CN115025128A - Application of nocardia rubra cell wall skeleton in treatment of cervical lesion - Google Patents

Abstract

The present disclosure relates to the use of nocardia rubra cell wall scaffolds in the treatment of cervical lesions. In particular, the present disclosure provides nocardia rubra cell wall skeleton for use in preventing or treating cervical lesions with infection by high risk HPV selected from any one or a combination of: 16. models 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68. Nocardia rubra cell wall skeleton is used as an immune activator with biological activity, and can be used for effective strategies aiming at HPV infection and induced lesions thereof.

Description

Application of nocardia rubra cell wall skeleton in treatment of cervical lesion

Technical Field

The present disclosure relates to the fields of medicine and biopharmaceutical technology. In particular, the disclosure relates to the use of nocardia rubra cell wall scaffolds in the treatment of cervical lesions.

Background

Cervical cancer is a common malignant tumor occurring in the female reproductive system, and the morbidity and mortality of the cervical cancer are on the 4 th level worldwide, which seriously affects the female health and the life quality worldwide. The major histological types include squamous cell carcinoma and glandular cell carcinoma, with squamous carcinoma accounting for about 90% of cervical cancer and adenocarcinoma accounting for about 10%.

Epidemiological studies have shown that HPV DNA can be detected in almost all specimens of cervical cancer. Thus, HPV infection is the most major risk factor for the development and progression of cervical cancer. Some of the genotypes with stronger oncogenicity are called high-risk HPV (hrHPV), which is associated with more than 85% of cervical cancer cases, with types 16 and 18 being the most common high-risk types. Squamous Intraepithelial Lesions (SIL) are the precursor lesions of cervical cancer and are classified into Low-grade Squamous intraepithelial lesions (LSIL) and High-grade Squamous intraepithelial lesions (HSIL) according to the severity of cervical dysplasia. The LSIL lesions also resolve spontaneously due to clearance of the majority of HPV infections by the host immune system. However, in the case of hrHPV persistent infection, invasive cancer may develop within years. Therefore, clearing hrHPV infection is the most critical problem for blocking the course of disease and preventing the occurrence of cervical cancer. HPV vaccines have a very good protective effect on the vaccinated population, but do not eliminate existing infections. Surgical resection remains the first choice for therapeutic intervention for patients who already have HPV-related high-grade cervical lesions. However, with the development of immunotherapy research, new therapeutic regimens are becoming possible, where cell-based immunotherapy is of interest.

Nocardia rubra (Nocardia rubra) is one of Nocardia. The red nocardia thallus is fermented, cell crushed and degraded by protease to obtain the red nocardia cell wall skeleton.

In the prior art, the Nocardia rubra cell wall skeleton is commercially available, specifically from Liaoning Gersted biopharmaceutical GmbH (Nr-CWS). Nocardia rubra cell wall skeleton has been used for the treatment of cervical erosion, precancerous lesions of cervical cancer (CN101073583A), anti-human papilloma virus (CN1935262A), skin lesions or skin ulcers (CN101209267A), fungal infections, herpes simplex, herpes zoster (CN 1879661A).

Disclosure of Invention

In a first aspect, the present disclosure provides a nocardia rubra cell wall skeleton.

Nocardia rubra refers to the genus Nocardia, the species Nocardia rubra (Nocardia rubra).

Identification of nocardia rubra: the skilled person can taxonomically identify a strain of bacteria according to known or future microbial identification techniques, e.g. available identification techniques include morphological, physiological and biochemical characteristics, 16S rRNA, etc. The skilled person understands that with the development of technology, identification techniques involve different approaches, and morphological and biochemical identification approaches have been mainly used in the early days, but the reliability of such methods is not high. After the advent of sequencing technology, the skilled artisan could identify strains in a more reliable manner. For example, when the DNA sequence of 16S rRNA is identified as having 97% (inclusive) or more similarity, two genera are judged to be of the same species. For nocardia rubra, known strains deposited in the international (or national grade) collection of species are used as model strains and compared therewith.

In the present disclosure, "nocardia rubra cell wall" can be understood as both an intact cell wall and an incomplete cell wall (e.g., disrupted, or partially degraded). The skilled artisan, in light of the present disclosure, will appreciate that the component exhibiting the desired activity is derived from the cell wall of nocardia rubra (e.g., is the cell wall itself or a constituent thereof). Therefore, various forms of intact cell walls, disrupted cell walls, incomplete degradation products of cell walls, constituents of cell walls, extracts of cell walls, etc., which are allowed to be used in clinical applications, are included in the scope of the present disclosure.

The cell wall skeleton of the present disclosure is not to be understood as merely representing a cross-linked network entity within the cell wall, and the skilled person will understand that the term does not exclude other cell wall components adsorbed, bound, carried on the cross-linked network entity.

In a specific example, the cell wall skeleton of the present disclosure is the product of bacteria after disruption and decontamination (protein, nucleic acid, cell membrane, lipid).

In a specific embodiment, the cell wall scaffold is nocardia rubra cell wall scaffold according to the national drug standard S20030009 or its update number.

The skilled artisan will appreciate that S20030009 is an administrative approval number issued by the administration of drug administration, which number will vary with the renewal of the certificate, the laws, and the adjustment of the numbering rules. However, the product standard, product parameters, production process and quality requirements represented by the changed number are not changed. Thus, S20030009 in this disclosure should be understood as S20030009 and equivalents thereof.

In some embodiments, the nocardia rubra cell wall skeleton is the nocardia rubra cell wall skeleton corresponding to the national drug standard S20030009 or renewal code thereof.

In other embodiments, the nocardia rubra cell wall skeleton is obtained by a method comprising or consisting of the steps of:

1) providing nocardia rubra;

2) crushing the nocardia rubra to obtain a crushed product;

3.1) removing lipids from the disrupted product;

3.2) removing nucleic acids from the disruption product;

3.3) removing proteins from the disruption product;

3.4) removing cell membranes from the disruption product;

3.5) obtaining a red nocardia cell wall skeleton;

4) optionally, subpackaging;

5) optionally, freeze-drying the nocardia rubra cell wall skeleton;

steps 3.1), 3.2), 3.3), 3.4) can be interchanged or parallel, and steps 4) and 5) can be interchanged.

For disruption of nocardia rubra, the aim is to remove intracellular material. Therefore, the techniques of ultrasonic crushing, high-pressure homogenizer crushing, lysozyme and the like can be adopted. The skilled artisan will appreciate that any known or future method suitable for disrupting gram-positive bacteria is suitable for use in the presently disclosed embodiments.

The skilled person has the ability to adapt the specific parameters and equipment of culturing, disruption, separation, collection, removal of impurities, packaging in response to the subsequent application (e.g. topical application) of the active ingredient (cell wall and its constituent components) in order to avoid introduction of factors in the preparation step that affect the subsequent application.

In some embodiments, the lipids in the disrupted product are removed using an organic solvent. In some embodiments, the DNA and RNA in the disruption products are removed using a nuclease. In some embodiments, the protein in the disruption product is degraded using a hydrolase. In some embodiments, the cell membranes in the disruption products are removed using a surfactant.

In some embodiments, the average particle size of the disruption is from 10nm 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 ranges between any two of the foregoing values. The methods of particle size testing are well known in the art.

In some specific embodiments, the average particle size of the disruption is from 10nm to 800 nm.

In other specific embodiments, the average particle size of the disruption is from 10nm to 500 nm.

In specific embodiments, the dispensing means into a bottle or ampoule. Just prior to use, a solvent (e.g., sterile water) is added to the vial or ampoule. As an example, the bottle is a vial (visual, made of borosilicate glass or soda lime glass).

