Human placenta tissue extraction and human placenta tissue injection preparation method

Abstract

The invention discloses a method for extracting human placenta tissue and preparing human placenta tissue injection, which comprises the following steps: screening placenta, unfreezing, soaking, rinsing, draining, weighing, preparing liquid, mincing, grinding and hydrolyzing, adding acid water to adjust the pH value for acid hydrolysis, adding alkaline water to adjust the pH value for alkaline hydrolysis after hydrolysis, and then inactivating viruses. The preparation method comprises the following steps: preparing a human placenta tissue solution, carrying out virus inactivation, carrying out rough filtration and fine ultrafiltration to remove bacteria and filtration, carrying out nanofiltration virus removal treatment by using a nano-membrane to obtain a sterile human placenta tissue solution stock solution, carrying out dilution preparation on the human placenta tissue solution stock solution according to the stock solution concentration, carrying out sterile subpackaging in bottles, sealing and detecting. The prepared human placenta tissue fluid is high in biological activity, the biological activity of the human placenta tissue fluid is effectively guaranteed, and meanwhile the biological safety of the human placenta tissue fluid is guaranteed by adopting a two-time virus inactivation and removal method.

Description

Technical Field

[0001] The invention relates to a method for extracting human placental tissue and preparing a human placental tissue injection, in particular to an injection containing a variety of substances beneficial to body functions. Background Art

[0002] Human placental tissue fluid is a kind of various polypeptides, various factors, amino acids and other substances extracted from the human placenta through biological extraction methods after passing inspection and quarantine. It is completely absorbed by the human body and has medical, health, beauty, and health-preserving functions. It can improve human immunity and is mainly used to treat gynecology, dermatology, postoperative adhesions, scar contracture, tracheitis and other chronic diseases. It can also be used for skin beauty and skin care.

[0003] The invention patent with the authorization announcement number CN103735577A discloses a process for extracting placental tissue fluid, which includes the following steps in sequence: placing the frozen placenta in drinking water and thawing it naturally; placing the thawed placenta in a 4% sodium chloride solution to extract the blood, rinsing it with drinking water until there is no obvious blood stain, and controlling the water; placing the rinsed blood-stained placenta in a steamer and steaming it with steam (steaming for 10 minutes) to shape it; placing the shaped placenta in an oven and drying it at 60-90°C to obtain the obtained placenta; crushing the dried placental tissue and weighing it, adding injection water at a ratio of 1:10 and boiling it , twice, the first time, add injection water at a ratio of 1:8, boil at 115-120℃ for 3 hours, separate by pressure filtration to obtain filtrate I, the second time, add injection water at a ratio of 1:2, boil at 115-120℃ for 1 hour, separate the liquid from the placental tissue residue by pressure filtration to obtain filtrate II, combine filtrates I and II, and store the filtrates in another container for standby use; collect the above filtrates, adjust the pH value to 3.0-3.1 with 2mol / L hydrochloric acid, add an appropriate amount of 3g / L phenol according to the purpose of the embryo extract, stir evenly, and boil at 100℃ for 1 hour for hydrolysis. When the hydrolyzate is cooled to about 80°C, adjust the pH value to 2.6-3.6 with 2mol / L hydrochloric acid, add an appropriate amount of 20g / L activated carbon according to the amount and purpose of the embryo extract, stir for 15 minutes, remove carbon and filter, and store the filtrate in another container for later use; collect the above filtrate, adjust the pH value to 6.8-8.0 with 2mol / L sodium hydroxide, add sodium chloride to 0.85% (W / V), heat and boil for 1 hour (sterilization), add needle-grade activated carbon (add about 6 grams per liter), stir for 15 minutes, remove carbon and filter, and store the filtrate in another container for later use; adjust the pH value of the above filtrate to 6.6-7.2 with 2mol / L hydrochloric acid, and filter the filtrate with a microporous filter membrane with a diameter of φ=0.65μm-0.45μm, which is the placental tissue extract. This method subjects the placenta to multiple high-temperature treatments at temperatures above 100°C and acid hydrolysis, which reduces the activity of the components in the extract and weakens the biological activity of the product, thereby affecting the final efficacy of the product. Summary of the invention

