CN111487398B - Biomarkers for hemangioma treatment - Google Patents
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- CN111487398B CN111487398B CN202010067261.0A CN202010067261A CN111487398B CN 111487398 B CN111487398 B CN 111487398B CN 202010067261 A CN202010067261 A CN 202010067261A CN 111487398 B CN111487398 B CN 111487398B
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention provides Sub>A biomarker for hemangiomSub>A treatment, and particularly provides Sub>A method and Sub>A kit for effectively evaluating and predicting the curative effect of itraconazole on infantile hemangiomSub>A treatment by using the expression level of Sub>A biological factor, wherein the biological factor is one or more of VEGF-A, PDGF-D, HES, HEY1, HIF3A, IL-8, TNF-A or FGF. Has important significance for the curative effect judgment, the prognosis evaluation and the medication course guidance of the infantile hemangioma.
Description
Technical Field
The invention relates to a biomarker for hemangioma treatment, in particular to a method and a kit for effectively evaluating and predicting the curative effect of itraconazole on hemangioma treatment by using a biological factor in blood as a marker, belonging to the field of biological medicines.
Background
Hemangioma is the most common benign tumor of skin in infants and young children, and the incidence rate is 8% -10%. It is good at head, face and limbs, can affect any part of body, rapidly proliferate within 1 year old, and gradually enter self-regressive stage for 5-9 years. Although most hemangiomas have pathophysiological processes that resolve themselves, pigmentation, telangiectasia, fibrous and adipose tissue deposits often remain. The tumor body of 10% -20% of children patients increases with age, and even serious complications such as ulcer, bleeding and infection occur, which leads to the consequences such as appearance damage and function loss: the central part of the face, the air passage, the skin fold, the perineum, the perianal area and the like are easy to rub or are difficult to self-heal after being subjected to the formation of the ulcer in sweat, urine and excrement dipping areas; hemangiomas located on the eyelids, conjunctiva can affect vision resulting in amblyopia, astigmatism and blindness; respiratory tract location can lead to respiratory disorders and affect cardiopulmonary function; some hemangiomas can lead to life-threatening Kasabach-Merritt syndrome (Kasabach-Merritt syndrome), congestive heart failure, etc.; the skin hemangioma seriously affects the image, and leads the infant to have a series of psychological obstacles such as inferior, inward, self-closing, extreme and the like.
Invasive treatment technology for treating hemangioma relates to surgical excision, dye pulse laser, intervention, freezing, fulguration and the like, and has certain side effect or limitation due to different requirements on compliance, cost and convenience of clinical skill, surgical conditions, instruments, children patients and family members of a surgeon, easy secondary infection and scar remaining. The non-invasive treatment comprises oral and external medicines, wherein the oral medicine mainly comprises glucocorticoid, alpha interferon, vincristine, propranolol, itraconazole and the like, and the external medicine mainly comprises beta receptor blockers such as timolol, carteolol and the like. The noninvasive treatment has the advantages of reducing the probability of secondary infection and scar, simple operation, relatively low price and high compliance of children patients and family members. But has the problems of long treatment time, side effect of the medicine and the like. The applicant finds that most family members of children patients prefer to treat hemangioma by non-invasive methods of firstly selecting oral or external medicines in the clinical treatment process. The operation or external application of some special parts such as the blood vessel tumor of eyelid, nasal cavity, vulva and perianal part is difficult, and only oral medicine can be selected.
The side effects caused by long-term use of oral drugs, such as the effects on liver and kidney functions and cardiac functions, are a major concern of the family members. If the side effects of the drugs are considered, the premature withdrawal of the drugs may cause the skin lesions to recur and rebound. Therefore, finding some biomarker factors related to hemangioma regression, evaluating the effectiveness or ineffectiveness of treatment according to the biomarkers and when to stop taking medicine is a matter of urgent concern for doctors and family members, and no such biomarker is reported at present.