In a second aspect, the present disclosure provides the use of the aforementioned nocardia rubra cell wall scaffold in the preparation of a medicament for preventing or treating cervical lesions with high risk HPV infection in a subject.

The high risk HPV is selected from any one or combination of the following: 16. types 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68;

the cervical lesion is selected from any one of: atypical squamous cell lesions, low-grade squamous intraepithelial lesions.

In a specific embodiment, the subject is 21 to 65 years old, preferably 21 to 40 years old.

In some embodiments, the medicament is prepared in a dosage form selected from any one of: injection, unguent, cream, lotion, suspension, paste, gel, lotion, tablet, aerosol, spray, liniment, powder, dressing, bandage, membrane, patch, and suppository.

In some embodiments, the medicament is capable of achieving any one or a combination selected from the group consisting of: reducing the expression level of a vimentin in cervical epithelium, increasing the expression level of IFN-gamma, reducing the expression level of IL-4, reducing the expression level of IL-17a, reducing the expression level of TGF-beta, reducing the number of Foxp3 positive cells, increasing the mRNA expression level of T-beta, reducing the mRNA expression level of GATA3, reducing the mRNA expression level of ROR gamma T, reducing the mRNA expression level of Foxp 3.

In a third aspect, the present disclosure provides a pharmaceutical composition or medicament for preventing or treating cervical lesions with high risk HPV infection in a subject, comprising: a pharmaceutically acceptable carrier and the nocardia rubra cell wall skeleton of the present disclosure.

The pharmaceutical composition or medicament of the present disclosure may be prepared in the form of a unit dose (or unit formulation).

In some embodiments, the pharmaceutical composition or medicament may be prepared in a liquid state (liquid formulation).

In other embodiments, the pharmaceutical composition or medicament may be prepared as a solid (dry powder formulation or lyophilized powder formulation).

The skilled person understands that liquid formulations and dry powder formulations (or lyophilized powder formulations), which can be interconverted, differ only in the water content. Removing most or all of water in the liquid preparation to obtain dry powder preparation (or lyophilized powder preparation). Dissolving (or redissolving) the dry powder preparation (or the freeze-dried powder preparation) to obtain a liquid preparation.

In some embodiments, the pharmaceutically acceptable carrier is selected from, but not limited to: fillers, stabilizers (e.g. trehalose, glycine), flavoring agents (e.g. xylitol), disintegrants (e.g. sodium carboxymethyl cellulose), binders (e.g. gelatin), lubricants (e.g. magnesium stearate).

In some embodiments, the stabilizing agent is selected from one or a combination of: glycine, lysine, arginine, hydroxyethyl starch, hydroxymethyl starch, trehalose, and dextran.

In some embodiments, the flavoring agent is selected from one or a combination of the following: sucrose, monosaccharide, saccharin sodium, aspartame, sorbitol, xylitol and mannitol.

In some embodiments, the binder is selected from one or a combination of: sodium carboxymethylcellulose, hypromellose, and gelatin.

In some embodiments, the lubricant is selected from one or a combination of: comprises talcum powder, magnesium stearate and superfine silica powder.

In some specific embodiments, 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, sodium carboxymethylcellulose, erythritol, gelatin, magnesium stearate, a propellant, a humectant, a solvent, a solubilizer, an emulsifier, an antioxidant, a pH regulator and a preservative. Specifically, non-limiting examples also include: white petrolatum, carbomer, hypromellose, methylcellulose, sodium carboxymethylcellulose, chitosan, sucralfate chitosan, polyvinylpyrrolidone, polyvinyl alcohol, sodium hyaluronate, dimethyl ether, tetrafluoroethane, hydrofluoroalkane, glycerol, propylene glycol, deionized water, water for injection, distilled water, ethanol, cetyl alcohol, stearyl alcohol, p-aminobenzoic acid, acetamide, isopropyl alcohol, tween, polyoxyethylene hydrogenated castor oil, stearic acid, glyceryl monostearate, triglycerol monostearate, sucrose fatty acid ester, sucrose acetate isobutyrate, sucrose anhydride tristearate, isopropyl myristate, cholesterol, squalene, squalane, n-butanol, ethylene glycol, ethanol, propylene glycol, polyglycerol ester, sulfite, cysteine, di-tert-butyl hydroxytoluene, potassium sorbate, phosphate buffer solution, Triethanolamine, sodium hydroxide, ethylenediamine, laurylamine, sodium bicarbonate, hydrochloric acid, parabens, thimerosal, chlorocresol, chlorobutanol, benzoic acid and its sodium salt.

In a fourth aspect, the present disclosure provides a method of preventing or treating cervical lesions with high risk HPV infection in a subject, comprising the steps of: administering to the subject a therapeutically effective amount of a nocardia rubra cell wall skeleton or pharmaceutical composition of the present disclosure.

"administering," "providing," "treating," when applied to an animal, human, cell, tissue, organ, or biological sample, means that the drug or medical device is in contact with the animal, human, cell, tissue, organ, or biological sample.

By "treating" is meant administering an internal or external drug (therapeutic agent, active ingredient or composition) (e.g., exosomes or pharmaceutical compositions of the present disclosure) or medical device to a subject who has been, suspected of having, or is susceptible to one or more diseases or symptoms thereof, in a subject (or population) being treated to alleviate (reduce, delay, ameliorate, cure) one or more symptoms of the disease, so as to achieve a clinically measurable degree.

The amount of drug (therapeutic agent, active ingredient or composition) that is effective to alleviate any symptoms of the disease is referred to as a therapeutically effective amount. May vary depending on a number of factors, such as the disease state, age and weight of the subject. It is understood that a drug (therapeutic agent, active ingredient or composition) may not be effective in alleviating a target disease or symptom thereof in an individual subject, but the drug (therapeutic agent, active ingredient or composition) is statistically effective against the target disease or symptom thereof as determined by any statistical test method known in the art, such as Student's T test, chi-square test, U-test by Mann and Whitney.

In some specific embodiments, the subject is an animal other than a human, e.g., a farm animal, a pet, a work animal, an ornamental animal, a production animal, a laboratory animal (e.g., rat, mouse, guinea pig, rabbit, dog, primate).

In some specific embodiments, the subject is a human. In some specific embodiments, the subject is suspected of having, diagnosed with, has had, or is susceptible to the target disease or a symptom thereof.

In some embodiments, the administration is 1-3 times a day, or once two days. Different dosages are adopted according to different areas and degrees of the focus of the patient.

In some embodiments, the administration cycle 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 more, and ranges between any two of the foregoing.

The same or different pharmaceutically active ingredients may be administered at once, or may be divided into a number of smaller unit doses to be administered at intervals. It will be understood that the exact dose, duration, and interval of treatment is a function of the disease being treated and can be determined using animal or clinical trial data inferences. The administration may comprise a single administration, or two or more administrations separated by a suitable time interval. Wherein two consecutive administrations are 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, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, one and a half of a day, 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.

In a fifth aspect, the present disclosure provides a method of inhibiting proliferation of Hela cells in vitro, comprising the steps of:

1) contacting an effective amount of nocardia rubra cell wall skeleton and dendritic cells for 1 to 5 days to obtain activated dendritic cells;

2) allowing the activated dendritic cells and naive CD4 ⁺ Contacting the T cells for 1 to 3 days (preferably 2 to 3 days) to obtain a mixed culture;

3) the mixed culture and Hela cells were contacted for 48 hours or more.

In some embodiments, the Hela cells are HPV18 positive cells.

In some embodiments, an effective amount of 30 μ g/ml nocardia rubra cell wall scaffold is contacted with the dendritic cells.

In some embodiments, the nocardia rubra cell wall skeleton is the nocardia rubra cell wall skeleton corresponding to the national drug standard S20030009 or renewal code thereof.