[0004] The purpose of the present invention is to provide a method for extracting human placental tissue and preparing a human placental tissue injection solution to solve the problems raised in the above-mentioned background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for extracting human placental tissue and preparing human placental tissue injection solution comprises the following steps:

[0007] Step 1, select a healthy human placenta (passed the virus test) and place the healthy placenta in a thawing room at 20°C to thaw overnight;

[0008] Step 2, removing the placental blood in step 1, manually picking out the blood clots and fascia in the placenta, then rinsing the placenta with drinking water until it is nearly flesh-colored, and then draining the rinsed placenta with a stainless steel perforated plate, and setting aside after draining;

[0009] Step 3, weighing the weight of the placenta to be used after draining in step 2, and the amount of water for injection that needs to be added to the placenta, the water for injection is weighed with treated warm water at a ratio of 1:1.5, that is, 1.5 kg of water for injection is required for every 1 kg of placenta;

[0010] Step 4, placing the weighed placenta in step 3 on a meat grinder and mincing it into a meat paste, then using the weighed water for injection to clean the residual material in the meat grinder, and mixing the water for injection with the minced material;

[0011] Step 5, add the placenta tissue minced material mixed with injection water in step 4 into the colloid mill at a volume of 4000-6000 ml / time, circulate the grinding for 3 minutes until it is completely ground into a pulp, and freeze the grinding liquid for more than 36 hours. The grinding is mainly to fully break the tissue and cells, and the time can be extended. Freeze at -20~-30 degrees for more than 48 hours, and then put it at room temperature to melt, so that ice crystals are formed in the tissue cells, the concentration of the cytosol in the cells is increased, and the cells are swollen and broken, thereby releasing the active ingredients such as polypeptides, nucleic acids, and cytokines. The high-temperature extraction used in the original process (which leads to inactivation of activity) is avoided, thereby ensuring the vitality of the active ingredients.

[0012] Step 6, thawing the grinding liquid frozen in step 5, transferring the thawed grinding liquid into a homogenization tank, opening the homogenization tank, heating the homogenization tank to 40°C~60°C, and maintaining the homogenization tank at 50±1°C during the homogenization process, heating and thawing to further break up the tissues and cells, and quickly release the effective ingredients, until the grinding liquid is homogenized to form a homogenate.

[0013] Step 7, after homogenizing the homogenate in step 6 for 1.5 hours, add pancreatin (according to 1 kg of homogenate: 0.1 g of pancreatin), stir evenly, and then adjust the pH to about 5 with 5 mol / L hydrochloric acid solution, adjust the pH, fully hydrolyze, and make some impurities reach the isoelectric point, fully remove impurities, and continue homogenizing for 30 minutes;

[0014] Step 8, the homogenate is homogenized with hydrochloric acid to form a supernatant, the supernatant is filtered with a filter cloth, and the pH value of the supernatant is adjusted to 8 with a 5 mol / L NaOH solution, alkaline hydrolysis is performed, and impurities are further removed.

[0015] Step 9, placing the supernatant of step 8 in a homogenization tank and heating it to 80° C., then stopping stirring and keeping warm for 5 minutes to inactivate the virus;

[0016] Step 10, transferring the supernatant of step 9 into a preparation tank, starting the composite membrane clarification filtration system, and ultrafiltering all the supernatant to achieve clarification;

[0017] Step 11, the supernatant clarified in step 10 enters an ultrafiltration system with a relative molecular weight cutoff of 5KD, the supernatant is completely ultrafiltered, the supernatant is checked, the supernatant must be in a clarified state, the pH value of the checked supernatant is adjusted to 6.2-7.2 with dilute hydrochloric acid, and then the adjusted supernatant is stored in a batching tank;

