CN115737699A - Stem cell derived preparation and preparation method and application thereof - Google Patents
Stem cell derived preparation and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of animal feeding, and discloses a stem cell derivative preparation as well as a preparation method and application thereof. The stem cell derived preparation contains camelina plant extract and mesenchymal stem cell extracellular product. The invention prolongs the storage time of the preparation, has no toxic or side effect and provides the function of protecting intestines and stomach; is beneficial to supplementing special nutrient substances required by human body, relaxing bowel, relieving intestinal spasm, relieving pain, halitosis, and nutrient deficiency, and maintaining intestinal flora balance; can be used for the comprehensive prevention and treatment of digestive system disorder diseases caused by common metabolic disturbance diarrhea, constipation, dyspepsia, distemper, parvovirus, infectious peritonitis, etc.
Description
Technical Field
The invention relates to the technical field of animal feeding, in particular to a stem cell derived preparation, a preparation method and application thereof.
Background
With the increasing living standard of people, the number of people raising pets is gradually increased, and people pay more and more attention to the pets. In the process of raising the pet, if the care for the pet is not weekly, the pet is easy to get ill. Among the diseases suffered by pets, the proportion of diseases related to diarrhea, enteritis, intestinal spasm and the like is the largest of all the diseases. Dog enteritis is acute or chronic inflammation of small intestinal mucosa and deep tissues, and is mainly seen in secondary infection of some infectious diseases, such as canine parvovirus disease, canine distemper and the like, and is a disease characterized by digestive disorder, vomiting, abdominal pain, diarrhea, body temperature rise and autointoxication signs. The feed mainly damages young dogs and old dogs, has high morbidity and relatively low mortality, seriously influences the growth and development of the dogs, further reduces the utilization rate of the feed, causes economic loss of dog farmers, and is frequently generated in spring and autumn.
Mesenchymal Stem Cells (MSCs) are important members of a stem cell family, and can be defined as adult stem cells with multi-differentiation potential, the Mesenchymal stem cells play an important role in the treatment of chronic inflammatory fibrosis and fibrotic diseases, the Mesenchymal stem cells have a strong paracrine effect, and the Mesenchymal stem cell secretion has the functions of inhibiting tissue fibrosis, promoting angiogenesis, participating in tissue repair and regeneration and the like. However, there are immunological rejection and side effects of mesenchymal stem cells in transplantation therapy, and the effect of mesenchymal stem cell secretion on intestinal fibrosis is still lack of research.
Alhagi pseudohagi (Alhagi pseudohagi) is a perennial root-planting half shrub plant of the subfamily Papili onaceae of Leguminosae (Leguminosae), sugar particles formed by condensing the secretion of branches and leaves of the Alhagi pseudohagi are acanthose, the appearance is yellowish white to brown, the sugar particles are elliptical or spherical small particles, the diameter is about 1-5 mm, the sugar particles have viscosity and sweet taste, the branches and leaves are round, the sugar particles are important national medicaments for Uygur families, and the single use of the Alhagi pseudohagi has effects on digestive system diseases, but has long effective slow treatment course and 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 of camel plants and mesenchymal stem cells when the camel plants and the 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 a camelid plant extract and an extracellular product of mesenchymal stem cells.
In a second aspect, the invention provides a method of preparing a stem cell derived preparation, the method comprising: mixing the camel thorn plant extract and the mesenchymal stem cell extracellular product.
In a third aspect, the invention provides a use of a stem cell derived preparation as described above and/or a stem cell derived preparation prepared by a method as described above in the preparation of a medicament or nutritional agent for alleviating a disease of the digestive system.
Through the technical scheme, the stem cell derivative preparation prepared by combining the camel thorn plant extract and the mesenchymal stem cell extracellular product can more efficiently treat digestive system diseases, promote small intestine movement, improve the speed of intestinal tract peristalsis, promote recovery of digestive system injury, relieve inflammation of the digestive system and avoid immunological rejection reaction and side effect; by the specific preparation method, the preservation time of the prepared stem cell derived preparation is prolonged, and the stability of active components in the stem cell derived preparation is ensured.
