CN110742906A - Application of mesenchymal stem cell paracrine factor in preparation of pain medicine - Google Patents
Application of mesenchymal stem cell paracrine factor in preparation of pain medicine Download PDFInfo
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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Abstract
An application of mesenchymal stem cell paracrine factors in preparing pain medicines relates to the application of the technical field of biological stem cells in pain. The invention provides a preparation method of mesenchymal stem cell paracrine factor; on the other hand, the invention discovers that the paracrine factor prepared by the method can be used for treating various pains, particularly pains which are subjectively felt by all patients, such as arthralgia, inflammatory pain, cancer pain and the like, and has application prospect in preparing pain medicaments. The invention can effectively relieve pains caused by various reasons. The cell protein preparation of the present invention exhibits excellent biological effects in treating pain.
Description
Technical Field
The invention relates to application of the technical field of biological stem cells in pain, in particular to a preparation method and application of mesenchymal stem cell paracrine factors for treating various pains.
Background
Pain is an unpleasant sensation and emotional experience caused by tissue damage or potential tissue damage, and is a subjective feeling. Pain is listed as the fifth vital sign together with four vital signs of human respiration, blood pressure, pulse and body temperature. It is known that some methods of physical therapy such as massage, infrared, etc., or anti-inflammatory and analgesic drugs are used to improve and treat pain. The physical therapy method can only relieve symptoms temporarily, and the drug analgesia not only produces dependence, but also has larger side effect, and the methods can not improve pain symptoms for a long time.
Mesenchymal stem cells are a class of adult stem cells that are present in a variety of tissues (e.g., bone marrow, umbilical cord blood and umbilical cord tissue, placental tissue, adipose tissue, etc.), have multipotent differentiation potential, and are not hematopoietic stem cells. The stem cells have the potential of differentiating into various mesenchymal series cells (such as osteogenic cells, chondrogenic cells, adipogenic cells and the like) or non-mesenchymal series cells, and have unique cytokine secretion functions. At present, mesenchymal stem cell transplantation has been successfully applied to the treatment of diseases of various systems including the nervous system, the digestive system, the immune system, and the like.
In recent years, mesenchymal stem cells have been successfully used as biopharmaceuticals for treating knee joint diseases, but no one has proposed a treatment purpose mainly for enhancing and improving knee joint movement or a treatment purpose for regenerating knee joint chondrocytes and treating pain. In addition, the field of the mesenchymal stem cells for treating pain is still blank, and no one finds that the mesenchymal stem cells have the function of resisting pain.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects in the prior art, firstly provide a method for obtaining more mesenchymal stem cell paracrine factors, and secondly provide the application of the mesenchymal stem cell paracrine factors in preparing the medicine for treating pain. The mesenchymal stem cells comprise umbilical cord, umbilical cord blood, fat, bone marrow, amnion and other mesenchymal stem cells obtained by culturing all mammals, particularly human. The mesenchymal stem cells are autologous or allogeneic mesenchymal stem cells.
The mesenchymal stem cell tissue treatment methods of different tissue sources are different, but the methods after primary cell culture are the same.
