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

Abstract

The application belongs to the field of biological medicine, and particularly relates to the use of recombinant humanized collagen type III in the treatment of ovarian cancer, and more particularly to the use of recombinant humanized collagen type III in the preparation of a drug for preventing and / or treating ovarian diseases; wherein the recombinant humanized collagen type III comprises n repeats of the sequence shown in SEQ ID No. 1, and n is an integer greater than or equal to 1, wherein when n is an integer greater than or equal to 2, the repeat sequences are directly connected. Research data shows that the recombinant humanized collagen type III can significantly inhibit the proliferation, migration, invasion and tumorigenicity of ovarian cancer cells, and the inhibitory effect is more significant under the condition of relatively high protein concentration, thereby providing a new idea for preparing a drug for preventing and / or treating ovarian cancer and other ovarian diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the use of recombinant type III humanized collagen in the treatment of ovarian cancer. Background Technology

[0002] Ovarian cancer is a malignant tumor of the ovary, referring to malignant tumors that grow on the ovary. 90%–95% of these are primary ovarian cancers, while the remaining 5%–10% are metastases from primary cancers in other sites. Because early-stage ovarian cancer often lacks symptoms, and even when symptoms are present, they are nonspecific, and screening methods have limited effectiveness, early diagnosis is difficult. By the time patients seek medical attention, 60%–70% are already at an advanced stage, and late-stage cases typically have poor treatment outcomes. Therefore, although the incidence of ovarian cancer is lower than that of cervical and endometrial cancer, ranking third among gynecological malignancies, its mortality rate exceeds the combined mortality rates of cervical and endometrial cancers, ranking first among gynecological cancers and posing a serious threat to women's health.

[0003] Collagen is the most abundant protein in animals, accounting for approximately 30% of total protein, and plays a vital role in physiological processes such as cell adhesion. Due to its excellent biocompatibility and biodegradability, collagen has been widely used in the medical field. To date, the vast majority of collagen used in various research studies is derived from animal tissues and skin extracts. Collagen extracted from animals typically has poor water solubility and limited processability, directly restricting the development of many of its potential applications. However, collagen produced using genetic engineering technology can effectively overcome these shortcomings of animal-derived collagen.

[0004] Patent application CN201811438582.6, entitled "Polypeptide, its Production Method and Use," describes recombinant type III humanized collagen previously researched and produced by the inventors of this invention. This recombinant type III humanized collagen is characterized by high water solubility and high bioactivity. Currently, there are no reports documenting a link between recombinant type III humanized collagen and ovarian cancer treatment. Summary of the Invention

[0005] The problem the invention aims to solve

[0006] This invention aims to evaluate the efficacy of recombinant type III humanized collagen in the treatment of ovarian cancer using the recombinant type III humanized collagen in the patent application with application number CN201811438582.6. Specifically, this invention studies the effects of recombinant type III humanized collagen on the proliferation, migration, invasion and tumorigenicity of ovarian cancer cells, thereby achieving the goal of preventing and / or treating ovarian cancer.

[0007] Solution for solving the problem

[0008] This invention provides the use of recombinant type III humanized collagen in the preparation of medicaments for the prevention and / or treatment of ovarian diseases; wherein the recombinant type III humanized collagen comprises n repeats of the sequence shown in SEQ ID No. 1, where n is an integer greater than or equal to 1, and when n is an integer greater than or equal to 2, the repeat sequences are directly connected.

[0009] Preferably, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 24, or 32.

[0010] Furthermore, the recombinant type III humanized collagen may contain the sequence shown in SEQ ID No. 2 or may not contain the sequence shown in SEQ ID No. 2.

