CN110945362A

CN110945362A - Application of CD93 in preparation of umbilical blood detection kit for early warning of infantile hemangioma and treatment medicine

Info

Publication number: CN110945362A
Application number: CN201980000578.8A
Authority: CN
Inventors: 江成鸿
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-04-23
Filing date: 2019-04-23
Publication date: 2020-03-31
Anticipated expiration: 2039-04-23
Also published as: CN110389165A; CN110945362B; WO2019206122A1

Abstract

Application of CD93 in preparing an umbilical blood detection kit for early warning Infantile Hemangioma (IH), preparing a medicament for treating or preventing infantile hemangioma and preparing a serum marker for preventing hemangioma. The cord blood detection kit using the CD93 as the prediction/early warning protein can early warn the generation of IH and carry out early monitoring, early finding and early treatment on the IH. Meanwhile, CD93 as a transmembrane protein can promote angiogenesis of hemangioma stem cells, has important significance of anti-angiogenesis therapeutic targets, and can be used for early treatment and prevention of IH.

Description

Application of CD93 in preparation of umbilical blood detection kit for early warning of infantile hemangioma and treatment medicine

Technical Field

The invention relates to the technical field of biology, in particular to application of CD93 in preparing an umbilical blood detection kit for early warning Infantile Hemangioma (IH).

Background

IH is the most common benign tumor in infants. In china, the incidence is as high as 4-10%, and epidemiological studies have found that it is more common in girls, premature and low-weight infants, with a male-female incidence ratio of about 1: 4. its unique rapid proliferation and natural regression course makes it the only natural model for human to observe tumorous angiogenesis. More importantly, in view of the high morbidity, high invasiveness, unique disease course and epidemiological representation and unsatisfactory treatment status of IH, the exploration of the etiology and the generation and development mechanism becomes a hot spot and a difficult point for studying by scholars at home and abroad.

However, the etiology and pathogenesis of IH are not elucidated, the treatment lacks specificity, the damage is not insignificant, and some IH is easily confused with body surface tumors with similar appearances such as venous malformation, which causes misdiagnosis and mistreatment, thus delaying the treatment and causing unnecessary iatrogenic injuries to the infant patient.

Currently, two major problems still face clinically: on the one hand, early detection of early treatment is an important prerequisite to avoid malformation residues, but unfortunately, no serum markers predictive for IH occurrence have been found so far. On the other hand, there is still a lack of clinically effective specific therapeutic targets.

CD93, also known as C1qRp, is a type I single transmembrane protein, and from N-terminus to C-terminus, CD93 is composed of a C-type lectin-like domain (CTLD, D1), an EGF-like domain (D2), a mucin-like domain (D3), a transmembrane domain (D4), a cytoplasmic domain (D5), and a domain located between D1 and D2 (DX). It is expressed in Endothelial Cells (ECs), stem cells, monocytes, etc., and is cleaved by matrix metalloproteases and stimulated by extracellular factors to form soluble CD93, which is released into the blood. CD93 has been suggested as a serum marker for acute asthma, acute myocardial infarction and coronary artery disease.

Recent studies have found that CD93 has a pro-angiogenic effect. First, CD93 binds specifically to its natural ligand polyprotein-2 (Multimerin 2, MMRN2) through the extracellular CTLD domain, and the interaction between the two plays a key role in the adhesion, migration, and angiogenesis of ECs. Second, CD93 is essential for tubular morphogenesis of ECs and plays a key role in organizing ECs scaffolds and cell junctions. Again, studies have shown that CD93 can mediate pro-angiogenic effects through a variety of pathways, including the MMRN2 pathway, the b-DG/Src/CD93/Cbl pathway, the activation of the PI3K/Akt/eNOS pathway, and the EGFR/EKR pathway. Finally, inhibition of CD93 expression reduces ECs migration and sprouting, thereby inhibiting the pro-angiogenic effects of VEGF, i.e. functions as an anti-angiogenic target.