The invention discloses a preparation method of a mesenchymal stem cell paracrine factor by taking an umbilical cord mesenchymal stem cell acquisition method as an example, which comprises the following steps:
(1) taking the umbilical cord of a healthy fetus in term, detecting the serum HBsAg, anti-HCV, anti-HDV, anti-HEV, anti-HIV-1/2, HCV-RNA, HIV-1-RNA and CMV-DNA of the umbilical cord, wherein the detection results are negative and can be used;
(2) wiping a normal saline bottle once by using gauze sprayed with alcohol, removing sealing plastic at the bottle mouth, and putting the bottle into a clean bench in an operating state;
(3) stripping and washing umbilical cord Wharton's jelly;
preferably, the cord is washed in normal saline to remove blood stain of the cord, then soaked in alcohol for disinfection, washed again by the normal saline, cut the cord into small sections of 2-3cm in a culture dish filled with the normal saline, and washed by the normal saline to remove extravasated blood and clots in the small sections of the cord; then stripping the Wharton's jelly and washing with normal saline;
(4) tissue mass preparation
Shearing umbilical cord Wharton's jelly: shearing the umbilical cord Wharton's jelly washed in the step (3) into tissue homogenate of 1-4 mm; washing the tissue homogenate with physiological saline, and centrifuging;
(5) inoculation of
Removing supernatant from the tissue homogenate obtained by centrifugation in the step (4), inoculating the tissue homogenate into an inoculation culture bottle such as T150, and spreading the tissue homogenate on the bottom surface of the whole inoculation culture bottle;
(6) culture of Wharton's jelly
Flatly placing the inoculation culture bottle in the step (5), placing the inoculation culture bottle in a CO2 culture box, slowly erecting the culture bottle after 6-12 hours, adding a basal medium (high-sugar DMEM medium) containing 10 wt% of serum substitute (preferably, the trade name: Ultroser G serum sublittate; the trade name: 15950-017; purchased from Pall company, USA) into the bottom of the culture bottle, and slowly infiltrating the whole tissue mass;
the culture conditions are as follows: 37.0 plus or minus 0.5 ℃ and the concentration of CO2 is 5.0 plus or minus 0.2 percent;
(7) liquid changing device
Liquid is changed for the first time: culturing umbilical cord Wharton's jelly till 6-7 days, and performing total liquid change; pouring out the old culture medium and the non-adherent tissue blocks, adding an equal amount of culture medium containing 10% serum substitute, horizontally placing a culture bottle, and placing the culture bottle in a CO2 incubator for continuous culture;
and (3) replacing liquid for the second time: repeating the first liquid changing operation after culturing the umbilical cord Wharton's jelly for 9-10 days;
and (3) liquid exchange for the third time: observing the growth condition of the cells under a mirror image, and if the cell fusion degree does not reach 70-80%, adding one full-amount liquid changing operation after the umbilical cord Wharton's jelly is cultured for 12-13 days;
(8) harvesting of Primary cells
And (3) cell observation: observing all culture bottles under a microscope, and if the cell fusion degree is observed to reach 70-80%, carrying out digestion and harvesting;
primary cell harvest
Preparing a cell digestive juice: mixing pancreatin and normal saline according to the volume ratio of 1:4 to obtain a mixed solution, namely the cell digestive juice.
Removing the old culture solution: slightly shaking the culture bottle to enable the non-adherent tissue blocks to fall off, and pouring the tissue blocks and the old culture solution into a waste liquid bottle;
washing: pouring normal saline into each bottle from which the old culture solution is removed, slightly shaking the culture bottle to enable the normal saline to infiltrate the bottom surface and the tissue blocks of the culture bottle, pouring the normal saline again, and repeatedly washing once;
digestion: adding digestive juice into each washed culture bottle to enable the digestive juice of each culture bottle to infiltrate the bottom surface of the whole culture bottle, standing for 1min, shaking the culture bottles up and down to enable the digestive juice to fully contact cells, and observing 90% of cells under a mirror to fall off and round, so that digestion can be stopped;
termination of digestion: adding a mesenchymal stem cell culture medium into each culture bottle capable of terminating digestion, shaking the culture bottles back and forth for full contact dilution, and then transferring the culture bottles into a centrifuge tube such as a 50ml centrifuge tube for centrifugation;
(9) passage of primary cells (P0 pass P1)
Removing the supernatant of the cells after the centrifugation in the previous step, adding a stem cell culture medium containing a serum substitute, uniformly resuspending the cell sediment, merging the cell sediment into a centrifuge tube, and counting by using a cell counting plate;
inoculating the resuspended cell suspension into a culture flask, and metering the volume to a certain volume by using a stem cell culture medium containing a serum substitute so as to enable the cells to reach a certain concentration; shaking up, putting into a CO2 incubator for culture, wherein the culture conditions are as follows: 37 +/-0.5 ℃ and 5.0 +/-0.2 percent of CO2, and culturing until the cell fusion degree reaches 80-90 percent.
A bottle is planted: 50000cells/cm2 were inoculated into T150 flasks, and 20ml of medium was added to each flask.