[0011] Furthermore, the recombinant type III humanized collagen comprises:

[0012] a) The amino acid sequence of SEQ ID No. 3;

[0013] b) An amino acid sequence that has 90% identity with the amino acid sequence of SEQ ID No. 3, which retains the therapeutic effect of the amino acid sequence of SEQ ID No. 3 on ovarian diseases;

[0014] c) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted, or inserted into the amino acid sequence of SEQ ID No. 3, while retaining the therapeutic effect of the amino acid sequence of SEQ ID No. 3 on ovarian diseases; or

[0015] d) An amino acid sequence encoded by a nucleotide sequence, wherein the nucleotide sequence hybridizes with the amino acid sequence encoding SEQ ID No. 3 under strict conditions, wherein the amino acid sequence retains the therapeutic effect of the amino acid sequence of SEQ ID No. 3 on ovarian diseases, wherein the strict conditions are moderately strict, moderately-highly strict, highly strict, or very highly strict.

[0016] Furthermore, the ovarian disease mentioned is ovarian cancer.

[0017] Furthermore, according to the classification of ovarian cancer, the ovarian cancer includes ovarian epithelial tumors, ovarian germ cell tumors, and ovarian sex cord-stromal tumors.

[0018] Furthermore, the recombinant type III humanized collagen can prevent and / or treat ovarian cancer by inhibiting the proliferation, migration, invasion, and / or tumorigenicity of ovarian cancer cells.

[0019] Furthermore, the ability to inhibit the proliferation of ovarian cancer cells includes inhibiting the DNA replication activity of ovarian cancer cells.

[0020] Furthermore, the ability to inhibit ovarian cancer cell tumorigenesis includes the ability to inhibit the formation of tumors by ovarian cancer cells.

[0021] Furthermore, the ability to inhibit the formation of tumors by ovarian cancer cells includes inhibiting the number of ovarian cancer cell clones and / or inhibiting the growth rate of ovarian cancer cells.

[0022] The effects of the invention

[0023] This invention provides the use of recombinant type III humanized collagen in the preparation of drugs for the prevention and / or treatment of ovarian diseases. Research data shows that recombinant type III humanized collagen can significantly inhibit the proliferation, migration, invasion, and tumorigenesis of ovarian cancer cells, and this inhibitory effect is more significant at relatively high protein concentrations, thus providing a new approach for the preparation of drugs for the prevention and / or treatment of ovarian cancer and other ovarian diseases. Furthermore, since the recombinant type III humanized collagen provided by this invention is a humanized collagen, it also has the advantages of convenient preparation and high safety, which helps in the preparation and widespread application of drugs for ovarian cancer and in the clinical treatment of ovarian cancer. Attached Figure Description

[0024] Figure 1 This is a statistical result showing the effect of different concentrations of recombinant type III humanized collagen on the survival ability of ovarian cancer cells at different time points.

[0025] Figure 2 The results show the effect of recombinant type III humanized collagen on the proliferation ability of ovarian cancer cells.

[0026] Figure 3 The results show the effect of recombinant type III humanized collagen on the migration ability of ovarian cancer cells.

[0027] Figure 4 The results show the effect of recombinant type III humanized collagen on the invasive ability of ovarian cancer cells.

[0028] Figure 5 The results show the effect of recombinant type III humanized collagen on the in vitro tumorigenicity of ovarian cancer cells.

[0029] Figure 6 The results show the effect of recombinant type III humanized collagen on the tumorigenicity of ovarian cancer cells in the subcutaneous tissue of nude mice. Detailed Implementation

[0030] The following describes embodiments of the present invention, but the present invention is not limited thereto.

[0031] In this invention, the terms "comprising," "having," "including," or "containing" can mean included or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising," "having," "including," or "containing" can also mean closed-ended, excluding additional, uncited elements or method steps.

[0032] In this invention, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both cases in which the event occurs and cases in which the event does not occur.

[0033] In this invention, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0034] In this invention, the terms "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to the described embodiment that are included in at least one of the embodiments described herein, and may or may not be present in other embodiments. Furthermore, it should be understood that the elements may be combined in any suitable manner in various embodiments.

[0035] In this invention, the range of values ​​represented by “value A ~ value B” or “value A - value B” refers to the range that includes the endpoint values ​​A and B.

