Application of recombinant III-type humanized collagen in treatment of cervical cancer

Abstract

The invention relates to application of recombinant III-type humanized collagen in treatment of cervical cancer, in particular to application of recombinant III-type humanized collagen in preparation of products for preventing and / or inhibiting and / or treating cervical cancer. The amino acid sequence of the recombinant III-type humanized collagen comprises n repetitions of the sequence as shown in SEQ ID NO.1, n is an integer greater than or equal to 1 and preferably is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 24 or 32, and when n is an integer greater than or equal to 2, the repetitions are directly connected. Research data shows that the recombinant III-type humanized collagen can obviously inhibit the proliferation capacity and migration capacity of cervical cancer cells, and help is provided for preparation and wide application of products for preventing and treating cervical cancer and clinical treatment of cervical cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to the use of recombinant type III humanized collagen in the treatment of cervical cancer. Background Art

[0002] Cervical cancer is the most common malignant tumor of the female reproductive tract, and its incidence rate 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 treatments for cervical cancer include surgery, radiotherapy, chemotherapy, and targeted drug 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 greater side effects such as reduced immunity of patients, and also have a certain impact on surrounding normal tissues; although targeted drug therapy can target specific molecular markers and reduce the impact on normal cells, the types of drugs currently available are limited, and specific biomarker expression is required for use. Therefore, finding more effective means of inhibiting cervical cancer has become a research focus.

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

[0004] Collagen is the most abundant and widely covered macromolecular functional protein in various tissues and organs of mammals, accounting for about 30% to 40% of the body's protein. It is widely distributed in mammalian tissues and organs in various forms, mainly including skin, bones and muscle tissues. So far, 28 different types of collagen have been found in the human body, which can be divided into two categories: fibrous and non-fibrous according to whether the structure is fibrous. Fibrous collagen mainly serves as a cell scaffold, which is used to fix the position of cells and play an anchoring role. It also provides tensile strength and stiffness for tissues. The main representative types are collagen types I, II, III, V, and XI. Non-fibrous collagen is further subdivided into basal collagen, short-chain collagen, transmembrane collagen, etc., each of which has different functions.

[0005] The unique mark of collagen is a right-handed triple helical domain, that is, a repeated Gly-XY triplet, in which proline and lysine usually appear at positions X and Y. Due to its unique biological structure, collagen has good biocompatibility, degradability and low antigenicity. In recent years, it has been widely used in many fields of production and life such as biomedicine, cosmetics, health products, and food.

[0006] The traditional method of producing collagen is to use acid, alkali and enzymatic hydrolysis to treat animal tissues and extract collagen derivatives. On the one hand, the collagen extracted by these methods has lost its original biological activity and cannot be used in the biomedical field to play a real role; on the other hand, the hidden dangers of animal-derived collagen in terms of purity, disease transmission and repeatability limit its application. If collagen is extracted from raw materials such as human placenta, not only is the source limited, but it is also unethical and does not comply with legal regulations. Therefore, with the development of modern biotechnology, people have begun to try to use transgenic technology to prepare recombinant human collagen in animal, plant and microbial expression systems, solving many shortcomings of traditional extraction processes. For example, a research institute has obtained milk containing human collagen by cultivating mice containing human collagen genes, but the cost of such production is too high and the production cycle is too long to be put into large-scale production. Therefore, at present, collagen can basically only be used in cosmetics and health products, and the original biological function of collagen cannot be fully exerted.

[0007] With the advancement of genetic engineering and synthetic biology, recombinant collagen with specific structure and function can be developed and shows potential as starting material for surgical implants and matrix for regenerative medicine. In general, recombinant collagen is prepared by transcribing specific gene fragments and expressed in yeast, bacteria or animal cells, with consistent formula 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 with broad medical application prospects. At present, recombinant humanized collagen is defined as a full-length or fragment functional amino acid sequence encoded by a specific type of human collagen gene, or a combination containing functional fragments of human collagen.

[0008] Chinese invention patent 201811438582.6 discloses recombinant humanized type III collagen (rhCOL III) developed by Shanxi Jinbo Biopharmaceutical Co., Ltd. This study is based on the original gene sequence of type III human collagen, and optimizes and selects the parts with strong water solubility and high biological activity for codon optimization and splicing recombination, and obtains a new recombinant humanized collagen sequence. Experiments have confirmed that this recombinant type III humanized collagen has a large expression amount, easy removal of endotoxins, good water solubility, high stability, high biological activity, and excellent adhesion effect. Its overall performance is better than that of natural human collagen. Studies have shown that rhCOL III has the effects of anti-photoaging, promoting wound repair, and promoting cell proliferation. Although there are reports on the treatment of HPV infection and cervical erosion with type III collagen gel, the connection between rhCOL III and cervical cancer treatment has not been reported. Summary of the invention

[0009] Problem that the invention aims to solve

[0010] In view of the problems existing in the above-mentioned prior art, the present invention studies the recombinant type III humanized collagen disclosed in ZL201811438582.6 in the treatment of cervical cancer. Specifically, the present invention studies the effect of recombinant type III humanized collagen on the proliferation and migration ability of human cervical cancer cells. Based on this, the present invention provides the use of recombinant type III humanized collagen in the prevention and / or inhibition and / or treatment of cervical cancer.

