CN113577248B - Use of recombinant type III humanized collagen - Google Patents
Use of recombinant type III humanized collagen Download PDFInfo
- Publication number
- CN113577248B CN113577248B CN202110864279.8A CN202110864279A CN113577248B CN 113577248 B CN113577248 B CN 113577248B CN 202110864279 A CN202110864279 A CN 202110864279A CN 113577248 B CN113577248 B CN 113577248B
- Authority
- CN
- China
- Prior art keywords
- type iii
- collagen
- recombinant type
- expression
- humanized
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 102000008186 Collagen Human genes 0.000 title claims abstract description 39
- 108010035532 Collagen Proteins 0.000 title claims abstract description 39
- 229920001436 collagen Polymers 0.000 title claims abstract description 39
- 230000014509 gene expression Effects 0.000 claims abstract description 24
- 210000002744 extracellular matrix Anatomy 0.000 claims abstract description 23
- 210000001519 tissue Anatomy 0.000 claims abstract description 20
- 208000030373 chronic endometritis Diseases 0.000 claims abstract description 17
- 230000002357 endometrial effect Effects 0.000 claims abstract description 12
- 230000002757 inflammatory effect Effects 0.000 claims abstract description 12
- 201000010099 disease Diseases 0.000 claims abstract description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 8
- 239000003814 drug Substances 0.000 claims abstract description 7
- 229940088592 immunologic factor Drugs 0.000 claims abstract description 7
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 claims description 22
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 claims description 22
- 238000011282 treatment Methods 0.000 claims description 17
- 108091081062 Repeated sequence (DNA) Proteins 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 108090001005 Interleukin-6 Proteins 0.000 claims description 3
- 239000003112 inhibitor Substances 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 2
- 230000003247 decreasing effect Effects 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims description 2
- 208000024891 symptom Diseases 0.000 claims description 2
- 102000005741 Metalloproteases Human genes 0.000 claims 2
- 108010006035 Metalloproteases Proteins 0.000 claims 2
- 239000011159 matrix material Substances 0.000 claims 1
- 241000700159 Rattus Species 0.000 abstract description 27
- 210000004696 endometrium Anatomy 0.000 abstract description 16
- 210000004291 uterus Anatomy 0.000 abstract description 16
- 210000004907 gland Anatomy 0.000 abstract description 6
- 239000000367 immunologic factor Substances 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 2
- 210000001161 mammalian embryo Anatomy 0.000 abstract 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 30
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 10
- 210000001015 abdomen Anatomy 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 239000008096 xylene Substances 0.000 description 10
- 108010000684 Matrix Metalloproteinases Proteins 0.000 description 8
- 102000002274 Matrix Metalloproteinases Human genes 0.000 description 8
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 6
- 102100030339 Homeobox protein Hox-A10 Human genes 0.000 description 6
- 101001083164 Homo sapiens Homeobox protein Hox-A10 Proteins 0.000 description 6
- 238000011552 rat model Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 239000012188 paraffin wax Substances 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 206010002091 Anaesthesia Diseases 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 230000037005 anaesthesia Effects 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000002513 implantation Methods 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 241000283707 Capra Species 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 239000008399 tap water Substances 0.000 description 3
- 235000020679 tap water Nutrition 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 102100026802 72 kDa type IV collagenase Human genes 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 208000004145 Endometritis Diseases 0.000 description 2
- 241000282414 Homo sapiens Species 0.000 description 2
- 102000004889 Interleukin-6 Human genes 0.000 description 2
- 102000003992 Peroxidases Human genes 0.000 description 2
- 239000013616 RNA primer Substances 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 108010005246 Tissue Inhibitor of Metalloproteinases Proteins 0.000 description 2
- 102000005876 Tissue Inhibitor of Metalloproteinases Human genes 0.000 description 2
- 206010046793 Uterine inflammation Diseases 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000032692 embryo implantation Effects 0.000 description 2
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 208000000509 infertility Diseases 0.000 description 2
- 230000036512 infertility Effects 0.000 description 2
