WO2023246293A1 - 红色诺卡氏菌细胞壁骨架在治疗宫颈病变中的应用 - Google Patents

红色诺卡氏菌细胞壁骨架在治疗宫颈病变中的应用 Download PDF

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WO2023246293A1
WO2023246293A1 PCT/CN2023/090846 CN2023090846W WO2023246293A1 WO 2023246293 A1 WO2023246293 A1 WO 2023246293A1 CN 2023090846 W CN2023090846 W CN 2023090846W WO 2023246293 A1 WO2023246293 A1 WO 2023246293A1
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nocardia
cell wall
cells
cws
wall skeleton
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French (fr)
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盖波
张轶
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辽宁天安生物制药股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/005—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor after treatment of microbial biomass not covered by C12N1/02 - C12N1/08
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
    • C12N2502/1114—T cells
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
    • C12N2502/1121—Dendritic cells
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    • C12N2502/30—Coculture with; Conditioned medium produced by tumour cells
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    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/365—Nocardia

Definitions

  • the present disclosure relates to the fields of medicine and biopharmaceuticals. Specifically, the present disclosure relates to the use of Nocardia rubrum cell wall scaffolds in the treatment of cervical lesions.
  • Cervical cancer is a common malignant tumor that occurs in the female reproductive system. Its incidence and mortality rates rank fourth in the world, seriously affecting the health and quality of life of women around the world.
  • the main histological types include squamous cell carcinoma and adenocarcinoma. Among them, squamous cell carcinoma accounts for about 90% of cervical cancers, and adenocarcinoma accounts for about 10%.
  • HPV DNA can be detected in almost all cervical cancer specimens. Therefore, HPV infection is the most important risk factor for the occurrence and development of cervical cancer.
  • Some genotypes with stronger carcinogenicity are called high-risk HPV (hrHPV).
  • hrHPV is associated with more than 85% of cervical cancer cases, of which types 16 and 18 are the most common high-risk types.
  • Squamous intraepithelial lesions are precursor lesions of cervical cancer. They are divided into low-grade squamous intraepithelial lesions (LSIL) and high-grade squamous epithelium according to the severity of cervical epithelial dysplasia.
  • High-quamous intraepithelial lesions HSIL
  • HPV infection Since most HPV infections are cleared by the host immune system, LSIL lesions also spontaneously resolve. However, in the case of persistent hrHPV infection, invasive cancer may develop over several years. Therefore, clearing hrHPV infection is the most critical issue to interrupt the course of the disease and prevent the occurrence of cervical cancer.
  • the HPV vaccine has a good protective effect on vaccinated people, but it cannot eliminate existing infections. For patients with existing HPV-related high-grade cervical lesions, surgical resection remains the first choice of therapeutic intervention. However, with the development of immunotherapy research, some new treatment options are becoming possible, among which cell-based immunotherapy has attracted much attention.
  • Nocardia rubra is a type of Nocardia.
  • the Nocardia rubrum cell wall skeleton can be obtained by fermentation, cell disruption and protease degradation of the Nocardia rubrum cells.
  • Nocardia rubrum In the prior art, the cell wall skeleton of Nocardia rubrum can be obtained commercially. Specifically, It is a product (Nr-CWS) produced by Liaoning Gristech Biopharmaceutical Co., Ltd. Nocardia rubrum cell wall skeleton has been used to treat cervical erosion, cervical precancerous lesions (CN101073583A), anti-human papillomavirus (CN1935262A), skin lesions or skin ulcers (CN101209267A), fungal infections, herpes simplex, herpes zoster ( CN1879661A).
  • Nocardia rubrum cell wall skeleton has been used to treat cervical erosion, cervical precancerous lesions (CN101073583A), anti-human papillomavirus (CN1935262A), skin lesions or skin ulcers (CN101209267A), fungal infections, herpes simplex, herpes zoster ( CN1879661A).
  • the present disclosure provides a Nocardia rubrum cell wall scaffold.
  • Nocardia rubra refers to Nocardia rubra species of the genus Nocardia.
  • Identification of Nocardia rubrum Based on known or future microbial identification technologies, technicians can conduct taxonomic identification of a strain of bacteria. For example, available identification technologies include morphology, physiological and biochemical characteristics, 16S rRNA, etc. Technicians understand that with the development of science and technology, identification technology involves different means. In earlier periods, morphological and biochemical identification methods were mainly used, but the reliability of this method is not high. The advent of sequencing technology has given technicians a more reliable way to identify strains. For example, when the DNA sequence of 16S rRNA is identified as having more than 97% (inclusive) similarity, the two bacteria are judged to belong to the same species. As for Nocardia rubrum, known strains deposited in international (or national) culture collection units are used as model strains and compared with them.
  • Nocardia rubrum cell wall can be understood as either a complete cell wall or an incomplete cell wall (for example, broken or partially degraded).
  • the component exhibiting the desired activity is derived from the cell wall of Nocardia rubrum (eg, the cell wall itself or a component thereof). Therefore, various forms such as intact cell walls, broken cell walls, incomplete cell wall degradation products, cell wall components, cell wall extracts, etc. are allowed to be used in clinical applications, all of which are included in the scope of this disclosure.
  • the cell wall skeleton of the present disclosure cannot be understood to only represent the cross-linked network entities in the cell wall. Skilled persons should understand that this term does not exclude other cell wall components that are adsorbed, combined, and carried on the cross-linked network entities.
  • the cell wall skeleton of the present disclosure is a product obtained by disrupting bacteria and removing impurities (proteins, nucleic acids, cell membranes, and lipids).
  • the cell wall skeleton is the Nocardia rubrum cell wall skeleton corresponding to the national drug approval number S20030009 or its updated number.
  • S20030009 is the administrative license number issued by the drug regulatory department. This number will change with the update of certificates, adjustments to laws and numbering rules. However, the product standards, product parameters, production processes, and quality requirements represented by the number change remain unchanged. Therefore, S20030009 in this disclosure should be understood as S20030009 and its equivalent numbers.
  • the Nocardia rubrum cell wall skeleton is the Nocardia rubrum cell wall skeleton corresponding to the national drug approval number S20030009 or its updated number.
  • Steps 3.1), 3.2), 3.3), and 3.4) can be interchanged or parallel, and steps 4) and 5) can be interchanged.
  • Nocardia rubrum disruption the purpose is to remove intracellular material. Therefore, technologies such as ultrasonic crushing, high-pressure homogenizer crushing, and lysozyme can be used.
  • any known or future method suitable for disrupting Gram-positive bacteria is applicable to the technical solution of the present disclosure.
  • organic solvents are used to remove lipids from the fragmentation product.
  • nucleases are used to remove DNA and RNA from fragmentation products.
  • hydrolytic enzymes are utilized to degrade proteins in the fragmentation product.
  • surfactants are used to remove cell membranes from the disrupted products.
  • the average particle size of the fragmentation is from 10 nm to 1000 nm; mention may be made of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190nm ⁇ 10nm, and the range between any two of the above values. Testing methods for particle size are well known in the art.
  • the average particle size of the fragmentation is from 10 nm to 800 nm.
  • the average particle size of the fragmentation is from 10 nm to 500 nm.
  • said dispensing refers to dispensing into bottles or ampoules. Just before use, add solvent (such as sterile water) to the bottle or ampoule.
  • solvent such as sterile water
  • the vial refers to a vial (made of borosilicate glass or soda-lime glass).
  • the present disclosure provides the use of the aforementioned Nocardia rubrum cell wall skeleton in the preparation of a medicament, wherein the medicament is used to prevent or treat cervical lesions associated with high-risk HPV infection in a subject.
  • the high-risk HPV is selected from any one or combination of the following: types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68;
  • the cervical lesions are selected from any one of the following: atypical squamous cell lesions and low-grade squamous intraepithelial lesions.
  • the subject is between 21 and 65 years old, preferably between 21 and 40 years old.
  • the drug is prepared in a dosage form selected from any one of the following: injections, ointments, creams, emulsions, suspensions, pastes, gels, lotions, tablets, aerosols, Sprays, liniments, powders, dressings, bandages, films, patches, suppositories.
  • the drug is capable of achieving any one or combination thereof selected from the following: reducing the expression level of vimentin in cervical epithelium, increasing the expression level of IFN- ⁇ , reducing the expression level of IL-4, reducing IL The expression level of -17a, reducing the expression level of TGF- ⁇ , reducing the number of Foxp3-positive cells, increasing the mRNA expression level of T-bet, reducing the mRNA expression level of GATA3, reducing the mRNA expression level of ROR ⁇ t, reducing the mRNA expression level of Foxp3 .
  • the present disclosure provides a pharmaceutical composition or medicament for preventing or treating cervical lesions associated with high-risk HPV infection in a subject, which includes: a pharmaceutically acceptable carrier and the red color of the present disclosure.
  • a pharmaceutically acceptable carrier and the red color of the present disclosure.
  • Nocardia cell wall skeleton Nocardia cell wall skeleton.
  • compositions or medicaments of the present disclosure may be prepared in unit dose (or unit preparation) form.
  • a pharmaceutical composition or drug may be prepared in a liquid state (liquid formulation).
  • the pharmaceutical composition or drug can be prepared as a solid (dry powder formulation or lyophilized powder formulation).
  • liquid preparations and dry powder preparations can be converted into each other, and the only difference lies in the water content. Most or all of the water in the liquid preparation is removed to obtain a dry powder preparation (or freeze-dried powder preparation). The dry powder preparation (or freeze-dried powder preparation) is dissolved (or reconstituted) to obtain a liquid preparation.
  • the pharmaceutically acceptable carrier is selected from, but is not limited to: fillers, stabilizers (such as trehalose, glycine), flavoring agents (such as xylitol), disintegrants (such as carboxylic acid) Sodium methylcellulose), binder (e.g. gelatin), lubricant (e.g. magnesium stearate).
  • the stabilizer is selected from one or a combination of the following: glycine, lysine, arginine, hydroxyethyl starch, hydroxymethyl starch, trehalose, dextran.
  • the flavoring agent is selected from one or a combination of sucrose, monosaccharide, sodium saccharin, aspartame, sorbitol, xylitol, mannitol.
  • the binder is selected from one or a combination of sodium carboxymethylcellulose, hypromellose, and gelatin.
  • the lubricant is selected from one or a combination of talc, magnesium stearate, and micronized silica gel.
  • pharmaceutically acceptable carriers suitable for use in the present disclosure may also be mentioned, such as but not limited to: dextran, lactose, microcrystalline cellulose, trehalose, glycine, xylitol, carboxymethyl Sodium cellulose, erythritol, gelatin, magnesium stearate, propellant, humectant, solvent, solubilizer, emulsifier, antioxidant, pH adjuster, preservative.
  • non-limiting examples also include: white petrolatum, carbomer, hypromellose, methylcellulose, sodium carboxymethylcellulose, chitosan, sucralfate chitosan, polyvinylpyrrolidone, Polyvinyl alcohol, sodium hyaluronate, dimethyl ether, tetrafluoroethane, hydrofluoroalkane, glycerin, propylene glycol, deionized water, water for injection, distilled water, ethanol, cetyl alcohol, stearyl alcohol, para-aminobenzoic acid, ethyl alcohol Amide, isopropyl alcohol, Tween, polyoxyethylated hydrogenated castor oil, stearin Acid, glyceryl monostearate, triglyceryl monostearate, fatty acid sucrose ester, sucrose ester, sucrose acetate isobutyrate, sorbitan tristearate, isopropyl myristate, cholesterol ,
  • the present disclosure provides a method for preventing or treating cervical lesions associated with high-risk HPV infection in a subject, comprising the steps of: administering to the subject a therapeutically effective amount of the Nocardia rubrum cell wall of the present disclosure Matrix or pharmaceutical composition.