Sequentially passaging according to the passaging method, and selecting the mesenchymal stem cells of P3-P6 generations as a source of the stem cell paracrine factors for treating pain. The purity of the obtained mesenchymal stem cells is identified by a flow cytometer, and the mesenchymal stem cells accord with the cell phenotype as follows: CD34/CD45/CD11b/CD19/HLADR (-), CD90/CD105/CD73 (+).
Obtaining mesenchymal stem cell paracrine factors:
selecting the mesenchymal stem cells of P3-P6 generation, removing the culture medium in a culture bottle, washing for 2-3 times by using a physiological saline buffer solution, completely absorbing the physiological saline in the culture bottle, adding a high-sugar DMEM culture medium (the same as above) containing 5 wt% of serum substitute, placing the culture bottle containing the cells into a low-oxygen culture box with 2% of oxygen concentration (the preferential culture condition is 37.0 +/-0.5 ℃, and the CO2 concentration is 5.0 +/-0.2%), continuously culturing for 48h, and then collecting the mesenchymal stem cell paracrine factor suspension;
and (3) putting the mesenchymal stem cell paracrine factor suspension into a centrifuge tube, centrifuging to collect supernatant, and then sterilizing and filtering by using an aseptic filter to obtain the mesenchymal stem cell paracrine factor for treating pain.
The method for obtaining the paracrine factor of the mesenchymal stem cells is not extracted from a culture solution of conventional culture, and the method promotes the mesenchymal stem cells to release a large amount of paracrine factors by carrying out hypoxia combined low-nutrition double stimulation on the mesenchymal stem cells and contains hundreds of protein combined components.
The method for obtaining the mesenchymal stem cell paracrine factor adopts a dual treatment mode of hypoxia and low nutrition, wherein the hypoxia is cultured by a hypoxia culture box, the concentration range of oxygen is different from 1-15%, the low nutrition mode is that serum or growth factor combination is added in a proportion of 1-5%, and the research and comparison show that when the concentration of the oxygen is 2% and the nutrient content accounts for 5% of a serum-free culture medium, the paracrine effect of the mesenchymal stem cell can be promoted to the maximum extent, and meanwhile, the activity of the mesenchymal stem cell is ensured. Including human placenta, umbilical cord blood, fat, bone marrow and other mesenchymal stem cells from human body tissues.
The invention has the beneficial effects that:
the invention discovers the new application of the mesenchymal stem cell paracrine factor in the medicine for treating pain for the first time, and is suitable for treating the pain caused by different causes, specifically comprising ① pain caused by all factors such as inflammation, rheumatism, fatigue and the like, pain in neck, shoulder, waist, leg and knee joint, ② neuropathic pain caused by unknown reasons, ③ muscular pain, ④ headache caused by various factors and ⑤ pain accompanied by various tumors.
The application of the pain medicine provided by the invention comprises the following steps: direct pain site injection, intravenous injection, intramuscular injection, intraosseous injection, intra-articular injection, subcutaneous injection or intradermal injection. Wherein the mesenchymal stem cell paracrine factor is administered by direct transplantation. Wherein the mesenchymal stem cell paracrine factor further comprises an injectable carrier. Wherein the injectable carrier is selected from hyaluronic acid, collagen, fibronectin, laminin, or other therapeutically relevant drugs.
Drawings
FIG. 1 is a photograph of umbilical cord mesenchymal stem cells prepared in example 1 of the present invention.
FIG. 2 is a photograph of flow cytometry identification of umbilical cord mesenchymal stem cells prepared in example 1 of the present invention.
Detailed Description
The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments. However, the present invention is not limited to the following examples.
Example 1 preparation of umbilical cord mesenchymal stem cells
The umbilical cord mesenchymal stem cells are mainly derived from human umbilical cord mesenchymal stem cells, but the source of the stem cells is not limited to umbilical cords and can be derived from a plurality of human tissue sources such as human placenta, bone marrow, dental pulp, fat and the like.
