Use of recombinant type iii humanized collagen in the treatment of cervical cancer

Abstract

The present application relates to the use of recombinant humanized collagen type III in the treatment of cervical cancer, in particular, the present application provides the use of recombinant humanized collagen type III in the preparation of products for preventing and / or inhibiting and / or treating cervical cancer; the amino acid sequence of the recombinant humanized collagen type III comprises n repeats of the sequence shown in SEQ ID NO. 1, n is an integer greater than or equal to 1, n is preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 24 or 32, wherein when n is an integer greater than or equal to 2, each repeat sequence is directly connected. Research data shows that the recombinant humanized collagen type III can significantly inhibit the proliferation and migration ability of cervical cancer cells, which provides help for the preparation and wide application of products for preventing and treating cervical cancer and the clinical treatment of cervical cancer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to the use of recombinant humanized collagen type III in the treatment of cervical cancer. BACKGROUND

[0002] Cervical cancer is the most common malignant tumor of the female reproductive tract, and its incidence ranks second among female malignant tumors. Globally, there are about 500,000 new cases of cervical cancer each year, accounting for 5% of all new cancer cases. At present, common treatment methods for cervical cancer include surgical treatment, radiotherapy, chemotherapy, and drug targeted therapy. However, these methods have certain side effects and limitations. For example, surgical treatment has a high risk and may cause bleeding, infection, or other complications; radiotherapy and chemotherapy have a greater side effect of reducing the patient's immunity, and also have a certain impact on the surrounding normal tissues; drug targeted therapy can target specific molecular markers and reduce the impact on normal cells, but the types of available drugs are limited, and specific biomarker expression is required for use. Therefore, finding more effective inhibition methods for cervical cancer has become a research focus.

[0003] In recent years, it has been found that the extracellular matrix in the tumor microenvironment not only provides a physical scaffold for cell adhesion, but also participates in the regulation of many biological processes, such as tumor cell growth, migration, differentiation, and homeostasis. The role of collagen, the most abundant protein in the extracellular matrix, in tumor occurrence and development is still controversial.

[0004] Collagen is the most abundant and widely distributed macromolecular functional protein in mammalian tissues and organs, accounting for about 30%-40% of the total body protein. It is widely distributed in mammalian tissues and organs in various forms, mainly including skin, bone, and muscle tissue. So far, 28 different types of collagen have been found in the human body, which can be divided into two categories according to whether they are fibrous or not. Fibrous collagen mainly serves as a cell scaffold to fix the position of cells and plays an anchoring role. It also provides tensile strength and stiffness to tissues. The main representative types are collagen types I, II, III, V, and XI. Non-fibrous collagen is further divided into basement collagen, short-chain collagen, and transmembrane collagen, each of which has different functions.

[0005] The unique marker of collagen is a right-handed triple helix domain, i.e., a repeating Gly-X-Y triplet, in which proline and lysine usually appear at the X and Y positions. Due to its unique biological structure, collagen has good biocompatibility, degradability, and low antigenicity, and has been widely used in many fields of production and life such as biomedical, cosmetics, health products, and food in recent years.

[0006] Traditional methods for producing collagen involve treating animal-derived tissues with acid, alkali, or enzymatic hydrolysis to extract collagen derivatives. On the one hand, these methods extract collagen that has already lost its original biological activity and cannot be applied to biomedical fields to perform its true functions. On the other hand, animal-derived collagen has hidden dangers in terms of purity, disease transmission, and repeatability, limiting its application. If collagen is extracted from raw materials such as human placenta, not only is the source limited, but it also does not meet ethical and legal requirements. Therefore, with the development of modern biotechnology, people have begun to try to use transgenic technology to produce recombinant human collagen in animal, plant, and microbial expression systems, solving many shortcomings of traditional extraction processes. For example, some research institutions have obtained human collagen-containing milk by breeding mice containing human collagen genes, but the cost of production is too high, and the production cycle is too long, making it impossible to be mass-produced. Therefore, collagen can only be used in cosmetics and health products at present, and its original biological functions cannot be fully utilized.