IH has unique rapid proliferation and natural regression course, and is the only natural model for observing tumorous angiogenesis, and excessive angiogenesis is the important mechanism of IH rapid proliferation. The CD93 is a potential new target for anti-tumor angiogenesis as a tumor angiogenesis characteristic gene. Thus, this project further validated CD93 histological examination and evaluated the potential of CD93 as a target for anti-IH angiogenesis by in vitro cell model studies.

Technical problem

The method finds the cord blood early warning protein, predicts the IH occurrence, and has extremely important clinical significance and social value for early monitoring, early treatment and even prevention of IH (especially severe IH). Meanwhile, the discovery of the early warning protein provides an important clue for the research of pathogenesis and provides a new entry point for developing related medicines and even preventive medicines.

Technical solution

The invention aims to provide application of CD93 in preparing an umbilical blood detection kit for early warning infantile hemangioma.

The second purpose of the invention is to provide the application of CD93 in preparing the medicine for treating infantile hemangioma.

The third purpose of the invention is to provide the application of CD93 in preparing the medicine for preventing infantile hemangioma.

The fourth purpose of the invention is to provide the application of CD93 in preparing hemangioma serum markers.

In order to realize the first purpose, the invention provides an application of CD93 in preparing an umbilical blood detection kit for early warning infantile hemangioma.

Preferably, the CD93 is detected in combination with cathepsin D.

In order to achieve the second purpose, the invention provides application of CD93 in preparing a medicine for treating infantile hemangioma.

In order to achieve the third object, the invention provides an application of CD93 in preparing a medicine for preventing infantile hemangioma.

In order to achieve the fourth object, the invention provides the application of CD93 in preparing hemangioma serum markers.

Advantageous effects

The invention has the advantages that the research finds that the newborn umbilical cord blood serum CD93 has an important function of predicting/early warning IH occurrence, has strong predicting/early warning value and is an IH tumor serum marker. Because the umbilical cord blood of the newborn is just easy to collect in a sufficient amount (the umbilical cord blood is mostly discarded) in the early stage of IH (infectious anemia) disease, has the advantages of no wound, easy acceptance by family members, convenience for general investigation and the like, the umbilical cord blood detection kit or test paper which takes CD93 as prediction/early warning protein is developed and put in each delivery room, is widely applied to the birth stage of the newborn, reduces the incidence rate of IH (infectious anemia) of the most common benign tumors of the infants, namely IH (infectious anemia) by carrying out early monitoring, early finding and early treatment to reduce the incidence rate of IH as far as possible and reduce misdiagnosis and treatment rate, has extremely important clinical significance and social value, has extremely wide demand range and low cost, and can embody considerable economic benefit.

Furthermore, after histological verification, in vitro cell model research in this subject group found that CD93 has the effect of promoting IH angiogenesis, and the effect was significantly reduced after interference, suggesting that CD93 has the function of anti-IH angiogenesis target, and can be applied to the preparation of related therapeutic drugs and prophylactic drugs to develop early treatment and prevention of IH. CD93 has never been reported in previous IH studies, so the discovery that CD93 has two major functions of predicting/warning IH occurrence and resisting an IH angiogenesis target at the same time belongs to source innovation. Because excessive angiogenesis is an important mechanism for IH rapid proliferation, if anti-angiogenesis targeted drugs are successfully developed, IH specific targeted therapy is promoted, so that the curative effect is improved, the side effects and iatrogenic injuries are reduced, and the teratogenesis rate is greatly reduced; if the serum CD93 concentration of the newborn is found to be too high when the tumor does not appear, the medicine can also be used prophylactically to prevent the occurrence of IH, and the research is further carried out. Meanwhile, IH has rapid proliferation and natural regression disease course, is the only natural model for observing angiogenesis of solid tumors, has universality on excessive angiogenesis, and has uniqueness on natural regression. The research on the IH angiogenesis resistant drug target provides reference for the anti-angiogenesis targeted treatment and induced regression of solid tumors, has very important clinical and social values, and cannot underestimate the economic benefit.

Drawings

FIG. 1 is a diagram of a cluster analysis of the results of quantitative mass spectrometry.

FIG. 2 is a sample-to-sample analysis for identifying protein identity.

