2. Taking out the sterile collection bottle, sterilizing the bottle with 75% alcohol, and placing the bottle into a biological safety cabinet. The umbilical cord collection cassette is opened in the biosafety cabinet. And (5) aseptically sucking 1ml of collection liquid into a sterile EP tube by using a 3ml plastic pipette, marking codes and carrying out asepsis detection.

3. The umbilical cord was transferred to a new sterile 100mm petri dish with autoclaved 18cm toothed forceps, washed 2-3 times with PBS phosphate buffered saline (phosphate buffer saline) to wash the residual blood, and the umbilical cord (about 10cm) was cut into small pieces of about 2-3cm in length with direct scissors. And (3) reserving 1 section of umbilical cord, putting the umbilical cord into a sterile 100mm culture dish, putting other umbilical cord sections into a 50ml centrifuge tube added with a basic RPMI-1640 culture medium, and reserving the coded mark as a specimen.

4. Taking a small section of the umbilical cord into a new culture dish each time, carefully distinguishing the positions of 2 arteries and 1 vein of the umbilical cord, cutting a small opening between the two arteries by using a direct shear, carefully spreading the umbilical cord along the cut small opening by using a toothed forceps, flatly laying the umbilical cord on the culture dish, carefully stripping the arteries and the veins, then stripping the colloid into the new culture dish, and infiltrating the new culture dish by using PBS.

5. Transferring the stripped colloid to 1And (3) washing the 00ml plastic measuring cup with PBS (phosphate buffer solution) for 2-3 times, transferring the plastic measuring cup into a 50ml centrifuge tube, and properly controlling water during transfer. Shearing the gel to about 1mm by a curved shear³The tissue mass of (1).

6. Inoculating the cut colloid into T75cm in a 10 × 10 arrangement with a stainless steel spoon²In the culture bottle, the bottom of the bottle is marked with date, generation and code. Put at 37 ℃ with 5% CO₂An incubator.

After 7.1-2 hours, 10ml of the culture broth was carefully added to each flask. Gently shaking the culture bottle to allow the culture solution to soak the bottom wall and tissue blocks, taking care not to allow the adherent tissue blocks to slide, placing the culture bottle into an incubator at 37 deg.C and 5% CO₂And (5) culturing.

8. Changing culture medium, culturing for 4 days, sucking 5ml of culture medium with 10ml pipette, and adding 6-7ml of fresh culture medium.

9. And (4) carrying out passage, wherein the growth condition of the cells is observed by paying attention every day, and the passage is carried out after the cells are confirmed to have good growth state and the cell density is about 80 percent of the bottom wall of the culture flask, namely the fusion degree of the cells is 80 to 90 percent.

10. Gently shaking the culture bottle to shake off adherent tissue blocks, and discarding the tissue blocks and the culture solution to a waste liquid tank.

11. 10ml of physiological saline (0.9% NaCl) was added to each flask, the flasks were washed, and the wash was discarded into a waste liquid tank.

12. 1ml of 0.25% pancreatin and 1ml of physiological saline were added to the culture flask and uniformly spread on the bottom surface of the flask. Standing for 2-5 min, observing under microscope, adding 1ml special fetal calf serum into culture bottle when the cells retract more than 80%, and stopping digestion.

13. The cell suspension was collected in a 50ml centrifuge tube, 10ml of physiological saline was added to the flask to gently rinse the flask, and the cells remaining in the flask were collected and centrifuged at 300G for 5 min.

14. After centrifugation, the supernatant was discarded, and the cells were resuspended in mesenchymal stem cell culture medium at 3-5X 10 per flask⁵Inoculating each cell, supplementing the culture solution to 10ml per culture flask, and marking cell generation number on the culture flaskP1, date, Place the flask in 5% CO₂The culture was continued at 37 ℃ in the incubator.

15. And (5) carrying out passage, repeating the operation processes of the steps 9-14, and culturing until the 3 rd generation.

16. Sampling for cell identification and sterility test. The detection of bacteria and endotoxin is negative, and no endogenous and exogenous pathogenic bacteria exist; the stem cell immunophenotyping detects that CD73, CD90 and CD105 are positive in expression, the positive rate is greater than 95%, CD34, CD45 and HLA-DR are negative in expression, and the positive rate is less than 2%.