Drawings
FIG. 1 is a diagram of MSC and Con group differential proteins Wann;
FIG. 2 is a heat map of differential protein expression between MSC and Con groups;
FIG. 3 is a graph of the effect of MSC/Alhagi on colon length in mice;
FIG. 4 is a graph of the effect of HE staining of MSC/Alhagi on colonic mucosal damage in enteritis mice;
FIG. 5 is a graph of the effect of HE-stained MSC/Alhagi on thickness of the colonic muscle layer in mice.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, one or more new ranges of values may be obtained from combinations of values between the endpoints of each range, the endpoints of each range and the individual values, and the individual values of the points, and these ranges of values should be considered as specifically disclosed herein.
In the present invention, without being stated to the contrary, "MSC" refers to a mesenchymal stem cell.
In a first aspect, the invention provides a stem cell derived preparation comprising a camelina plant extract and an extracellular product of a mesenchymal stem cell.
According to the invention, the weight ratio of the mesenchymal stem cell extracellular product to the alhagi plant extract in the stem cell derived preparation 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 therebetween.
According to the invention, the camelid plant extract is a water extract of camelid plants, and is preferably a freeze-dried substance of the water extract of camelid plants in order to further prolong the storage time of the stem cell derivative preparation. The active ingredients in the alhagi plant extract are mainly polysaccharides, and the content of the polysaccharides in the alhagi plant extract can be 695.2 +/-13.15 mg/g. Controlling the polysaccharide content within this range enables the stem cell-derived preparation to be more effective. The method for testing the polysaccharide content is phenol-sulfuric acid method.
According to a preferred embodiment of the invention, the camelina plant is at least one of camelina and hippophae rhamnoides, more preferably camelina (Alhagi spisifolia sharp). In particular, the alhagi sparsifolia extract is extracted from alhagi sparsifolia fruits. The invention particularly discovers that the mesenchymal stem cells and the alhagi sparsifolia have more obvious synergistic effect when being used in combination.
According to the invention, the extracellular product of the mesenchymal stem cell is culture supernatant of the mesenchymal stem cell, and in order to further prolong the storage time of the stem cell derived preparation, the mesenchymal stem cell extracellular product is preferably freeze-dried substance of the culture supernatant of the mesenchymal stem cell.
According to the present invention, the mesenchymal stem cell may be derived from at least one of fat (particularly mesenteric fat), bone marrow, umbilical cord, placenta, and the like of human and animal. The animal is preferably a feline and/or canine animal, and can be any of the various commonly occurring feline and/or canine animals, particularly a cat, a dog (Canis lupis), a wolf (Canis lupis), a jackal (Cuon alpinus) and a fox (Vulpes). More preferably, the mesenchymal stem cell source is canine fat. The active ingredients in the extracellular products of the mesenchymal stem cells are mainly cytokines, such as Vascular Endothelial Growth Factor (VEGF).
According to the present invention, the preparation method of the camelid plant extract can be obtained by various conventional preparation methods of plant extracts. However, in order to better obtain the effective components in the extract and avoid the pollution of impurity components, the preparation method of the camel thorn plant extract comprises the following steps: mixing the camelia plant with water and then extracting, wherein the extraction comprises the following steps: soaking extraction, ultrasonic extraction and hot reflux extraction. 3. The extraction can be performed in any order.
According to a preferred embodiment of the invention, said camelid plant extract is prepared in a manner comprising: mixing the camelia plant with water and then sequentially carrying out soaking extraction, ultrasonic extraction and hot reflux extraction or mixing the camelia plant with water and then sequentially carrying out soaking extraction, reflux extraction and ultrasonic extraction. The preparation of the camelid plant extract according to the specific mode can further ensure the content of active ingredients in the camelid plant extract so as to further improve the effect of the stem cell derived preparation on relieving digestive system diseases.
According to a preferred embodiment of the present invention, the conditions of said soaking extraction comprise: the temperature is 15-40 deg.C, and the time is 40-50h. Such as: the temperature is 15 deg.C, 16 deg.C, 18 deg.C, 20 deg.C, 23 deg.C, 26 deg.C, 30 deg.C, 35 deg.C, 40 deg.C or any value therebetween. The time is 41h, 43h, 44h, 45h, 48h, 50h or any value in between the above values.
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 temperature is 50 deg.C, 51 deg.C, 53 deg.C, 55 deg.C, 58 deg.C, 60 deg.C, 65 deg.C, 70 deg.C or any value in between. 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 of the hot reflux extraction include: the temperature is 90-95 ℃ and the time is 1-5h. Such as: the temperature is 90 deg.C, 91 deg.C, 92 deg.C, 92.5 deg.C, 93 deg.C, 93.8 deg.C, 95 deg.C or any value in between. 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 camelina plants to water is 1: 5-15.