[0011] Solutions for solving problems

[0012] The first aspect of the present invention provides the use of recombinant type III humanized collagen in the preparation of a product for preventing and / or inhibiting and / or treating cervical cancer; wherein the recombinant type III humanized collagen comprises n repetitions 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 repeated sequence is directly connected.

[0013] In some embodiments, the recombinant humanized type III collagen 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 type III collagen 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 as shown in SEQ ID NO.2, and which retains the cell adhesion effect of the sequence as shown in SEQ ID NO.2;

[0018] (d) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the sequence shown in SEQ ID NO.2 under stringent conditions, and the amino acid sequence retains the cell adhesion effect of the sequence shown in SEQ ID NO.2, wherein the stringent conditions are medium stringent conditions, medium-high stringent conditions, high stringent conditions or very high stringent conditions.

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

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

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

[0022] In some embodiments, the cervical cancer includes 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 with high-risk human papillomavirus.

[0026] In some specific embodiments, the high-risk human papillomavirus includes any one or more of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59 and HPV68.

[0027] In some specific embodiments, the high-risk human papillomavirus includes any one or more of HPV18, HPV39 and HPV68.

[0028] In some specific embodiments, the high-risk human papillomavirus is HPV18, HPV39 or HPV68.

[0029] In some specific embodiments, the high-risk human papillomavirus is HPV18, HPV39 and HPV68.

[0030] In some embodiments, the prevention and / or inhibition and / or treatment of cervical cancer includes any one or more of the following manifestations: inhibiting the viability of cervical cancer cells, inhibiting the proliferation ability of cervical cancer cells, and inhibiting the migration ability of cervical cancer cells.

[0031] In some embodiments, the prevention and / or inhibition and / or treatment of cervical cancer simultaneously includes the following performances: inhibiting the viability of cervical cancer cells, inhibiting the proliferation ability of cervical cancer cells, and inhibiting the migration ability of cervical cancer cells.

[0032] In some embodiments, the product is preferably a drug, a medical device, or a combination of a drug and a 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 type III collagen and optional acceptable excipients.

[0036] The second aspect of the present invention provides the use of recombinant humanized type III collagen for preventing and / or inhibiting and / or treating cervical cancer; wherein the recombinant humanized type III collagen and the cervical cancer are as described in the first aspect of the present invention.

[0037] The third aspect of the present invention provides a product for preventing and / or inhibiting and / or treating cervical cancer, wherein the product is prepared according to the use described in the first aspect of the present invention, and the product includes a pharmaceutical composition, a medical device or a drug-device combination.

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

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

[0040] In some embodiments, the pharmaceutical composition comprises, in addition to the recombinant humanized type III collagen as described in the first aspect of the present invention, a pharmaceutically acceptable excipient or carrier.

[0041] The 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 type III humanized collagen or a product containing recombinant type III humanized collagen to an individual in need thereof; wherein the recombinant type III humanized collagen and the cervical cancer are as described in the first aspect of the present invention, and the product containing recombinant type III humanized collagen is as described in the third aspect of the present invention.

[0042] Effects of the Invention

[0043] The present invention provides the use of recombinant humanized type III collagen in the preparation of products for preventing and / or inhibiting and / or treating cervical cancer. Research data show that recombinant humanized type III collagen can significantly inhibit the proliferation and migration abilities of cervical cancer cells, and the inhibitory effect is more significant when the collagen concentration is relatively high, thus providing a new idea for the preparation of products for preventing and / or inhibiting and / or treating cervical cancer.

