阅读说明：本技术 Fgl2在制备妊娠期糖尿病诊断和预后的试剂中的应用 (Application of FGL2 in preparation of reagent for diagnosis and prognosis of gestational diabetes ) 是由 漆洪波 栾晓瑾 付雍 童超 于 2021-07-19 设计创作，主要内容包括：本发明涉及FGL2在制备妊娠期糖尿病诊断和预后的试剂中的应用,属于疾病诊断领域。本发明通过检测FGL2基因和蛋白表达水平,可以及早判断和预示是否存在GDM,并且通过监测FGL2表达水平的变化可以判断GDM的进程,预示是否存在GDM子代胚胎和胎盘发育不良,以及反映GDM患者治疗效果和预后,为患者的疾病风险及治疗方案提供重要依据。(The invention relates to an application of FGL2 in preparation of a reagent for diagnosis and prognosis of gestational diabetes, belonging to the field of disease diagnosis. According to the invention, by detecting the expression level of FGL2 gene and protein, whether GDM exists can be judged and predicted as early as possible, and the process of GDM can be judged by monitoring the change of the expression level of FGL2, whether the embryo and placenta dysplasia of GDM filial generation exists is predicted, the treatment effect and prognosis of GDM patients are reflected, and an important basis is provided for the disease risk and treatment scheme of the patients.)

The application of FGL2 in preparing a reagent for diagnosing and prognosing gestational diabetes is characterized in that the nucleotide sequence of FGL2 is SEQ ID NO.1, and FGL2 is derived from blood.

2. The use of claim 1, wherein the protein amino acid sequence encoding FGL2 is SEQ ID No. 2.

3. The use according to claim 1, wherein the expression levels of genes and proteins of FGL2 are positively correlated with the development of gestational diabetes.

4. The use of claim 1, wherein the expression levels of genes and proteins of FGL2 are negatively correlated with the development of intrauterine fetuses and placental function of pregnant diabetic pregnant women.

5. Use according to any one of claims 1 to 4, wherein: the diagnosis and prognosis are specifically screening, auxiliary diagnosis, curative effect evaluation, prognosis evaluation or relapse monitoring of gestational diabetes.

Technical Field

The invention belongs to the field of disease diagnosis, and particularly relates to application of FGL2 in preparation of a reagent for diagnosis and prognosis of gestational diabetes.

Background

Gestational Diabetes Mellitus (GDM) refers to impaired glucose tolerance or elevated blood glucose that occurs first during pregnancy. GDM is one of the most common complications of pregnancy, with a prevalence of about 16.9% in women of the world's reproductive age. Currently, the recommended GDM diagnostic criteria are: the pregnant woman is subjected to 75g Oral Glucose Tolerance Test (OGTT) at 24-28 weeks of gestation, fasting blood glucose is less than or equal to 5.1mmol/L, blood glucose is less than or equal to 10.0mmol/L after 1 hour of oral glucose, and blood glucose is less than or equal to 8.5mmol/L after 2 hours of oral glucose, and GDM can be diagnosed when the blood glucose value at any point reaches or exceeds the above standard. To date, research on GDM pathogenesis has focused mainly on: insulin resistance during pregnancy and islet beta cell dysfunction lead to insufficient insulin secretion. It is known that elevated levels of estrogen, progesterone and cortisol during pregnancy induce insulin resistance. In addition, dysfunction of various immune cells (including, e.g., macrophages, dendritic cells and Th1 cells) and release of inflammatory mediators in the maternal decidua and placenta can also induce insulin resistance and impair beta cell function, leading to the development of GDM. GDM seriously threatens the health of mother and infant, GDM pregnant women are susceptible to gestational hypertension and preeclampsia, and newborns are susceptible to hyperbilirubinemia, giant children, respiratory distress syndrome and the like. Therefore, early diagnosis and intervention are critical to the prevention of GDM. Because of the complex pathogenesis of GDM and the lack of reliable biological indicators for screening and monitoring during pregnancy, the search for potential molecular targets for the prevention, diagnosis and treatment of GDM is of great importance.

Disclosure of Invention

In view of the above, an object of the present invention is to provide an application of FGL2 in preparing a reagent for diagnosis and prognosis of gestational diabetes.

In order to achieve the purpose, the invention provides the following technical scheme:

1. the application of FGL2 in preparing a reagent for diagnosing and prognosing gestational diabetes, wherein the nucleotide sequence of FGL2 is SEQ ID NO.1, and FGL2 is derived from blood.

As one of the preferable technical schemes, the amino acid sequence of the protein coded by FGL2 is SEQ ID NO.2

As one of the preferable technical schemes, the expression level of the FGL2 gene and protein is positively correlated with the development of gestational diabetes.

As one of the preferable technical schemes, the expression level of the FGL2 gene and protein is in negative correlation with the development and the placenta function of the intrauterine fetus of the pregnant diabetic pregnant woman.