[0007] With the progress of genetic engineering and synthetic biology, recombinant collagen with specific structure and function can be developed and shown to have potential as a starting material for surgical implants and regenerative medicine matrices. In general, recombinant collagen is prepared by transcribing specific gene fragments and expressed in yeast, bacteria, or animal cells, with consistent formulation yield and high productivity. In order to be biocompatible with the human body, a human collagen gene is mainly selected to encode a specific type of human collagen to produce predictable and reliable recombinant collagen, which has broad prospects for medical applications. Currently, recombinant humanized collagen is defined as a functional amino acid sequence of full-length or fragments encoded by a specific type of human collagen gene, or a combination containing a functional fragment of human collagen.

[0008] Chinese invention patent 201811438582.6 discloses recombinant humanized collagen type III (rhCOL III) developed by Shanxi Jinbo Biomedical Co., Ltd. Based on the original gene sequence of human collagen type III, the study optimally selected a portion with strong water solubility and high biological activity for codon optimization and splicing recombination, obtaining a new recombinant humanized collagen sequence. Experiments have confirmed that the recombinant collagen type III has a large expression amount, is easy to remove endotoxins, has good water solubility, high stability, and high biological activity, and has superior adhesion effect. Its overall performance is superior to that of human natural collagen. Studies have shown that rhCOL III has the effects of anti-photoaging, promoting wound repair, and promoting cell proliferation. Although there have been reports of collagen type III gel in the treatment of HPV infection and cervical erosion, the relationship between rhCOL III and cervical cancer treatment has not been reported. SUMMARY

[0009] Problem to be solved by the invention

[0010] In view of the problems in the prior art, the present application studies the recombinant humanized collagen type III disclosed in ZL201811438582.6 in the treatment of cervical cancer. Specifically, the present application studies the effect of the recombinant humanized collagen type III on the proliferation and migration ability of human cervical cancer cells. Based on this, the present application provides the use of the recombinant humanized collagen type III in preventing and / or inhibiting and / or treating cervical cancer.

[0011] Solution for solving the problem

[0012] The first aspect of the present application provides the use of the recombinant humanized collagen type III in preparing a product for preventing and / or inhibiting and / or treating cervical cancer; wherein the recombinant humanized collagen type III comprises n repeats of the sequence shown in SEQ ID NO. 1, n is an integer greater than or equal to 1, n is preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 24 or 32, wherein when n is an integer greater than or equal to 2, each repeat sequence is directly connected.

[0013] In some embodiments, the recombinant humanized collagen type III comprises 16 repeats of the sequence shown in SEQ ID NO. 1, and each repeat sequence is directly connected.

[0014] In some specific embodiments, the amino acid sequence of the recombinant humanized collagen type III comprises any one of the following (a) to (d):

[0015] (a) the sequence shown in SEQ ID NO. 2;

[0016] (b) a sequence having 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with the sequence shown in SEQ ID NO. 2, and which retains the cell adhesion effect of the sequence shown in SEQ ID NO. 2;

[0017] (c) a sequence in which one or more amino acid residues are added, substituted or deleted in the sequence shown in SEQ ID NO. 2, and which retains the cell adhesion effect of the sequence shown in SEQ ID NO. 2;

[0018] (d) an amino acid sequence encoded by a nucleotide sequence that hybridizes to a polynucleotide sequence encoding the sequence as set forth in SEQ ID NO. 2 under stringent conditions, and the amino acid sequence retains the cell adhesion effect of the sequence as set forth in SEQ ID NO. 2, the stringent conditions being medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions.

[0019] In some specific embodiments, the amino acid sequence of the recombinant humanized collagen type III comprises the sequence as set forth in SEQ ID NO. 2.

[0020] In some specific embodiments, the amino acid sequence of the recombinant humanized collagen type III is the sequence as set forth in SEQ ID NO. 2.

[0021] The recombinant humanized collagen type III described herein can be produced by methods conventional in the art. For example, it can be produced by the following steps: (1) construction of an engineered E. coli bacterium; (2) fermentation culture of the engineered E. coli bacterium; (3) induced expression of the recombinant humanized collagen type III; and (4) purification and optional enzymatic cleavage of the recombinant humanized collagen type III.

[0022] In some embodiments, the cervical cancer comprises any one or more of squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, clear cell carcinoma, small cell carcinoma, and sarcoma.