The preparation process of the umbilical cord mesenchymal stem cells comprises the following steps: see step (1) -step (9) of the summary of the invention;
fresh umbilical cords are cut, cleaned by sterile normal saline, further visible blood vessels and Fahrenheit glue tissues are removed, the umbilical cords are cut into 3 size of about 1mm, then placed into a cell culture dish, a basal medium (high-sugar DMEM medium) containing 10 wt% of serum substitutes (trade name: Ultroser G serum sublittate; trade name: 15950-. After about 10-14 days, spindle cells can be seen to climb out of the tissue block, namely the umbilical cord mesenchymal stem cells, and after several passages, the spindle cells can be used for preparing cell medicines for treating pain when 3-6 passages are reached. As shown in fig. 1 and 2, fig. 1 and 2 are a culture picture and a flow cytometer identification picture of umbilical cord mesenchymal stem cells, respectively.
(2) The preparation of the umbilical cord mesenchymal stem cell paracrine factor as a pain treatment medicine comprises the following steps: selecting the mesenchymal stem cells of P3-P6 generation, removing the culture medium in a culture bottle, washing for 2-3 times by using a physiological saline buffer solution, completely absorbing the physiological saline in the culture bottle, adding a high-sugar DMEM culture medium (the same as above) containing 5 wt% of serum substitute, placing the culture bottle containing the cells into a low-oxygen culture box with 2% of oxygen concentration (the preferential culture condition is 37.0 +/-0.5 ℃, and the CO2 concentration is 5.0 +/-0.2%), continuously culturing for 48h, and then collecting the mesenchymal stem cell paracrine factor suspension; and (3) putting the mesenchymal stem cell paracrine factor suspension into a centrifuge tube, centrifuging for 5 minutes at 1000rpm, collecting supernatant, and sterilizing and filtering by using a 0.22 sterile filter to obtain the mesenchymal stem cell paracrine factor for treating pain.
The injection amount of the cells is selected according to the pain part and the pain degree, wherein the injection carrier can be selected from hyaluronic acid, collagen, fibronectin, laminin or other related medicaments for treatment. The means by which the cells are used for therapy are: direct pain site injection, intravenous injection, intramuscular injection, intraosseous injection, intra-articular injection, subcutaneous injection, intradermal injection.
Example 2 evaluation experiment of mesenchymal Stem cell paracrine factor on stiff neck pain
Stiff neck is a common disease, which is better in young and strong years and is common in winter and spring. The common onset of stiff neck is no symptoms before sleep, and the neck and back feel sore obviously and the neck movement is limited after morning. Generally, the patients who suffer from stiff neck need to recover from the stiff neck for 3 to 7 days. Selecting 10 patients with stiff neck, performing local multi-point subcutaneous injection of the mesenchymal stem cell paracrine factor on the second day after stiff neck generation, and after pain evaluation, filling the patients with pain evaluation scales on the 2 nd, 4 th and 7 th days after injection by locally injecting 2ml of human umbilical cord mesenchymal stem cell paracrine factor in total. Pain improvement was reduced in 10 patients on day 2 post injection, with 7 patients having total pain loss. In 3 additional patients, the pain score decreased from 5. + -.2 to 1. + -.1, with all patients having no pain on day 4 post-injection and no pain on day 7 when all patients were evaluated for pain. The paracrine stem cell factor relieves the pain symptoms of the patient.
Example 3 evaluation experiment of mesenchymal Stem cells for cervical spondylosis pain
Chronic pain refers to pain lasting for more than one month (three months or half a year before), and as society progresses and computer networks develop, chronic pain caused by cervical spondylosis is increasing. 8 patients with chronic pain from cervical spondylosis were selected and the pain of the individuals was evaluated using the numerical pain assessment scale. After the evaluation of pain, a total amount of 2ml of human umbilical cord mesenchymal stem cell paracrine factor was injected by local multi-point injection. Patients were filled out pain assessment scales on days 3, 7, 14 and 30 post injection, respectively. The pain improvement was reduced in 8 patients on the second 2 days post injection, with a pain score decreasing from 7 ± 3 to 6 ± 2 points, a further pain score decreasing to 4 ± 2 points after 7 days, and a further pain score decreasing to 2 ± 1 points after 14 days, with pain disappearing for 4 patients on day 14 and pain disappearing for the remaining 4 patients on a 1 month score. The paracrine factor of the stem cells relieves the pain symptoms of patients and does not cure cervical spondylosis.