[0044] At the same time, the collagen provided by the present invention for preventing and / or inhibiting and / or treating cervical cancer is recombinant type III humanized collagen, which has the advantages of convenient production and high safety during use, and overcomes the problems that natural collagen is insoluble in water, has uneven properties, is difficult to be used by the human body, and needs to be treated by chemical means before use. It also overcomes the problems that common animal-derived collagen (for example, collagen taken from animal tissues such as pigs, cattle, and fish) may be infected with viruses, is incompatible with the human body, and is prone to immune rejection and allergic symptoms when used. This further provides favorable assistance for the preparation and wide application of products for preventing and / or inhibiting and / or treating cervical cancer and the clinical treatment of cervical cancer. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0047] Figure 2B: Statistical results of the EdU proliferation assay to detect the effect of rhCol III on the proliferation ratio of ME-180 cells; compared with the control group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0048] Figure 3A : The experimental results of scratch assay to detect the effect of rhCol III on the migration ability of ME-180 cells.

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

[0050] Figure 4A : Transwell chamber migration assay to detect the effect of rhCol III on the migration ability of ME-180 cells.

[0051] Figure 4B : The experimental results of Transwell chamber migration assay to detect the effect of rhCol III on the migration ability of ME-180 cells; compared with the negative control group, **p<0.01, ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION

[0052] The following describes the embodiments of the present invention, but the present invention is not limited thereto. Various changes can be made within the scope of the present invention, and the embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention.

[0053] In the present invention, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0054] In the present invention, the word "may" includes both performing a certain process and not performing a certain process.

[0055] In the present invention, the terms "comprising", "having", "including" or "containing" may mean inclusive or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising", "having", "including" or "containing" may also mean closed, excluding additional, uncited elements or method steps.

[0056] In the present invention, the term "optional" or "optionally" means that the event or situation described below may or may not occur, or the step or element described below may or may not be included in the embodiment described herein, and the description includes the case where the event occurs and the case where the event does not occur, and also includes the case where the embodiment includes and does not include the step or element.

[0057] In the present invention, the term "individual", "patient" or "subject" includes mammals. Mammals include, but are not limited to, domestic 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 invention, the term "prevention" means that before a subject develops a disease, the probability of developing a disease and / or the symptoms after developing a disease are reduced by exposing the subject to the recombinant humanized type III collagen described in the present invention, compared with when the subject does not develop the disease, and does not mean that the disease must be completely suppressed. Developing a disease means that symptoms of the disease appear in the body.

[0059] In the present invention, the term "treatment" means that after suffering from a disease, a subject is contacted with (e.g., administered) the recombinant humanized type III collagen described in the present invention, thereby alleviating the symptoms of the disease compared to when not contacted, and does not necessarily mean that the symptoms of the disease must be completely suppressed.

[0060] In the present invention, the terms "administering", "administering" and "treating" when applied to animals, humans, experimental subjects, cells, tissues or organs, etc., refer to the contact of exogenous therapeutic agents, diagnostic agents or compositions with animals, humans, experimental subjects, cells, tissues or organs.

[0061] In the present invention, 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 may vary depending on factors such as the condition to be treated, the subject's overall health, the method and dosage of administration, and the severity of side effects. The effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0062] In the present invention, the term "identity" refers to the percentage of identical amino acids 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 containing identical amino acid residues in the aligned polypeptides and comparing them to the number of positions containing different amino acid residues in the aligned polypeptides. 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 polypeptide.

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

[0064] In the present invention, amino acid substitution may refer to the replacement of an amino acid at a certain position of an amino acid sequence by another amino acid, as long as the altered sequence retains the activity of the original amino acid sequence in whole or in part. Amino acid substitution may be a conservative amino acid substitution, which means that compared with the original amino acid sequence, several amino acids are replaced by amino acids with similar or similar properties to form a peptide (conservative variant peptide). Exemplarily, these conservative variant peptides can be produced according to the following amino acid substitutions: substitution of Ala by Val, Leu or Ile, substitution of Arg by Lys, Gln, Asn or His, substitution of Asn by Gln, His, Lys or Arg, substitution of Asn by Glu or Asn, substitution of Cys by Ser or Ala, substitution of Gln by Asn or Glu, substitution of Glu by Asp or Gln, substitution of Gly by Ala, substitution of His by Asn, Lys, Gln or Arg, substitution of Leu, Met, Ala, Val, Phe or norleucine by The amino acid substitutions may also be non-conservative amino acid substitutions.

[0065] In the present invention, amino acid deletion may refer to deleting 1, 2 or 3 or more amino acids from an amino acid sequence, as long as the altered sequence fully or partially retains the activity of the original amino acid sequence.