FIG. 3 is a graph of frequency of difference.

Figure 4 is a differential protein volcano plot analysis.

Fig. 5 is a serological validation of CD93 ELISA (performed in duplicate), suggesting that there was a significant difference in CD93 expression levels between the two sera (P <0.01), and the IH group was higher than the normal group (left panel). ROC curve analysis suggests that the area under the curve is 0.9097, specificity is 91.67%, sensitivity is 75% (P ═ 0.0007), and the curve has strong diagnostic value (right panel).

Fig. 6 is a serological validation of Cathepsin D (Cathepsin D, CTSD) ELISA (duplicate) suggesting significant differences in CTSD expression levels between the two sera (P <0.05), with the IH group being lower than the normal group (left panel). CTSD ROC curve analysis suggests: AUC 0.7649, specificity 91.67%, sensitivity 50% (P0.0221), with moderate predictive value (right panel).

Fig. 7 shows that the concentration ratio of CD93 to CTSD (CD93/CTSD) in IH group is significantly higher (P <0.001) (left panel) than that in normal group, and ROC curve analysis shows: AUC is up to 0.96, specificity is up to 91.67%, sensitivity is up to 83.33%, prediction efficiency is further improved, and the prediction method has a great value in predicting IH occurrence (right side of the figure).

FIG. 8 shows that the curve of CD93/CTSD is closest to the upper left by superposing three ROC curves of CD93, CTSD and CD93/CTSD, and the AUC value is the highest and reaches 0.96, so the prediction efficiency is extremely strong.

FIG. 9 shows immunohistochemical analysis of CD93 expression in IH tissues of different stages and normal skin: (A) and (B) respectively 200 times and 400 times of vision field of IH in proliferative stage, and CD93 can be seen to be expressed in HemECs in large quantity and is mainly located in cytoplasm and cell membrane; (C) and (D) respectively shows 200 times of visual field and 400 times of visual field of IH in the regression period, so that lumen differentiation in IH tissues is obvious, IH enters the degeneration stage, and CD93 expression is very little; (E) and (F) are respectively 200 times and 400 times of normal skin tissues, the expression condition of CD93 is similar to that of IH in a regression phase, and the expression quantity is very small.

FIG. 10 is immunofluorescence laser confocal imaging of CD133+ HemSCs, CD133-, and HUVEC cells CD 93: CD93 was most expressed in HUVEC; CD93 and MMRN2 were expressed more in CD133+ HemSCs than in CD 133-cells, and were expressed mainly in the cytoplasm and the cell membrane. Merge map is the synthesis map of three proteins, DAPI, CD93 and MMRN 2.

Fig. 11 shows immunofluorescence laser confocal imaging of IH tissue with normal skin: the expression level of CD93 in IH tissue is obviously higher than that in normal skin tissue. (A) - (D), (E) - (H) are 200 times and 630 times of the field of view of normal skin tissue, respectively; (I) - (L), (M) - (P) are 200 times and 630 times of IH tissues visual field respectively.

FIG. 12 shows the expression of CD93 protein and mRNA in IH tumor body and paraneoplastic tissue: (A) - (F) measuring the expression of the CD93 protein in 6 cases of IH in the proliferative stage and paraneoplastic tissues by a WB method (each sample is repeated for 2-4 times), and indicating that the expression level of the CD93 protein in each case of IH tissues in the proliferative stage is higher than that in the paraneoplastic tissues; (G) (H) grayscale analysis of WB results indicated that the expression level of CD93 protein in IH tissue in proliferative stage was higher than that in paraneoplastic tissue (P ═ 0.0003), which was 33.78 times higher than that in paraneoplastic tissue on average; (I) - (J) relative expression of CD93 mRNA in the proliferative IH tissue was measured by qPCR (2 replicates per sample, using paraneoplastic tissue as a control), indicating that CD93 mRNA levels in the proliferative IH tissue were higher than those in paraneoplastic tissue (P0.0133), averaging 26.07 times that in paraneoplastic tissue.

