阅读说明：本技术 重组iii型人源化胶原蛋白的用途 (Use of recombinant type III humanized collagen ) 是由 杨霞 兰小宾 何振瑞 王建 王玲玲 张玢玢 闫欣 胡丽娜 游爽 于 2021-07-29 设计创作，主要内容包括：本发明属于生物医药领域,涉及重组III型人源化胶原蛋白的用途。具体而言,本发明提供了该种重组III型人源化胶原蛋白在制备用于预防和/或治疗子宫相关疾病(尤其是慢性子宫内膜炎)的药物中的用途。本发明在实验中证明重组III型人源化胶原蛋白可以增大慢性子宫内膜炎患病大鼠的子宫内膜厚度并使其腺体数量增多、参与维持细胞外基质的动态稳定、降低炎症免疫因子的表达、减少内膜组织中CD138的表达、改善子宫内膜容受性从而促进胚胎种植。(The invention belongs to the field of biomedicine, and relates to application of recombinant III-type humanized collagen. Specifically, the invention provides application of the recombinant type III humanized collagen in preparing a medicament for preventing and/or treating uterus related diseases (especially chronic endometritis). The invention proves that the recombinant III-type humanized collagen can increase the thickness of endometrium and the number of glands of a rat with chronic endometritis, participate in maintaining the dynamic stability of extracellular matrix, reduce the expression of inflammatory immune factors, reduce the expression of CD138 in endometrial tissue, improve the receptivity of endometrium and promote embryo planting in experiments.)

1. Use of a recombinant type III humanized collagen comprising n repeats of a sequence represented by SEQ ID No.1, n being an integer of 1 or more, wherein when n is an integer of 2 or more, direct linkage between the respective repeated sequences, in the manufacture of a medicament for the prevention and/or treatment of uterine related diseases.

2. The use according to claim 1, wherein 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.

3. Use according to claim 1 or 2, wherein n is 16 and the repeats are directly linked.

4. Use according to any one of claims 1 to 3, wherein the uterus-related disease is metritis.

5. The use according to claim 4, wherein the metritis is endometritis.

6. Use according to claim 5, wherein the endometritis is chronic endometritis.

7. Use according to claim 6, wherein the chronic endometritis is asymptomatic.

8. Use according to claim 6, characterized in that the chronic endometritis has at least one of the following symptoms: endometrial thinning, uterine extracellular matrix changes, increased expression of inflammatory immune factors, and decreased endometrial receptivity.

9. Use according to claim 8, characterized in that the uterine extracellular matrix change is an increase in tissue inhibitor of metalloproteinases factor 1 and/or a decrease in matrix metalloproteinases.

10. The use according to claim 8, wherein the inflammatory immune factor is selected from at least one of IL-1b and IL-6.

Technical Field

The invention belongs to the field of biomedicine, and relates to application of recombinant III-type humanized collagen.

Background

Chronic Endometritis (CE) is a persistent inflammatory change in endometrial structure caused by various causes, often accompanied by chronic cervicitis and chronic salpingitis. Although chronic endometritis may be asymptomatic, its presence is found in up to 40% of patients with infertility and the disease is one of the causes of repeated implantation failure and repeated miscarriage, studies in recent years have shown that CE prevalence rates in patients with repeated transplantation failure are as high as 57.11%. Although there are many treatments for CE, the results vary widely due to population inclusion choices and post-healing criteria. Currently, oral antibiotic therapy is commonly used in clinic, but no unified operating instruction is given.

Collagen, one of the major components of extracellular matrix (ECM), is the most abundant protein in the animal body, accounts for about 30% of the total protein, and plays an important role in physiological processes such as cell adhesion. Collagen has a relatively high affinity, relatively weak antigenicity, good biocompatibility and biodegradation safety to protein molecules, and can form fibers with extremely high strength and stability through self-crosslinking, and the collagen serving as a protein with special biocompatibility and physicochemical properties has been widely applied to the field of medicine: including treatment of myocardial infarction, joint diseases, etc., and as surgical sutures, hemostats, cell culture media, artificial blood vessels, artificial valves, etc.

