CN116173178A - Umbilical cord mesenchymal stem cell preparation for gout - Google Patents

Umbilical cord mesenchymal stem cell preparation for gout Download PDF

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CN116173178A
CN116173178A CN202310047279.8A CN202310047279A CN116173178A CN 116173178 A CN116173178 A CN 116173178A CN 202310047279 A CN202310047279 A CN 202310047279A CN 116173178 A CN116173178 A CN 116173178A
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张正亮
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Anhui Kemen Biotechnology Co ltd
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Abstract

The invention discloses an umbilical cord mesenchymal stem cell preparation for gout, which comprises umbilical cord mesenchymal stem cells 1-5 multiplied by 10 6 individual/mL, plantain seed polysaccharide 5-20mg/mL, mannatide 200-300 mu g/mL, tea polyphenol 50-100 mu g/mL, and balance of physiological saltAnd (3) water. The stem cell preparation provided by the invention can inhibit the generation of pro-inflammatory factors, effectively relieve symptoms such as joint swelling and pain caused by gout, and has the advantages of good curative effect and low adverse reaction.

Description

Umbilical cord mesenchymal stem cell preparation for gout
Technical Field
The invention relates to the technical field of stem cells, in particular to an umbilical mesenchymal stem cell preparation for gout.
Background
Gout is a disease in which purine metabolism and/or uric acid excretion are impaired, resulting in elevated blood uric acid. Gout is clinically manifested as hyperuricemia, gouty arthritis, and also causes nephritis and kidney uric acid calculus. Because gout is frequently repeatedly used, joint swelling and pain and even deformation are caused, the life and the work of a patient are seriously influenced, and the life quality of the patient is obviously reduced. At present, the medicine applied to gout treatment or symptom relief often brings adverse reaction after long-term administration, so that development of a treatment mode with lower adverse reaction and good curative effect becomes an important direction of gout treatment research.
The stem cells have high regeneration capacity and can repair damaged body tissues. The stem cells can also secrete various cytokines, and play roles in increasing anti-inflammatory cytokines and immune regulation factors and inhibiting the generation of pro-inflammatory factors, so that inflammation is effectively relieved. The symptoms of gout are closely related to the massive secretion of inflammatory factors, and researches show that the stem cell preparation has a certain curative effect on the treatment of gout. Because the stem cell preparation has the characteristic of low adverse reaction, the development of the stem cell preparation for treating gout with good curative effect has great application potential.
Disclosure of Invention
Based on the technical problems in the background technology, the invention provides an umbilical mesenchymal stem cell preparation for gout.
The invention provides an umbilical cord mesenchymal stem cell preparation for gout, which comprises 1-5 multiplied by 10 umbilical cord mesenchymal stem cells 6 The total weight of the composition comprises 5-20mg/mL of semen plantaginis polysaccharide, 200-300 mug/mL of mannatide, 50-100 mug/mL of tea polyphenol and the balance of physiological saline.
Preferably, the preparation method of the umbilical cord mesenchymal stem cells comprises the following steps:
(1) Sterilizing fresh umbilical cord, cleaning, removing blood vessel, cutting into blocks, sequentially digesting the obtained tissue blocks with trypsin and collagenase, washing with PBS, placing in culture dish, adding DMEM culture medium containing 10% foetal calf serum at 37deg.C and 5% CO 2 Culturing in an incubator, and after the cell fusion degree reaches 80-90%, carrying out passage according to the proportion of 1 (2-4), and marking as generation P1;
(2) And (3) subculturing the P1 generation cells to P3-P5 generation cells by adopting a DMEM culture medium containing 10% fetal bovine serum, removing the culture medium, digesting with pancreatin, and centrifugally collecting the cells to obtain the recombinant strain.
