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

Abstract

The present application belongs to the field of biological medicine, and particularly relates to the use of recombinant humanized collagen type XVII in the treatment of cervical cancer, and more particularly to the use of recombinant humanized collagen type XVII in the preparation of a drug for preventing and / or treating cervical diseases; the recombinant humanized collagen type XVII comprises or consists of a sequence represented by (A)m, wherein each A is selected from the amino acid sequence represented by any one of SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3; m is an integer between 1 and 10; each A is the same or different, and two adjacent As are directly connected by a peptide bond or connected by one or more amino acid residues. Research data shows that the recombinant humanized collagen type XVII can significantly inhibit the proliferation, migration, invasion and tumorigenicity of cervical cancer cells, and the inhibitory effect is dose-dependent.

Description

[0001] Priority and Related Applications

[0002] This application claims priority to the Chinese patent application No. 202310961277.X, filed on August 1, 2023, entitled “Use of Recombinant Humanized Collagen XVII in Treatment of Cervical Cancer”, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application belongs to the field of biological medicine, and specifically relates to the use of recombinant humanized collagen XVII in the treatment of cervical cancer. BACKGROUND

[0004] Cervical cancer is the fourth most common cancer in women. In 2018, an estimated 57 million women were diagnosed with cervical cancer worldwide, of which about 311,000 women died. Cervical cancer occurs in the cervix of women (the entrance from the vagina into the uterus). Almost all cases of cervical cancer (99%) are associated with high-risk human papillomavirus (HPV) infection, which is an extremely common virus transmitted through sexual contact. Although most human papillomavirus infections will resolve spontaneously without causing any symptoms, persistent infection can lead to cervical cancer. The current methods that can effectively prevent most cases of cervical cancer mainly include primary prevention (HPV vaccination) and secondary prevention methods (screening and treatment of precancerous lesions).

[0005] Collagen is the most abundant protein in the animal body, accounting for about 30% of total protein, and plays an important role in physiological processes such as cell adhesion. Due to its good biocompatibility and biodegradable safety, collagen has been widely used in the medical field. Until now, the collagen used by various studies is mostly from animal tissues, skin extracts. However, collagen extracted from animals has poor water solubility and weak processability, which directly limits the development of many potential uses of collagen. Collagen produced by genetic engineering technology can effectively overcome the above shortcomings.

[0006] The Chinese patent application No. 201911051106.3, entitled “Human collagen type 17 polypeptide, method for producing same, and use thereof”, discloses the recombinant humanized collagen XVII produced by the present inventors in previous research, which has the characteristics of high water solubility and high biological activity. There is no report so far to suggest the connection between the recombinant humanized collagen XVII and the treatment of cervical cancer. SUMMARY

[0007] Problem to be solved by the invention

[0008] The present application intends to use the recombinant humanized collagen type XVII in the Chinese patent application No. 201911051106.3 as a raw material to evaluate its effectiveness in the treatment of cervical diseases such as cervical cancer. Specifically, the present application intends to use the recombinant humanized collagen type XVII to inhibit the proliferation, migration, invasion and tumorigenic ability of cervical cancer cells, so as to achieve the effect of preventing and / or treating cervical cancer.

[0009] Solution for solving the problem

[0010] The present application provides the use of the recombinant humanized collagen type XVII in the preparation of a drug for preventing and / or treating cervical diseases; wherein,

[0011] The recombinant humanized collagen type XVII comprises or consists of a sequence represented by (A)m, wherein each A is selected from the amino acid sequence represented by any one of SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, or an amino acid sequence in which 1 or more amino acid residues are substituted, added, inserted or deleted from any one of SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, or a sequence having at least 83% sequence identity with the amino acid sequence represented by any one of SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3; m is an integer between 1 and 10; wherein each A is the same or different and adjacent two As are directly connected by a peptide bond or connected by 1 or more amino acid residues.

[0012] Further, the recombinant humanized collagen type XVII comprises or consists of the amino acid sequence represented by SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 or SEQ ID No. 5.

[0013] Further, the cervical disease includes cervical cancer.

[0014] Further, according to the pathological typing of cervical cancer, the cervical cancer includes squamous carcinoma, adenocarcinoma and adenosquamous carcinoma.