[0036] In this invention, the terms "a", "an", or "the" may refer to "one", "one or more", "at least one", or "one or more".

[0037] In this invention, the terms "individual," "patient," or "subject" include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0038] In this invention, the term "working concentration" refers to the final concentration of a substance in a working system.

[0039] In this invention, "prevention" means that before the onset of a disease, by exposing the subject to the recombinant type III humanized collagen described in this invention, the probability of developing the disease and / or the symptoms of the disease after onset are reduced compared to when there is no exposure, but does not mean that it is necessary to completely suppress the disease.

[0040] In this invention, "treatment" means, after contracting a disease, exposing (e.g., administering medication) the recombinant type III humanized collagen described in this invention to a subject, thereby alleviating the symptoms of the disease compared to when not exposed, without implying the necessity of completely suppressing the symptoms of the disease. Contracting a disease means that the body exhibits symptoms of a disease.

[0041] In this invention, "recombinant humanized collagen" refers to a full-length or partial amino acid sequence fragment encoded by a specific type of human collagen gene prepared by DNA recombination technology, or a combination containing functional fragments of human collagen. Further, "recombinant type III humanized collagen" refers to a combination containing functional fragments of type III human collagen prepared by DNA recombination technology.

[0042] In this invention, recombinant type III humanized collagen is described in patent application CN201811438582.6, entitled "Polypeptide, its production method and use".

[0043] In some embodiments, the recombinant type III humanized collagen of the present invention comprises n repeats of the sequence shown in SEQ ID No. 1, where n is an integer greater than or equal to 1, wherein when n is an integer greater than or equal to 2, the repeat sequences are directly connected.

[0044] SEQ ID No. 1: GERGAPGFRGPAGPNGIPGEKGPAGERGAP. It is derived from the amino acid sequence of human type III collagen.

[0045] The amino acid sequence of the recombinant type III humanized collagen of the present invention may include the C-terminal sequence GPP GPCCGGG (SEQ ID No. 2). Those skilled in the art typically consider adding this sequence to increase the stability of the expressed peptide when expressing proteins containing multiple repetitive sequences. Preferably, the protein sequence may not include this C-terminal sequence, maintaining sequence consistency with human type III collagen and avoiding the introduction of additional amino acids.

[0046] In some specific embodiments, the recombinant type III humanized collagen of the present invention comprises 16 repeats of the sequence shown in SEQ ID No. 1, and the repeat sequences are directly linked together.

[0047] In some embodiments, the recombinant type III humanized collagen of the present invention comprises the sequence shown in SEQ ID No. 3: GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGA PGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPG EKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGIPPNGGEKGPAGERGAP(SEQ IDNo.3).

[0048] In some embodiments, the recombinant type III humanized collagen of the present invention comprises an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequence of SEQ ID No. 3, and retains the therapeutic effect of the amino acid sequence of SEQ ID No. 3 on ovarian diseases.

[0049] In this invention, amino acid addition refers to adding an amino acid to the C-terminus or N-terminus of an amino acid sequence, such as SEQ ID No. 3, as long as the recombinant type III humanized collagen of this invention retains the activity of the amino acid sequence of SEQ ID No. 3.

[0050] In this invention, amino acid insertion refers to the insertion of amino acid residues at appropriate positions within an amino acid sequence, such as the sequence in SEQ ID No. 3. The inserted amino acid residues may be all or partly adjacent to each other, or none of the inserted amino acids may be adjacent to each other, as long as the recombinant type III humanized collagen of this invention retains the activity of the amino acid sequence in SEQ ID No. 3. In this document, the amino acid insertion positions are not between repeating sequences.

[0051] In this invention, amino acid deletion refers to the deletion of 1, 2 or more amino acids from an amino acid sequence, such as the sequence of SEQ ID No. 3, as long as the recombinant type III humanized collagen of this invention retains the activity of the amino acid sequence of SEQ ID No. 3.