Disclosure of Invention
The invention provides a method for evaluating, predicting or optimizing the treatment effectiveness of itraconazole on hemangioma, which comprises the following steps:
a) Obtaining a test sample from a patient having a hemangioma, and detecting the expression level of a biological factor in said test sample;
b) The hemangioma patient is treated by an effective dose of itraconazole;
c) Comparing the change of the expression level of the biological factor before and after the treatment, and achieving the treatment effect when the expression level of the biological factor is up-regulated or down-regulated.
The test sample is normal tissue, tumor tissue, cell line, plasma, serum, whole blood, cerebrospinal fluid, lymph fluid, circulating tumor cells, cell lysate, tissue lysate, urine and/or aspirate, preferably serum or tumor tissue; the biological factor is one or more of VEGF-A, PDGF-D, HES, HEY1, HIF3A, IL-8, TNF-A or FGF.
In some embodiments, the expression level of the biological factor is the mRNA expression level of the corresponding biological factor in the test sample.
In some preferred embodiments, sub>A therapeutic effect is achieved when the mRNA expression level of one or more of PDGF-A, VEGF-A, HES, HEY1, IL-8 or TNF-A is upregulated by Sub>A fold difference greater than or equal to 2, 3, 5, 8, 10 or 20, more preferably greater than or equal to 10 or 20, after treatment; in other preferred embodiments, a therapeutic effect is achieved when the mRNA expression level of one or more of PDGF-D, HIF3A, or FGF after treatment is downregulated by a factor greater than or equal to 2, 3, 5, 8, 10, or 20, preferably by a factor greater than or equal to 10 or 20, as compared to prior treatment; preferably, the biological factor is one or more of PDGF-A, PDGF-D or FGF; more preferably, the biological factor is PDGF-D.
In still other embodiments, the expression level of the biological factor is the protein level of the corresponding biological factor in the test sample, preferably, the therapeutic effect is achieved when the protein level of the corresponding biological factor decreases by greater than or equal to 10%, 30%, 50% or 70%, more preferably, by greater than or equal to 50% or 70% before and after the treatment. More preferably, the biological factor is PDGF-D or PDGF-A.
In some embodiments, the hemangiomatosis is in an infant.
In some embodiments, the hemangioma is a proliferative hemangioma.
The invention also provides Sub>A detection kit comprising reagents for measuring the mRNA expression level or protein level of Sub>A biological factor in Sub>A test sample of Sub>A subject, wherein the test sample is normal tissue, tumor tissue, cell line, plasmSub>A, serum, whole blood, cerebrospinal fluid, lymph fluid, circulating tumor cells, cell lysate, tissue lysate, urine and/or aspirate, preferably serum or tumor tissue, and the biological factor is one or more of VEGF-A, PDGF-D, HES, HEY1, HIF3A, IL-8, TNF-A or FGF.
In some embodiments, the test kit further comprises instructions for a purpose selected from the group consisting of: determining whether the patient with the hemangioma will respond to itraconazole treatment by said biological factor expression level or a change thereof, monitoring the disease progression of the hemangioma patient treated with itraconazole by said biological factor expression level or a change thereof, demonstrating the biological activity of itraconazole in the patient administered with itraconazole by said biological factor expression level or a change thereof, or a combination thereof.
The invention also provides application of the detection kit in evaluating, predicting or optimizing the treatment effectiveness of itraconazole on patients with hemangioma.
In some embodiments, the expression level of the biological agent is the mRNA expression level of the corresponding biological agent; preferably, sub>A therapeutic effect is achieved when the mRNA expression level of one or more of PDGF-A, VEGF-A, HES, HEY1, IL-8 or TNF-A after treatment is greater than or equal to 2, 3, 5, 8, 10 or 20 fold difference in upregulation prior to treatment, more preferably greater than or equal to 10 or 20 fold difference in upregulation; preferably, a therapeutic effect is achieved when the mRNA expression level of one or more of PDGF-D, HIF3A or FGF after treatment is downregulated by a fold difference greater than or equal to 2, 3, 5, 8, 10 or 20, more preferably by a fold difference greater than or equal to 10 or 20, as compared to prior treatment; more preferably, the biological factor is one or more of PDGF-D, HIF3A or FGF; more preferably, the biological factor is PDGF-D.