FIG. 13 shows the construction of CD93^OEHemSCs、CD93^shHemSCs stable transformants: CD93 was obtained by drug (puromycin, 0.8ug/ml) screening^OEHemSCs (panel A), interference group one (panel B), interference group two (panel C), interference group three (panel D) HemSCs, panels E-H are fluorescence images corresponding to panels A-D (x 100), respectively; WB results significant differences in the expression level of CD93 protein in each group were observed from the bands (FIG. J shows the enhancement of each band on the basis of FIG. I): CD93 in contrast to WT HemSCs (panel J, panel C)^OEThe HemSCs (lane 1 in FIG. I, J) bands were too strong to quantify, the interference group one, two, three (

lanes

2, 3, 4 in FIG. J, respectively) bands were too weak, and quantification was inaccurate; (K) the qPCR detection of the expression level of the CD93 gene of each group of stable transgenic cells indicates that the overexpression group, the interference group I, the interference group II and the interference group are respectively 2701779.1%, 14%, 9.4% and 13.5% of WT HemSCs, and the CD93 is indicated^OEThe expression of HemSCs CD93 was significantly increased, with minimal interference with group II CD93 expression.

FIG. 14 shows the tube formation experiment (triplicate wells) for stable CD93 transformants: (A) - (E) are respectively CD93^OEDuctal images of HemSCs, CD93 interference group one, interference group two, interference group three, and WT HemSCs; (F) - (J) analyzing the images for each group of cell lines ImageJ respectively, and observing 3 indexes of the number of nodes, the number of meshes and the total length of the blood vessel; (K) CD93 for ImageJ analysis results^OEThe tube forming effect of the HemSCs is obviously stronger than that of the WT HemSCs, while the tube forming effect of the three groups of interfering cell strains is obviously weaker than that of the WT HemSCs, wherein the tube forming capability of the interfering group II is weakest in the interfering group.

FIG. 15 shows that the CD93 antibody blocks WT HemSCs and CD93^OEHemSCs tube formation experiment: (A) - (D) respectively WT HemSCs, CD93^OEHemSCs, WT HemSCs + antibody (antibody ab), CD93^OE6-hour images of HemSCs + ab tubes and ImageJ software analysis images corresponding to the images; (E) - (H) results of ImageJ software analysis, CD93^OEHemSCs group cells have the strongest tube forming efficacy, CD93^OEHemSCs + ab-tubulating potency weakest, CD93^OEThe efficacy of the HemSCs constitutive tube is stronger than that of the WT HemSCs group, and the efficacy of the WT HemSCs constitutive tube is only slightly stronger than that of the WT HemSCs + ab group, which indicates that the CD93 antibody can obviously inhibit CD93^OETubulogenesis of HemSCs, but for WT HemSCsThe suppression of energy is weak.

Modes for carrying out the invention

Hereinafter, the technique of the present invention will be described in detail with reference to specific embodiments. It should be understood that the following detailed description is only for the purpose of assisting those skilled in the art in understanding the present invention, and is not intended to limit the present invention.

Example 1 newborn umbilical blood serum CD93 has important function of predicting/early warning IH occurrence

For searching the cord blood early warning protein, the following experimental sample collection and research are carried out:

1. about 1100 samples of the blood serum of the umbilical cord of the newborn are prospectively collected and reserved in 2014 to 2016, the blood serum is stored in a refrigerator at the temperature of 80 ℃, and repeated freezing and thawing of the samples are avoided in the experimental process.

2. Each neonate was observed for half a year of follow-up and found to have 12 IH, thus dividing the reserved serum samples into two groups: the serum of cord blood in which IH occurred (hereinafter referred to as IH group) and a normal control group in which IH did not occur (hereinafter referred to as normal group) were followed.

3. Quantitative mass spectrometry was performed using a Label-free quantitative proteomics technology (Label-free quantitative proteomics technology) for 9 pairs of IH group and normal group serum samples to obtain differential proteins between the two groups.