[0018] Step 11 uses an ultrafiltration system with a relative molecular weight cutoff of 5KD for ultrafiltration, which can cut off macromolecules with a molecular weight greater than 5KD, thereby ensuring the presence of safe and effective small molecules and avoiding safety reactions such as allergies caused by macromolecules;

[0019] Step 12, taking the supernatant stored in the batching tank in step 11 under the condition of a Class B environment and a local Class A environment, sterilizing the supernatant with a 0.22 μm sterilizing filter that has been sterilized, and then using a nanofiltration system with a nanofiltration membrane to perform nanofiltration virus removal on the sterilized supernatant to obtain a human placenta tissue extract. After the human placenta tissue extract is virus-free, it is aseptically packaged in bottles;

[0020] Steps 9 and 12 introduce two virus inactivation steps, namely, 80-degree temperature virus inactivation and 20nm nano-membrane virus interception to remove the virus, ensuring product safety;

[0021] Step 13, preparation of preparation: dilute the human placenta tissue extract according to the concentration of the original solution, add appropriate auxiliary materials to prepare a certain solution, sterilize and filter in a Class B environment and local Class A, divide into containers, and seal to obtain human placenta tissue injection.

[0022] Furthermore, the stock solution is diluted, and suitable auxiliary materials are added to prepare a certain solution, which is sterilized and filtered in a Class B environment and partially Class A, and packaged in containers.

[0023] Furthermore, the temperature of the water for injection in step 3 needs to be controlled below 40°C.

[0024] Furthermore, the homogenization tank in step 6 is heated to 50°C.

[0025] Furthermore, the virus inactivation temperature in step 9 is 80 degrees, and the temperature is kept for 5 minutes.

[0026] Furthermore, the pore size of the nanofiltration membrane in step 12 is 20 nm.

[0027] Beneficial effects of the present invention:

[0028] The present invention provides a method for extracting human placental tissue and preparing a human placental tissue injection solution. The human placental tissue solution is prepared under low temperature conditions. The prepared human placental tissue solution has high biological activity, thereby effectively ensuring the biological activity of the product and the biological vitality effect of the product. The two virus removal processes (removal processes for lipid enveloped viruses and non-lipid enveloped viruses, respectively) ensure the safety of the product and eliminate the risk of virus contamination.

[0029] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 : Rosette diagram of viability determination of human placental tissue fluid using traditional heating process.

[0031] Figure 2 : Rosette diagram of viability determination of human placental tissue fluid using traditional heating process.

[0032] Figure 3 : Preparation flow chart of the present invention.

[0033] Figure 4 : Component atlas 1 in the tissue fluid of the present invention.

[0034] Figure 5 : Component atlas 2 in the tissue fluid of the present invention.

[0035] Figure 6 : Component atlas three in the tissue fluid of the present invention.

[0036] Figure 7 : Component atlas four in the tissue fluid of the present invention.

[0037] Figure 8 : Component atlas five in the tissue fluid of the present invention.

[0038] Fig. 9 : Vitality assay rosette diagram of the present invention.

[0039] Fig.10 : Vitality assay rosette diagram of the present invention. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0041] Please refer to Figure 1-10 As shown;

[0042] A method for extracting human placental tissue and preparing human placental tissue injection, which does not require multiple high-temperature treatments above 100°C, but is prepared under low-temperature conditions, so that the content of active ingredients in the product is high and the biological activity of the product is high, thereby effectively ensuring the biological activity of the ingredients in the product and ensuring the biological vitality effect of the product:

[0043] Step 1, screening healthy placentas;

[0044] Healthy human placentas are collected and tested for hepatitis B, hepatitis C, AIDS, syphilis and other viruses. Placentas with negative virus test results are selected as raw materials and placed in a 20℃ thawing room for thawing;

[0045] Step 2, soaking, rinsing and draining the placenta;