  • administer when applied to an animal, human, cell, tissue, organ or biological sample means that the drug or medical device comes into contact with the animal, human, cell, tissue, organ or biological sample.
  • Treatment means administering to a subject an internal or external drug (therapeutic agent, active ingredient or composition) (e.g., exosomes or pharmaceutical compositions of the present disclosure) or medical device, and in the subject being treated ( Alleviating (mitigating, delaying, ameliorating, curing) one or more disease symptoms to a clinically measurable extent (or group), wherein said subject has suffered from, is suspected of suffering from, or is susceptible to a disease one or more diseases or their symptoms.
  • an internal or external drug e.g., active ingredient or composition
  • therapeutic agent e.g., exosomes or pharmaceutical compositions of the present disclosure
  • the amount of a drug (therapeutic agent, active ingredient or composition) that is effective in alleviating the symptoms of any disease is called a therapeutically effective amount.
  • a drug therapeutic agent, active ingredient or composition
  • the subject is an animal other than a human, such as a farm animal, a pet, a working animal, an ornamental animal, a production animal, a laboratory animal (such as a rat, mouse, guinea pig, rabbit, dog, primates).
  • a farm animal such as a farm animal, a pet, a working animal, an ornamental animal, a production animal, a laboratory animal (such as a rat, mouse, guinea pig, rabbit, dog, primates).
  • the subject is human. In some specific embodiments, the subject is suspected of having, diagnosed with, has suffered from, or is susceptible to the target disease or symptoms thereof.
  • the administration is 1-3 times a day, or once a day, or every other day. Use once. Depending on the area and degree of the patient's lesions, different doses are used.
  • the administration period lasts from 2 days to 6 months, e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks or longer, and the range between any two of the aforementioned values.
  • the same pharmaceutical active ingredient or different pharmaceutical active ingredients may be administered at once, or may be divided into many smaller unit doses and administered at regular intervals. It is understood that the exact dosage, duration, and intervals of treatment are a function of the disease being treated and can be determined using extrapolation from animal or clinical trial data.
  • the administration may comprise a single administration, or two or more administrations spaced at appropriate intervals.
  • two consecutive administrations can be separated by 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, one and a half days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months.
  • the present disclosure provides a method for inhibiting HeLa cell proliferation in vitro, including the steps:
  • the HeLa cells are HPV18 positive cells.
  • an effective amount of 30 ⁇ g/ml of Nocardia rubrum cell wall scaffold is contacted with dendritic cells.
  • the Nocardia rubrum cell wall skeleton is the Nocardia rubrum cell wall skeleton corresponding to the national drug approval number S20030009 or its updated number.
  • the high-risk HPV types are selected from any one or combination of the following: types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68.
  • the Nocardia rubrum cell wall skeleton activates dendritic cells and Th1 cells in the same, or different, containers.
  • Nocardia rubrum cell wall skeleton activated dendritic cells and Th1 cells are administered simultaneously or sequentially.
  • Combination refers to a method of providing two or more active compounds to a subject simultaneously or successively for the purpose of treatment. When “combined” administration is involved, the time between each administration is sufficient to achieve a synergistic effect between the active compounds administered.
  • Optional means that the events described subsequently may occur, but do not have to occur; it depends on the circumstances.
  • dispensed means that the product is allowed to be dispensed, but is not required to be dispensed.
  • Figure 3A to Figure 3E Effect of Nr-CWS-intervention DC on naive CD4 + T cell factor expression. *p ⁇ 0.05 indicates the significance level between the intervention group and the control group.
  • FIG. 4A to Figure 4B Effect of Nr-CWS-intervention DC on T cell proliferation and its effect on Hela cell proliferation. *p ⁇ 0.05 indicates the significance level between the intervention group and the control group.
  • Figure 5 qPCR detection and analysis of FPR3mRNA expression in dendritic cells.
  • FIG. 7 Effect of Nr-CWS on inhibiting the proliferation of HeLa cells by DC-activated T cells transfected with FPR3siRNA (p ⁇ 0.05).
  • Example 1 Commercially available cell wall scaffold of Nocardia rubrum
  • Nocardia rubrum cell wall skeleton (referred to as Nr-CWS in this article) was purchased from Liaoning Gristech Biopharmaceutical Co., Ltd., national drug approval number S20030009 (the solid content of the cell wall skeleton in each bottle should be no less than 60 ⁇ g, of which muramic acid The content is not less than 1.0 ⁇ g, the sugar content is not less than 4.0 ⁇ g; the reconstitution volume is 2.0ml).
  • Lipid removal Add organic reagents (such as one or a combination of acetone, ether, ethanol) to the precipitate, and remove lipids according to conventional operations in this field.
  • organic reagents such as one or a combination of acetone, ether, ethanol
  • the pellet was reconstituted in Injection Use water and set aside. Optionally, it can be sterilized at 115°C for 20-30 minutes as a stock solution of the cell wall skeleton.
  • Example 1 Coat the product obtained in Example 1 or 2 on a dressing (such as sterile gauze) to prepare an external medicine.
  • a dressing such as sterile gauze
  • lotions mostly use water and alcohol as the dispersion medium; they are made from active ingredients, electrolytes, isotonic regulators, etc. in the dispersion medium.
  • Test Example 1 The efficacy and safety of Nr-CWS in treating HPV infection and its relationship with age factors
  • Nr-CWS Study the therapeutic effect and safety of Nr-CWS on persistent cervical HPV infection and abnormal cervical cytology, and explore its relationship with the patient's age.
  • Nr-CWS Nr-CWS
  • HPV subtypes include types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68.
  • TBS Cervical liquid-based cytology test results were recorded according to The Bethesda system (TBS).
  • TBS is used to report cervical or vaginal cytology diagnosis.
  • the results of this diagnostic system include: non-intrusive load monitoring (NILM), atypical squamous cells of undetermined significance, ASC-US), atypical squamous cells-cannot exclude HSIL (ASC-H), low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), squamous cell carcinoma (squamous cell cancer (SCC), atypical glandular cells-not otherwise specified (AGC-NOS), atypical glandular cells (AGC), adenocarcinoma in situ (AIS).
  • NILM non-intrusive load monitoring
  • ASC-US atypical squamous cells of undetermined significance
  • ASC-US atypical squamous cells-cannot exclude H
  • Pathological examination is the gold standard for judging the degree of cervical lesions. Abnormal results for this diagnosis include: CIN-I, CIN-II, CIN-III (glandular involvement), squamous cell carcinoma, and adenocarcinoma. wait.
  • the treatment uses the Nocardia rubrum cell wall scaffold (Example 1).
  • the subjects came to the outpatient clinic to take medication within 2 days after their menstruation was clear.
  • the researchers first obtained epithelial tissue biopsy specimens from the subject's cervical area (i.e., 0-month sample). Starting from D1, the subjects came to the outpatient clinic every other day, and the doctor administered Nr-CWS. Instruct the patient to take the lithotomy position and open the vaginal dilator to expose the cervix. First use a cotton ball to remove the secretions from the surface of the cervix and the cervix, then rub the surface of the cervix with dry gauze to create a minimally invasive surface.
  • Nr-CWS The therapeutic effect of Nr-CWS on patients with persistent HPV infection and cervical lesions was evaluated based on the outcome of HPV infection clearance.
  • the evaluation outcomes are as follows:
  • HPV infection still persists within 12 months of treatment, or there are other types of new HPV infections.
  • Nr-CWS The therapeutic effect of Nr-CWS on patients with persistent HPV infection and cervical lesions was evaluated based on the therapeutic effectiveness of cervical lesions.
  • the evaluation outcomes are as follows:
  • Cervical liquid-based cytology continues to be abnormal within 12 months of treatment, or may develop further.
  • the clinical remission rates in the younger group after receiving Nr-CWS treatment were 89.3% and 92.8% at 3-month and 6-month follow-up, respectively.
  • the clinical remission rate in the elderly group after receiving Nr-CWS treatment was 72.7% at 3-month and 6-month follow-up.
  • the clinical remission rate of the younger age group was higher than that of the older age group, but the difference between the two groups was not statistically significant (p>0.05).
  • Nr-CWS can effectively eliminate persistent HPV infection and treat abnormal cytology of low-grade cervical epithelial lesions. Compared with older patients, Nr-CWS has a better therapeutic effect on HPV infection and abnormal cervical cytology in younger patients. During the treatment period, the patient tolerated the drug well and no obvious adverse reactions occurred.
  • Nr-CWS treatment To explore the repairing effect of Nr-CWS treatment on the epithelial-mesenchymal transition of cervical tissue in patients with HPV infection and cervical lesions and the changes in CD4 + T subset cells in the local immune environment.
  • Cervical tissue samples were obtained from patients with HPV infection and cervical lesions before Nr-CWS treatment and 1 month or 3 months after treatment.
  • HE staining was used to observe the tissue structure; immunohistochemical staining was used to observe the expression of Vimentin and CD4. + Changes in T subgroup cytokines IFN- ⁇ , IL-4, IL-17a, and TGF- ⁇ .
  • the immunohistochemical score consists of two parts: staining intensity and proportion of positive cells. Observe under an optical microscope. At the typical cervical epithelial site with no tissue folding or edge effect, randomly select several high-power fields and count 100 cells in each field. cell. No positive cells were scored as 0 points, the number of positive cells ⁇ 25% was scored as 1 point, 26%-50% was scored as 2 points, 51%-75% was scored as 3 points, and >75% was scored as 4 points; the staining intensity was calculated as The score is based on the depth of the brown-yellow particles displayed by the majority of positive cells: no coloring is 0 points, light yellow is 1 point, brown is 2 points, and tan is 3 points.
  • the final score is the product of the positive cell percentage score and the staining intensity score: the score ranges from 0 to 9 points, with a score of 0-1 scored as "-”, a score of 2-3 points scored as "+”, and a score of 4-5 points. Marked as “++”, score ⁇ 6 points as “+++”.
  • Method for determining CD4 + T cell factor results observe under a light microscope, and randomly select 5 different fields of view for image collection under a high-power microscope. The counting and scoring methods are the same as above. IFN- ⁇ , IL-4, IL-17, and TGF- ⁇ are mainly expressed in the cytoplasm. The product of the positive cell percentage score and the staining intensity score is the final score: ⁇ 3 points are negative, and ⁇ 3 points are positive expression. Foxp3 is expressed in the nucleus, and the number is relatively small. The number of positive cells in each high-power field is used as statistical data.
  • Real-time fluorescence quantitative PCR was used to detect the mRNA expression levels of key transcription factors T-bet, GATA3, ROR ⁇ t, and Foxp3 for each subset of CD4 + T cells in cervical tissue.
  • Cervical tissue is mainly composed of epithelium and connective tissue.
  • the cervical mucosa is a single layer of columnar epithelium, and the cervix and vagina are covered by stratified squamous epithelium.
  • the junction area between single-layer columnar epithelium and stratified squamous epithelium at the external cervical os, that is, the squamous-columnar epithelial transition zone, is a common site for HPV infection and cervical cancer.
  • the cervical tissue structure of the normal group was clear, and cuboidal immature cells with strong basophilic and deep blue staining could be seen in the basal layer of the epithelium, neatly arranged.
  • Polygonal epithelial cells can be seen in the middle layer, and there are several layers of spindle or flat epithelial cells on the surface.
  • the flat cells on the surface degenerate and fall off.
  • the lamina basement of the mucosa below the epithelium is mainly connective tissue, with abundant fibers, cells and blood vessels, and stromal cells are loosely and evenly distributed. The connection between the epithelium and deep connective tissue is uneven.