In a sixth aspect, the present disclosure provides the use of a nocardia rubra cell wall skeleton-activated dendritic cell in combination with a Th1 cell in the manufacture of a medicament for preventing or treating a cervical lesion associated with a high risk type HPV infection in a subject, the cervical lesion selected from any one of: atypical squamous cell lesions, low-grade squamous intraepithelial lesions.

In some embodiments, the high risk HPV is selected from any one or a combination of: 16. models 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68.

In some embodiments, the nocardia rubra cell wall skeleton-activated dendritic cells and the Th1 cells are in the same, or different, containers.

In some embodiments, the nocardia rubra cell wall scaffold-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, either simultaneously or sequentially, for therapeutic purposes. When reference is made to "combined" administration, the time interval between each administration is sufficient to achieve a synergistic effect between the active compounds administered.

"optional" means that the subsequently described event can, but need not, occur; as the case may be. For example, "optionally, portioned" means that product is allowed to be portioned, but is not necessarily.

The terms "a", "an", "the", and "the" include plural references unless expressly stated otherwise.

When referring to a range of values (e.g., 60 μ g to 120 μ g), this is intended to be a shorthand way of referring explicitly to each value falling within the range, including both fractional and integer values.

Drawings

Fig. 1A to 1D: q-PCR detection of cervical tissue CD4 ⁺ T cell subsets of transcription factors. (. p < 0.01,. p < 0.001,. p < 0.0001).

FIG. 2: qPCR validation of differential genes.

Fig. 3A to 3E: Nr-CWS intervened DC vs. naive CD4 ⁺ Effect of T-cytokine expression. P < 0.05 indicates the level of significance of the intervention group versus the control group.

Fig. 4A to 4B: the effect of Nr-CWS-intervened DCs on T cell proliferation and its effect on Hela cell proliferation. P < 0.05 indicates the level of significance of the intervention group versus the control group.

FIG. 5: the qPCR assay analyzes FPR3 mRNA expression in dendritic cells.

Fig. 6A to 6E: ELISA assays the effect of Nr-CWS on cytokine secretion by DC activated T cells transfected with FPR3 siRNA (/ p < 0.05,/p < 0.01,/p < 0.001).

FIG. 7: the effect of Nr-CWS on the inhibition of Hela cell proliferation by DC-activated T cells transfected with FPR3 siRNA (p < 0.05).

Detailed Description

The present disclosure is further described below in conjunction with the examples. These examples are not intended to limit the scope of the disclosure. When the specific conditions are not specified, the operation is carried out under the conventional conditions, as recommended by the raw material supplier. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

Examples

Example 1 commercially available Nocardia rubra cell wall skeleton

Red nocardia rubra cell wall skeleton (herein abbreviated as Nr-CWS) is purchased from Liaoning Gehrist biopharmaceutical GmbH, and is of Chinese medicine standard S20030009 (the solid content of the cell wall skeleton in each bottle should be not less than 60 μ g, wherein the muramic acid content is not less than 1.0 μ g, the sugar content is not less than 4.0 μ g, and the redissolution volume is 2.0 ml).

Example 2 preparation of cell wall skeleton of Nocardia erythraea

The preparation method of the cell wall skeleton of the Chinese medicine standard character S20030009 is basically not obviously different from the following steps, but can be adjusted due to different production scales.

Thus, as an alternative, the cell wall skeleton can be prepared by:

1. the cells were cultured according to a known method and collected. The cells are disrupted (e.g., by sonication or high pressure homogenizer). It is also permissible to crush the cells by any suitable method known in the art. The broken condition is checked under a microscope, the number of the visible bacteria in each visual field is not more than 5, and the standard is met by checking a plurality of visual fields (10 to 30) to be qualified.

2. Removing nucleic acid: the disrupted supernatant was centrifuged, and DNase and RNase were added to the obtained precipitate to remove nucleic acids according to the procedures recommended by the enzyme supplier.

3. Removing protein: the precipitate is added with a common protease (e.g. trypsin) and the protein is removed according to the procedures recommended by the supplier of the enzyme.

4. Removing lipid: adding organic reagent (such as one or combination of acetone, diethyl ether, and ethanol) into the precipitate, and removing lipid according to conventional operation in the art.

5. Removing cell membranes: TritonX-100 was added to the precipitate, and the precipitate was collected by centrifugation and rinsed with PBS.

It should be understood that between the above steps of removing impurities, the skilled person can adjust the order of the steps to make the steps compatible. After removing the non-cell wall components, the precipitate was redissolved in water for injection for use. Optionally, it can be sterilized at 115 ℃ for 20-30 minutes as a stock solution of cell wall matrix.

Example 3 an exemplary method for preparing a pharmaceutical composition

1. The product obtained in example 1 or 2 is applied to a dressing (e.g., sterile gauze) to prepare a drug for external use.

2. The product obtained in example 2 was made into lyophilized powder.

3. Lotion preparation methods well known in the art may also be employed, for example: the lotion mostly takes water and alcohol as dispersion media; is prepared from active component, electrolyte, isoosmotic regulator, etc. in dispersing medium.

4. The product obtained in example 1 or 2 was prepared into capsules.

5. The product obtained in example 1 or 2 is dissolved in water for injection to prepare injection.

6. The product obtained in example 1 or 2 was prepared into pessaries.

Test example

Test example 1 efficacy and safety of Nr-CWS in the treatment of HPV infection and its relationship to age factors

I. Purpose(s) to

The treatment effect and safety of the Nr-CWS on the persistent infection of cervical HPV and cervical cytological abnormality are researched, and the relation between the Nr-CWS and the age of a patient is explored.

Process II

1. Subjects meeting the criteria were enrolled from the fourth hospital of north Hebei medical university. After informed consent, the patients enter into Nr-CWS cervical administration treatment for 20 days, and follow-up till 12 months after the administration, and respectively take HPV infection conditions and cervical fluid-based cytology as outcome indexes. The test example was approved by the ethical committee of the executive hospital and signed with an informed consent form after the patients entered the group had given full informed consent.

2. Inclusion and exclusion criteria

Patients with HPV infection meeting the standard are screened from patients who are treated by a gynecological colposcopy clinic in hospitals such as comprehensive grade-A, and the like, and the patients with age between 21 and 65 years are included in the screening, wherein the hr-HPV persistent infection is accompanied with SIL or SIL history. Subjects who met all inclusion criteria and who did not meet exclusion criteria may participate in the trial. Subjects followed voluntary principles before taking part in the screening and signed informed consent.

72 patients were included in the analysis. To compare the effectiveness of the Nr-CWS on patients of different ages, groups were made according to patient age: patients 21-40 years of age were in the low age group (50, 69.4%); patients between 41 and 65 years of age were in the advanced age group (22, 30.6%).

3. Cervical HPV typing detection

More than 200 HPV subtypes are determined according to a human papilloma virus laboratory manual issued by the world health organization in 11 months in 2010 in Switzerland, and are classified into high-risk types and low-risk types according to the carcinogenic capacity of the HPV subtypes. Among them, high risk types of HPV include types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68.

4. Cervical fluid-based cytology test (TCT)

Cervical fluid-based cytology results were recorded according to The Bethesda System (TBS). TBS is used to report cervical or vaginal cytological diagnoses, the results of which include: there are no such endothelial lesions and malignant cells (NILM), Atypical squamous cells of unknown significance (ASC-US), Atypical squamous cells-not excluding HSIL (ASC-H), low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSIL), Squamous Cell Carcinoma (SCC), unspecified Atypical glandular cells (AGC-NOS), Atypical glandular cells (AIS).