[0046] First, the thawed placenta is immersed in a sodium chloride solution to remove the blood in the placenta, and the blood clots and fascia of the placenta are manually picked out. After being picked out, the placenta is rinsed with drinking water until it is nearly flesh-colored, and the rinsed placenta is drained using a stainless steel perforated plate, and the placenta is set aside after the draining is completed;

[0047] Step 3, weighing and preparing liquid;

[0048] Weigh the weight of the placenta to be used after draining, and the amount of water for injection that needs to be added to the placenta. The temperature of the water for injection is controlled below 40°C. Then weigh the weight of the placenta and the water for injection at a ratio of 1:1.5, that is, 1.5 kg of water for injection is required for every 1 kg of placenta.

[0049] Step 4, mincing the placenta;

[0050] The weighed placenta is placed on a meat grinder for mincing. After the meat grinder minces the placenta into a meat paste, the residual material in the meat grinder is cleaned with the weighed injection water, and the injection water is mixed with the minced material;

[0051] Step 5, grinding;

[0052] The minced placenta tissue mixed with water for injection is added to the colloid mill at a volume of 4000~6000ml / time, and the colloid mill is used to grind it for 3 minutes until it is completely ground into a pulp. Then the grinding liquid is frozen for more than 36 hours, frozen at -20~-30 degrees for more than 48 hours, and then thawed at room temperature to form ice crystals in the tissue cells, increase the concentration of intracellular cytosol, and cause the cells to swell and break, thereby releasing the active ingredients such as polypeptides, nucleic acids, and cytokines. The high-temperature extraction used in the original process (which leads to inactivation of activity) is avoided, thereby ensuring the vitality of the active ingredients;

[0053] Step 6, homogenization;

[0054] Thawing the frozen grinding liquid, transferring the thawed grinding liquid into a homogenizing tank, opening the homogenizing tank, heating the homogenizing tank to 50°C, and maintaining the temperature of the homogenizing tank at 50±1°C during the homogenization process until the grinding liquid is homogenized to form a homogenate, heating and thawing to further break up the tissues and cells, and quickly release the effective ingredients, until the grinding liquid is homogenized to form a homogenate;

[0055] Step 7, acid hydrolysis;

[0056] After 1.5 hours of homogenization, add pancreatic enzyme (0.1g pancreatic enzyme for 1kg homogenate) and stir evenly. In a low pH environment, the protein may be denatured. Use pancreatic enzyme first. Through the digestion of pancreatic enzyme, the placental tissue structure can be more effectively decomposed without destroying other important proteins. Then use 5mol / L hydrochloric acid solution to adjust the pH to about 5 to obtain an acid-hydrolyzed homogenate. Then continue to homogenize the acid-hydrolyzed homogenate for 30 minutes, adjust the pH to reach the isoelectric point of some impurities, fully remove impurities, and continue to homogenize for 30 minutes.

[0057] Step 8, alkaline hydrolysis;

[0058] The homogenate is homogenized with hydrochloric acid and centrifuged to form a supernatant, the supernatant is filtered with a filter cloth, and the pH value of the supernatant is adjusted to 8 with a 5 mol / L NaOH solution to perform alkaline hydrolysis and further remove impurities;

[0059] Step 9, virus inactivation;