[0023] In some specific embodiments, the cervical cancer comprises squamous cell carcinoma.

[0024] In some specific embodiments, the cervical cancer is squamous cell carcinoma.

[0025] In some embodiments, the cervical cancer is associated with persistent infection of a high-risk type of human papillomavirus.

[0026] In some specific embodiments, the high-risk type of human papillomavirus comprises any one or more of HPV type 16, HPV type 18, HPV type 31, HPV type 33, HPV type 35, HPV type 39, HPV type 45, HPV type 51, HPV type 52, HPV type 56, HPV type 58, HPV type 59, and HPV type 68.

[0027] In some specific embodiments, the high-risk type of human papillomavirus comprises any one or more of HPV type 18, HPV type 39, and HPV type 68.

[0028] In some specific embodiments, the high-risk type of human papillomavirus is HPV type 18, HPV type 39, or HPV type 68.

[0029] In some specific embodiments, the high-risk type of human papillomavirus is HPV type 18, HPV type 39 and HPV type 68.

[0030] In some embodiments, the prevention and / or inhibition and / or treatment of cervical cancer comprises any one or more of the following: inhibition of the viability of cervical cancer cells, inhibition of the proliferative capacity of cervical cancer cells and inhibition of the migratory capacity of cervical cancer cells.

[0031] In some embodiments, the prevention and / or inhibition and / or treatment of cervical cancer comprises all of the following: inhibition of the viability of cervical cancer cells, inhibition of the proliferative capacity of cervical cancer cells and inhibition of the migratory capacity of cervical cancer cells.

[0032] In some embodiments, the product is preferably a pharmaceutical, a medical device or a combination of a pharmaceutical and a medical device.

[0033] In some embodiments, the product comprises an injection.

[0034] In some specific embodiments, the injection comprises any one or more of an injection solution and a lyophilized preparation for injection.

[0035] In some specific embodiments, the product comprises the recombinant humanized collagen type III and optionally acceptable adjuvants.

[0036] The second aspect of the present application provides the use of a recombinant humanized collagen type III for the prevention and / or inhibition and / or treatment of cervical cancer; wherein the recombinant humanized collagen type III and the cervical cancer are as described in the first aspect of the present application.

[0037] The third aspect of the present application provides a product for the prevention and / or inhibition and / or treatment of cervical cancer, the product being prepared according to the use described in the first aspect of the present application, the product comprising a pharmaceutical composition, a medical device or a combination of a pharmaceutical and a medical device.

[0038] In some embodiments, the dosage form of the pharmaceutical composition is a tablet, a capsule, a dripping pill, an aerosol, a pill, a powder, a solution, a suspension, an emulsion, a granule, a liposome, a transdermal agent, a gel, an infiltrating agent, a suppository or a lyophilized powder injection.

[0039] In some embodiments, the pharmaceutical composition is administered by injection, by administration to a cavity, by administration to the respiratory tract, by administration to the mucosa or by administration to the surface; the injection includes any one or more of subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intracisternal injection and infusion; the administration to a cavity includes any one or more of rectal administration, vaginal administration and sublingual administration; the administration to the respiratory tract includes nasal administration.

[0040] In some embodiments, the pharmaceutical composition, in addition to comprising recombinant type III humanized collagen as described in the first aspect of the invention, also comprises pharmaceutically acceptable excipients or carriers.

[0041] A fourth aspect of the present invention provides a method for preventing and / or inhibiting and / or treating cervical cancer, the method comprising administering an effective amount of recombinant humanized type III collagen or a product containing recombinant humanized type III collagen to an individual in need; wherein the recombinant humanized type III collagen and the cervical cancer are as described in the first aspect of the present invention, and the product containing recombinant humanized type III collagen is as described in the third aspect of the present invention.

[0042] Effects of the invention

[0043] This invention provides the use of recombinant type III humanized collagen in the preparation of products for the prevention and / or inhibition and / or treatment of cervical cancer. Research data shows that recombinant type III humanized collagen can significantly inhibit the proliferation and migration of cervical cancer cells, and this inhibitory effect is more pronounced at relatively high collagen concentrations, thus providing a new approach for the preparation of products for the prevention and / or inhibition and / or treatment of cervical cancer.