5. Cervical tissue biopsy pathology diagnosis

Recording pathological detection results of cervical biopsy, wherein the pathological examination is a gold standard for judging the cervical lesion degree, and the abnormal results of diagnosis comprise: CIN-I, CIN-II, CIN-III (affected glands), squamous cell carcinoma, adenocarcinoma, etc.

6. Therapeutic intervention

Treatment was with nocardia rubra cell wall skeleton (example 1). The subjects were out-patient within 2 days of menstruation. Prior to treatment, the investigator first obtained a biopsy specimen of epithelial tissue from the cervical region of the subject (i.e., a 0 month sample). The subjects were outpatient every other day from D1 and were dosed with Nr-CWS by a physician. Order the patient to take the lithotomy position, open the vaginal dilator and expose the cervix. Firstly, the secretion on the surface of the cervix and the cervical orifice is cleared by a cotton ball, and the surface of the cervix is rubbed with dry gauze to form a micro wound surface. Taking the cell wall skeleton of the externally used nocardia rubra, dissolving 60 mu g of the medicine by 2mL of sterile water, and slowly injecting the liquid medicine into a cervical canal by using an injector; dissolving 60 μ g of the medicine in 2mL of sterile water, soaking the tail-carrying cotton ball, tightly attaching the tail-carrying cotton ball to the surface of the cervix with long forceps, and slightly squeezing for 3 minutes to ensure that the cotton ball soaked with the liquid medicine is fully attached to the lesion part; the dilator is gently taken out to avoid taking the cotton ball with the tail out of the surface of the cervix. The cotton ball with the tail is placed in the cervical orifice and is left for 24 hours, and then is taken out by the patient. The preparation is applied every other day, 10 times of the preparation form 1 course of treatment, and the total course of treatment is 20 days. The patient is instructed to forbid sexual life, forbid bath, swim and avoid strenuous exercise during the period of taking medicine.

7. Therapeutic Effect on HPV infection

The treatment effect of Nr-CWS on patients with HPV persistent infection and cervical lesion is evaluated by taking the elimination of HPV infection as a result, and the result is evaluated as follows:

1) and (4) completely curing: HPV infection turned negative within 12 months after treatment.

2) And (3) partial cure: HPV infection partially turned negative within 12 months of treatment.

3) No response: HPV infection persists for 12 months of treatment, or there are other types of new HPV infections.

8. Therapeutic effect on cervical lesions

The treatment effect of the Nr-CWS on patients with HPV persistent infection and cervical lesions is evaluated by taking the treatment effectiveness of the cervical lesions as the result, and the result is evaluated as follows:

1) and (3) curing: there were no abnormalities in cervical fluid-based cytology within 12 months after treatment.

2) No response: cervical fluid-based cytology persisted abnormally, or had further progression, within 12 months of treatment.

Results III

1. A total of 72 patients with persistent HPV infection were included in this test. The subjects were divided into two groups according to their age. The age of 21-40 years is the low age group, and the total number is 50; the age was between 41 and 65 years and was the elderly group, totaling 22 people (table 1).

TABLE 1 basic information for patients enrolled in the study

Therapeutic effectiveness of Nr-CWS on cervical epithelial cytological abnormalities

The follow-up clinical remission rates at 3 months and 6 months after treatment with Nr-CWS in the low age group were 89.3% and 92.8%, respectively. The follow-up clinical remission rates of the high-age group were 72.7% at 3 and 6 months after Nr-CWS treatment. Clinical remission rates were higher in the low age group compared to the high age group, but the difference between the two groups was not statistically significant (p > 0.05).

3. The complete negative conversion rate of HPV infection of patients in the low-age group is 58%, 64% and 66% respectively at the follow- up 3, 6 and 12 months, the partial remission rate is 33.3%, 38.9% and 44.4% respectively, and the total effective rate is 70%, 78% and 82%; the complete negative conversion rate of HPV infection of patients in an advanced group is 18.2 percent in 3, 6 and 12 months of follow-up, the partial remission rate is 28.6 percent, and the total effective rate is 27.3 percent. The comparison between the two groups showed significant differences (p < 0.05).

TABLE 2 HPV infection Change after Nr-CWS treatment

4. The subjects had slight adverse events during the period of Nr-CWS treatment and there were no cases where the patients discontinued medication or withdrawn from the study due to adverse events. No serious Nr-CWS related adverse events were observed within 12 months of follow-up after treatment.

Conclusion IV

The Nr-CWS can effectively eliminate HPV persistent infection and treat low-level cervical epithelial lesion cytological abnormality. The Nr-CWS has a better therapeutic effect on HPV infection and cervical cytological abnormalities in patients of lower age than in patients of higher age. The tolerance of the patient to the medicine is better during the treatment period, and no obvious adverse reaction occurs.

Test example 2 Effect of Nr-CWS on cervical tissue and CD4+ T cell subsets of patients with HPV infection with cervical lesions

I. Purpose(s) to

Researches the repairing effect of Nr-CWS treatment on the epithelial-mesenchymal transition of the cervical tissue of a patient with HPV infection and cervical lesion and CD4 in a local immune environment ⁺ Change in T subset cells.

Process II

1. Patients with high risk HPV infections were enrolled for Nr-CWS administration therapy. Subject inclusion and exclusion criteria are given in test example 1. Cervical epithelial tissue of the subject was obtained as study samples before Nr-CWS treatment and 30 or 90 days after completion of treatment, respectively. The samples were divided into case groups (72 cases), treatment groups for 30 days (41 cases), and treatment groups for 90 days (31 cases) according to the sample acquisition time. Normal control cervical specimens were obtained from HPV-negative factor hysteromyoma patients with total hysterectomy, for a total of 8 cases.

2. Histochemical test

Respectively obtaining cervical tissue samples of a patient before and 1 month or 3 months after Nr-CWS treatment is carried out on the patient with HPV infection and cervical lesion, and observing the tissue structure by HE staining; observation of Vimentin (Vimentin) expression and CD4 by immunohistochemical staining ⁺ The T subgroup cytokines IFN-gamma, IL-4, IL-17a, TGF-beta.

The determination method of the vimentin result comprises the following steps: immunohistochemical scores consisted of staining intensity and positive cell proportion. Observing under an optical microscope, randomly selecting a plurality of high power fields at a typical cervical epithelial part without tissue folding and edge effect, and counting 100 cells in each field. The number of the positive cells is 0, 1 for less than or equal to 25%, 2 for 26-50%, 3 for 51-75% and 4 for more than 75%; the intensity of staining was scored as the shade of the brown-yellow particles presented by the majority of positive cells: no coloration is 0 min, light yellow is 1 min, tan is 2 min, and tan is 3 min. The final score is the product of the percentage positive cells score and the staining intensity score: the score range is 0-9 points, the score of 0-1 points is marked as < - >, the score of 2-3 points is marked as < + >, the score of 4-5 points is marked as < + >, and the score of more than or equal to 6 points is marked as < + >.

CD4 ⁺ T cell factor result determination method: observing under an optical microscope, randomly selecting 5 different visual fields under a high power microscope for image acquisition, and carrying out the counting and grading method as above. IFN-gamma, IL-4, IL-17, TGF-beta are expressed predominantly in the cytoplasm, with the final score being the product of the percentage positive cells score and the staining intensity score: negative expression is classified as < 3, and positive expression is classified as > 3. Foxp3 was expressed in nuclei and was relatively small in number, with the number of positive cells per high power field being used as a statistic.

3. PCR assay of mRNA transcription factor in cervical tissue

Real-time fluorescent quantitative PCR detection of CD4 in cervical tissue ⁺ The expression levels of the key transcription factors T-beta, GATA3, ROR gamma T and Foxp3 mRNA of each subgroup of T cells.