Preferably, in the step (1), the specific steps of digesting the obtained tissue block with trypsin and collagenase in sequence are as follows: placing the tissue block in a culture dish, adding trypsin with concentration of 0.125%, digesting for 20-30min in 37 ℃ water bath, centrifuging, discarding supernatant, washing the obtained tissue block with PBS, placing in the culture dish, adding collagenase with concentration of 0.2%, digesting for 1.5-2h in 37 ℃ water bath, centrifuging, and discarding supernatant.
Preferably, in the step (2), trypsin with the concentration of 0.125% is added, and after digestion for 10-15min in a water bath at 37 ℃, the mixture is centrifuged for 5-10min at 1000-1500 r/min.
Preferably, the preparation method of the plantain seed polysaccharide comprises the following steps: soaking dried semen plantaginis with 8-10 times of ethanol overnight, filtering, collecting the residue, adding 10-15 times of water, extracting at 90-95deg.C for 3-5 hr, concentrating the filtrate, precipitating with ethanol, washing the precipitate with anhydrous ethanol and acetone sequentially, freeze drying, dissolving the obtained crude polysaccharide with 10-15 times of water, removing impurity protein, dialyzing with water, concentrating, precipitating with ethanol, and drying.
The preparation method of the umbilical cord mesenchymal stem cell preparation for gout comprises the following steps:
s1, respectively dissolving mannatide and tea polyphenol in a proper amount of physiological saline to obtain a mannatide solution and a tea polyphenol solution; uniformly mixing the mannatide solution and the tea polyphenol solution, heating and stirring, and cooling to room temperature to obtain a component A;
s2, dissolving semen plantaginis polysaccharide in a proper amount of physiological saline to obtain a component B;
s3, re-suspending umbilical cord mesenchymal stem cells obtained by centrifugal collection after culture by using a proper amount of physiological saline to obtain a component C;
s4, mixing the component A, the component B and the component C, and then fixing the volume to a certain volume by using normal saline to obtain the compound.
Preferably, in S1, the conditions of heating and stirring are: stirring at 30-40deg.C for 30-60min.
An umbilical mesenchymal stem cell preparation for gout is prepared by the preparation method.
The beneficial effects of the invention are as follows:
the stem cell preparation takes umbilical cord mesenchymal stem cells, plantain seed polysaccharide, mannatide and tea polyphenol as main components, wherein the umbilical cord mesenchymal stem cells can play roles in repairing damaged organism tissues, increasing anti-inflammatory cytokines and immune regulation factors, inhibiting the generation of pro-inflammatory factors and effectively relieving inflammation; the plantain seed polysaccharide and the tea polyphenol not only have the anti-oxidation function and improve the activity of umbilical mesenchymal stem cells, but also can reduce the generation of inflammatory factors IL-1 beta, thus having good anti-inflammatory function; the semen plantaginis polysaccharide, the mannatide and the tea polyphenol are compounded, so that a synergistic effect can be achieved, the stability of a stem cell preparation system is improved, and the effects of anti-inflammatory and repairing can be better exerted. In the preparation process, the tea polyphenol and the mannatide are firstly mixed, so that a polyphenol-polypeptide covalent complex can be formed between the tea polyphenol and the mannatide, and then the complex is mixed with the plantain seed polysaccharide and the umbilical cord mesenchymal stem cells, the stability and the bioactivity of the tea polyphenol and the plantain seed polysaccharide in the system can be improved, the anti-inflammatory effect can be better exerted, and the composite system formed by the method is favorable for maintaining the bioactivity of the umbilical cord mesenchymal stem cells, so that the effects of repairing tissues and inhibiting the generation of pro-inflammatory factors of the umbilical cord mesenchymal stem cells can be better exerted. Therefore, the stem cell preparation provided by the invention can inhibit the generation of pro-inflammatory factors, effectively relieve symptoms such as joint swelling and pain caused by gout, and has the advantages of good curative effect and low adverse reaction.
Detailed Description
The technical scheme of the invention is described in detail through specific embodiments.