- 231100000535 infertility Toxicity 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000007634 remodeling Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000012192 staining solution Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 230000008719 thickening Effects 0.000 description 2
- 210000002993 trophoblast Anatomy 0.000 description 2
- 101710151806 72 kDa type IV collagenase Proteins 0.000 description 1
- BEMGNWZECGIJOI-WDSKDSINSA-N Ala-Gly-Glu Chemical compound [H]N[C@@H](C)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O BEMGNWZECGIJOI-WDSKDSINSA-N 0.000 description 1
- AQPVUEJJARLJHB-BQBZGAKWSA-N Arg-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](N)CCCN=C(N)N AQPVUEJJARLJHB-BQBZGAKWSA-N 0.000 description 1
- 235000011330 Armoracia rusticana Nutrition 0.000 description 1
- 240000003291 Armoracia rusticana Species 0.000 description 1
- 206010014733 Endometrial cancer Diseases 0.000 description 1
- 206010014759 Endometrial neoplasm Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- QXPRJQPCFXMCIY-NKWVEPMBSA-N Gly-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN QXPRJQPCFXMCIY-NKWVEPMBSA-N 0.000 description 1
- DHDOADIPGZTAHT-YUMQZZPRSA-N Gly-Glu-Arg Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N DHDOADIPGZTAHT-YUMQZZPRSA-N 0.000 description 1
- FXLVSYVJDPCIHH-STQMWFEESA-N Gly-Phe-Arg Chemical compound [H]NCC(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O FXLVSYVJDPCIHH-STQMWFEESA-N 0.000 description 1
- CAVKXZMMDNOZJU-UHFFFAOYSA-N Gly-Pro-Ala-Gly-Pro Natural products C1CCC(C(O)=O)N1C(=O)CNC(=O)C(C)NC(=O)C1CCCN1C(=O)CN CAVKXZMMDNOZJU-UHFFFAOYSA-N 0.000 description 1
- SCJJPCQUJYPHRZ-BQBZGAKWSA-N Gly-Pro-Asn Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O SCJJPCQUJYPHRZ-BQBZGAKWSA-N 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 108700005087 Homeobox Genes Proteins 0.000 description 1
- 101000627872 Homo sapiens 72 kDa type IV collagenase Proteins 0.000 description 1
- 101000669513 Homo sapiens Metalloproteinase inhibitor 1 Proteins 0.000 description 1
- KCTIFOCXAIUQQK-QXEWZRGKSA-N Ile-Pro-Gly Chemical compound CC[C@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O KCTIFOCXAIUQQK-QXEWZRGKSA-N 0.000 description 1
- 208000012659 Joint disease Diseases 0.000 description 1
- RFQATBGBLDAKGI-VHSXEESVSA-N Lys-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CCCCN)N)C(=O)O RFQATBGBLDAKGI-VHSXEESVSA-N 0.000 description 1
- 102100039364 Metalloproteinase inhibitor 1 Human genes 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 208000007893 Salpingitis Diseases 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 206010000210 abortion Diseases 0.000 description 1
- 231100000176 abortion Toxicity 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 229940030225 antihemorrhagics Drugs 0.000 description 1
- 108010091092 arginyl-glycyl-proline Proteins 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 206010008323 cervicitis Diseases 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 201000003988 chronic cervicitis Diseases 0.000 description 1
- 201000010309 chronic salpingitis Diseases 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 201000002595 endometriosis of ovary Diseases 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000000762 glandular Effects 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 239000002874 hemostatic agent Substances 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 238000013115 immunohistochemical detection Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 210000002570 interstitial cell Anatomy 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 229940127249 oral antibiotic Drugs 0.000 description 1
- 208000030747 ovarian endometriosis Diseases 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000003761 preservation solution Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108010029020 prolylglycine Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 238000012453 sprague-dawley rat model Methods 0.000 description 1
- 210000004231 tunica media Anatomy 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
Abstract
The invention belongs to the field of biological medicine, and relates to application of recombinant type III humanized collagen. In particular, the invention provides the application of the recombinant type III humanized collagen in preparing medicaments for preventing and/or treating uterus-related diseases (especially chronic endometritis). Experiments prove that the recombinant III type humanized collagen can increase the endometrium thickness of rats suffering from chronic endometritis, increase the number of glands, participate in maintaining the dynamic stability of extracellular matrixes, reduce the expression of inflammatory immune factors, reduce the expression of CD138 in endometrium tissues and improve the endometrial receptivity so as to promote embryo planting.