In other embodiments, the expression level of the biological factor is the protein level of the corresponding biological factor; preferably, a therapeutic effect is achieved when the protein level of the corresponding biological factor decreases by greater than or equal to 10%, 30%, 50% or 70% after treatment compared to before treatment; more preferably, the biological factor is PDGF-A or PDGF-D.
In some embodiments, the hemangioma is a proliferative hemangioma.
In some preferred embodiments, the hemangiomatosis is in an infant.
The invention has the beneficial effects that: at present, no biomarker for effectively evaluating and predicting the curative effect of infantile hemangioma treatment by using biological factors is published. The biomarker has important significance in the treatment effect judgment, prognosis evaluation and medication course guidance of the infantile hemangioma.
Description of the drawings:
FIG. 1 shows the trend of VEGF and PDGF-AA level changes in the serum of infants before and after treatment.
Figure 2 mRNA expression profile of infantile hemangiomas treated with itraconazole and significantly altered first 20 relevant biological processes, cellular components, molecular functions and pathways. (a) Scattergrams of mRNA expression levels between endothelial cells from infant hemangiomas (HemEC) treated with itraconazole (10 μ M) and DMSO control. Red dots indicate up-regulated mRNA, green dots indicate down-regulated mRNA (fold change > 2.0). (b) Hierarchical clustering showed distinguishable mRNA expression patterns between itraconazole and controls. In HemEC, a total of 172 mrnas were up-regulated (fold change > 2.0) and 819 mrnas were down-regulated (fold change > 2.0) after itraconazole treatment. Corresponding to the differentially expressed mRNA in itraconazole-treated HemEC, the first 20 gene ontology terms for biological processes (c), the first 20 cellular components (d), the first 20 molecular functions (e), and the first 20 pathway terms (f).
FIG. 3 PI3K/Akt/mTOR signaling in infant hemangiomas was inhibited by itraconazole in vitro. (a) Expression of PDGF-D mRNA in HemEC treated with 10. Mu.M itraconazole for 48 hours was detected by reverse transcription PCR. (b) Expression of PDGF-D protein in HemEC treated with 10. Mu.M itraconazole for the indicated time as detected by immunoblotting. (c) Phosphorylation levels of p-Akt, p-p70S6K, p-4E-BP1 and T-Akt were examined in HemEC treated with 10. Mu.M itraconazole for 2 hours, 24 hours and 48 hours, respectively. (d) Levels of p-Akt, p-p70S6K, p-4E-BP1 and β -actin in 2 and 24 hour mouse hemangioma endothelial cells (EOMA) were treated with 0.3 μ M or 1 μ M itraconazole.
Figure 4 comparison of the effect of itraconazole and the PDGFR- β inhibitor CP-673451 on hemangiomas in infants HemECs. (a) HemECs were treated with itraconazole or CP-673451 at the indicated concentrations for 72 hours and then cell viability was determined by MTS assay. (b) After 48 hours of treatment with 3. Mu.M itraconazole and 5. Mu.M CP-673451, annexin V/PI staining was performed on HemEC. (c) The effect of 10 μ M itraconazole and 10 μ M CP-673451 on HemEC was determined by tube format. Scale bar =0.1mm. (d) Phosphorylation levels of p-PDGFR-beta, p-Akt, p-p70S6K, p-4E-BP1, PDGFR-beta and T-Akt in HemEC treated with 10. Mu.M itraconazole or 10. Mu.M CP-673451 for 48 hours were examined by Western blotting.
The specific implementation mode is as follows:
the methods and techniques of the present invention are generally performed according to conventional methods known in the art, unless otherwise indicated. Nomenclature related to biology, pharmacology, and medical and medicinal chemistry described herein, and laboratory procedures and techniques are those well known and commonly employed in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and testing or testing.
Unless defined otherwise, scientific and technical terms used herein shall have the meanings that are commonly understood by those of ordinary skill in the art. The following terms have the following definitions:
the term "effective dose", also commonly referred to as "therapeutically effective dose", refers to any amount of a drug that, when used alone or in combination with another therapeutic agent, promotes disease regression as manifested by a decrease in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of disease, or the prevention of a disorder or disability resulting from the disease. The "effective dose" of the drug of the present invention also includes a "prophylactically effective dose", which is any amount of the drug that, when administered alone or in combination with another therapeutic agent to a patient at risk of developing a disease or suffering from a recurrence of a disease, inhibits the occurrence or recurrence of the disease.