17. Centrifuging the mesenchymal stem cells obtained in step 15 at 300G for 5min, discarding supernatant, re-suspending the collected mesenchymal stem cells with physiological saline (0.9% sodium chloride), sampling, and counting to obtain 6 × 10 cells⁷The preparation method comprises evenly dividing into two parts, adding physiological saline to desired volume of 100ml, adding 1ml human serum albumin injection with concentration of 20%, mixing, and injecting into two cell transfer bags with cell number of 3 × 10⁷。

Second, programmed hypoxia treatment is carried out to culture mature high-activity umbilical cord mesenchymal stem cells

1. Pretreating one bag of cells by gradient oxygen concentration, and putting the bag of cells into a three-gas culture box:

(1) conditioning cell incubators to CO₂Concentration of 5%, O₂Concentration 20%, N₂The concentration is 75%;

(2) after 2h the cell incubator was adjusted to CO₂Concentration of 5%, O₂Concentration 15%, N₂The concentration is 80%

(3) After 4h, adjust the cell incubator to CO₂Concentration of 5%, O₂Concentration 10%, N₂The concentration is 85%;

(4) after 6h, adjust the cell incubator to CO₂Concentration of 5%, O₂Concentration of 5%, N₂The concentration is 90%;

(5) after 8h, adjust the cell incubator to CO₂Concentration of 5%, O₂Concentration 2%, N₂The concentration is 93%; the cell culture chamber was maintained at CO at all times₂Concentration of 5%, O₂Concentration 2%, N₂The concentration is 93%Until 12 h.

2. The umbilical cord mesenchymal stem cell preparation is prepared for standby.

Third, building of PBMC humanized mouse spinal cord injury model

1. Reagent consumable

1.1 reagents

75% medical alcohol 500ml 0.9% physiological saline TAKARA Baozi GT551

Hualan biological 20% human serum albumin TBD human lymphocyte separation liquid

BI Special grade fetal bovine serum Cryosure-DMSO

1.2 consumable

Kangning 175cm2 culture bottle kangning 50ml centrifuge tube, 10ml centrifuge tube, 5ml pipette

10ml pipette NEST 3ml pipette 100. mu.l tip sterile 2ml Ep tube

2ml freezes and deposits 70 μm cell filter scissors dust-free cloth hemostatic forceps of pipe 5ml syringe

1.3 instrumentation

Esco biosafety cabinet Thermo ST40 centrifuge Esco CO₂Culture box

Portable liquid nitrogen tank of refrigerator Thermo F2 micro liquid-transfering gun water bath for medical use of Haixin

Merrill BC-30S hematology analyzer Thermo S1 electric pipettor BD Accuri C6 plus flow cytometer

2. Specimen hand-over

The preparation of 50ml of autologous blood of a volunteer, which was confirmed to have no infectious disease and to have acceptable physiological indexes, and was checked to confirm that the specimen package was not damaged and the specimen was not agglutinated, was carried into a GMP laboratory, and the preparation was started.

3. Cell preparation

3.2.1 clean the biological safety counter with dust-free cloth sprayed with 75% alcohol, a pair of hemostats and 2 50ml centrifuge tubes.

3.2.2 with 75% alcohol will take the surface of the blood collection tube into the biological safety cabinet after disinfection, then will use the hemostatic clamp to hold the alcohol cotton ball to wipe the upper end of the blood collection tube, gently pull off the cap of the blood collection tube, will be the average pouring of blood into 2 prepared 50ml centrifuge tubes, each tube 25 ml.

3.2.3 draw 1ml of blood into a sterile EP tube with a 3ml pipette, and leave the sample code for sterility testing.

3.2.4 screw the tube cap tightly, carefully and symmetrically put into the centrifuge, and centrifuge for 10min with a centrifugal force of 1300G.

3.2.5A 500ml bottle of 0.9% physiological saline and two 15ml portions of the separated human lymphocyte solutions were prepared for use.

3.2.6 after the centrifugation is finished, the two centrifuge tubes are stably taken into the biological safety cabinet. The upper plasma layer was gently removed by pipetting with an electric pipette equipped with a 10ml pipette.

3.2.7 the lower layer of the precipitated cells is made to be 35ml by using 0.9 percent of normal saline, and is gently blown and beaten by using an electric pipette to be evenly suspended.

3.2.8 the cell suspension is slowly added to the upper part of the human lymphocyte separation liquid by an electric pipettor, and the membrane layer is formed on the surface of the human lymphocyte separation liquid by dripping without causing the fluctuation of the interface. The suspension was then slowly added to 50ml per tube.

