CN115737699B - Stem cell derivative preparation and preparation method and application thereof - Google Patents
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Abstract
The invention relates to the technical field of animal feeding, and discloses a stem cell derivative preparation, a preparation method and application thereof. The stem cell derived preparation contains an extract of a plant of the genus Alhagi and an extracellular product of mesenchymal stem cells. The invention prolongs the preservation time of the preparation, has no toxic or side effect and provides the function of gastrointestinal protection; is beneficial to supplementing special nutrient substances required by the human body, relaxing bowel, intestinal spasm, pain relieving, halitosis, insufficient nutrition and balance of intestinal flora; can be used for comprehensively preventing and treating digestive system disorder diseases caused by common metabolic disorder diarrhea, constipation, dyspepsia, pestilence, parvovirus, infectious peritonitis and other infectious diseases.
Description
Technical Field
The invention relates to the technical field of animal feeding, in particular to a stem cell derivative preparation and a preparation method and application thereof.
Background
With the continuous improvement of the living standard of people, the number of people raising pets is gradually increased, and people pay more attention to the pets. In the process of raising the pet, if the pet is careless, the pet is easy to get ill. The rate of diarrhea, enteritis, intestinal cramp and other related diseases among the diseases of pets is the largest among all diseases. Canine enteritis is an acute or chronic inflammation of the mucous membrane and deep tissues of the small intestine, and is mainly found in secondary infections of some infectious diseases, such as canine parvovirus disease, canine distemper and the like, and is characterized by digestive disorders, vomiting, abdominal pain, diarrhea, elevated body temperature and autologous toxic signs. Mainly endangering young dogs and old dogs, has high morbidity and relatively low mortality, seriously affects the growth and development of dogs, further reduces the utilization rate of feed, causes economic loss of dogs breeders, and is frequent in spring and autumn.
Mesenchymal stem cells (Mesenchymal stem cells, MSC) are important members of the stem cell family, and mesenchymal stem cells can be defined as adult stem cells with multi-differentiation potential, play an important role in the treatment of chronic inflammatory fibrosis and fibrotic diseases, have a strong paracrine effect, and have the functions of inhibiting tissue fibrosis, promoting angiogenesis, participating in tissue repair regeneration and the like. However, mesenchymal stem cells have immunological rejection and side effects at the time of transplantation therapy, and the effect of mesenchymal stem cell secretion on intestinal fibrosis is still lack of study.
The alhagi (Alhagi pseudalhagi) is a perennial root-planting half shrub plant of the subfamily Papili (Papili onaae) of the family Leguminosae, sugar particles formed by coagulating the secretion of branches and leaves of alhagi is spiny, light yellow-white to brown-yellow in appearance, elliptic or spherical small particles, has the diameter of about 1-5 mm, is sticky and sweet, has round branches and leaves, is used for important national medicines of Uygur, has slow effect and long treatment course although being used independently for treating digestive system diseases, and has certain defects in effect.
Disclosure of Invention
The invention aims to solve the problems of insufficient treatment effect and slow effect on digestive system diseases when camel thorn plants and mesenchymal stem cells are used independently in the prior art, and provides a stem cell derivative preparation and a preparation method and application thereof.
In order to achieve the above object, the present invention provides in a first aspect a stem cell derived preparation comprising an extract of a plant of the genus alhagi and an extracellular product of mesenchymal stem cells.
In a second aspect, the invention provides a method of preparing a stem cell derived formulation, the method comprising: mixing the extract of Alhagi plant and the extracellular product of mesenchymal stem cells.
In a third aspect, the present invention provides the use of a stem cell derived formulation as described above and/or a stem cell derived formulation prepared by a method as described above in the manufacture of a medicament or nutritional agent for alleviating disorders of the digestive system.
Through the technical scheme, the stem cell derivative preparation prepared by specifically combining the alhagi plant extract and the mesenchymal stem cell extracellular product can treat digestive system diseases more efficiently, promote small intestine movement, improve intestinal peristalsis speed, promote recovery of digestive system injury, relieve inflammation of the digestive system and avoid generating immune rejection and side effects; by the specific preparation method, the preservation time of the prepared stem cell derivative preparation is prolonged, and the stability of active components in the stem cell derivative preparation is ensured.
