CN116987171A - Preparation method of placenta-derived cytokine for preventing and treating herpes virus infection and application of preparation thereof - Google Patents
Preparation method of placenta-derived cytokine for preventing and treating herpes virus infection and application of preparation thereof Download PDFInfo
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- CN116987171A CN116987171A CN202310170253.2A CN202310170253A CN116987171A CN 116987171 A CN116987171 A CN 116987171A CN 202310170253 A CN202310170253 A CN 202310170253A CN 116987171 A CN116987171 A CN 116987171A
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Abstract
The invention discloses a preparation method of placenta-derived cytokines for preventing and treating herpes virus infection and application of a preparation thereof. Specifically, the invention relates to a method for preparing a placenta tissue-degrading enzyme, which comprises the steps of adopting collagenase I, trypsin and papain trigeminal enzyme, carrying out grading and cleavage of placenta tissue by adjusting the composition, pH value, temperature and time of the enzyme, collecting placenta tissue dissociation liquid, and separating active substances in the placenta tissue dissociation liquid by using a Sephadex gel column of cross-linked dextran to obtain a plurality of necessary amino acids, small molecular active peptides, immunoglobulins, lipopolysaccharides and the like; meanwhile, collecting primary placenta-derived mesenchymal stem cells from placenta tissue dissociation solution in an adherent way, and inducing the mesenchymal stem cells to highly express active factors, mainly Interleukin (IL), colony Stimulating Factor (CSF), tumor Necrosis Factor (TNF), interferon (IFN), epidermal Growth Factor (EGF), transforming Growth Factor (TGF) and the like, through in vitro proliferation culture, and combining and collecting the primary placenta-derived mesenchymal stem cells, which are called placenta-derived cytokines. The placenta-derived cytokines are freeze-dried to prepare samples and preparations, and the placenta-derived cytokines prepared by extracting active cytokines from placenta tissues and combining placenta-derived mesenchymal stem cells are shown by the in vitro HELFF cell infection herpesvirus test, mouse testis infection herpesvirus simplex virus test and clinical herpesvirus infection patient anti-herpesvirus effect, so that lesions of HELFF cells and mouse testis caused by the herpesvirus can be obviously inhibited, HELFF cell necrosis and mouse testis tissue injury caused by the herpesvirus infection can be repaired, and the clinical application shows stronger anti-herpesvirus effect, especially on drug-resistant strain infected patients.
Description
Technical Field
The invention relates to a preparation method of placenta-derived cytokines for preventing and treating herpes virus infection and application of a preparation thereof.
Background
Human herpesvirus (Human herpes virus, HHV) infections are very common, with infection rates of 40-60% in developed countries and 100% in countries with late development. In China, the infection rate of children is about 80 percent. HHV is a weak causative agent, normal people are usually latent infection or asymptomatic subclinical infection, but for patients with low body immunity (such as infants and old people), immunosuppression or deficiency, primary infection or latent infection HHV can activate and cause active HHV infection, cause various common diseases, exacerbate symptoms of the original diseases of some patients with low immunity, and even cause death.
In the past four and fifty years, HHV infection is more common due to the massive increase of organ transplantation, tumor patients and AIDS patients, the mortality rate of HHV infection is obviously increased, and simultaneously, HHV is one of the most common causes of congenital infection.
There are mainly 8 common human herpesviruses, of which humans are the only hosts for Herpes Simplex Virus (HSV) and cytomegalovirus (HCMV).
Currently, herpes viruses mainly invade ectoderm-derived tissues including skin, mucous membrane and nerve tissues, and the sites of infection and the diseases caused are diverse and have a tendency to latent infection, severely threatening human health. The diseases associated with herpes virus infection are several of the following: 1) Primary hypertensive patients present with higher active herpes virus infections; 2) Herpes virus infection causes damage to the cell endothelium and thus participates in the pathogenesis of coronary heart disease; 3) Pulmonary infectious diseases complicated with herpes virus infection; 4) Chronic hepatitis b complicated with herpes virus infection can exacerbate liver dysfunction; 5) The infection rate of the herpesvirus of patients with renal failure dialysis is obviously increased; 6) The infection rate of human herpesvirus after burn is obviously increased, especially for patients with large-area burn; 7) Other patients receiving immunosuppressant therapy have a concomitant human herpesvirus infection, with chemotherapy having the malignant tumor most common.
In addition, HHV is also associated with transverse myelitis, polyneuritis, guillain-Barre syndrome, myocarditis, thrombocytopenia, hemolytic anemia, various types of cytopenias, secondary sclerosing cholangitis, venous thrombosis, joubert's syndrome, diabetes, systemic lupus erythematosus, nephrotic syndrome, meniere's disease, rheumatoid arthritis, chronic periodontitis, and the like.
HHV infection is continually becoming newly recognized in clinical pathogenesis, and more human diseases are considered to be associated with its infection. For herpes virus infection, no effective treatment measures are available at present, and clinically common anti-herpes virus medicines comprise acyclovir, ganciclovir, valacyclovir, interferon, immunoglobulin, monoclonal antibodies and the like, but the medicines cannot be subjected to targeted effective treatment, viruses still relapse easily, and drug resistance is also easy to generate.
