CN110772535A - Application of multisystem differentiation sustained stress cells in preparation of analgesic drugs - Google Patents
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
Abstract
The invention discloses an application of multisystem differentiation sustained stress cells in preparing an analgesic drug. The multi-lineage differentiation sustained stress cells can be obtained from various mesenchymal tissues such as bone marrow, fat, umbilical cord, dermis and the like. The research of the invention shows that the multi-differentiated sustained stress cells can effectively treat various pains, such as various diseases and symptoms including neuropathic pain, inflammatory pain, arthralgia, cancer pain, chemotherapy pain, diabetic hyperalgesia, hyperalgesia and the like, have long action time and have great clinical application value.
Description
Technical Field
The invention belongs to the field of stem cell biology and biomedicine, and particularly relates to application of multisystem differentiation sustained stress cells in preparation of analgesic drugs
Background
Pain is an unpleasant sensation and emotional feeling with substantial or potential tissue damage, and often the patient has negative emotional reactions such as depression and anxiety, which seriously affect the daily life of the patient and also bring heavy burden to family and society. 30% of the adult population in the world suffers from chronic pain, and according to the statistics in the united states, the number of patients with chronic pain has exceeded the sum of patients with diabetes, cardiovascular disease and cancer, and the american society for pain 2014 counts the economic losses due to chronic pain in the united states as high as $ 6000 billion in the year 5000-. Conservative estimates, there are more than two hundred million pain patients in our country. The current research on chronic pain treatment drugs has mainly focused on the improvement of traditional analgesics such as opioid receptor drugs and the development of sodium channel blockers, bradykinin blockers, 5-HT blockers, growth factor inhibitors, glutamate inhibitors, adenosine inhibitors, and the like. However, these drugs have short action time, and can cause serious side effects such as tolerance, addiction, interference with normal physiological functions of the nervous system and other systems, and the like after long-term use, and even affect life safety. At present, no medicine can treat chronic pain thoroughly, long-acting and without side effect. More effective treatments are urgently needed by patients. In order to alleviate and treat the medical problem of pain patients and the social problems caused by the medical problem, the 'pain' research is taken as one of the key support fields of the national natural science development planning in China. Therefore, the search and development of new analgesic drugs have important medical significance and profound social significance.
Disclosure of Invention
The multisystem differentiation continuous stress cell is an adult stem cell newly found in adult bone marrow and dermis tissues in 2010, and has the following characteristics: 1) stage-specific embryonic antigen 3(stage specific monoclonal antibodies-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. The research of the invention finds that the multi-series differentiation continuously stressed cells can effectively treat various acute and chronic pains. The injection of the multi-differentiated sustained stress cells can obviously treat the allodynia, has long action time, has obvious treatment effect on various diseases and symptoms including neuropathic pain, inflammatory pain, cancer pain, chemotherapy pain, diabetic hyperalgesia, hyperalgesia and the like, and has great clinical application value.
The invention aims to provide application of multisystem differentiation sustained stress cells in preparation of an analgesic drug, and provides a new choice for clinical analgesic drugs.
In order to solve the technical problems, the specific technical scheme of the invention is as follows:
the invention provides an application of multisystem differentiation sustained stress cells in preparing an analgesic drug. Preferably, the analgesic drug is a drug for treating chronic pain.
Further, the pain of the invention is selected from one or more of neuropathic pain, inflammatory pain, cancer pain, chemotherapy pain, diabetic hyperalgesia, hyperalgesia and the like.
Furthermore, the multi-differentiation continuous stress cells are derived from mesenchymal tissues such as bone marrow, fat, umbilical cord, dermis and the like, can grow in a suspended ball or attached wall mode, express SSEA-3, Oct3/4, Sox2, CD105 and CD90, and do not express CD34, CD45 and CD11 b. A specific embodiment of the invention uses multi-lineage differentiated continuously stressed cells derived from bone marrow.
The multi-lineage differentiation sustained-stress cell of the present invention can be prepared by a method described in the literature (Kuroda, et al., Uniquemultipotent cells in adult human mesenchymel cells. PNAS,2010,107: 8639-.