As one of the preferred technical schemes, the diagnosis and prognosis are specifically screening, auxiliary diagnosis, curative effect evaluation, prognosis evaluation or relapse monitoring of gestational diabetes.

The invention has the beneficial effects that:

the invention provides a novel GDM effective detection marker. The invention can judge and predict whether GDM exists in an early stage by detecting the Protein expression level of FGL2(Fibrinogen-Like Protein 2), and judge the process of GDM by monitoring the change of the expression level of FGL 2. Moreover, by detecting the change of FGL2 gene and protein expression level, the existence of GDM progeny embryo and placenta dysplasia can be predicted, and the treatment effect and prognosis of GDM patients can be reflected. The invention can carry out detection only by preparing serum, and patients can carry out routine blood examination in hospital without special equipment, thus having simple and convenient operation. The invention provides a target molecule for deeply researching GDM pathogenesis by detecting the expression level of FGL2 gene or FGL2 protein in the decidua of GDM mice and normal pregnant mice.

Drawings

For the purposes of promoting a better understanding of the objects, aspects and advantages of the invention, reference will now be made to the following detailed description taken in conjunction with the accompanying drawings in which:

FIG. 1 shows the FGL2 expression level in the serum of GDM patients.

FIG. 2 is the analysis of the correlation between the OGTT blood glucose level of GDM patients and the expression level of FGL 2.

FIG. 3 shows the FGL2 expression level in GDM mouse serum.

FIG. 4 is the correlation analysis of the OGTT blood glucose level of GDM mice and the expression level of FGL 2.

FIG. 5 shows the uptake of the offspring embryos of GDM mice.

FIG. 6 is a correlation analysis of the expression level of FGL2 in GDM mouse progeny embryos and placentas.

FIG. 7 shows FGL2 gene and protein expression levels in GDM mouse decidua.

Detailed Description

The invention will be further illustrated with reference to specific preferred embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not indicated in the examples, are generally carried out according to conventional conditions, such as those described in the molecular cloning protocols (third edition, sambrook et al), or according to the manufacturer's recommendations.

Example 1

Expression of FGL2 protein in normal pregnant women and GDM pregnant women

(1) Establishment of GDM animal model

The experimental animals comprise 20 healthy female C57 mice at 6-8 weeks and 10 healthy male C57 mice at 6-8 weeks.

Female mice were randomly divided into 2 groups, GDM group (n ═ 10) and control group (n ═ 10). After adaptive feeding for 1 week, the male mice are combined with the adaptive feeding according to the ratio of 2: 1, the next morning is checked for vaginal embolus, and if vaginal embolus is observed, the pregnancy is marked as 0.5 d. The model group was injected with Streptozotocin (STZ) 150mg/kg i.p. at 12.5 days of pregnancy, and the control group was injected with an equal volume of PBS buffer i.p. at the same time. And performing an OGTT test on 16.5 days of pregnancy, and if the blood sugar of the model group is abnormally increased, successfully molding.

(2) Preparation of test serum

Blood samples of GDM pregnant women and normal pregnant women were from the obstetrics department of the first hospital affiliated to the university of Chongqing medicine. Placing blood samples of GDM pregnant women and normal pregnant women, GDM pregnant mice and normal pregnant mice in an incubator at 37 ℃ for 20min, then centrifuging at 3000rpm for 10min, and carefully collecting supernatant clear liquid for later use.

(3) Detecting FGL2 protein expression level

This was done using Biolegend kit instructions. The operation steps are as follows: all reagents were returned to room temperature before use and allowed to stand at room temperature for 20 min. Preparing a protein standard product and a detection sample: the protein standard substance is subjected to gradient dilution by adopting Assay Buffer A, and the concentrations are respectively 40ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml and 0 ng/ml; the serum to be tested was diluted 10-fold with Assay Buffer A and mixed well. ② immune reaction: the sample on ELISA was washed 4 times with 300. mu.L of washing solution, and then 50. mu.L of Assay buffer A and 50. mu.L of the prepared standard and test sample were added, mixed well, sealed and incubated at room temperature for 2 h. Next, discard the waste from each well, wash the plate 4 times with 300. mu.L, pat dry, add 100. mu.L FGL2 detection antibody to each well, seal the plate and incubate it for 1h on a shaker at room temperature. Then, each well was discarded and washed, patted dry, and 100. mu.L of Anti-rabbitIgG-HRP secondary antibody was added, followed by shaking incubation at room temperature for 1h after sealing. After washing the plate, 100 μ L of Substrate Solution F was added to each well, after closing the plate, incubation was performed for 30min in the dark at room temperature, and 100 μ L of Stop Solution was added to each well to terminate the reaction. Detecting an OD value: and immediately detecting and recording the absorbance value at the position of 450nm of wavelength, and finally drawing a standard curve according to the standard substance and calculating the concentration of the sample.