Currently, acyclovir and ganciclovir are the first drugs to treat HHV infection, mainly by inhibiting DNA polymerase to exert antiviral effects. Clinically acyclovir and ganciclovir have definite therapeutic effects on retinitis of HHV infection, but have insignificant therapeutic effects on interstitial pneumonia of HHV infection and are more toxic, and some patients are forced to discontinue treatment due to serious side effects such as granulocytopenia. In addition, in recent years, HHV has a growing tendency for drug-resistant strains of acyclovir and ganciclovir, and studies have found that drug-resistant strains often have mutations such as substitution or deletion of the UL97 region, and that acyclovir and ganciclovir Wei Jiage are expensive, which have not been overcome, and thus have a great limitation in clinical application. Other drugs against HHV infection, such as sodium fosfomate, cidofovir, also present similar problems. Therefore, the search and research of drugs for treating HHV infection with definite curative effect and drugs for treating and preventing diseases caused by HHV infection have important innovative significance and clinical value.
The invention relates to a method for preparing a placenta tissue, which comprises the steps of adopting collagenase I, trypsin and papain trigeminy enzyme, carrying out grading dissociation on placenta tissue by adjusting the composition, pH value, temperature and time of the enzyme, collecting placenta tissue dissociation liquid, and separating active substances in the placenta tissue dissociation liquid by using a Sephadex gel column of cross-linked dextran to obtain various essential amino acids, small molecular active peptides, immunoglobulins, lipopolysaccharides and the like; meanwhile, collecting primary placenta-derived mesenchymal stem cells from placenta tissue dissociation solution in an adherent way, and inducing the mesenchymal stem cells to highly express active factors, mainly Interleukin (IL), colony Stimulating Factor (CSF), tumor Necrosis Factor (TNF), interferon (IFN), epidermal Growth Factor (EGF), transforming Growth Factor (TGF) and the like, through in vitro proliferation culture, and combining and collecting the primary placenta-derived mesenchymal stem cells, which are called placenta-derived cytokines. The placenta-derived cytokine is freeze-dried to prepare samples and preparations, and the new findings are obtained through in vitro and in vivo anti-human herpesvirus activity screening experimental research, so that the placenta-derived cytokine can obviously inhibit HELFF cells and mouse testis lesions caused by human herpesvirus, has stronger anti-human herpesvirus infection effect, and is especially suitable for drug-resistant strain infected persons. Furthermore, toxicity test results show that oral administration of the maximum tolerated amount of placenta-derived cytokines did not appear to have toxic effects in animals. Compared with the prior clinically applied medicaments acyclovir and ganciclovir for resisting human herpesvirus infection, the placenta-derived cytokine has the advantages of more obvious effect, better safety and lower cost for resisting herpesvirus infection, so the placenta-derived cytokine has good application prospect in resisting human herpesvirus infection.
Disclosure of Invention
The inventionThe aim of the invention is to provide a preparation method of placenta-derived cytokines for preventing and treating herpes virus infection and application of the preparation thereof. The method comprises the steps of dissociating placenta tissues by a trigeminy enzyme fractionation method, collecting placenta tissue dissociation liquid, and separating active substances in the placenta tissue dissociation liquid by a Sephadex gel column of cross-linked dextran to obtain various essential amino acids, small molecule active peptides, immunoglobulin, lipopolysaccharide and the like; primary placenta-derived mesenchymal stem cells are collected from placenta tissue dissociation solution in an adherent way, and the mesenchymal stem cells are induced to highly express active factors, namely, interleukin (IL), colony Stimulating Factor (CSF), tumor Necrosis Factor (TNF), interferon (IFN), epidermal Growth Factor (EGF), transforming Growth Factor (TGF) and the like, through in vitro proliferation culture, and are collected in a combined way, and are called placenta-derived cytokines. The placenta-derived cytokines are freeze-dried and prepared into medicines, health-care products, foods and/or external preparations, tissue preservation solutions and the like, and the placenta-derived cytokines prepared by extracting active cytokines from placenta tissues and combining placenta-derived mesenchymal stem cells are shown by activity measurement results through in vitro HELFF cell infection herpesvirus tests, mouse testis infection herpesvirus simplex virus tests and anti-herpesvirus effects of clinical herpesvirus infected patients, so that lesions of HELFF cells and mouse testis caused by herpesvirus can be remarkably inhibited, HELFF cell necrosis and mouse testis tissue injury caused by herpesvirus infection can be repaired, and the clinical application of the placenta-derived cytokines also shows stronger anti-herpesvirus effects, especially for drug-resistant strain infected patients, compared with the clinically applied anti-herpesvirus infection drugs acyclovir and ganciclovir at present, the effect of inhibiting the development of the herpes simplex virus infection is more remarkable. Furthermore, toxicity test results showed that mice were lavaged for LD with placenta-derived cytokines 50 >1.8g/kg; the maximum tolerated dose of mice was 400 times the oral dose of humans, and no animal death was seen.
For a better understanding of the essence of the invention, the following is specifically presented: 1) Dissociation of placenta tissue by a triple enzyme fractionation method; 2) Separating active substances in placenta tissue dissociation solution by using a Sephadex gel column of cross-linked dextran; 3) Collecting primary placenta source mesenchymal stem cells by adherence and performing in-vitro proliferation culture to induce the mesenchymal stem cells to highly express active factors; 4) Pharmacological and pharmacodynamic tests and safety tests of placenta-derived cytokines against human herpesvirus infection, 5) preparation tests of formulations.