Example 4 mesenchymal stem cell paracrine factor treats pain caused by the formation of gonitis.
Gonarthritis is a disease based on degenerative pathological changes, is mostly suffered from middle-aged and elderly people, and has symptoms of red and swollen knee pain, pain of going up and down stairs, soreness and discomfort of the knee when sitting up and standing, and the like, which seriously affects the life quality of patients. 10 cases of knee joint pain patients are selected, the average pain cycle is 12 +/-2 months, 2ml of stem cell paracrine factor is injected into the knee joint cavity, 5 cases of patients have obvious improvement of pain symptoms after 10 days, the pain score is 4 +/-2 points and is reduced to 2 +/-1 points, and after 20 days, the pain of the 5 cases of patients is improved. In addition, the pain degree of 5 patients is severe, walking is seriously influenced, the pain score before treatment is 6 +/-3 points, the pain improvement is relieved by 5 +/-2 points after the first treatment, 2ml of mesenchymal stem cell paracrine factor is repeatedly injected into the patients after 2 weeks, the pain score of the patients after 10 days is relieved to 3 +/-1 points, the pain of 3 patients after 20 days is disappeared, the pain of 2 patients after the operation is relieved, 2ml of mesenchymal stem cell paracrine factor is injected into the patients for the third time, and the pain of 2 patients disappears after 10 days. It can be seen that the mesenchymal stem cell paracrine factor is directly related to the severity of the gonarthritis, and the severe disease condition needs to be improved after multiple injections.
The application of the mesenchymal stem cell paracrine factor in the pain field is only a preferred embodiment of the invention, and therefore, the scope of the invention should not be limited by the application, and the equivalent changes and modifications made according to the patent scope and the content of the specification should be covered by the invention.
Claims (10)
1. Application of mesenchymal stem cell paracrine factor in preparing medicine for treating pain is provided.
2. The use according to claim 1, wherein the mesenchymal stem cells are derived from any tissue. The source of the mesenchymal stem cells comprises umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, adipose mesenchymal stem cells, placenta mesenchymal stem cells, amniotic mesenchymal stem cells and the like, wherein the mesenchymal stem cells are derived from all tissues of mammals, particularly human beings.
3. The use according to claim 1, wherein said pain medication is suitable for the treatment of pain of various etiologies, including ① pain of neck, shoulder, waist, leg and knee joints due to inflammation, rheumatism, fatigue, etc., ② neuropathic pain of unknown origin, ③ muscular pain, ④ headache due to various factors, ⑤ pain associated with various tumors.
4. The use according to claim 1, wherein the pain medication is administered by: direct pain site injection, intravenous injection, intramuscular injection, intraosseous injection, intra-articular injection, subcutaneous injection or intradermal injection.
5. Use according to claim 4, wherein the carrier is selected from hyaluronic acid, collagen, fibronectin, laminin, or other therapeutically relevant drugs.