The invention relates to a specific preparation method of multisystem differentiation sustained stress cells, which comprises the following steps:
the reference (Kuroda, et al, PNAS,2010,107:8639-8643.) discloses a method for preparing bone marrow cells, and expressing SSEA-3 by using multi-lineage differentiation continuous stress cells
+/CD105
+/CD45
-Is characterized in thatThe method comprises the steps of sorting bone marrow cells by adopting a flow cell sorting technology, carrying out suspension culture on the sorted multi-differentiation continuously stressed cells, using a-MEM + 0.9% MethoCult H4100+ 20% FBS culture medium, starting cell aggregation after 1d of culture, aggregating single cells to form cell colonies, then dividing and growing the cell colonies again to form stem cell balls, and gradually increasing the number of cells in 3-5d to gradually increase the cell balls 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 1) the cell ball can still form suspension growth; 2) has the same analgesic effect with MUSE cells within passage 5; 3) protein mass spectrometry shows that compared with MUSE cells of passage 5, the protein expression has no statistical difference in various aspects such as cytokines, chemokines, receptors and the like. The stable passage characteristic of the multisystem differentiation continuous stress cells is extremely beneficial to preparing analgesic drugs by providing a large number of standardized cell products.
Further, the multi-lineage differentiation stress cells of the present invention are administered in the form of a cell suspension prepared as follows: digesting the cultured multisystem differentiation continuous stress cells with 0.25% trypsin-EDTA to prepare single cell suspension, washing with normal saline, and suspending with normal saline to obtain multisystem differentiation continuous stress cell suspension.
Furthermore, the cell concentration of the multisystem differentiation continuous stress cell suspension is 1-10 multiplied by 10
7One per ml.
Furthermore, the dosage of the drug of the multisystem differentiation continuous stress cell suspension is 5-10 multiplied by 10 counted by the number of multisystem differentiation continuous stress cells
6Per kg body weight, local injection (intrathecal injection, intra-articular injection, subcutaneous injection, intramuscular injection, etc.) 1Secondly; or 5 to 10 x 10
7One/kg body weight, 1 intravenous infusion.
The medicine of the invention can further contain pharmaceutically acceptable auxiliary materials.
The invention utilizes various acute and chronic pain mouse models, and firstly proves that the transplantation of multi-differentiation continuous stress cells in a mode of intrathecal injection can obviously inhibit mechanical pain and thermal pain allergy of various pain model mice for a long time and can correct the pain sense disturbance of diabetes model mice.
Drawings
FIG. 1 shows the results of the preparation and identification of human multisystem differentiated continuously stressed cells (MUSE cells). (wherein, panel A is the result of collecting human bone marrow cells cultured to the 3 rd generation and sorting by flow cell sorting technique using the characteristics of multiscale differentiation continuous stress cells SSEA-3+/CD105 +. Panel B is the cell ball formed by single cells sorted from sorted multiscale differentiation continuous stress cells under suspension culture; Panel C is the cell morphology when adherent culture is performed after the suspension cell ball of multiscale differentiation continuous stress cells is digested into single cells).
FIG. 2 shows the analgesic effect of human Muse cells on mechanical pain and thermal pain allergy of mice as model of chronic compressive injury of the sciatic nerve. (wherein, the graph A is the mechanical foot-shrinking reflex threshold of the sciatic nerve chronic compressive injury model mouse injected with human Muse cells intrathecally, and the graph B is the heat-shrinking foot-reflecting latency of the sciatic nerve chronic compressive injury model mouse injected with human Muse cells intrathecally).
FIG. 3 is a graph of the analgesic effect of human Muse cells on diabetic model mice. (wherein, the graph A is the mechanical foot-shrinking reflex threshold value of the diabetes model mouse injected with the human Muse cells intrathecally, and the graph B is the heat-shrinking foot-reflecting latency period of the diabetes model mouse injected with the human Muse cells intrathecally).
FIG. 4 is a graph showing the analgesic effect of human Muse cells on mechanical pain hypersensitivity of mice injected with Paclitaxel (PTX), a chemotherapeutic drug.