[0060] The supernatant was placed in a homogenization tank and heated to 80°C. After reaching 80°C, stirring was stopped and kept warm for 5 minutes. The sample was kept warm at 80°C for 5 minutes to inactivate the virus. The virus inactivation verification experiment was as follows: about 90 ml of human placental tissue centrifuge was placed in a water bath and heated to 80°C with stirring at 100 rpm. About 10 ml of PRV or VSV virus was added according to 9:1 (centrifuge: virus, V:V) and mixed. Place in an 80℃ water bath for 5 min, take samples at 0.5min, 2min and 5min, dilute 10 times and measure the virus titer after heating. After the virus is kept in the sample at 80℃ for 5min, the PRV and VSV titers in the three batches of samples are reduced to below the detection limit of 1.0 log, the average decrease in PRV titer is ≥ 4.5logs, and the average decrease in VSV titer is ≥ 5.0 logs. Refer to the standards of 2.1.1.10 Virus Inactivation Test in Technical Specifications for Disinfection (2002 Edition) and "Animal Virology" (Second Edition) (IV) Titration of Virus Infectivity, and use the endpoint dilution method to measure the virus titer in each batch of samples before and after treatment. The samples taken at different times were filtered with a 0.22 μm disposable filter, and then diluted 10-fold with cell maintenance medium (MEM culture medium containing 2% FBS), and immediately added to a 96-well plate inoculated with Vero cells (PPV inoculated with ST cells), placed in a 5% CO2 incubator, incubated at 37°C for 1 hour, and then the cell maintenance medium was replaced, and incubated for another 72 hours (ST cells were incubated for 120 hours, and the medium was replaced once every 48 hours), and the cell lesions were observed under an inverted microscope. The virus titer was calculated according to the Reed-Muench method (expressed as half-cell infectious dose TCID50, TCID50 logarithm = logarithm of the dilution in the group with a pathology rate higher than 50% + distance ratio), and the decrease in virus titer before and after treatment was calculated. Referring to the "Technical Methods and Validation Guidelines for Removal / Inactivation of Viruses in Blood Products", which stipulates that "virus reduction (log10) ≥ 4 logs indicates that the step is effective in removing / inactivating the virus", the 80-degree Celsius insulation treatment in this validation is an effective process step for inactivating lipid enveloped viruses (indicator viruses PRV and VSV).

[0061] Step 10, clarification and filtration;

[0062] The supernatant after the virus inactivation treatment is transferred to the preparation tank, and the composite membrane clarification filtration system is turned on. The composite membrane clarification filtration system ultrafilters all the supernatant to a clarified state.

[0063] Step 11, fine ultrafiltration;

[0064] The clarified supernatant enters the ultrafiltration system with a relative molecular weight cutoff of 5KD. The supernatant is completely ultrafiltered through the ultrafiltration system to check whether the supernatant is in a clarified state. When the supernatant is in a clarified state, its pH value is adjusted to 6.2~7.2 with dilute hydrochloric acid, and the adjusted supernatant is stored in a batching tank.

[0065] Step 12, remove viruses again and pack;

[0066] S1, virus removal;

[0067] Under the condition of Grade B environment and Grade A locally, the supernatant stored in the batching tank was taken out, and the supernatant was sterilized with a 0.22μm sterilizing filter that had been sterilized. Then, the sterilized supernatant was subjected to nanofiltration and virus removal by a nanofiltration system with a 20nm membrane, which was the human placental tissue extract. In this operation, about 1000g of the sample was pre-filtered with a 0.22 um filter membrane, and about 10ml of PPV or EMCV virus was added at 1% and mixed. Sartorius Virosart CPV (virus removal filter membrane, membrane area 5 cm2) was used to filter 800g within 12 hours at a pressure of 3.0 bar. The filtrate was collected in separate tubes, with 50g of filtrate collected in each tube. Samples were taken at loads of 100, 800, 1400, 1500, and 1600 L / m2, respectively. The virus titer was determined after dilution 10 times. After EMCV and PPV viruses were added to the three batches of samples, 5cm 2 Virosart CPV virus removal membrane filtration, within a total time of 720-790min, 814-976g of filtrate were obtained, respectively, with a maximum load of about 976g / 5cm2, (i.e. 1952 L / m2). The average PPV titer of the samples after three batches of nanofiltration decreased by ≥ 4.4 logs, and the PPV titer of the non-nanofiltration control group decreased by only 0.2 log; the average EMCV titer of the samples after three batches of nanofiltration decreased by ≥ 5.1 logs, and the EMCV titer of the non-nanofiltration control group decreased by only 0.6 log. The samples with a load of 1600 L / m2 and the filtration end point in the three batches of nanofiltration treatment removal verification were blindly propagated for three generations, and no cytopathic effect was observed, proving that the virus removal was completed. Referring to the "Technical Methods and Validation Guidelines for Removal / Inactivation of Viruses in Blood Products", "Virus reduction (log10) ≥ 4 logs indicates that the step is effective in removing / inactivating viruses", 20nm membrane filtration treatment is an effective process step for removing non-lipid envelope viruses (indicator viruses EMCV and PPV).