As will be apparent to those skilled in the art, the effective dosage and particular mode of administration in vivo will vary depending upon the species, weight and age of the mammal being treated, the particular compound employed and the particular purpose for which it is employed. Effective dosage levels (i.e., the dosage levels necessary to achieve the desired effect) can be determined by one of skill in the art based on routine pharmacological procedures. Generally, the human clinical application of the product starts with a lower dosage level, followed by increasing dosage levels until the desired effect is achieved. Alternatively, acceptable in vitro studies can be employed to establish useful dosages and routes of administration of the compositions identified by the present methods by available pharmacological methods.
The term "drug" is any chemical substance recognized in the art as being a biologically, physiologically or pharmacologically active substance. Drugs are also referred to as "therapeutic agents," examples of which are described in known references (e.g., merck Index, physicians Desk Reference, and The Pharmacological Basis of therapeutics), and include, but are not limited to, drugs, vitamins, mineral supplements, substances for treating, preventing, diagnosing, curing, or alleviating a disease or condition, substances that affect body structure or function, or prodrugs that are biologically active or more active when placed in a physiological environment. Various forms of therapeutic agents may be used, wherein upon administration to a subject, the composition is capable of being released from the subject into adjacent tissue or fluid. The drug in the invention refers to itraconazole and comprises various pharmaceutical preparations or pharmaceutical compositions which take itraconazole as an active ingredient.
The term "hemangioma" refers to a congenital benign tumor or vascular malformation, commonly found in skin and soft tissue, formed by the proliferation of angioblasts during the embryo, most commonly found in infants at or shortly after birth. The residual embryo hemangioblast and active endothelial embryo invade to the adjacent tissue to form an endothelial cord, which is connected with the left blood vessel after canalization to form hemangioma, and the blood vessel in the hemangioma is self-organized and is not connected with the peripheral blood vessel. Hemangiomas can occur throughout the body, most of which occur in facial skin, subcutaneous tissue, and oral mucosa, tissues such as tongue, lips, and floor of mouth, and few of which occur in the jaw bone or deep tissues. The vascular endothelial cells of hemangiomas have proliferative properties, and the natural course of disease can be divided into proliferative, stationary and regressive phases. Hemangiomas can be further classified into capillary hemangiomas, cavernous hemangiomas, mixed hemangiomas, and tendrilous hemangiomas according to histological structure and clinical manifestations.
The codes and Chinese and English names of the biological factors are shown in Table 1.
TABLE 1 biological factor code and its Chinese and English name comparison table
The term "PDGF", a platelet-derived growth factor, is a growth factor that regulates cell growth and differentiation and plays an important role in angiogenesis, which often leads to cancer. There are four known PDGF proteins encoded by four genes, PDGF-A, PDGF-B, PDGF-C and PDGF-D. PDGF is produced by discrete cell populations and secreted as disulfide-linked homodimers or heterodimers, including PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD, and PDGF-AB. PDGF dimers act primarily in a paracrine manner by binding to the PDGF receptor. There are two known PDGF receptors, PDGFR- α and PDGFR- β, which can form both heterodimers and homodimers. Ligand binding promotes receptor dimerization, autophosphorylation, and activation of multiple intracellular signaling cascades downstream, thereby stimulating actin filament rearrangement, disruption of gap junction communication, initiation of gene transcription, and cell survival.
The "test sample" may be any biological material isolated from the body of a patient or subject, such as normal tissue, tumor tissue, cell lines, plasma, serum, whole blood, cerebrospinal fluid, lymph fluid, circulating tumor cells, cell lysate, tissue lysate, urine, and aspirates; preferably, the test sample is derived from normal tissue, tumor tissue, cell lines, circulating tumor cells, serum, plasma, or whole blood; more preferably, the test sample is derived from serum, tumor tissue. Methods for removing a sample are well known in the art, and it may be removed from a subject, for example, by biopsy, e.g., by needle biopsy, hollow needle biopsy, or fine needle aspiration biopsy, endoscopic biopsy, or surface biopsy; or by venipuncture, whole blood, plasma or serum samples are collected and further processed according to standard techniques. After obtaining a test sample from a patient or subject, the sample is subjected to method steps that encompass measuring the level of expression of the biological factor. In the present invention, the expression level of the biological factor mainly refers to the mRNA expression level and the protein level.