Example 1
An umbilical cord mesenchymal stem cell preparation for gout comprises umbilical cord mesenchymal stem cells 3×10 6 individual/mL, 15mg/mL of plantain seed polysaccharide, 250 mug/mL of mannatide, 80 mug/mL of tea polyphenol and the balance of physiological saline.
The preparation method comprises the following steps:
s1, respectively dissolving 250mg of mannatide in 100mL of physiological saline, and dissolving 80mg of tea polyphenol in 100mL of physiological saline to obtain a mannatide solution and a tea polyphenol solution; uniformly mixing the mannatide solution and the tea polyphenol solution, stirring for 40min at 35 ℃, and cooling to room temperature to obtain a component A;
s2, soaking dried semen plantaginis with 9 times of ethanol with the concentration of 80% for overnight, filtering, taking filter residues, adding 12 times of water, extracting for 4 hours at 90 ℃, concentrating filtrate, precipitating with ethanol with the concentration of 80%, washing precipitate with absolute ethanol and acetone in sequence, freeze-drying, adding 12 times of water into the obtained crude polysaccharide for dissolving, adding 1/3 volume of chloroform-n-butanol mixed solution (obtained by mixing chloroform and n-butanol according to the volume ratio of 4:1), shaking and centrifuging to remove protein layers at the intersection of water phase and organic phase, re-multiplexing chloroform-n-butanol mixed solution to remove foreign proteins until no protein layer exists at the intersection of water phase and organic phase, dialyzing the water phase, concentrating, precipitating with ethanol with the concentration of 80%, and drying to obtain semen plantaginis polysaccharide;
dissolving 15g of semen plantaginis polysaccharide in 300mL of physiological saline to obtain a component B;
s3, taking fresh umbilical cord, removing blood vessels, cutting the umbilical cord into blocks after disinfection and cleaning, and sequentially digesting the obtained tissue blocks by trypsin and collagenase, wherein the specific steps are as follows: placing the tissue block in a culture dish, adding trypsin with concentration of 0.125%, digesting for 20min at 37deg.C in water bath, centrifuging, discarding supernatant, washing the obtained tissue block with PBS, placing in the culture dish, adding collagenase with concentration of 0.2%, digesting for 1.5 hr at 37deg.C in water bath, centrifuging, discarding supernatant, washing with PBS, placing in the culture dish, adding DMEM medium containing 10% foetal calf serum at 37deg.C and 5% CO 2 Culturing in an incubator, and after the cell fusion degree reaches 80%, carrying out passage according to the proportion of 1:3, and marking as generation P1;
subculturing the P1 generation cells to P3 generation cells by adopting a DMEM culture medium containing 10% of fetal bovine serum, removing the culture medium, adding trypsin with the concentration of 0.125%, digesting for 10min in a water bath at 37 ℃, centrifuging for 5min at 1000r/min, and collecting umbilical cord mesenchymal stem cells;
the collected umbilical cord mesenchymal stem cells were resuspended to a density of 3X 10 with 100mL of physiological saline 7 Obtaining component C by using the total volume of the components per mL;
s4, mixing the component A, the component B and the component C, and then fixing the volume to 1000mL by using normal saline to obtain the compound.
Example 2
An umbilical cord mesenchymal stem cell preparation for gout comprises umbilical cord mesenchymal stem cells 1×10 6 The total weight of the polysaccharide is 5mg/mL, the total weight of the polysaccharide is 200 mu g/mL, the total weight of the polysaccharide is 50 mu g/mL, and the balance is physiological saline.
