CN112587632A - Biological preparation for preventing and treating HPV (human papillomavirus) infection and preparation method thereof - Google Patents
Biological preparation for preventing and treating HPV (human papillomavirus) infection and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a biological preparation for preventing and treating HPV virus infection, which comprises the following components in parts by weight: 0.5-1.0 part of sodium hyaluronate, 10-20 parts of hydroxypropyl methyl cellulose, 1-5 parts of matrine, 5-15 parts of glycerol, 1-5 parts of aloin, 0.1-0.5 part of rose essential oil, 1-5 parts of radix stemonae extract, 1-5 parts of saffron crocus, 1-10 parts of coptis chinensis, 20-30 parts of NK cell derivative liquid and 50-70 parts of ultrapure water. The invention discloses a preparation method of a biological agent for preventing and treating HPV virus infection, which comprises the following steps: (1) preparing a first component; (2) preparing NK cell derived liquid; (3) preparing a second component; (4) and fully stirring and uniformly mixing the first component, the NK cell derivative liquid with the formula amount and the second component under the overall B-level local A-level laboratory environment to obtain the biological preparation for preventing and treating HPV virus infection.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a biological preparation for preventing and treating HPV (human papillomavirus) virus infection and a preparation method thereof.
Background
Cervical cancer is a common malignant tumor in women, and epidemiological and molecular biological data show that Human Papilloma Virus (HPV) infection is closely related to cervical cancer. Almost all epidemiological data, combined with laboratory data, strongly support that persistent infection with HPV of high-risk type is an essential condition for the development of uterine cervical cancer:
(1) high-risk HPV infection can be found in 99.7% of cervical cancer, about 97% of high-grade lesions (HSIL) are positive, and the positive rate of low-grade lesions (LSIL) reaches 61.4%;
(2) the research of experimental animals and tissue specimens shows that the titer of HPV-DNA is positively correlated with the degree of cervical precancerous lesion;
(3) HPV infection is in a time sequence relation with the occurrence of cervical cancer, and the time interval from the infection to the cervical cancer development is 10-15 years, which accords with the biological pathogenesis.
Screening for cervical cancer can reduce the risk of developing cervical cancer, but cannot prevent HPV infection. At present, no definite treatment method for HPV infection exists, and how to quickly convert positive HPV patients into negative HPV patients is an important means for blocking HPV infection and preventing cervical cancer.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the technical problems in the prior art, the invention provides a biological agent for preventing and treating HPV virus infection and a preparation method thereof, and the biological agent has a high-efficiency inhibiting effect on HPV virus. Particularly, the gel preparation prepared by nutrient solution containing high-concentration cell factors is obtained in the process of carrying out mass culture and amplification on natural killer cells (NK cells) in vitro, and has obvious clinical effect on preventing and treating HPV virus infection or vagina and cervical inflammation patients.
The technical scheme is as follows: a biological preparation for preventing and treating HPV virus infection comprises the following components in parts by weight:
0.5-1.0 part of sodium hyaluronate, 10-20 parts of hydroxypropyl methyl cellulose, 1-5 parts of matrine, 5-15 parts of glycerol, 1-5 parts of aloin, 0.1-0.5 part of rose essential oil, 1-5 parts of radix stemonae extract, 1-5 parts of saffron crocus, 1-10 parts of coptis chinensis, 20-30 parts of NK cell derivative liquid and 50-70 parts of ultrapure water.
Further, the medicine comprises 0.8 part of sodium hyaluronate, 15 parts of hydroxypropyl methyl cellulose, 2 parts of matrine, 10 parts of glycerol, 3 parts of aloin, 0.3 part of rose essential oil, 2 parts of radix stemonae extract, 3 parts of saffron crocus, 4 parts of coptis chinensis, 25 parts of NK cell derivative liquid and 50 parts of ultrapure water.
The NK cell derived liquid is a main active ingredient of the invention and is obtained by in vitro culture and mass amplification of human NK cells.