4. Clustering analysis mass spectrometry detection results, clustering IH group sera and normal group sera into two groups respectively, and drawing a cluster map (figure 1), wherein each group has some proteins with equivalent expression levels. The identity protein identity analysis between samples (fig. 2a and 2b) suggested that the identity was above 80%, indicating that the reproducibility of sample preparation and analysis was good. The difference frequency of two groups of serum proteins accords with normal distribution (figure 3), and the reliability is high.

5. The serum samples of the IH group and the normal group of 9 cases are detected and compared, 517 differential proteins are identified together, a volcano graph of the differential proteins is drawn (figure 4), red (upper right corner region) and green (upper left corner region) scatter points in the graph indicate that the serum of the IH group and the normal group has significant quantitative differential proteins (P <0.05, change multiple >2.5 times), wherein the red point is increased by more than 2.5 times, and the green point is reduced by more than 2.5 times. A total of 19 differential proteins with an increase of more than 2.5 fold are represented by red dots, from which a secondary screen is performed: and (3) analyzing the correlation between the 19 differential proteins and the pathogenesis of the hemangioma by combining the biological information analysis result of the early-stage hemangioma tissue specimen gene chip data IPA (insulin Pathway analysis), and screening out 3 differential proteins with the increase of more than 2.5 times and 1 differential protein with the decrease of more than 2.5 times.

The 4 differential proteins were further serologically verified by ELISA. Two repeated ELISA assays were performed to screen out 2 significantly different proteins: CD93 and Cathepsin D (Cathepsin D, CTSD). The IH group CD93 concentration was significantly higher (P <0.01) than the normal group (left in fig. 5), while the IH group CTSD concentration was significantly lower (P <0.05) than the normal group (left in fig. 6). CD93 ROC curve analysis suggests: the Area Under the Curve (AUC) reaches 0.91(AUC >0.9, namely, the specificity is 91.67%, the sensitivity is 75% (P ═ 0.0007), and the diagnostic value is very strong (right in figure 5). CTSD ROC curve analysis suggests: AUC 0.7649, specificity 91.67%, sensitivity 50% (P0.0221), with moderate predictive value (fig. 6 right). Significantly, the IH group concentration ratio of CD93 to CTSD (CD93/CTSD) was significantly higher (P <0.001) than the normal group (fig. 7 left), and ROC curve analysis showed: AUC is as high as 0.96, specificity is 91.67%, sensitivity is 83.33%, prediction efficiency is further improved, and the prediction method has great value in predicting IH occurrence (figure 7 right). The curves of CD93, CTSD and CD93/CTSD superimposed by three ROC curves are near the upper left of the curve of CD93/CTSD, the AUC value is the highest (0.96), and the prediction efficiency is extremely strong (FIG. 8).

The results, which were repeatedly verified, illustrate that: in the stage of birth of the infant with IH, the expression level of the cord blood serum CD93 of the infant with IH is increased, which shows that the cord blood serum CD93 has the function of predicting/early warning the occurrence of IH. Besides, the AUC of the ROC curve reaches 0.91(AUC is greater than 0.9, namely, the ROC curve has strong diagnostic value), and the specificity reaches 91.67%, so that the factor has strong diagnostic value and high early warning significance. More importantly, ROC curve analysis of the concentration ratio of CD93 to CTSD (CD93/CTSD) showed that: AUC is up to 0.96, specificity is up to 91.67%, sensitivity is up to 83.33%, and IH occurrence prediction value is extremely high. The above data indicate that cord blood CD93 concentration is closely related to the onset of IH and is a good predictor serum marker (or serum marker) for IH.

The research finds that the newborn umbilical cord blood serum CD93 has an important function of predicting/early warning IH occurrence and has strong predicting/early warning value, so that the advantages of easiness in sufficient acquisition of newborn umbilical cord blood, no wound, easiness in acceptance of family members and the like can be utilized, a cord blood detection kit or test paper taking CD93 as prediction/early warning protein is developed, the cord blood detection kit or test paper is widely applied to newborns, early monitoring, early finding and early treatment are carried out on IH (especially serious IH) with the early warning incidence rate of 4-10%, the teratogenesis rate of the serious IH is reduced as far as possible, the misdiagnosis and treatment rate is reduced, and the cord blood serum CD93 has important clinical significance and social value.