[0044] Meanwhile, the collagen provided by this invention for the prevention and / or inhibition and / or treatment of cervical cancer is recombinant type III humanized collagen, which has the advantages of convenient production and high safety during use. It also overcomes the problems of natural collagen being insoluble in water, having uneven properties, being difficult for the human body to utilize, and requiring chemical treatment before use. It also overcomes the problems of common animal-derived collagen (such as collagen taken from animal tissues such as pigs, cattle, and fish) potentially being infected with viruses, not being compatible with the human body, and easily causing immune rejection and allergic symptoms when used. This further provides beneficial assistance for the preparation and widespread application of products for the prevention and / or inhibition and / or treatment of cervical cancer, as well as the clinical treatment of cervical cancer. Attached Figure Description

[0045] Figure 1 Results of CCK-8 assay to detect the effect of rhCol III on the survival of ME-180 cells; among them, compared with the negative control group, **p<0.01, ***p<0.001, ****p<0.0001.

[0046] Figure 2A The results of the EdU proliferation assay to detect the effect of rhCol III on the proliferation ratio of ME-180 cells.

[0047] Figure 2BStatistical results of the effect of rhCol III on the proliferation ratio of ME-180 cells in the EdU proliferation experiment; wherein, compared with the control group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0048] Figure 3A Experimental results of the effect of rhCol III on the migration ability of ME-180 cells in the scratch experiment.

[0049] Figure 3B Statistical results of the effect of rhCol III on the migration ability of ME-180 cells in the scratch experiment; wherein, compared with the negative control group, ns represents no statistical difference; *p<0.05, **p<0.01, ****p<0.0001.

[0050] Figure 4A Experimental results of the effect of rhCol III on the migration ability of ME-180 cells in the Transwell chamber migration experiment.

[0051] Figure 4B Statistical results of the effect of rhCol III on the migration ability of ME-180 cells in the Transwell chamber migration experiment; wherein, compared with the negative control group, **p<0.01, ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION

[0052] The embodiments of the present application are described below, but the present application is not limited thereto. Various modifications can be made within the scope of the present application, and the embodiments obtained by appropriately combining the technical means disclosed in each of the embodiments are also included in the technical scope of the present application.

[0053] In the present application, "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", and the like mean that the specific elements (for example, features, structures, properties, and / or characteristics) described in relation to the embodiments are included in at least one of the embodiments described herein, and can be present in other embodiments or can not be present in other embodiments. In addition, it should be understood that the elements can be combined in various embodiments in any suitable manner.

[0054] In the present application, the meaning indicated using "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0055] In the present application, the terms "comprise", "have", "include" or "contain" can refer to inclusion or open-ended, and do not exclude additional, unrecited elements or method steps. At the same time, "comprise", "have", "include" or "contain" can also mean closed, excluding additional, unrecited elements or method steps.

[0056] In the present application, the term "optional" or "optionally" means that the event or circumstance described immediately below can or can not occur, or the step or element described immediately below can or can not be included in the embodiments described herein, and the description includes both the case where the event occurs and the case where the event does not occur, and both the case where the embodiment includes the step or element and the case where the embodiment does not include the step or element.

[0057] In the present application, the term "individual", "patient" or "subject" includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0058] In the present application, the term "prevent" means, before the onset of a disease, by contacting (e.g., administering) the recombinant humanized collagen type III described in the present application to a subject, thereby reducing the probability of the onset of the disease and / or alleviating the symptoms after the onset of the disease, compared with the case of not contacting, and does not mean that it is necessary to completely inhibit the disease. The onset of the disease means that the body has symptoms of the disease.

[0059] In the present application, the term "treat" means, after the onset of a disease, contacting (e.g., administering) the recombinant humanized collagen type III described in the present application to a subject, thereby alleviating the symptoms of the disease, compared with the case of not contacting, and does not mean that it is necessary to completely inhibit the symptoms of the disease.

[0060] In the present application, the terms "administer", "administering", and "treat" when applied to an animal, a human, an experimental subject, a cell, a tissue or an organ, etc., mean the contact of an exogenous therapeutic agent, a diagnostic agent or a composition with the animal, the human, the experimental subject, the cell, the tissue or the organ.