[0015] Further, the recombinant humanized collagen type XVII prevents and / or treats cervical cancer by inhibiting the proliferation ability of cervical cancer cells, inhibiting the migration ability of cervical cancer cells, inhibiting the invasion ability of cervical cancer cells and / or inhibiting the tumorigenic ability of cervical cancer cells.

[0016] Preferably, the recombinant type XVII humanized collagen prevents and / or treats cervical cancer by simultaneously inhibiting the proliferation, migration, invasion, and tumorigenesis of cervical cancer cells.

[0017] Furthermore, the ability to inhibit the proliferation of cervical cancer cells includes inhibiting the DNA replication activity of cervical cancer cells.

[0018] Furthermore, the ability to inhibit cervical cancer cell tumorigenesis includes the ability to inhibit the formation of cervical cancer cell clones.

[0019] Furthermore, the ability to inhibit the formation of cervical cancer cell clones includes inhibiting the number of cervical cancer cell clones and / or inhibiting the growth rate of cervical cancer cells.

[0020] Effects of the invention

[0021] This invention provides the use of recombinant type XVII humanized collagen in the preparation of drugs for the prevention and / or treatment of cervical diseases. Research data from this invention show that recombinant type XVII humanized collagen can significantly inhibit the proliferation, migration, invasion, and tumorigenesis of cervical cancer cells, and this inhibitory effect is dose-dependent, thus providing a new approach for the preparation of drugs for the prevention and / or treatment of cervical cancer and other cervical diseases. Furthermore, since the collagen provided by this invention is recombinant type XVII humanized collagen, it has the advantages of convenient preparation and high safety, thereby contributing to the preparation and application of drugs for cervical cancer and the clinical treatment of cervical cancer. Attached Figure Description

[0022] Figure 1 This is a statistical result showing the effect of different concentrations of recombinant type XVII humanized collagen on the survival ability of cervical cancer cells at different time points.

[0023] Figure 2 The results show the effect of recombinant type XVII humanized collagen on the proliferation ability of cervical cancer cells.

[0024] Figure 3 The results show the effect of recombinant type XVII humanized collagen on the migration ability of cervical cancer cells.

[0025] Figure 4 The results show the effect of recombinant type XVII humanized collagen on the invasive ability of cervical cancer cells.

[0026] Figure 5 The results show the effect of recombinant type XVII humanized collagen on the tumorigenicity of cervical cancer cells.

[0027] Figure 6 This refers to C33A tumor-bearing mice with cervical cancer cells treated with recombinant type XVII humanized collagen and saline.

[0028] Figure 7 The results of tumor observation in mice bearing cervical cancer cells C33A under treatment with recombinant type XVII humanized collagen and saline.

[0029] Figure 8 The results show the tumor volume of C33A cervical cancer cells in mice treated with recombinant type XVII humanized collagen and saline. Detailed Implementation

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

[0031] In this invention, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[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 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.

[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, the term "treatment" means, after a person has contracted a disease, exposing (e.g., administering medication) the recombinant type XVII humanized collagen described in this invention to reduce 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 the appearance of disease symptoms in the body.

[0040] In this invention, the term "prevention" means that, before the onset of a disease, subjecting the subject to the recombinant type XVII humanized collagen described in this invention reduces the probability of developing the disease and / or alleviates the symptoms of the disease compared to when no exposure is made, without implying the necessity of completely suppressing the disease.

[0041] In this invention, the recombinant type XVII humanized collagen is described in the patent application number 201911051106.3, entitled "Human Collagen Type 17 Polypeptide, Its Production Method and Use".

[0042] In some embodiments, the recombinant type XVII humanized collagen of the present invention comprises or is composed of the sequence shown in (A)m, wherein each A is an amino acid sequence selected from any one of SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 or an amino acid sequence of any one of SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 with one or more amino acid residues substituted, added or deleted, or a sequence having at least 83% sequence identity with any one of SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3; m is an integer between 1 and 10; wherein each A is the same or different and adjacent A are directly linked by a peptide bond or by one or more amino acid residues.

[0043] The SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 described in this invention are derived from the human type 17 collagen COL17A1 sequence, which can be found at https: / / www.ncbi.nlm.nih.gov / protein / Q9UMD9.3.

[0044] SEQ ID No. 1: GSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQG PKGQKGSVGDPGMEGPMGQRGREGPMGPRGEA.