Until now, most of the collagen used in various studies is derived from animal tissues and skin extracts. Extraction of collagen from animals has poor water solubility and poor processability, directly limiting the development of many potential uses. Therefore, developing a safe, effective and biologically functional collagen material is receiving more and more extensive attention in the fields of tissue engineering, medical cosmetology, etc. The application of xenogeneic and xenogeneic collagen in human body has high infection rate, high rejection risk, and the extraction method has the fatal defects of high cost, heavy pollution, limited sources and limited extraction amount. Therefore, the collagen protein is produced by utilizing the genetic engineering technology, and the defects can be effectively overcome. Patent application No. 201811438582.6 entitled "polypeptide, method for producing the same, and use thereof" (CN 109593126 a) describes recombinant type III humanized collagen previously studied and produced by the present inventors, 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 and evaluate the effectiveness of the recombinant type III humanized collagen for repairing endometrium. In particular, the invention intends to provide the use of the recombinant humanized type III collagen of CN 109593126 a for preventing and/or treating uterine related diseases (especially chronic endometritis), for improving the inflammatory state of endometrium, and further increasing the pregnancy rate of CE patients.

Means for solving the problems

The invention provides application of recombinant III type humanized collagen in preparing a medicament for preventing and/or treating uterus related diseases, wherein the recombinant III type humanized collagen comprises n repeats of a sequence shown as SEQ ID No.1, n is an integer which is more than or equal to 1, and when n is an integer which is more 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 repeated sequences are directly connected.

More preferably, n is 16 and the repeated 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, uterine extracellular matrix changes, increased expression of inflammatory immune factors, and decreased endometrial receptivity.

Further, in the above use, the uterine extracellular matrix change is an increase in tissue inhibitor of metalloproteinases 1(TIMP-1) and/or a decrease in Matrix Metalloproteinases (MMPs).

Further, in the above use, the inflammatory immune factor is at least one selected from the group consisting of 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 the endometrial tissue, promote the endometrial thickening and the gland generation, inhibit the expression of related inflammatory immune factors, increase the endometrial receptivity and maintain the extracellular matrix steady state, thereby laying a foundation for the preparation of the medicine for treating the chronic endometritis and being used for treating the chronic endometritis.

Drawings

FIG. 1 shows the results of different-stage uterine section analyses in four rat models.

FIG. 2 shows the results of detecting the expression levels of extracellular matrix-related genes at different stages in four groups of rat models.

FIG. 3 shows the results of testing the expression levels of the inflammatory immune-related factors at different stages in four groups of rat models.

FIG. 4 shows the results of the endometrial receptivity analysis at different periods in four groups of rat models.

FIG. 5 shows the results of measurements of CD138 expression levels at different stages in four groups of rat models.

Detailed Description

The technical solution of the present invention will be further described with reference to specific examples.

The recombinant type III humanized collagen of the present invention is described in patent application No. 201811438582.6 entitled "polypeptide, method for producing the same, and use thereof".

In one embodiment, the recombinant type iii humanized collagen of the present invention comprises N repeats of the sequence represented by SEQ ID No.1 (consisting of 30 amino acids, GERGAPGFRGPAGPNGIPGEKGPAGERGAP from segment N to segment C), 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 humanized type iii collagen of the present 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 present invention comprises 16 repeats of the sequence shown in SEQ ID No.1, with direct linkage between the repeats.

Unless otherwise indicated, instruments, reagents, materials, laboratory animals and the like used in the present invention are commercially available in a conventional manner.

Example 1: recombinant type III humanized collagen for promoting morphological recovery of CE rat endometrium

80 Sprague Dawley rats (220-. After 1 week of feeding, the rats were randomly divided into a normal control group (n-20, 40 uteri), a sham-operated group (n-20, 40 uteri), a model group (n-20, 40 uteri), and a treatment group (n-20, 40 uteri).

Normal Control (NC) rats were not treated at all. In SHAM (SHAM) rats only, after anaesthesia and abdominal sterilisation, were opened, the abdomen closed after exposure of the uterus to air for 30min and the same procedure was performed after 24h anaesthesia. MODEL group (MODEL) rats were anesthetized, sterilized at their abdomens, opened, 150. mu.l of LPS solution (5mg/ml) was injected into each of the uterine cavities from the uterine horn, and then the abdomens were sutured, and 24 hours were anesthetized, sterilized, opened, and 150. mu.l of sterile PBS solution was injected into the same site, and then the abdomens were sutured. After anesthesia and abdominal sterilization, treatment group (COL) rats were opened, 150. mu.l each of LPS solution (5mg/ml) was injected into both uterine cavities from the uterine horn and then the abdomen was closed, and after 24 hours, 150. mu.l of recombinant type III humanized collagen (containing 16 repetitive sequences) solution (10mg/ml) was injected into the same site and then the abdomen was closed. In each group 4 rats were sacrificed at 1d, 4d, 7d, 14d, 28d treatment, respectively, and bilateral uteri of the rats were removed. The rat uterus was stored in RNA storage solution and paraformaldehyde, respectively, for subsequent experiments.