A method for preparing a biological agent for preventing and treating HPV virus infection comprises the following steps:
(1) preparing a first component:
(11) heating ultrapure water with the formula amount of 70-90% to above 90 ℃ in a water bath heating mode;
(12) respectively adding hydroxypropyl methyl cellulose, glycerol and sodium hyaluronate according to the formula ratio into ultrapure water, stirring and fully dissolving, then naturally cooling to 50-60 ℃, keeping the temperature unchanged, stirring for 2-4 hours in a water bath environment, fully and uniformly mixing, sterilizing at high temperature and high pressure, and naturally cooling overnight to obtain a first component;
(2) preparing NK cell derived liquid:
(21) the method comprises the following steps of collecting a proper amount of umbilical cord blood in the umbilical cord of a healthy newborn, transferring the umbilical cord blood into a centrifugal tube for centrifugal separation, and obtaining upper plasma and lower blood cells, wherein:
absorbing upper plasma for inactivation;
the lower layer of blood cells are diluted by normal saline and then transferred into a centrifuge tube containing lymphocyte separating medium for centrifugation, and the centrifugation is divided into four layers:
the upper layer is a mixed solution of physiological saline and blood plasma;
the second layer is lymphocytes;
the third layer is separation liquid;
the fourth layer is red blood cells;
(22) collecting the second layer of lymphocytes, centrifuging, adding a lymphocyte culture medium containing an induction factor, suspending, inoculating into a culture bottle, placing in a 37 ℃ and 5% carbon dioxide incubator, and supplementing the lymphocyte culture medium every 1-2 days until the 14 th day;
(23) and on 17-20 days, transferring the cell suspension into a centrifuge tube for centrifugation, collecting supernatant, transferring the supernatant into a disposable sterile plate, and carrying out freeze drying treatment on the plate for 24 hours by adopting a low-temperature freeze drying method to obtain a concentrated solution, namely the NK cell derivative solution.
(3) And preparing a second component:
dissolving matrine, aloin, rose essential oil, radix stemonae extract, saffron and coptis chinensis in the balance of ultrapure water to obtain a mixed solution, heating the mixed solution to 40-50 ℃ in a water bath heating mode, fully dissolving, and filtering to remove bacteria to obtain a second component;
(4) and fully stirring and uniformly mixing the first component, the NK cell derivative liquid with the formula amount and the second component under the overall B-level local A-level laboratory environment to obtain the biological preparation for preventing and treating HPV virus infection.
Further, the inducing factors in step (22) include IL-2, IL-15, CD3 monoclonal antibody and CD52 monoclonal antibody.
Further, lymphocyte culture medium was purchased from LONZA, USA, serum-free medium X-VIVO 15.
Further, the matrine, aloin, rose essential oil, stemona root extract, saffron and coptis chinensis in the step (3) are all nano-powder materials.
The functions of the components are as follows:
hydroxypropyl methylcellulose is a semi-synthetic, inactive, viscoelastic polymer that is commonly used in ophthalmic applications as a lubricant, or as an adjuvant or excipient in oral pharmaceuticals; the invention mainly plays roles of thickening, lubricating and binding.
Sodium hyaluronate is an inherent component in human body, is a glucan aldehyde acid, has no species specificity, and is widely present in tissues such as placenta, amniotic fluid, crystalline lens, articular cartilage, skin dermis and the like; it is distributed in the cytoplasm and the intercellular substance of the organ, and plays a role in lubricating and nourishing the cells and the organelles contained therein.
Glycerol is a thick, colorless, odorless, sweet, clear liquid, and is a commonly used lubricant. It has effects of isolating air and preventing water evaporation, and can be applied on skin surface exposed in air such as hand and face to keep skin soft and elastic, and has moisture keeping effect without damage of dust and climate.
The matrine is prepared by extracting dried root, plant and fruit of Sophora flavescens ait of Leguminosae with organic solvent such as ethanol. Has effects of clearing heat, promoting urination, killing parasite, eliminating dampness, resisting virus, tumor and allergy.
The aloin has effects of resisting virus infection, promoting wound healing and recovery, diminishing inflammation, sterilizing, absorbing heat, relieving swelling, softening skin, and maintaining cell activity, and the combination of curdlan and wound healing acid also has wound healing activity.