[0052] In this invention, amino acid substitution refers to the replacement of a certain amino acid residue at a certain position in an amino acid sequence, such as the sequence of SEQ ID No. 3, with another amino acid residue, as long as the recombinant type III humanized collagen of this invention retains the activity of the amino acid sequence of SEQ ID No. 3.

[0053] In this invention, substitution can be a conserved amino acid substitution, meaning that compared to the amino acid sequence of SEQ ID No. 3, three, more preferably two, or one amino acid are replaced by amino acids with similar or related properties to form a peptide. These conserved variant peptides can be generated by amino acid substitution according to Table 1.

[0054] Table 1 Conserved Substitutions of Amino Acids

[0055] The initial residues Representative substitution Preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0056] In some embodiments, the recombinant type III humanized collagen of the present invention comprises an amino acid sequence encoded by a nucleotide sequence, the nucleotide sequence hybridizing with the amino acid sequence encoding SEQ ID No. 3 under stringent conditions, the amino acid sequence retaining the ovarian disease treatment effect of the amino acid sequence of SEQ ID No. 3, the stringent conditions being moderately stringent, moderately-highly stringent, highly stringent, or very highly stringent.

[0057] As used herein, the terms “medium stringent conditions,” “medium-high stringent conditions,” “high stringent conditions,” or “very high stringent conditions” describe the conditions for nucleic acid hybridization and washing. For guidance on performing hybridization reactions, see Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1–6.3.6, which is incorporated herein by reference. Aqueous and non-aqueous methods are described in that literature, and either method may be used. For example, specific hybridization conditions are as follows: (1) Low-toughness hybridization conditions: 6× sodium chloride / sodium citrate (SSC) at about 45°C, then at at least 50°C, washed twice in 0.2× SSC, 0.1% SDS (for low-toughness conditions, the washing temperature can be increased to 55°C); (2) Medium-toughness hybridization conditions: 6× SSC at about 45°C, then at 60°C, washed once or more in 0.2× SSC, 0.1% SDS; (3) High-toughness hybridization conditions: 6× SSC at about 45°C, then at 65°C, washed once or more in 0.2× SSC, 0.1% SDS, preferably; (4) Very high-toughness hybridization conditions: 0.5M sodium phosphate, 7% SDS at 65°C, then at 65°C, washed once or more in 0.2× SSC, 1% SDS.

[0058] In some embodiments, the recombinant type III humanized collagen of the present invention comprises an amino acid sequence that is 90% identical to the amino acid sequence of SEQ ID No. 3, and retains the cell adhesion effect of the amino acid sequence of SEQ ID No. 3.

[0059] The "percentage of amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after aligning the candidate sequence with the reference polypeptide sequence, introducing vacancies where necessary to obtain the maximum percentage of sequence identity, and without considering any conserved substitutions as part of the sequence identity. Alignment used to determine the percentage of amino acid sequence identity can be performed in various ways well known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0060] In this invention, recombinant type III humanized collagen can be produced using conventional methods in the art. Exemplarily, it can be produced through the following steps: ① construction of genetically engineered *Escherichia coli*; ② fermentation culture of genetically engineered *Escherichia coli*; ③ induction and expression of recombinant type III humanized collagen; and ④ purification and optional enzymatic digestion of the recombinant collagen.

[0061] Unless otherwise defined, other technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0062] Example

[0063] The present invention is further illustrated by the following embodiments, but any embodiment or combination thereof should not be construed as limiting the scope or implementation of the present invention. The scope of the present invention is defined by the appended claims, and those skilled in the art will clearly understand the scope defined by the claims in conjunction with this specification and common knowledge in the art. Without departing from the spirit and scope of the present invention, those skilled in the art can make any modifications or changes to the technical solutions of the present invention, and such modifications and changes are also included within the scope of the present invention.

[0064] Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments without a specified manufacturer are commercially available, conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. Those skilled in the art should understand that the invention can be practiced even without certain specific details. In other embodiments, methods, means, equipment, and steps well-known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0065] The amino acid sequence of the recombinant type III humanized collagen used in the examples is shown in SEQ ID No. 3. The human ovarian cancer cells used in the examples are A2780 cells.