  • Immunohistochemical staining was used to detect IFN- ⁇ and IL-4 staining in cervical tissue in the normal group and enrolled patients before Nr-CWS treatment, 30 days after treatment, and 90 days after treatment, and their expression levels were analyzed. IFN- ⁇ and IL-4 are mainly expressed in the cytoplasm of inflammatory cells. According to the IFN- ⁇ immunohistochemical analysis results, the positive expression rate in the normal group was 62.5%, and the positive expression rate before Nr-CWS treatment was 15.4%; the positive expression rate 30 days after treatment was 65.9%; and the positive expression rate 90 days after treatment is 87.1%. Statistical analysis showed that compared with before treatment, the expression level of IFN- ⁇ in cervical tissue of patients with high-risk HPV infection and cervical lesions increased after Nr-CWS treatment, and the difference was statistically significant (p ⁇ 0.05).
  • the positive expression rate in the normal group was 25%; the positive expression rate before Nr-CWS treatment was 94.4%; the positive expression rate 30 days after treatment was 48.7%; and the positive expression rate 90 days after treatment.
  • the expression rate is 25.8%.
  • Statistical analysis showed that compared with the normal group, the expression level of IL-4 in the cervical tissue of patients with high-risk HPV infection and cervical lesions increased significantly, and 30 and 90 days after Nr-CWS treatment, the expression level of IL-4 gradually decreased. decreased, the difference was statistically significant (p ⁇ 0.05).
  • Immunohistochemical staining method was used to detect IL-17a, TGF- ⁇ , and Foxp3 staining of cervical tissue in the normal group and enrolled patients before Nr-CWS treatment, 30 days after treatment, and 90 days after treatment. situation and analyze expression levels. IL-17a and TGF- ⁇ are mainly expressed in the cytoplasm of inflammatory cells and appear as brown to tan particles.
  • the positive expression rate in the normal group was 0, and the positive expression rate before Nr-CWS treatment was 77.8%; the positive expression rate 30 days after treatment was 31.7%; and the positive expression rate 90 days after treatment 16.1%.
  • Statistical analysis showed that compared with the normal group, the expression level of IL-17a in the cervical tissue of patients with high-risk HPV infection and cervical lesions increased, while the expression level of IL-17a gradually decreased 30 and 90 days after Nr-CWS treatment. , the difference is statistically significant (p ⁇ 0.05).
  • the positive expression rate in the normal group was 12.5%
  • the positive expression rate before Nr-CWS treatment was 84.7%
  • the positive expression rate 30 days after treatment was 46.3%
  • the positive expression rate 90 days after treatment is 12.9%.
  • Statistical analysis showed that compared with the normal group, the expression level of TGF- ⁇ in the cervical tissue of patients with high-risk HPV infection and cervical lesions increased, while the expression level of TGF- ⁇ gradually decreased 30 and 90 days after Nr-CWS treatment. , the difference is statistically significant (p ⁇ 0.05).
  • Foxp3 is expressed in the nuclei of inflammatory cells and appears as round tan particles.
  • Immunohistochemical analysis used positive cell counting under high-power fields. The results showed that the number of Foxp3-positive cells in the cervical tissue of the normal group was 1.25 ⁇ 1.21; the number of positive cells before receiving Nr-CWS treatment was 10.75 ⁇ 3.82; the number of positive cells 30 days after treatment was 5.1 ⁇ 2.40; the number of positive cells 90 days after treatment was 1.4 ⁇ 0.99.
  • Statistical analysis showed that compared with the normal group, the number of Foxp3-positive cells in the cervical tissue of patients with high-risk HPV infection and cervical lesions increased significantly. However, 30 and 90 days after Nr-CWS treatment, the number of Foxp3-positive cells gradually decreased, and the difference was as follows Statistical significance (p ⁇ 0.05).
  • the qPCR method was used to detect the expression levels of T-bet, GATA3, ROR ⁇ t, and Foxp3 mRNA in the cervical tissue of the normal group and enrolled patients before Nr-CWS treatment, 30 days after treatment, and 90 days after treatment ( Figure 1A to Figure 1D).
  • SIL is the early stage of cervical cancer and is divided into multiple types based on the abnormal cells in cervical smears or the extent of epithelial infiltration of atypical hyperplasia cells in pathological examination. Mild dysplasia is called LSIL, and severe dysplasia is called HSIL. Among the patients recruited in this test case, the diagnosis of SIL is based on the pathological results. After Nr-CWS treatment, pathological results showed that cervical intraepithelial dysplasia was significantly reduced, and epithelial cell abnormalities were significantly improved. This is consistent with the follow-up results in Test Example 1.
  • epithelial-to-mesenchymal transition plays an important role in the development of cervical cancer.
  • Epithelial cells gain better migration ability and invasiveness after undergoing EMT.
  • EMT persists and is abnormally activated.
  • EMT Epithelial calcium-dependent adhesion
  • N-cadherins neurocalcium-dependent adhesion
  • vimentin fibronectin
  • Fn fibronectin
  • Nr-CWS has a significant role in reversing the occurrence of cervical EMT caused by HPV and blocking the progression of cervical lesions.
  • Nr-CWS treatment repaired the local tissue structure of cervical lesions in patients with persistent hrHPV infection, reduced the expression level of vimentin in the cervical epithelium of patients, and had a significant reversal effect on EMT caused by persistent HPV infection.
  • it also increased the number of Th1 type CD4 + T cells in cervical tissue and induced the secretion of IFN- ⁇ , helping to reverse the immunosuppressive microenvironment, which may play a key role in the immune response against HPV infection.
  • Test Example 3 Microarray-based analysis of the mRNA expression profile of cervical tissue of patients treated with Nr-CWS
  • High-throughput transcriptome chip technology was used to analyze the differentially expressed genes in mRNA in cervical tissue caused by Nr-CWS treatment, and to preliminarily explore the possible immune mechanism of Nr-CWS.
  • Test Example 1 Select 3 patients who were recruited and followed up for 3 months after Nr-CWS administration and turned negative for HPV. The inclusion and exclusion criteria for subjects are shown in Test Example 1. Cervical tissues from 3 subjects were obtained as research samples before Nr-CWS treatment and 90 days after completion of treatment. According to the time when the samples were obtained, they were divided into pre-treatment group (3 cases) and post-treatment group (3 cases).
  • RNA screening results of differentially expressed RNA showed that there were 215 differentially expressed mRNAs in the cervical tissue of HPV-infected patients after Nr-CWS treatment compared with before treatment, including 33 up-regulated genes and 182 down-regulated genes.
  • a high-throughput mRNA expression profiling chip was used to differentially screen 6 samples of cervical tissue from HPV-infected patients before and after Nr-CWS treatment. A total of 215 differential genes were screened in the cervical tissue after treatment.
  • the Blood Transfusion Department of the Blood Center obtained a total of 20 human blood leukocyte-removing filters for the preparation of dendritic cells.
  • the Nr-CWS intervention concentration is set to 6 groups:
  • Nr-CWS Dissolve Nr-CWS and add it to the culture medium of DC cultured for 5 days in a six-well plate, 0.5 ml per well, the total volume of culture medium is 2.5 ml, the final concentration of Nr-CWS is grouped as above, the blank group is 2.5 ml without Complete culture medium for cells. Place in a cell culture incubator at 37°C and 5% CO2 and continue culturing for 48 hours.
  • Nr-CWS Incubate Nr-CWS with multiple dilutions of 3.75-60 ⁇ g/ml with dendritic cells for 48 hours, and detect cell viability with CCK-8 at 490 nm to determine the optimal Nr-CWS concentration for dendritic cell stimulation. Based on the results of the CCK-8 experiment, the optimal intervention concentration of Nr-CWS was determined to be 30 ⁇ g/ml.
  • DC cells cultured to day 5 were divided into three groups:
  • Nr-CWS Use nanoferric oxide particles to label Nr-CWS and interfere with DC for 48 hours. Observe the phagocytosis of Nr-CWS by DC under a transmission electron microscope. Use Prussian blue-nuclear fast red staining to observe the combination of DC and Nr-CWS under a light microscope. Condition.
  • Nr-CWS or LPS-treated iDCs were mixed with naive CD4 + T cells for 3 days, and the cell morphology was observed under light microscopy and scanning electron microscopy. The results showed that dendritic cells in the iDC group were scattered and did not attract T cells. Both Nr-CWS and LPS-treated DCs can attract naive T cells to gather around them and connect through cell processes.
  • Nr-CWS is an extract of Nocardia rubrum and may be recognized by APC as a pathogen antigen.
  • Nr-CWS labeled with DMSA-Fe 3 O 4 nanomagnetic beads was used to interfere with DC for 48 hours, stained with Prussian blue-nuclear fast red, and observed under a microscope. The results showed that the nuclei of DCs were round or oval and were magenta; the Nr-CWS labeled with DMSA-Fe 3 O 4 nanomagnetic beads was in the shape of blue fragments, and the red nuclei and blue-dyed Nr-CWS fragments were visible. Attached to each other and tightly combined.
  • Nr-CWS labeled with DMSA-Fe 3 O 4 nanomagnetic beads was used to interfere with DCs for 48 hours.
  • the cells were collected and the phagocytosis of Nr-CWS by DCs was observed under a transmission electron microscope. Under the electron microscope, it can be seen that a large number of magnetic bead particles are connected to the dendritic cells, and the cell membrane is indented into the cytoplasm to form a phagocytosis vacuole, which contains a large number of magnetic bead particles.
  • Nr-CWS-treated DC Effects of Nr-CWS-treated DC on the growth and differentiation of naive CD4 + T cells
  • this test example compared DC cells from the control group, Nr-CWS intervention group, and LPS intervention group.
  • CD4+T cells were co-cultured for 2 days, and the expression levels of IL-2, IFN- ⁇ , IL-4, TGF- ⁇ , and IL-17a cytokines in the cell culture supernatant were detected by ELISA.
  • Nr-CWS can directly activate dendritic cells induced in vitro and enable it to induce the differentiation of naive CD4 + T cells into Th1 type.
  • Nr-CWS can be used as an effective strategy against HPV infection and its induced lesions.
  • DCs were cultured to D5, and the cell transfection efficiency was observed under a fluorescence microscope. Add 30 ⁇ g/ml Nr-CWS to intervene DC, and the cells are grouped as follows: 1sico DC; 2siFPR3 DC; 3sico DC+Nr-CWS; 4siFPR3 DC+Nr-CWS.
  • the above-mentioned cultured cells were divided into groups and CD4 + T cells were co-cultured at a 1:10 ratio for 48 hours.
  • the cultured cell supernatants were collected on D7, and the changes in IL-2, IFN- ⁇ , IL-4, TGF- ⁇ , and IL-17a in the cell culture supernatants of the three groups were detected by ELISA.
  • iDCs were obtained from PBMCs isolated from human peripheral blood and induced with IL-4 and GM-CSF for 5 days. The cells were stimulated with or without Nr-CWS for 2 days. Harvest cells. The expression level of FPR3 in DCs was detected by immunohistochemistry, flow cytometry and qPCR.
  • FPR3 plays a positive regulatory role in the differentiation of Th1 cells during the process of DC-activated T cell immunity induced by Nr-CWS.
  • CCK-8 method detects the effect of Nr-CWS on the growth of Hela cells through siFPR3 DC and T cell co-culture model
  • Nr-CWS-treated DC and naive CD4 + T cells can inhibit Hela cell proliferation after mixed culture.
  • siFPR3 and its blank control NC were transfected twice into DC on D3 and D5. After transfection, they were stimulated with Nr-CWS for 2 days and cultured mixed with naive CD4 + T cells.