Drawings
FIG. 1 is an electrophoresis diagram of a sample B polypeptide fragment (Sephadex G-50 gel column);
FIG. 2 is P 0 Substituted by P 4 A growth morphology diagram of the placenta-derived mesenchymal cells;
FIG. 3 is P 3 substituted-P 10 A cell surface expression pattern of the placenta-derived mesenchymal stem cells;
FIG. 4 is a quantitative standard graph of human IL-2 and IFN-gamma ELISA;
FIG. 5 is a graph of HELF cytopathic effects under various intervention conditions;
FIG. 6 is a graph showing the results of pathological sections of the testis of mice under different intervention conditions (HE staining);
FIG. 7 is a graph of mouse testis HSV2 infection results (in situ hybridization) under different intervention conditions;
Detailed Description
EXAMPLE 1 preparation of active substances in placenta tissue dissociation solution by triple enzyme fractionation of placenta tissue and Sephadex gel column
Taking the human placenta which meets the national relevant legal regulations and ethical requirements, obtaining informed consent, quarantining one of the qualified, fresh and frozen human placenta, putting the human placenta into a refrigerator at 4 ℃ for thawing one day in advance, and cleaning with normal saline, removing impurities and cutting. The umbilical cord, the endometrium and the umbilical artery and vein are stripped, the placenta tissue is sheared into paste by small scissors and transferred into a 50 ml centrifuge tube, and the centrifuge tube is centrifuged at 2000 rpm for 5min, so that red blood cells are removed, and the placenta tissue is repeatedly cleaned by normal saline. PBS buffer was added at 1:2 (W/V) and homogenized for 15min with a tissue homogenizer. Freezing overnight at-20deg.C, thawing in refrigerator at 4deg.C, homogenizing for 15min. Repeating the steps of freezing and thawing, and homogenizing for three times. Then sterilizing at 114 ℃ for 30min.
Adding sterilized ultra-pure water according to the ratio of 3:7, adjusting the pH value to 7.0-7.6 by using a sodium carbonate solution, adding collagenase I (1 mg/mL) to the final concentration of 200U/mL, standing at 37 ℃ for 24 h, and replacing collagenase I digestive juice. Namely, 3000 r/min for 25min, collecting supernatant A, heating at 100deg.C for 10min, and inactivating. The tissue is resuspended with new collagenase I at a concentration of 200U/mL, the pH is adjusted to 7.0-7.6 with sodium carbonate solution, and the tissue is left to stand at 37℃for 24 h. pH below 6.5 is avoided during digestion. 3000 Centrifuging at r/min for 25min, collecting supernatant B, and heating at 100deg.C for 10min for inactivation.
Adding sterilized ultra-pure water into the rest tissues according to the proportion of 3:7, adding trypsin-EDTA digestive juice according to the proportion of 26 mg/300mL (which is equivalent to 1500 vitality units of trypsin), adjusting the pH value to 7.4-8.0 by using sodium carbonate solution, standing at the temperature of 2-8 ℃ for 36 h, and then digesting at the temperature of 37 ℃ for 2 h.3000 Centrifuging at r/min for 25min, collecting supernatant C, and heating at 100deg.C for 10min for inactivation.
Adding sterilized ultra-pure water into the rest tissues according to the proportion of 3:7, adding double enzymes (papain 8.1 mg/300mL and pancreatin 24.3 mg/300 mL) for enzymolysis, adjusting the pH value to 7.0-6.5 by using a sodium carbonate solution, performing shake digestion on the rest tissues at 46 ℃, stopping digestion when the amino nitrogen/total nitrogen reaches 30%, and standing at room temperature overnight. 3000 Centrifuging at r/min for 25min, collecting supernatant D, and heating at 100deg.C for 10min for inactivating.
And (3) mixing the supernatant A-D, adding 1:1 into a sterilized super-diluted gel column, passing through a Sephadex G-25 gel column, a Sephadex G-50 gel column and a Sephadex G-75 gel column in sequence, and separating amino acids, small molecular peptides, polypeptides and proteins with molecular weight ranges of 1 KD-80 KD. Mixing the filtrates, packaging in 500ml saline bottle, capping, and sterilizing at 121deg.C for 20 min. The sterilized stock solution is subpackaged into 15mL centrifuge tubes, and freeze-dried to obtain white powder S1, which is 21.34g in total.
S1, the content of polysaccharide is 0.116 mg/mg, and the content of nucleic acid is 0.469 mg/mg; the electrophoresis diagram of the polypeptide fragment is measured, and the result is shown in figure 1 of the specification.
Example 2 in vitro induced proliferation and analysis of placenta-derived mesenchymal Stem cells
Run 1 isolation of P 0 Placenta-substituted mesenchymal stem cells
The supernatant A described in example 1 was taken, placed in a 50 ml centrifuge tube, centrifuged at 2000 rpm for 5min, the supernatant was discarded, and the pellet was repeatedly washed with PBS buffer. 2mL of the solution was diluted with PBS buffer and inoculated to T75cm in an amount of 0.5. 0.5 mL/bottle 2 In the culture flask, add per flask5ml with serum medium (DMEM/F12 (1:1) +10% FBS) was added and the tissue mass was homogenized. The liquid was changed every 3 days, and the cell was observed for climbing out, and whether contamination was present was observed.
Culturing for about 12 days, allowing mesenchymal stem cells to climb out of the wall, removing the supernatant, washing with physiological saline (3 ml/bottle), digesting with 0.25% pancreatin (2 ml/bottle) for 1min, stopping digestion with stopping pancreatin digestion solution, collecting cell suspension, and centrifuging at 2000 rpm for 5 min; removing supernatant, adding appropriate amount of physiological saline, mixing, and filtering with 70 μm filter screen to remove residual substances to obtain P 0 And replacing the placenta mesenchymal stem cells.