[0066] Example 1: The ability of recombinant type III humanized collagen to inhibit tumor cell proliferation

[0067] (1) CCK-8 experiment:

[0068] After setting up experimental groups (experimental group: ovarian cancer cells (human ovarian cancer cells A2780 (derived from ATCC)) + 10% FBS medium + recombinant type III humanized collagen; negative control group: ovarian cancer cells (A2780) + 10% FBS medium, without collagen co-culture; blank control group: no cells, 10% FBS medium, without collagen co-culture), cells were seeded at an appropriate density into 96-well plates at a volume of 100 μL / well; each group had 3-5 replicates. 96-well culture plates were placed in a cell culture incubator and pre-cultured at 37°C and 5% CO2 for 6-8 hours. After cell attachment, recombinant type III humanized collagen was added at working concentrations of 0 mg / mL (negative control group), 0.5 mg / mL, 1 mg / mL, 4 mg / mL, and 8 mg / mL. After co-culturing cells and collagen for 24 h, 48 h, and 72 h, the 96-well plates were transferred to a clean bench in the cell culture room. The culture medium was discarded, residual liquid was aspirated, and the plates were protected from light. 100 μL of serum-free culture medium (from Sigma) and 10 μL of CCK-8 (GK10001, GLPBIO) were added to each well, and PBS was added around the perimeter of the 96-well plate to prevent reagent evaporation. The 96-well plates were then incubated at 37°C in the dark for 1 hour, and the absorbance (OD) was measured using a microplate reader. 450 Repeat the measurement three times and take the average value. Calculate the cell viability according to the formula in the kit instructions: Cell viability = (OD value of experimental group - OD value of blank control group) / (OD value of negative control group - OD value of blank control group) × 100%.

[0069] (2) Edu proliferation experiment:

[0070] Ovarian cancer cell culture: A2780 cells in logarithmic growth phase were seeded at approximately 2000 cells per well in 96-well plates and cultured in a cell culture incubator until normal growth was achieved. Cell suspension was then added to each well using a pipette, 100 μL per well. Each group had 3-5 replicates. The 96-well plates were placed in a cell culture incubator and pre-cultured at 37°C and 5% CO2 for 6-8 hours. After cell attachment, the control group received fresh culture medium (from Sigma), while the experimental group received an equal volume of fresh culture medium and recombinant type III humanized collagen (working concentration 8 mg / mL), and incubated for 72 hours. EdU solution (from C0075, Beyotime, China) was diluted 1000:1 in DMEM medium containing 10% fetal bovine serum. 100 μL of the prepared EdU-containing medium was added to each well, and the cells were incubated in a cell culture incubator for 2 hours, then the medium was discarded. Cells were then washed twice with PBS for 5 minutes each time. Cell fixation was performed by adding 100 μL of cell fixative containing 4% (w / v) paraformaldehyde (from P0099, Beyotime, China) to each well and incubating at room temperature for 10 minutes, then the fixative was discarded. 100 μL of 2 mg / mL glycine was added to each well, and the cells were incubated on a shaker for 5 minutes, then the glycine solution was discarded. 100 μL of PBS was added to each well, and the cells were washed on a shaker for 5 minutes to remove the stain, then the PBS was discarded. Add 100 μL of PBS containing 0.5% (v / v) Triton X-100 to each well, then incubate on a shaker for 10 minutes. Wash twice with PBS, 5 minutes each time. Apollo staining: Add 100 μL of Apollo staining reaction solution (from CA1170, Solarbio) to each well, incubate at room temperature in the dark for 30 minutes, then add 100 μL of permeabilizer containing 0.5% (v / v) Triton X-100 (from P0096, Beyotime, China), and wash three times. DNA staining: Dilute Hoechst 33342 DNA staining solution (from C1029, Beyotime, China) with double-distilled water at a ratio of 50:1 to prepare an appropriate amount of 1×Hoechst 33342 DNA staining solution, and store in the dark. Add 200 μL of 1×Hoechst 33342 DNA staining solution to each well, incubate at room temperature in the dark for 20 minutes, and then discard the staining reaction solution. Then, each well was washed three times with 100 μL of PBS. Images were taken under a fluorescence microscope; Hoechst 33342 showed blue fluorescence, with a maximum excitation wavelength of 346 nm and a maximum emission wavelength of 460 nm. Counts were performed, and the results were analyzed.