  • the Transwell chamber was used to co-culture with Hela cells for 48 hours, and the proliferation of Hela cells was detected by CCK-8 method. The results showed ( Figure 7) that the Hela cell viability in the siFPR3+Nr-CWS group was significantly higher than that in the NC+Nr-CWS group, and the difference was statistically significant (p ⁇ 0.05).
  • Nr-CWS regulates the differentiation of CD4 + T cells in the direction of Th1 by stimulating DCs, and DC-related genes UBD, MARCO, Upregulation of FPR3.
  • FPR3-silenced DCs were co-cultured with naive CD4 + T cells treated with Nr-CWS.
  • the culture supernatant was detected by ELISA and the concentrations of Th1 cytokines IL-2 and IFN- ⁇ were restored to low levels.
  • Nr-CWS lost the ability to induce Th1 differentiation of naive CD4 + T cells through DCs.
  • FPR3-silenced DC were treated with Nr-CWS and co-cultured with naive CD4 + T cells. Hela cells co-cultured with MLR were detected by CCK-8 method, and the proliferation activity of Hela cells returned to normal. It was shown that after FPR3 silencing in DCs, Nr-CWS could not inhibit HeLa cell proliferation through the MLR model of DC-CD4 + T cells.

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Abstract

红色诺卡氏菌细胞壁骨架在治疗宫颈病变中的应用。具体用于在受试者中预防或治疗伴有高危型HPV感染的宫颈病变，所述高危型HPV选自以下的任一项或组合：16、18、31、33、35、39、45、51、52、56、58、59、66、68型。红色诺卡氏菌细胞壁骨架作为具有生物活性的免疫激活剂，可用于HPV感染及其诱发病变。

Description

红色诺卡氏菌细胞壁骨架在治疗宫颈病变中的应用 技术领域
本公开涉及医学、生物制药领域。具体而言，本公开涉及红色诺卡氏菌细胞壁骨架在治疗宫颈病变中的应用。
背景技术
宫颈癌是发生于女性生殖系统的常见的恶性肿瘤，发病率和死亡率均居全球第4位，严重影响了全球女性健康和生活质量。其主要的组织学类型包括鳞状细胞癌和腺细胞癌，其中，鳞癌约占宫颈癌的90％，腺癌约占10％。
流行病学研究显示，在宫颈癌的所有标本中几乎都能检测到HPV DNA。因此，HPV感染是宫颈癌发生和发展的最主要危险因素。部分具有更强的致癌性的基因型被称为高危型HPV(high-risk HPV，hrHPV)，hrHPV与85％以上的宫颈癌病例相关，其中16型和18型是最常见的高危类型。鳞状上皮内病变(Squamous intraepithelial lesions，SIL)是宫颈癌的前期病变，根据宫颈上皮异型增生严重程度分为低级别鳞状上皮内病变(Low-quamous intraepithelial lesions，LSIL)和高级别鳞状上皮内病变(High-quamous intraepithelial lesions，HSIL)。由于大部分HPV感染后被宿主免疫系统清除，LSIL病变也随之自发消退。然而，在hrHPV持续感染的情况下，可能在数年内发展为浸润性癌。因此，清除hrHPV感染是阻断病程、预防宫颈癌发生的最关键问题。HPV疫苗对接种人群有很好的保护作用，但不能消除已存在的感染。对于已存在HPV相关高级别宫颈病变的患者，手术切除仍然是治疗干预的首选。然而随着免疫治疗研究的发展，一些新的治疗方案正在成为可能，其中以细胞为基础的免疫治疗备受瞩目。
红色诺卡氏菌(Nocardia rubra)是诺卡氏菌中的一种。红色诺卡氏菌的菌体经发酵、细胞破碎、蛋白酶降解后可制得红色诺卡氏菌细胞壁骨架。
现有技术中，红色诺卡氏菌细胞壁骨架可以是市售获得，具体而 言由辽宁格瑞仕特生物制药有限公司生产的商品(Nr-CWS)。红色诺卡氏菌细胞壁骨架已用于治疗宫颈糜烂、宫颈癌前病变(CN101073583A)、抗人乳头瘤病毒(CN1935262A)、皮肤损伤或皮肤溃疡(CN101209267A)、真菌感染、单纯疱疹、带状疱疹(CN1879661A)。
发明内容
第一方面，本公开提供了一种红色诺卡氏菌细胞壁骨架。
红色诺卡氏菌是指Nocardia属的红色诺卡氏菌种(Nocardia rubra)。
红色诺卡氏菌的鉴定：根据已知的或未来的微生物鉴定技术，技术人员可以对一株细菌进行分类学鉴定，例如可用的鉴定技术包括形态学、生理生化特征、16S rRNA等。技术人员理解，随着科技的发展，鉴定技术涉及不同的手段，在较早的时期主要采用形态学和生化鉴定方式，但是这种方法的可靠程度不高。测序技术出现后，技术人员可以利用更为可信的方式鉴定菌株。例如，当16S rRNA的DNA序列被鉴定为具有97％(含)以上相似性时，判定两个菌属于相同的种。就红色诺卡氏菌而言，将保藏在国际(或国家级)菌种保藏单位中的已知菌株作为模式菌株，并与其进行比对。
本公开中，“红色诺卡氏菌细胞壁”既可以理解为完整的细胞壁，也可以理解为不完整的细胞壁(例如，破碎的、或部分降解的)。在本公开的教导下，技术人员将理解，显示出所需活性的成分来自红色诺卡氏菌的细胞壁(例如，是细胞壁本身或其组成成分)。因此，在临床应用中允许采用完整的细胞壁、经破碎的细胞壁、细胞壁的不完全降解产物、细胞壁的组成成分、细胞壁的提取物等各种形式，这些都包含在本公开范畴之内。
本公开的细胞壁骨架不能理解为仅仅表示细胞壁当中的交联网状实体，技术人员应理解该术语不排除交联网状实体上所吸附、结合、携带的其他细胞壁成分。
在具体的示例中，本公开的细胞壁骨架是细菌经破碎、除杂(蛋白、核酸、细胞膜、脂质)后所得的产物。
在具体的实施方案中，细胞壁骨架是国药准字S20030009或其更新编号对应的红色诺卡氏菌细胞壁骨架。
技术人员应当理解，S20030009是药监管理部门发放的行政许可编号，此号码会随着证件的更新、法律、编号规则的调整而变化。然而，号码变化后所代表的产品标准、产品参数、生产工艺、质量要求是不变的。因此，本公开中S20030009应理解为S20030009及其等同编号。
在一些实施方案中，所述红色诺卡氏菌细胞壁骨架是国药准字S20030009或其更新编号对应的红色诺卡氏菌细胞壁骨架。
在另一些实施方案中，所述红色诺卡氏菌细胞壁骨架通过以下方法获得，所述方法包括以下步骤或由以下步骤组成：
1)提供红色诺卡氏菌；
2)破碎所述红色诺卡氏菌，得到破碎产物；
3.1)对所述破碎产物去除脂质；
3.2)对所述破碎产物去除核酸；
3.3)对所述破碎产物去除蛋白质；
3.4)对所述破碎产物去除细胞膜；
3.5)得到红色诺卡氏菌细胞壁骨架；
4)任选地，分装；
5)任选地，对所述红色诺卡氏菌细胞壁骨架进行冷冻干燥；
步骤3.1)、3.2)、3.3)、3.4)能够互换或并行，步骤4)和步骤5)能够互换。
对于红色诺卡氏菌的破碎，其目的在于去除细胞内的物质。因此可以采用超声破碎、高压均质机破碎、溶菌酶等技术。技术人员理解，任何适用于破碎革兰氏阳性菌的已知或未来方法，均适用于本公开的技术方案。
技术人员有能力根据活性成分(细胞壁及其组成成分)的后续应用(例如外敷)，来调整培养、破碎、分离、收集、除杂质、分装的具体参数和设备，以免制备步骤中引入影响后续应用的因素。
在一些实施方案中，利用有机溶剂去除破碎产物中的脂质。在一些实施方案中，利用核酸酶去除破碎产物中的DNA和RNA。在一些实施方案中，利用水解酶降解破碎产物中的蛋白质。在一些实施方案 中，利用表面活性剂去除破碎产物中的细胞膜。
在一些实施方案中，破碎的平均粒度为10nm至1000nm；可以提及10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190nm±10nm，以及上述任意两个数值之间的范围。粒度的测试方法是现有技术公知的。
在一些具体的实施方案中，破碎的平均粒度为10nm至800nm。
在另一些具体的实施方案中，破碎的平均粒度为10nm至500nm。
在具体的实施方案中，所述分装是指分装至瓶或安瓿中。临用前，向瓶或安瓿中添加溶剂(如无菌水)。作为一个示例，瓶是指西林瓶(vial，由硼硅玻璃或钠钙玻璃制成)。
第二方面，本公开提供了前述红色诺卡氏菌细胞壁骨架在制备药物中的用途，其中所述药物用于在受试者中预防或治疗伴有高危型HPV感染的宫颈病变。
所述高危型HPV选自以下的任一项或组合：16、18、31、33、35、39、45、51、52、56、58、59、66、68型；
所述宫颈病变选自以下的任一项：非典型鳞状细胞病变、低级别鳞状上皮内病变。
在具体的实施方案中，受试者是21岁至65岁，优选21岁至40岁。
在一些实施方案中，所述药物制备成选自以下任一项的剂型：注射剂、膏剂、霜剂、乳液、混悬剂、糊剂、凝胶剂、洗剂、片剂、气雾剂、喷雾剂、搽剂、粉剂、敷料、绷带、膜、贴片、栓剂。
在一些实施方案中，所述药物能够实现选自以下的任一项或其组合：降低宫颈上皮中波形蛋白的表达水平、提高IFN-γ的表达水平、降低IL-4的表达水平、降低IL-17a的表达水平、降低TGF-β的表达水平、降低Foxp3阳性细胞的数量、提高T-bet的mRNA表达水平、降低GATA3的mRNA表达水平、降低RORγt的mRNA表达水平、降低Foxp3的mRNA表达水平。