FIG. 5 shows the analgesic effect of Muse cells (SSEA-3positive) and non-Muse cells on mechanical pain in mice as model of chronic compressive injury of the sciatic nerve.
Detailed Description
The following examples illustrate specific steps of the present invention, but are not intended to limit the invention.
Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.
The invention is described in further detail below with reference to specific examples and data, it being understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.
Example 1: preparation and identification of multisystem differentiation sustained stress 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 5mm, 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% CO
2. After 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. After 3 rd generation, the cells were essentially single in morphology and spindle-shaped, and about 95% of the cells were CD90 detected by flow cytometry
+/CD45
-/CD11b
-. CollectingCells cultured to the 3 rd generation using Muse cells SSEA-3
+/CD105
+The characteristics of the method are that the flow cytometry sorting technology is adopted for sorting, the sorted Muse cells are subjected to suspension culture, a-MEM + 0.9% MethoCultH4100+ 20% FBS culture medium is used, the cells begin to aggregate after 1d, the single cells aggregate to form cell colonies, then the cell colonies divide and grow again to form stem cell balls, the number of the cells is gradually increased after 3-5d, and the cell balls gradually increase and present a large and transparent 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. Muse cells were identified using the pluripotent stem cell markers SSEA-3, Nanog, Oct4 and Sox 2. The results are shown in FIG. 1, in which Panel A shows the collection of human bone marrow cells cultured to passage 3 using multi-lineage differentiation continuous stress cells SSEA-3
+/CD105
+The characteristics of (a) are obtained by sorting the mixture by adopting a flow cytometry sorting technology. This result indicates that approximately 0.89% of the cells in cultured bone marrow cells are multisystem differentiation-sustaining stress cells. Panel B is a diagram showing that sorted multilineage differentiation sustainably stressed cells can form a cell pellet from sorted single cells in suspension culture. This result indicates that cells with continued stress in multisystem differentiation can be expanded in vitro in large numbers. FIG. C shows the cell morphology after digesting suspension cell beads of multi-lineage differentiation-persistently stressed cells into single cells and performing adherent culture. 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 detection of analgesic Activity of multisystem differentiated continuously stressed cells
1) Mouse pain model:
① model of chronic compressive injury of sciatic nerve (CCI) mice were anesthetized continuously with isoflurane gas on an operating platform, shaved and disinfected, then the skin was incised longitudinally over the lateral aspect of the femur, muscles were bluntly separated along the muscle striations, sciatic nerve was exposed, peripheral tissues were dissociated, and the skin was sutured after closing the incision at the proximal end of the trigeminal branch of sciatic nerve approximately 5mm with three ligatures of 6.0 surgical suture at a distance of approximately 1mm to avoid affecting the blood transport of the adventitia.
② model of selective nerve injury in sciatic nerve Branch (SNI) mice were anesthetized continuously with isoflurane gas on a surgical platform, shaved and disinfected, then the skin was incised longitudinally over the lateral aspect of the femur, muscles were bluntly separated along the muscle striations, sciatic nerve was exposed, peripheral tissues were freed, the common peroneal nerve and the tibial nerve were ligated and severed at the trigeminal branch of the sciatic nerve, the sural nerve was retained, and the skin was sutured after closing the incision.
③ Neuromycin (STZ) injection induced diabetic neuropathic pain model, i.e. injecting STZ 60mg/kg intraperitoneally, continuously administering for 5 days, 20 days after 5 times of administration, detecting blood sugar value, and using mice with blood sugar value more than 25mM/L for experiment.
④ model of neuropathic pain caused by Paclitaxel (PTX) injection, PTX 2mg/kg is intraperitoneally injected, and the administration is carried out every other day for 4 times.
2) Mouse pain-sensation behavior detection: all behavioral experiments were performed in a "double-blind" experimental design.