The rose essential oil has the functions of diminishing inflammation, sterilizing, preventing infectious diseases, inflammation and spasm, and promoting cell metabolism and cell regeneration. Regulating female endocrine, nourishing uterus, relieving dysmenorrhea, and improving sexual coldness and climacteric discomfort.
The radix Stemonae extract is effective component extracted from dried rhizome of Stemonaceae plant. Has inhibitory effect on most pathogenic bacteria and dermatophytes.
The Coptidis rhizoma extract contains berberine, coptisine, methyl coptisine, tetrandrine, etc. Can be used for treating skin inflammation, eczema, wound healing, etc., and has strong antibacterial property; has effects in promoting collagen production, and activating skin, and can be used in antiaging cosmetic by combining their antioxidant effects; also has effects in inhibiting allergy, whitening skin, and keeping moisture.
The NK cell derived liquid is a concentrated liquid containing a plurality of cell factors obtained by separation, screening and purification in NK cell culture, and the paracrine effect and the immune effect of various cell factors in the concentrated liquid are utilized to regulate immune response, so that local inflammatory response is promoted to be reduced, and tissue inflammatory injury is reduced. On one hand, the method can promote the proliferation and differentiation of cells, so that the new cells are used for replacing aged and dead cells, repairing damaged parts, promoting the healing of mucosal wounds, improving the activity of capillary vessels and inhibiting pigmentation.
The using method comprises the following steps: and (3) disinfecting vulva and vagina conventionally, smearing and filling the biological preparation of the invention on cervix, and lying statically for 3-5 minutes.
Compared with the prior art, the biological agent for preventing and treating HPV virus infection and the preparation method thereof disclosed by the invention have the beneficial effects that:
1. the biological preparation contains various plant extracts, has no irritation to vagina, is safe and has no side effect;
2. hydroxypropyl methylcellulose is carried, sodium hyaluronate and glycerol are compounded into a biological adhesion carrier to carry multieffect factors secreted by NK cells and a gelatinous biological preparation prepared from various plant extracts, the biological adhesion carrier is convenient to apply and administer, has good retention and long action time, and enables antiviral and antibacterial components of the biological adhesion carrier to quickly permeate skin to protect mucosal cells from being invaded by viruses;
3. the factors of the tissue repair liquid fully play a role in regulating the reaction through the paracrine effect and the immune effect, promote the local inflammatory reaction to be reduced, relieve the inflammatory injury of the tissue and improve the regeneration capability of the wound tissue.
4. In the using process, the obvious antiviral effect and the repairing effect of gynecological inflammation wounds are obtained.
The specific implementation mode is as follows:
the following describes in detail specific embodiments of the present invention.
Detailed description of the preferred embodiment 1
A biological preparation for preventing and treating HPV virus infection comprises the following components in parts by weight:
0.8 part of sodium hyaluronate, 15 parts of hydroxypropyl methylcellulose, 2 parts of matrine, 10 parts of glycerol, 3 parts of aloin, 0.3 part of rose essential oil, 2 parts of radix stemonae extract, 3 parts of saffron crocus, 4 parts of coptis chinensis, 25 parts of NK cell derived liquid and 50 parts of ultrapure water.
The NK cell derived liquid is a main active ingredient of the invention and is obtained by in vitro culture and mass amplification of human NK cells.
A method for preparing a biological agent for preventing and treating HPV virus infection comprises the following steps:
(1) preparing a first component:
(11) heating ultrapure water with the formula amount of 80% to 95 ℃ in a water bath heating mode;
(12) respectively adding hydroxypropyl methyl cellulose, glycerol and sodium hyaluronate according to the formula amount into ultrapure water, stirring and fully dissolving, then naturally cooling to 55 ℃, keeping the temperature unchanged, stirring for 3 hours in a water bath environment, fully and uniformly mixing, sterilizing at high temperature and high pressure, and naturally cooling overnight to obtain a first component;
(2) preparing NK cell derived liquid:
(21) the method comprises the following steps of collecting a proper amount of umbilical cord blood in the umbilical cord of a healthy newborn, transferring the umbilical cord blood into a centrifugal tube for centrifugal separation, and obtaining upper plasma and lower blood cells, wherein:
absorbing upper plasma for inactivation;
the lower layer of blood cells are diluted by normal saline and then transferred into a centrifuge tube containing lymphocyte separating medium for centrifugation, and the centrifugation is divided into four layers:
the upper layer is a mixed solution of physiological saline and blood plasma;
the second layer is lymphocytes;
the third layer is separation liquid;
the fourth layer is red blood cells;
(22) collecting the second layer of lymphocytes, centrifuging, adding a lymphocyte culture medium containing an induction factor, suspending, inoculating into a culture bottle, placing in a 5% carbon dioxide incubator at 37 ℃, and supplementing the lymphocyte culture medium every 1.5 days until the 14 th day;
(23) and (4) transferring the cell suspension to a centrifuge tube for centrifugation, collecting supernatant, transferring the supernatant to a disposable sterile plate, and performing freeze drying treatment for 24 hours by adopting a low-temperature freeze drying method to obtain a concentrated solution, namely the NK cell derivative solution.
