Placenta-derived cytokine gel composition, preparation method therefor and use thereof

Abstract

Disclosed are a placenta-derived cytokine gel composition, a preparation method therefor and a use thereof, relating to the technical field of bioengineering. Based on a preparation method for a placenta-derived cytokine, placental tissue is extracted and cleaned, then minced; a digestive enzyme is added to perform a digestion reaction; a human lymphocyte separation liquid is added to a cell suspension and centrifuged, then a white film layer located in the middle of the centrifuged product is suctioned out, resuspended and centrifuged; a precipitate is resuspended and cells are counted, to obtain mononuclear cells having cell activity; the cells are then ultrasonically crushed and lysed under ice bath conditions, to obtain a cell lysate; the cell lysate is filtered and centrifuged, and a supernatant is collected and resuspended, to obtain a cytokine suspension. In the preparation method of the present invention, after digesting the placental tissue, a large number of mononuclear cells are separated by means of human lymphocyte separation liquid, and then the cells are lysed by means of ultrasonic disruption, thereby obtaining a large amount of highly active cytokine.

Description

Placenta-derived cytokine gel complex and its preparation method and application Technical Field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to a placenta-derived cytokine gel complex and a preparation method and application thereof. Background Art

[0002] The placenta is a crucial organ for the exchange of substances between the fetus and the mother. It is the tissue-to-tissue connection between the mother and the fetus, formed during pregnancy by the combined growth of the embryonic membrane and the maternal endometrium. Reports indicate that placental tissue contains a variety of cytokines, such as EGF, bFGF, TGF-β, VEGF, NGF, and IL-6. These cytokines can effectively regulate signal transduction and activate existing cells, thereby achieving wound repair and anti-aging effects. However, existing techniques for extracting placental-derived cytokines have low extraction efficiency and activity.

[0003] Currently, cytokines extracted from placental tissue are mainly in the form of liquid and lyophilized powder. The fluidity of the preparations formed by these two forms is relatively large, which is not conducive to the full play of the role of cytokines and the maintenance of the activity of cytokines. Gel preparations are a new drug dosage form that has emerged in China in recent years. They are transparent or translucent semi-solid preparations that are made of drugs and suitable excipients in a uniform or suspended state. Gel preparations have good biocompatibility and good stability. They have sustained-release and controlled-release effects on drug release and can effectively protect the activity of proteins. They are ideal wound repair drug preparations. However, there are no reports on the preparation of placenta-derived cytokines into gel preparations.

[0004] The present invention combines these two approaches to provide a method for preparing a gel complex containing placenta-derived cytokines. The gel in this complex effectively maintains the activity of cytokines and can be more effectively used to treat various difficult-to-heal wounds, such as skin burns, diabetic foot, venous ulcers, and anal fistulas.

[0005] The present invention fully combines cytokines with gel preparations to prepare gel complexes, which can not only more effectively maintain the activity of cytokines, but also better combine cytokines with wound surfaces and thus give full play to their wound repairing effect. Summary of the Invention

[0006] Based on this, the present invention provides a method for preparing placenta-derived cytokines, which obtains highly active placenta-derived active cytokines with high extraction efficiency. At the same time, further preparation of a gel complex can more effectively protect the activity of the cytokines and effectively repair refractory wounds.

[0007] The first aspect of the present invention is to provide a method for preparing placenta-derived cytokines, comprising the following steps:

[0008] S1, taking placental tissue, cleaning it, mincing it into a minced state, adding digestive enzymes to perform a digestion reaction, then passing the digestion fluid through a cell sieve, collecting the filtrate after the sieve, centrifuging it, and resuspending the precipitate to obtain a first suspension;

[0009] S2, adding human lymphocyte separation fluid to the first cell suspension, centrifuging, then aspirating the buffy coat layer in the middle of the centrifuged product, and resuspending to obtain a second suspension;

[0010] S3, centrifuging the second suspension, resuspending the precipitate and counting the cells to obtain mononuclear cells with cell activity, and then lysing the cells in an ice bath to obtain a cell lysate;

[0011] S4, filtering the cell lysate and centrifuging it, collecting the supernatant, and resuspending it to obtain a cytokine suspension.