[0045] SEQ ID No. 2: GLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPA.

[0046] SEQ ID No. 3: GADFAGDLDYNELAVRVSESMQRQGLLQGMAYTVQ GPPGQPGPQGPPGISKVFSAYSNVTADLMDFFQTYGAIQGPPGQKGEMGT PGPKGDRGPAGPPGHPGPPGPRGHKGEKGDKGDQ.

[0047] In some specific embodiments, the recombinant type XVII humanized collagen of the present invention comprises or is composed of sequences as shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3.

[0048] In other specific embodiments, the recombinant type XVII humanized collagen of the present invention comprises or consists of the sequence shown in SEQ ID No. 4, which is repeated three times with SEQ ID No. 1 as the repeating unit.

[0049] SEQ ID No.4: GSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQG PKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEA.

[0050] In some other preferred embodiments, the recombinant type XVII humanized collagen of the present invention comprises or consists of the sequence shown in SEQ ID No. 5, which is repeated three times with SEQ ID No. 2 as the repeating unit.

[0051] SEQ ID No.5: GLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGP RGLTGEPGMRGLPGAVGEPGAKGAMGPAGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPA.

[0052] 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.

[0053] Amino acid addition refers to the addition of an amino acid to the C-terminus or N-terminus of an amino acid sequence, such as SEQ ID No. 1, provided that the recombinant type XVII humanized collagen has the activity of the amino acid sequence of SEQ ID No. 1.

[0054] Amino acid insertion refers to the insertion of an amino acid residue at an appropriate position in an amino acid sequence, such as SEQ ID No. 1. 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 XVII humanized collagen of the present invention retains the activity of the amino acid sequence of SEQ ID No. 1. Preferably, the insertion position of the amino acid is not between repeating sequences.

[0055] Amino acid deletion refers to the deletion of one, two, or three or more amino acids from an amino acid sequence, such as the sequence of SEQ ID No. 1, as long as the recombinant type XVII humanized collagen of the present invention retains the activity of the amino acid sequence of SEQ ID No. 1.

[0056] Amino acid substitution refers to the replacement of an amino acid residue at a certain position in an amino acid sequence, such as the sequence of SEQ ID No. 1, with another amino acid residue, as long as the recombinant type XVII humanized collagen of the present invention retains the activity of the amino acid sequence of SEQ ID No. 1.

[0057] In this invention, substitution can be a conserved amino acid substitution, for example, three, more preferably two, or one amino acid is replaced by an amino acid with similar or analogous properties to form a peptide compared to the amino acid sequence of SEQ ID No. 1. These conserved variant peptides can be generated by amino acid substitution according to Table 1.

[0058] Table 1 Conserved Substitutions of Amino Acids

[0059] Original residues Representative substitutions Preferred substitutions Ala (A) Val; Leu; lie Val Arg (R) Lys; Gin; Asn Lys Asn (N) Gin; His; Lys; Arg Gin Asp (D) Glu Glu Cys (C) Ser Ser Gin (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala Ala His (H) Asn; Gin; Lys; Arg Arg lie (I) Leu; Val; Met; Ala; Phe Leu Leu (L) lie; Val; Met; Ala; Phe lie Lys (K) Arg; Gin; Asn Arg Met (M) Leu; Phe; lie Leu Phe (F) Leu; Val; lie; 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) lie; Leu; Met; Phe; Ala Leu

[0060] All amino acids in the amino acid sequence of this invention can be L-type amino acids. One or more of these amino acids (e.g., 2-5, 2-4, or 2-3) can be replaced with D-type amino acids, artificially modified amino acids, or naturally occurring rare amino acids to improve the bioavailability, stability, and / or antiviral activity of the peptide. D-type amino acids refer to amino acids corresponding to the L-type amino acids that make up proteins; artificially modified amino acids refer to common L-type amino acids that make up proteins after modification such as methylation or phosphorylation; naturally occurring rare amino acids include uncommon amino acids that make up proteins and amino acids that do not make up proteins, such as 5-hydroxylysine, methylhistidine, γ-aminobutyric acid, and homoserine.

[0061] In this invention, recombinant type XVII 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 XVII humanized collagen; and ④ purification and optional enzymatic digestion of the recombinant humanized collagen.

[0062] 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.

[0063] Example

[0064] 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.

[0065] 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.