Results III

1. The cervical tissue is mainly composed of epithelium and connective tissue, the mucosa of cervical canal is a single-layer columnar epithelium, and the surface of the cervicovaginal part is covered by a multiple-layer squamous epithelium. The junction area of the monolayer columnar epithelium and the multilayer squamous epithelium of the external cervical orifice, namely the squamous-columnar epithelial transitional area, is a good part for HPV infection and cervical cancer. After HE staining, the normal group of cervix uteri has clear tissue structure, strong alkalophilicity can be seen in the epithelial basal layer, and the cubic juvenile cells deeply stained blue are regularly arranged. The middle layer can be seen as polygonal epithelial cells, the surface layer is a plurality of spindle-shaped or flat epithelial cells, and the flat cells on the surface layer are degenerated and shed. The inherent layer of the mucosa under the epithelium is mainly connective tissue, rich fibers, cells and blood vessels can be seen, and the stromal cells are distributed loosely and uniformly. The junction between the epithelium and the deep connective tissue is uneven.

The cervical tissue before Nr-CWS treatment has increased number of epithelial-basal layer immature cells, disordered arrangement, migration and extension to the middle layer, and a large amount of lymphocyte infiltration can be seen. The connective tissues of the inherent layer of the mucosa are arranged more densely and inflammatory cells infiltrate and gather. The junction between the epithelium and the deep connective tissue tends to be gentle; 30 days and 90 days after Nr-CWS treatment, the structure level of the cervical epithelial layer gradually tends to a normal tissue along with the time, the connective tissue of the mucosa lamina propria gradually turns from compact to loose and uniform, the number of lymphocytes is reduced, and the distribution basically tends to normal. The connection between connective tissue and epithelium restores the rugged normal morphology.

3. Immunohistochemistry detects the expression level of vimentin (vimentin) in cervical tissue. The results showed that vimentin was mainly expressed in the cytoplasm and was in the form of brown-yellow granules. Almost no expression of the vimentin in the epithelium of normal group cervical tissue; a large amount of vimentin positive cell infiltration can be seen in cervical tissue epithelium before Nr-CWS treatment; 30 days after Nr-CWS treatment, the vimentin positive cells in the cervical epithelium are scattered; 90 days after Nr-CWS treatment, the quantity of the positive cells of the vimentin in the cervical epithelium is obviously reduced.

4. Further statistical results show that the vimentin is expressed negatively in the epithelium of the normal cervix, and the expression rate is 0; the high expression rate of vimentin in cervical epithelium before treatment of patients with HPV infection and cervical lesion is 84.7%, and the low expression rate is 15.3%; the high expression rate of the vimentin in the cervical tissue is 4.8 percent, the low expression rate is 51.2 percent and the negative rate is 43.9 percent 30 days after Nr-CWS treatment; after 90 days of Nr-CWS treatment, the high expression rate of the vimentin in the cervical tissue is 3.2%, the low expression rate is 19.3%, and the negative rate is 77.4%. Statistical analysis shows that compared with the prior treatment, the expression level of vimentin in cervical epithelium of a patient with high-risk HPV infection and cervical lesion is obviously reduced after Nr-CWS treatment, and the difference has statistical significance (p is less than 0.01).

5. And (3) detecting the staining conditions of IFN-gamma and IL-4 in cervical tissues before, 30 days after and 90 days after the Nr-CWS treatment of a normal group and a group-entering patient respectively by an immunohistochemical staining method, and analyzing the expression levels of the IFN-gamma and IL-4. IFN-gamma and IL-4 are expressed predominantly in the cytoplasm of inflammatory cells. According to the IFN-gamma immunohistochemical analysis result, the positive expression rate of the normal group is 62.5 percent, and the positive expression rate before receiving Nr-CWS treatment is 15.4 percent; the positive expression rate 30 days after treatment is 65.9%; the positive expression rate 90 days after treatment was 87.1%. Statistical analysis shows that compared with the prior treatment, the expression level of IFN-gamma in cervical tissues of patients with high-risk HPV infection and cervical lesion is increased after Nr-CWS treatment, and the difference has statistical significance (p is less than 0.05).

6. According to the IL-4 immunohistochemical analysis result, the positive expression rate of the normal group is 25 percent; the positive expression rate before receiving Nr-CWS treatment is 94.4 percent; the positive expression rate 30 days after treatment is 48.7%; the positive expression rate 90 days after treatment was 25.8%. Statistical analysis shows that compared with a normal group, the expression level of IL-4 in cervical tissues of patients with high-risk HPV infection and cervical lesions is remarkably increased, while the expression level of IL-4 is gradually reduced 30 days and 90 days after Nr-CWS treatment, and the difference has statistical significance (p is less than 0.05).

7. Immunohistochemical staining method for detecting staining conditions of cervical tissues IL-17a, TGF-beta and Foxp3 and analyzing the expression level of the cervical tissues before, 30 days after and 90 days after Nr-CWS treatment in normal group and group-in patients respectively. IL-17a and TGF-. beta.are expressed predominantly in the cytoplasm of inflammatory cells, in the form of tan to tan granules.

According to the result of IL-17a immunohistochemical analysis, the positive expression rate of the normal group is 0, and the positive expression rate before receiving Nr-CWS treatment is 77.8%; the positive expression rate 30 days after treatment is 31.7%; the positive expression rate 90 days after treatment was 16.1%. Statistical analysis shows that compared with a normal group, the expression level of IL-17a in cervical tissues of patients with high-risk HPV infection and cervical lesions is increased, while the expression level of IL-17a is gradually reduced 30 days and 90 days after Nr-CWS treatment, and the difference is statistically significant (p is less than 0.05).

According to the result of TGF-beta immunohistochemical analysis, the positive expression rate of the normal group is 12.5 percent, and the positive expression rate before receiving Nr-CWS treatment is 84.7 percent; and the positive expression rate 30 days after treatment is 46.3 percent; and the positive expression rate is 12.9% 90 days after treatment. Statistical analysis shows that compared with a normal group, the expression level of TGF-beta in cervical tissues of patients with high-risk HPV infection and cervical lesion is increased, and 30 days and 90 days after Nr-CWS treatment, the expression level of TGF-beta is gradually reduced, and the difference has statistical significance (p is less than 0.05).

Foxp3 is expressed in the nucleus of inflammatory cells as rounded tan granules. The immunohistochemical analysis adopts positive cell counting under a high-power visual field, and the result shows that the number of positive cells of the cervical tissue Foxp3 of the normal group is 1.25 +/-1.21; the number of positive cells before receiving Nr-CWS treatment is 10.75 +/-3.82; the number of positive cells 30 days after treatment is 5.1 +/-2.40; the number of positive cells was 1.4. + -. 0.99 days after treatment. Statistical analysis shows that compared with a normal group, the number of Foxp3 positive cells in cervical tissues of patients with high-risk HPV infection and cervical lesions is obviously increased, and Foxp3 positive cells are gradually reduced 30 days and 90 days after Nr-CWS treatment, and the difference is statistically significant (p is less than 0.05).

qPCR method to detect the mRNA expression levels of cervical tissue T-beta, GATA3, ROR γ T, Foxp3 (FIG. 1A-FIG. 1D) before treatment, 30 days after treatment, and 90 days after treatment of normal and group patients, respectively.

The results show that: compared with the Nr-CWS treatment of normal group and group-entering patients before the Nr-CWS treatment, the T-beta expression level is obviously increased after the Nr-CWS treatment, and the difference has statistical significance (p is less than 0.0001). Compared with a normal group, the expression levels of GATA3, ROR gamma t and Foxp3 in the cervical tissues of the patients with high-risk HPV infection and cervical lesions are increased, the expression levels are gradually reduced after Nr-CWS treatment, and the difference has statistical significance (p is less than 0.01).