According to a preferred embodiment of the invention, in order to further extend the storage time of the stem cell derived preparation, the process for the preparation of the camelid plant extract further comprises drying, more preferably freeze-drying, under conditions comprising: the vacuum pressure is 0.01-0.05mbar; the temperature is between-90 ℃ and-80 ℃; the time is 20-30h.
According to the invention, the preparation method of the extracellular product of the mesenchymal stem cell can 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 are more directed to influence the digestive system, and avoid the defects of immunological rejection and side effects of the mesenchymal stem cells, preferably, the preparation method of the extracellular product of the mesenchymal stem cells of the present invention comprises: subculturing the mesenchymal stem cells, collecting the culture of the cells of any generation after the 3 rd generation or the 3 rd generation, and taking the supernatant for drying.
According to a preferred embodiment of the invention, the culture is a culture of cells of any of passages 3-5 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 reaction 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 with high purity. In the present invention, the medium used for subculture may be a medium commonly used in the art for culturing mesenchymal stem cells, such as α -MEM medium and/or DMEM high-sugar medium.
According to a preferred embodiment of the present invention, in order to further extend the storage time of the stem cell-derived preparation, the supernatant is dried by freeze-drying under conditions comprising: 0.01-0.05mbar; the temperature is-90 ℃ to-80 ℃; the time is 40-50h. The freeze-drying technology used in the invention can dehydrate more thoroughly, the freeze-drying method can remove the water, and the stem cell active factors can be stored for a long time without deterioration; and the high-activity protein extracted under vacuum and low temperature not only contains antifungal protein, but also more importantly, the structure of protein and enzyme, active factors and growth factors are not damaged, so that the defect of immunological rejection reaction during mesenchymal stem cell treatment can be further avoided, and the storage time of the stem cell derived preparation can be further prolonged.
According to a preferred embodiment of the present invention, the stem cell-derived preparation may further contain at least one of a preservative, a stabilizer and an antioxidant to further improve the stability of the stem cell-derived preparation, thereby prolonging the preservation time.
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 preparation; the content of the stabilizer may be 0.1 to 0.8% by weight, preferably 0.2 to 0.5% by weight; 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 substance having a preservative function commonly known in the art, and according to a preferred embodiment of the present invention, the preservative is at least one of sodium p-hydroxybenzoate, sodium ethylparaben and chlorobutanol.
The stabilizer may be a substance having a function of stabilizing the formulation, which is generally known in the art, and according to a preferred embodiment of the present invention, the stabilizer is at least one of sodium carboxymethyl cellulose, gum tragacanth and povidone.
The antioxidant may be a substance having an antioxidant function, which is generally known 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 butylated hydroxyanisole.
According to the present invention, the stem cell-derived preparation may be in various forms of preparations such as a powder preparation, a gel preparation or a liquid preparation, and preferably, the stem cell-derived preparation is a liquid preparation.
In a second aspect, the invention provides a method of preparing a stem cell derived preparation, the method comprising: mixing the extract of the camelia plant and the extracellular product of the mesenchymal stem cells. The specific selection of the alhagi plant extract and the mesenchymal stem cell extracellular product is as described above, and is not described in detail herein.
According to a preferred embodiment of the invention, the method of preparing the stem cell derived preparation further comprises (simultaneously) mixing a preservative, a stabilizer, an antioxidant and optionally water with the camelina plant extract and the extracellular product of mesenchymal stem cells.
According to the preferred embodiment of the present invention, the preparation of the camelid plant extract and the mesenchymal stem cell extracellular product as described above is also included, and the details are not repeated herein.
In a third aspect, the invention provides a stem cell derived preparation as described above and/or the use of a stem cell derived preparation as prepared by the method described above in the preparation of a medicament or nutritional agent for alleviating a disease of the digestive system.
In the present invention, the digestive system diseases may include: intestinal spasm, halitosis, malnutrition, intestinal flora imbalance, and digestive system disorder caused by bacteria or virus, such as 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 human or animals, and may be 150 to 300mg/kg, for example, in a mouse having a body weight of about 25 g.
In the present invention, the stem cell-derived preparation may be administered orally, intravenously, and the like.
In the present invention, the pharmaceutical or nutraceutical is primarily intended for use in humans, felines or canines, particularly companion canines and/or felines.