The preparation method comprises the following steps:
s1, respectively dissolving 200mg of mannatide in 100mL of physiological saline, and dissolving 50mg of tea polyphenol in 100mL of physiological saline to obtain a mannatide solution and a tea polyphenol solution; uniformly mixing the mannatide solution and the tea polyphenol solution, stirring for 60min at 30 ℃, and cooling to room temperature to obtain a component A;
s2, soaking dried semen plantaginis with 80% ethanol by weight of 10 times of the weight of the dried semen plantaginis overnight, filtering, taking filter residues, adding 15 times of water, extracting for 3 hours at 95 ℃, concentrating filtrate, precipitating with 80% ethanol, washing precipitate with absolute ethanol and acetone in sequence, freeze-drying, adding 15 times of water into the obtained crude polysaccharide to dissolve, adding 1/3 volume of chloroform-n-butanol mixed solution (obtained by mixing chloroform and n-butanol according to the volume ratio of 4:1), shaking and centrifuging to remove protein layers at the interface of water phase and organic phase, re-multiplexing chloroform-n-butanol mixed solution to remove foreign proteins until no protein layer exists at the interface of water phase and organic phase, dialyzing the water phase, concentrating, precipitating with 80% ethanol, and drying to obtain semen plantaginis polysaccharide;
dissolving 5g of semen plantaginis polysaccharide in 300mL of physiological saline to obtain a component B;
s3, taking fresh umbilical cord, removing blood vessels, cutting the umbilical cord into blocks after disinfection and cleaning, and sequentially digesting the obtained tissue blocks by trypsin and collagenase, wherein the specific steps are as follows: placing the tissue block in a culture dish, adding trypsin with concentration of 0.125%Digesting for 20min in 37deg.C water bath, centrifuging, removing supernatant, washing the obtained tissue block with PBS, placing in culture dish, adding collagenase with concentration of 0.2%, digesting for 1.5 hr in 37deg.C water bath, centrifuging, discarding supernatant, washing with PBS, placing in culture dish, adding DMEM medium containing 10% foetal calf serum at 37deg.C and 5% CO 2 Culturing in an incubator, and after the cell fusion degree reaches 80%, carrying out passage according to the proportion of 1:2, and marking as generation P1;
subculturing the P1 generation cells to P3 generation cells by adopting a DMEM culture medium containing 10% of fetal bovine serum, removing the culture medium, adding trypsin with the concentration of 0.125%, digesting for 10min in a water bath at 37 ℃, centrifuging for 5min at 1000r/min, and collecting umbilical cord mesenchymal stem cells;
the collected umbilical cord mesenchymal stem cells were resuspended to a density of 1X 10 with 100mL of physiological saline 7 Obtaining component C by using the total volume of the components per mL;
s4, mixing the component A, the component B and the component C, and then fixing the volume to 1000mL by using normal saline to obtain the compound.
Example 3
An umbilical cord mesenchymal stem cell preparation for gout comprises umbilical cord mesenchymal stem cells 5×10 6 The total weight of the composition is 20mg/mL, 20mg/mL of plantain seed polysaccharide, 300 mug/mL of mannatide, 100 mug/mL of tea polyphenol and the balance of physiological saline.
The preparation method comprises the following steps:
s1, respectively dissolving 300mg of mannatide in 100mL of physiological saline, and dissolving 100mg of tea polyphenol in 100mL of physiological saline to obtain a mannatide solution and a tea polyphenol solution; uniformly mixing the mannatide solution and the tea polyphenol solution, stirring for 30min at 40 ℃, and cooling to room temperature to obtain a component A;
s2, soaking dried semen plantaginis with 8 times of ethanol with the concentration of 80% for overnight, filtering, taking filter residues, adding 10 times of water, extracting for 5 hours at 90 ℃, concentrating filtrate, precipitating with ethanol with the concentration of 80%, washing precipitate with absolute ethanol and acetone in sequence, freeze-drying, adding 10 times of water into the obtained crude polysaccharide for dissolving, adding 1/3 volume of chloroform-n-butanol mixed solution (obtained by mixing chloroform and n-butanol according to the volume ratio of 4:1), shaking and centrifuging to remove protein layers at the intersection of water phase and organic phase, re-multiplexing chloroform-n-butanol mixed solution to remove mixed proteins until no protein layer exists at the intersection of water phase and organic phase, dialyzing the water phase, concentrating, precipitating with ethanol with the concentration of 80%, and drying to obtain semen plantaginis polysaccharide;