Description
Technical Field
The invention belongs to the field of biological medicine, and relates to application of recombinant type III humanized collagen.
Background
Chronic endometritis (chronic endometritis, CE) is a persistent inflammatory change in endometrium structure caused by various causes, often accompanied by chronic cervicitis, chronic salpingitis. Although chronic endometritis may be asymptomatic, its presence is found in up to 40% of infertility patients, and the disease is one of the causes of repeated implantation failure and repeated abortion, and recent studies have shown that CE prevalence is as high as 57.11% in patients with repeated implantation failure. Although there are many treatments for CE, the differences in outcome are large due to differences in inclusion selection and post-healing criteria for the population. At present, oral antibiotic treatment is commonly used clinically, but a unified operation guideline is not given.
Collagen, one of the main components of extracellular matrix (extracellular matrix, ECM), is the most abundant protein in animals, and it is about 30% of the total protein, and plays an important role in physiological processes such as cell adhesion. Collagen has a large affinity to protein molecules, weak antigenicity, good biocompatibility and biodegradability safety, can form fibers with extremely high strength and stability through self-crosslinking, and is widely applied to the field of medicine as a protein with special biocompatibility and physicochemical properties: including the treatment of myocardial infarction, joint diseases, etc., as surgical sutures, hemostatic agents, cell culture media, vascular prostheses, prosthetic valve materials, etc.
Up to now, most of the collagen used in various studies is derived from animal tissue, skin extracts. Collagen extracted from animals is poorly water-soluble and poorly processable, directly limiting the development of many potential uses. Therefore, development of a safe, effective and good-biological collagen material has been receiving more and more attention in the fields of tissue engineering, medical cosmetology and the like. The application of heterologous and allogenic collagens in human bodies has the risks of high infection rate and high rejection, and the extraction method has the fatal defects of high cost, heavy pollution, limited sources and limited extraction amount. Therefore, the collagen is produced by utilizing the genetic engineering technology, and the defects can be effectively overcome. Patent application number 201811438582.6 entitled "polypeptide, method for its production and use" (CN 109593126A) describes recombinant type III humanized collagen that the inventors of the present invention have previously studied and produced, but does not directly disclose its specific use in the medical field.
Disclosure of Invention
Problems to be solved by the invention
The invention is intended to use the recombinant type III humanized collagen in CN 109593126A as a raw material to evaluate the effectiveness of the collagen for endometrial repair. In particular, the present invention is intended to provide the use of recombinant type III humanized collagen in CN 109593126A for the prevention and/or treatment of uterine related diseases (in particular chronic endometritis) for improving the inflammatory state of the endometrium, further increasing the pregnancy rate in CE patients.
Solution for solving the problem
The invention provides an application of recombinant III type humanized collagen in preparing medicaments for preventing and/or treating uterus-related diseases, wherein the recombinant III type humanized collagen comprises n repeats of a sequence shown in SEQ ID No.1, n is an integer greater than or equal to 1, and when n is an integer greater than or equal to 2, the repeated sequences are directly connected.
Preferably, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 24 or 32, wherein when n is an integer of 2 or more, the repeating sequences are directly linked.
More preferably, n is 16 and the repeat sequences are directly linked.
Further, in the above use, the uterine related disease is metritis.
Preferably, in the above use, the metritis is endometritis.
More preferably, in the above use, the endometritis is chronic endometritis.
Further, in the above use, the chronic endometritis is asymptomatic or has at least one of the following symptoms: endometrial thinning, extracellular matrix changes, increased inflammatory immune factor expression, and decreased endometrial receptivity.
Further, in the above-mentioned uses, the extracellular matrix changes are an increase in metalloproteinase tissue inhibitor 1 (TIMP-1) and/or a decrease in Matrix Metalloproteinases (MMPs).
Further, in the above-mentioned use, the inflammatory immune factor is at least one selected from IL-1b and IL-6.