Experiment 2 induced high expression of placental mesenchymal stem cells
The above P is added 0 The placental mesenchymal stem cells were diluted 1 mL with serum culture medium (DMEM/F12 (1:1) +10% FBS) and inoculated to T75cm in an amount of 0.5 ml/bottle 2 Adding 15ml serum culture solution (DMEM/F12 (1:1) +10% FBS+1% L-Glutamine+1% MEM NEAA+10 ng/ml bFGF) into each flask, changing the culture solution once every 3 days, and adding pancreatin for digestion when cell fusion rate reaches about 80%, to obtain P 1 And replacing the placenta mesenchymal stem cells. Continuing to press 1×10 6 Bottle inoculation to T75cm 2 15ml serum culture medium (DMEM/F12 (1:1) +10% FBS+1% L-Glutamine+1% MEM NEAA+10 ng/ml bFGF) was added to each flask and the flask was changed every 3 days; when the cell fusion rate reaches about 80%, pancreatin is added for digestion, and the cells are passaged according to 1:3. Collecting the merged P 3 substituted-P 10 And replacing the placenta mesenchymal stem cells S2.
Test 3 cell growth morphology analysis
Observing the morphology of cells by a microscope, and performing stem cell adherent growth to obtain primary P 0 And P 1 The stem cells are in the shape of irregular angles, and some of the stem cells are in the shape of circles, triangles and polygons, and rarely protrude, so that the cell bodies are not obvious; with slow passage from P 3 →P 4 Starting from the beginning, the cells develop into long fusiform shape, the center of the cells is provided with oval nuclei, and cytoplasm extends outwards to form protrusions with different lengths, so that P is reached 4 The stem cells exhibit a very uniform long fusiform shape; in spiral running water arrangement growthThe cell bodies are obvious, the cell vitality is vigorous, and the result is shown in figure 2 of the specification.
In addition, the placenta-derived mesenchymal stem cells are continuously passaged to the 25 th generation, and we find that the placenta-derived mesenchymal stem cells of different generations have different proliferation capacities and P 4 Substitution, P 5 Proliferation potency significantly higher than P 7 Substitution and P 10 Passage to P 7 At the time of passage, the cell proliferation rate begins to slow down, P 10 The proliferation rate of cells is significantly slowed down after the generation, and the cell morphology is kept stable in long fusiform.
Assay 4 surface expression analysis
Collecting the merged P 3 substituted-P 10 And (3) detecting the placenta-derived mesenchymal stem cells S2 by a flow cytometer. The results are shown in figure 3 of the specification.
P 3 substituted-P 10 The surface of the placental mesenchymal stem cells express CD105, CD73 and CD90 (more than or equal to 95 percent), and do not express CD34 and HLA-DR (less than or equal to 2 percent).
Assay 5 cytokine detection assay
Interlukin-2, IL-2, is a cytokine that plays an important regulatory function in the immune system, and is secreted mainly by Th1 cells, acting on macrophages and natural killer cells (natural kiler cell, NK cells) to infect cells against viruses.
Human IFN-gamma is a highly species-specific protein whose biological activity only acts on human and primate cells. IFN-gamma was originally recognized for its antiviral activity. The protein has proliferation inhibiting effect, immunoregulation and anti-inflammatory activity, and thus is important for host defense mechanism.
Thus, IL-2 and IFN-gamma were selected for the assay.
Experimental materials: human interleukin 2 (IL-2) ELISA quantitative detection kit (48T/96T, shanghai Yupingbio-technology Co., ltd.), human gamma interferon (IFN-gamma) ELISA kit (product number: ELSH007, shanghai Jinma Biotechnology Co., ltd.), 37℃water bath incubator, enzyme-labeled instrument (MK 3, thermo Co., ltd.).
S1 sample solution 10. Mu.g/L.
The experimental method comprises the following steps: the operation was performed according to the kit instructions. The kit was removed from the refrigerator and equilibrated at room temperature for 30 minutes. Diluting the 20-time concentrated washing solution into original-time washing solution by distilled water, and diluting the IL-2 standard substance to the concentration of: 320. 160, 80, 40, 20, 10 ng/L for standby. In addition, IFN-gamma standard was diluted to a concentration of: 400. 200, 100, 50, 25, 12.5 and ng/L for standby.
And (3) taking an enzyme-labeled coating plate, fixing the enzyme-labeled coating plate on a frame, respectively setting a standard substance hole (6 holes multiplied by 2), a sample hole to be tested (2 holes) and a blank control hole (2 holes), and recording the positions of the holes. 50 mu L of standard substance is added into a standard substance hole, 10 mu L of sample to be detected is added into a sample hole to be detected, 40 mu L of sample diluent is added (namely, the sample is diluted 5 times by 2 mu g/L), and a blank control hole is not added. Incubation was carried out in a 37℃water bath incubator for 30min.
Removing liquid, beating to dry on the water-absorbing paper, filling the washing liquid in each hole, standing for 1min, throwing away the washing liquid, beating to dry on the water-absorbing paper, and repeating the plate washing for 4 times.
50 mu L of enzyme-labeled working solution is added to each well, and blank control wells are not added. Incubate in a 37℃water bath or incubator for 30min.
Removing liquid, beating to dry on the water-absorbing paper, filling the washing liquid in each hole, standing for 1min, throwing away the washing liquid, beating to dry on the water-absorbing paper, and repeating the plate washing for 4 times.
50 mu L of a color reagent A solution and 50 mu L of a color reagent B solution are added into each hole, the mixture is gently shaken and mixed for 30 seconds, and the mixture is developed for 15 minutes at 37 ℃ in a dark place. The ELISA plate was removed, and 50. Mu.L of stop solution was added to each well to terminate the reaction (color changed from blue to yellow).
