CN111388649A - Platelet-rich plasma placenta polypeptide composition and application thereof - Google Patents
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Abstract
The invention discloses a platelet-rich plasma placental polypeptide composition and application thereof in preparation of an immunomodulator, wherein the composition is prepared by mixing platelet-rich plasma and placental polypeptide in a volume ratio of 1: 1-4.
Description
(I) technical field
The invention relates to a polypeptide composition, in particular to a platelet-rich plasma placenta polypeptide composition for injection and a preparation method and application thereof.
(II) background of the invention
At present, a mature extraction technology of placenta polypeptide (patent application No. 201510995668.9) is available, the placenta polypeptide contains various active ingredients, especially cell factors and growth factors, has obvious curative effects on oxidation resistance, aging delay, tumor resistance, infection resistance, damaged tissue repair, endocrine regulation and various gynecological diseases treatment, and also has the effects of inhibiting the peroxidation of the body and eliminating free radicals to achieve the aim of aging resistance.
Platelet-rich plasma (PRP) is a highly concentrated product obtained by centrifuging and separating whole blood, and the level of platelets is usually 4-5 times that of normal whole blood, and PRP contains various growth factors such as platelet-derived growth factor, β -transforming growth factor, vascular endothelial growth factor, epidermal growth factor, insulin-like growth factor, fibroblast growth factor and the like.
Disclosure of the invention
The invention aims to provide a platelet-rich plasma placental polypeptide composition and a preparation method thereof, and solves the problems of single efficacy and unstable effect of the conventional single-use placental polypeptide injection or PRP (platelet-rich plasma) so as to enable the treatment effect to be more obvious and improve the immunity.
The technical scheme adopted by the invention is as follows:
the invention provides a platelet-rich plasma placental polypeptide composition, which is prepared by mixing platelet-rich plasma and placental polypeptide in a volume ratio of 1: 1-4.
Further, the platelet rich plasma is prepared as follows:
(1) adding whole blood (preferably peripheral blood) containing 10% anticoagulant by volume concentration into a horizontal centrifuge for centrifugation to obtain supernatant and precipitate, and respectively recording as supernatant A and precipitate B; the anticoagulant is sodium citrate;
(2) adding physiological saline in a volume ratio of 1:1, diluting the precipitate B, centrifuging, and collecting supernatant, and recording as supernatant C;
(3) uniformly mixing the supernatant A and the supernatant C, and placing the mixture in a refrigerator at 4 ℃ for 25 minutes to obtain a mixed solution, and recording the mixed solution as a mixed solution D;
(4) and centrifuging the mixed solution D, and collecting supernatant to obtain the platelet-rich plasma.
Further, the centrifugation conditions in step (1) and step (2) were 1000rpm and 4 ℃ for 10 minutes.
Further, the centrifugation conditions in the step (4) were 2000rpm and 5 minutes at 4 ℃.
Further, the placenta polypeptide is placenta polypeptide injection, preferably placenta polypeptide injection produced by Tai Pont biological products, Guizhou, with a product batch number of 201803014.
The invention also provides application of the platelet-rich plasma placental polypeptide composition in preparation of an immunomodulator, wherein the immunomodulator is a preparation for reducing expression of I L-4 or I L-5, the adjustor is a preparation for improving expression of IFN-gamma, and the adjustor is a preparation for improving expression of IgG, IgE, IgM or IgA.
Compared with the prior art, the platelet-rich plasma placental polypeptide composition has the main beneficial effects that the expression of I L-4 and I L-5 can be obviously reduced, the expression of IFN-gamma is improved, the aim of treating asthma is fulfilled, the effect is more obvious than that of a single-use group, and the effect of better enhancing immunity can be achieved.
(IV) description of the drawings
FIG. 1: comparison of immunoglobulin Change before and after treatment
The alphabetical differences represent significant differences using L SD one-way variance multiple comparisons.
FIG. 2: comparison of immunoglobulin Change before and after treatment
The alphabetical differences represent significant differences using L SD one-way variance multiple comparisons.
FIG. 3: comparison of immunoglobulin Change before and after treatment
The alphabetical differences represent significant differences using L SD one-way variance multiple comparisons.
FIG. 4 serum I L-4, I L-5, IFN- γ content comparison (x. + -.s) (pg/ml) for each group of rats, letter differences represent significant differences using L SD one-way variance multiple comparisons.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1:
1. preparation of platelet rich plasma:
(1) adding 30ml of peripheral blood whole blood containing 10% sodium citrate (anticoagulant) by volume into a horizontal centrifuge, and centrifuging at the centrifugal condition of 1000rpm and 4 ℃ for 10 minutes to obtain 8ml of supernatant (marked as supernatant A) and 22ml of sediment (marked as sediment B);
(2) the mixture is dissolved in 22ml of physiological saline in a volume ratio of 1:1, then centrifuging for 10 minutes at 1000rpm and 4 ℃, and collecting 10ml of supernatant, and recording as supernatant C;
(3) after 8ml of the supernatant A and 10ml of the supernatant C were mixed uniformly, the mixture was placed in a refrigerator at 4 ℃ for 25 minutes to obtain 18ml of a mixed solution, which was designated as a mixed solution D.