① the 50% foot contraction threshold value is calculated by up-down method using von Frey fiber filament in mechanical algesia experiment, an organic transparent glass box is placed on a metal screen, after the mouse adapts in the organic glass box for 30min, the von Frey fiber filament is used to stimulate the middle part of the hind limb sole of the mouse vertically, the duration is 1-2s, the foot raising or licking behavior of the mouse is regarded as positive reaction, otherwise, the negative reaction is regarded as negative reaction.
② thermal hyperalgesia experiment, the plexiglass box was placed on a 3mm thick glass plate, and the foot of the mouse was irradiated to the portion of the glass plate closely contacting the foot by thermal radiation stimulation according to Hargreaves' method, and the time from the start of irradiation to the occurrence of lifting of the foot of the mouse was recorded, with the cut-off time being 20 s.
The results of the analgesic activity assay of multisystem differentiated continuously stressed cells on the mouse pain model are shown in FIGS. 2 to 5. FIG. 2 shows the analgesic effect of human Muse cells on mechanical pain and thermal pain allergy of mice as model of chronic compressive injury of the sciatic nerve. (wherein, the picture A is the mechanical foot-contracting reflex threshold value of the sciatic nerve chronic compressive injury model mouse injected with the human Muse cells in the sheath, and the picture B is the thermal foot-contracting reflex latency period of the sciatic nerve chronic compressive injury model mouse injected with the human Muse cells in the sheath.
FIG. 3 is a graph of the analgesic effect of human Muse cells on diabetic model mice. (wherein, the graph A is the mechanical foot-shrinking reflex threshold value of the mice of the diabetes model injected with the human Muse cells intrathecally, and the result shows that the intrathecally injected human Muse cells can obviously inhibit the phenomena of early mechanical pain hypersensitivity and late mechanical pain insensitivity (dysalgesia) of the mice of the diabetes model.
FIG. 4 is a graph of the analgesic effect (mechanical paw withdrawal reflex threshold) of human Muse cells on mechanical pain hypersensitivity of chemotherapy drug Paclitaxel (PTX) injected model mice. The results show that single intrathecal injection of human Muse cells can inhibit the mechanical pain allergy of model mice injected with chemotherapeutic drug Paclitaxel (PTX) for a long time.
FIG. 5 shows the analgesic effect of Muse cells (SSEA-3positive) and non-Muse cells on mechanical pain (mechanical paw withdrawal reflex threshold) in mice as model of chronic compressive injury to the sciatic nerve. The result shows that the Muse cells (SSEA-3positive) and the non-Muse cells (SSEA-3 negative, from the SSEA-3 negative cells in the bone marrow cell culture sorted by the flow cytometer) with the same number are injected into a single intrathecal injection to influence the mechanical pain of the mouse of the model of the chronic compressive injury of the sciatic nerve, and the Muse cells have better analgesic effect.
In conclusion, the implantation of the multi-differentiated continuously stressed cells by the intrathecal injection mode can remarkably inhibit the mechanical pain and the thermal pain hypersensitivity of various pain model mice for a long time and can correct the pain sense disturbance of the diabetes model mice.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (8)
1. Application of multisystem differentiation sustained stress cells in preparing analgesic drugs.
2. Use according to claim 1, characterized in that the analgesic drug is a drug for the treatment of chronic pain.
3. Use according to claim 2, wherein the pain is selected from the group consisting of neuropathic pain, inflammatory pain, joint pain, cancer pain, chemotherapy pain, diabetic hyperalgesia and hyperalgesia.
4. The use according to claim 1, wherein the multi-lineage differentiated continuously stressed cells are derived from mesenchymal tissues such as bone marrow, adipose, umbilical cord, dermis, etc.
5. An analgesic medicament, characterised by comprising a cell suspension of a plurality of differentiated continuously stressed cells.
6. The pharmaceutical composition according to claim 5, wherein the cell concentration of said multi-lineage differentiated continuously stressed cells is 1 to 10X 10
7One per ml.
7. The medicament of claim 5, further comprising a pharmaceutically acceptable excipient.
8. The medicament of claim 5, wherein said medicament is administered by subcutaneous injection, intramuscular injection, intraperitoneal injection, intra-articular injection, intravenous injection, intrathecal injection, or the like.
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