Drawings
FIG. 1 is a diagram of the MSC and Con group differential protein Wen;
FIG. 2 is a heat map of MSC and Con group differential protein expression;
FIG. 3 is a graph of the effect of MSC/Alhagi on the colon length of mice;
FIG. 4 is a graph showing the effect of HE-stained MSC/Alhagi on colonic mucosal lesions in enteritis mice;
FIG. 5 is a graph showing the effect of HE-stained MSC/Alhagi on colon muscle layer thickness in mice.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In the present invention, "MSC" refers to mesenchymal stem cells unless otherwise stated.
In a first aspect the present invention provides a stem cell derived preparation comprising an extract of a plant of the genus alhagi and an extracellular product of mesenchymal stem cells.
According to the invention, the weight ratio of the mesenchymal stem cell extracellular product and the alhagi plant extract of the stem cell derived preparation on a dry basis is 1:1.5 to 150, preferably 1:2-100, such as 1:2. 1:2.1, 1:2.2, 1:2.5, 1: 3. 1:4. 1:4.8, 1: 5. 1: 6. 1: 8. 1: 10. 1: 50. 1: 80. 1: 90. 1:100 or any value between the above values.
According to the present invention, the alhagi plant extract is an aqueous alhagi plant extract, preferably a lyophilized extract of an aqueous alhagi plant extract, in order to further extend the shelf life of the stem cell derived preparation. The active ingredient in the alhagi plant extract is mainly polysaccharide, and the content of the polysaccharide in the alhagi plant extract can be 695.2 +/-13.15 mg/g. The control of the polysaccharide content in this range can make the stem cell derived preparation more effective. The polysaccharide content test method is a phenol sulfuric acid method.
According to a preferred embodiment of the present invention, the plants of the genus alhagi are at least one of alhagi and seabuckthorn, more preferably alhagi (Alhagi sparsifolia shape). In particular, the alhagi extract is extracted from alhagi fruit. The invention particularly discovers that the mesenchymal stem cells and the alhagi have more obvious synergistic effect when being matched.
According to the present invention, the extracellular product of mesenchymal stem cells is a supernatant of mesenchymal stem cell culture, and preferably a lyophilized product of the supernatant of mesenchymal stem cell culture for further prolonging the preservation time of the stem cell-derived preparation.
According to the present invention, the mesenchymal stem cells may be derived from at least one of human and animal fat (especially mesenteric fat), bone marrow, umbilical cord, placenta, and the like. The animals are preferably felines and/or canines, and can be any of the usual felines and/or canines, especially cats, dogs (Canis lupus familiaris), wolves (Canis lupus), jackals (Cuon alpinus) and foxes (Vulpes). More preferably, the mesenchymal stem cell source is canine fat. The active ingredient in the extracellular product of the mesenchymal stem cells is mainly a cytokine such as Vascular Endothelial Growth Factor (VEGF).
According to the present invention, the preparation method of the camelina plant extract can be obtained by various conventional preparation methods of plant extracts. However, in order to obtain the active ingredient therein better and avoid contamination of the impurity ingredient, the preparation method of the extract of the plant belonging to the genus Alhagi comprises: mixing a camel thorn plant with water and extracting, wherein the extracting comprises the following steps: soaking extraction, ultrasonic extraction and thermal reflux extraction. The three extraction modes can be carried out in any order.
According to a preferred embodiment of the present invention, the preparation of the alhagi plant extract comprises: the method comprises the steps of mixing the alhagi plant with water and then sequentially carrying out soaking extraction, ultrasonic extraction and thermal reflux extraction, or mixing the alhagi plant with water and then sequentially carrying out soaking extraction, reflux extraction and ultrasonic extraction. The preparation of the extract of the plants of the genus Alhagi in this particular manner can further ensure the content of the active ingredient in the extract of the plants of the genus Alhagi and thus further enhance the effect of the stem cell derived preparation in alleviating disorders of the digestive system.
According to a preferred embodiment of the present invention, the conditions for the soaking extraction include: the temperature is 15-40 ℃ and the time is 40-50h. Such as: the temperature is 15 ℃, 16 ℃, 18 ℃, 20 ℃, 23 ℃, 26 ℃, 30 ℃, 35 ℃, 40 ℃ or any value between the above values. Times were 41h, 43h, 44h, 45h, 48h, 50h or any value in between.