Discussion IV

Persistent infection with high-risk HPV types can cause abnormalities in cervical epithelial cells, including SIL and cervical cancer. SIL is a pre-stage in the development of cervical cancer and is classified into various types according to the extent to which abnormal cells in cervical smears or atypical proliferating cells in pathological examination infiltrate the epithelium. Mild dysplasia is called LSIL and severe dysplasia is called HSIL. Among the enrolled patients in this test example, SIL was diagnosed based on the pathological outcome. After Nr-CWS treatment, pathological results show that the cervical intraepithelial atypical hyperplasia is obviously reduced, and epithelial cell abnormality is obviously improved. This is consistent with the follow-up results in test example 1.

In the process of development and progression of tumors of epithelial origin, cells often lose epithelial characteristics and acquire a mesenchymal phenotype, a process known as epithelial-mesenchymal transition, which plays an important role in the development of cervical cancer. Epithelial cells acquire better migration capacity and invasiveness after EMT. When epithelial cells are exposed to pathogens for prolonged periods, EMTs persist and are abnormally activated.

Molecular markers of EMT occurrence include down-regulation of Epithelial cadherins (E-cadherins), increased expression of neurophysis (N-cadherins), vimentin, and Fibronectin (Fn). Vimentin is one of the most widely expressed and highly conserved proteins in the type III intermediate filament cytoskeletal protein family, responsible for changing cell shape and strengthening cytoskeleton, and is mainly expressed in cells of mesenchymal origin. In recent years, up-regulation of vimentin has been recognized as a typical biomarker for the occurrence of EMT.

Compared with the normal group, the positive expression level of vimentin in the cervical epithelial tissue of the patient infected by Hr-HPV and accompanied with cervical lesion is obviously increased, and the positive expression level is reduced after Nr-CWS treatment. The Nr-CWS is shown to have obvious effects on reversing cervical EMT generation caused by HPV and blocking the progression of cervical lesions.

The present test example observed Nr-CWS on CD4 in cervical tissue of a patient ⁺ The regulation and control of the expression change of the T cell subset shows that the IFN-gamma expression level is obviously increased after treatment, the IL-4, IL-17a and TGF-beta expression level is reduced averagely, and the change trend of the corresponding key transcription factors is consistent.

In conclusion, the Nr-CWS treatment restores the local tissue structure of cervical lesion of the hrHPV persistent infection patient, reduces the expression level of vimentin in cervical epithelium of the patient and has obvious reversal effect on EMT caused by HPV persistent infection. In addition, Th1 type CD4 in cervical tissue is increased ⁺ The number of T cells and the induction of IFN- γ secretion contribute to the reversal of the immunosuppressive microenvironment, which may play a key role in the immune response against HPV infection.

Test example 3 mRNA expression profiling of cervical tissue of Nr-CWS-treated patients based on microarray analysis

I. Purpose(s) to

Adopts high-throughput transcriptome chip technology to analyze mRNA differential expression gene of Nr-CWS treatment in cervical tissue, and preliminarily discusses Nr-CWS possible immune mechanism.

Process II

1. 3 patients were selected for enrollment and were reviewed for HPV turning negative at follow-up 3 months after Nr-CWS administration. Subject inclusion and exclusion criteria are given in test example 1. Cervical tissue from 3 subjects was obtained as study samples before and 90 days after the Nr-CWS treatment. The samples were divided into a pre-treatment group (3 cases) and a post-treatment group (3 cases) according to their time of acquisition.

2. The Agilent whole genome expression profiling chip is adopted to detect and screen the differential expression mRNA.

3. GO/KEGG enrichment analysis was performed on 33 differentially expressed up-regulated mrnas and 182 down-regulated mrnas, respectively. Amplification of sample size PCR verified immune-related differential gene expression. 5 genes related to immune function, FPR3, MARCO, UBD, CLECL1 and CHIT1, were selected from the up-regulated differential mRNA selected on the chip and subjected to qPCR in cervical tissue samples of 10 patients in the group.

Results III

1. The screening result of the data of the differential expression RNA shows that the cervical tissues of the HPV infected patients treated by the Nr-CWS have 215 differential expression mRNAs compared with the cervical tissues before treatment, wherein 33 genes are up-regulated and 182 genes are down-regulated.

TABLE 3 partial genes upregulated in Nr-CWS-treated cervical tissue

TABLE 4 partial genes downregulated in Nr-CWS-treated cervical tissue

2. The enrichment analysis result shows that the 33 Nr-CWS groups of high-expression differential genes are enriched to obtain 8 paths with p less than 0.05. The 182 Nr-CWS groups enrich 14 paths with p less than 0.05.

3. The real-time fluorescent quantitative PCR (polymerase chain reaction) verification difference gene result shows that the expression levels of FPR3, MARCO, UBD and CHIT1 in cervical tissues of patients in the group are obviously increased after Nr-CWS treatment, the result is consistent with the result of an mRNA expression profile chip, and the significant difference is not detected in a sample by CLECL1 (figure 2).

Conclusion IV

1. The high-throughput mRNA expression profiling chip carries out differential screening on 6 samples of cervical tissues of HPV infected patients before and after Nr-CWS treatment, and 215 differential genes are screened in the treated cervical tissues.

2. GO/KEGG enrichment analysis of the differential gene shows that the immune action mechanism of the differential gene is possibly related to the activation of immune cells with phagocytic function.

3. The expression level of the dendritic cell related genes FPR3, MARCO, UBD and CHIT1 in cervical tissues after Nr-CWS treatment is obviously increased by verifying the sample size of the screened differential gene amplification.

Test example 4 Nr-CWS vs CD4 by dendritic cells ⁺ Regulation of T cell differentiation I

Analysis of the effects of Nr-CWS on dendritic cell morphology, validation of binding, and intervention thereof in DC on CD4 ⁺ Modulation of T cell differentiation.

Process II

1. 20 human leukocyte-removing filters were obtained from blood center transfusions and used to prepare dendritic cells.

2. Induction and culture of human PBMC-derived dendritic cells

Adding RPMI1640 double-antibody complete culture medium containing 30ng/ml rhIL-4 and 30ng/ml rhGM-CSF into a six-well plate or culture dish in which PBMC is placed, placing at 37 deg.C and 5% CO ₂ The cells in the cell culture box are continuously cultured.

Half of the medium was changed on day 3 of culture and cytokines were replenished to 30 ng/ml.

And continuing culturing until the 5 th day, wherein most of the cells grow in suspension, the volume is increased, and the cell surface protrudes and forms colonies, namely the dendritic cells are obtained.

3, detecting the influence of different concentrations of Nr-CWS on the activity of dendritic cells by a CCK-8 method

The Nr-CWS intervention concentration was set to 6 groups:

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。

Nr-CWS was dissolved and added to a medium of 5d DCs cultured in six-well plates at 0.5ml per well, with a total volume of 2.5ml, and the final Nr-CWS concentrations were grouped as above, with a blank of 2.5ml of complete medium without cells. Adding 5% CO at 37 deg.C ₂ The cell culture chamber of (2) was continued for 48 hours.

To each well of the six well plate, 250. mu.l of CCK-8 solution was added, taking care not to generate bubbles. Followed by incubation in an incubator at 37 ℃ for 4 hours. The cell culture medium supernatant after reaction was added to a 96-well plate at 100. mu.l/well, with 8 duplicate wells per group. Absorbance at 450nm was measured with a microplate reader.