[0061] In the present application, the term "effective amount" includes an amount sufficient to improve or prevent the symptoms or conditions of a medical disease. The effective amount for a particular subject can vary depending on factors such as the condition to be treated, the overall health status of the subject, the method route and dosage of administration, and the severity of side effects, etc. The effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxic effects.

[0062] In the present application, the term "identity" refers to the percentage of amino acids that are the same between two or more polypeptides. The sequence identity between two or more polypeptides can be determined by aligning the amino acid sequences of the polypeptides and scoring the number of positions in the aligned polypeptides that contain identical amino acid residues, and comparing it to the number of positions in the aligned polypeptides that contain different amino acid residues. The sequence identity can be calculated by dividing the number of positions containing identical amino acid residues by the total number of amino acid residues in the polypeptides.

[0063] In the present application, the amino acid addition can refer to the addition of 1, 2, or more than 3 amino acids at the C-terminus, the N-terminus, or any position in between the C-terminus and the N-terminus of the amino acid sequence, as long as the altered sequence completely or partially retains the activity of the original amino acid sequence.

[0064] In the present application, the amino acid substitution can refer to the substitution of an amino acid at a certain position of the amino acid sequence with another amino acid, as long as the altered sequence completely or partially retains the activity of the original amino acid sequence. The amino acid substitution can be a conservative amino acid substitution, which refers to the substitution of several amino acids with amino acids of similar or close properties to form a peptide (conservatively varied peptide) compared to the original amino acid sequence. Illustratively, these conservatively varied peptides can be generated according to the following amino acid substitutions: substitution of Val, Leu, or Ile for Ala, substitution of Lys, Gin, Asn, or His for Arg, substitution of Gin, His, Lys, or Arg for Asn, substitution of Glu or Asn for Asp, substitution of Ser or Ala for Cys, substitution of Asn or Glu for Gin, substitution of Asp or Gin for Glu, substitution of Ala for Gly, substitution of Asn, Lys, Gin, or Arg for His, substitution of Leu, Met, Ala, Val, Phe, or norleucine for Ile, substitution of Ile, Met, Ala, Val, Phe, or norleucine for Leu, substitution of Asn, Gin, or Arg for Lys, substitution of Ile, Leu, or Phe for Met, substitution of Leu, Val, Ile, Ala, or Tyr for Phe, substitution of Ala for Pro, substitution of Thr for Ser, substitution of Ser or Val for Thr, substitution of Phe or Tyr for Trp, substitution of Trp, Phe, Thr, or Ser for Tyr, and substitution of Phe, Ala, Met, Ile, Leu, or norleucine for Val. The amino acid substitution can also be a non-conservative amino acid substitution.

[0065] In the present application, the amino acid deletion can refer to the deletion of 1, 2, or more than 3 amino acids from the amino acid sequence, as long as the altered sequence completely or partially retains the activity of the original amino acid sequence.

[0066] In the present application, "hybridization" means the ability of a polynucleotide or oligonucleotide to bind to a substantially complementary sequence under stringent conditions, without non-specific binding to non-complementary objects occurring under these conditions. The terms "moderate stringency conditions", "moderate-high stringency conditions", "high stringency conditions" or "very high stringency conditions" as used in the present application describe the conditions of nucleic acid hybridization and washing. For example, specific hybridization conditions are as follows: (1) low stringency hybridization conditions in 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by at least 50°C in 0.2x SSC, 0.1% SDS, (for low stringency conditions, the wash temperature can be increased to 55°C); (2) moderate stringency hybridization conditions in 6x SSC at about 45°C, followed by 60°C in 0.2x SSC, 0.1% SDS, one or more times; (3) high stringency hybridization conditions in 6x SSC at about 45°C, followed by 65°C in 0.2x SSC, 0.1% SDS, one or more times and preferably; (4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by 65°C in 0.2x SSC, 1% SDS, one or more times.

[0067] In the present application, the cell adhesion activity can refer to the cell adhesion activity measured by the activity detection method of the collagen disclosed in the reference Juming Yao, Satoshi Yanagisawa, Tetsuo Asakura, Design, Expression and Characterization of Collagen-Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from Native Collagens, J Biochem. 136, 643-649 (2004).