3.2.9 the tube cap was screwed on and carefully placed symmetrically in the centrifuge and centrifuged at 750G for 18 min. During centrifugation, the temperature should rise and fall quickly.

3.2.11 after the centrifugation, the biosafety cabinet was carefully and smoothly taken in, and the position of the leucoderma layer was observed. Gently sucking off the liquid on the upper layer of the leucoderma layer by using a 10ml pipette, sucking the leucoderma layer cells into a new 50ml centrifuge tube, fixing the volume to 45ml by using 0.9% physiological saline, screwing a centrifuge tube cover, carefully and symmetrically placing the centrifuge tube cover into a centrifuge, and centrifuging for 8min at the centrifugal force of 700G.

3.2.12 after centrifugation, the two tubes were carefully and smoothly placed in a biosafety cabinet, the supernatant was discarded, about 15ml of 0.9% saline was added to each tube, and the suspension cells were pipetted and combined into one tube.

3.2.13 diluting with 0.9% physiological saline to 50ml, counting 100ul cell suspension, screwing the centrifugal tube cover, balancing, and centrifuging for 5min at 500G centrifugal force.

Obtaining a viable cell count of 9.0X 10 for PBMC⁷

3.2.14 preparation of frozen stock solution, 1 10ml centrifuge tube is prepared, 7ml of BI special grade fetal calf serum is added firstly, 1ml of DMSO is added, the mixture is shaken gently and mixed evenly for standby.

3.2.18 after step 3.2.13, the centrifuge tube is securely loaded into the biosafety cabinet. And (3) sucking 1ml of supernatant into a sterile EP tube by using a 3ml pipette, reserving a sample code, and sampling to carry out cell identification and sterile detection. And (3) displaying a detection result: the detection of bacteria and endotoxin is negative, and no endogenous and exogenous pathogenic bacteria exist; the stem cell immunophenotyping detects that CD90 and CD105 are positive in expression, the positive rate is greater than 95%, CD34, CD45 and HLA-DR are negative in expression, and the positive rate is less than 2%.

3.2.19 discarding supernatant, adding 8ml of prepared cryopreservation solution, sufficiently and slowly mixing with 3ml of straw, adding into 42 ml cryopreservation tubes, placing into a programmed cooling box, transferring into a refrigerator at-80 deg.C, transferring into a liquid nitrogen tank after 12 hr, and keeping.

4. Establishment of humanized PBMC mouse model

Injecting PBMC into tail vein of mouse, injecting PBMC into tail vein of mouse at 1d, 3d, 5d, and 7d after preparing and detecting qualified cells respectively at 2.0 × 10⁷0.3ml, the specific implementation steps are as follows:

4.1 the temperature of the thermostatic water bath is adjusted to 37 ℃.

4.2 checking the corresponding cell number and the cell storage position, wearing the anti-freezing thickened gloves with two hands, taking out the cryopreservation tube 1 tube to be recovered from the liquid nitrogen, immediately putting the tube into warm water at 37-40 ℃ and uniformly shaking until most of the cryopreservation liquid is dissolved, taking out the tube until the surface of the tube is disinfected by 75% medical alcohol.

4.3 prepare a centrifuge tube containing about 10ml of 4 ℃ brine ice in a biological safety cabinet, transfer the frozen suspension into the centrifuge tube containing brine ice by using a 3ml plastic pipette, and lightly blow and mix the frozen suspension evenly. The tube containing the cell suspension was trimmed, centrifuged at 300G for 5min and the supernatant discarded.

4.4 repeated washing of the cells with ice saline 2-3 times, the supernatant was discarded. Finally, 300ul of physiological saline and 6ul of 20% human serum albumin were added with a microsyringe, and the cells were mixed thoroughly and slowly and injected via the tail vein of the mouse. Regulating the cells injected each time due to the damage of the cells during the process of freezing and thawingThe concentration is about 2.0X 10⁷0.3 ml.

4.5 after 4 PBMC injections, 2 weeks, collecting peripheral blood of the mice, detecting the percentage of hCD45+ cells in the peripheral blood of the mice in mCD45+ cells and hCD45+ cells at different time points, and achieving immune reconstruction mainly by using human T cells.