6. The application of claim 1, wherein the preparation method of the mesenchymal stem cell paracrine factor is illustrated by taking a method for obtaining umbilical cord mesenchymal stem cells as an example, and comprises the following steps:
(1) taking the umbilical cord of a healthy fetus in term, detecting the serum HBsAg, anti-HCV, anti-HDV, anti-HEV, anti-HIV-1/2, HCV-RNA, HIV-1-RNA and CMV-DNA of the umbilical cord, wherein the detection results are negative and can be used;
(2) wiping a normal saline bottle once by using gauze sprayed with alcohol, removing sealing plastic at the bottle mouth, and putting the bottle into a clean bench in an operating state;
(3) stripping and washing umbilical cord Wharton's jelly;
preferably, the cord is washed in normal saline to remove blood stain of the cord, then soaked in alcohol for disinfection, washed again by the normal saline, cut the cord into small sections of 2-3cm in a culture dish filled with the normal saline, and washed by the normal saline to remove extravasated blood and clots in the small sections of the cord; then stripping the Wharton's jelly and washing with normal saline;
(4) tissue mass preparation
Shearing umbilical cord Wharton's jelly: shearing the umbilical cord Wharton's jelly washed in the step (3) into tissue homogenate of 1-4 mm; washing the tissue homogenate with physiological saline, and centrifuging;
(5) inoculation of
Removing supernatant from the tissue homogenate obtained by centrifugation in the step (4), inoculating the tissue homogenate into an inoculation culture bottle such as T150, and spreading the tissue homogenate on the bottom surface of the whole inoculation culture bottle;
(6) culture of Wharton's jelly
Horizontally placing the inoculation culture bottle in the step (5), placing the inoculation culture bottle in a CO2 culture box, slowly erecting the culture bottle after 6-12 hours, adding a basic culture medium containing 10 wt% of serum substitute, namely a high-glucose DMEM culture medium, into the bottom of the culture bottle, and slowly infiltrating the whole tissue block;
the culture conditions are as follows: 37.0 plus or minus 0.5 ℃ and the concentration of CO2 is 5.0 plus or minus 0.2 percent;
(7) liquid changing device
Liquid is changed for the first time: culturing umbilical cord Wharton's jelly till 6-7 days, and performing total liquid change; pouring out the old culture medium and the non-adherent tissue blocks, adding an equal amount of culture medium containing 10% serum substitute, horizontally placing a culture bottle, and placing the culture bottle in a CO2 incubator for continuous culture;
and (3) replacing liquid for the second time: repeating the first liquid changing operation after culturing the umbilical cord Wharton's jelly for 9-10 days;
and (3) liquid exchange for the third time: observing the growth condition of the cells under a mirror image, and if the cell fusion degree does not reach 70-80%, adding one full-amount liquid changing operation after the umbilical cord Wharton's jelly is cultured for 12-13 days;
(8) harvesting of Primary cells
And (3) cell observation: observing all culture bottles under a microscope, and if the cell fusion degree is observed to reach 70-80%, carrying out digestion and harvesting;
primary cell harvest
Preparing a cell digestive juice: mixing pancreatin and normal saline according to the volume ratio of 1:4 to obtain a mixed solution, namely the cell digestive juice.
Removing the old culture solution: slightly shaking the culture bottle to enable the non-adherent tissue blocks to fall off, and pouring the tissue blocks and the old culture solution into a waste liquid bottle;
washing: pouring normal saline into each bottle from which the old culture solution is removed, slightly shaking the culture bottle to enable the normal saline to infiltrate the bottom surface and the tissue blocks of the culture bottle, pouring the normal saline again, and repeatedly washing once;
digestion: adding digestive juice into each washed culture bottle to enable the digestive juice of each culture bottle to infiltrate the bottom surface of the whole culture bottle, standing for 1min, shaking the culture bottles up and down to enable the digestive juice to fully contact cells, and observing 90% of cells under a mirror to fall off and round, so that digestion can be stopped;
termination of digestion: adding a mesenchymal stem cell culture medium into each culture bottle capable of terminating digestion, shaking the culture bottles back and forth for full contact dilution, and then transferring the culture bottles into a centrifuge tube such as a 50ml centrifuge tube for centrifugation;
(9) passage of primary cells (P0 pass P1)
Removing the supernatant of the cells after the centrifugation in the previous step, adding a stem cell culture medium containing a serum substitute, uniformly resuspending the cell sediment, merging the cell sediment into a centrifuge tube, and counting by using a cell counting plate;
inoculating the resuspended cell suspension into a culture flask, and metering the volume to a certain volume by using a stem cell culture medium containing a serum substitute so as to enable the cells to reach a certain concentration; shaking up, putting into a CO2 incubator for culture, wherein the culture conditions are as follows: 37 +/-0.5 ℃ and 5.0 +/-0.2 percent of CO2, and culturing until the cell fusion degree reaches 80-90 percent.