(3) And preparing a second component:
dissolving matrine, aloin, rose essential oil, radix Stemonae extract, stigma croci Sativi, and Coptidis rhizoma in the rest amount of ultrapure water to obtain mixed solution, heating the mixed solution to 45 deg.C by water bath heating, dissolving completely, filtering, and sterilizing to obtain the second component;
(4) and fully stirring and uniformly mixing the first component, the NK cell derivative liquid with the formula amount and the second component under the overall B-level local A-level laboratory environment to obtain the biological preparation for preventing and treating HPV virus infection.
Further, the inducing factors in step (22) include IL-2, IL-15, CD3 monoclonal antibody and CD52 monoclonal antibody.
Further, lymphocyte culture medium was purchased from LONZA, USA, serum-free medium X-VIVO 15.
Further, the matrine, aloin, rose essential oil, stemona root extract, saffron and coptis chinensis in the step (3) are all nano-powder materials. Can improve the bioavailability and simultaneously ensure that the contained liquid is easy to filter and sterilize.
Application of the biological preparation prepared in example 1 to HPV infected patients
The biological preparation prepared in this example 1 was clinically applied to 10 patients with HPV virus infection, and the treatment process was performed in a gynecological treatment room. Lying the patient on the treatment bed with the lithotomy position, disinfecting the vulva and the vagina by a conventional method, and paving a towel; wiping the disinfectant with the dry cotton ball, and washing cervix, vagina and vulva with the nutrient solution; the cervix is smeared and perfused with the biological preparation of the invention. The operation is continuously carried out for 7 days according to the medicine feeding flow.
The curative effect observation after one month: among 10 cases of high-risk low-level multi-type HPV infection, 4 cases of low-level negative conversion, 3 cases of high-risk negative conversion, and 10 cases of high-risk negative conversion are rechecked after one month of treatment; 10 patients have cervicitis, cervical erosion and hypertrophy with different degrees, and after treatment for 7 days continuously, the symptoms are obviously improved, and moderate erosion becomes mild and mild smooth; the symptoms of patients suffering from acute and chronic vaginitis are improved after 1 day of treatment, and the patients are cured after 3 days; patients without any symptoms feel comfortable and moist vagina after 1 treatment.
In conclusion, the biological preparation obtained in the embodiment has no irritation, is viscous, has good biological adhesion effect and low fluidity, cannot run off and cause discomfort after being smeared and poured into a vagina, can form a layer of protective film of a physical barrier on the smearing surface, maintains the cervical surface or the ulcer wound surface from being secondarily cross-infected, and effectively ensures the drug effect action time. Because the unique component NK cell derived liquid contains a large amount of cell factors, the invention has the functions of inhibiting inflammation, regulating immunity and promoting the rapid tissue repair of a diseased region. The NK cell derived liquid is concentrated by a freeze-drying technology, so that the functions of various active protein molecules in the NK cell culture solution are protected from being lost due to protein denaturation in the concentration process.
Specific example 2
A biological preparation for preventing and treating HPV virus infection comprises the following components in parts by weight:
0.5 part of sodium hyaluronate, 10 parts of hydroxypropyl methylcellulose, 1 part of matrine, 5 parts of glycerol, 1 part of aloin, 0.1 part of rose essential oil, 1 part of radix stemonae extract, 1 part of saffron crocus, 1 part of coptis chinensis, 20 parts of NK cell derived liquid and 55 parts of ultrapure water.