[0012] In some embodiments, in step S1, the reaction temperature of the digestion reaction is 36-38° C., the reaction time is 100-140 min, and the mixture is shaken every 25-35 min; and the pore size of the cell sieve is 70-100 mesh.

[0013] In some embodiments, in step S2, the volume ratio of the human lymphocyte separation fluid to the first cell suspension is 1:(1.5-3), the centrifugal speed is 1900-2200 rpm, the centrifugal time is 18-25 min, and the centrifugal speed increase and decrease are both 5.

[0014] In some embodiments, in step S3, the cell lysis is performed by ultrasonic disruption, and the treatment conditions include: temperature of 2-8°C, ultrasonic power of 90-110W, ultrasonic operation for 5-7 seconds followed by a 2-4 seconds pause and cycle treatment, and a total ultrasonic time of 18-22 minutes.

[0015] In some embodiments, in step S4, the pore size of the filtration is 35-45 μm;

[0016] In steps S1, S3 and S4, the centrifugal speed is 1700-1900 rpm and the centrifugal time is 4-6 min.

[0017] The second aspect of the present invention provides a cytokine gel complex, comprising cytokines prepared based on the preparation method of placenta-derived cytokines.

[0018] In some embodiments, the volume ratio of the cytokine suspension to the mixed gel is 1:(1-2), wherein the cytokine suspension is composed of cells with a concentration of (0.8-1.2)×10 6The mixed gel is prepared by lysing a cell suspension of 100 cells / ml. The mixed gel includes the following components in percentage by mass:

[0019] Sodium alginate 2~5%, collagen 0.5~3%, human serum albumin 3~8%, vitamin C 0.1~2%, glycerol 1~5%, and normal saline as the balance.

[0020] The third aspect of the present invention provides a method for preparing a cytokine gel complex, comprising the following steps:

[0021] 1) Dissolve the formulated amount of sodium alginate in physiological saline until swelled, then stir thoroughly. Then add collagen, human serum albumin, vitamin C, and glycerol and stir thoroughly to obtain a mixed gel.

[0022] 2) Evenly mixing the mixed gel and the cytokine to obtain a cytokine gel complex.

[0023] In some embodiments, the swelling temperature is 23-28° C., and the swelling time is 24-48 hours.

[0024] The fourth aspect of the present invention provides a use of a cytokine gel complex in the preparation of a drug for treating skin burns, diabetic foot, venous ulcers or anal fistulas.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention provides a method for preparing placenta-derived cytokines. After digesting the placenta tissue, human lymphocytes are stimulated to produce a large amount of cytokines, which are then further extracted to obtain mononuclear cells, which are then lysed by ultrasonication to obtain a large amount of highly active cytokines.

[0027] The cytokine gel complex of the present invention can more effectively protect the activity of cytokines. Cell activity is not affected when placed at 2-8°C for 3 months. The highly active cytokines rich in the complex can effectively repair refractory wounds and can be effectively used to treat various refractory wound diseases such as skin burns, diabetic foot, venous ulcers, and anal fistulas. Implementation Method

[0028] The technical solutions of the present invention are further illustrated by specific examples below. Those skilled in the art should understand that the examples are only for helping to understand the present invention and should not be regarded as specific limitations of the present invention.

[0029] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0030] A method for preparing placenta-derived cytokines comprises the following steps:

[0031] S1, taking placental tissue, cleaning it, mincing it into a minced state, adding digestive enzymes to perform a digestion reaction, then passing the digestion fluid through a cell sieve, collecting the filtrate after the sieve, centrifuging it, and resuspending the precipitate to obtain a first suspension;

[0032] S2, adding human lymphocyte separation fluid to the first cell suspension, centrifuging, then aspirating the buffy coat layer in the middle of the centrifuged product, and resuspending to obtain a second suspension;

[0033] S3, centrifuging the second suspension, resuspending the precipitate and counting the cells to obtain mononuclear cells with cell activity, and then lysing the cells in an ice bath to obtain a cell lysate;

[0034] S4, filtering the cell lysate and centrifuging it, collecting the supernatant, and resuspending it to obtain a cytokine suspension.

[0035] In some embodiments, in step S1, the reaction temperature of the digestion reaction is 36-38° C., the reaction time is 100-140 min, and the mixture is shaken every 25-35 min; and the pore size of the cell sieve is 70-100 mesh.

[0036] In some embodiments, in step S2, the volume ratio of the human lymphocyte separation fluid to the first cell suspension is 1:(1.5-3), the centrifugal speed is 1900-2200 rpm, the centrifugal time is 18-25 min, and the centrifugal speed increase and decrease are both 5.

[0037] In some embodiments, in step S3, the cell lysis is performed by ultrasonic disruption, and the treatment conditions include: temperature of 2-8°C, ultrasonic power of 90-110W, ultrasonic operation for 5-7 seconds followed by a 2-4 seconds pause and cycle treatment, and a total ultrasonic time of 18-22 minutes.

[0038] In some embodiments, in step S4, the pore size of the filtration is 35-45 μm;

[0039] In steps S1, S3 and S4, the centrifugal speed is 1700-1900 rpm and the centrifugal time is 4-6 min.

[0040] A cytokine gel complex comprises cytokines prepared by a preparation method of placenta-derived cytokines.

[0041] In some embodiments, the following components are included in weight percentage:

[0042] The invention comprises a cytokine suspension and a mixed gel at a volume ratio of 1:(1-2), wherein the cytokine suspension is composed of cells with a concentration of (0.8-1.2)×10 6 The mixed gel is prepared by lysing a cell suspension of 100 cells / ml. The mixed gel includes the following components in percentage by mass:

[0043] Sodium alginate 2~5%, collagen 0.5~3%, human serum albumin 3~8%, vitamin C 0.1~2%, glycerol 1~5%, and normal saline as the balance.

[0044] A method for preparing a cytokine gel complex comprises the following steps:

[0045] 1) Dissolve the formulated amount of sodium alginate in physiological saline until swelled, then stir thoroughly. Then add collagen, human serum albumin, vitamin C, and glycerol and stir thoroughly to obtain a mixed gel.

[0046] 2) Evenly mixing the mixed gel and the cytokine to obtain a cytokine gel complex.

[0047] In some embodiments, the swelling temperature is 23-28° C., and the swelling time is 24-48 hours.

[0048] The invention discloses an application of a cytokine gel complex in the preparation of a medicine for treating skin burns, diabetic foot, venous ulcer or anal fistula. Example 1

[0049] A method for preparing placenta-derived cytokines comprises the following steps:

[0050] S1: Remove the placenta from the sample collection bag and place it in a sterilized stainless steel tray. Wash it 2-3 times with normal saline to remove contamination and large blood clots. Then, cut 250g of placental tissue into 0.3cm × 0.3cm tissue pieces and repeatedly wash them with normal saline to obtain clean placental tissue.

[0051] S2: Place clean placental tissue in a 50 mL centrifuge tube and mince until finely minced. Place the minced tissue in a 500 mL blue-capped bottle and add digestive enzymes. Digest the tissue in a 37°C water bath for 120 min, shaking every 30 min.

[0052] S3, then collecting the digestion fluid and passing it through an 80-mesh cell sieve, collecting the filtrate after sieving, centrifuging at 1800 rpm for 5 minutes, discarding the supernatant, and resuspending the precipitate with physiological saline to obtain a first suspension;

[0053] S4, adding human lymphocyte separation solution to the cell suspension, centrifuging at 2000 rpm for 20 min with slow ascent and descent, then aspirating the buffy coat layer in the middle of the centrifuged product and resuspending with physiological saline to obtain a second suspension;

[0054] S5, the second suspension was centrifuged at 1800 rpm for 5 min, the precipitate was resuspended and the cells were counted to obtain mononuclear cells with cell activity, and the number of cells was calculated as 1×10 6 Cells were divided into 50 ml centrifuge tubes at a concentration of 100 cells / ml and then lysed using a cell ultrasonic disruptor in an ice bath to obtain a cell lysate. The ultrasonic disruptor was set to the following parameters: temperature 2-8°C, ultrasonic wave 100W, ultrasonic action 6s, rest 3s, and ultrasonication time 20 min.

[0055] S6. After filtering the cell lysate through a 40 µm mesh, centrifuge at 1800 rpm for 5 min to remove the precipitate, collect the supernatant, and resuspend in physiological saline to obtain a cytokine suspension. Example 2

[0056] A method for preparing a cytokine gel complex comprises the following steps:

[0057] 1) Dissolve sodium alginate powder in physiological saline, allow to swell at room temperature for 36 hours, then stir thoroughly to prepare a mixed gel according to the following weight percentage formula:

[0058] Sodium alginate 3%, collagen 1%, human serum albumin 5%, vitamin C 1%, glycerol 3%, and normal saline as the balance.

[0059] 2) The mixed gel and the cytokine suspension described in Example 1 were mixed evenly at a volume ratio of 1:1 to obtain a cytokine gel complex. Example 3

[0060] The difference between this embodiment and embodiment 2 is that the mixed gel comprises the following components in weight percentage:

[0061] Sodium alginate 2%, collagen 0.5%, human serum albumin 8%, vitamin C 0.1%, glycerol 5%, and normal saline as the balance. Example 4

[0062] The difference between this embodiment and embodiment 2 is that the mixed gel comprises the following components in weight percentage:

[0063] Sodium alginate 5%, collagen 3%, human serum albumin 3%, vitamin C 2%, glycerol 1%, and normal saline as the balance.

[0064] Comparative Example 1

[0065] The difference between this comparative example and Example 1 is that in step S5, the cell lysis is performed by repeated freezing and thawing. The specific steps include placing the cell suspension at -20°C, freezing it completely, and then placing it at 2-8°C, repeating this three times to obtain a cell lysate.

[0066] Comparative Example 2

[0067] A method for preparing placenta-derived cytokines comprises the following steps:

[0068] S1: Remove the placenta from the sample collection bag and place it in a sterilized stainless steel tray. Wash it 2-3 times with normal saline to remove contamination and large blood clots. Then, cut 250g of placental tissue into 0.3cm × 0.3cm tissue pieces and repeatedly wash them with normal saline to obtain clean placental tissue.

[0069] S2: Place clean placental tissue in a 50 mL centrifuge tube and mince until finely minced. Place the minced tissue in a 500 mL blue-capped bottle and add digestive enzymes. Digest the tissue in a 37°C water bath for 120 min, shaking every 30 min.

[0070] In step S3, the digestion fluid was collected and centrifuged, the supernatant was removed, and the pellet was resuspended with physiological saline. The resuspended solution was then aliquoted into 50-ml centrifuge tubes. The cells were then lysed using a cell ultrasonic disruptor in an ice bath to obtain cell lysate. The ultrasonic disruptor settings were: temperature 2–8°C, ultrasonic wave 100W, sonication for 6 seconds, rest for 3 seconds, and sonication time for 20 minutes.

[0071] S4. After filtering the cell lysate through a 40 µm mesh, centrifuge at 1800 rpm for 5 min, remove the precipitate, collect the supernatant, and resuspend in physiological saline to obtain a cytokine suspension.

[0072] Performance Testing

[0073] 1. Cytokine content test

[0074] The cell lysates obtained in Example 1 and Comparative Examples 1-2 were used to detect cytokines using an ELISA kit. The results are shown in Table 1.

[0075] Table 1

[0076] EGF (pg / ml) VEGF (pg / ml) BFGF (pg / ml) TGF-β (pg / ml) NGF (pg / ml) Example 1 2 7 4.45 1 3 6 2.8 4 9 0 7.56 2 0 5 7.89 3 2 0.97 Comparative Example 1 1 0 5.16 5 8 7.6 3 3 6 2.47 9 76.19 8 7.96 Comparative Example 2 8 6.21 4 8 5.59 4 46.98 8 47.21 7 3.59

[0077] As can be seen from Table 1, compared with the existing freeze-thaw cycle method, the ultrasonic disruption method used in the present invention can effectively increase the cytokine content. In Comparative Example 2, the tissue fluid was digested and then directly lysed, resulting in a product with more impurities and a significantly lower cytokine content.

[0078] 2. Cytokine activity test

[0079] The cytokine gel complex of Example 2 was placed at 5°C for half a month, one month, two months, and three months, and the contents of EGF, VEGF, BFGF, TGF-β, and NGF cytokines in the cytokine gel complex were detected using an ELISA kit and compared with the contents of the cytokines just prepared. The results are shown in Table 2.

[0080] Table 2

[0081] Storage time EGF (pg / ml) VEGF (pg / ml) BFGF (pg / ml) TGF-β (pg / ml) NGF (pg / ml) 0 day 274.451362.84907.562057.89320.97 15 day 278.011341.02896.082014.49311.45 30 day 267.351327.39890.432007.12319.97 60 day 269.081339.65904.272028.16309.48 90 day 271.641355.68901.712015.83310.46

[0082] As can be seen from Table 2, the gel complex of the present invention has no significant effect on cell activity after being placed at low temperature for 3 months, which proves that the gel complex of the present invention can more effectively protect the activity of cytokines.

[0083] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0084] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for preparing placenta-derived cytokines, characterized in that: The following steps are involved: S1, taking the placental tissue, cleaning it, cutting it into minced pieces, adding digestive enzymes to perform digestion reaction, then passing the digestion liquid through a cell sieve, collecting the filtrate after sieving, resuspending the precipitate after centrifugation, and obtaining a first suspension; S2, adding the human lymphocyte separation solution to the first cell suspension, centrifuging, then aspirating the buffy coat layer in the middle of the centrifuged product, and resuspending to obtain a second suspension; S3, centrifuging the second suspension, resuspending the precipitate and counting the cells to obtain mononuclear cells with cell activity, and then lysing the cells in an ice bath to obtain a cell lysate; S4, filtering the cell lysate and centrifuging it, collecting the supernatant, and resuspending it to obtain a cytokine suspension.

2. The method for preparing placenta-derived cytokines according to claim 1, characterized in that: In step S1, the reaction temperature of the digestion reaction is 36-38°C, the reaction time is 100-140 min, and shaking is performed every 25-35 min; the pore size of the cell screen is 70-100 mesh.

3. The method for preparing placenta-derived cytokines according to claim 1, characterized in that: In step S2, the volume ratio of the human lymphocyte separation solution to the first cell suspension is 1:(1.5-3), the centrifugal speed is 1900-2200 rpm, and the centrifugal time is 18-25 min.

4. The method for preparing placenta-derived cytokines according to claim 1, characterized in that: In step S3, the cell lysis is ultrasonic disruption, and the treatment conditions include: temperature of 2-8°C, ultrasonic power of 90-110W, ultrasonic work for 5-7s followed by a 2-4s pause and cyclic treatment, and a total ultrasonic time of 18-22 min.

5. The method for preparing placenta-derived cytokines according to claim 1, characterized in that: In step S4, the pore size of the filtration is 35-45 μm; In steps S1, S3 and S4, the centrifugal speed is 1700-1900 rpm and the centrifugal time is 4-6 min.

6. A cytokine gel complex, characterized in that: The invention relates to a cytokine prepared by the method for preparing a placenta-derived cytokine according to any one of claims 1 to 5.

7. The cytokine gel complex according to claim 6, characterized in that: The invention comprises a cytokine suspension and a mixed gel in a volume ratio of 1:(1-2), wherein the cytokine suspension is composed of cells with a concentration of (0.8-1.2)×10 6 The cell suspension of 100 cells / ml was lysed and prepared. The mixed gel comprises the following components in percentage by mass: Sodium alginate 2~5%, collagen 0.5~3%, human serum albumin 3~8%, vitamin C 0.1~2%, glycerol 1~5%, and the balance is normal saline.

8. A method for preparing the cytokine gel complex according to claim 6 or 7, characterized in that: The following steps are involved: 1) Dissolve the formulated amount of sodium alginate in physiological saline until it swells, stir it thoroughly, then add collagen, human albumin, vitamin C and glycerol and stir them evenly to obtain a mixed gel; 2) Evenly mixing the mixed gel and the cytokine to obtain a cytokine gel complex.

9. A method for preparing the cytokine gel complex according to claim 8, characterized in that: The swelling temperature is 23-28° C., and the swelling time is 24-48 hours.

10. Use of the cytokine gel complex according to claim 6 or 7 in the preparation of a drug for treating skin burns, diabetic foot, venous ulcer or anal fistula.