ADVANTAGEOUS EFFECTS OF INVENTION
The research result of the invention shows that the recombinant III-type humanized collagen can reduce the expression of CD138 in endometrium tissue, promote the generation of endometrium thickening and glandular body, inhibit the expression of related inflammatory immune factors, increase the endometrial receptivity and maintain the extracellular matrix steady state, thereby laying the foundation for the preparation of medicines for treating chronic endometritis and being used for treating chronic endometritis.
Drawings
FIG. 1 shows the results of uterine section analysis of four groups of rat models at different periods.
FIG. 2 shows the results of the detection of extracellular matrix-related gene expression levels in four groups of rat models at different periods.
FIG. 3 shows the results of detecting the expression level of the inflammatory immune related factor gene in four groups of rat models at different periods.
Fig. 4 shows the results of endometrial receptivity analysis for the various periods of the four groups of rat models.
FIG. 5 shows the results of the detection of CD138 expression levels in four groups of rat models at different periods.
Detailed Description
The technical scheme of the invention will be further described below in conjunction with specific examples.
The recombinant III type humanized collagen is described in patent application with the application number of 201811438582.6 and the invention of 'polypeptide, production method and application'.
In one embodiment, the recombinant type III humanized collagen of the present invention comprises N repeats of the sequence shown in SEQ ID No.1 (consisting of 30 amino acids, GERGAPGFRGPAGPNGIPGEKGPAGERGAP in sequence from N-segment to C-segment), N being an integer of 1 or more; wherein, when n is an integer of 2 or more, the repeated sequences are directly connected.
In a preferred embodiment, the recombinant type III humanized collagen of the invention comprises n repeats of the sequence shown in SEQ ID No.1, n being 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 24 or 32; wherein, when n is an integer of 2 or more, the repeated sequences are directly connected.
In a more preferred embodiment, the recombinant type III humanized collagen of the invention comprises 16 repeats of the sequence set forth in SEQ ID No.1, with direct linkage between each repeat.
Unless otherwise indicated, the instruments, reagents, materials, laboratory animals, etc. used in the present invention are all available through conventional commercial means.
Example 1: recombinant type III humanized collagen for promoting morphological restoration of endometrium of CE rat
80 rats (220-260 g) of Sprague Dawley, 6-8 weeks old, were purchased and kept in Chongqing university animal center. After 1 week of feeding, rats were randomly divided into normal control group (n=20, 40 uterus), sham operation group (n=20, 40 uterus), model group (n=20, 40 uterus), treatment group (n=20, 40 uterus).
Normal Control (NC) rats were not treated at all. SHAM group (SHAM) only rats were anesthetized and the abdomen was sterilized, opened, the uterus was exposed to air for 30min, and the abdomen was sutured, and the same procedure was performed after 24h of anesthesia. After the MODEL group (MODEL) rats were anesthetized and the abdomen was disinfected, the abdomen was opened, 150. Mu.l of LPS solution (5 mg/ml) was injected into each of the uterine cavities at both sides from the uterine corners, the abdomen was sutured, and after 24 hours anesthesia and disinfection, 150. Mu.l of sterile PBS solution was injected at the same position, the abdomen was sutured. After anesthesia and abdomen sterilization of the treatment group (COL) rats, the abdomen was opened, 150. Mu.l of LPS solution (5 mg/ml) was injected into each of uterine cavities at both sides from the uterine horn, the abdomen was sutured, after 24 hours, 150. Mu.l of recombinant type III humanized collagen (containing 16 repeated sequences) solution (10 mg/ml) was injected at the same position after anesthesia and sterilization, and the abdomen was sutured. 4 rats were sacrificed in treatment 1d, 4d, 7d, 14d, 28d, respectively, and bilateral uterus was taken from the rats. Rat uterus was stored in RNA preservation solution and paraformaldehyde, respectively, for subsequent experiments.
The uterus of the 0/1/4/7/14/28 day rat was taken and paraffin embedded and sectioned, followed by the following operations:
(1) Dewaxing paraffin sections to water: the slices were sequentially put into xylene I and xylene II, each immersed for 10min. Sequentially adding absolute ethanol I, absolute ethanol II, 90% ethanol, 80% ethanol, and 70% ethanol for 5min, and washing with distilled water for 2-3min.