Example 2 evaluation of the potential of CD93 as a target for anti-IH angiogenesis by in vitro cell model studies

Firstly, histological verification is carried out, if the expression level of CD93 protein and mRNA in IH tumor bodies in a proliferation stage is definitely higher than that of tissues beside the tumor, CD93 is suggested to participate in the proliferation process of the IH tumor bodies, and the potential of CD93 for resisting an IH angiogenesis target is further evaluated. Histological validation the following experimental specimen collections and studies were performed:

1. immunohistochemical staining

Selecting IH paraffin tissue specimens comprising hyperplastic IH (n-14), degenerative IH (n-14) and normal skin (n-13), carrying out immunohistochemical staining on CD93 and CTSD, and observing staining results.

And (4) prompting by a result: CD93 was localized to the HemECs cytoplasm and membrane, with high expression in IH tissue during the hyperplastic phase, minimal expression during the regressive phase and in normal skin (fig. 9). Whereas CTSD showed no difference in expression between groups (fig. 9), the focus of the subsequent study was around CD 93.

2. Immunofluorescence laser confocal imaging

Immunofluorescence laser confocal imaging of CD133+ HemSCs, CD 133-cells, umbilical vein endothelial cells (HUVEC), IH tumor bodies and normal skin tissues revealed that CD133+ HemSCs expressed more abundant CD93 and MMRN2 proteins than CD 133-cells, mainly localized in the cytoplasm and the cell membrane (FIG. 10); the expression level of CD93 in IH tumor body tissues is obviously higher than that in normal skin (FIG. 11).

WB and qPCR detection

Surgical acquisition of proliferative phase ICutting H tumor body and peritumoral tissue into 2-3mm³Size. Quickly freezing part of the liquid nitrogen, and transferring to a refrigerator at the temperature of-80 ℃ for storage for WB detection; the remaining tissue was added to RNA later and left to stand overnight at 4 ℃ before removing RNA later and transferred to a refrigerator at-80 ℃ for storage for qPCR detection.

WB and qPCR detection were performed on 6 pairs of proliferative IH and paraneoplastic tissues, respectively, (2-4 replicates per specimen) to compare the expression levels of CD93 protein and mRNA in the neoplastic tissues and paraneoplastic tissues, and the results suggest that the expression level of CD93 protein in the proliferative IH tissues is higher than that in the paraneoplastic tissues, with significant difference (P ═ 0.0003), which is 33.78 times that in the paraneoplastic tissues on average (fig. 12G); CD93 mRNA levels in the hyperplastic IH tumor were higher than in paraneoplastic tissue (P ═ 0.0133), averaging 26.07 times higher than in paraneoplastic tissue (fig. 12I). These experimental results further suggest that CD93 may play an important role in the IH proliferation process.

In conclusion, the above histological verification shows that the expression level of CD93 protein and mRNA in IH tumor body in the proliferation stage is higher than that of the tissues beside the tumor, which indicates that CD93 participates in the proliferation process of IH tumor body.

Second, construction of CD93 overexpressing HemSCs (CD 93) using successfully cultured hemangio stem cells (HemSCs)^OEHemSCs) and CD93 interference with HemSCs (CD 93)^shHemSCs) to evaluate the potential of CD93 against the IH angiogenic target. The method comprises the following specific steps:

1. construction of CD93^OEHemSCs、CD93^shHemSCs stable transformants

After preliminary confirmation of high expression of CD93 mRNA levels in proliferating tumors and HemSCs, construction of CD93 by adenoviral transfection was initiated^OEHemSCs、CD93^shHemSCs stable transformants. And (4) prompting by a result: a. the virus transduction effect of each experimental group is good, and the fluorescence rate reaches more than 80% (FIG. 13A-H). Wb results significant differences in CD93 protein levels were seen for each group from the bands: CD93 in contrast to WT HemSCs^OECD93 is abundantly expressed in HemSCs, the overexpression effect is good, and the band is too strong to be quantified (FIG. 13I, J); the expression of CD93 in all three interference groups is obviously reduced, the interference effect is good, and the quantification is inaccurate (FIG. 13I, J). qPCR detection shows that the CD93 gene expression quantity of each group of cells is as follows: 2701779.1 for the over-expression group, the interference group I, II and III were WT HemSCs% 14%, 9.4%, 13.5%, suggesting CD93^OEExpression of HemSCs CD93 was significantly increased with minimal interference with group two CD93 expression (fig. 13K). The above results suggest CD93^OEHemSCs、CD93^shHemSCs stable transformants were successfully constructed.