The uterus of 0/1/4/7/14/28-day rats was taken and paraffin-embedded sections were taken, followed by the following procedures:

(1) paraffin section dewaxing and dewatering: placing the slices into xylene I and xylene II in sequence, and soaking for 10min respectively. Then sequentially adding anhydrous ethanol I, anhydrous ethanol II, 90% ethanol, 80% ethanol, and 70% ethanol for 5min, respectively, and washing with distilled water for 2-3 min.

(2) Hematoxylin staining of cell nucleus: the slices are stained with hematoxylin for 3-5min, washed with tap water for 2-3min, differentiated with 1% hydrochloric acid alcohol for 3-5s, and washed with tap water for 2-3 min.

(3) Eosin staining: and (4) placing the section into eosin staining solution for staining for 1-3 min.

(4) Dewatering and sealing: placing the slices in 70% ethanol, 80% ethanol, 90% ethanol, anhydrous ethanol II, anhydrous ethanol I, xylene II, and xylene I for 5min respectively, taking out, 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, endometrial thickness and number of glands counted using Image pro-plus Image processing software, and the average of the 5 fields was taken.

As shown in FIG. 1, the endometrium of the rat was thinned and the number of glands was reduced due to chronic endometritis in the model group. After the treatment group is perfused with the recombinant type III humanized collagen, the endometrium of the rat is gradually thickened, and becomes normal after 14 days of treatment, and the number of glands is gradually increased.

Example 2: recombinant type III humanized collagen for promoting extracellular matrix remodeling

A rat experimental model was established in the same manner as in example 1.

The uterus of 0/1/4/7/14/28-day rats was taken and treated as follows:

(1) total RNA extraction

Weighing tissue, adding 1ml TRIzol into 100mg tissue, grinding the tissue uniformly by a homogenizer at low temperature, and standing for 3-5min on ice.

② adding 200 mul chloroform, shaking vigorously for 15s, and standing on ice for 3-5 min.

③ 4 ℃, 13000rpm/min for 15min, sucking the supernatant into a new centrifugal tube, and standing for 10min on ice.

Fourthly, centrifuging for 10min at 13000rpm/min at 4 ℃, and removing the supernatant after a white precipitate is seen.

Fifthly, adding 1ml of precooled 75% ethanol, centrifuging at 4 ℃ and 7000rpm/min for 5min, and discarding the supernatant.

Sixthly, air-drying the clean bench for 5min, and adding 25 mu l of RNase-free water.

(2) Reverse transcription of cDNA

The total RNA extracted was assayed, the concentration was determined, and then the concentration was trimmed to 1. mu.g/. mu.l.

Preparing RNA-primer Mix:

③ centrifuging RNA-primer Mix with a low-speed centrifuge at 65 ℃ for 10min to denature the RNA-primer Mix, cooling and placing on ice.

Preparing a cDNA reverse transcription reaction solution:

fifthly, incubating for 1h at 37 ℃ and inactivating for 5min at 85 ℃. Storing at-20 deg.C.

(3) PCR reaction

Preparing a reaction solution:

centrifugation, setting a PCR reaction detector program:

analysis of dissolution curve after the reaction is finished:

it is generally believed that chronic endometritis causes an increase in extracellular matrix (ECM) in rat uterus, while slowing its degradation. While implantation of the embryo is mediated by trophoblasts, the aggressive trophoblasts can encounter a large amount of extracellular matrix during implantation, and the excessive extracellular matrix expression of the secreted middle and late endometrium prevents implantation of the blastocyst to cause infertility.

The results of qPCR detection of gene expression of ECM components in tissues are shown in fig. 2, and after treatment with recombinant type III humanized collagen, there was a tendency for ECM components to increase between D1-D4, but gradually decrease after D7, and D28 returned to near normal levels, indicating that recombinant type III humanized collagen is involved in the process of dynamic regulation of extracellular matrix.

TIMP1 and MMP-2 are extracellular matrix related factors. Tissue inhibitor of matrix metalloproteinases factor 1(TIMP-1) is a tissue inhibitor of matrix metalloproteinases and is 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 the MMPs family function, regulating the latter to function normally. Both maintain extracellular matrix homeostasis. The results in fig. 2 show a significant increase in MMP2 in the treated group, which greatly reduced the extracellular matrix. While TIMP-1, an inhibitor of MMP2, was significantly less in the treatment group than in the model group.

Example 3: recombinant type III humanized collagen for inhibiting generation of inflammatory factor

A rat experimental model was established 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 in example 2.