(2) Hematoxylin-stained nuclei: the slices are placed into hematoxylin for dyeing for 3-5min, and are washed for 2-3min by tap water, differentiated for 3-5s by 1% hydrochloric acid alcohol, and washed for 2-3min by tap water.
(3) Eosin staining: the sections were stained in eosin staining solution for 1-3min.
(4) And (3) removing the water sealing piece: sequentially adding 70% ethanol, 80% ethanol, 90% ethanol, absolute ethanol II, absolute ethanol I, xylene II, and xylene I into the slices for 5min, taking out the slices, air drying, and sealing with neutral resin.
(5) Image acquisition analysis
After staining, each section was photographed under a 100-fold high power microscope, 5 fields were randomly selected, the endometrial thickness and the number of counted glands were measured with Image pro-plus Image processing software, and the average of 5 fields was taken.
As a result, as shown in FIG. 1, the model group had a reduced number of glands due to the thinning of rat endometrium caused by chronic endometritis. After the recombinant III type humanized collagen is infused into the treatment group, the endometrium of the rat becomes thicker gradually, the treatment group tends to be normal after 14 days of treatment, and the number of glands is gradually increased gradually.
Example 2: recombinant type III humanized collagen to promote extracellular matrix remodeling
A rat experimental model was constructed in the same manner as in example 1.
The uterus of the 0/1/4/7/14/28 day rat was taken and treated as follows:
(1) Total RNA extraction
(1) Weighing the tissue, adding 1ml TRIzol into 100mg tissue, grinding the tissue uniformly with a refiner at low temperature, and standing on ice for 3-5min.
(2) 200 μl of chloroform was added, vigorously shaken for 15s, and left on ice for 3-5min.
(3) Centrifuge at 13000rpm/min at 4deg.C for 15min, aspirate supernatant into fresh centrifuge tube, and rest on ice for 10min.
(4) Centrifugation was carried out at 13000rpm/min at 4℃for 10min, white precipitation was seen and the supernatant was discarded.
(5) 1ml of pre-chilled 75% ethanol was added and centrifuged at 7000rpm/min at 4℃for 5min, and the supernatant was discarded.
(6) The super clean bench was air dried for 5min and 25. Mu.l RNase-free water was added.
(2) Reverse transcription of cDNA
(1) The total RNA extracted was assayed, the concentration was determined, and then trimmed to 1. Mu.g/. Mu.l.
(2) Preparing an RNA-primer Mix:
(3) the RNA-primer Mix was centrifuged at 65℃for 10min in a low-speed centrifuge to denature it, cooled and placed on ice.
(4) Preparing cDNA reverse transcription reaction liquid:
(5) incubation at 37℃for 1h and inactivation at 85℃for 5min. Preserving at-20 ℃.
(3) PCR reaction
(1) Preparing a reaction solution:
(2) centrifuging, and setting a PCR reaction detector program:
(3) after the reaction was completed, the dissolution profile was analyzed:
chronic endometritis is thought to cause an increase in the extracellular matrix (ECM) of the rat uterus, while slowing its degradation. Whereas implantation of embryos is mediated by trophoblasts, aggressive trophoblasts encounter large amounts of extracellular matrix during implantation, and excessive extracellular matrix expression in the medium and late endometrium is secreted to prevent blastocyst implantation leading to infertility.
The results of qPCR detection of the gene expression of ECM components in tissues are shown in FIG. 2, and the components of ECM have a tendency to increase between D1 and D4 after treatment of recombinant type III humanized collagen, but gradually decrease after D7, and D28 returns to a near normal level, which indicates that the recombinant type III humanized collagen participates in the dynamic regulation process of extracellular matrix.
TIMP1 and MMP-2 are extracellular matrix related factors. Metalloproteinase tissue inhibitor 1 (TIMP-1) is a tissue inhibitor of matrix metalloproteinases, a glycoprotein commonly expressed in tissues. Matrix Metalloproteinases (MMPs) are a family of polypeptides that degrade the extracellular matrix. High expression of MMPs can promote cell migration and proliferation. The TIMPs family inhibits MMPs family function, regulating the latter to function normally. Together, both maintain extracellular matrix homeostasis. The results in fig. 2 show that MMP2 content is significantly increased in the treated group, which significantly reduced the extracellular matrix. Whereas TIMP-1 was significantly less in the treated group than in the model group as an inhibitor of MMP 2.