2. Comparing the tube forming function of three groups of cells, the CD93 is proved to have the function of anti IH angiogenesis target

Successful construction of CD93^OEHemSCs、CD93^shAfter HemSCs were stably transformed, a tube formation experiment was performed using WT HemSCs as a control to compare the tube formation ability of each group of cell lines (FIGS. 14A-E). The major observations were made by ImageJ software analysis of 3 indices of node number (Junctions), mesh number (meshes), total vessel length (top. length) (fig. 14F-J). The results show that CD93^OEThe tube forming effect of the HemSCs is obviously stronger than that of the WT HemSCs, and the tube forming effect of the three groups of interfering cell strains is obviously weaker than that of the WT HemSCs; the nodes and meshes of the interference group two are less than those of the other 2 interference groups, and the total length of the blood vessels is not greatly different (fig. 14K). The result proves that the CD93 has the effect of promoting IH angiogenesis, and the effect is obviously weakened after interference, namely the CD93 has the function of resisting the IH angiogenesis target.

Finally, the tube forming effect of the CD93 antibody is further utilized to try to block, and from another angle, the CD93 is proved to have the effect of promoting IH angiogenesis and can be used as a target for resisting the IH angiogenesis, and the CD93 antibody can block the angiogenesis promoting effect of CD93, so that a brand-new specific treatment means is provided for IH treatment.

The experimental scheme is as follows: for CD93^OEAddition of anti-human CD93 antibodies (R) to HemSCs and WT HemSCs&D, Polyclonal coat IgG antibody, AF2379) blocks tubulogenic effects cell grouping is as follows, ① WT HemSCs ② CD93^OEHemSCs③WT HemSCs+ab④CD93^OEHemSCs + ab. The intervention concentration of the antibody is 100ug/ml (calculated by the goat IgG molecular weight of about 144kDa, which is about equal to 700nmol/L), and the tube forming effect and the inhibition by the antibody of the two groups of cell strains after the addition of the antibody are observed.

The method comprises the following specific operations: the matrigel was spread evenly on a cover glass (200. mu.l/slide) placed in a 12-well plate and allowed to stand for 30 min. After the matrigel is solidified, diluting 4 groups of cells with EBM-2 culture solutionInto 10⁵Perml, 500. mu.l per well, 50ug antibody per well of the antibody plus panel. Culturing at 37 deg.C under 5% CO2, and performing mapping at 1h, 2h, 4h, 6h, 8h, and 10 h.

The experimental results are as follows: WT HemSCs, CD93^OEHemSCs、WT HemSCs+ab、CD93^OEImages of tubes were taken at 6 hours after inoculation of the HemSCs + ab 4 group of cells, and the tube forming ability of the 4 groups of cells was compared by ImageJ software, and three indexes of the number of nodes (Nb Junctions), the number of meshes (Nb meshes) and the total length of blood vessels (tot. length) were mainly observed (FIG. 15). The results show CD93^OEThe 3 tube forming indexes of the HemSCs group are slightly higher than those of the WT HemSCs group, which indicates that the over-expression of CD93 can enhance the tube forming efficiency of the HemSCs. CD93 in group 4^OEHemSCs constitute the most potent tube, while CD93^OEThe HemSCs + ab has the weakest tube forming capability, and the CD93 antibody can obviously inhibit CD93^OEThe tube forming effect of the HemSCs is weaker to inhibit the tube forming effect of the WT HemSCs.