The NK cell derived liquid is a main active ingredient of the invention and is obtained by in vitro culture and mass amplification of human NK cells.
A method for preparing a biological agent for preventing and treating HPV virus infection comprises the following steps:
(1) preparing a first component:
(11) heating ultrapure water with the formula amount of 70% to 90 ℃ in a water bath heating mode;
(12) adding hydroxypropyl methyl cellulose, glycerol and sodium hyaluronate according to the formula amount into ultrapure water respectively, stirring and fully dissolving, naturally cooling to 50 ℃, keeping the temperature unchanged, stirring for 4 hours in a water bath environment, fully and uniformly mixing, sterilizing at high temperature and high pressure, and naturally cooling overnight to obtain a first component;
(2) preparing NK cell derived liquid:
(21) collecting a proper amount of umbilical cord blood in the umbilical cord of a healthy newborn, transferring the umbilical cord blood into a centrifugal tube for centrifugal separation, and obtaining upper plasma and lower blood cells, wherein:
absorbing upper plasma for inactivation;
the lower layer of blood cells are diluted by normal saline and then transferred into a centrifuge tube containing lymphocyte separating medium for centrifugation, and the centrifugation is divided into four layers:
the upper layer is a mixed solution of physiological saline and blood plasma;
the second layer is lymphocytes;
the third layer is separation liquid;
the fourth layer is red blood cells;
(22) collecting the second layer of lymphocytes, centrifuging, adding a lymphocyte culture medium containing an induction factor, suspending, inoculating into a culture bottle, placing in a 5% carbon dioxide incubator at 37 ℃, and supplementing the lymphocyte culture medium every 1 day until the 14 th day;
(23) and (4) transferring the cell suspension to a centrifuge tube for centrifugation, collecting supernatant, transferring the supernatant to a disposable sterile plate, and performing freeze drying treatment for 24 hours by adopting a low-temperature freeze drying method to obtain a concentrated solution, namely the NK cell derivative solution.
(3) And preparing a second component:
dissolving matrine, aloin, rose essential oil, radix Stemonae extract, stigma croci Sativi, and Coptidis rhizoma in the rest amount of ultrapure water to obtain mixed solution, heating the mixed solution to 40 deg.C by water bath heating, dissolving completely, filtering, and sterilizing to obtain the second component;
(4) and fully stirring and uniformly mixing the first component, the NK cell derivative liquid with the formula amount and the second component under the overall B-level local A-level laboratory environment to obtain the biological preparation for preventing and treating HPV virus infection.
Further, the inducing factors in step (22) include IL-2, IL-15, CD3 monoclonal antibody and CD52 monoclonal antibody.
Further, lymphocyte culture medium was purchased from LONZA, USA, serum-free medium X-VIVO 15.
Further, the matrine, aloin, rose essential oil, stemona root extract, saffron and coptis chinensis in the step (3) are all nano-powder materials. Can improve the bioavailability and simultaneously ensure that the contained liquid is easy to filter and sterilize.
Clinical examples of the biological preparations prepared in this example:
liu certain, female, 30 years old, positive cervical HPV59 and HPV6 found by examination on 8 months and 5 days in 2020, and a report of cervical fluid-based cytology TBS examination: the biological preparation prepared by the embodiment is used for carrying out local related treatment on the cervix on 11 days in 9 months in 2020 to 14 days in 9 months in 2020, and the cervix is changed from moderate hypertrophy to smoothness in four days of local treatment; after one month, the result of HPV reexamination is that the HPV6 (low-risk) is positive, and the high-risk HPV59 is negative.
Specific example 3
A biological preparation for preventing and treating HPV virus infection comprises the following components in parts by weight:
1.0 part of sodium hyaluronate, 20 parts of hydroxypropyl methylcellulose, 5 parts of matrine, 15 parts of glycerol, 5 parts of aloin, 0.5 part of rose essential oil, 5 parts of radix stemonae extract, 5 parts of saffron crocus, 10 parts of coptis chinensis, 30 parts of NK cell derived liquid and 70 parts of ultrapure water.