4. The optimal Nr-CWS concentration for dendritic cell stimulation was determined by incubating dendritic cells with Nr-CWS at a dose of 3.75-60 μ g/ml fold dilution for 48h and measuring cell viability at 490nm using CCK-8. According to the result of the CCK-8 experiment, the optimal intervention concentration of the Nr-CWS is determined to be 30 mu g/ml. DC cells cultured to day 5 were divided into three groups:

①iDC，

②iDC+30μg/ml Nr-CWS，

③iDC+100μg/ml LPS。

after 48 hours of intervention, the cell status was observed and recorded photographically.

5. And (5) observing the morphology of the three groups of dendritic cells by a scanning electron microscope, and comparing the change of the cell morphology after Nr-CWS intervention.

6. Marking Nr-CWS by adopting nano ferroferric oxide particles and intervening DC48 hours, observing phagocytosis of the Nr-CWS by the DC through a transmission electron microscope, dyeing by adopting Prussian blue-nuclear fast red, and observing the combination condition of the DC and the Nr-CWS under a light microscope.

7. Establishment of DC-naive CD4 ⁺ The concentration changes of IL-2, IFN-gamma, IL-4, TGF-beta and IL-17a in the supernatant of the DC-T mixed culture cell are detected by an ELISA method through a mixed lymphocyte culture Model (MLR).

8. The Nr-CWS-treated mixed lymphocytes were co-cultured with Hela cells (HPV18+) in a Transwell chamber, and proliferation of Hela cells was detected by the CCK-8 method.

Results III

1. And observing the surface morphology of the dendritic cells of the control group, the Nr-CWS group and the LPS group by a scanning electron microscope. The iDC of the control group is circular or elliptical, the volume is small, and a small amount of burr-like short protrusions can be seen on the surface; the volume of the dendritic cells of the Nr-CWS group is large, the cell surface is uneven, a plurality of thick pseudopodous-like protrusions can be seen, and the length can be twice as long as that of the cell body; the mature dendritic cells induced by LPS are increased in volume, and a large number of slender burr-like protrusions are visible on the surface.

After DC activation, the antigen uptake capacity is weakened, but the DC activation has strong attraction and activation of the naive CD4 ⁺ T cells and the ability to promote T cell proliferation. Combining Nr-CWS or LPS treated iDC with naive CD4 ⁺ T cells are mixed and cultured for 3 days, and the cell morphology is observed by an optical microscope and a scanning electron microscope. The results showed that the iDC dendritic cells were scattered and not attracting T cells. Both Nr-CWS and LPS treated DCs attract naive T cells to accumulate around them and to become connected by the cell process.

2. Prussian blue staining observation of Nr-CWS and DC binding condition

DCs are the major antigen presenting cells and have powerful antigen recognition functions. Nr-CWS is an extract of nocardia rubra, possibly recognized by APC as a pathogen antigen. To verify the binding of Nr-CWS to DC, DMSA-Fe was used ₃ O ₄ And (3) interfering the DC for 48 hours by Nr-CWS marked by nano magnetic beads, dyeing the DC by Prussian blue-nuclear fast red, and observing the DC under a microscope. The result shows that the cell nucleus of the DC is circular or elliptical and is magenta; DMSA-Fe ₃ O ₄ Nr-CWS (nano magnetic bead) blue fragment marked by nano magnetic beadsAs a result, red nuclei and blue-stained Nr-CWS fragments were observed to attach to each other and tightly bind to each other.

3. Transmission electron microscope observation of phagocytosis of Nr-CWS by dendritic cells

To further observe whether DCs phagocytose Nr-CWS, DMSA-Fe was used ₃ O ₄ And (4) interfering the DC for 48 hours by the Nr-CWS marked by the nano magnetic beads, collecting the cells, and observing the phagocytosis of the DC on the Nr-CWS by a transmission electron microscope. Under an electron microscope, a large number of magnetic bead particles are connected with dendritic cells, cell membranes are sunken into cytoplasm to form swallow bubbles, and a large number of magnetic bead particles are wrapped in the swallow bubbles.

4.Nr-CWS treated DC vs. naive CD4 ⁺ Growth and differentiation effects of T cells

To explore the Nr-CWS treated DC versus naive CD4 ⁺ Effect of T cell growth and differentiation, control, Nr-CWS and LPS Stem control and control groups

CD4+ T cells were co-cultured for 2 days, and expression levels of IL-2, IFN-. gamma., IL-4, TGF-. beta.and IL-17a cytokines in cell culture supernatants were measured by ELISA. The results show (fig. 3A to fig. 3E) that the IL-2 and IFN- γ expression levels were significantly increased in the Nr-CWS intervention group and LPS intervention group compared to the control group; the expression levels of IL-4 and TGF-beta are reduced, and the difference has statistical significance (p is less than 0.05). IL-17a showed no significant difference between the groups.

Discussion IV

The test demonstrates that Nr-CWS can directly activate dendritic cells induced in vitro and make the dendritic cells have the function of inducing naive CD4 ⁺ The ability of T cells to differentiate towards the Th1 type. As an immune activator with biological activity, Nr-CWS can be used for an effective strategy against HPV infection and induced lesions thereof.

Test example 5 mechanism of action of Nr-CWS in binding to FPR3 in dendritic cells to mediate differentiation of Th1 cells

I. Object(s) to

The role of FPR3 in Nr-CWS through DC-mediated T cell differentiation was demonstrated.

Process II

DC culture and induction are described in test example 4. Dendritic cells cultured to D5 were divided into 3 groups, and the cells were treated with the corresponding drugs: first, iDC; ② iDC +30 mu g/ml Nr-CWS, continuously culturing until D7, and collecting each group of cells.

2. The FPR3 expression level in the DC interfered by the Nr-CWS is detected by immunohistochemistry, real-time fluorescence quantitative PCR and flow cytometry.

FPR3 in DC in Nr-CWS mediated CD4 ⁺ Role in T cell differentiation

The DCs were cultured to D5 and the transfection efficiency of the cells was observed by fluorescence microscopy. Add 30. mu.g/ml Nr-CWS intervention DCs, and the cell groupings were as follows: firstly, sico DC; ② siFPR3 DC; ③ sico DC + Nr-CWS; and siFPR3 DC + Nr-CWS.

ELISA method for detecting influence of Nr-CWS on cytokines in siFPR3-DC and T cell co-culture model

The cultured cells are grouped into groups

CD4 ⁺ T cells were co-cultured at a ratio of 1:10 for 48 hours 4. D7 collecting the culture cell supernatant, and detecting the changes of IL-2, IFN-gamma, IL-4, TGF-beta and IL-17a of the three groups of cell culture supernatants by an ELISA method.

5. Mixed culture lymphocytes of DCs after silencing FPR3 were co-cultured with Hela cells in a Transwell chamber, and proliferation of Hela cells was detected by the CCK-8 method (FIGS. 4A to 4B).

Results III

Effect of Nr-CWS on FPR3 expression in DCs

To examine the effect of Nr-CWS on FPR3 expression in DCs, iDCs were obtained by inducing PBMCs isolated from human peripheral blood with IL-4 and GM-CSF for 5 days, and the cells were harvested after stimulating the cells for 2 days with or without Nr-CWS. The expression level of FPR3 in DCs was detected by immunohistochemistry, flow cytometry and qPCR, respectively.

The expression level of the dendritic cell FPR3 is detected by an immunohistochemical method. It can be seen that FPR3 is expressed mainly in cell membranes and cytoplasm and is distributed uniformly in the form of brown-yellow granules. Through statistical analysis, the average optical density value of FPR3 in the Nr-CWS group DC is obviously increased (0.113 +/-0.003 vs 0.527 +/-0.013) compared with that of the control group, and the difference is statistically significant (p is less than 0.001).