The results of qPCR detection of the expression levels of IL-1b and IL-6 genes of the immune-related factors in tissues are shown in FIG. 3, and it is found that in the model group, both the immune-related factors show an upward trend, while the expression of the related factors in the treatment group is also in the upward trend, but is significantly lower than the relative expression of the control group compared with the control group, and shows a downward trend in D7-D14. Therefore, it can be shown that the recombinant type III humanized collagen can reduce the expression of inflammatory immune factors, thereby regulating the cellular immune function.

Example 4: recombinant type III humanized collagen for improving expression of factors related to endometrial receptivity of rats

A rat experimental model was established in the same manner as in example 1.

The uterus of 0/1/4/7/14/28-day rats was collected and sectioned in paraffin-embedded sections, which were treated as follows:

(1) and (5) baking the paraffin sections for 2 hours at 60 degrees.

(2) Placing the slices into xylene I and xylene II in sequence, and soaking for 20min respectively. Then, absolute ethyl alcohol I, 95% ethyl alcohol and 85% ethyl alcohol are sequentially added for 5min respectively, and the mixture is washed for 3minx3 times by PBS.

(3) Soaking the tissue slices in sodium citrate buffer solution, heating to boil with high fire in microwave oven, heating with medium fire for 5minx3 times, and allowing rest for 3min each time. Then taken out and kept stand to room temperature. Then washed 3minx3 times with PBS.

(4) Wiping off water around the tissue, adding 100 μ L endogenous peroxidase blocking agent dropwise to cover the tissue, and incubating in a wet box for 10min to completely inactivate endogenous peroxidase. Then washed 3minx3 times with PBS.

(5) Wiping off water around the tissue, adding 100 μ L of the above solution, sealing, covering the tissue with normal goat serum working solution, and incubating in a wet box for 15 min.

(6) The goat serum working solution was spin-dried and 100. mu.L of HOXA10 antibody dilution (dilution ratio 1:300) was added dropwise to each tissue. Wet box incubate 4 degrees overnight.

(7) The wet box was placed at 37 degrees for 30 min. Wash 5minx3 times with PBS. 100 mu L or a proper amount of biotin-labeled goat anti-mouse/rabbit IgG polymer is added dropwise, and the mixture is incubated at room temperature for 15 min. PBS buffer washing 5min x3 times.

(8) And (3) dripping 100 mu L or a proper amount of horseradish enzyme labeled streptavidin working solution, and incubating for 15min at room temperature. PBS buffer washing 5min x3 times.

(9) Color development: adding a proper amount of freshly prepared DAB, and incubating for 3min at room temperature.

(10) Counterdyeing: washing with tap water, and incubating with hematoxylin staining solution for 1 min. Differentiation with 1% hydrochloric acid alcohol for 2s, washing and returning blue.

(11) And (5) baking the tissue sections for 1 hour at 60 degrees. And soaking the xylene I and the xylene II for 20min respectively. Then sequentially adding 85% ethanol, 95% ethanol, and anhydrous ethanol I for 5min, 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 lens, 5 fields were randomly selected, and the average absorbance value of the section was measured and analyzed using Image pro-plus Image processing software.

HOXA10 is an important member of homeobox gene family, is expressed in both epithelial and mesenchymal cells of endometrial gland of normal human, and plays roles in coding transcription factor, promoting cell differentiation and regulating embryonic development. Studies have shown that abnormal expression of HOXA10 is detected in the intima of patients with endometrial, ovarian, endometriosis and other gynecological diseases.

The results of the HOXA10 test are shown in FIG. 4, the expression of rat uterus HOXA10 in the model group is reduced, while the HOXA10 in the treatment group shows an ascending trend, which indicates that the recombinant type III humanized collagen can improve the endometrial receptivity and promote the embryo planting.

Example 5: recombinant type III humanized collagen for inhibiting expression of CD138

A rat experimental model was established in the same manner as in example 1.

The rat uterus from 0/1/4/7/14/28 days was collected and sectioned by paraffin embedding, and the same procedure as in example 4 was carried out, 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 CD138 immunohistochemical detection result is shown in figure 5, the rat endometrial CD138 expression of the model group is increased, and the rat endometrial CD138 expression of the treatment group is reduced after the treatment group is treated by the recombinant type III humanized collagen.

To summarize: in a rat model of chronic endometritis constructed by LPS, the recombinant type III humanized collagen can reduce the expression of CD138 in an inner membrane tissue and promote the thickening of endometrium and the generation of glands damaged after modeling. Meanwhile, the composition can inhibit the generation of inflammatory factors and increase the endometrial receptivity. Its action may be related to the remodeling of extracellular matrix components.

Sequence listing

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