The NK cell derived liquid is a main active ingredient of the invention and is obtained by in vitro culture and mass amplification of human NK cells.
A method for preparing a biological agent for preventing and treating HPV virus infection comprises the following steps:
(1) preparing a first component:
(11) heating ultrapure water with the formula amount of 90% to above 90 ℃ in a water bath heating mode;
(12) adding hydroxypropyl methyl cellulose, glycerol and sodium hyaluronate according to the formula amount into ultrapure water respectively, stirring and fully dissolving, naturally cooling to 60 ℃, keeping the temperature unchanged, stirring for 2 hours in a water bath environment, fully and uniformly mixing, sterilizing at high temperature and high pressure, and naturally cooling overnight to obtain a first component;
(2) preparing NK cell derived liquid:
(21) collecting a proper amount of umbilical cord blood in the umbilical cord of a healthy newborn, transferring the umbilical cord blood into a centrifugal tube for centrifugal separation, and obtaining upper plasma and lower blood cells, wherein:
absorbing upper plasma for inactivation;
the lower layer of blood cells are diluted by normal saline and then transferred into a centrifuge tube containing lymphocyte separating medium for centrifugation, and the centrifugation is divided into four layers:
the upper layer is a mixed solution of physiological saline and blood plasma;
the second layer is lymphocytes;
the third layer is separation liquid;
the fourth layer is red blood cells;
(22) collecting the second layer of lymphocytes, centrifuging, adding a lymphocyte culture medium containing an induction factor, suspending, inoculating into a culture bottle, placing in a 5% carbon dioxide incubator at 37 ℃, and supplementing the lymphocyte culture medium every 2 days until the 14 th day;
(23) and (4) transferring the cell suspension to a centrifuge tube for centrifugation, collecting supernatant, transferring the supernatant to a disposable sterile plate, and performing freeze drying treatment for 24 hours by adopting a low-temperature freeze drying method to obtain a concentrated solution, namely the NK cell derivative solution.
(3) And preparing a second component:
dissolving matrine, aloin, rose essential oil, radix Stemonae extract, stigma croci Sativi, and Coptidis rhizoma in the rest amount of ultrapure water to obtain mixed solution, heating the mixed solution to 50 deg.C by water bath heating, dissolving completely, filtering, and sterilizing to obtain the second component;
(4) and fully stirring and uniformly mixing the first component, the NK cell derivative liquid with the formula amount and the second component under the overall B-level local A-level laboratory environment to obtain the biological preparation for preventing and treating HPV virus infection.
Further, the inducing factors in step (22) include IL-2, IL-15, CD3 monoclonal antibody and CD52 monoclonal antibody.
Further, lymphocyte culture medium was purchased from LONZA, USA, serum-free medium X-VIVO 15.
Further, the matrine, aloin, rose essential oil, stemona root extract, saffron and coptis chinensis in the step (3) are all nano-powder materials. Can improve the bioavailability and simultaneously ensure that the contained liquid is easy to filter and sterilize.
Clinical examples of the biological preparations prepared in this example:
zhangiao, female, 34 years old, secondary examination of HPV52 (high risk) positivity on 2019.3.28 days, vaginal drug application treatment for two months by recombinant human interferon alpha 2b vaginal effervescent capsule, secondary examination on 2019.10.30 days still shows HPV52 positivity, interferon treatment is ineffective, thus drug administration is stopped. Reexamination at 9 and 30 days 2020 shows that HPV52 and HPV35 are high-risk positive, when the biological preparation prepared in the embodiment is subjected to cervical local related treatment from 10 and 10 days in 2020 to 16 days in 10 and 2020, the cervix is changed from hypertrophy to smoothness after the local treatment for seven days; after one month, the result of HPV reexamination is that the HPV52 is of high-risk positive type, and the high-risk HPV35 is changed into negative.
The embodiments of the present invention have been described in detail. However, the present invention is not limited to the above-described embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention.