According to a preferred embodiment of the present invention, the conditions of the ultrasonic extraction include: the ultrasonic power is 100-300W, the ultrasonic frequency is 50-200Hz, the temperature is 50-70 ℃ and the time is 0.2-5h. Such as: the ultrasonic power is 100W, 101W, 102W, 105W, 107W, 110W, 115W, 120W, 150W, 200W, 250W, 300W or any value in between. The ultrasound frequency is 50Hz, 51Hz, 52Hz, 55Hz, 58Hz, 60Hz, 65Hz, 70Hz, 80Hz, 100Hz, 150Hz, 200Hz or any value in between the above values. The temperature is 50 ℃, 51 ℃, 53 ℃, 55 ℃, 58 ℃,60 ℃, 65 ℃, 70 ℃ or any value between the above values. The time is 0.2h, 0.21h, 0.25h, 0.26h, 0.3h, 0.5h, 1h, 1.5h, 1.65h, 1.8h, 2h, 4h, 5h, or any value in between.
According to a preferred embodiment of the present invention, the conditions for the thermal reflux extraction include: the temperature is 90-95 ℃ and the time is 1-5h. Such as: the temperature is 90 ℃, 91 ℃, 92 ℃, 92.5 ℃, 93 ℃, 93.8 ℃, 95 ℃ or any value between the above values. The time is 1h, 1.5h, 1.65h, 1.8h, 2h, 4h, 5h or any value in between.
According to a preferred embodiment of the invention, the weight ratio of the camelid to water is 1:5-15.
According to a preferred embodiment of the present invention, in order to further extend the shelf life of the stem cell derived preparation, the process for the preparation of the extract of the plant species alhagi additionally comprises drying, more preferably freeze-drying, the conditions of which comprise: vacuum pressure is 0.01-0.05mbar; the temperature is between-90 ℃ and-80 ℃; the time is 20-30h.
According to the present invention, the preparation method of the mesenchymal stem cell extracellular product may be obtained by various cell culture methods. However, in order to better retain active factors in the mesenchymal stem cells, so that the mesenchymal stem cells have more specific effects on a digestive system, and the defects of immune rejection and side effects existing in the mesenchymal stem cells are avoided, preferably, the preparation method of the extracellular product of the mesenchymal stem cells comprises the following steps: mesenchymal stem cells are subcultured, and cultures of cells of 3 rd generation or any generation after 3 rd generation are collected and supernatant is taken for drying.
According to a preferred embodiment of the invention, the culture is a culture of any of the 3 rd to 5 th generation cells in order to increase the active factor in the stem cell derived preparation.
According to a preferred embodiment of the present invention, in order to avoid immune rejection and side effects of the mesenchymal stem cells themselves, the cell growth density in the culture is 80-90%, and the mesenchymal stem cells are obtained in high purity. In the present invention, the medium used for subculture may be a medium commonly used in the art for mesenchymal stem cell culture, such as an α -MEM medium and/or DMEM high sugar medium.
According to a preferred embodiment of the present invention, in order to further extend the shelf life of the stem cell derived preparation, the supernatant is dried by freeze-drying under conditions comprising: 0.01-0.05mbar; the temperature is between-90 ℃ and-80 ℃; the time is 40-50h. The freeze-drying technology used in the invention can dehydrate more thoroughly, the freeze-drying method can exclude the active factors of stem cells, and the active factors of the stem cells can be stored for a long time without deterioration; the high-activity protein extracted in vacuum and at low temperature not only contains antifungal protein, but also has undamaged structure, active factors and growth factors of protein and enzyme, so that the defect of immune rejection reaction in mesenchymal stem cell treatment can be further avoided, and meanwhile, the preservation time of the stem cell derivative preparation can be further prolonged.
According to a preferred embodiment of the present invention, the stem cell derived formulation may further contain at least one of a preservative, a stabilizer and an antioxidant to further improve the stability of the stem cell derived formulation, thereby extending the shelf life.
According to a preferred embodiment of the present invention, the preservative may be present in an amount of 0.01 to 0.1 wt%, preferably 0.02 to 0.05 wt%, based on the total weight of the stem cell derived formulation; the stabilizer may be present in an amount of 0.1 to 0.8 wt%, preferably 0.2 to 0.5 wt%; the antioxidant may be present in an amount of 0.01 to 0.8 wt.%, preferably 0.02 to 0.5 wt.%.