And (4) conclusion: the experiment further proves that the CD93 has the effect of promoting IH angiogenesis, is a potential drug target for resisting the IH angiogenesis, and can block the angiogenesis promoting effect of CD93, inhibit the IH proliferation and promote the IH regression by applying the CD93 antibody. Therefore, the preparation of the CD93 blocking monoclonal antibody drug can specifically block the angiogenesis promoting effect of CD93, possibly provides a brand new specific means for IH clinical treatment, and has value for preventing IH from occurring if the drug is used before the IH occurs.

And (3) prospect: in recent years, cryoelectron microscopy (cryo-EM) has revolutionized the analysis of protein structure, function and mechanism of action, and the nobel prize of chemistry in 2017. The advanced technology can be used for analyzing the CD93 specific target to provide sufficient protein conformation and blocking target information for new drug research and development, and the technology can be used for analyzing and optimizing candidate monoclonal antibodies, so that the preparation period of high-specificity immune protein and high-efficiency blocking monoclonal antibodies can be greatly shortened, and the success rate of screening target monoclonal antibodies at the later stage is obviously improved. Meanwhile, the prepared monoclonal antibody is subjected to screening optimization on a CD93OE/CD93sh HemSCs stable transformant, an NOD/SCID mouse IH tumor implantation model and an IH three-dimensional angiogenesis model which are established through earlier research to obtain a blocking monoclonal antibody with high specificity, high affinity and high efficiency, so that a basis is provided for developing IH anti-angiogenesis targeted therapeutic drugs, and reference is provided for monitoring solid tumors and anti-angiogenesis therapy.

In conclusion, the subject group applies protein mass spectrometry to detect the serum of the cord blood of the IH group and the serum of the cord blood of the normal group, two groups of differential proteins are analyzed, and the serological verification of the parallel ELISA method finds that CD93 is a serum marker for predicting the generation of IH, the prediction efficiency of the CD93/CTSD ratio is extremely strong, the AUC is as high as 0.96, the specificity is 91.67%, and the sensitivity is 83.33%; the protein and mRNA expression level of CD93 in IH tissue is obviously higher than that in paraneoplastic tissue through further walk histological verification; then in vitro cell model research finds that the CD93 has the effect of promoting IH angiogenesis, and the effect is obviously weakened after interference, which indicates that the CD93 has the function of resisting the IH angiogenesis target; and the CD93 monoclonal antibody can block the angiogenesis promoting effect caused by CD93 overexpression, which indicates that the preparation of the treatment medicament and the prevention medicament related to the CD93 blocking monoclonal antibody is helpful for developing the early treatment and prevention of IH. Since CD93 has never been reported in previous IH studies, the discovery that it has two functions of predicting IH occurrence and resisting IH angiogenesis target at the same time is also a source innovation.

Because excessive angiogenesis is an important mechanism for IH rapid proliferation, if anti-angiogenesis targeted drugs are successfully developed, IH specific targeted therapy is promoted, so that the curative effect is improved, the side effects and iatrogenic injuries are reduced, and the teratogenesis rate is greatly reduced; if the serum CD93 concentration of the newborn is found to be too high when the tumor does not appear, the medicine can also be used prophylactically to prevent the occurrence of IH, and the research is further carried out. Meanwhile, IH has rapid proliferation and natural regression disease course, is the only natural model for observing angiogenesis of solid tumors, has universality on excessive angiogenesis, and has uniqueness on natural regression. The research on the IH angiogenesis resistant drug target provides reference for the anti-angiogenesis targeted treatment and induced regression of solid tumors, has very important clinical and social values, and cannot underestimate the economic benefit.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Industrial applicability

The main body of the present application can be industrially produced and used, and has industrial applicability.

Claims

Application of CD93 in preparing an umbilical blood detection kit for early warning of infantile hemangioma.
The use of CD93 in the preparation of a cord blood test kit for warning infantile hemangiomas according to claim 1, wherein CD93 is detected in combination with cathepsin D.
Application of CD93 in preparing medicine for treating infantile hemangioma is provided.
Application of CD93 in preparing medicine for preventing infantile hemangioma is provided.
Application of CD93 in preparing hemangioma serum marker.