The present invention will be described in detail below by way of examples. In the following examples, the cell growth density parameter was measured by cell counting method, and the obtained cell number was absolute number; centrifuging through a TD-4M centrifugal machine; subculturing the cells in a Thermo type incubator; proteome high throughput sequencing was determined by Orbitrap Fusion Lumos; observing tissues and organs by using an upright microscope; the alpha-MEM medium is a commercial product of Gibco company with the trade name of Eagle's; the collagenase I is a commercial product with the brand number of SCR103 of Sigma company; ultra GRo-Advanced is a commercial product of Bioscience, inc. under the brand name GMP; trypsin is a commercial product sold by Gibco corporation under the trademark TrypLETM Express; kunbai male mice were purchased from Dao laboratory animals, inc.
Preparation example 1
Preparing mesenchymal stem cell culture supernatant:
(1) Subjecting 1 year old female hybrid test dog to Sutai 50 general anesthesia, opening abdominal cavity by strict aseptic technique, cleaning mesenteric adipose tissue 50g in alpha-MEM for 3 times, and cutting to 1mm with scissors 3 In the form of powder;
(2) The adipose tissues were aspirated repeatedly through 30mL of α -MEM solution mixed with 0.1% collagenase type I, previously prepared, filtered through a 0.22 micron filter and preheated at 37 ℃, and 50mg of collagenase type I was mixed with 50mL of α -MEM solution in a centrifuge tube, and finally the whole adipose tissues were aspirated into the centrifuge tube.
(3) Sealing with alcohol-sprayed sealing film, sterilizing in 37 deg.C constant temperature water bath oscillator for 1h, and repeatedly inverting the centrifuge tube every 10min to uniformly digest adipose tissue.
(4) After 1h, the collagenase type I was neutralized with an equivalent amount of α -MEM culture medium, and centrifuged at 1000rpm for 5min after complete neutralization by repeated aspiration, and the mature adipose tissue was isolated.
(5) Discarding the upper suspension and the floating white fat, washing with 20mL alpha-MEM culture solution, pumping uniformly, filtering with 100 μm filter screen, 1000rpm, and centrifuging at 25 deg.C for 5min.
(6) Centrifuging, removing the supernatant, uniformly blowing with 1mL of 5% ultra GRo-Advanced alpha-MEM culture medium, transferring into 6-well plate, and adding 5% CO 2 And culturing in a constant-temperature incubator at 37 ℃. After the cells had grown to 90% cell fusion, the cells were digested with 0.25% trypsin, the trypsin was discarded when the cells were microscopically observed to be separated from each other, the cells were resuspended in alpha-MEM medium, and the volume ratio of the alpha-MEM medium to the cell pellet was adjustedIs 1:3, subculturing.
(7) When the growth of the adipose-derived mesenchymal primary stem cells is close to 80-90% fusion, replacing a fresh culture medium, carrying out subculture, collecting cell culture supernatant of the 3 rd generation, centrifuging the supernatant for 10min at 1500rpm by using an aseptic centrifugal tube, removing precipitates, taking the supernatant, and pre-freezing the supernatant in an ultra-low temperature refrigerator at-80 ℃ for 20 h.
(8) Placing frozen solid at-80 deg.C in a freeze drier with vacuum pressure of 0.030mbar, freeze drying at-88 deg.C for 50 hr to remove ice crystal, making into lyophilized powder, weighing, wrapping with tinfoil, registering, and storing at-80 deg.C.
Preparing the alhagi sparsifolia extract:
(1) Pulverizing semen Camellia sinensis (Bull.) Merr at 25 deg.C to 100g, soaking in 10 times of distilled water for 45 hr, ultrasonic extracting at 200W,100Hz and 60 deg.C for 1 hr, ultrasonic extracting for 3 times, and reflux extracting at 93 deg.C for 2 hr. Filtering, concentrating the filtrate under reduced pressure, and freezing at-80 deg.C for use.
(2) Freeze-drying the frozen body at-80 deg.C in a freeze-drying machine with vacuum pressure of 0.030mbar at-88 deg.C for 24 hr to remove ice crystal, making into lyophilized powder, weighing, wrapping with tinfoil, and refrigerating in a refrigerator at 4 deg.C for use.