The preservative may be a material having a preservative function, which is common in the art, and according to a preferred embodiment of the present invention, the preservative is at least one of sodium paraben, sodium ethylparaben, and chlorobutanol.
The stabilizer may be a material commonly used in the art having a function of stabilizing a formulation, and according to a preferred embodiment of the present invention, the stabilizer is at least one of sodium carboxymethyl cellulose, tragacanth, and povidone.
The antioxidant may be a material having an antioxidant function, which is common in the art, and according to a preferred embodiment of the present invention, the antioxidant is at least one of ascorbic acid, propyl gallate and butyl hydroxy anisole.
According to the present invention, the stem cell-derived formulation may be in various forms of formulation, such as a powdery formulation, a gel formulation or a liquid formulation, and preferably, the stem cell-derived formulation is a liquid formulation.
In a second aspect, the invention provides a method of preparing a stem cell derived formulation, the method comprising: mixing the extract of Alhagi plant and the extracellular product of mesenchymal stem cells. The specific selection of the extract of the plants of the genus Alhagi and the extracellular product of the mesenchymal stem cells is as described above, and will not be described here again.
According to a preferred embodiment of the present invention, the method of preparing the stem cell derived formulation further comprises mixing a preservative, a stabilizer, an antioxidant and optionally water with the extract of the plant species alhagi and the extracellular product of mesenchymal stem cells (simultaneously).
According to a preferred embodiment of the present invention, the preparation of the extract of the plants of the genus Alhagi and the extracellular product of mesenchymal stem cells as described above is also included, and will not be described in detail herein.
In a third aspect, the present invention provides the use of a stem cell derived formulation as described above and/or a stem cell derived formulation prepared by a method as described above in the manufacture of a medicament or nutritional agent for alleviating disorders of the digestive system.
In the present invention, the digestive system diseases may include: intestinal cramps, bad breath, malnutrition of the intestinal flora, digestive system disorders caused by bacteria or viruses, etc., including diarrhea, constipation, dyspepsia, etc.
In the present invention, the amount of the stem cell-derived preparation used may be determined according to the physical condition, body weight, etc. of a human or animal, and in the case of a mouse having a body weight of about 25g, the amount of the stem cell-derived preparation may be 150 to 300mg/kg.
In the present invention, the stem cell-derived preparation administration method may be oral administration, intravenous injection, or the like.
In the present invention, the medicament or nutritional agent is primarily for use in humans, felines or canines, particularly pet dogs and/or cats.
The present invention will be described in detail by examples. In the following examples, the cell growth density parameter was measured by the cell counting method, and the number of cells obtained was an absolute number; centrifuging by a TD-4M type centrifuge; the cells are subcultured in a Thermo-type incubator; proteome high throughput sequencing was measured by Orbitrap Fusion Lumos; tissue and organs were observed by a positive-working microscope; alpha-MEM medium is commercially available under the trademark Eagle's from Gibco; type I collagenase is commercially available from Sigma under the trade designation SCR 103; ultra GRo-Advanced is commercially available under the trade name GMP from bioscience; trypsin is a commercial product available from Gibco under the trademark TrypLETM Express; male Kunbai mice were purchased from Dacron laboratory animals Inc.
Preparation example 1
Preparation of mesenchymal stem cell culture supernatant:
(1) After general anesthesia of a 1 year old female hybrid test dog with Shutai 50, the abdominal cavity was opened by strict asepsis, 50g of mesenteric adipose tissue was washed 3 times in alpha-MEM, and minced with scissors to 1mm 3 Is a crushed form of (2);
(2) The pre-prepared 30mL of the 0.1% type I collagenase-mixed alpha-MEM solution, which had been filtered through a 0.22 μm filter and preheated at 37℃was sucked up repeatedly to the adipose tissue, 50mg of type I collagenase was mixed with 50mL of the alpha-MEM solution in a centrifuge tube, and finally the adipose tissue was sucked up entirely into the centrifuge tube.
(3) Sealing with sealing film sprayed with alcohol, placing in a 37deg.C constant temperature water bath vibrator, digesting for 1 hr, repeatedly reversing centrifuge tube every 10min, and uniformly digesting fat tissue.
(4) After 1h, the type I collagenase was neutralized with an equal amount of alpha-MEM culture medium, and after repeated blowing and suction, the medium was centrifuged at 1000rpm for 5min to separate mature adipose tissues.