A bottle is planted: 50000cells/cm2 is inoculated in a T150 culture flask, and 20ml of culture medium is added into each flask;
sequentially passaging according to the passaging method, and selecting mesenchymal stem cells of P3-P6 generations;
obtaining mesenchymal stem cell paracrine factors:
selecting mesenchymal stem cells of P3-P6 generation, removing culture medium in a culture bottle, washing for 2-3 times by using a physiological saline buffer solution, completely sucking the physiological saline in the culture bottle, adding a high-sugar DMEM culture medium containing 5 wt% of serum substitute, and placing the culture bottle containing the cells into a 2% oxygen concentration hypoxia incubator under the culture conditions: culturing at 37.0 + -0.5 deg.C and CO2 concentration of 5.0 + -0.2% for 48 hr, and collecting the suspension of paracrine factor of mesenchymal stem cell;
and (3) putting the mesenchymal stem cell paracrine factor suspension into a centrifuge tube, centrifuging to collect supernatant, and then sterilizing and filtering by using an aseptic filter to obtain the mesenchymal stem cell paracrine factor for treating pain.
7. The use of claim 6, wherein the mesenchymal stem cells of P3-P6 generation are selected, and the obtained mesenchymal stem cells are identified with purity by a flow cytometer, and the phenotype of the mesenchymal stem cells is as follows: CD34/CD45/CD11b/CD19/HLADR (-), CD90/CD105/CD73 (+).
8. A pain medication comprising mesenchymal stem cell paracrine factors; wherein the mesenchymal stem cells comprise umbilical cord, umbilical cord blood, fat, bone marrow, amnion and other mesenchymal stem cells obtained by culturing all mammals, particularly human; the mesenchymal stem cells are autologous or allogeneic mesenchymal stem cells.
9. The pain medication of claim 8 wherein the mesenchymal stem cell paracrine factor is used as an analgesic component.
10. A preparation method of pain medicine, taking an obtaining method of umbilical cord mesenchymal stem cells as an example, explains a preparation method of mesenchymal stem cell paracrine factors, and comprises the following steps:
(1) taking the umbilical cord of a healthy fetus in term, detecting the serum HBsAg, anti-HCV, anti-HDV, anti-HEV, anti-HIV-1/2, HCV-RNA, HIV-1-RNA and CMV-DNA of the umbilical cord, wherein the detection results are negative and can be used;
(2) wiping a normal saline bottle once by using gauze sprayed with alcohol, removing sealing plastic at the bottle mouth, and putting the bottle into a clean bench in an operating state;
(3) stripping and washing umbilical cord Wharton's jelly;
preferably, the cord is washed in normal saline to remove blood stain of the cord, then soaked in alcohol for disinfection, washed again by the normal saline, cut the cord into small sections of 2-3cm in a culture dish filled with the normal saline, and washed by the normal saline to remove extravasated blood and clots in the small sections of the cord; then stripping the Wharton's jelly and washing with normal saline;
(4) tissue mass preparation
Shearing umbilical cord Wharton's jelly: shearing the umbilical cord Wharton's jelly washed in the step (3) into tissue homogenate of 1-4 mm; washing the tissue homogenate with physiological saline, and centrifuging;
(5) inoculation of
Removing supernatant from the tissue homogenate obtained by centrifugation in the step (4), inoculating the tissue homogenate into an inoculation culture bottle such as T150, and spreading the tissue homogenate on the bottom surface of the whole inoculation culture bottle;
(6) culture of Wharton's jelly
Horizontally placing the inoculation culture bottle in the step (5), placing the inoculation culture bottle in a CO2 culture box, slowly erecting the culture bottle after 6-12 hours, adding a basic culture medium containing 10 wt% of serum substitute, namely a high-glucose DMEM culture medium, into the bottom of the culture bottle, and slowly infiltrating the whole tissue block;
the culture conditions are as follows: 37.0 plus or minus 0.5 ℃ and the concentration of CO2 is 5.0 plus or minus 0.2 percent;
(7) liquid changing device
Liquid is changed for the first time: culturing umbilical cord Wharton's jelly till 6-7 days, and performing total liquid change; pouring out the old culture medium and the non-adherent tissue blocks, adding an equal amount of culture medium containing 10% serum substitute, horizontally placing a culture bottle, and placing the culture bottle in a CO2 incubator for continuous culture;
and (3) replacing liquid for the second time: repeating the first liquid changing operation after culturing the umbilical cord Wharton's jelly for 9-10 days;
and (3) liquid exchange for the third time: observing the growth condition of the cells under a mirror image, and if the cell fusion degree does not reach 70-80%, adding one full-amount liquid changing operation after the umbilical cord Wharton's jelly is cultured for 12-13 days;
(8) harvesting of Primary cells
And (3) cell observation: observing all culture bottles under a microscope, and if the cell fusion degree is observed to reach 70-80%, carrying out digestion and harvesting;
primary cell harvest
Preparing a cell digestive juice: mixing pancreatin and normal saline according to the volume ratio of 1:4 to obtain a mixed solution, namely the cell digestive juice.