(4) Results

As shown in FIGS. 1, 2 and 3, NP (normal pregnancy) is a sample of a pregnant woman of normal pregnancy, and GDM is a sample of a patient who was confirmed to be GDM by the OGTT test. As can be seen from fig. 1, the expression level of FGL2 in the serum of GDM patients was significantly increased compared to normal pregnant women, and the difference was statistically significant. Furthermore, the expression level of FGL2 was analyzed by correlation with the blood glucose levels at 0h, 1h, and 2h after the OGTT test, and the expression level of FGL2 was found to be proportional to the blood glucose level at each time point (fig. 2). Furthermore, the animal experimental results also showed that the expression level of FGL2 was significantly increased in GDM mice compared to control group pregnant mice (fig. 3), and that the expression level of FGL2 was also proportional to the blood glucose value at each time point (fig. 4). This result demonstrates that by detecting the expression level of FGL2 protein, the presence or absence of GDM can be judged and predicted early, and the progress of GDM can be judged by monitoring the change of the expression level of FGL 2.

Example 2

Relationship between FGL2 gene and protein expression level and GDM progeny embryo and placenta development

(1) Evaluation of the development of the embryo and placenta of the offspring of GDM mice

GDM and control animal models were constructed as in example 1. Two groups of pregnant mice were euthanized at 18.5d of pregnancy, embryos and placentas of the pregnant mice were removed, and the offspring embryos and placentas were observed for absorption and weighed.

(2) Detecting FGL2 mRNA expression level in ecdysis tissue of pregnant mouse

Decidua tissues of pregnant mice in GDM group and control group were separated, total RNA was extracted by TRIzol method, and RNA concentration was measured by UV spectrophotometer. cDNA was synthesized using a reverse transcription kit (TAKARA). The PCR reaction system is prepared by using TB Green fluorescent dye (TAKARA), cDNA and primers.

The reaction condition is pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 15s and annealing at 60 ℃ for 20s for 40 cycles. Ct value application 2^-ΔΔCtThe method is characterized in that the relative expression level of FGL2 is normalized according to the expression level of internal reference (GAPDH).

The FGL2 primer sequence is:

5'-CTCAAAGAAGTGCGGACCCTCAAG-3'(SEQ ID NO.3)；

3'-TCGGCTGTCTCTGCTCCATTCC-5'(SEQ ID NO.4。

(3) detecting FGL2 protein expression level in ecdysis tissue of pregnant mouse

The collected decidua tissues of pregnant mice were extracted for total protein using RIPA lysate (Beyotime Biotechnology) and the total protein concentration was determined by BCA method. Preparing 10% separation gel and 4% concentrated gel, adding the prepared protein sample into a sample hole on the gel, adjusting the electrophoresis initial voltage to be 80V after the protein sample is electrophoresed to the separation gel, adjusting the voltage to be 110V after the protein sample is electrophoresed to the separation gel, stopping the electrophoresis until bromophenol blue just comes out, and performing membrane transfer. The membrane is rotated for 90min by using a constant current of 250mA, and the target protein is transferred to the PVDF membrane. The membranes were blocked with 5% BSA for 1h at room temperature and incubated with FGL2 murine monoclonal antibody (1:200, Abnova), Tubulin antibody (1:1000, Beyotime Biotechnology) overnight at 4 ℃. TBS-T membranes were washed and incubated with horseradish peroxidase-labeled goat anti-mouse secondary antibody (1:5000, Beyotime Biotechnology) for 1h at room temperature. Protein content was determined by chemiluminescence (ECL, Vazyme), exposed to a video camera and photographed. The grey values of each band were quantitatively analyzed using ImageJ software.

(4) Results

As a result, as shown in fig. 5, 6 and 7, the child embryo absorption rate of the pregnant mice in the GDM group was increased compared to that in the control group (fig. 5), and the expression level of FGL2 in the two groups of pregnant mice was correlated with the weight of the child embryo and the placenta, respectively, and the result showed that the expression level of FGL2 was inversely proportional to the weight of both the embryo and the placenta (fig. 6), and the expression level of FGL2 protein increased as the weight of the embryo and the placenta decreased. In addition, the relative expression levels of FGL2 mRNA and protein were elevated in GDM decidua tissue compared to control decidua tissue, with the difference being statistically significant (fig. 7). The results show that by detecting the expression level of the FGL2 protein, whether the development and the placenta function of the intrauterine fetus of the GDM pregnant woman are abnormal can be judged, or the treatment effect and the prognosis of the GDM patient can be reflected by monitoring the expression level of the FGL2 protein.

Finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the claims of the present invention.

Sequence listing

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Application of <120> FGL2 in preparation of reagent for diagnosis and prognosis of gestational diabetes

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