5. Establishment of humanized mouse Acute Spinal Cord Injury (ASCI) animal model

5.1 preoperative preparation

Sterilizing 84 and an ultraviolet lamp for more than 30min in a laboratory before operation, and after a fresh air conditioner runs for 30min, keeping the indoor temperature at 23-25 ℃ and the humidity at 45-65%; sterilizing the surgical operation instrument with high-pressure steam at 121 deg.C for 30min, and drying in a drying oven at 60 deg.C; the mice are forbidden to drink for more than 8-10 h before operation, and all operations are carried out under the anesthesia state of the mice. Strict heat preservation is needed during and after the operation, the temperature is maintained at 36.5 ℃, the temperature range during clamping is 36.6-37.1 ℃, and the heat preservation is continuously carried out for 24 hours after the operation.

5.2 anesthetizing mice

The method comprises the steps of putting a cotton ball soaked with ether into a wide-mouth glass bottle (with a cover) for anesthetizing a mouse, selecting a humanized SPF SCID mouse with the same size and weight or similar health, putting the male SCID mouse, the age of 6-8 weeks and the weight of about 40g into the wide-mouth bottle soaked with ether cotton ball, covering the wide-mouth bottle with the cover, and giving attention to general anesthesia of the mouse due to ether inhalation within a short period of time, wherein the cotton ball soaked with ether is not suitable for being deeply anesthetized, so that the mouse is prevented from dying. Generally, 2-3min, the mice gradually become anesthetized and breathe slowly, and the anesthesia box is taken out for dorsal skin preparation, wherein the range of the skin preparation is about 0.6cm of a side-opened spine at two sides of C7-L5. Cleaning the skin preparation area with warm water, wiping with dry gauze, and cutting off excessive hair. The mouse is fixed on the stereotaxic apparatus, and the front teeth and the mouth are firstly fixed. The second is to fix the skull, and the last is to fix the tail, the principle of fixation is to keep the position of the spine straight.

5.3 surgical procedures

Before operation, the level of T10 is determined, iodine tincture in operation area is disinfected 3 times (T10 is taken as the center, the range of C7-L5), and then alcohol is disinfected 3 times. The gas flow of the gas anesthesia machine is adjusted to ensure that the gas flow is minimum to maintain the anesthesia state of the mice. Adjusting the visual field of a microscope, cutting back skin by a T2-T12 sharp knife to avoid damaging subcutaneous fat pads, carefully separating subcutaneous fascia tissues by a No. 5 forceps, exposing muscle tissues to see the termination of superficial paravertebral aponeurosis, carefully separating paravertebral muscles by using a cotton ball as a center, exposing a T9-T11 vertebral body, cutting a T10 vertebral plate from an intervertebral facet joint by using fiber scissors, exposing spinal cord tissues to avoid damaging dura mater, fixing T9 vertebral spinous process and paravertebral tissues by using a toothed fixing forceps to keep the vertebral column at a straight line position, connecting an operating rod by using a special No. 5 forceps, fixing the forceps to the operating rod by using the special No. 5 forceps, carefully placing the front ends of the forceps on two sides of the exposed T10 spinal cord after the correction position, downwards moving by about 0.4mm, confirming that no bone block exists, tightly contacting the sharp ends of the forceps with the spinal cord part after the forceps are completely clamped, clamping time is 5s, immediately releasing the timer after sounding, removing the clamping device to stop bleeding sufficiently, wherein the root of the rat tail is in a state of spasmodic tilting or swinging; the double hind limbs have the phenomenon of shaking or transient twitching, the purplish red blood stasis after bleeding is obviously seen under the hard spinal membrane of T10 and the clamp mark is obviously clamped, the bleeding condition is checked again, and subcutaneous and skin tissues are sutured layer by layer after no active bleeding is ensured. After the small anesthesia is recovered, the two hind limbs are in a complete paralysis state. On the BBB score, it is now at score 0 with no visible hind limb movement.

5.4 postoperative Care

Weighing after operation, recording the operation condition, placing the mice in a cage after the mice are awakened from anesthesia, feeding water, and feeding the mice properly, wherein the room temperature is controlled to be about 25 ℃, and the humidity is 45-65%. The wound part is disinfected every day to assist the mouse to urinate. After urination, care was taken to keep clean and dry. The body position of the mouse is adjusted regularly, and secondary injury caused by long-term local compression is avoided.