The absorbance (OD) of each well was measured at 450nm wavelength in 15 minutes after termination with a blank Kong Diaoling. And drawing a standard curve according to the concentration of the standard substance and the corresponding OD value by taking the OD value as an ordinate and the concentration of the standard substance as an abscissa, wherein the result is shown in figure 4 of the specification. And calculating the corresponding sample concentration on a regression equation according to the OD value of the sample, wherein the final concentration of the sample is the actual measured concentration multiplied by the dilution multiple. The results are shown in tables 1 and 2.
Table 1 IL-2 Standard, sample to be tested and blank ELISA test results
TABLE 2 IFN-gamma Standard, sample to be tested and blank ELISA detection results
By detection and calculation, each 100 g placenta-derived cytokine contained 11.27 g IL-2; contains 5.80 g IFN-gamma.
EXAMPLE 3 in vitro anti-human herpesvirus Activity assay of placenta-derived cytokines
Test 1 Human Cytomegalovirus (HCMV) AD169 Strain infectivity assay
Preparation of cell density 1X 10 5 In (2) HELF cell suspension, inoculating 100 μl per well in sterile 96-well plate, and placing CO 2 Culturing in an incubator until the culture medium is a single layer. Continuously diluting the HCMV virus solution by 10 times with a maintaining solution, wherein the dilution is 10 -1 ~10 -12 . The culture broth was discarded, 100. Mu.L of virus dilution was inoculated per well, 4 wells were inoculated per dilution, CO 2 The incubator was allowed to adsorb for 1h, and 4-well normal cell controls were set. Removing liquid in the culture well, replacing with 2ml of low melting point agar maintaining solution containing 0.5% and covering, and placing 5% CO 2 Culturing for 5-7 days at 37 ℃, taking out the culture plate, fixing with 10% formaldehyde for 30min, removing agar blocks in the holes, dyeing with 0.5% crystal violet for 5min, flushing with distilled water, and counting plaques under an inverted microscope.
Test 2 sample solution formulation
The placenta tissue active factor S1, the placenta source mesenchymal stem cells S2 and the placenta source cytokines S1+S2 are respectively prepared into mother solutions by DMSO, the concentrations are all 10mg/mL, and the concentrations are respectively 100, 50, 25, 12.5, 6.25 and 3.1 mu mol/mL by diluting the maintenance solution.
Test 3 cytotoxicity test of placenta-derived cytokine
HELF cells at 1X 10 5 Density inoculation in 96 well culture platesIn each well, 100. Mu.L was placed with 5% CO 2 Culturing in a 37 ℃ incubator until the culture medium is a single layer, adding the sample DMSO solution with the concentration, wherein each concentration is 4 times of wells, and 4 times of untreated cells with the wells are additionally arranged as a control. The maintenance solution containing the drug was changed every 3 days, the growth of cells was observed, the culture solution was discarded after 7 days, 100. Mu.L of serum-free culture solution containing 0.5mg/mL MTT was added to each well, 4. 4 h was incubated in a CO2 incubator, the culture solution was discarded, 100. Mu.L of DMSO was added to each well, and absorbance (OD value) was measured at 570nm wavelength by an ELISA meter after thoroughly mixing. The test was repeated 3 times and the average value was taken as the test result.
Cell viability (%) = experimental OD value/control OD value x 100% was calculated.
The cell viability of ganciclovir was measured by the same method and the TC of the drug was calculated 50 And TC 0 The test was repeated 3 times, and an average of 3 times was taken as a test result.
The results show that the lowest nontoxic concentrations of the placenta tissue active factor S1, the placenta-derived mesenchymal stem cells S2 and the placenta-derived cytokines S1+S2 are respectively 50 mu mol/mL, 50 mu mol/mL and 60 mu mol/mL; the minimum non-toxic concentration of ganciclovir is 12.5. Mu. Mol/mL.
Test 4 inhibition of herpesvirus proliferation by placenta-derived cytokine in vitro
HELF with good growth is taken at 1×10 5 Inoculating into 96-well culture plate at a density of 100 μl/well, and placing 5% CO 2 Culturing in incubator at 37deg.C to single layer, synchronizing 24 h, and mixing with about 100 TCID per well 50 The amount of virus challenge cells, placed in 5% CO 2 Adsorbing 1h in an incubator at 37 ℃, and discarding the supernatant; treating placenta-derived cytokine DMSO solution with TD 0 Adding 3 dilution concentrations for the lowest dilution into cell wells respectively, wherein each well is 150 mu L, 8 wells are arranged for each dilution, and a cell control group and a virus control group are simultaneously established; changing the maintenance liquid for 1 time every other day; at 5% CO 2 Culturing at 37deg.C in incubator of about 8 d, adding 100 μl of neutral red dye solution of 5mg/mL into each hole when the cytopathy of virus control group reaches 70-80%, and placing 5% CO 2 Culturing in incubator at 37deg.C for 2h, taking out, washing with physiological saline for 3 times, adding 100 μl of decolorized solution, standing at room temperature for 10min, and placing enzymeThe absorbance (OD value) was measured at the wavelength of the label 570nm, and the cell viability was calculated according to the following formula. The test was repeated 3 times and the average value was taken as the test result.
Cell viability (%) = OD value of experimental group (or virus control group)/OD value of control group x 100%.
The cell viability of ganciclovir was measured by the same method and the TC of the drug was calculated 50 And TC 0 The test was repeated 3 times, and an average of 3 times was taken as a test result.
The results are shown in tables 3 and 4 and in FIG. 5 of the specification.