[0071] Experimental results:

[0072] The results of the CCK-8 experiment are as follows: Figure 1 As shown, the results of the Edu proliferation experiment are as follows: Figure 2 As shown, the results indicated that treatment with recombinant type III humanized collagen for 72 hours significantly inhibited the growth of ovarian cancer cells, and the inhibitory effect increased significantly with increasing collagen concentration. After group culture, compared with the control group, the ovarian cancer cells in the proliferative activity group after treatment with recombinant type III humanized collagen (…) Figure 2 The proportion of Edu red fluorescence decreased significantly, indicating that recombinant type III humanized collagen has an inhibitory effect on DNA replication activity and proliferation of ovarian cancer cells.

[0073] Example 2: The ability of recombinant type III humanized collagen to inhibit tumor migration

[0074] Scratch test:

[0075] First, draw straight lines on the back of a six-well plate using a marker pen. Seed an appropriate amount of A2780 ovarian cancer cells into the six-well plate, ensuring the cells cover the bottom of the plate after attachment. After cell attachment, serum-free culture medium is added to the control group, while an equal volume of recombinant type III humanized collagen (working concentration 8 mg / mL) mixed with serum-free culture medium is added to the experimental group. Once the cells have covered the bottom of the plate, use a 10 μL pipette tip to draw cell scratches perpendicular to the wells, ensuring the scratches are as uniform in width as possible. Pour off the cell culture medium and rinse the wells three times with PBS to remove as much cell debris as possible from the scratches. Simultaneously, photograph the scratches under an inverted microscope and record the width as the scratch width at 0 hours. Place the plate in a cell incubator and photograph again after 24 hours.

[0076] Experimental results:

[0077] Experimental results are as follows Figure 3 As shown, ovarian cancer cells treated with recombinant type III humanized collagen showed significantly reduced cell migration ability compared to the control group that was not treated with collagen.

[0078] Example 3: The ability of recombinant type III humanized collagen to inhibit tumor invasion

[0079] Transwell chamber invasion experiment:

[0080] Before the experiment, ovarian cancer cells A2780 were starved for 24 hours using serum-free medium to eliminate the influence of serum. The cells were digested, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in serum-free medium to adjust the cell concentration to 2 × 10⁶ cells / mL. 4The control group had 500 μL of DMEM medium containing 10% fetal bovine serum added to the lower chamber, while the experimental group had an equal volume of DMEM medium containing 10% fetal bovine serum added to the lower chamber. Recombinant type III humanized collagen (working concentration 8 mg / mL) was then placed in a Transwell chamber (724101, NEST), taking care to avoid air bubbles forming at the bottom of the chamber and the contact surface with the culture medium. 5 × 10⁻⁶ cells / mL were added to each chamber. 4 Cells were cultured in a cell culture incubator for 48 hours. The chamber was removed, and the cells inside were wiped away with a cotton swab, leaving the cells on the outer side. The cells were then fixed with 4% (w / v) paraformaldehyde for 10 minutes. Hematoxylin staining was performed for 10 minutes, followed by rinsing with tap water for 5 minutes to ensure no hematoxylin staining remained. The chamber was dried in an oven, and then the membrane was cut off along the edge of the chamber with a blade. A drop of neutral resin was added to a clean glass slide, with the cell side facing down. A coverslip was then placed on top, and excess resin was gently pressed out. The cells were observed and counted under a microscope. Five fields of view (top, bottom, left, right, and center) were randomly selected under the microscope to count the number of cells.