2. Flow cytometry for detecting the effect of Nr-CWS on FPR3 expression in DCs

Flow cytometry analysis showed an increase in the positive rate of FPR3 in the Nr-CWS group DCs compared to the control group (18.4% vs 42.8%).

3. Real-time fluorescent quantitative PCR method for detecting influence of Nr-CWS on FPR3 expression in DC

qPCR detects dendritic cell FPR3 mRNA expression levels. The results showed that the expression level of FPR3 was increased in the Nr-CWS group of DCs compared to the control group, with the difference being statistically significant (p < 0.05) (FIG. 5).

ELISA method for detecting influence of Nr-CWS on cytokines in siFPR3 DC and T cell co-culture model

To explore the influence of Nr-CWS on CD4 by DC ⁺ Whether DC FPR3 plays a key regulatory role in the T cell differentiation process, 2 siFPR3 and its blank control NC were transfected into DC D3 and D5, and after transfection, Nr-CWS was used to stimulate for 2 days, and the DC and naive CD4 ⁺ T cells are cultured for 2 days, and the expression difference of the cell factors in the supernatant of the culture medium is detected. The ELISA result shows that compared with the NC + Nr-CWS group, the concentrations of IL-2 and IFN-gamma in the culture supernatant of the siFPR3+ Nr-CWS group are obviously reduced, and the difference has statistical significance (p is less than 0.001); while there was no significant difference in the concentrations of IL-4, TGF-. beta.and IL-17a (FIGS. 6A to 6E).

The above results indicate that FPR3 exerts a positive regulatory effect on the differentiation of Th1 cells during Nr-CWS-induced DC-activated T cell immunity.

CCK-8 method for detecting influence of Nr-CWS on Hela cell growth through siFPR3 DC and T cell co-culture model

Nr-CWS treated DC and naive CD4 ⁺ The proliferation of Hela cells can be inhibited after the mixed culture of T cells. To investigate the role of FPR3 in this process in DCs, siFPR3 and its blank control NC were transfected 2 times into DCs at D3 and D5, stimulated with Nr-CWS for 2 days after transfection, and naive CD4 ⁺ T cells were cultured in a mixed manner for 2 days, and then cultured together with Hela cells in a Transwell chamber for 48 hours, and proliferation of Hela cells was detected by the CCK-8 method. The results show (FIG. 7) that the activity of Hela cells in siFPR3+ Nr-CWS group is significantly higher than that in NC + Nr-CIn WS group, the difference was statistically significant (p < 0.05).

Conclusion IV

In the previous test examples, it has been demonstrated that Nr-CWS regulates CD4 by stimulating DC ⁺ T cells are differentiated towards Th1, and the upregulation of DC-related genes UBD, MARCO and FPR3 is found in the mRNA expression profiling chip of cervical lesion tissue treated by Nr-CWS.

1. Immunohistochemistry, real-time fluorescence quantitative PCR and flow cytometry detection show that the expression of FPR3 protein and mRNA in the DC stimulated by the Nr-CWS is up-regulated.

2. Treatment of FPR3 silenced DCs and naive CD4 with Nr-CWS ⁺ T cell co-culture, ELISA method detection culture medium supernatant, Th1 type cell factor IL-2, IFN-gamma concentration recovery low level. Upon targeted silencing of FPR3 expression in DCs, Nr-CWS lost the induction of naive CD4 by DCs ⁺ The ability of T cells to differentiate towards Th 1.

Nr-CWS treatment of FPR3 silenced DCs and naive CD4 ⁺ And (4) co-culturing the T cells, and detecting Hela cells co-cultured with the MLR by a CCK-8 method, wherein the proliferation activity of the Hela cells is recovered to be normal. After FPR3 silencing in DC, Nr-CWS can not pass through DC-CD4 ⁺ The MLR model of T cells inhibited Hela cell proliferation.

Claims (9)

1. Use of a nocardia rubra cell wall scaffold in the preparation of a medicament, wherein:

the medicament is for use in preventing or treating cervical lesions associated with high risk type HPV infection in a subject;

the high risk HPV is selected from any one or combination of the following: 16. types 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68;

the cervical lesion is selected from any one of: atypical squamous cell lesions, low-grade squamous intraepithelial lesions.

2. The use of claim 1, wherein the subject is 21 to 40 years old.

3. The use according to claim 1 or 2, the medicament being prepared in a dosage form selected from any one of: ointment, cream, suppository, emulsion, suspension, paste, gel, lotion, tablet, aerosol, spray, liniment, powder, dressing, bandage, film, patch, and injection.

4. The use according to any one of claims 1 to 3, wherein the Nocardia rubra cell wall skeleton is the national drug standard S20030009 or Nocardia rubra cell wall skeleton corresponding to its accession number.

5. Use according to any one of claims 1 to 3, wherein the Nocardia rubra cell wall skeleton is obtained by a method comprising or consisting of:

1) providing nocardia rubra;

2) crushing the nocardia rubra to obtain a crushed product;

3.1) removing lipids from the disrupted product;

3.2) removing nucleic acids from the disruption product;

3.3) removing proteins from the disruption product;

3.4) removing cell membranes from the disruption product;

3.5) obtaining a red nocardia cell wall skeleton;

4) optionally, subpackaging;

5) optionally, freeze-drying the nocardia rubra cell wall skeleton;

wherein,

steps 3.1), 3.2), 3.3), 3.4) can be interchanged or in parallel,

step 4) and step 5) can be interchanged;

the average particle size of the fractures is from 10nm to 1000nm, preferably from 10nm to 800nm, more preferably from 10nm to 500 nm.

6. The use according to any one of claims 1 to 5, wherein the medicament is for any one or combination selected from the group consisting of:

reducing the expression level of a vimentin in cervical epithelium, increasing the expression level of IFN-gamma, reducing the expression level of IL-4, reducing the expression level of IL-17a, reducing the expression level of TGF-beta, reducing the number of Foxp3 positive cells, increasing the mRNA expression level of T-beta, reducing the mRNA expression level of GATA3, reducing the mRNA expression level of ROR gamma T, reducing the mRNA expression level of Foxp 3.

7. In vitro induced juvenile CD4 ⁺ A method of differentiating T cells to Th1 type comprising the steps of:

1) contacting an effective amount of nocardia rubra cell wall skeleton and dendritic cells for 1 to 5 days to obtain activated dendritic cells;

2) allowing the activated dendritic cells and naive CD4 ⁺ T cells are contacted for 1 to 3 days (preferably 2 to 3 days) to obtain a mixed culture;

the effective amount is 30 μ g/ml nocardia rubra cell wall skeleton;

the red nocardia cell wall skeleton is a red nocardia cell wall skeleton corresponding to the national standard character S20030009 or the updated number thereof.

8. A method for inhibiting proliferation of Hela cells in vitro comprising the steps of:

contacting the mixed culture as defined in claim 7 with HeLa cells for 48 hours or more; the HeLa cells were positive for HPV 18.

9. Use of a combination of nocardia rubra cell wall skeleton-activated dendritic cells and Th1 cells in the manufacture of a medicament, wherein:

the medicament is for use in preventing or treating cervical lesions associated with high risk type HPV infection in a subject;

the high risk HPV is selected from any one or combination of the following: 16. types 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68;

the cervical lesion is selected from any one of: atypical squamous cell lesions, low-grade squamous intraepithelial lesions;

the subject is 21 to 40 years old;

the medicament is prepared into a dosage form selected from any one of the following: ointment, cream, suppository, emulsion, suspension, paste, gel, lotion, tablet, aerosol, spray, liniment, powder, dressing, bandage, film, patch, injection;

preferably, the nocardia rubra cell wall skeleton is the nocardia rubra cell wall skeleton corresponding to the national standard character S20030009 or the update number thereof.

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