TABLE 3 Effect of different doses of placenta-derived cytokines on HCMV Activity
| Group of | OD value | Cell viability (%) |
| Cell normal control group | 0.222±0.037 | 100 |
| Virus control group | 0.113±0.043 # | 50.9 |
| 25 mu mol/L ganciclovir control group | 0.221±0.031* | 100 |
| 12.5 mu mol/L ganciclovir control group | 0.223±0.025* | 100 |
| 6.25 mu mol/L ganciclovir control group | 0.225±0.042* | 100 |
| 50 mu mol/L placenta-derived cytokine S1+S2 | 0.231±0.024* | 100 |
| 25 mu mol/L placenta-derived cytokine S1+S2 | 0.232±0.043* | 100 |
| 12.5. Mu. Mol/L placenta-derived cytokine S1+S2 | 0.226±0.041* | 100 |
| 6.25 mu mol/L placenta-derived cytokine S1+S2 | 0.207±0.044* | 93.2 |
| 3.1. Mu. Mol/L placenta-derived cytokine S1+S2 | 0.167±0.044 | 75.2 |
# P<0.01 Virus control groupvsA normal control group of cells; * P (P)<0.01 vsVirus control group
TABLE 4 Effect of different placenta-derived cytokines and placenta-derived mesenchymal Stem cells on HCMV Activity
| Group of | OD value | Cell viability (%) |
| Cell normal control group | 0.222±0.037 | 100 |
| Virus control group | 0.113±0.043 # | 50.9 |
| 12.5 mu mol/L ganciclovir control group | 0.223±0.025* | 100 |
| 12.5 mu mol/L placenta tissue active factor S1 | 0.221±0.031* | 99.6 |
| 12.5 mu mol/L placenta-derived mesenchymal stem cells S2 | 0.223±0.034* | 100 |
| 12.5. Mu. Mol/L placenta-derived cytokine S1+S2 | 0.226±0.041* | 100 |
# P<0.01 Virus control groupvsA normal control group of cells; * P (P)<0.01 vsVirus control group
As can be seen from the results in tables 3 and 4, compared with the cells of the normal control group, the OD value is obviously reduced and the cell survival rate is obviously reduced after the cells of the virus control group are infected by HCMV; compared with virus infection groups, the placenta tissue active factor S1, the placenta source mesenchymal stem cells S2 and the placenta source cytokines S1+S2 can all improve the OD value, obviously improve the survival rate of the infected HCMV cells, and the lowest effective dose is 6.25 mu mol/L.
Fig. 5 shows that cells developed lesions abnormal after infection of HCMV with HELF cells, while placenta-derived cytokine s1+s2 significantly inhibited the occurrence of HELF cytopathy caused by HCMV, with a minimum effective dose of 12.5 μmol/L.
EXAMPLE 4 investigation of the in vivo anti-human herpesvirus infection by placenta-derived cytokines
Mouse embryonic lung fibroblasts NIH/3T3, HSV2 were purchased from the cell institute of Chinese medical science. The cells and viruses were cultured and passaged by conventional methods with a virus titer of 10 4.9 TCID 50 0.1. 0.1 mL. The experimental animals were clean BALB/c male mice 8-10 weeks old, weighing 20+ -2 g, purchased from Beijing Vitolihua technology Co.
Establishment of a mouse testis herpes simplex virus infection model: the experimental animals are divided into 6 groups, namely a normal control group, a virus control group, an acyclovir group and a placenta-derived cytokine S1+S2 high, medium and low dose groups. The normal control group was inoculated with 25 μl DMEM at the testes on both sides, and the remaining groups were each inoculated with an equal amount of HSV2 suspension. Administration was initiated 24 h after virus inoculation by intraperitoneal injection (0.5 mL/dose). Wherein, normal control group is not inoculated with virus, and physiological saline is administered for 15 days. The virus control group was inoculated with virus, and physiological saline was administered for 15 days without administration. Acyclovir was inoculated with virus and acyclovir was administered 60 mg/Kg/time for 15 days. The placenta-derived cytokine S1+S2 was inoculated with the virus in high, medium and low doses of 60 mg/Kg/time, 30 mg/Kg/time, 15 mg/Kg/time, respectively, for 15 days.
15 days after administration, the mice were sacrificed after 12 h without water withdrawal after fasting, and bilateral testicular paraffin embedding was used as a pathological section.
Testis tissue was HE stained and observed under a microscope for pathological changes of the tissue. The results are shown in figure 6 of the specification.
The HSV2 infection of testis tissue is detected by adopting a single-phase oligonucleotide probe mRNA in situ hybridization method, digoxin is used for marking, in situ hybridization is carried out according to instructions, and DAB staining is carried out. As a result of the analysis under the light microscope, the presence of brown-yellow particles in the cells was judged as positive hybridization signals. The results are shown in figure 7 of the specification.
HE staining results show that the testis tissue spermatogenic cells and the interstitial cells of the normal control group mice are orderly arranged; the viral control group, the seminiferous tubules, were necrotic in basal membrane, peritubular myoid cells were denatured, the arrangement of sperm cells was disordered, and most of them were necrotic in coagulation. The placenta-derived cytokine high dose group still showed slight atrophy change of local seminiferous tubules, but compared with the virus control group, the lesions of seminiferous tubules were significantly reduced and the arrangement of sperm cells was ordered. Acyclovir group showed some atrophy and change of local seminiferous tubules, and the arrangement of sperm cells was slightly disturbed, and compared with virus control group, the lesions were remarkably reduced.