[0081] Experimental results:

[0082] Experimental results are as follows Figure 4 As shown, after treatment with recombinant type III humanized collagen, the number of invasive tumor cells in ovarian cancer cells was significantly reduced compared with the control group that was not treated with collagen, indicating that the cell invasion ability was significantly weakened.

[0083] Example 4: Recombinant type III humanized collagen inhibits tumor cell tumorigenesis

[0084] Cloning experiments:

[0085] Ovarian cancer cells A2780 in the logarithmic growth phase were collected, digested, and centrifuged according to standard procedures to collect the cell pellet. The cell pellet was resuspended, and cell counts were performed to adjust the cell concentration to 1×10⁻⁶. 5 / mL, serially diluted to 1×10⁻⁶ 3 / mL; take 200μL of cell suspension and seed it into a petri dish (i.e., 200 cells per dish), add culture medium to 10mL, add culture medium to the control group, and add an equal volume of recombinant type III humanized collagen (working concentration of 8mg / mL) mixed with culture medium to the experimental group; shake in a "+" shape to disperse the cells evenly, and incubate in an incubator for 2-3 weeks as usual; stop the culture when visible clones appear and discard the culture medium; wash twice with PBS; fix with 5mL of methanol for 15min, discard the fixative; add Giemsa stain (C0131, Beyotime, China) for 20min, rinse with tap water, air dry, and take pictures.

[0086] Experimental results:

[0087] Experimental results are as follows Figure 5 As shown, ovarian cancer cells treated with recombinant type III humanized collagen showed slower cell growth and a significantly smaller number of clones compared to the control group that was not treated with collagen.

[0088] Example 5: Subcutaneous Tumor Formation Experiment in Nude Mice

[0089] Ovarian cancer cells A2780 (derived from ATCC) in the logarithmic growth phase were digested, centrifuged, and resuspended in PBS. Six nude mice were randomly selected and divided into two groups. Each mouse was subcutaneously injected with 100 μL of the cell suspension (approximately 5 × 10⁻⁶ cells). 6 The control group (A2780-NC or NC) received PBS, while the experimental group (A2780-Col3 or Col3) received subcutaneous injections of recombinant type III humanized collagen (8 mg / ml). Tumor growth in nude mice was observed daily, and the volume of subcutaneous tumors and the weight of the mice were measured. After 28 days, the mice were euthanized by cervical dislocation, and the tumor tissue was removed under aseptic conditions. The longest diameter (b) and shortest diameter (b) of the tumor were measured with calipers, and the tumor volume and mass were also measured. Tumor volume (mm²) was recorded. 3 V = 1 / 2 × a × b 2 .

[0090] Experimental results:

[0091] Experimental results are as follows Figure 6 As shown, in vivo animal experiments indicate that, compared with the control group, recombinant type III humanized collagen can inhibit the tumorigenic ability of tumor cells in vivo.

[0092] in conclusion

[0093] Collagen, as an important component of the extracellular matrix, has a complex structure and function. Current research indicates that collagen plays a role in the tumor microenvironment, and recombinant collagen may achieve anti-cancer effects by remodeling the tumor microenvironment. The research data of this invention demonstrate that recombinant type III humanized collagen inhibits the proliferation, migration, invasion, and tumorigenicity of ovarian cancer cells, with more significant inhibitory effects at high concentrations. Furthermore, in vivo animal experiments show that recombinant type III humanized collagen can inhibit the tumorigenicity of tumor cells in vivo. This will provide new theoretical basis and support for the clinical treatment of ovarian cancer and drug development.

Claims

1. The use of recombinant type III humanized collagen in the preparation of drugs for the treatment of ovarian cancer or for inhibiting the proliferation, migration, invasion, and / or tumorigenesis of ovarian cancer cells; wherein, The amino acid sequence of the recombinant type III humanized collagen is shown in SEQ ID No. 3; the ovarian cancer is an epithelial tumor of the ovary.

Images