(5) The supernatant suspension and the floating white fat were discarded, washed with 20mL of an alpha-MEM culture solution, blown uniformly, filtered through a 100 μm sieve, and centrifuged at 1000rpm at 25℃for 5min.
(6) Centrifuging, removing upper suspension, blowing with 1mL alpha-MEM culture solution containing 5% ultra GRO-Advanced, transferring into 6-well plate, and adding 5% CO 2 Culturing in a constant temperature incubator at 37 ℃. After cells were grown to 90% confluence, the cells were digested with 0.25% trypsin, pancreatin was discarded when microscopic observation that the cells were separated from each other, and the cells were resuspended in alpha-MEM culture medium such that the volume ratio of alpha-MEM culture medium to cell pellet was 1:3, subculturing.
(7) After the growth of the adipose-derived mesenchymal primary stem cells is approximately 80-90% fused, replacing a fresh culture medium, subculturing, collecting cell culture supernatant of the 3 rd generation, centrifuging the supernatant for 10min by using a sterile centrifuge tube at 1500rpm, discarding the precipitate, taking the supernatant, and pre-freezing for 20h in an ultralow temperature refrigerator at-80 ℃.
(8) Freeze-drying the frozen body at-80deg.C in a freeze dryer with vacuum pressure of 0.030mbar for 50 hr at-88deg.C to remove ice crystal to obtain lyophilized powder, weighing, wrapping with tinfoil, registering, and preserving at-80deg.C.
Preparation of Alhagi sparsifolia extract:
(1) Pulverizing Alhagi sparsifolia fruit 100g at 25deg.C, soaking in 10 times of distilled water for 45 hr, ultrasonic extracting at 200W,100Hz and 60deg.C for 1 hr, ultrasonic extracting for 3 times, and reflux extracting at 93 deg.C for 2 hr. The filtrate is concentrated under reduced pressure after filtration, and is frozen at-80 ℃ for standby.
(2) Freeze-drying the frozen body at-80deg.C in a vacuum pressure 0.030mbar freeze dryer at-88deg.C for 24 hr to obtain freeze-dried powder, weighing, and wrapping with tinfoil and refrigerating in a refrigerator at 4deg.C.
Preparation of a liquid stem cell derived formulation:
adding 30mL of distilled water into 20g of the alhagi sparsifolia freeze-dried powder prepared by the method and 10g of the mesenchymal stem cell freeze-dried powder, uniformly mixing, adding 0.04g of ascorbic acid, 0.04g of sodium parahydroxybenzoate and 0.02g of sodium carboxymethyl cellulose, uniformly mixing, adding distilled water to complement to 100mL, and mechanically stirring for 10min to fully and uniformly mix, thereby obtaining the liquid stem cell derivative preparation.
Preparation example 2
The procedure of preparation example 1 was followed, except that the stem cell derived preparation was prepared as follows: uniformly mixing 20g of the alhagi sparsifolia freeze-dried powder prepared by the method, 2g of the freeze-dried powder of the mesenchymal stem cell culture supernatant prepared by the method, 0.04g of ascorbic acid, 0.04g of sodium parahydroxybenzoate and 0.22g of sodium carboxymethylcellulose, adding distilled water to complement to 100mL, and mechanically stirring for 10min to fully and uniformly mix the materials.
Preparation example 3
The procedure of preparation example 1 was followed, except that the preparation method of the stem cell derived preparation was modified to prepare a powdery preparation: 25g of the alhagi sparsifolia lyophilized powder prepared by the method, 250mg of the lyophilized powder of the mesenchymal stem cell culture supernatant, 0.04g of ascorbic acid, 0.04g of sodium paraben and 0.2g of sodium carboxymethyl cellulose are mixed to obtain a powdery stem cell derivative preparation.
Preparation example 4
The procedure of preparation example 1 was followed, except that the vacuum pressure of the freeze-drying in step (8) in the preparation method of the mesenchymal stem cell culture supernatant was 0.04mbar, the temperature was-60℃and the freeze-drying was performed for 30 hours. The vacuum pressure of the freeze drying in step (2) in the preparation method of the extract is 0.02mbar, the temperature is-80 ℃, and the freeze drying is carried out for 40 hours.