dissolving 20g of semen plantaginis polysaccharide in 500mL of physiological saline to obtain a component B;
s3, taking fresh umbilical cord, removing blood vessels, cutting the umbilical cord into blocks after disinfection and cleaning, and sequentially digesting the obtained tissue blocks by trypsin and collagenase, wherein the specific steps are as follows: placing the tissue block in a culture dish, adding trypsin with concentration of 0.125%, digesting for 30min in 37 deg.C water bath, centrifuging, discarding supernatant, washing the obtained tissue block with PBS, placing in the culture dish, adding collagenase with concentration of 0.2%, digesting for 2 hr in 37 deg.C water bath, centrifuging, discarding supernatant, washing with PBS, placing in the culture dish, adding DMEM medium containing 10% fetal bovine serum at 37deg.C and 5% CO 2 Culturing in an incubator, and after the cell fusion degree reaches 80%, carrying out passage according to the proportion of 1:4, and marking as generation P1;
subculturing the P1 generation cells to P3 generation cells by adopting a DMEM culture medium containing 10% of fetal bovine serum, removing the culture medium, adding trypsin with the concentration of 0.125%, digesting for 15min in a water bath at 37 ℃, centrifuging for 10min at 1500r/min, and collecting umbilical cord mesenchymal stem cells;
the collected umbilical cord mesenchymal stem cells were resuspended to a density of 5X 10 with 100mL of physiological saline 7 Obtaining component C by using the total volume of the components per mL;
s4, mixing the component A, the component B and the component C, and then fixing the volume to 1000mL by using normal saline to obtain the compound.
Comparative example 1
The only differences between comparative example 1 and example 2 are: no mannatide is contained; the method comprises the following steps:
an umbilical cord mesenchymal stem cell preparation comprises umbilical cord mesenchymal stem cells 1×10 6 Each mL (volume),Semen plantaginis polysaccharide 5mg/mL, tea polyphenols 50 μg/mL, and rest physiological saline.
The preparation method comprises the following steps:
s1, dissolving 50mg of tea polyphenol in 100mL of physiological saline to obtain a component A;
s2, soaking dried semen plantaginis with 80% ethanol by weight of 10 times of the weight of the dried semen plantaginis overnight, filtering, taking filter residues, adding 15 times of water, extracting for 3 hours at 95 ℃, concentrating filtrate, precipitating with 80% ethanol, washing precipitate with absolute ethanol and acetone in sequence, freeze-drying, adding 15 times of water into the obtained crude polysaccharide to dissolve, adding 1/3 volume of chloroform-n-butanol mixed solution (obtained by mixing chloroform and n-butanol according to the volume ratio of 4:1), shaking and centrifuging to remove protein layers at the interface of water phase and organic phase, re-multiplexing chloroform-n-butanol mixed solution to remove foreign proteins until no protein layer exists at the interface of water phase and organic phase, dialyzing the water phase, concentrating, precipitating with 80% ethanol, and drying to obtain semen plantaginis polysaccharide;
dissolving 5g of semen plantaginis polysaccharide in 300mL of physiological saline to obtain a component B;
s3, taking fresh umbilical cord, removing blood vessels, cutting the umbilical cord into blocks after disinfection and cleaning, and sequentially digesting the obtained tissue blocks by trypsin and collagenase, wherein the specific steps are as follows: placing the tissue block in a culture dish, adding trypsin with concentration of 0.125%, digesting for 20min at 37deg.C in water bath, centrifuging, discarding supernatant, washing the obtained tissue block with PBS, placing in the culture dish, adding collagenase with concentration of 0.2%, digesting for 1.5 hr at 37deg.C in water bath, centrifuging, discarding supernatant, washing with PBS, placing in the culture dish, adding DMEM medium containing 10% foetal calf serum at 37deg.C and 5% CO 2 Culturing in an incubator, and after the cell fusion degree reaches 80%, carrying out passage according to the proportion of 1:2, and marking as generation P1;
subculturing the P1 generation cells to P3 generation cells by adopting a DMEM culture medium containing 10% of fetal bovine serum, removing the culture medium, adding trypsin with the concentration of 0.125%, digesting for 10min in a water bath at 37 ℃, centrifuging for 5min at 1000r/min, and collecting umbilical cord mesenchymal stem cells;
the collected umbilical cord mesenchymal stem cells were resuspended to a density of 1X 10 with 100mL of physiological saline 7 Obtaining component C by using the total volume of the components per mL;
s4, mixing the component A, the component B and the component C, and then fixing the volume to 1000mL by using normal saline to obtain the compound.