Removing the old culture solution: slightly shaking the culture bottle to enable the non-adherent tissue blocks to fall off, and pouring the tissue blocks and the old culture solution into a waste liquid bottle;
washing: pouring normal saline into each bottle from which the old culture solution is removed, slightly shaking the culture bottle to enable the normal saline to infiltrate the bottom surface and the tissue blocks of the culture bottle, pouring the normal saline again, and repeatedly washing once;
digestion: adding digestive juice into each washed culture bottle to enable the digestive juice of each culture bottle to infiltrate the bottom surface of the whole culture bottle, standing for 1min, shaking the culture bottles up and down to enable the digestive juice to fully contact cells, and observing 90% of cells under a mirror to fall off and round, so that digestion can be stopped;
termination of digestion: adding a mesenchymal stem cell culture medium into each culture bottle capable of terminating digestion, shaking the culture bottles back and forth for full contact dilution, and then transferring the culture bottles into a centrifuge tube such as a 50ml centrifuge tube for centrifugation;
(9) passage of primary cells (P0 pass P1)
Removing the supernatant of the cells after the centrifugation in the previous step, adding a stem cell culture medium containing a serum substitute, uniformly resuspending the cell sediment, merging the cell sediment into a centrifuge tube, and counting by using a cell counting plate;
inoculating the resuspended cell suspension into a culture flask, and metering the volume to a certain volume by using a stem cell culture medium containing a serum substitute so as to enable the cells to reach a certain concentration; shaking up, putting into a CO2 incubator for culture, wherein the culture conditions are as follows: 37 +/-0.5 ℃ and 5.0 +/-0.2 percent of CO2, and culturing until the cell fusion degree reaches 80-90 percent.
A bottle is planted: 50000cells/cm2 is inoculated in a T150 culture flask, and 20ml of culture medium is added into each flask;
sequentially passaging according to the passaging method, and selecting mesenchymal stem cells of P3-P6 generations;
obtaining mesenchymal stem cell paracrine factors:
selecting the mesenchymal stem cells of the P3-P6 generation, identifying the purity of the obtained mesenchymal stem cells by a flow cytometer, and conforming to the cell phenotype as follows: CD34/CD45/CD11b/CD19/HLADR (-), CD90/CD105/CD73 (+);
selecting mesenchymal stem cells of P3-P6 generation, removing culture medium in a culture bottle, washing for 2-3 times by using a physiological saline buffer solution, completely sucking the physiological saline in the culture bottle, adding a high-sugar DMEM culture medium containing 5 wt% of serum substitute, and placing the culture bottle containing the cells into a 2% oxygen concentration hypoxia incubator under the culture conditions: culturing at 37.0 + -0.5 deg.C and CO2 concentration of 5.0 + -0.2% for 48 hr, and collecting the suspension of paracrine factor of mesenchymal stem cell;
and (3) putting the mesenchymal stem cell paracrine factor suspension into a centrifuge tube, centrifuging to collect supernatant, and then sterilizing and filtering by using an aseptic filter to obtain the mesenchymal stem cell paracrine factor for treating pain.
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