[0066] The amino acid sequence of the recombinant type XVII humanized collagen used in the examples is shown in SEQ ID No. 1.

[0067] Example 1: Effects of recombinant type XVII humanized collagen on the growth and proliferation of cervical cancer cells

[0068] (1) CCK-8 experiment:

[0069] After setting up experimental groups (experimental group: cervical cancer cells (human cervical squamous cell carcinoma cells Siha) + 10% FBS medium + recombinant type XVII humanized collagen; negative control group: cervical cancer cells (Siha) + 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 humanized type XVII collagen was added to the experimental groups 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 LCK-8 (from GK10001, GLPBIO) were added to each well, and PBS was added around the perimeter of the 96-well plates 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%.

[0070] (2) Edu proliferation experiment:

[0071] Cervical cancer cell culture: Siha 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 humanized collagen type XVII (working concentration 8 mg / mL), and incubated for 72 hours. Dilute EdU solution (from C0075, Beyotime) in DMEM medium containing 10% fetal bovine serum at a ratio of 1000:1. Add 100 μL of the prepared EdU-containing medium to each well and incubate in a cell culture incubator for 2 hours, then discard the medium. Wash cells twice with PBS for 5 minutes each time. Fix cells: Add 100 μL of cell fixative containing 4% (w / v) paraformaldehyde (from P0099, Beyotime) to each well and incubate at room temperature for 10 minutes, then discard the fixative. Add 100 μL of 2 mg / mL glycine to each well and incubate on a shaker for 5 minutes, then discard the glycine solution. Add 100 μL of PBS to each well and wash on a shaker for 5 minutes to decolorize, then discard the PBS. 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), and wash three times. DNA staining: Dilute the DNA concentration staining solution (from C1029, Beyotime) with double-distilled water at a ratio of 50:1 according to the kit instructions to prepare an appropriate amount of 1×Hoechst33342 DNA staining solution, and store in the dark. Add 200 μL of 1×Hoechst33342 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 exhibited blue fluorescence, with a maximum excitation wavelength of 346 nm and a maximum emission wavelength of 460 nm. The cells were counted, and the results were analyzed.

[0072] Experimental results:

[0073] 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 CCK-8 and Edu proliferation experiments indicated that treatment of cervical cancer cells with recombinant type XVII humanized collagen for 72 hours significantly inhibited tumor cell growth, and the inhibitory effect increased significantly with increasing collagen concentration. After group culture, compared with the control group, the number of cervical cancer cells in proliferative activity after treatment with recombinant type XVII humanized collagen (…) Figure 2 The proportion of Edu red fluorescent cells decreased significantly, indicating that recombinant type XVII humanized collagen has an inhibitory effect on the DNA replication activity and proliferation of cervical cancer cells.

[0074] Example 2: Effect of recombinant type XVII humanized collagen on the migration ability of cervical cancer cells (scratch assay):

[0075] First, draw straight lines on the back of a six-well plate using a marker pen. Seed an appropriate amount of cervical cancer cells (Siha) into the six-well plate, ensuring the cells cover the bottom of the plate after adhesion. After cell adhesion, serum-free culture medium is added to the control group, while an equal volume of recombinant humanized collagen type XVII (working concentration 8 mg / mL) mixed with serum-free culture medium is added to the experimental group. Once the cells cover 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 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, cervical cancer cells treated with recombinant type XVII humanized collagen showed significantly reduced cell migration ability compared to the control group that was not treated with collagen.

[0078] Example 3: Effect of recombinant type XVII humanized collagen on the invasive ability of cervical cancer cells

[0079] Transwell chamber invasion experiment:

[0080] Before the experiment, cervical cancer cells (Siha) 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 XVII 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 cervical cancer cells were treated with recombinant type XVII humanized collagen, the invasive ability of tumor cells in the experimental group was significantly reduced compared with that in the control group without collagen treatment.

[0083] Example 4: Effect of recombinant type XVII humanized collagen on the tumorigenicity of cervical cancer cells (clonal formation experiment):

[0084] Siha cervical cancer cells 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 XVII 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, and discard the fixative; add Giemsa stain (C0131, Beyotime) for 20min, rinse with tap water, air dry, and take pictures.