第三方面，本公开提供了一种用于在受试者中预防或治疗伴有高危型HPV感染的宫颈病变的药物组合物或药物，其包含：药学上可接受的载体和本公开的红色诺卡氏菌细胞壁骨架。
本公开的药物组合物或药物可以制备成单位剂量(或单元制剂)的形式。
在一些实施方案中，药物组合物或药物可以制备成液态的(液体制剂)。
在另一些实施方案中，药物组合物或药物可以制备成固体的(干粉制剂或冻干粉制剂)。
技术人员理解，液体制剂和干粉制剂(或冻干粉制剂)，二者可以相互转化，差别仅在于含水量。除去液体制剂中的绝大部分或全部水，得到干粉制剂(或冻干粉制剂)。干粉制剂(或冻干粉制剂)溶解(或复溶)后得到液体制剂。
在一些实施方案中，所述药学上可接受的载体选自，但不限于：填充剂、稳定剂(例如海藻糖、甘氨酸)、矫味剂(例如木糖醇)、崩解剂(例如羧甲基纤维素钠)、粘合剂(例如明胶)、润滑剂(例如硬脂酸镁)。
在一些实施方案中，稳定剂选自以下的一种或组合：甘氨酸、赖氨酸、精氨酸、羟乙基淀粉、羟甲基淀粉、海藻糖、葡聚糖。
在一些实施方案中，矫味剂选自以下的一种或组合：蔗糖、单糖、糖精钠、阿斯巴甜、山梨醇、木糖醇、甘露醇。
在一些实施方案中，粘合剂选自以下的一种或组合：羧甲基纤维素钠、羟丙甲纤维素、明胶。
在一些实施方案中，润滑剂选自以下的一种或组合：有滑石粉、硬脂酸镁、微粉硅胶。
在一些具体的实施方案中，适用于本公开的药学上可接受的载体还可以提及，例如但不限于：右旋糖酐、乳糖、微晶纤维素、海藻糖、甘氨酸、木糖醇、羧甲基纤维素钠、赤藓糖醇、明胶、硬脂酸镁、抛射剂、保湿剂、溶剂、增溶剂、乳化剂、抗氧化剂、pH调节剂、防腐剂。具体而言，非限制实例还包括：白凡士林、卡波姆、羟丙甲纤维素、甲基纤维素、羟甲基纤维素钠、壳聚糖、硫糖铝壳聚糖、聚乙烯吡咯烷酮、聚乙烯醇、玻璃酸钠、二甲醚、四氟乙烷、氢氟烷烃、甘油、丙二醇、去离子水、注射用水、蒸馏水、乙醇、十六醇、十八醇、对氨基苯甲酸、乙酰胺、异丙醇、吐温、聚氧乙基氢化蓖麻油、硬脂 酸、单硬脂酸甘油酯、三聚甘油单硬脂酸酯、脂肪酸蔗糖酯、蔗糖酯、乙酸异丁酸蔗糖糖酯、山梨醇酐三硬脂酸酯、肉豆蔻酸异丙酯、胆固醇、角鲨烯、角鲨烷、正丁醇、乙二醇、乙醇、丙二醇、聚甘油酯、亚硫酸盐、半胱氨酸、二叔丁基羟基甲苯、山梨酸钾、磷酸缓冲溶液、三乙醇胺、氢氧化钠、乙二胺、月桂胺、碳酸氢钠、盐酸、尼泊金类、硫柳汞、氯甲酚、三氯叔丁醇、苯甲酸及其钠盐。
第四方面，本公开提供一种在受试者中预防或治疗伴有高危型HPV感染的宫颈病变的方法，包括步骤：向受试者施用治疗有效量的本公开的红色诺卡氏菌细胞壁骨架或药物组合物。
“施用”、“提供给”、“处理”当应用于动物、人、细胞、组织、器官或生物样品时，是指药物或医疗装置与动物、人、细胞、组织、器官或生物样品接触。
“治疗”意指给予受试者内用或外用药物(治疗剂、活性成分或组合物)(如，本公开的外泌体或药物组合物)或医疗装置，在被治疗的受试者(或群体)中缓解(减轻、延迟、改善、治愈)一种或多种疾病症状，以至于达到临床可测量的程度，其中所述的受试者已经患有、疑似患有或易感于一种或多种疾病或其症状。
有效缓解任何疾病症状的药物(治疗剂、活性成分或组合物)的量，称为治疗有效量。可根据多种因素变化，例如受试者的疾病状态、年龄和体重。应当理解，在缓解单个受试者的目标疾病或其症状时，药物(治疗剂、活性成分或组合物)可能无效，但是根据本领域已知的任何统计学检验方法(如Student T检验、卡方检验、依据Mann和Whitney的U检验)确定，药物(治疗剂、活性成分或组合物)在统计学意义上对目标疾病或其症状是有效的。
在一些具体的实施方案中，受试者是人以外的动物，例如用于农场动物、宠物、工作动物、观赏动物、生产动物、实验动物(如大鼠、小鼠、豚鼠、兔、犬、灵长类)。
在一些具体的实施方案中，受试者是人。在一些具体的实施方案中，受试者疑似患有、确诊患有、已经患有、或易感于目标疾病或其症状。
在一些实施方案中，一天施用1-3次，或一天施用一次，或二天施 用一次。视患者病灶的面积及程度不同，采用不同的剂量。
在一些实施方案中，施用周期持续2天至6月，例如，1周、2周、3周、4周、5周、6周、7周、8周、9周、10周、11周、12周、13周、14周、15周、16周、17周、18周或更长、以及前述任意两个数值之间的范围。
同一药物活性成分或不同药物活性成分可一次性施用，或者可分成许多更小的单位剂量，以一定时间间隔施用。应理解治疗的确切剂量、持续时间、间隔时间是所治疾病的函数，并且可使用动物或临床试验数据推断而确定。所述施用可包括单次施用，或者间隔适当时间间隔的两次或更多次施用。其中，相邻两次的施用可间隔30分钟、40分钟、50分钟、60分钟、2小时、3小时、4小时、5小时、6小时、7小时、8小时、9小时、10小时、12小时、14小时、16小时、18小时、20小时、22小时、24小时、一天半、2天、3天、4天、5天、6天、7天、8天、9天、10天、1周、2周、3周、4周、5周、6周、1个月、2个月、3个月、4个月、5个月、6个月、7个月、8个月、9个月、10个月、11个月或12个月。
第五方面，本公开提供了一种体外抑制Hela细胞增殖的方法，包括步骤：
1)使有效量的红色诺卡氏菌细胞壁骨架和树突状细胞接触1至5天，获得激活的树突状细胞；
2)使所述激活的树突状细胞和幼稚CD4+T细胞接触1至3天(优选2至3天)，获得混合培养物；
3)使所述混合培养物和Hela细胞接触48小时或以上。
在一些实施方案中，所述Hela细胞是HPV18阳性的细胞。
在一些实施方案中，有效量为30μg/ml的红色诺卡氏菌细胞壁骨架和树突状细胞接触。
在一些实施方案中，所述红色诺卡氏菌细胞壁骨架是国药准字S20030009或其更新编号对应的红色诺卡氏菌细胞壁骨架。
第六方面，本公开提供了红色诺卡氏菌细胞壁骨架激活的树突状细胞和Th1细胞的联合在制备药物中的用途，其中所述药物用于在受试者中预防或治疗伴有高危型HPV感染的宫颈病变，所述宫颈病变选 自以下的任一项：非典型鳞状细胞病变、低级别鳞状上皮内病变。
在一些实施方案中，高危型HPV选自以下的任一项或组合：16、18、31、33、35、39、45、51、52、56、58、59、66、68型。
在一些实施方案中，红色诺卡氏菌细胞壁骨架激活的树突状细胞和Th1细胞在相同的、或不同的容器。
在一些实施方案中，红色诺卡氏菌细胞壁骨架激活的树突状细胞和Th1细胞同时或顺序施用。
“联合”是指为了达到治疗目的，而将两种或两种以上活性化合物同时或先后提供给受试者的方法。当涉及“联合”施用时，每次施用之间的时间间隔，足以实现施用的各活性化合物之间的协同作用。
“任选”意味着其随后所描述的事项可以发生，但不必须发生；需要视情况而定。例如，“任选地，进行分装”意味着允许对产品进行分装，但是不是必须进行分装。
“一个”、“一”、“单个”、“该”，如果没有明确说明，也包括复数形式。
当提及数值范围时(比如60μg至120μg)，这是一种简写方式，视为将范围内的每一个值被明确提及，包括小数和整数。
附图说明
图1A至图1D：q-PCR检测宫颈组织CD4+T细胞各亚群转录因子。(*p＜0.01，**p＜0.001，***p＜0.0001)。
图2：差异基因的qPCR验证。
图3A至图3E：Nr-CWS干预的DC对幼稚CD4+T细胞因子表达的影响。*p＜0.05表示干预组与对照组的显著性水平。
图4A至图4B：Nr-CWS干预的DC对T细胞增殖的影响及其对Hela细胞增殖的影响。*p＜0.05表示干预组与对照组的显著性水平。
图5：qPCR检测分析FPR3mRNA在树突状细胞中表达情况。
图6A至图6E：ELISA检测Nr-CWS对转染FPR3siRNA的DC活化T细胞分泌细胞因子的影响(*p＜0.05，**p＜0.01，***p＜0.001)。
图7：Nr-CWS对转染FPR3siRNA的DC活化T细胞抑制Hela细胞增殖的影响(p＜0.05)。
具体实施方式
以下结合实施例进一步描述本公开。但这些实施例并非限制着本公开的范围。当未注明具体条件时，按照常规条件、按照原料供应商所建议的条件操作。未注明具体来源的试剂，为市场购买的常规试剂。
实施例
实施例1.市售的红色诺卡氏菌细胞壁骨架
红色诺卡氏菌细胞壁骨架(本文中简称Nr-CWS)购自辽宁格瑞仕特生物制药有限公司，国药准字S20030009(每瓶中细胞壁骨架的固体含量应不少于60μg，其中胞壁酸含量不低于1.0μg，糖含量不低于4.0μg；复溶体积为2.0ml)。
实施例2.红色诺卡氏菌细胞壁骨架的制备
国药准字S20030009细胞壁骨架的制备方法与以下步骤本质上无显著差异，但因为生产规模不同，可进行调整。
因而，作为一个替代，可以采用以下步骤制备细胞壁骨架：
1.按照公知的方法培养菌体，并收集。对细胞进行破碎(例如超声波破碎或高压均质机破碎)。也允许采用本领域任何适当的公知方法对菌体进行破碎。显微镜下检查破碎的情况，每个视野有形菌不得超过5个，检查若干(10至30个)视野均达到此标准为合格。
2.除核酸：将破碎上清液进行离心，获得的沉淀物中加入DNA酶和RNA酶，按照酶的供应商建议的操作去除核酸。
3.除蛋白质：沉淀物加入常见的蛋白酶(例如胰蛋白酶)，按照酶的供应商建议的操作去除蛋白质。
4.除脂质：沉淀物中加入有机试剂(如丙酮、乙醚、乙醇中的一种或组合)，按照本领域常规操作去除脂质。
5.除细胞膜：沉淀物中加入TritonX-100，离心收集沉淀物，用PBS漂洗。
应当理解，上述除去杂质的步骤之间，技术人员可以调整先后顺序，使得步骤之间兼容。去除非细胞壁成分后，将沉淀物复溶于注射 用水，待用。任选地，可以在115℃下灭菌20-30分钟，作为细胞壁骨架的原液。
实施例3.药物组合物的示例性制备方法
1.将实施例1或2所得产物涂覆在敷料(例如无菌纱布)上，制备成外用药物。
2.将实施例2所得产物制成冻干粉。
3.也可以采用本领域公知的洗剂制备方法，例如：洗剂多以水和醇为分散介质；由活性成分、电解质、等渗调节剂等在分散介质中制成。
4.将实施例1或2所得产物制备成胶囊。
5.将实施例1或2所得产物溶于注射用水制成注射剂。
6.将实施例1或2所得产物制备成阴道栓。
测试例
测试例1.Nr-CWS治疗HPV感染的疗效与安全性及其与年龄因素的关系
I.目的
研究Nr-CWS对宫颈HPV持续感染及宫颈细胞学异常的治疗效果和安全性，并探究其与患者年龄的关系。
II.方法
1.从河北医科大学第四医院纳入符合标准的受试者。患者经知情同意后进入为期20天的Nr-CWS宫颈施用治疗，并随访至施用后12个月，分别以HPV感染情况和宫颈液基细胞学为结局指标。本测试例通过执行医院的伦理委员会批准，并经入组患者充分知情同意后签署知情同意书。