Example 3: recombinant type III humanized collagen inhibiting the production of inflammatory factors
A rat experimental model was constructed in the same manner as in example 1.
The uterus of the 0/1/4/7/14/28 day rat was removed and treated as in example 2.
The results of qPCR detection of the levels of immune-related factors IL-1b and IL-6 gene expression in tissues are shown in FIG. 3, and it is found that in the model group, both immune-related factors have an upward trend, while the expression of the related factors in the treatment group is also in an upward trend, but is significantly lower than the relative expression level in the control group compared with the control group, and has a downward trend in the case of D7-D14. Thus, it can be demonstrated that recombinant type III humanized collagen can reduce the expression of inflammatory immune factors, thereby modulating cellular immunity.
Example 4: recombinant type III humanized collagen improving expression of rat endometrium tolerance related factors
A rat experimental model was constructed in the same manner as in example 1.
The uterus of the 0/1/4/7/14/28 day rats was removed and sectioned for paraffin embedding and the following treatments were performed:
(1) Paraffin sections were baked at 60 degrees for 2h.
(2) The slices were sequentially put into xylene I and xylene II, and immersed for 20min each. Then sequentially adding absolute ethanol I, 95% ethanol and 85% ethanol for 5min, and washing with PBS for 3min x3 times.
(3) The tissue slices are soaked in sodium citrate buffer solution, heated to boiling by high fire in a microwave oven, heated for 5min x3 times by medium fire, and rested for 3min each time. And then taking out and standing to room temperature. Then washed 3minx3 times with PBS.
(4) The tissue was wiped clean of surrounding water, 100 μl of endogenous peroxidase blocking agent was added dropwise to cover the tissue, and incubated in a wet box for 10min to fully inactivate endogenous peroxidase. Then washed 3minx3 times with PBS.
(5) The tissue was wiped clean of surrounding water, 100 μl of blocking was applied to cover the tissue with normal goat serum working solution and incubated in a wet box for 15min.
(6) Goat serum working solution was spun dry and 100 μl of HOXA10 antibody dilution (dilution ratio 1:300) was added dropwise to each tissue. Wet boxes were incubated for 4 degrees overnight.
(7) The wet box was allowed to rewet at 37 degrees for 30min. Wash 5minx3 times with PBS. 100 μl or an appropriate amount of biotin-labeled goat anti-mouse/rabbit IgG polymer was added dropwise and incubated for 15min at room temperature. The PBS buffer was washed 5min X3 times.
(8) 100 mu L or a proper amount of horseradish enzyme-labeled streptavidin working solution is dripped, and the mixture is incubated for 15min at room temperature. The PBS buffer was washed 5min X3 times.
(9) Color development: add the appropriate amount of freshly prepared DAB and incubate for 3min at room temperature.
(10) Counterstaining: washing with tap water, and incubating hematoxylin staining solution for 1min. Differentiation of 1% hydrochloric acid alcohol for 2s and flushing to return to blue.
(11) The tissue sections were baked at 60 degrees for 1h. Xylene I and xylene II, each soaked for 20min. Then sequentially adding 85% ethanol, 95% ethanol and absolute ethanol I for 5min, and xylene II and xylene I for 10min, and sealing with neutral resin.
(12) Image acquisition analysis
After staining, each section was photographed under a 200-fold high power microscope, 5 fields were randomly selected, and the average absorbance values of the sections were measured and analyzed using Image pro-plus Image processing software.
HOXA10 is an important member of the homeobox gene family, and is expressed in endometrial gland epithelium and interstitial cells of normal people, so that the functions of encoding transcription factors, promoting cell differentiation and regulating embryo development are achieved. Studies have shown that abnormal expression of HOXA10 is detected in the endometrium of patients with endometrial cancer, ovarian cancer, endometriosis and other gynaecological diseases.