Preparation of a liquid stem cell-derived preparation:
20g of alhagi sparsifolia freeze-dried powder prepared by the method and 10g of mesenchymal stem cell freeze-dried powder are added with 30mL of distilled water to be uniformly mixed, then 0.04g of ascorbic acid, 0.04g of sodium p-hydroxybenzoate and 0.02g of sodium carboxymethyl cellulose are added to be uniformly mixed, the distilled water is added to be complemented to 100mL, and then the mechanical stirring is carried out for 10min to ensure that the mixture is fully and uniformly mixed, thus 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 by: uniformly mixing 20g of the lyophilized powder of alhagi sparsifolia prepared by the method, 2g of the lyophilized powder of mesenchymal stem cell culture supernatant prepared by the method, 0.04g of ascorbic acid, 0.04g of sodium p-hydroxybenzoate and 0.22g of sodium carboxymethyl cellulose, adding distilled water to complement to 100mL, and mechanically stirring for 10min to fully and uniformly mix.
Preparation example 3
The method of preparation example 1 was followed except that the preparation method of the stem cell-derived preparation was modified to the preparation of a powdery preparation: mixing 25g of lyophilized alhagi sparsifolia powder prepared by the method, 250mg of lyophilized powder of mesenchymal stem cell culture supernatant, 0.04g of ascorbic acid, 0.04g of sodium p-hydroxybenzoate and 0.2g of sodium carboxymethylcellulose to obtain a powdery stem cell derivative preparation.
Preparation example 4
The method of preparation example 1 was followed except that the freeze-drying of step (8) in the preparation method of mesenchymal stem cell culture supernatant was modified to a vacuum pressure of 0.04mbar at a temperature of-60 ℃ for 30 hours. The extract is prepared by freeze drying step (2) under vacuum pressure of 0.02mbar at-80 deg.C for 40 hr.
Preparation example 5
The method of preparation example 1 was followed except that the step (1) of the preparation method of the alhagi sparsifolia extract was sequentially carried out by soaking extraction, reflux extraction and ultrasonic extraction.
Preparation example 6
The procedure of preparation example 1 was followed except that the mesenteric adipose tissue mass was replaced with an umbilical cord tissue mass.
Preparation example 7
According to the method of preparation example 1, except that the alhagi sparsifolia is replaced by sea buckthorn.
Test example 1
The polysaccharide content of the alhagi sparsifolia extract was measured according to the phenol-sulfuric acid method and the results are shown in table 2.
Proteome high-throughput sequencing: the sequencing analysis of the MSC (culture supernatant from preparation example 1) group and the Con (negative solution/control group, no MSC cells) group showed that the results of the clustering maps of differential protein expression are shown in FIGS. 1 and 2. As can be seen from fig. 1, there were 3364 proteins differentially expressed, of which 778 were the number of differential genes expressed in both MSC and Con groups, 336 were only expressed in Con group, and 2250 were only expressed in MSC group. As can be seen from FIG. 2, in the MSC group, compared with the Con group, the expressions of F1PHY1 and F1Q3I5 of the collagen family are obviously up-regulated, and compared with the expression of normal cells, the expression is up-regulated by more than 50 times, so that the mesenchymal stem cell culture supernatant prepared by the specific method has excellent platelet aggregation performance, good hemostatic effect and better smoothness and elasticity. The heparan sulfate proteoglycan family A0A5F4C710, A0A5F4CLC9 and A0A5F4CRW6 are also highly expressed, can interact with proteins such as various growth factors, chemokine enzymes and the like, influence various signal pathways, participate in metabolism, transportation, information transmission, regulation and structure maintenance of all organ systems, and play an important role in organ development.
Test example 2
Selecting 60 healthy Kunzai male mice, 5-7 weeks old, 25-28g in weight, randomly dividing the mice into 5 groups, each group comprising 12 mice: control group (NC), model group (Model), stem cell group (MSC), camel thorn group (Alhagi), preparation example 1 group (Alhagi/MSC). The Kunbai mice were bred adaptively for 7 days with intragastric intervention on day 8. MSC group was gavaged with 200mg/kg of the culture supernatant of mesenchymal stem cells of preparation 1, alhagi group was gavaged with 300mg/kg of the extract of Camellia sinensis of preparation 1, alhagi/MSC group was gavaged with 0.2mL of the stem cell-derived preparation of preparation 1, and NC group and Model group were given 0.2mL of physiological saline simultaneously. On the 15 th day of the experiment, 4% DSS was used to establish an enteritis Model, and the drinking water of Model group, MSC group, alhagi group and Alhagi/MSC group mice was changed to 4% DSS solution, and the mice were allowed to drink freely, during which time corresponding preparations were normally gazed. Focusing on the establishment of the enteritis model of the mice at the time of the establishment of the enteritis model, the mice were sacrificed on day 21 and the corresponding tissues and organs were observed, and the results are shown in FIGS. 3 to 5.