2.纳入标准和排除标准
从综合性三级甲等医院妇科阴道镜门诊就诊的患者中筛选符合标准的HPV感染患者，年龄在21-65岁之间，hr-HPV持续感染伴SIL或SIL史的患者将纳入筛选。符合全部入选标准且不符合排除标准的受试者方可参加试验。受试者参加筛选前均遵循自愿原则签署知情同意书。
72例患者被纳入分析。为比较Nr-CWS对不同年龄患者的有效性，依据患者年龄进行分组：21-40岁患者为低龄组(50人，69.4％)；41-65岁患者为高龄组(22人，30.6％)。
3.宫颈HPV分型检测
依据世界卫生组织于2010年11月在瑞士发布的《人乳头状瘤病毒实验室手册》确定了200多种HPV亚型，根据其致癌能力分为高危型和低危型。其中，高危型HPV包括16、18、31、33、35、39、45、51、52、56、58、59、66和68型。
4.宫颈液基细胞学检测(Thinprep cytologic test，TCT)
依据贝塞斯达系统(The Bethesda system，TBS)记录宫颈液基细胞学检测结果。TBS用于报告宫颈或阴道细胞学诊断，该诊断系统的结果包括：未见上皮内病变及恶性细胞(Non-intrusive load monitoring，NILM)、意义不明的非典型鳞状细胞(atypical squamous cells of undetermined significance，ASC-US)、非典型鳞状细胞-不能排除HSIL(ASC-H)、低级别鳞状上皮内病变(LSIL)、高级别鳞状上皮内病变(HSIL)、鳞状细胞癌(squamous cell cancer，SCC)、未特指的非典型腺细胞(Atypical glandular cells-not otherwise specified，AGC-NOS)、非典型腺细胞(atypical glandular cells，AGC)、原位腺癌(adenocarcinoma in situ，AIS)。
5.宫颈组织活检病理学诊断
记录宫颈活检病理检测结果，病理学检查为判断宫颈病变程度的金标准，该诊断的异常结果包括：CIN-I，CIN-II，CIN-III(累及腺体)，鳞状细胞癌，腺癌等。
6.治疗干涉
治疗采用红色诺卡氏菌细胞壁骨架(实施例1)。受试者于月经干净后2天内来门诊用药。治疗前，研究者先获取受试者宫颈部位的上皮组织活检标本(即0个月样本)。从D1开始受试者隔日来门诊，由医生进行Nr-CWS给药。嘱患者取截石位，打开阴道扩张器暴露宫颈。先用棉球清除宫颈表面及宫颈口的分泌物，用干纱布重搓宫颈表面，使其形成微创面。取外用红色诺卡氏菌细胞壁骨架，用2mL无菌水溶解60μg药物，使用注射器缓慢将药液推注到宫颈管里；再取2mL无 菌水溶解60μg药物，将其浸湿带尾棉球，用长镊子将带尾棉球紧贴宫颈表面，稍用力挤压3分钟，使浸润药液的棉球与病变部位充分贴合；轻柔取出扩张器，避免把带尾棉球带离宫颈表面。带尾棉球置于宫颈口保留24小时后，由患者自行取出。隔日施用一次，10次为1个疗程，总疗程共20天。嘱咐患者用药期间禁止性生活、禁止盆浴、游泳、避免剧烈运动。
7.对HPV感染的治疗效果
以HPV感染的清除为结局评价Nr-CWS对HPV持续感染伴宫颈病变患者的治疗效果，评价结局如下：
1)完全治愈：治疗后12个月内HPV感染转为阴性。
2)部分治愈：治疗12个月内HPV感染部分转为阴性。
3)无应答：治疗12个月内HPV感染仍持续存在，或有其他类型的新发HPV感染。
8.对宫颈病变的治疗效果
以宫颈病变的治疗有效性为结局评价Nr-CWS对HPV持续感染伴宫颈病变患者的治疗效果，评价结局如下：
1)治愈：治疗后12个月内宫颈液基细胞学无异常。
2)无应答：治疗12个月内宫颈液基细胞学持续异常，或有进一步发展。
III.结果
1.本测试例共纳入72例高危型HPV持续感染患者。根据受试者年龄将其分为两组。年龄在21-40岁之间的为低龄组，共计50人；年龄在41-65岁之间为老年组，共计22人(表1)。
表1.纳入研究的患者基本信息

2.Nr-CWS对宫颈上皮细胞学异常的治疗有效性
低龄组接受Nr-CWS治疗后3个月和6个月随访临床缓解率分别为89.3％和92.8％。高龄组接受Nr-CWS治疗后3个月和6个月随访临床缓解率均为72.7％。低龄组的临床缓解率相对高龄组较高，但两组间差异无统计学意义(p＞0.05)。
3.低龄组患者在随访3、6、12个月时HPV感染完全转阴率分别为58％、64％、66％，部分缓解率分别为33.3％、38.9％、44.4％，总有效率为70％、78％、82％；高龄组患者在随访3、6、12个月时HPV感染完全转阴率均为18.2％，部分缓解率均为28.6％，总有效率均为27.3％。两组间比较具有显著差异(p＜0.05)。
表2.Nr-CWS治疗后的HPV感染变化

4.受试者在接受Nr-CWS治疗期间不良反应轻微，没有患者因不良反应而中止用药或退出研究的情况。治疗后随访12个月内未观察到Nr-CWS相关的严重不良事件。
IV.结论
Nr-CWS可有效清除HPV持续感染、治疗低级别宫颈上皮病变细胞学异常。与高龄患者相比，Nr-CWS对低龄患者HPV感染和宫颈细胞学异常有更好的治疗效果。治疗期间患者对药物的耐受性较好，无明显不良反应发生。
测试例2.Nr-CWS对HPV感染伴宫颈病变患者宫颈组织及CD4+T细胞亚群的影响
I.目的
探究Nr-CWS治疗对HPV感染伴宫颈病变患者宫颈组织上皮间质转化的修复作用及局部免疫环境中CD4+T亚群细胞的变化。
II.方法
1.招募高危型HPV感染的患者入组进行Nr-CWS施用治疗。受试者纳入及排除标准见测试例1。分别于Nr-CWS治疗前和完成治疗后30天或90天获取受试者宫颈上皮组织作为研究样本。根据样本获得时间将其分为病例组(72例)，治疗30天组(41例)、治疗90天组(31例)。正常对照组宫颈标本取自HPV阴性的因子宫肌瘤行全子宫切除术的患者，共计8例。
2.组织化学测试
在对HPV感染伴宫颈病变患者实施Nr-CWS治疗前和治疗后1个月或3个月分别获取患者宫颈组织样本，HE染色观察组织结构；免疫组化染色观察波形蛋白(Vimentin)表达及CD4+T亚群细胞因子IFN-γ、IL-4、IL-17a、TGF-β的变化。
波形蛋白结果判定方法：免疫组化评分由染色强度和阳性细胞比例两部分组成。光学显微镜下观察，在无组织折叠、无边缘效应的典型宫颈上皮部位，随机选取数个高倍视野，每视野分别计数100个细 胞。无阳性细胞计为0分，阳性细胞数≤25％计为1分，26％-50％计为2分，51％-75％计为3分，＞75％计为4分；着色强度以多数阳性细胞呈现的棕黄色颗粒的深浅程度来计分：无着色为0分，淡黄色为1分，棕黄色为2分，棕褐色为3分。以阳性细胞百分比评分和着色强度评分的乘积为最终评分：评分范围为0～9分，得分0-1分记为“-”，得分2-3分记为“+”，得分4-5分记为“++”，得分≥6分记为“+++”。
CD4+T细胞因子结果判定方法：光学显微镜下观察，高倍镜下随机选取5个不同视野进行图像采集，计数和评分方法同上。IFN-γ、IL-4、IL-17、TGF-β主要表达在细胞质内，以阳性细胞百分比评分和着色强度评分的乘积为最终评分：＜3分为阴性，≥3分为阳性表达。Foxp3表达在细胞核，且数量相对较少，以每个高倍视野内阳性细胞个数作为统计数据。
3.宫颈组织进行mRNA转录因子PCR测定
实时荧光定量PCR检测宫颈组织中CD4+T细胞各亚群关键转录因子T-bet、GATA3、RORγt、Foxp3mRNA表达水平。
III.结果
1.宫颈组织主要由上皮和结缔组织构成，宫颈管粘膜为单层柱状上皮，宫颈阴道部由复层鳞状上皮覆盖表面。宫颈外口单层柱状上皮与复层鳞状上皮的交界区，即鳞-柱状上皮移行区是HPV感染和宫颈癌的好发部位。经HE染色后，正常组宫颈组织结构清晰，上皮基底层可见嗜碱性较强、蓝色深染的立方形幼稚细胞整齐排列。中间层可见多边形的上皮细胞，表层为数层梭形或扁平形的上皮细胞，表层的扁平细胞退化并脱落。上皮下方的粘膜固有层主要为结缔组织，可见丰富的纤维、细胞和血管，基质细胞分布疏松、均匀。上皮与深部结缔组织间的连接凹凸不平。
2.Nr-CWS治疗前宫颈组织的上皮基底层幼稚细胞数量增多、排列紊乱，向中间层移行延伸，并可见大量淋巴细胞浸润。粘膜固有层结缔组织排列更加致密，且有炎性细胞浸润聚集。上皮与深部结缔组织间的连接趋于平缓；经过Nr-CWS治疗后30天和90天，宫颈上皮结构层次随时间的推移逐渐趋于正常组织，粘膜固有层结缔组织由致密逐渐转为疏松、均匀，淋巴细胞数量减少，且分布基本趋于正常。结 缔组织和上皮间的连接恢复凹凸不平的正常形态。
3.免疫组化法检测宫颈组织中波形蛋白(波形蛋白)表达水平。结果显示，波形蛋白主要表达在细胞质中，呈棕黄色颗粒状。正常组宫颈组织上皮中波形蛋白几乎不表达；Nr-CWS治疗前宫颈组织上皮中可见大量波形蛋白阳性细胞浸润；经过Nr-CWS治疗后30天，宫颈上皮中可见波形蛋白阳性细胞散在分布；经过Nr-CWS治疗后90天，宫颈上皮中波形蛋白阳性细胞数量明显减少。
4.进一步统计结果显示，波形蛋白在正常宫颈的上皮中呈阴性表达，表达率为0；而HPV感染伴宫颈病变患者治疗前宫颈上皮中波形蛋白高表达率为84.7％，低表达率为15.3％；Nr-CWS治疗后30天宫颈组织中波形蛋白高表达率为4.8％，低表达率为51.2％，阴性率43.9％；Nr-CWS治疗后90天宫颈组织中波形蛋白高表达率为3.2％，低表达率为19.3％，阴性率77.4％。统计学分析显示，与治疗前相比，高危型HPV感染伴宫颈病变患者经Nr-CWS治疗后宫颈上皮中波形蛋白的表达水平明显降低，差异有统计学意义(p＜0.01)。
5.免疫组化染色法分别检测正常组、入组患者接受Nr-CWS治疗前、治疗后30天及治疗后90天宫颈组织IFN-γ、IL-4染色情况，并分析其表达水平。IFN-γ和IL-4主要表达在炎性细胞的细胞质中。根据IFN-γ免疫组化分析结果，正常组阳性表达率为62.5％、接受Nr-CWS治疗前阳性表达率为15.4％；治疗后30天阳性表达率为65.9％；治疗后90天阳性表达率为87.1％。统计学分析显示，与治疗前相比，高危型HPV感染伴宫颈病变患者经Nr-CWS治疗后宫颈组织中IFN-γ的表达水平增加，差异有统计学意义(p＜0.05)。
6.根据IL-4免疫组化分析结果，正常组阳性表达率为25％；接受Nr-CWS治疗前阳性表达率为94.4％；治疗后30天阳性表达率为48.7％；治疗后90天阳性表达率为25.8％。统计学分析显示，与正常组相比，高危型HPV感染伴宫颈病变患者宫颈组织中IL-4表达水平显著增加，而经Nr-CWS治疗后30天和90天，IL-4的表达水平逐渐降低，差异有统计学意义(p＜0.05)。