[0068] Step 13, preparation of preparation: dilute the human placenta tissue extract according to its original concentration, add appropriate auxiliary materials to prepare a certain solution, sterilize and filter in a Class B environment or a local Class A environment, dispense into containers, and seal to obtain a human placenta tissue injection solution.

[0069] Step 9 and step 12 introduce two virus inactivation steps, namely, 80-degree temperature virus inactivation and 20nm nano-membrane virus interception to remove the virus, thereby ensuring the safety of the product.

[0070] In step 11, an ultrafiltration system with a relative molecular weight cutoff of 5KD is used for ultrafiltration, which can intercept macromolecular substances with a molecular weight greater than 5KD, thereby ensuring the presence of safe and effective small molecular substances, avoiding adverse reactions such as allergies caused by macromolecules, and improving the safety of the product.

[0071] S1, examination of human placental tissue fluid;

[0072] Step 1, using UPLC-MS to analyze the components in human placental tissue fluid, the two batches of samples are No. 3 and No. 4;

[0073] like Figure 4 As shown, the mass spectra of the two batches of samples No. 3 and No. 4 are very similar, with a series of peaks in the m / z range of 500-1200;

[0074] like Figure 5 As shown in the partial magnified mass spectrum of sample No. 3-4, judging from the retention time, molecular weight and distribution of mass spectrum peaks, it may be a polypeptide substance;

[0075] like Figure 6 As shown in the partial magnified mass spectrum of sample No. 3-4, judging from the retention time, molecular weight and distribution of mass spectrum peaks, it may be a polypeptide substance;

[0076] like Figure 7 As shown, the local enlarged mass spectrum of the unretained or weakly retained components of sample No. 3 shows that it contains a variety of amino acids;

[0077] like Figure 8 As shown, the local enlarged mass spectrum of the unretained or weakly retained components of sample No. 4 shows that it contains multiple amino acids.

[0078] Step 2, using the Folin phenol method to detect the polypeptide content in human placental tissue fluid, the detection method comprises the following steps;

[0079] First, select reagents to prepare an alkaline copper test solution, specifically: take 10g of sodium hydroxide and 50g of sodium carbonate, and add 400ml of water to dissolve them, as liquid A; then take 0.5g of potassium tartrate, add 50ml of water to dissolve it, and take 0.25g of copper sulfate, add 30ml of water to dissolve it, and mix the two dissolved liquids to form liquid B. Before use, mix liquid A and liquid B, and add water until the mixed liquid reaches 500ml, that is, the liquid is an alkaline copper test solution;

[0080] Take the bovine serum albumin reference substance and add water to make a solution containing 0.3 mg per 1 ml to prepare the reference substance solution;

[0081] Then take 1 ml of human placental tissue fluid and dilute it to 100 ml with normal saline to prepare the nutrient solution;

[0082] Then accurately measure 0.0, 0.1, 0.3, 0.5, 0.7, and 0.9 ml of the reference solution, place them in stoppered test tubes, add water to 1.0 ml each, then add 1.0 ml of alkaline copper test solution, shake well, add 4.0 ml of Folin phenol test solution (take the stock solution 1→16 in Folin test solution), mix well immediately, place in a 55°C water bath for accurate reaction for 5 minutes, place in a cold water bath for 10 minutes, and measure the absorbance at a wavelength of 650 nm; at the same time, use tube No. 0 as a blank, draw a standard curve with absorbance as the ordinate and the concentration of the reference solution as the abscissa to prepare the standard curve;

[0083] Accurately measure 1.0 ml of the test solution and determine it according to the method under the preparation of the standard curve, starting from "adding alkaline copper test solution". The concentration of the test solution is found from the standard curve and multiplied by the dilution factor. The results show that the contents of the three batches are 12.7 mg / ml, 12.7 mg / ml, and 13.1 mg / ml, respectively.