Comparative example 2
Comparative example 2 differs from example 2 only in that: does not contain tea polyphenol; the method comprises the following steps:
an umbilical cord mesenchymal stem cell preparation comprises umbilical cord mesenchymal stem cells 1×10 6 individual/mL, psyllium polysaccharide 5mg/mL, mannatide 200 μg/mL, balance physiological saline.
The preparation method comprises the following steps:
s1, dissolving 200mg of mannatide in 100mL of physiological saline to obtain a component A;
s2, soaking dried semen plantaginis with 80% ethanol by weight of 10 times of the weight of the dried semen plantaginis overnight, filtering, taking filter residues, adding 15 times of water, extracting for 3 hours at 95 ℃, concentrating filtrate, precipitating with 80% ethanol, washing precipitate with absolute ethanol and acetone in sequence, freeze-drying, adding 15 times of water into the obtained crude polysaccharide to dissolve, adding 1/3 volume of chloroform-n-butanol mixed solution (obtained by mixing chloroform and n-butanol according to the volume ratio of 4:1), shaking and centrifuging to remove protein layers at the interface of water phase and organic phase, re-multiplexing chloroform-n-butanol mixed solution to remove foreign proteins until no protein layer exists at the interface of water phase and organic phase, dialyzing the water phase, concentrating, precipitating with 80% ethanol, and drying to obtain semen plantaginis polysaccharide;
dissolving 5g of semen plantaginis polysaccharide in 300mL of physiological saline to obtain a component B;
s3, taking fresh umbilical cord, removing blood vessels, cutting the umbilical cord into blocks after disinfection and cleaning, and sequentially digesting the obtained tissue blocks by trypsin and collagenase, wherein the specific steps are as follows: placing the tissue block in a culture dish, adding trypsin with concentration of 0.125%, digesting for 20min in water bath at 37deg.C, centrifuging, removing supernatant, and collecting the final productWashing tissue block with PBS, placing in culture dish, adding collagenase with concentration of 0.2%, digesting for 1.5 hr in water bath at 37deg.C, centrifuging, discarding supernatant, washing with PBS, placing in culture dish, adding DMEM medium containing 10% foetal calf serum at 37deg.C and 5% CO 2 Culturing in an incubator, and after the cell fusion degree reaches 80%, carrying out passage according to the proportion of 1:2, and marking as generation P1;
subculturing the P1 generation cells to P3 generation cells by adopting a DMEM culture medium containing 10% of fetal bovine serum, removing the culture medium, adding trypsin with the concentration of 0.125%, digesting for 10min in a water bath at 37 ℃, centrifuging for 5min at 1000r/min, and collecting umbilical cord mesenchymal stem cells;
the collected umbilical cord mesenchymal stem cells were resuspended to a density of 1X 10 with 100mL of physiological saline 7 Obtaining component C by using the total volume of the components per mL;
s4, mixing the component A, the component B and the component C, and then fixing the volume to 1000mL by using normal saline to obtain the compound.
Comparative example 3
Comparative example 3 differs from example 2 only in that: no psyllium polysaccharide; the method comprises the following steps:
an umbilical cord mesenchymal stem cell preparation comprises umbilical cord mesenchymal stem cells 1×10 6 Individual/mL, 200. Mu.g/mL of mannatide, 50. Mu.g/mL of tea polyphenol, and the balance of physiological saline.