(4) The mixture D was centrifuged at 2000rpm at 4 ℃ for 5 minutes, and the supernatant was collected to obtain 16ml of platelet-rich plasma.
2. Placenta polypeptide:
the placenta polypeptide is purchased from placenta polypeptide injection produced by Tai Pont biological products, Guizhou, and the product batch number is 201803014.
3. Preparation of platelet rich plasma placental polypeptide composition:
mixing the platelet-rich plasma prepared in the step 1 and the placenta polypeptide prepared in the step 2 in a volume ratio of 1:1, mixing to obtain the platelet-rich plasma placental polypeptide composition, wherein the platelet-rich plasma is 18ml, and the placental polypeptide injection is 18 ml.
The asthma patient takes a sitting position, 2-4 acupuncture points are taken each time, the skin is disinfected conventionally, about 2ml of the platelet-rich plasma placental polypeptide composition (combined group) is extracted by a 5ml syringe, about 10-15 mm of the platelet-rich plasma placental polypeptide composition is obliquely pricked on the acupuncture points (Feishu, Dingchuan and Zusanli), the patient is slowly lifted and inserted until the acupuncture feels, after the suction syringe has no blood return, the liquid medicine is injected (0.2-0.4 ml of the liquid medicine is injected into each acupuncture point), every other day is carried out once, 6-10 times are taken as a treatment course, the immunoglobulin change condition is detected before treatment and 1 month after treatment respectively, the platelet-rich plasma (PRP group) prepared in the step 1 and the placental polypeptide (placental polypeptide group) in the step 2 are taken as a control under the same condition, and the result is shown in the. Four immunological indexes show that the three treatment groups have the function of improving the immunoglobulin indexes at different degrees. Wherein the PRP group and the placenta polypeptide group have obvious effect on IgE, have certain effect on other indexes but are not statistically significant; the combined group has obvious effects on IgG, IgE, IgM and IgA, the effect is superior to that of a PRP group and a placental peptide group, and the treatment advantages of the combined group are reflected.
Example 2:
1. preparation of platelet rich plasma:
(1) adding 20ml of peripheral blood whole blood containing 10% sodium citrate anticoagulant by volume into a horizontal centrifuge for centrifugation, and centrifuging at the centrifugation condition of 1000rpm and 4 ℃ for 10 minutes to obtain 6ml of supernatant (marked as supernatant A) and 14ml of precipitate (marked as precipitate B);
(2) using 14ml of physiological saline in a volume ratio of 1:1, then centrifuging for 10 minutes at 1000rpm and 4 ℃, and collecting 8ml of supernatant, and recording as supernatant C;
(3) after 6ml of the supernatant A and 8ml of the supernatant C were mixed uniformly, the mixture was placed in a refrigerator at 4 ℃ for 25 minutes to obtain 14ml of a mixed solution, which was designated as a mixed solution D.
(4) The mixture D was centrifuged at 2000rpm at 4 ℃ for 5 minutes, and the supernatant was collected to obtain 12ml of platelet-rich plasma.
2. Placenta polypeptide:
the placenta polypeptide is purchased from placenta polypeptide injection produced by Tai Pont biological products, Guizhou, and the product batch number is 201803014.
3. Preparation of platelet rich plasma placental polypeptide composition:
and (3) mixing the platelet-rich plasma prepared in the step (1) and the placental polypeptide in the step (2) according to the volume ratio of 1:2 to obtain the platelet-rich plasma placental polypeptide composition, wherein the volume of the platelet-rich plasma is 14ml, and the volume of the placental polypeptide injection is 28 ml.
The asthma patient takes a sitting position, 2-4 acupuncture points are taken each time, the skin is disinfected conventionally, about 2ml of platelet-rich plasma placenta polypeptide composition (combined group) for injection is extracted by a 5ml syringe, about 10-15 mm of the platelet-rich plasma placenta polypeptide composition is obliquely pricked on the acupuncture points (same as the embodiment 1), the composition is slowly extracted and inserted until the acupuncture feeling is achieved, after the blood return of a suction syringe is avoided, liquid medicine is injected (0.2-0.4 ml of the liquid medicine is injected into each acupuncture point), the medicine is injected once every other day, and 6-10 times are taken as a treatment course. Immunoglobulin changes were measured before and 1 month after treatment, and the results of platelet-rich plasma (PRP group) prepared in step 1 and placental polypeptide (placental polypeptide group) prepared in step 2 were used as controls under the same conditions, respectively, and are shown in fig. 2. Four immunological indexes show that the three treatment groups have the function of improving the immunoglobulin indexes at different degrees. Wherein the PRP group and the placenta polypeptide group have obvious effect on IgE, the placenta polypeptide group has obvious effect on IgA, and other indexes of the PRP group and the placenta polypeptide group have certain effect but are not statistically significant; the combined group has obvious effects on IgG, IgE, IgM and IgA, the effect is superior to that of a PRP group and a placenta polypeptide group, and the treatment advantages of the combined group are reflected.
Example 3:
1. preparation of platelet rich plasma:
(1) adding 10ml of peripheral blood whole blood containing anticoagulant with volume concentration of 10% into a horizontal centrifuge, and centrifuging at the centrifugal condition of 1000rpm and 4 ℃ for 10 minutes to obtain 3ml of supernatant (marked as supernatant A) and 7ml of sediment (marked as sediment B);
(2) adding 7ml of physiological saline in a volume ratio of 1:1, then centrifuging for 10 minutes at 4 ℃ at 1000rpm, and collecting 5ml of supernatant, which is recorded as supernatant C;
(3) after 3ml of the supernatant A and 5ml of the supernatant C were mixed uniformly, the mixture was placed in a refrigerator at 4 ℃ for 25 minutes to obtain 8ml of a mixed solution, which was designated as a mixed solution D.
(4) The mixture D was centrifuged at 2000rpm at 4 ℃ for 5 minutes, and the supernatant was collected to obtain 6ml of platelet-rich plasma.
2. Placenta polypeptide:
the placenta polypeptide is purchased from placenta polypeptide injection produced by Tai Pont biological products, Guizhou, and the product batch number is 201803014.
3. Preparation of platelet rich plasma placental polypeptide composition:
mixing the platelet-rich plasma prepared in the step 1 and the placenta polypeptide prepared in the step 2 in a volume ratio of 1: 4, mixing to obtain the platelet-rich plasma placental polypeptide composition, wherein the platelet-rich plasma is 8ml, and the placental polypeptide injection is 32 ml.
The asthma patient takes a sitting position, 2-4 acupuncture points are taken each time, the skin is disinfected conventionally, about 2ml of the platelet-rich plasma placenta polypeptide composition for injection is extracted by a 5ml syringe, the acupuncture points (same as example 1) are pricked obliquely by about 10-15 mm, the patient is slowly lifted and inserted until the acupuncture feeling exists, after the blood return of a suction syringe does not exist, liquid medicine (0.2-0.4 ml of liquid medicine is injected into each acupuncture point) is injected, the medicine is injected every other day, and 6-10 times are taken as a treatment course. The results of examining the immunoglobulin changes before and 1 month after the treatment using the platelet-rich plasma prepared in step 1 (PRP group) and the placental polypeptides prepared in step 2 (placental polypeptide group) as controls under the same conditions are shown in fig. 3. Four immunological indexes show that the three treatment groups have the function of improving the immunoglobulin indexes at different degrees. Wherein the PRP group and the placenta polypeptide group have obvious effect on IgE, and other indexes of the PRP group and the placenta polypeptide group have certain effect but are not statistically significant; the combined group has obvious effects on IgG, IgE, IgM and IgA, the effect is superior to that of a PRP group and a placenta polypeptide group, and the treatment advantages of the combined group are reflected.
Example 4
40 SPF male BA L B/c mice of 6 weeks old are selected, the body mass (20.50 +/-2.15) g is randomly divided into 5 groups (a control group, a model group, a placental polypeptide group, a PRP group and a combined group), each group comprises 8 mice, the mice are raised in the animal center of Zhejiang Chinese medicine university, the experiment is started after the mice are fed for 3 days in a normal adaptive manner, and the mice are fed with ordinary feed and free drinking water.
A bronchial asthma model was established in mice of the groups other than the control group by intraperitoneally injecting Ovalbumin (OVA) suspension (aluminum hydroxide 400. mu.g and OVA 100. mu.g formulated with 0.1 mol/L PBS) at a concentration of 50. mu.g/L on days 1, 4, 7, 10, and 13, respectively, 1 time per day, 1ml each time, starting on day 14, administering 50. mu.l of nebulized inhalation OVA solution (OVA1mg dissolved with 0.1 mol/L PBS) 30min each time, 1 time per day, and continuously stimulating for 1 week.
After atomization excitation, continuously observing the itch condition of the mouse head and face, respiration and behavior change for 10min, scoring the following indexes by a superposition quantitative scoring method, wherein the total score is greater than 5 to indicate the behavior change of an animal model, and the behavior change accords with the behavior change of an allergic asthma animal model. First, the condition of facial itching: 0 point for head and face without scratching, 1 point for 1-2 times for head and face with light itching, and 2 points for head and face with intense scratching; second, breathing situation: stable 0min, tachypnea 1 min, dyspnea 2 min, dyspnea with wheezing sound or mucus secretion 3 min, irregular respiration or sudden death 4 min; third, action case: the free movement is 0 minutes, the quiet small movement is 1 minute, the action retardation is 2 minutes, and the depression immobility is 3 minutes.
The administration of the placental peptide group, PRP group and combination group was started on day 17, the injection of acupoints was performed, 2 acupoints were injected each time, the acupoints were alternately selected on both Feishu and Shenshu, each acupoint was injected with 0.4m L of placental peptide of step 2 of example 1 (placental peptide group), 0.4m L of platelet-rich plasma (PRP group) prepared in example 1, 0.4m L of the platelet-rich plasma placental peptide composition (combination group) prepared in example 1, 1 acupoint injection every other day, 6 times before and after each treatment for 1 treatment period, after the administration, the heart was bled, serum was extracted, and the contents of I L-4, I L-5 and IFN-. gamma.in serum were measured using E L ISA, and under the same conditions, physiological saline acupoints were used for the control group and model group, and the results are shown in FIG. 4.
The attack process of bronchial asthma is mainly completed by a plurality of receptors on the surfaces of inflammatory cells and inflammatory mediators and cytokines produced by the inflammatory cells through a series of complex antigen presentation processes, which can be called as an immune-inflammatory mechanism of asthma attack, wherein the functional imbalance of helper T cell 1(Th1)/Th2 cells is one of important immunological mechanisms of the bronchial asthma attack, and is mainly represented by low function of Th1 cells, Th2 cell hyperfunction IFN-gamma is a representative cytokine of Th1 cells, can promote differentiation of Th1 cells, block formation of Th2 cells, reduce secretion of main cytokine I L-4, reduce IgE level and airway hyperreactivity, and I L-4 is the most representative Th2 cytokine and can stimulate transformation and proliferation of B lymphocytes, promote IgE synthesis, promote chemotaxis and infiltration of EOS and release active substances such as histamine and leukotriene by mast cells, and the like, and I L-5 can also promote basic granulocytes to release inflammatory mediators such as histamine and leukotriene and the like.
From figure 4, it is seen that the PRP and placental polypeptide combined group can significantly reduce the expression of I L-4 and I L-5, increase the expression of IFN-gamma, achieve the purpose of treating asthma, and the effect is more obvious than that of the single group.
Claims (10)
1. The composition is characterized by being prepared by mixing platelet-rich plasma and placenta polypeptide in a volume ratio of 1: 1-4.
2. The platelet rich plasma placental polypeptide composition of claim 1, wherein said platelet rich plasma is prepared by the following method:
(1) centrifuging whole blood containing anticoagulant with volume concentration of 10% to obtain supernatant and precipitate, and respectively recording as supernatant A and precipitate B;
(2) adding physiological saline in a volume ratio of 1:1, diluting the precipitate B, centrifuging, and collecting supernatant, and recording as supernatant C;
(3) uniformly mixing the supernatant A and the supernatant C, and placing the mixture in a refrigerator at 4 ℃ for 25 minutes to obtain a mixed solution, and recording the mixed solution as a mixed solution D;
(4) and centrifuging the mixed solution D, and collecting supernatant to obtain the platelet-rich plasma.
3. The platelet rich plasma placental polypeptide composition of claim 2, wherein the centrifugation conditions of step (1) and step (2) are 1000rpm at 4 ℃ for 10 minutes.
4. The platelet rich plasma placental polypeptide composition of claim 2, wherein the centrifugation conditions of step (4) are 2000rpm for 5 minutes at 4 ℃.
5. The platelet rich plasma placental polypeptide composition of claim 2, wherein the anticoagulant is sodium citrate.
6. The platelet rich plasma placental polypeptide composition of claim 2, wherein said placental polypeptide is placental polypeptide injection.
7. Use of the platelet rich plasma placental polypeptide composition of claim 1 in the preparation of an immunomodulator.
8. Use according to claim 7, characterized in that the modulator is an agent which reduces the expression of I L-4 or I L-5.
9. Use according to claim 7, characterized in that the modulator is an agent that increases the expression of IFN- γ.
10. Use according to claim 7, characterized in that the modulator is an agent which increases the expression of IgG, IgE, IgM or IgA.
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| CN111803520A (en) * | 2020-07-27 | 2020-10-23 | 杭州嘉耀健康管理有限公司 | Acupoint injection for improving immune function |
| RU2752747C1 (en) * | 2020-10-01 | 2021-08-02 | федеральное государственное бюджетное учреждение "Национальный медицинский исследовательский центр имени В.А. Алмазова" Министерства здравоохранения Российской Федерации | Method for treating trophic skin disorders in patients with severe brain damage |
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