[0066] In the present invention, "hybridization" means the ability of a polynucleotide or oligonucleotide to bind to a substantially complementary sequence under stringent conditions, while non-specific binding with non-complementary objects does not occur under these conditions. As used in the present invention, the terms "moderate stringency conditions", "medium-high stringency conditions", "high stringency conditions" or "very high stringency conditions" describe conditions for nucleic acid hybridization and washing. For example, specific hybridization conditions are as follows: (1) low stringency hybridization conditions are in 6× sodium chloride / sodium citrate (SSC) at about 45°C, followed by two washes in 0.2×SSC, 0.1% SDS at at least 50°C (for low stringency conditions, the washing temperature can be increased to 55°C); (2) moderate stringency hybridization conditions are in 6×SSC at about 45°C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 60°C; (3) high stringency hybridization conditions are in 6×SSC at about 45°C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 65°C and preferably; (4) very high stringency hybridization conditions are 0.5 M sodium phosphate, 7% SDS at 65°C, followed by one or more washes in 0.2×SSC, 1% SDS at 65°C.

[0067] In the present invention, the cell adhesion activity may refer to the cell adhesion activity measured by the activity detection method of 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 invention, the amino acid sequence involved is as follows:

[0069] SEQ ID NO. 1: GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0070] SEQ ID NO.2: GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAP GFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIP GEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP.

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

[0072] Example

[0073] The embodiments of the present invention will be described in detail below in conjunction with examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the materials or instruments used in the examples are all conventional products that can be used commercially.

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

[0075] Example 1: rhCol III's ability to inhibit tumor cell proliferation

[0076] (1) CCK-8 experiment

[0077] After setting the experimental groups (negative control group NC: human cervical cancer cell ME-180 + 10% FBS medium (GibicoMcCoy's 5A (modified) medium, HEPES), no collagen co-culture; positive control group PC: human cervical cancer cell ME-180 + 10% FBS medium + 8% NaCl, no collagen co-culture; blank control group: no cell 10% FBS medium, no collagen co-culture; experimental group: human cervical cancer cell ME-180 + 10% FBS medium, rhCol III co-culture), 5000 cells / well were inoculated in a 96-well plate, so that the cell suspension volume was 100 μL, and 6 replicates were set for each group. The 96-well plate was placed in an incubator at 37°C and 5% CO 2 Pre-cultured for 6 to 8 hours under the conditions, after the cells adhered to the wall, replaced with 0.4% FBS medium for starvation treatment, and replaced with rhCol III dissolved in 0.4% FBS medium after 24 hours, with concentrations of 0.5, 1, 2, 5, and 10 mg / mL, respectively. After the cells were co-cultured with collagen for 48 hours, replaced with 5% CCK-8 solution prepared with basal medium (Gibico McCoy's 5A (modified) medium, HEPES), incubated in a 37°C incubator in the dark for 2 hours, and the absorbance value OD450 was detected using an enzyme marker, and the cell survival rate was calculated as (OD value of the experimental group-OD value of the blank control group) / (OD value of the negative control group-OD value of the blank control group)×100%.

[0078] (2) EdU proliferation assay

[0079] ME-180 cells in the logarithmic growth phase were taken and 2000 cells were seeded in each well of a 96-well plate at 100 μL. The plates were incubated at 37°C and 5% CO. 2 The cells were pre-cultured for 6-8 hours under the same conditions. After the cells attached to the wall, the medium was replaced with 0.4% FBS for starvation treatment. After 24 hours, the medium was replaced with rhCol III dissolved in 0.4% FBS at concentrations of 0.5, 1, 2, 5, and 10 mg / mL. At the same time, cells without rhCol III were used as the control group (Control). After the cells were co-cultured with collagen for 48 hours, BeyoClick TMEdU-555 cell proliferation detection kit was used to detect cell proliferation. The specific method is as follows: Add EdU working solution and incubate for 2 hours, then replace 4% paraformaldehyde fixative and fix at room temperature for 15 minutes. Then pour out the fixative, wash each well with 100 μL washing solution 3 times, 3-5 minutes each time, then replace the permeabilization solution and incubate at room temperature for 10-15 minutes; wash 1-2 times with washing solution, 3-5 minutes each time. Remove the washing solution, add Click reaction solution to each well, incubate at room temperature in the dark for 30 minutes, then discard the reaction solution, wash 3 times with washing solution, 3-5 minutes each time. Then replace Hoechst 33342 solution, incubate at room temperature in the dark for 10 minutes, then wash 3 times with washing solution, and then take pictures under a fluorescence microscope. EdU is red fluorescence, Hoechst 33342 is blue fluorescence, the maximum excitation wavelength is 346nm, and the maximum emission wavelength is 460nm. ImageJ counts and analyzes the results.