The preparation method comprises the following steps:
s1, respectively dissolving 200mg of mannatide in 100mL of physiological saline, and dissolving 50mg of tea polyphenol in 100mL of physiological saline to obtain a mannatide solution and a tea polyphenol solution; uniformly mixing the mannatide solution and the tea polyphenol solution, stirring for 60min at 30 ℃, and cooling to room temperature to obtain a component A;
s2, taking fresh umbilical cord, removing blood vessels, cutting the umbilical cord into blocks after disinfection and cleaning, and sequentially digesting the obtained tissue blocks by trypsin and collagenase, wherein the specific steps are as follows: placing the tissue block in a culture dish, adding trypsin with concentration of 0.125%, digesting for 20min in water bath at 37deg.C, centrifuging, removing supernatant, washing the obtained tissue block with PBSPlacing in a culture dish, adding collagenase with concentration of 0.2%, digesting for 1.5 hr in 37deg.C water bath, centrifuging, discarding supernatant, washing with PBS, placing in culture dish, adding DMEM medium containing 10% foetal calf serum at 37deg.C and 5% CO 2 Culturing in an incubator, and after the cell fusion degree reaches 80%, carrying out passage according to the proportion of 1:2, and marking as generation P1;
subculturing the P1 generation cells to P3 generation cells by adopting a DMEM culture medium containing 10% of fetal bovine serum, removing the culture medium, adding trypsin with the concentration of 0.125%, digesting for 10min in a water bath at 37 ℃, centrifuging for 5min at 1000r/min, and collecting umbilical cord mesenchymal stem cells;
the collected umbilical cord mesenchymal stem cells were resuspended to a density of 1X 10 with 100mL of physiological saline 7 Obtaining a component B by the number of the components per mL;
s4, mixing the component A and the component B, and then, fixing the volume to 1000mL by using normal saline to obtain the compound.
Test examples
Gout is a crystal-associated arthropathy caused by deposition of monosodium urate (MSU). Its clinical symptoms are closely related to the formation and deposition of MSU crystals in tissues, as well as acute and/or chronic inflammatory responses to MSU crystals. The MSU crystals can trigger the release of inflammatory factors such as IL-1β, which is an important mediator in the inflammatory response caused by the MSU crystals. Thus, detection of IL-1β can be used to assess the severity of gout.
The method utilizes the method of injecting MSU crystals to construct the gout model of the mice. The specific method comprises the following steps: the right ankle joint of C57/BL6 mice was injected with 50. Mu.L of MSU crystal suspension (dose 1 mg). After the injection, 100. Mu.L of the stem cell preparations of example 2 and comparative examples 1 to 3, designated as experimental group, comparative group 1, comparative group 2, comparative group 3, were subcutaneously injected into the tail of the mice at the 1d, 7d, respectively; 100. Mu.L of physiological saline was injected subcutaneously into the tail of the mice at 1d and 7d, respectively, and the mice were designated as a blank group. Wherein the experimental group, the comparison groups 1-3 and the blank control group are all 10 mice.
The effect of each group of stem cell preparations on gout was evaluated by measuring the swelling degree of the toe part of the mouse and the IL-1 beta content in serum 10d after the injection. The calculation formula of the toe swelling degree is as follows: toe swelling = (post-test mouse toe volume-pre-test mouse toe volume)/pre-test mouse toe volume x 100%, where the pre-test mouse toe volume measurement time point is 0h post-molding and the post-test mouse toe volume measurement time point is 10d post-molding. The evaluation results are shown in table 1:
TABLE 1
Figure BDA0004056103500000121
Therefore, the stem cell preparation can effectively reduce the release amount of the gout key inflammatory factor IL-1 beta, obviously relieve the symptoms such as joint swelling and pain caused by gout, and has good curative effect.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.