The results of HOXA10 detection are shown in fig. 4, in the model group, rat uterus HOXA10 expression was reduced, while the treatment group HOXA10 showed an upward trend, indicating that recombinant type III humanized collagen can improve endometrial receptivity, promoting embryo implantation.
Example 5: recombinant type III humanized collagen inhibiting CD138 expression
A rat experimental model was constructed in the same manner as in example 1.
The uterus of the 0/1/4/7/14/28 day rats was removed and sectioned for paraffin embedding, which was treated as in example 4, wherein the antibody dilution in step (6) was CD138 antibody dilution (dilution ratio 1:300).
CD138 positive expression is commonly used to diagnose chronic endometritis. The results of the CD138 immunohistochemical detection are shown in fig. 5, and the expression of the rat endometrium CD138 in the model group is increased, and the expression of the rat endometrium CD138 in the treatment group is reduced after the treatment of the recombinant type III humanized collagen.
Summarizing: in a rat model of chronic endometritis constructed by LPS, the recombinant type III humanized collagen can reduce the expression of CD138 in endometrium tissues and promote the thickening of endometrium and the generation of glands damaged after molding. Meanwhile, can inhibit the generation of inflammatory factors and increase the endometrial receptivity. Its effect may be related to remodeling of extracellular matrix components.
Sequence listing
<110> Shanxi brocade biological medicine Co., ltd
Auxiliary second hospital for Chongqing medical university
<120> use of recombinant type III humanized collagen
<130> 6C39-2163547I
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> PRT
<213> Homo sapiens
<400> 1
Gly Glu Arg Gly Ala Pro Gly Phe Arg Gly Pro Ala Gly Pro Asn Gly
1 5 10 15
Ile Pro Gly Glu Lys Gly Pro Ala Gly Glu Arg Gly Ala Pro
20 25 30
Claims (5)
1. Use of recombinant type III humanized collagen comprising n repeats of the sequence shown in SEQ ID No.1 in the manufacture of a medicament for the prevention and/or treatment of a uterine related disease; n is 16, and each repeated sequence is directly connected;
and the uterine related disease is chronic endometritis.
2. The use according to claim 1, wherein the chronic endometritis is asymptomatic.
3. The use according to claim 1, wherein the chronic endometritis has at least one of the following symptoms: endometrial thinning, extracellular matrix changes, increased inflammatory immune factor expression, and decreased endometrial receptivity.
4. The use according to claim 3, wherein the extracellular matrix change is an increase in metalloprotease tissue inhibitor 1 and/or a decrease in matrix metalloprotease.
5. The use according to claim 3, wherein the inflammatory immune factor is at least one selected from the group consisting of IL-1b and IL-6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110864279.8A CN113577248B (en) | 2021-07-29 | 2021-07-29 | Use of recombinant type III humanized collagen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110864279.8A CN113577248B (en) | 2021-07-29 | 2021-07-29 | Use of recombinant type III humanized collagen |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113577248A CN113577248A (en) | 2021-11-02 |
| CN113577248B true CN113577248B (en) | 2024-02-02 |
Family
ID=78251939
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110864279.8A Active CN113577248B (en) | 2021-07-29 | 2021-07-29 | Use of recombinant type III humanized collagen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113577248B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114671946B (en) * | 2021-12-24 | 2022-11-01 | 河北纳科生物科技有限公司 | Recombinant human III-type collagen and preparation method and application thereof |
| CN114920827B (en) * | 2022-06-29 | 2023-06-09 | 山西锦波生物医药股份有限公司 | Polypeptides and uses thereof |
| CN115581762A (en) * | 2022-10-31 | 2023-01-10 | 湖南中禧医疗科技有限公司 | Medical bioprotein repair gel for clearing HPV virus infection and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104127258A (en) * | 2014-08-11 | 2014-11-05 | 青岛农业大学 | Mouse uterus perfusion apparatus and method for establishing mouse endometritis model |