Preparation example 5
According to the method of preparation example 1, except that step (1) in the preparation method of the alhagi sparsifolia extract is sequentially subjected to soaking extraction, reflux extraction and ultrasonic extraction.
Preparation example 6
The procedure of preparation 1 was followed except that the mesenteric adipose tissue mass was replaced with umbilical cord tissue mass.
Preparation example 7
The procedure of preparation 1 was followed except that the alhagi was replaced with seabuckthorn.
Test example 1
The content of polysaccharide in the alhagi extract was measured according to the phenol sulfuric acid method, and the results are shown in table 2.
Proteome high throughput sequencing: the results of cluster map of differential protein expression are shown in FIGS. 1 and 2, after sequencing analysis of MSC (supernatant of MSC culture prepared in preparation example 1) group and Con (negative fluid/control group, no MSC cells) group. As can be seen from FIG. 1, there were a total of 3364 proteins differentially expressed, wherein the number of differential genes expressed in both MSC and Con groups was 778, 336 differential proteins were expressed only in Con group, and 2250 differential proteins were expressed only in MSC group. According to FIG. 2, compared with the Con group, the MSC group has obviously up-regulated expression of F1PHY1 and F1Q3I5 of collagen family, and compared with the normal cell, the up-regulated expression of F1PHY1 and F1Q3I5 is more than 50 times, so that the mesenchymal stem cell culture supernatant prepared by the specific method of the invention has excellent platelet aggregation performance, good hemostatic effect and better smoothness and elasticity. The A0A5F4C710, A0A5F4CLC9 and A0A5F4CRW6 of the heparan sulfate proteoglycan family are also highly expressed, can interact with proteins such as various growth factors and chemotactic factor enzymes, influence various signal paths, participate in metabolism, transportation, information transmission, regulation and structural maintenance of all organ systems, and play an important role in organ development.
Test example 2
Healthy Kunbai male mice were selected for 60, 5-7 weeks of age, body weight 25-28g, and randomly divided into 5 groups of 12 mice each: control (NC), model (Model), stem cell (MSC), alhagi (Alhagi), preparation 1 (Alhagi/MSC). Mice were acclimatized for 7 days, and on day 8, gastric lavage intervention was used. Mesenchymal stem cell culture supernatant of preparation example 1 was lavaged with 200mg/kg of MSC group, alhagi group was lavaged with 300mg/kg of the extract of Alhagi group of preparation example 1, alhagi/MSC group was lavaged with 0.2mL of the stem cell-derived preparation of preparation example 1, and NC group and Model group were simultaneously administered with 0.2mL of physiological saline. On experiment day 15, enteritis Model establishment was performed using 4% DSS, and Model group, MSC group, alhagi group, and Alhagi/MSC group mice were completely replaced with 4% DSS solution for free drinking, and corresponding preparations were normally perfused during the period. The mice were sacrificed on day 21 during enteritis model establishment and the corresponding tissues and organs were observed, the results of which are shown in fig. 3-5.
The enteritis pathology scores of the mice were scored according to the enteritis pathology tissue microscopy of the mice, and the scoring criteria are shown in table 1.
TABLE 1
| Scoring of | Inflammation | Depth of lesions | Pit destruction | Lesion extent (%) |
| 0 | Without any means for | Without any means for | Without any means for | Without any means for |
| 1-2 | Light weight | Submucosa layer | 1/3 destruction of substrate | 1-25 |
| 2-4 | In (a) | Myolayer | Substrate 2/3 destruction | 26-50 |
| 3-5 | Heavy weight | Serosal layer | Only intact epithelium | 51-75 |
As shown in fig. 3, the NC group mice had a long and thin colon with uniform thickness, a smooth and pale red colonic mucosa, no congestion and edema, and a large amount of oval feces were visible in the intestinal lumen. The colonic mucosa of mice in Model group showed various degrees of congestion and swelling, and the mucosa surface had bleeding points, the intestinal lumen had blood content, and the colon length was significantly shortened. The MSC group was similar to the Alhagi group, with longer colon and glossy intestinal surface compared to the Model group mice. The Alhagi/MSC group has obviously better effect than the MSC group and the Alhagi group, the surface of the colon of the mouse is glossy and complete, atrophy, congestion, ulcer, necrosis and the like do not occur, the colon is not obviously shortened, the colon of the mouse is longer, and the colon is more similar to the colon shape of the NC group.
As shown in fig. 4, the colon mucosa structure of NC group mice is complete, glands are aligned in order, a large number of cup-shaped cells are visible, and no obvious immune cell infiltration is found in the mucosa lamina propria and submucosa. The colonic mucosal structures of mice in Model groups are destroyed and completely disappeared, and a great amount of neutrophil infiltration and lymphocyte infiltration of the mucosa lamina propria and submucosa appear, so that inflammatory exudation is visible. The colon mucous membrane structure of the mice in the MSC group and the Alhagi group is complete, the damage of glands and infiltration of immune cells are relieved, and goblet cells can be seen. The colon mucosa of the Alhagi/MSC group mice is relatively complete, the ulcer and erosion degree is reduced, the goblet cells and crypt morphology are partially intact, the infiltration degree of mucosal inflammatory cells is reduced, and the pathological degree is lighter than that of the MSC group and the Alhagi group. Further scoring according to the injury degree of the colonic mucosa and the inflammatory cell infiltration degree, the Model group DSS pathology score is obviously increased compared with the NC group, and the MSC group and the Alhagi group pathology score after separate stem prognosis of the MSC and the Alhagi are reduced, but the effect of reducing by more than 50% cannot be achieved, which indicates that the alleviation effect is general when the Model group DSS pathology score is singly used. The Alhagi/MSC group has the effect of obviously reducing pathological scoring, can be reduced by at least more than 60 percent, and has obviously better alleviation capability on pathological degree than the MSC group and the Alhagi group.
As shown in fig. 5, both of which were observed at 20-fold magnification, the Model group had a significantly thinner colon muscle layer thickness than the NC group, and the MSC group and the Alhagi group had a thickened dry prognosis muscle layer thickness alone, but the effect of restoring the muscle layer thickness was still poor as compared to the Alhagi/MSC group combined intervention.
The stool characteristics, pathology scores, and muscle layer thicknesses of the other preparations were examined in a similar manner as above and the results are shown in table 2.
TABLE 2 polysaccharide content and intestinal tract status (control muscle layer thickness of about 0.8-1mm, model group pathology score of 5) results
From preparation examples 1 and 6, it can be seen that the synergistic therapeutic effect of the combination of the mesenchymal stem cells obtained from mesenteric adipose tissue mass and the alhagi was superior to that of the mesenchymal stem cells obtained by using umbilical cord tissue mass in combination. Although not shown, the effect of the invention when the mesenchymal stem cells obtained from mesenteric adipose tissue blocks are used for being matched with the alhagi is also superior to the effect of the invention when the mesenchymal stem cells obtained from peritoneal adipose tissue are matched with the alhagi, and the invention has more obvious synergistic effect.
The invention prolongs the preservation time of the preparation, has no toxic or side effect and provides the function of gastrointestinal protection; is beneficial to supplementing special nutrient substances required by the human body, relaxing bowel, intestinal spasm, pain relieving, halitosis, insufficient nutrition and balance of intestinal flora; can be used for comprehensively preventing and treating digestive system disorder diseases caused by common metabolic disorder diarrhea, constipation and dyspepsia, and various infectious diseases such as (canine) pestilence, (canine) parvovirus, (feline) infectious peritonitis, etc.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (10)
1. A stem cell derived preparation having an enteritis alleviation function, characterized in that the stem cell derived preparation comprises an extract of a plant of the genus alhagi and an extracellular product of mesenchymal stem cells;
the Alhagi plant extract is water extract of Alhagi plant;
the plant of Alhagi is Alhagi sparsifolia;
the preparation method of the mesenchymal stem cell extracellular product comprises the following steps: subculturing the mesenchymal stem cells, collecting cultures of cells of 3 rd generation or any generation after 3 rd generation, and taking supernatant for drying; the mesenchymal stem cells are derived from at least one of the fat, bone marrow, umbilical cord and placenta of a dog; the cell growth density in the culture is 80-90%; the supernatant is dried by freeze drying;
wherein, the weight ratio of the stem cell derived preparation intermediate mesenchymal stem cell extracellular product to the alhagi plant extract is 1:2-100.
2. The stem cell derived formulation of claim 1, wherein the alhagi plant extract is a lyophilizate of an aqueous alhagi plant extract.
3. The stem cell derived formulation of claim 1, wherein the process for preparing the camelid extract comprises: mixing the alhagi plant with water, and extracting, wherein the extraction comprises soaking extraction, ultrasonic extraction and thermal reflux extraction;
wherein, the conditions of soaking and extracting comprise: the temperature is 15-40 ℃ and the time is 40-50h;
the conditions of the ultrasonic extraction include: the ultrasonic power is 100-300W, the ultrasonic frequency is 50-200Hz, the temperature is 50-70 ℃ and the time is 0.2-5h;
the conditions of the thermal reflux extraction include: the temperature is 90-95 ℃ and the time is 1-5h.
4. A stem cell derived formulation according to claim 3, wherein the process for the preparation of the camelid extract further comprises drying after extraction.
5. The stem cell derived formulation of claim 4, wherein the method of preparing the camelina plant extract is by freeze-drying under conditions comprising: vacuum pressure is 0.01-0.05mbar; the temperature is between-90 ℃ and-80 ℃; the time is 20-30h.
6. The stem cell derived formulation of claim 1, wherein the culture is a culture of any of the 3 rd-5 th generation cells;
and/or, the conditions for freeze-drying the supernatant include: 0.01-0.05mbar; the temperature is between-90 ℃ and-80 ℃; the time is 40-50h.
7. A method of preparing a stem cell derived formulation according to any one of claims 1 to 6, comprising: mixing the extract of the plants of the genus Alhagi with the extracellular product of the mesenchymal stem cells;
the Alhagi plant extract is water extract of Alhagi plant.
8. The method according to claim 7, wherein the method further comprises the step of preparing a camelina plant extract in the following manner: mixing a camel thorn plant with water and extracting, wherein the extracting comprises the following steps: soaking extraction, ultrasonic extraction and thermal reflux extraction;
wherein, the conditions of soaking and extracting comprise: the temperature is 15-40 ℃ and the time is 40-50h;
the conditions of the ultrasonic extraction include: the ultrasonic power is 100-300W, the ultrasonic frequency is 50-200Hz, the temperature is 50-70 ℃ and the time is 0.2-5h;
the conditions of the thermal reflux extraction include: the temperature is 90-95 ℃ and the time is 1-5h.
9. The method according to claim 7 or 8, wherein the preparation of the camelina plant extract is dried by freeze-drying under conditions comprising: vacuum pressure is 0.01-0.05mbar; the temperature is between-90 ℃ and-80 ℃; the time is 20-30h.
10. Use of a stem cell derived preparation according to any one of claims 1 to 6 and/or a stem cell derived preparation prepared by a method according to any one of claims 7 to 9 in the manufacture of a medicament for alleviating enteritis.
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| CN101862366A (en) * | 2009-11-19 | 2010-10-20 | 新疆维吾尔自治区中药民族药研究所 | Method for extracting medicament for treating allergic colitis from Alhagi sparsifolia plant and medicament and application thereof |
| CN102920734A (en) * | 2012-11-14 | 2013-02-13 | 青岛奥克生物开发有限公司 | Mesenchymal stem cell injection and preparation method thereof as well as application in preparation of medicine for treating ulcerative colitis |
| CN112618515A (en) * | 2020-12-29 | 2021-04-09 | 江南大学 | Preparation method of exosome-loaded oral colon-targeted drug delivery polymer |
| CN113304103A (en) * | 2021-04-23 | 2021-08-27 | 西北农林科技大学 | Preparation method and application of human mesenchymal stem cell supernatant dry powder gel |
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| CN101862366A (en) * | 2009-11-19 | 2010-10-20 | 新疆维吾尔自治区中药民族药研究所 | Method for extracting medicament for treating allergic colitis from Alhagi sparsifolia plant and medicament and application thereof |
| CN102920734A (en) * | 2012-11-14 | 2013-02-13 | 青岛奥克生物开发有限公司 | Mesenchymal stem cell injection and preparation method thereof as well as application in preparation of medicine for treating ulcerative colitis |
| CN112618515A (en) * | 2020-12-29 | 2021-04-09 | 江南大学 | Preparation method of exosome-loaded oral colon-targeted drug delivery polymer |
| CN113304103A (en) * | 2021-04-23 | 2021-08-27 | 西北农林科技大学 | Preparation method and application of human mesenchymal stem cell supernatant dry powder gel |
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