7.免疫组化染色法分别检测正常组、入组患者接受Nr-CWS治疗前、治疗后30天及治疗后90天宫颈组织IL-17a、TGF-β、Foxp3染色 情况并分析表达水平。IL-17a和TGF-β主要表达在炎性细胞的细胞质中，呈棕黄色至棕褐色颗粒。
根据IL-17a免疫组化分析结果，正常组阳性表达率为0、接受Nr-CWS治疗前阳性表达率为77.8％；治疗后30天阳性表达率为31.7％；治疗后90天阳性表达率为16.1％。统计学分析显示，与正常组相比，高危型HPV感染伴宫颈病变患者宫颈组织中IL-17a表达水平增加，而经Nr-CWS治疗后30天和90天，IL-17a的表达水平逐渐减少，差异有统计学意义(p＜0.05)。
根据TGF-β免疫组化分析结果，正常组阳性表达率为12.5％、接受Nr-CWS治疗前阳性表达率为84.7％；、治疗后30天阳性表达率为46.3％；、治疗后90天阳性表达率为12.9％。统计学分析显示，与正常组相比，高危型HPV感染伴宫颈病变患者宫颈组织中TGF-β表达水平增多，而经Nr-CWS治疗后30天和90天，TGF-β的表达水平逐渐降低，差异有统计学意义(p＜0.05)。
Foxp3表达于炎性细胞的细胞核中，呈圆形的棕褐色颗粒。免疫组化分析采用高倍视野下阳性细胞计数，结果显示，正常组宫颈组织Foxp3阳性细胞数为1.25±1.21；接受Nr-CWS治疗前阳性细胞数为10.75±3.82；治疗后30天阳性细胞数为5.1±2.40；治疗后90天阳性细胞数为1.4±0.99。统计学分析显示，与正常组相比，高危型HPV感染伴宫颈病变患者宫颈组织中Foxp3阳性细胞数量明显增多，而经Nr-CWS治疗后30天和90天，Foxp3阳性细胞逐渐减少，差异有统计学意义(p＜0.05)。
8.qPCR法分别检测正常组、入组患者接受Nr-CWS治疗前、治疗后30天、治疗后90天宫颈组织T-bet、GATA3、RORγt、Foxp3mRNA表达水平(图1A至图1D)。
结果显示：与正常组和入组患者Nr-CWS治疗前相比，经Nr-CWS治疗后T-bet表达水平明显升高，差异有统计学意义(p＜0.0001)。与正常组相比，高危型HPV感染伴宫颈病变患者宫颈组织中GATA3、RORγt、Foxp3表达水平增加，经Nr-CWS治疗后表达水平逐渐降低，差异具有统计学意义(p＜0.01)。
IV.讨论
高危型HPV持续感染会引发宫颈上皮细胞异常，包括SIL及宫颈癌。SIL是宫颈癌发生的前期阶段，根据宫颈涂片中的异常细胞或病理检查中不典型增生细胞浸润上皮的范围分为多种类型。轻度不典型增生称为LSIL，重度不典型增生称为HSIL。本测试例招募的入组患者中，SIL的诊断以病理结果为准。在经过Nr-CWS治疗后，病理结果显示宫颈上皮内不典型增生明显减少，上皮细胞异常得到明显改善。这和测试例1中的随访结果一致。
在上皮来源肿瘤的发生和进展过程中，细胞通常会失去上皮特征并获得间充质表型，这一过程称为上皮-间质转化，在宫颈癌的发生中起到重要作用。上皮细胞经过EMT后获得了更好的迁移能力和侵袭性。当上皮细胞长期暴露于病原体时，EMT会持续存在并异常激活。
EMT发生的分子标志包括上皮钙黏蛋白(Epithelial calcium-dependent adhesion，E-cadherins)的下调，神经钙黏蛋白(Neuro calcium-dependent adhesion，N-cadherins)、波形蛋白和纤连蛋白(Fibronectin，Fn)的表达增加。波形蛋白是III型中间丝细胞骨架蛋白家族中表达最广泛和高度保守的蛋白质之一，负责改变细胞形状和加强细胞骨架，主要表达于间充质来源的细胞。近年来，波形蛋白的上调被公认为EMT发生的典型生物学标志。
与正常组相比，Hr-HPV感染伴宫颈病变患者的宫颈上皮组织中波形蛋白阳性表达水平明显上升，而经过Nr-CWS治疗后下调。表明Nr-CWS在逆转HPV引起的宫颈EMT发生、阻断宫颈病变的进展中有明显作用。
本测试例观察了Nr-CWS对患者宫颈组织中CD4+T细胞亚群表达变化的调控，发现治疗后IFN-γ表达水平显著升高，IL-4、IL-17a和TGF-β表达水平均降低，与之对应的关键转录因子变化趋势一致。
综上，Nr-CWS治疗修复了hrHPV持续感染患者宫颈病变的局部组织结构，降低了患者宫颈上皮中波形蛋白的表达水平，对HPV持续感染造成的EMT有明显逆转作用。此外，还增加了宫颈组织中Th1型CD4+T细胞的数量并诱导IFN-γ的分泌，有助于逆转免疫抑制微环境，这可能在抗HPV感染的免疫反应中发挥了关键作用。
测试例3.基于微阵列分析Nr-CWS治疗患者宫颈组织的mRNA表达谱
I.目的
采用高通量转录组芯片技术分析Nr-CWS治疗对宫颈组织中mRNA的差异表达基因，并初步探讨Nr-CWS可能的免疫机制。
II.方法
1.选取招募入组并进行Nr-CWS施用治疗后随访3个月时复查HPV转阴的患者3例。受试者纳入及排除标准见测试例1。分别于Nr-CWS治疗前和完成治疗后90天获取3名受试者宫颈组织作为研究样本。根据样本获得时间将其分为治疗前组(3例)，治疗后组(3例)。
2.采用Agilent全基因组表达谱芯片检测和筛选差异表达mRNA。
3.对差异表达的33条上调的mRNA和182条下调的mRNA分别进行GO/KEGG富集分析。扩大样本量PCR验证免疫相关的差异基因表达。从芯片筛选的上调差异mRNA中筛选到5条免疫功能相关的基因FPR3、MARCO、UBD、CLECL1、CHIT1，并在10名入组患者宫颈组织样本中进行qPCR验证。
III.结果
1.差异表达RNA的数据筛选结果显示，HPV感染患者使用Nr-CWS治疗后的宫颈组织与治疗前相比，差异表达的mRNA共有215条，其中上调基因33条，下调基因182条。
表3.经Nr-CWS治疗的宫颈组织中上调的部分基因
表4.经Nr-CWS治疗的宫颈组织中下调的部分基因
2.富集分析结果显示33条Nr-CWS组高表达的差异基因富集出p＜0.05的通路共计8条。182条Nr-CWS组低表达的差异基因富集出p＜0.05的通路共计14条。
3.实时荧光定量PCR验证差异基因结果显示，入组患者经Nr-CWS治疗后宫颈组织中FPR3、MARCO、UBD、CHIT1表达水平明显升高，与mRNA表达谱芯片结果一致，CLECL1在样本中未检测到显著差异(图2)。
IV.结论
1.高通量mRNA表达谱芯片对HPV感染患者经过Nr-CWS治疗前后的宫颈组织的6例样本进行了差异筛选，在治疗后的宫颈组织中共筛选到215条差异基因。
2.对差异基因进行GO/KEGG富集分析发现其免疫作用机制可能与具有吞噬功能的免疫细胞的激活相关。
3.对筛选的差异基因扩大样本量进行验证，树突状细胞相关基因FPR3、MARCO、UBD、CHIT1在Nr-CWS治疗后的宫颈组织中表达水平明显升高。
测试例4.Nr-CWS通过树突状细胞对CD4+T细胞分化的调节作用
I.目的
分析Nr-CWS对树突状细胞形态影响、验证结合作用，及其干预DC对CD4+T细胞分化的调节。
II.方法
1.血液中心输血科取得人血去白细胞过滤器共20个，用于制备树突状细胞。
2.人PBMC来源树突状细胞的诱导及培养
向PBMC所在的六孔板或培养皿中加入含有30ng/ml rhIL-4和30ng/ml rhGM-CSF的RPMI1640双抗完全培养基，放入37℃、5％CO2的细胞培养箱中继续培养。
培养第3天进行半量换液，并补足细胞因子至30ng/ml。
继续培养至第5天，可见细胞大部分呈悬浮生长，体积增大，细胞表面突起并有集落形成，即为树突状细胞。
3.CCK-8法检测不同浓度Nr-CWS对树突状细胞活力的影响
Nr-CWS干预浓度设为6组：
①DC+PBS；
②DC+60μg/ml Nr-CWS；
③DC+30μg/ml Nr-CWS；
④DC+15μg/ml Nr-CWS；
⑤DC+7.5μg/ml Nr-CWS；
⑥DC+3.75μg/ml Nr-CWS。
将Nr-CWS溶解并加入在六孔板中培养至5d的DC的培养基中，每孔0.5ml，培养基总体积2.5ml，Nr-CWS终浓度如上述分组，空白组为2.5ml不含细胞的完全培养基。放入37℃、5％CO2的细胞培养箱中，继续培养48小时。
向六孔板中每孔加入250μl CCK-8溶液，注意不要产生气泡。随后在37℃培养箱中孵育4小时。取反应后的细胞培养基上清液，分别加入96孔板中，100μl/孔，每组设8个复孔。用酶标仪测定在450nm处的吸光度。
4.剂量为3.75-60μg/ml倍数稀释的Nr-CWS与树突状细胞孵育48h，在490nm处用CCK-8检测细胞活力，以测定树突状细胞刺激的最佳Nr-CWS浓度。根据CCK-8实验的结果，确定Nr-CWS最佳干预浓度为30μg/ml。将培养至第5天的DC细胞分为三组：
①iDC，
②iDC+30μg/ml Nr-CWS，
③iDC+100μg/ml LPS。
干预48小时后，观察并拍照记录细胞状态。
5.扫描电镜观察三组树突状细胞形态，比较Nr-CWS干预后细胞形态的变化。
6.采用纳米四氧化三铁颗粒标记Nr-CWS并干预DC48小时，透射电镜观察DC对Nr-CWS的吞噬，并采用普鲁士蓝-核固红染色，光镜下观察DC与Nr-CWS的结合情况。
7.建立DC-幼稚CD4+T细胞混合淋巴细胞培养模型(mixed lymphocyte reaction，MLR)，ELISA法检测DC-T混合培养细胞上清IL-2、IFN-γ、IL-4、TGF-β、IL-17a的浓度变化。
8.通过Transwell小室将Nr-CWS处理的混合淋巴细胞与Hela细胞(HPV18+)共培养，CCK-8法检测Hela细胞的增殖。
III.结果
1.扫描电镜观察对照组、Nr-CWS组、LPS组树突状细胞的表面形态。对照组iDC呈圆形或椭圆形，体积较小，表面可见少量毛刺样短小突起；Nr-CWS组树突状细胞体积较大，细胞表面凹凸不平，可见多个粗壮的伪足样突起，长度可达胞体两倍；LPS诱导的成熟树突状细胞体积增大，表面可见大量细长毛刺样突起。
DC激活后，抗原摄取能力减弱，但拥有了强大的吸引和活化幼稚CD4+T细胞和促进T细胞增殖的能力。将Nr-CWS或LPS处理的iDC与幼稚CD4+T细胞混合培养3天，光学显微镜和扫描电镜观察细胞形态。结果显示，iDC组树突状细胞散在分布，没有吸引T细胞。Nr-CWS和LPS处理的DC均能吸引幼稚的T细胞聚集在其周围并通过细胞突起发生连接。