[0084] S2, abnormal toxicity detection;

[0085] Abnormal toxicity test: For the acute toxicity experimental reaction in mice, 60 mice that passed the quarantine were selected, half of which were male and half were female, weighing 19.0g~21.4g, and were randomly divided into 4 groups according to gender and weight, namely, negative control group (0.9% sodium chloride injection), test sample low-dose group (nitrogen content 2mg / ml) and test sample high-dose group (nitrogen content 4mg / ml). Each mouse was injected with 0.5ml human placental tissue fluid through the tail vein, and the results were observed after 48 hours. If there was no death in the negative control group, the test sample low-dose group (nitrogen content 2mg / ml) and the test sample high-dose group (nitrogen content 4mg / ml), it proved that the human placental tissue fluid did not cause toxic reactions in mice, and the human placental tissue fluid passed the abnormal toxicity test.

[0086] S3, allergic reaction:

[0087] Active systemic allergic reaction of guinea pigs: 24 guinea pigs that passed the quarantine were selected, half male and half female, weighing 280.5-320.0g, and were randomly divided into 4 groups according to gender and weight, namely, negative control group (5% glucose injection), positive control group (100mg / ml egg protein solution), low-dose group of test article (nitrogen 2mg / ml) and high-dose group of test article (nitrogen 4mg / ml), with 6 animals in each group. Each group was given the corresponding drug by intraperitoneal injection, once every other day, for a total of 3 times. The sensitization drug volume is 0.5 ml / animal. On the 14th and 21st days after the last sensitization drug administration, the animals are challenged by injection of the drug solution of the same concentration as that used in sensitization through the toe vein. The attack dose is twice the sensitization dose, and the attack volume is 1.0 ml / animal. The animals are observed for 30 minutes immediately after the stimulation. The reaction of each animal, the appearance and disappearance time of the symptoms are observed in detail for 3 hours. A comprehensive judgment is made based on the incidence and degree of the allergic reaction. If the allergic reaction of the negative control group is negative and the allergic reaction of the positive control group is positive, all the animals in the low-dose group and the high-dose group of guinea pigs in the human placental tissue fluid have no allergic reaction symptoms within 3 hours after the intravenous stimulation, and the allergic reaction is negative, which proves that the allergy test of the human placental tissue fluid is qualified.

[0088] S4, viability assay;

[0089] According to the T cell activity assay - E receptor removal method, the experiment was conducted according to the method of sample activity = test sample test tube E rosette percentage minus control tube E rosette percentage. It was concluded that the cell activity of human placental tissue fluid prepared by this method was above 50%, which was much higher than the 15% activity of other processes. Specifically, after multiple tests, such as Fig. 9 As shown, the number of rosettes is 19, such as Fig.10 As shown, the number of rosettes is 13, which is the same as Figure 1 , Figure 2 Comparing the traditional rosette numbers of 2 and 3, the activity test results of the human placental tissue fluid prepared by the present invention are much higher than those of other processes.

[0090] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0091] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementations that can be understood by those skilled in the art.