Pathological scoring of enteritis in mice was performed according to microscopic examination of pathological tissues of enteritis in mice, and the scoring criteria are shown in table 1.
TABLE 1
| Scoring | Inflammation(s) | Depth of lesion | Crypt disruption | Extent of disease (%) |
| 0 | Is free of | Is free of | Is free of | Is free of |
| 1-2 | Light and lightweight | Submucosa | 1/3 destruction of substrate | 1-25 |
| 2-4 | In (1) | Muscular layer | Substrate 2/3 destruction | 26-50 |
| 3-5 | Heavy load | Serous layer | Only the intact epithelium | 51-75 |
As shown in figure 3, the colon of NC mice group is slender and uniform in thickness, the colon mucosa is smooth and light red, congestion and edema are avoided, and a large amount of oval feces are visible in the intestinal cavity. The colon mucosa of a Model group mouse can be seen to have congestion and swelling with different degrees, bleeding spots are formed on the surface of the mucosa, blood contents are contained in the intestinal cavity, and the colon length is obviously shortened. The MSC group was similar to the Alhagi group in that it had longer colon and glossy intestinal surface compared to the Model group. The effect of the Alhagi/MSC group is obviously better than that of 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 appear, the colon is not obviously shortened, and the colon of the mouse is longer and is closer to the colon form of the NC group.
As shown in FIG. 4, the colon mucosa of the mice in the NC group has a complete structure, the glands are arranged regularly, a large number of goblet cells can be seen, and no obvious immune cell infiltration is seen in the mucosa lamina propria and the submucosa. The colon mucosa structure of the Model group mouse is destroyed and completely disappears, and a large amount of neutrophil and lymphocyte infiltration appears in the mucosa lamina propria and submucosa, so that inflammatory exudation can be seen. The colon mucosa structure of mice in the MSC group and the Alhagi group is complete, gland damage and immune cell infiltration 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 form parts are intact, the infiltration degree of mucosa inflammatory cells is reduced, and the pathological degree is lighter than that of the MSC group and the Alhagi group. Further, the grading is carried out according to the damage degree of the colon mucous membrane and the infiltration degree of inflammatory cells, so that compared with an NC group, the Model group DSS pathological score is obviously increased, and the MSC group and Alhagi group pathological score for independent stem prognosis of MSC and Alhagi are reduced, but the effect of reducing by more than 50% cannot be achieved, which indicates that the relieving effect is common when the single medicine is used alone. The Alhagi/MSC group significantly reduced the effect of pathological scoring by at least 60% and was significantly better in its ability to alleviate pathological conditions than the MSC and Alhagi groups.
As shown in FIG. 5, both at 20-fold magnification, the Model group showed significant reduction in colonic muscularis thickness compared to the NC group mice, and the MSC and Alhagi groups showed thickening after intervention alone, but still had less effect on restoring muscularis thickness compared to the Alhagi/MSC group combination.
Stool characteristics, pathological scores and muscle layer thickness of other preparations were measured in a similar manner as above, and the results are shown in Table 2.
TABLE 2 results of polysaccharide content and intestinal tract status (control group having a muscle layer thickness of about 0.8-1mm, model group having a pathological score of 5)
It can be seen from preparation examples 1 and 6 that the synergistic therapeutic effect of the mesenchymal stem cells obtained from mesenteric adipose tissue mass in combination with camel thorn is superior to that of the mesenchymal stem cells obtained by using umbilical cord tissue mass in combination. Although not shown, the effect of the combination treatment of the camel thorn and the mesenchymal stem cells obtained from the mesenteric adipose tissue mass is also better than the treatment effect of the camel thorn and the mesenchymal stem cells obtained from the peritoneal adipose tissue, and the synergistic effect is more obvious.