2.普鲁士蓝染色观察Nr-CWS与DC结合情况
DC为主要的抗原呈递细胞，具有强大的抗原识别功能。Nr-CWS为红色诺卡氏菌的提取物，可能作为病原体抗原被APC识别。为验证Nr-CWS与DC的结合情况，以DMSA-Fe3O4纳米磁珠标记的Nr-CWS干预DC 48小时，普鲁士蓝-核固红染色，显微镜下观察。结果显示DC的细胞核为圆形或椭圆形，呈绛红色；DMSA-Fe3O4纳米磁珠标记的Nr-CWS呈蓝色碎片状，可见红色的细胞核与蓝染的Nr-CWS碎片 相互附着并紧密结合。
3.透射电镜观察树突状细胞对Nr-CWS的吞噬
为进一步观察DC是否对Nr-CWS有吞噬作用，使用DMSA-Fe3O4纳米磁珠标记的Nr-CWS干预DC 48小时后收集细胞，透射电镜观察DC对Nr-CWS的吞噬作用。电镜下可见大片磁珠颗粒与树突状细胞相连，细胞膜向胞质内凹陷形成吞饮泡，其中包裹大量磁珠颗粒。
4.经Nr-CWS处理的DC对幼稚CD4+T细胞的生长及分化影响
为探究经Nr-CWS处理后的DC对幼稚CD4+T细胞的生长及分化的影响作用，本测试例将对照组、Nr-CWS干预组、LPS干预组DC细胞与CD4+T细胞共培养2天，ELISA法检测细胞培养上清液中IL-2、IFN-γ、IL-4、TGF-β、IL-17a细胞因子的表达水平。结果显示(图3A至图3E)，与对照组相比，Nr-CWS干预组和LPS干预组IL-2、IFN-γ表达水平显著升高；IL-4、TGF-β表达水平降低，差异均有统计学意义(p＜0.05)。IL-17a在各组之间未见明显差异。
IV.讨论
本测试例证明了Nr-CWS可直接激活体外诱导的树突状细胞，并使其具有诱导幼稚CD4+T细胞向Th1型分化的能力。作为具有生物活性的免疫激活剂，Nr-CWS可用于针对HPV感染及其诱发病变的有效策略。
测试例5.Nr-CWS与树突状细胞中FPR3结合介导Th1细胞分化的作用机制探究
I.目的
验证FPR3在Nr-CWS通过DC介导的T细胞分化中的作用。
II.方法
1.DC培养及诱导见测试例4。培养至D5的树突状细胞分为3组，用相应药物处理细胞：①iDC；②iDC+30μg/ml Nr-CWS，继续培养至D7，收集各组细胞。
2.通过免疫组化、实时荧光定量PCR、流式细胞术检测Nr-CWS干预的DC中FPR3表达水平。
3.DC中FPR3在Nr-CWS介导的CD4+T细胞分化中的作用
DC培养至D5，荧光显微镜观察细胞转染效率。加入30μg/ml Nr-CWS干预DC，细胞分组如下：①sico DC；②siFPR3 DC；③sico DC+Nr-CWS；④siFPR3 DC+Nr-CWS。
4.ELISA法检测Nr-CWS对siFPR3-DC和T细胞共培养模型中细胞因子的影响
将上述培养细胞按照分组分别与CD4+T细胞以1:10的比例共培养48小时4。D7收集培养细胞上清，ELISA法检测三组细胞培养上清IL-2、IFN-γ、IL-4、TGF-β、IL-17a的变化。
5.用Transwell小室将沉默FPR3后DC的混合培养淋巴细胞与Hela细胞共培养，CCK-8法检测Hela细胞增殖(图4A至图4B)。
III.结果
1.Nr-CWS对DC中FPR3表达的影响
为验证Nr-CWS对DC中FPR3表达的影响作用，从人外周血中分离的PBMC经IL-4、GM-CSF诱导5天获得iDC，在有或没有Nr-CWS的条件下刺激细胞2天后收获细胞。分别通过免疫组化法、流式细胞学和qPCR的方法检测了DC中FPR3的表达水平。
采用免疫组化法检测树突状细胞FPR3表达水平。可见，FPR3主要表达于细胞膜和细胞质，呈棕黄色颗粒状均匀分布。经统计分析，与对照组相比，Nr-CWS组DC中FPR3平均光密度值显著增加(0.113±0.003vs 0.527±0.013)，差异有统计学意义(p＜0.001)。
2.流式细胞术检测Nr-CWS对DC中FPR3表达的影响
流式细胞学分析结果显示，与对照组相比，Nr-CWS组DC中FPR3阳性率增加(18.4％vs 42.8％)。
3.实时荧光定量PCR法检测Nr-CWS对DC中FPR3表达的影响
qPCR检测树突状细胞FPR3mRNA表达水平。结果显示与对照组相比，Nr-CWS组DC中FPR3表达水平增加，差异具有统计学意义(p＜0.05)(图5)。
4.ELISA法检测Nr-CWS对siFPR3 DC和T细胞共培养模型中细胞因子的影响
为了探究Nr-CWS通过DC影响CD4+T细胞分化过程中DC的FPR3是否起到关键调节作用，于D3和D5向DC中转染了2次siFPR3 及其空白对照NC，转染后用Nr-CWS刺激2天，与幼稚CD4+T细胞共培养2天，检测培养基上清细胞因子的表达差异。ELISA结果显示，与NC+Nr-CWS组相比，siFPR3+Nr-CWS组培养基上清中IL-2、IFN-γ的浓度显著降低，差异有统计学意义(p＜0.001)；而IL-4、TGF-β、IL-17a的浓度无显著差异(图6A至图6E)。
以上结果说明FPR3在Nr-CWS诱导的DC活化T细胞免疫的过程中，对Th1细胞的分化发挥正向调节作用。
5.CCK-8法检测Nr-CWS通过siFPR3 DC和T细胞共培养模型对Hela细胞生长的影响
Nr-CWS处理的DC和幼稚CD4+T细胞混合培养后可抑制Hela细胞增殖。为研究DC中FPR3在此过程中的作用，于D3和D5向DC中转染了2次siFPR3及其空白对照NC，转染后用Nr-CWS刺激2天，与幼稚CD4+T细胞混合培养2天，再使用Transwell小室与Hela细胞共培养48小时，CCK-8法检测Hela细胞增殖。结果显示(图7)，siFPR3+Nr-CWS组Hela细胞活力显著高于NC+Nr-CWS组，差异有统计学意义(p＜0.05)。
IV.结论
在前面的测试例中，已经证实Nr-CWS通过刺激DC调控CD4+T细胞向Th1方向分化，并在经Nr-CWS治疗的宫颈病变组织mRNA表达谱芯片中发现了DC相关基因UBD、MARCO、FPR3的上调。
1.免疫组化、实时荧光定量PCR、流式细胞学检测发现Nr-CWS刺激的DC中FPR3蛋白和mRNA表达上调。
2.用Nr-CWS处理FPR3沉默的DC与幼稚CD4+T细胞共培养，ELISA法检测培养基上清，Th1型细胞因子IL-2、IFN-γ浓度恢复低水平。通过靶向沉默DC中FPR3的表达后，Nr-CWS失去通过DC诱导幼稚CD4+T细胞向Th1分化的能力。
3.Nr-CWS处理FPR3沉默的DC并与幼稚CD4+T细胞共培养，CCK-8法检测与MLR共培养的Hela细胞，Hela细胞的增殖活性恢复正常。表明DC中FPR3沉默后，Nr-CWS不能通过DC-CD4+T细胞的MLR模型抑制Hela细胞增殖。

Claims (9)

  1. 红色诺卡氏菌细胞壁骨架在制备药物中的用途，其中：
    所述药物用于在受试者中预防或治疗伴有高危型HPV感染的宫颈病变；
    所述高危型HPV选自以下的任一项或组合：16、18、31、33、35、39、45、51、52、56、58、59、66、68型；
    所述宫颈病变选自以下的任一项：非典型鳞状细胞病变、低级别鳞状上皮内病变。
  2. 根据权利要求1所述的用途，所述受试者是21岁至40岁。
  3. 根据权利要求1或2所述的用途，所述药物制备成选自以下任一项的剂型：膏剂、霜剂、栓剂、乳液、混悬剂、糊剂、凝胶剂、洗剂、片剂、气雾剂、喷雾剂、搽剂、粉剂、敷料、绷带、膜、贴片、注射剂。
  4. 根据权利要求1至3任一项所述的用途，其中所述红色诺卡氏菌细胞壁骨架是国药准字S20030009或其更新编号对应的红色诺卡氏菌细胞壁骨架。
  5. 根据权利要求1至3中任一项所述的用途，其中所述红色诺卡氏菌细胞壁骨架通过以下方法获得，所述方法包括以下步骤或由以下步骤组成：
    1)提供红色诺卡氏菌；
    2)破碎所述红色诺卡氏菌，得到破碎产物；
    3.1)对所述破碎产物去除脂质；
    3.2)对所述破碎产物去除核酸；
    3.3)对所述破碎产物去除蛋白质；
    3.4)对所述破碎产物去除细胞膜；
    3.5)得到红色诺卡氏菌细胞壁骨架；
    4)任选地，分装；
    5)任选地，对所述红色诺卡氏菌细胞壁骨架进行冷冻干燥；
    其中，
    步骤3.1)、3.2)、3.3)、3.4)能够互换或并行，
    步骤4)和步骤5)能够互换；
    所述破碎的平均粒度为10nm至1000nm，优选10nm至800nm，更优选10nm至500nm。
  6. 根据权利要求1至5中任一项所述的用途，其中所述药物用于选自以下的任一项或其组合：
    降低宫颈上皮中波形蛋白的表达水平、提高IFN-γ的表达水平、降低IL-4的表达水平、降低IL-17a的表达水平、降低TGF-β的表达水平、降低Foxp3阳性细胞的数量、提高T-bet的mRNA表达水平、降低GATA3的mRNA表达水平、降低RORγt的mRNA表达水平、降低Foxp3的mRNA表达水平。
  7. 一种体外诱导幼稚CD4+T细胞向Th1型分化的方法，包括步骤：
    1)使有效量的红色诺卡氏菌细胞壁骨架和树突状细胞接触1至5天，获得激活的树突状细胞；
    2)使所述激活的树突状细胞和幼稚CD4+T细胞接触1至3天(优选2至3天)，获得混合培养物；
    所述有效量是30μg/ml红色诺卡氏菌细胞壁骨架；
    所述红色诺卡氏菌细胞壁骨架是国药准字S20030009或其更新编号对应的红色诺卡氏菌细胞壁骨架。
  8. 一种体外抑制Hela细胞增殖的方法，包括步骤：
    使权利要求7所限定的混合培养物和Hela细胞接触48小时或以上；所述Hela细胞是HPV18阳性的。
  9. 红色诺卡氏菌细胞壁骨架激活的树突状细胞和Th1细胞的联合在制备药物中的用途，其中：
    所述药物用于在受试者中预防或治疗伴有高危型HPV感染的宫颈 病变；
    所述高危型HPV选自以下的任一项或组合：16、18、31、33、35、39、45、51、52、56、58、59、66、68型；
    所述宫颈病变选自以下的任一项：非典型鳞状细胞病变、低级别鳞状上皮内病变；
    所述受试者是21岁至40岁；
    所述药物制备成选自以下任一项的剂型：膏剂、霜剂、栓剂、乳液、混悬剂、糊剂、凝胶剂、洗剂、片剂、气雾剂、喷雾剂、搽剂、粉剂、敷料、绷带、膜、贴片、注射剂；
    优选地，所述红色诺卡氏菌细胞壁骨架是国药准字S20030009或其更新编号对应的红色诺卡氏菌细胞壁骨架。
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