[0080] Experimental results:

[0081] CCK-8 test results Figure 1 The results of the EdU proliferation assay are shown in Figure 2A and 2B The results showed that after human cervical cancer cell ME-180 was treated with rhCol III for 48 hours, the growth ability of tumor cells was significantly inhibited, and the inhibitory effect increased significantly with the increase of collagen concentration. In the EdU proliferation experiment, the proportion of human cervical cancer cells in proliferation activity decreased significantly after rhCol III treatment, indicating that rhCol III has a significant inhibitory effect on the proliferation and DNA replication activity of human cervical cancer cells.

[0082] Example 2: rhCol III inhibits tumor cell migration

[0083] (1) Scratch test:

[0084] First, use a marker to draw a straight line mark on the back of the 12-well plate, then inoculate an appropriate amount of ME-180 cells in the 12-well plate, add 0.4% FBS medium for starvation treatment after the cells adhere to the wall and cover the bottom of the plate, incubate for 24 hours, use a 200μL gun tip to scratch the plate perpendicular to the well plate, try to keep the width of each scratch consistent, and rinse the well plate 1-3 times with PBS to remove the cell debris produced by the scratch, replace the corresponding culture medium (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), culture in an incubator, and take pictures at 0h and 12h.

[0085] (2) Transwell chamber migration assay

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

[0087] Experimental results:

[0088] The scratch test results are as follows Figure 3A and 3B As shown, after human cervical cancer cell ME-180 was treated with rhCol III, the cell migration ability was significantly weakened compared with the control group without collagen treatment.

[0089] Transwell chamber migration assay results Figure 4A and 4B As shown, compared with the control group without collagen treatment, the number of migrating human cervical cancer cell ME-180 in the experimental group with rhCol III added to the lower chamber was significantly reduced, indicating that the cell migration ability was weakened.

Claims

1. Use of recombinant humanized type III collagen in the preparation of products for preventing and / or inhibiting and / or treating cervical cancer; in, The amino acid sequence of the recombinant type III humanized collagen comprises n repetitions of the sequence shown in SEQ ID NO.1, where n is an integer greater than or equal to 1, and 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 repeated sequence is directly connected.

2. The use according to claim 1, characterized in that The amino acid sequence of the recombinant humanized type III collagen comprises any one of the following (a) to (d): (a) the sequence shown in SEQ ID NO.2; (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; (c) a sequence in which one or more amino acid residues are added, substituted or deleted in the sequence as shown in SEQ ID NO.2, and which retains the cell adhesion effect of the sequence as shown in SEQ ID NO.2; (d) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the sequence shown in SEQ ID NO.2 under stringent conditions, and the amino acid sequence retains the cell adhesion effect of the sequence shown in SEQ ID NO.2, wherein the stringent conditions are medium stringent conditions, medium-high stringent conditions, high stringent conditions or very high stringent conditions.

3. The use according to claim 1 or 2, characterized in that The cervical cancer includes any one or more of squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, clear cell carcinoma, small cell carcinoma and sarcoma.

4. The use according to any one of claims 1 to 3, characterized in that The cervical cancer includes squamous cell carcinoma.

5. The use according to any one of claims 1 to 4, characterized in that: The cervical cancer is associated with persistent infection with high-risk human papillomavirus.

6. The use according to claim 5, characterized in that The high-risk human papillomavirus includes any one or more of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59 and HPV68.

7. The use according to claim 5 or 6, characterized in that The high-risk human papillomavirus includes any one or more of HPV18, HPV39 and HPV68.

8. The use according to any one of claims 1 to 7, characterized in that The prevention and / or inhibition and / or treatment of cervical cancer includes any one or more of the following manifestations: inhibiting the viability of cervical cancer cells, inhibiting the proliferation ability of cervical cancer cells, and inhibiting the migration ability of cervical cancer cells.

9. A product for preventing and / or inhibiting and / or treating cervical cancer, characterized in that: The product is prepared according to the use of any one of claims 1 to 8, and the product includes a pharmaceutical composition, a medical device, or a drug-device combination; Preferably, the dosage form of the pharmaceutical composition is tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, gels, infiltration preparations, suppositories or lyophilized powder injections; Preferably, the pharmaceutical composition is administered by the following methods: injection, cavity administration, respiratory administration, mucosal administration or surface administration; the injection administration includes any one or more of subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intracisternal injection and infusion; the cavity administration includes any one or more of rectal administration, vaginal administration and sublingual administration; the respiratory administration includes nasal administration; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient or carrier.

10. A method for preventing and / or inhibiting and / or treating cervical cancer, the method comprising administering an effective amount of recombinant type III humanized collagen or a product containing recombinant type III humanized collagen to an individual in need thereof; wherein: The recombinant humanized type III collagen is as defined in claim 1 or 2, and the product containing the recombinant humanized type III collagen is the product for preventing and / or inhibiting and / or treating cervical cancer as described in claim 9.

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