Example 1 clinical Observation of curative Effect of itraconazole on infantile hemangioma treatment and Effect of itraconazole on levels of VEGF and PDGF-AA in serum of infants suffering from hemangioma
17 patients with infantile hemangioma are collected in an outpatient clinic, and after signing an informed consent, parents or legal guardians of the infants take itraconazole 5mg/kg/d orally, wherein the treatment course is 2 weeks to 12 weeks. Changes in skin lesions (tumor body area, color flattening, lightening, etc.) were recorded every 4 weeks from the treatment, and the treatment effect was judged and evaluated from a clinical point of view. 17 blood samples of children with hemangioma before taking medicine, 4 weeks after taking medicine and 12 weeks after taking medicine are collected, and the changes of the levels of VEGF and PDGF-AA in serum are detected by an ELISA method and are quantitatively analyzed. Statistical data were analyzed using SPSS software. The results of the study are shown in FIG. 1.
The study included 17 children, 6 men and 11 women, aged 1-12 months (average 5.4 months), with a weight of 5-10.5kg, 17 samples collected before treatment and 17 samples after treatment, with an interval of 13-56 days (average 31 days) between before and after treatment, and a total dose of oral itraconazole of 390-2240mg (average 1076 mg). The total follow-up time is 0.5-6 months. Of these 17 cases, 12 were hemangiomas in infants at the proliferative stage, of which 7 had a tendency to regress during the follow-up period with a regression rate of 58.3% (7/12).
(1) Serum VEGF (vascular endothelial cell growth factor) assays before and after treatment (fig. 1A).
The serum VEGF concentration of the infant before treatment is 214.82 +/-135.58 pg/mL, after treatment is 390.81 +/-649.18 pg/mL, after treatment is increased by 175.98 +/-551.17 pg/mL compared with before treatment (wherein VEGF is increased after 8 patients are treated, 9 patients are reduced), and the difference between the two groups has no statistical significance (p > 0.05) through T test analysis. Of 7 therapeutically effective children with proliferative hemangiomas, there were 4 and 3 post-treatment increases and decreases in VEGF levels.
(2) Serum PDGF-AA (platelet derived growth factor) assay before and after treatment (FIG. 1B).
Before treatment, the PDGF-AA concentration in the serum of the infant patient is 52879.92 +/-31435.65 pg/mL, after treatment is 26289.34 +/-26095.77 pg/mL, after treatment, the PDGF-AA concentration is 26590.57 +/-35627.71 pg/mL (wherein 2 patients are increased, 15 patients are decreased) than before treatment, and the difference between the two groups has statistical significance (p is less than 0.05) through T test analysis. Of 7 therapeutically effective patients with proliferative hemangiomas, there were 1 (14.3) and 6 (85.7) post-treatment increases in PDGF-AA levels.
Therefore, the above 17 infants with infantile hemangioma had no statistical significance for the change difference of VEGF level in serum before and after itraconazole treatment, but PDGF-AA level in serum was in the trend of decrease, and the difference had statistical significance. In the treatment of the effective proliferative stage of infantile hemangioma, the PDGF-AA level is reduced and the hemangioma is regressed with close correlation. Since PDGF-AA is Sub>A dimer of PDGF-A, changes in PDGF-AA reflect changes in PDGF-A protein levels, and it is known that Sub>A decrease in PDGF-A protein levels is closely related to resolution of hemangiomas.