The invention prolongs the storage time of the preparation, has no toxic or side effect and provides the function of protecting intestines and stomach; is beneficial to supplementing special nutrient substances required by human body, relaxing bowel, relieving intestinal spasm, relieving pain, halitosis, and nutrient deficiency, and maintaining intestinal flora balance; can be used for the comprehensive prevention and treatment of digestive system disorder diseases caused by various infectious diseases such as common metabolic disturbance diarrhea, constipation and dyspepsia, canine distemper, canine parvovirus and feline infectious peritonitis.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including various technical features combined in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (10)
1. A stem cell-derived preparation comprising an extract from a plant of the genus Camellia and an extracellular product of a mesenchymal stem cell.
2. The stem cell derived preparation of claim 1, wherein the weight ratio of mesenchymal stem cell extracellular product to camelina plant extract in the stem cell derived preparation on a dry basis is 1:1.5 to 150, preferably 1:2-100.
3. A stem cell derived preparation according to claim 1 or 2, wherein the camelid plant extract is a water extract of a camelid plant, preferably a lyophilisate of a water extract of a camelid plant;
preferably, the camelid plant is at least one of camel thorn and sea buckthorn.
4. A stem cell derived preparation according to claim 1 or 2, wherein the extracellular product of mesenchymal stem cells is a mesenchymal stem cell culture supernatant, preferably a lyophilisate of a mesenchymal stem cell culture supernatant;
and/or, the mesenchymal stem cells are derived from mesenchymal stem cells of at least one of fat, bone marrow, umbilical cord and placenta of canines;
preferably, the canine is a dog.
5. The stem cell derived preparation according to claim 1, wherein the camelid plant extract is prepared by a method comprising: mixing the camel thorn plants with water and then extracting, wherein the extraction comprises soaking extraction, ultrasonic extraction and hot reflux extraction;
wherein, the soaking and extracting conditions comprise: the temperature is 15-40 ℃, and the time is 40-50h;
the ultrasonic extraction conditions comprise: 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 hot reflux extraction include: the temperature is 90-95 ℃ and the time is 1-5h;
preferably, the preparation method of the camelid plant extract further comprises drying after the extraction, preferably freeze-drying, wherein the conditions of freeze-drying comprise: the vacuum pressure is 0.01-0.05mbar; the temperature is between-90 ℃ and-80 ℃; the time is 20-30h.
6. The stem cell derived preparation of claim 1, wherein the preparation method of the extracellular product of mesenchymal stem cells comprises: subculturing the mesenchymal stem cells, collecting the culture of the cells of any generation after the 3 rd generation or the 3 rd generation, and taking the supernatant for drying.
7. The stem cell-derived preparation according to claim 6, wherein the culture is a culture of cells of any of passages 3-5, and the cell growth density in the culture is 80-90%;
and/or the way of drying the supernatant is freeze drying, and the conditions of freeze drying comprise: 0.01-0.05mbar; the temperature is between-90 ℃ and-80 ℃; the time is 40-50h.
8. A method of preparing a stem cell-derived preparation, the method comprising: mixing the alhagi plant extract with the extracellular product of mesenchymal stem cell.
9. The process according to claim 8, wherein the process further comprises the step of preparing a camelid plant extract in the following manner: mixing camelia plants with water, and extracting, wherein the extraction comprises: soaking extraction, ultrasonic extraction and hot reflux extraction;
wherein, the soaking and extracting conditions comprise: the temperature is 15-40 ℃, and the time is 40-50h;
the ultrasonic extraction conditions comprise: 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 hot reflux extraction include: the temperature is 90-95 ℃ and the time is 1-5h;
preferably, the preparation method of the camel thorn plant extract further comprises drying, preferably freeze-drying, under the conditions comprising: the vacuum pressure is 0.01-0.05mbar; the temperature is between-90 ℃ and-80 ℃; the time is 20-30h;
alternatively, the method further comprises the step of preparing extracellular products of the mesenchymal stem cells in the following manner: subculturing the mesenchymal stem cells, collecting the culture of the cells of any generation of 3 rd generation or 3 rd generation later, and taking supernatant for drying;
preferably, the culture is a culture of cells of any of generations 3-5, with a cell growth density in the culture of 80-90%;
preferably, the way of drying the supernatant is freeze-drying, and the conditions of freeze-drying include: 0.01-0.05mbar; the temperature is-90 ℃ to-80 ℃; the time is 40-50h.
10. Use of a stem cell-derived preparation according to any one of claims 1 to 7 and/or a stem cell-derived preparation produced by a method according to any one of claims 8 and 9 in the manufacture of a medicament or nutritional agent for the alleviation of digestive system disorders.
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