[0085] Experimental results:

[0086] Experimental results are as follows Figure 5As shown, cervical cancer cells treated with recombinant type XVII humanized collagen showed slower cell growth and a significantly smaller number of clones compared to the control group that was not treated with collagen.

[0087] 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 humanized collagen may achieve anti-cancer effects by remodeling the tumor microenvironment. This invention demonstrates that recombinant type XVII humanized collagen inhibits the proliferation, migration, invasion, and tumorigenicity of cervical cancer cells, with more significant inhibitory effects at high concentrations. This will provide new theoretical basis and support for clinical treatment of cervical cancer and drug development.

[0088] Example 5: Effect of recombinant type XVII humanized collagen on the tumorigenicity of cervical cancer cells: Subcutaneous tumorigenicity in nude mice:

[0089] (1) Preparation: Prepare tumor cells (cervical cancer cells C33A) that are in the logarithmic growth phase and have a cell density of about 80-90%.

[0090] (2) Cell Collection: Discard the old culture medium, wash the cells 1-2 times with PBS (to remove serum from the cells), digest the cells with trypsin to prepare a cell suspension, centrifuge, discard the supernatant, then resuspend the cells in PBS or serum-free culture medium and count the cells to calculate the required final concentration. Typically, for subcutaneous tumor inoculation, the cell quantity used is (1-5) × 10⁶ cells per tumor. 6 With 10 cells and an inoculation volume of 0.1 mL, the concentration of cervical cancer cell C33A is approximately (1-5) × 10⁻⁶. 7 Cells / mL.

[0091] (3) Nude mouse inoculation: Ten female nude mice aged 4-6 weeks were used and ear-tagged. After 3 days of SPF-controlled rearing, the mice were restrained with the left hand, and a subcutaneous injection was performed in the groin area of ​​the left thigh. During injection, the needle was inserted approximately 1 cm subcutaneously, with an injection volume of 100 μL. The cell suspension should be placed on ice during inoculation to slow down cell apoptosis. The weight and tumor volume (length × width) of the nude mice were measured weekly. 2 ÷2).

[0092] (4) Drug administration: 3-5 days after tumor cell inoculation, the subcutaneous tumor volume of each nude mouse was examined and randomly divided into two groups: a control group (NC) of 5 mice and an experimental group (rhCOL17) of 5 mice (biological replicates). Each mouse in the control group received an intraperitoneal injection of 100 μl of physiological saline, while each mouse in the experimental group received an intraperitoneal injection of 100 μl of recombinant type XVII humanized collagen (concentration 8 mg / ml) dissolved in physiological saline. The drug was administered every 3 days for a total of 7 injections. During this period, the weight and tumor volume (length × width) of the nude mice were regularly monitored. 2 ÷2).

[0093] (5) Eradication: According to the experimental design, tumors with a volume not exceeding 1000 mm² were sacrificed. 3 Previously, nude mice needed to be euthanized, and the tumors were removed and photographed approximately 21 days later.

[0094] Experimental results:

[0095] Experimental results are as follows Figures 6 to 8 As shown, cervical cancer cells C33A can form tumors in nude mice. After intraperitoneal injection of recombinant type XVII humanized collagen, the tumor volume was significantly smaller compared with the control group injected with physiological saline. This demonstrates that recombinant type XVII humanized collagen has a significant inhibitory effect on the tumorigenicity of cervical cancer cells.

Claims

1. The use of recombinant type XVII humanized collagen in the preparation of drugs for the treatment of cervical cancer; wherein, The recombinant type XVII humanized collagen consists of the amino acid sequence shown in SEQ ID No. 1; the cervical cancer is cervical squamous cell carcinoma.

2. The use according to claim 1, characterized in that, The recombinant type XVII humanized collagen treats cervical cancer by inhibiting the proliferation, migration, invasion, and / or tumorigenesis of cervical cancer cells.

3. The use according to claim 2, characterized in that, The ability to inhibit the proliferation of cervical cancer cells includes inhibiting the DNA replication activity of cervical cancer cells.

4. The use according to claim 2, characterized in that, The ability to inhibit cervical cancer cell tumorigenesis includes the ability to inhibit the formation of cervical cancer cell clones.

5. The use according to claim 4, characterized in that, The ability to inhibit the formation of cervical cancer cell clones includes inhibiting the number of cervical cancer cell clones and / or inhibiting the growth rate of cervical cancer cells.

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