[0068] In the present application, the amino acid sequence involved is as follows:

[0069] SEQ ID NO. 1: GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0070] SEQ ID NO. 2: GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAP GFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGP AGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNG IPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFR GPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEK GPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGP AGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGE RGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGP AGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGN GIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPG FRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGA PGERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0072] Examples

[0073] The embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are for illustrative purposes only and should not be construed as limiting the scope of the present application. In the examples, unless otherwise specified, the operations were performed under conventional conditions or conditions recommended by the manufacturer. In the examples, unless otherwise specified, the materials or instruments used were commercially available conventional products that can be used.

[0074] The amino acid sequence of the recombinant humanized collagen type III used in the examples is shown in SEQ ID NO. 2.

[0075] Example 1: Tumor cell proliferation inhibitory ability of rhCol III

[0076] (1) CCK-8 experiment

[0077] After setting experimental groups (negative control group NC: human cervical cancer cells ME-180 + 10% FBS medium (Gibico McCoy's 5A (modified) medium, HEPES), no collagen co-culture; positive control group PC: human cervical cancer cells ME-180 + 10% FBS medium + 8% NaCl, no collagen co-culture; blank control group: no cells 10% FBS medium, no collagen co-culture; experimental group: human cervical cancer cells ME-180 + 10% FBS medium, rhCol III co-culture), 5000 cells / well were inoculated in a 96-well plate, and the cell suspension volume was 100 μL, and 6 replicate wells were set for each group. The 96-well plate was pre-cultured in a incubator at 37°C, 5% CO2 for 6-8 hours, and after the cells adhered, 0.4% FBS medium was replaced for starvation treatment, and after 24h, 0.4% FBS medium dissolved rhCol III was replaced, and the concentration was 0.5, 1, 2, 5, 10 mg / mL respectively. After the cells were co-cultured with collagen for 48h, 5% CCK-8 solution prepared with basic medium (Gibico McCoy's 5A (modified) medium, HEPES) was replaced, and incubated in a 37°C incubator for 2h in the dark, and the absorbance value OD450 was detected by an enzyme label instrument, and the cell survival rate was calculated = (experimental group OD value-blank control group OD value) / (negative control group OD value-blank control group OD value) x 100%.

[0078] (2) EdU proliferation experiment

[0079] Logarithmic growth phase ME-180 cells were taken, 100 μL 2000 cells per well were inoculated in a 96-well plate, and pre-cultured in a incubator at 37°C, 5% CO2 for 6-8 hours, and after the cells adhered, 0.4% FBS medium was replaced for starvation treatment, and after 24h, 0.4% FBS medium dissolved rhCol III was replaced, and the concentration was 0.5, 1, 2, 5, 10 mg / mL respectively, and at the same time, cells without the addition of rhCol III were used as a control group (Control). After the cells were co-cultured with collagen for 48h, BeyoClick TMEdU-555 Cell Proliferation Detection Kit was used to detect cell proliferation. The specific method was as follows: EdU working solution was added for incubation for 2 h, and then 4% paraformaldehyde fixing solution was replaced for room temperature fixing for 15 min. Then the fixing solution was poured out, and each well was washed with 100 μL of washing solution for 3-5 min, and then the permeation solution was replaced and incubated at room temperature for 10-15 min; the washing solution was washed for 1-2 times, each for 3-5 min. The washing solution was removed, and Click reaction solution was added to each well, which was incubated at room temperature for 30 min in the dark, and then the reaction solution was discarded, and the washing solution was washed for 3 times, each for 3-5 min. Then Hoechst 33342 solution was replaced, and incubated at room temperature for 10 min in the dark, and then washed with the washing solution for 3 times. Then the results were photographed under a fluorescence microscope. EdU was red fluorescence, and Hoechst 33342 was blue fluorescence, the maximum excitation wavelength was 346 nm, and the maximum emission wavelength was 460 nm. ImageJ was used for counting and then analyzing the results.