Claims

1. A method for extracting human placenta and preparing human placental tissue injection, characterized in that: The steps include: Step 1, select a healthy placenta (passed the virus test) and place the placenta in a 20°C thawing room to thaw overnight; Step 2, removing the placental blood in step 1, manually picking out the blood clots and fascia in the placenta, then rinsing the placenta with drinking water until it is nearly flesh-colored, and then draining the rinsed placenta with a stainless steel perforated plate, and setting aside after draining; Step 3, weighing the weight of the placenta to be used after draining in step 2, and the amount of water for injection that needs to be added to the placenta, the water for injection is weighed with treated warm water at a ratio of 1:1.5, that is, 1.5 kg of water for injection is required for every 1 kg of placenta; Step 4, placing the weighed placenta in step 3 on a meat grinder and mincing it into a meat paste, then using the weighed water for injection to clean the residual material in the meat grinder, and mixing the water for injection with the minced material; Step 5, adding the placental tissue minced material mixed with injection water in step 4 into a colloid mill in an amount of 4000-6000 ml / time, circulating grinding for 3 minutes until it is completely ground into a slurry, and freezing the grinding liquid for more than 36 hours; Step 6, thawing the grinding liquid frozen in step 5, transferring the thawed grinding liquid into a homogenization tank, opening the homogenization tank, heating the homogenate to 40°C to 60°C, and maintaining the homogenization tank at 50±1°C during the homogenization process until the grinding liquid is homogenized to form a homogenate; Step 7, after homogenizing the homogenate in step 6 for 1.5 hours, add pancreatin (according to 1 kg of homogenate: 0.1 g of pancreatin), stir evenly, and then adjust the pH to about 5 with 5 mol / L hydrochloric acid solution, continue homogenizing for 30 minutes, and centrifuge to form a supernatant; Step 8, filtering the supernatant with a filter cloth, and then adjusting the pH value of the supernatant to about 8 with a 5 mol / L NaOH solution; Step 9, placing the supernatant of step 8 in a homogenization tank and heating it to 80° C., then stopping stirring and keeping warm for 5 minutes to inactivate the virus; Step 10, transferring the supernatant of step 9 into a preparation tank, starting the composite membrane clarification filtration system, and ultrafiltering all the supernatant to achieve clarification; Step 11, the supernatant clarified in step 10 enters an ultrafiltration system with a relative molecular weight cutoff of 5KD, the supernatant is completely ultrafiltered, the supernatant is checked, the supernatant must be in a clarified state, the pH value of the checked supernatant is adjusted to 6.2-7.2 with dilute hydrochloric acid, and then the adjusted supernatant is stored in a batching tank; Ultrafiltration is performed using an ultrafiltration system with a relative molecular weight cutoff of 5KD, which can intercept macromolecular substances with a molecular weight greater than 5KD, thereby ensuring the presence of safe and effective small molecular substances and avoiding safety adverse reactions such as allergies caused by macromolecules; Step 12, taking the supernatant stored in the batching tank in step 11 under the condition of a Class B environment and a local Class A environment, sterilizing the supernatant with a 0.22 μm sterilizing filter that has been sterilized, and then using a nanofiltration membrane to remove viruses from the sterilized supernatant by nanofiltration, that is, a human placenta tissue extract, which is then aseptically packaged in bottles; Steps 9 and 12 introduce two virus inactivation steps, namely, 80-degree temperature virus inactivation and 20nm nano-membrane virus interception to remove the virus, ensuring product safety; Step 13, preparation of preparation: dilute the human placenta tissue extract according to the concentration of the original solution, add appropriate auxiliary materials to prepare a certain solution, sterilize and filter in a Class B environment and local Class A, divide into containers, and seal to obtain human placenta tissue fluid injection.

2. The method for extracting human placental tissue and preparing human placental tissue injection according to claim 1, characterized in that: The temperature of the water for injection in step 3 needs to be controlled below 40°C.

3. The method for extracting human placental tissue and preparing human placental tissue injection according to claim 1, characterized in that: The homogenization tank in step 6 is heated to 50°C.

4. The method for extracting human placental tissue and preparing human placental tissue injection according to claim 1, characterized in that: The virus inactivation temperature in step 9 is 80 degrees, and the temperature is kept for 5 minutes.

5. The method for extracting human placental tissue and preparing human placental tissue injection according to claim 1, characterized in that: The pore size of the nanofiltration membrane in step 12 is 20 nm.

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