HSV in situ hybridization results show that testis sperm cells and interstitial cells of a normal group are orderly arranged, and no brown yellow staining particles appear in the cells; the basilar membrane of the seminiferous tubule of the virus control group is necrotized, peritubular myoid cells are denatured, the arrangement of sperm cells is disordered, brown yellow granular positive staining is visible in the cytoplasm of the testicular interstitial cells and the sperm cells, and strong positive hybridization signals appear in each layer of blood vessels, peritubular myoid cells of the seminiferous tubules, testicular envelopes and the like in tissues; the placenta source cytokine high dose group testis seminiferous cells and interstitial cells are orderly arranged, and no positive cells of brown yellow staining particles appear in the cells; acyclovir groups still showed a degree of peritubular myoid cell degeneration and disorder of sperm cell arrangement, and small amounts of brown-yellow granular positive cells were still seen in the testicular interstitial cells and sperm cell cytoplasm. It can be seen that the effect of the placenta-derived cytokine high dose group is evident due to the acyclovir group.
EXAMPLE 5 clinical external use action of placenta tissue-derived cytokines for treating Herpes Simplex Virus (HSV) infection
Patients with clinical diagnosis of Herpes Simplex Virus (HSV) infection are selected to be 53 persons, randomly divided into three groups and continuously take the medicines for 8 days. Group a 16 (7 of whom were laser physical treated) using acyclovir ointment; group B, 18 (8 of whom had been laser physical treated), used placenta-derived cytokine ointment; group C19 (7 of which were laser physically treated) used placenta-derived cytokine sprays. Each group is applied three times daily, and is applied topically on affected parts. The results showed that the average recovery time of group B and group C using the placenta-derived cytokine external preparation was 4.37 days and 4.51 days, respectively, while the average recovery time of group a using acyclovir ointment was 6.23 days, which showed a significant difference in the effects of both. It can be seen that the placenta-derived cytokine is clinically applied to the treatment of Herpes Simplex Virus (HSV) infection, and the effect of the placenta-derived cytokine is obviously better than that of acyclovir. The results are shown in tables 5 and 6.
TABLE 5 action of clinically external placenta-derived cytokine on treatment of herpes simplex virus infection (cases number n: several)
TABLE 6 effects of external use placenta source cytokine adjuvant therapy/prevention of recurrence after laser removal of condyloma acuminatum
| Therapeutic method | Time of administration | Recurrence rate after 3 months (n=number of cases) |
| Simple carbon dioxide laser for removing wart | 0 week | 67.7%（n=31） |
| After removing wart body by laser, the wart body is coated with acyclovir ointment | For 12 weeks | 28.6%（n=7） |
| External coating with placenta cytokine after removing wart body by laser | For 12 weeks | 6.7%（n=15） |
The results show that the simple carbon dioxide laser can remove condyloma acuminatum very easily and recur, and the carbon dioxide laser can remove wart body after wound healing, and the acyclovir ointment can obviously control virus development, but can not completely inhibit and kill viruses. The carbon dioxide laser is used for removing warts and carrying out adjuvant therapy by using placenta-derived cytokines after wound healing, and the recurrence rate is reduced to 6.7% after 3 months, which proves that the placenta-derived cytokines can not only remarkably control the development of HSV infection, but also completely resist and kill HSV viruses.
EXAMPLE 6 acute toxicity study of placenta-derived cytokines
60 ICR mice for health experiments were obtained, and 20 ICR mice were obtained from the university of Beijing department of medicine, department of laboratory animal science, each group of which had a weight of 20.+ -.2 g. Placenta-derived cytokines were prepared as a suspension at a concentration of 0.5. 0.5 g/mL with distilled water, and administered by one-time gavage according to 0.8. 0.8 mL/20. 20 g, followed by continuous observation for 7 days.
The results show that at this dose, mice were well-conditioned and free from poisoning and death. ICR mice maximum tolerance test shows that the dose is 400 times of the effective dose taken by human mouth, and the mice are infused with LD of placenta-derived cytokines 50 >1.8 g/Kg. Thus, the safety of the placenta-derived cytokine was good.
In conclusion, experimental results show that the placenta-derived cytokine has obvious anti-herpesvirus infection effect, and compared with the clinically applied medicaments acyclovir and ganciclovir, the placenta-derived cytokine has the advantages of obvious anti-herpesvirus infection effect, better safety, lower cost and the like, and especially has good prospect for drug-resistant strain infected persons, so that the new application of the placenta-derived cytokine has good prospect for anti-herpesvirus infection.
EXAMPLE 7 study of placenta-derived cytokine external preparation
Test 1 preparation of placenta-derived cytokine ointment
The formula comprises the following components in percentage by weight: placenta-derived cytokine 2g, beeswax 6 g, glycerol 5 g, cetyl olive oil ester (emulsifier) 3 g, betaine 5 g, acrylamide-dimethyl taurate/VP copolymer 1 g, distilled water 28 mL.
The preparation method comprises the following steps:
1) 6 g beeswax and 3 g cetyl esters of olive oil (emulsifier) were melted by heating in a water bath at 90 ℃.
2) The 1 g acrylamide dimethyl taurate ammonium/VP copolymer was dissolved in 10 mL water, stirred slowly, and swollen by heating in a water bath at 90℃for 1 hour with slow stirring until completely dissolved.
3) 2g placenta-derived cytokine was poured into 18 mL water, 5 g glycerol was added, and then 5 g betaine was added, and the mixture was stirred slowly until complete dissolution.
4) Mixing 1) and 2) together in a water bath at 90 ℃ and stirring thoroughly and uniformly.