Example 2 molecular mechanisms of itraconazole treatment of hemangiomas
(1) Materials and methods
Reagents and cell culture
Itraconazole, ketoconazole and propranolol were purchased from Sigma-Aldrich. CP-67345 is purchased from MedChem Express. EOMA was obtained from the vascular biology program of Catherine Butterfield bos, a pediatric hospital surgical department of harvard medical school (boston, usa). Cultured in RPMI 1640 (GIBCO) containing 10% fetal bovine serum and penicillin/streptomycin/glutamine. After obtaining written informed consent from parents for the use of infant hemangioma specimens, we collected the proliferative tissue of infant hemangiomas. They received surgical treatment for hemangioma. In vitro infant hemangioma endothelial cells (HemEC) were successfully cultured using a tissue blocking approach with proliferating IH tissue. Then cultured in RPMI 1640 (GIBCO) containing 10% fetal bovine serum, 10ng/ml basic fibroblast growth factor and penicillin/streptomycin/glutamine. All cells were 5% CO at 37 ℃ 2 Culturing in an incubator.
Cell viability assay
For cell viability analysis, cells were seeded in 96-well plates and treatment was started the next day with itraconazole or CP-673451. After 72 hours of incubation, MTS analysis (Promega, madison, WD) was performed according to the manufacturer's instructions and normalized cell viability curves were fitted using GraphPad Prism software (version 6).
Apoptosis assay
Cells were treated with itraconazole or CP-673451 for 48 hours. Apoptotic cells were analyzed by Annexin V/PI staining (Annexin V FITC and PI kit, thermo Scientific) using a Canto flow cytometer (Becton Dickinson, franklin Lakes, N.J.) according to the manufacturer's instructions. Furthermore, nuclei of EOMA and HemEC were stained with Hoechst33258 at a concentration of 5. Mu.g/ml for 5 minutes at 37 ℃. The cells were washed to remove unbound dye, and then cell viability was observed by fluorescence microscopy (Olympus).
Angiogenesis assays
HemECs were seeded at a density of 1.5 × 10 cells/well in 96-well plates on Matrigel surfaces. Treating the cells with 0.1% dmso, itraconazole, propanol or CP-673451. After 3 hours of treatment, 6 randomly selected microscope fields were photographed (200 x magnification). The manifold length and The density of The capillary network were evaluated by Axiovision software (The Math Works, inc., of pedicure, ma).
HemECs mRNA array and data analysis
HemEC of 2 infants were treated with 10. Mu.M itraconazole or 0.1% DMSO for 48 hours. Total mRNA was extracted for mRNA array analysis of agilent human (V2) gene expression microarrays (8 × 60K chips) according to the manufacturer's instructions.
Reverse transcription PCR
Total RNA was extracted from itraconazole treated HemEC. Itraconazole or 0.1% dmso was used as a control treatment using Trizol reagent (Invitrogen, 15596-026) according to the manufacturer's instructions. The primer sequences are as follows: PDGF-D,5 'CCCAGAGAATTACTCGGTCAA-3' (F) (SEQ ID No.: 1) and 5 'ACAGCACACACTATTTCCTCCAC-3' (R) (SEQ ID No.: 2); ACTB,5 'GGACTTCGAGCAAGAGAGATGG-3' (F) (SEQ ID No.: 3) and 5 'AGCACTGTTGTTGGCGTACAG-3' (R) (SEQ ID No.: 4).
Western blot
Western blotting was performed with the following antibodies, referred to the reference (Zheng et al, 2009): p-p70S6K (product number 9272), pAKT-T308 (9275), T-AKT (4691), p-4E-BP1-S65 (9451), PDGFR (3169), p-PDGFR-B (3161) and B-actin (4970) were purchased from Cell Signaling Technologies; PDGF-D was purchased from R & D Systems (product number AF 1159).
Statistical analysis
Quantitative data are expressed as mean values of n =3 ± standard deviation (s.d.). Two groups were compared by student's t-test, and multiple groups were compared to Tukey correction by one-way anova with mismatch, using the mean of each column in GraphPad Prism compared to the mean of each other column (version 6 la jolla.ca).
(2) Potential target gene and biological factor of itraconazole function
Applicants explored the potential targeting genes of itraconazole in vitro infant HemEC by using the whole genome gene expression profile (GEO accession No.: GSE 123108). Scatter plots and hierarchical clustering analysis were performed to identify differentially expressed mrnas (see figure 2). HemECs mRNA array and data analysis HemECs from 2 infant patients were treated with 10 μ M itraconazole or 0.1% dmso for 48 hours. Total mRNA was extracted for mRNA array analysis using an Agilent human (V2) gene expression microarray (8X 60K chip) according to the manufacturer's instructions.
From the MAS biofunctional annotation system manual, the first 20 significantly enhanced bioprocess gene ontology terms (GO terms) (fig. 2 c), the first 20 significantly enhanced cellular component gene ontology terms (fig. 2 d), the first 20 significantly enhanced molecular functional gene ontology terms (fig. 2 e) and the first 20 significantly enhanced pathway terms (fig. 2 f) were determined, respectively.
Meanwhile, the genes corresponding to the biological factors of PDGF-A, VEGF-A, HES, HEY1, IL-8 or TNF-A are found to be remarkably up-regulated in mRNA with differential expression, and the difference multiple is more than 2; the genes corresponding to PDGF-D, HIF3A or FGF biological factors are down-regulated obviously in the mRNA with differential expression, the difference multiple is more than 2, and the specific difference condition is shown in a table 2:
TABLE 2 genes with significant expression differences and their corresponding biological factor names
Note: in the above fold differences, positive numbers indicate up-regulation, negative numbers indicate down-regulation, and the fold differences are expressed as absolute values of the numbers in the table.
(3) Itraconazole significantly reduced PDGF-D levels, thereby inhibiting activation of PDGFR- β and inhibiting its downstream effectors.
In vitro infant hemangioma endothelial cells (HemEC) were significantly inhibited PDGF-D protein levels following itraconazole treatment (FIGS. 3a, b). The activity of the PI3K/Akt/mTOR pathway, an important downstream of PDGF/PDGF receptor- β (PDGFR- β) signaling and a key signaling pathway involved in infantile hemangiomas, was further analyzed using western blotting. As shown in fig. 3c, itraconazole treatment was found to induce a decrease in the level of Akt (p-Akt) phosphorylation and two downstream effectors of Akt/mTOR, p70S6 kinase (p-p 70S 6K) and 4E binding protein 1 (p-4E-BP 1) in HemEC (fig. 3 c) and EOMA (fig. 3 d) cells.
(4) The activity of itraconazole on HemEC was similar to that of CP-673451 (PDGFR-beta inhibitor)
To confirm whether itraconazole can inhibit infantile hemangiomas by inhibiting PDGF-D/PDGFR- β signaling, the activity of itraconazole was compared to the PDGFR- β inhibitor CP-673451 in HemEC, and the results showed that both itraconazole and CP-673451 inhibited cell proliferation (fig. 4 a), promoted apoptosis (fig. 4 b), reduced angiogenesis (fig. 4 c), inhibited phosphorylated PDGFR- β and its downstream effectors, including p-Akt, p-p70S6K, and p-4E-BP1 in HemEC (fig. 4D).
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the embodiments, it will be apparent to those skilled in the art that the technical solutions described in the foregoing embodiments may be modified or some technical features may be substituted equally; such modifications and substitutions do not depart from the spirit and scope of the corresponding claims.
Reference documents:
Zheng B,Jeong JH,Asara JM,Yuan Y-Y,Granter SR,Chin L,et al.Oncogenic B-RAF negatively regulates the tumor suppressor LKB1 to promote melanoma cell proliferation.Mol Cell 2009；33:237-47.
sequence listing
<110> Sichuan university Hospital in Huaxi
<120> biomarkers for hemangioma treatment
<130> P2020-0091
<150> CN201910074711.6
<151> 2019-01-25
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<170> PatentIn version 3.5
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Claims (3)
1. Use of Sub>A detection reagent for PDGF-A protein for preparing Sub>A kit for evaluating, predicting or optimizing the effectiveness of itraconazole in treatment of patients with hemangiomas, said hemangiomas being infants.
2. The use of claim 1, wherein the therapeutic effect is achieved when the level of PDGF-A protein after treatment is reduced by Sub>A ratio greater than or equal to 10% compared to the level of PDGF-A protein before treatment.
3. The use of claim 1, wherein the hemangioma is a proliferative hemangioma.
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