Claims (7)
1. A biological agent for preventing and treating HPV virus infection is characterized by comprising the following components in parts by weight:
0.5-1.0 part of sodium hyaluronate, 10-20 parts of hydroxypropyl methyl cellulose, 1-5 parts of matrine, 5-15 parts of glycerol, 1-5 parts of aloin, 0.1-0.5 part of rose essential oil, 1-5 parts of radix stemonae extract, 1-5 parts of saffron crocus, 1-10 parts of coptis chinensis, 20-30 parts of NK cell derivative liquid and 50-70 parts of ultrapure water.
2. The biological agent for preventing and treating HPV viral infection according to claim 1, comprising 0.8 parts of sodium hyaluronate, 15 parts of hydroxypropyl methylcellulose, 2 parts of matrine, 10 parts of glycerin, 3 parts of aloin, 0.3 part of rose essential oil, 2 parts of stemona root extract, 3 parts of saffron crocus, 4 parts of coptis chinensis, 25 parts of NK cell derived liquid and 50 parts of ultrapure water.
3. The biological agent for preventing and treating HPV viral infection according to claim 2 wherein the NK cell derived fluid is derived from human NK cells cultured in vitro and amplified in large quantities.
4. A method for preparing a biological agent for preventing and treating HPV virus infection is characterized by comprising the following steps:
(1) preparing a first component:
(11) heating ultrapure water with the formula amount of 70-90% to above 90 ℃ in a water bath heating mode;
(12) respectively adding hydroxypropyl methyl cellulose, glycerol and sodium hyaluronate according to the formula ratio into ultrapure water, stirring and fully dissolving, then naturally cooling to 50-60 ℃, keeping the temperature unchanged, stirring for 2-4 hours in a water bath environment, fully and uniformly mixing, sterilizing at high temperature and high pressure, and naturally cooling overnight to obtain a first component;
(2) preparing NK cell derived liquid:
(21) the method comprises the following steps of collecting a proper amount of umbilical cord blood in the umbilical cord of a healthy newborn, transferring the umbilical cord blood into a centrifugal tube for centrifugal separation, and obtaining upper plasma and lower blood cells, wherein:
absorbing upper plasma for inactivation;
the lower layer of blood cells are diluted by normal saline and then transferred into a centrifuge tube containing lymphocyte separating medium for centrifugation, and the centrifugation is divided into four layers:
the upper layer is a mixed solution of physiological saline and blood plasma;
the second layer is lymphocytes;
the third layer is separation liquid;
the fourth layer is red blood cells;
(22) collecting the second layer of lymphocytes, centrifuging, adding a lymphocyte culture medium containing an induction factor, suspending, inoculating into a culture bottle, placing in a 37 ℃ and 5% carbon dioxide incubator, and supplementing the lymphocyte culture medium every 1-2 days until the 14 th day;
(23) and on the 17 th to 20 th days, transferring the cell suspension into a centrifuge tube for centrifugation, collecting supernatant, transferring the supernatant into a disposable sterile plate, and carrying out freeze drying treatment on the supernatant for 24 hours by adopting a low-temperature freeze drying method to obtain a concentrated solution, namely an NK cell derivative solution;
(3) and preparing a second component:
dissolving matrine, aloin, rose essential oil, radix stemonae extract, saffron and coptis chinensis in the balance of ultrapure water to obtain a mixed solution, heating the mixed solution to 40-50 ℃ in a water bath heating mode, fully dissolving, and filtering to remove bacteria to obtain a second component;
(4) and fully stirring and uniformly mixing the first component, the NK cell derivative liquid with the formula amount and the second component under the overall B-level local A-level laboratory environment to obtain the biological preparation for preventing and treating HPV virus infection.
5. The method of claim 4, wherein the inducing factors of step (22) comprise IL-2, IL-15, CD3 mAb and CD52 mAb.
6. The method for preparing a biological agent for the prevention and treatment of HPV viral infection according to claim 4, wherein the lymphocyte culture medium is purchased from LONZA, USA, serum-free medium X-VIVO 15.
7. The method of claim 4, wherein the matrine, aloin, rose essential oil, stemona root extract, saffron and coptis chinensis in step (3) are all nano-sized powders.
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| CN113318126A (en) * | 2021-06-15 | 2021-08-31 | 海南启研干细胞抗衰老医院有限公司 | Technology for treating HPV infected patient |
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