5) In a water bath at 90 ℃, 3) is poured into 4), and the mixture is fully stirred to a completely homogenized state by a homogenizer.
6) Cooling to below 40 ℃, and adding an antibacterial agent to obtain the placenta-derived mesenchymal stem cell ointment of which the placenta tissue active factors are combined with 50 g.
Test 2 preparation of placenta-derived cytokine spray
The formula comprises the following components in percentage by weight: placenta tissue active factor combined with placenta source mesenchymal stem cell 2g, carbomer 940 1 g, glycerin 5 g, sorbitol (solid state) 6 g, appropriate amount of antibacterial agent, distilled water 36 mL.
The preparation method comprises the following steps:
1) 1 g carbomer was slowly stirred in 10 mL water and swollen by heating in a water bath at 90℃for 1 hour with slow stirring until completely dissolved uniformly.
2) Combining 10 g placenta tissue active factors, pouring the placenta-derived mesenchymal stem cells into 26 mL water, adding 3 g glycerin, adding 6 g sorbitol (solid state), and slowly stirring until the placenta-derived mesenchymal stem cells are completely dissolved.
3) Mixing 1) and 2) together in a water bath at 90 ℃, and fully stirring to a completely homogeneous state by using a homogenizer.
4) Cooling to below 40 ℃, and adding an antibacterial agent to obtain the placenta-derived mesenchymal stem cell spray of 50 g placenta tissue active factors combined with placenta.
Claims (8)
1. A preparation method of placenta-derived cytokine for preventing and treating herpesvirus infection and its preparation application are provided.
2. Use according to claim 1, of a placenta-derived cytokine line to extract active cytokines from placenta tissue, combined with placenta-derived mesenchymal stem cells obtained by in vitro proliferation, characterized in that: every 0.5-30 g of placenta tissue is prepared into every 10-30 g of placenta-derived cytokine, wherein the placenta-derived cytokine contains 0.5-1.5 g of Interleukin (IL), 0.1-1.0 g of Colony Stimulating Factor (CSF), 0.1-1.0 g of Tumor Necrosis Factor (TNF), 0.5-1.5 g of Interferon (IFN), 0.5-1.5 g of Epidermal Growth Factor (EGF), 0.1-1.0 g of Transforming Growth Factor (TGF), 1.0-5.0 g of a plurality of essential amino acids, 1.0-5.0 g of small molecule active peptide, 0.5-1.5 g of immunoglobulin, 1.0-5.0 g of lipopolysaccharide and the like.
3. Use according to claim 1, 2, characterized in that: dissociating placenta tissue by adopting a trigeminy enzyme fractionation method, collecting placenta tissue dissociation solution, and separating active substances in the placenta tissue dissociation solution by using a Sephadex gel column of cross-linked dextran to mainly obtain a plurality of essential amino acids, small molecule active peptides, immunoglobulin, lipopolysaccharide and the like; the primary placenta source mesenchymal stem cells are collected from placenta tissue dissociation liquid in an adherence way, and the mesenchymal stem cells are induced to highly express active factors, mainly Interleukin (IL), colony Stimulating Factor (CSF), tumor Necrosis Factor (TNF), interferon (IFN), epidermal Growth Factor (EGF), transforming Growth Factor (TGF) and the like through in vitro proliferation culture.
4. Use according to claim 3, characterized in that: the trigeminy enzyme is 0.03% -0.3% of collagenase I, 0.05% -0.5% of trypsin and 0.1% -0.5% of papain; the grading method is 1) enzymolysis grading, namely firstly using collagenase I, then using trypsin, and finally combining trypsin and papain; 2) Classifying the pH values, wherein the pH value is 7.0-7.6, the pH value is 7.4-8.0, and the pH value is 7.0-6.5; 3) The temperature and time are graded, the temperature is set at 2-8 ℃ for 6-48 hours, the temperature is set at 30-40 ℃ for 0.5-48 hours, the temperature is set at 45-60 ℃ for 2-6 hours.
5. Use according to claim 3, 4, characterized in that: culturing 0.03% -0.3% collagenase I at a pH value of 7.0-7.6 and a temperature of 37 ℃ for 4-48 hours without shaking, so that the pH value is prevented from being lower than 6.5; placing 05% -0.5% trypsin at pH 7.4-8.0 for 6-48 h at 2-8 ℃ and culturing at 37 ℃ for 0.5-2h; the papain with the pH value of 0.1% -0.5% is placed for 2-6 hours at 45-60 ℃ and the pH value is 7.0-6.5.
6. Use according to claim 3, characterized in that: the Sephadex gel column is Sephadex G-25, sephadex G-50 and Sephadex G-75, and is used for separating active substances such as proteins, polypeptides, small molecular peptides, amino acids and other small molecules.
7. Use according to claim 1, characterized in that: the herpes viruses are Herpes Simplex Virus (HSV), human Cytomegalovirus (CMV) and the like; herpes virus infection and related diseases refer to the invasion of viruses into the body through the way of oral cavity, respiratory tract, genital tract mucosa, damaged skin and the like, the occurrence of herpes, ulcer and inflammation which are locally gathered on the mucosa or skin, common diseases such as gingivitis, herpes labialis, eczematoid herpes, herpetic keratitis, herpetic encephalitis and the like, the severe inflammation is accompanied with high fever and body pain, and serious systemic diseases occur occasionally, and internal organs are involved.
8. Use according to claim 1, characterized in that: the preparation is applied to medicines, health products, foods, external preparations, tissue preservation solution and the like.
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