Fourthly, the UCMSCs treated by programmed hypoxia are given to the treatment of spinal cord injury of mice

The method comprises the following steps: 30 mice, approximately 40 g/mouse, were randomly assigned to 2 groups: and respectively administering the umbilical cord mesenchymal stem cell injection solution group and the 0.9% sodium chloride injection solution group to the tail vein injection of the umbilical cord mesenchymal stem cell injection solution prepared by the second part:2×10⁵cells/mouse, 0.9% sodium chloride injection. The administration volume is 5ml, 1 time every 2 weeks, 4 times in total; after the last administration, the mice were recovered for 2 weeks, and observed for general conditions, body weight, food intake, etc., and for immunological indicators such as hematology, hematochemistry, histopathology, and subpopulations of serum cytokines, IgG, and T lymphocytes.

In the study of spinal cord injury animal models, hind or lower limb motor function assessment is often performed on animal models, of which the BBB scoring system is most commonly used. The invention adopts a rat spinal cord injury model, applies the tail vein of stem cells to inject the stem cells to repair the injured spinal cord, observes the motion condition of bilateral hind limbs and carries out BBB scoring.

Evaluation of the motor function of the rat postoperative bilateral hind limb:

the BBB scoring and the inclined plate test are respectively carried out according to the methods of Basso, Tarlov and the like, 1 time is carried out every 2 weeks, 5min is continuously observed every time or 5 rounds of back and forth crawling are carried out, and the average score is taken until 14 weeks after the operation. The scorers were trained and well known for BBB scoring criteria. The sloping plate is made by self, and the plate length is 80cm, and the width is 60 cm. Excluding other interference factors such as attraction, actuation and the like.

The main observation indexes are as follows: evaluation of hindlimb motor function was performed 1 time every 2 weeks after surgery using BBB scoring and the oblique plate test, respectively.

Design, implementation and evaluation: the experimental design, intervention implementation and evaluation are all different people, and the scoring is carried out by three different people at the same time, and the average value is taken.

TABLE 1 evaluation results of hindlimb nerve function BBB scores of mice of each group after operation

Group of	1d	2wk	4wk	6wk	8wk	10wk	12wk	14wk
									A	21	21	21	21	21	21	21	21
B	0	3	7	7	9	9	9	9
									C	0	4	12	14	15	15	16	16

Note: group a placebo was healthy SPF grade SCID mice; group B is 0.9% sodium chloride injection group; and the group C is an umbilical cord mesenchymal stem cell injection group.

TABLE 2 postoperative inclined plate test results of hindlimb nerve function of each group of mice

Group of

1d

2wk

4wk

6wk

8wk

10wk

12wk

14wk

A

63.20±1.17

63.80±1.72

63.8±1.60

62.60±1.85

63.20±1.72

62.60±1.36

62.80±1.60

62.40±1.02

B

26.20±1.72

27.20±1.17

29.20±0.98

28.40±1.02

28.60±1.02

29.00±1.41

28.60±1.02

30.20±1.17

C

27.20±0.75

28.20±0.75

31.20±1.33

46.20±2.32

51.00±1.41

52.20±0.75

52.80±0.75

53.00±1.10

And (4) conclusion:

the blank control and the normal saline group show that after the mice with acute spinal cord injury are treated by UCMSCs treated by programmed hypoxia, under the evaluation of nerve function BBB and the detection of a slant plate test, the spinal cord function can be respectively recovered to be 76.1 percent and 84.1 percent of normal after 14 weeks of operation, which are obviously higher than 42.9 percent and 47.6 percent of the normal saline group, and therefore, the UCMSC with hypoxia tolerance capability has great clinical application value in the aspect of treating acute spinal cord injury.

Claims

1. The application of the umbilical cord mesenchymal stem cells in preparing the medicine for repairing acute spinal cord injury is characterized in that the umbilical cord mesenchymal stem cells have hypoxia tolerance.

2. The use of umbilical cord mesenchymal stem cells according to claim 1 for the preparation of a medicament for the repair of acute spinal cord injury, wherein the umbilical cord mesenchymal stem cells are subjected to a gradient hypoxic concentration treatment.

3. The use of umbilical cord mesenchymal stem cells according to claim 1 for the preparation of a medicament for the repair of acute spinal cord injury, wherein the gradient hypoxia treatment is: placing the umbilical cord mesenchymal stem cells into a cell culture box, wherein the initial concentration of oxygen is 20%, and then gradually reducing the oxygen concentration every 2 hours until the final concentration of oxygen is below 2%.

4. The use of umbilical cord mesenchymal stem cells according to claim 3, wherein the gradient decreasing oxygen concentration has a magnitude within 5% for the preparation of a medicament for the repair of acute spinal cord injury.