Claims (8)

1. An umbilical mesenchymal stem cell preparation for gout is characterized by comprising umbilical mesenchymal stem cells 1-5 multiplied by 10 6 The total weight of the composition comprises 5-20mg/mL of semen plantaginis polysaccharide, 200-300 mug/mL of mannatide, 50-100 mug/mL of tea polyphenol and the balance of physiological saline.
2. The umbilical cord mesenchymal stem cell preparation for gout according to claim 1, wherein the umbilical cord mesenchymal stem cell preparation method comprises the following steps:
(1) Sterilizing fresh umbilical cord, cleaning, removing blood vessel, cutting into blocks, sequentially digesting the obtained tissue blocks with trypsin and collagenase, washing with PBS, placing in culture dish, adding DMEM culture medium containing 10% foetal calf serum at 37deg.C and 5% CO 2 In the incubatorCulturing, and after the cell fusion degree reaches 80-90%, carrying out passage according to the proportion of 1 (2-4), and marking as generation P1;
(2) And (3) subculturing the P1 generation cells to P3-P5 generation cells by adopting a DMEM culture medium containing 10% fetal bovine serum, removing the culture medium, digesting with pancreatin, and centrifugally collecting the cells to obtain the recombinant strain.
3. The umbilical cord mesenchymal stem cell preparation for gout according to claim 2, wherein in the step (1), the specific steps of sequentially digesting the obtained tissue mass with trypsin and collagenase are as follows: placing the tissue block in a culture dish, adding trypsin with concentration of 0.125%, digesting for 20-30min in 37 ℃ water bath, centrifuging, discarding supernatant, washing the obtained tissue block with PBS, placing in the culture dish, adding collagenase with concentration of 0.2%, digesting for 1.5-2h in 37 ℃ water bath, centrifuging, and discarding supernatant.
4. The umbilical mesenchymal stem cell preparation for gout according to claim 2, wherein in the step (2), trypsin with a concentration of 0.125% is added, and after digestion for 10-15min in a water bath at 37 ℃, the mixture is centrifuged for 5-10min at 1000-1500 r/min.
5. The umbilical mesenchymal stem cell preparation for gout according to claim 1, wherein the preparation method of the plantain seed polysaccharide is as follows: soaking dried semen plantaginis with 8-10 times of ethanol overnight, filtering, collecting the residue, adding 10-15 times of water, extracting at 90-95deg.C for 3-5 hr, concentrating the filtrate, precipitating with ethanol, washing the precipitate with anhydrous ethanol and acetone sequentially, freeze drying, dissolving the obtained crude polysaccharide with 10-15 times of water, removing impurity protein, dialyzing with water, concentrating, precipitating with ethanol, and drying.
6. A method of preparing an umbilical mesenchymal stem cell preparation for gout according to any one of claims 1 to 5, comprising the steps of:
s1, respectively dissolving mannatide and tea polyphenol in a proper amount of physiological saline to obtain a mannatide solution and a tea polyphenol solution; uniformly mixing the mannatide solution and the tea polyphenol solution, heating and stirring, and cooling to room temperature to obtain a component A;
s2, dissolving semen plantaginis polysaccharide in a proper amount of physiological saline to obtain a component B;
s3, re-suspending umbilical cord mesenchymal stem cells obtained by centrifugal collection after culture by using a proper amount of physiological saline to obtain a component C;
s4, mixing the component A, the component B and the component C, and then fixing the volume to a certain volume by using normal saline to obtain the compound.
7. The method for preparing an umbilical cord mesenchymal stem cell preparation for gout according to claim 6, wherein in S1, the heating and stirring conditions are as follows: stirring at 30-40deg.C for 30-60min.
8. An umbilical mesenchymal stem cell preparation for gout, characterized by being prepared by the preparation method of claim 6 or 7.
CN202310047279.8A 2023-01-31 2023-01-31 Umbilical cord mesenchymal stem cell preparation for gout Pending CN116173178A (en)

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