[0080] Experimental results:

[0081] The CCK-8 detection results are shown in Figure 1 The EdU proliferation experiment results are shown in Figure 2A and 2B The results show that after the human cervical cancer cells ME-180 are treated with rhCol III for 48 h, the growth ability of the tumor cells is obviously inhibited, and the inhibition effect is significantly increased with the increase of the collagen concentration. In the EdU proliferation experiment, the proportion of human cervical cancer cells in the proliferation activity is significantly decreased after the treatment with rhCol III, which indicates that rhCol III has an obvious inhibitory effect on the proliferation and DNA replication activity of human cervical cancer cells.

[0082] Example 2: rhCol III inhibits the migration ability of tumor cells

[0083] (1) Scratch experiment:

[0084] First, a straight line mark was drawn on the back of a 12-well plate with a marker pen, and then a proper amount of ME-180 cells were inoculated into the 12-well plate. After the cells adhered and covered the bottom of the plate, 0.4% FBS medium was added for starvation treatment. After 24 h of incubation, 200 μL of gun head was used to draw a scratch vertically to the well plate, and the width of each scratch was kept as consistent as possible. The well plate was washed with PBS for 1-3 times to remove the cell fragments generated by the scratch. The corresponding culture solution was replaced (negative control group control: 0.4% FBS medium; positive control group EGF: growth factor EGF + 0.4% FBS medium; experimental group: collagen + 0.4% FBS medium), and incubated in the incubator. Photographs were taken at 0 h and 12 h.

[0085] (2) Transwell chamber migration experiment

[0086] Before the experiment, human cervical cancer cells ME-180 were starved for 24 h with 0.4% FBS medium to eliminate the influence of serum on the experiment. Using 24-well plates and Transwell cell chambers, the lower chamber was added with corresponding culture medium (negative control group control: 10% FBS medium; experimental group: collagen + 10% FBS medium), and the upper chamber was added with 200 μL 10 x 10 5 / mL cell suspension resuspended with 0.4% FBS medium. After 24 h of culture, the chamber was transferred to a well pre-filled with 800 μL methanol, fixed at room temperature for 30 min, and then washed with PBS twice. Subsequently, it was transferred to a well containing 200 μL DAPI, stained for 15 min, and then the upper layer of un-migrated cells were gently wiped off with a cotton swab and washed with PBS three times. Photographs were taken under a fluorescence microscope and counted using ImageJ.

[0087] Experimental results:

[0088] The results of the scratch test are shown in Figure 3A and 3B After treatment with rhCol III, the migration ability of human cervical cancer cells ME-180 was significantly weakened compared with the control group without collagen treatment.

[0089] The results of the Transwell chamber migration experiment are shown in Figure 4A and 4B Compared with the control group without collagen treatment, the number of migrated human cervical cancer cells ME-180 in the experimental group with rhCol III added in the lower chamber was significantly reduced, indicating that the migration ability of the cells was weakened.

Claims

1. Use of a recombinant humanized collagen type III in the preparation of a product for treating cervical cancer; wherein an amino acid sequence of the recombinant humanized collagen type III is a sequence as shown in SEQ ID NO. 2; the cervical cancer is squamous cell carcinoma.

2. Use according to claim 1, characterized in that, the cervical cancer is associated with persistent infection of high-risk human papillomavirus, the high-risk human papillomavirus including any one or more of HPV type 18, HPV type 39 and HPV type 68.

3. Use according to claim 1 or 2, characterized in that, the treatment of cervical cancer includes any one or more of inhibition of viability of cervical cancer cells, inhibition of proliferation of cervical cancer cells and inhibition of migration of cervical cancer cells.

4. Use according to claim 1 or 2, characterized in that, the product includes a pharmaceutical composition, a medical device or a combination of the two.

5. Use according to claim 4, characterized in that, a dosage form of the pharmaceutical composition is a tablet, a capsule, an aerosol, a pill, a powder, a solution, a suspension, an emulsion, a granule, a liposome, a transdermal agent, a gel, a suppository or a lyophilized powder injection.

6. Use according to claim 4, characterized in that, the pharmaceutical composition is administered by injection, administration through a body cavity or mucosal administration; the injection includes any one or more of subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection; the administration through a body cavity includes any one or more of transrectal administration and transvaginal administration.

7. Use according to claim 4, characterized in that, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant or carrier.

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