| CN104548071A (en) * | 2015-01-27 | 2015-04-29 | 太原锦波生物医药科技有限公司 | Recombinant human-derived collagen vagina gel for vaginal dryness and preparation method thereof |
| CN105988010A (en) * | 2015-03-03 | 2016-10-05 | 南京鼓楼医院 | Method for detecting endometrial receptivity with phosphorylated HOXA10 |
| CN109575126A (en) * | 2018-07-06 | 2019-04-05 | 山西锦波生物医药股份有限公司 | Polypeptide, its production method and purposes |
-
2021
- 2021-07-29 CN CN202110864279.8A patent/CN113577248B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104127258A (en) * | 2014-08-11 | 2014-11-05 | 青岛农业大学 | Mouse uterus perfusion apparatus and method for establishing mouse endometritis model |
| CN104548071A (en) * | 2015-01-27 | 2015-04-29 | 太原锦波生物医药科技有限公司 | Recombinant human-derived collagen vagina gel for vaginal dryness and preparation method thereof |
| CN105988010A (en) * | 2015-03-03 | 2016-10-05 | 南京鼓楼医院 | Method for detecting endometrial receptivity with phosphorylated HOXA10 |
| CN109575126A (en) * | 2018-07-06 | 2019-04-05 | 山西锦波生物医药股份有限公司 | Polypeptide, its production method and purposes |
Non-Patent Citations (2)
| Title |
|---|
| Intravaginal Administration of Human Type III Collagen-Derived Biomaterial with High Cell-Adhesion Activity to Treat Vaginal Atrophy in Rats;Shuang You et al;《American Chemical Society》;第第6卷卷(第第4期期);第1977-1988页 * |
| 慢性子宫内膜炎中MMP-2和TIMP-2表达的研究;孔庆亮;孙少华;;中国妇幼健康研究(03);第25-27页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113577248A (en) | 2021-11-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113577248B (en) | Use of recombinant type III humanized collagen | |
| Gualillo et al. | Elevated serum leptin concentrations induced by experimental acute inflammation | |
| Woessner et al. | Formation and breakdown of collagen and elastin in the human uterus during pregnancy and post-partum involution | |
| Ghellai et al. | Role of transforming growth factor beta-l in peritonitis-induced adhesions | |
| AU647120B2 (en) | Use of protease nexin-I to mediate wound healing | |
| US20030203008A1 (en) | Preparation of collagen | |
| JP2001519786A (en) | Use of a substance having oxytocin activity for the manufacture of a medicament for wound healing | |
| WO2014152234A1 (en) | Improved method of making extracellular matrix compositions | |
| US5196196A (en) | Use of protease nexin-I in wound dressings | |
| US5155038A (en) | Use of thrombospondin to promote wound healing | |
| Ngamkitidechakul et al. | Maspin: synthesis by human cornea and regulation of in vitro stromal cell adhesion to extracellular matrix | |
| CN111298104A (en) | Application of exenatide injection in medicine for treating and preventing intrauterine adhesion | |
| Gallorini et al. | Serum markers of hepatic fibrogenesis in chronic hepatitis type C treated with alfa‐2A interferon | |
| US5202118A (en) | Method for promoting wound healing using IL-1 | |
| KR20030035047A (en) | Use of BMP-4 gene and its gene product for treatment and diagnosis of Lichen Planus | |
| AU2013353957B2 (en) | Protein slurp-1 for use in the treatment of ocular diseases | |
| CN116474178A (en) | Recombinant human collagen peptide lubricant and preparation method thereof | |
| Peacock | Collagenolysis: The other side of the equation | |
| Ji et al. | Comparing the characteristics of amniotic membrane-, endometrium-, and urinary-derived ECMs and their effects on endometrial regeneration in a rat uterine injury model | |
| CN105396136B (en) | CCN1（Cyr61）Application in treatment skin injury and atrophoderma relevant disease | |
| Manthorpe et al. | Long-term effect of glucocorticoid on connective tissue of aorta and skin Morphological and biochemical studies of tissues from rabbits with intact and injured aortas | |
| Chen et al. | Protective effect of sulodexide on podocyte injury in adriamycin nephropathy rats | |
| CN115337260B (en) | Composite acellular scaffold/hyaluronic acid temperature-sensitive hydrogel and application thereof | |
| CN114558115B (en) | Use of ELABELA in improving adipose-derived stem cell survival and migration | |
| Ali et al. | Wound healing effect of a one-week Aloe Vera mouthwash: a pilot study |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |