CN113186275A

CN113186275A - Application of serum amyloid A1 in preparation of biomarker for diagnosing polycystic ovarian syndrome

Info

Publication number: CN113186275A
Application number: CN202110665056.9A
Authority: CN
Inventors: 孙贇
Original assignee: Renji Hospital
Current assignee: Renji Hospital
Priority date: 2021-06-16
Filing date: 2021-06-16
Publication date: 2021-07-30

Abstract

The invention provides application of serum amyloid A1 in preparation of a biomarker for diagnosing polycystic ovary syndrome. The invention also provides application of a reagent for detecting serum amyloid A1 in preparing a diagnostic composition for in vitro diagnosis of a biomarker of polycystic ovarian syndrome, wherein the upstream primer sequence for detecting serum amyloid A1 is shown as SEQ ID NO: 2, the sequence of the downstream primer for detecting serum amyloid A1 is shown as SEQ ID NO: 3, the diagnosis comprises: detecting the level of serum amyloid a1 biomarker from the patient. The experimental research result of the invention indicates that SAA1 plays an important role in the occurrence and development of PCOS disease, and SAA1 can be used as a biomarker for evaluating the PCOS disease state.

Description

Application of serum amyloid A1 in preparation of biomarker for diagnosing polycystic ovarian syndrome

Technical Field

The invention belongs to the field of biological medicines, and relates to serum amyloid A1, in particular to application of serum amyloid A1 as a biomarker in diagnosis of polycystic ovarian syndrome.

Background

Polycystic ovary syndrome (PCOS) is the most common disorder of endocrine reproduction and metabolism, and the incidence rate of women in the reproductive age is 5% -10%, accounting for about 75% of anovulatory infertility. PCOS is characterized primarily by rare or no ovulation, hyperandrogenism and or clinical hyperandrogenism, with ovarian polycystic manifestations as typified by infertility, obesity, impaired glucose tolerance and insulin resistance. To date, the etiology and pathogenesis of PCOS remains unclear.

Serum Amyloid A (SAA) is a reactive protein in the acute phase of inflammation or injury, is secreted by the liver in a large amount in a short time, is 1000 times higher than the normal value, and is one of the most sensitive inflammatory reaction markers at present. SAA is also expressed in extrahepatic tissues such as macrophages and adipocytes, and is also used as a marker reflecting the degree of chronic inflammatory response in the body. SAA is mainly divided into four subtypes of SAA1, SAA2, SAA3 and SAA4, wherein SAA1 and SAA2 are acute phase reaction proteins, SAA3 is mainly expressed in mice but not in human bodies, and SAA4 is a constitutive gene and is not regulated by inflammation and stress. PCOS is a chronic inflammatory disease and the role of SAA in the development and progression of PCOS is not clear.

The amino acid sequence of SAA1 is shown in SEQ ID NO. 5.

Disclosure of Invention

In view of the above technical problems in the prior art, the present invention provides the use of serum amyloid a1 in the preparation of a biomarker for diagnosing polycystic ovarian syndrome, and the use is to solve the technical problem that no good method for diagnosing polycystic ovarian syndrome exists in the prior art.

The invention provides application of serum amyloid A1 in preparation of a biomarker for diagnosing polycystic ovary syndrome.

The invention also provides application of a reagent for detecting serum amyloid A1 in preparing a diagnostic composition for in vitro diagnosis of a biomarker of polycystic ovarian syndrome, wherein the upstream primer sequence for detecting serum amyloid A1 is shown as SEQ ID NO: 1, and the sequence of the downstream primer for detecting serum amyloid A1 is shown as SEQ ID NO: 2, the diagnosis comprising: detecting the level of serum amyloid a1 biomarker from the patient.

The invention discovers that SAA1 (the most important acute phase reaction protein of SAA) is remarkably increased in the serum and follicular fluid of a patient with PCOS, and the serum SAA1 level is closely related to the clinical characteristics of PCOS, such as high LH blood disease, high androgen blood disease, insulin resistance and the like. Meanwhile, the ovarian local SAA1 level is positively correlated with the serum SAA1 level, but the ovarian local SAA1 level is obviously higher than the serum SAA1 level, which indicates that the synthesis of the ovarian local SAA1 and the serum-derived SAA1 are both reasons for the increase of the PCOS ovarian local SAA1, and the ovarian local increased SAA1 can cause the insulin resistance of granulosa cells, thereby causing the dysfunction of the granulosa cells and influencing the follicular development.

Compared with the prior art, the invention has remarkable technical progress. The experimental research result of the invention indicates that SAA1 plays an important role in the occurrence and development of PCOS disease, and SAA1 can be used as a biomarker for evaluating the PCOS disease state.

Drawings

Figure 1 shows that serum SAA1 is significantly elevated in the PCOS population; the figure is as follows: p < 0.001.

Fig. 2 shows that follicular fluid SAA1 is significantly elevated locally in the PCOS ovary; the figure is as follows: p < 0.001.

Figure 3 shows the correlation analysis of follicular fluid with serum SAA 1.

FIG. 4 shows SAA1 expression in ovarian granulosa cells; the figure is as follows: a: SAA1 PCR gel map; b: SAA1 immunofluorescence represents the graph.

FIG. 5 shows that granulosa cells have the ability to synthesize SAA1 de novo; the figure is as follows: a: IL-1 β promotes granulosa cell SAA1mRNA expression,/vs 0, P < 0.05; b IL-1 β promotes synthesis and secretion of granulocyte SAA1 protein,. v s0, P < 0.01.

Figure 6 shows that SAA1 causes granulocytic insulin resistance; the figure is as follows: a: PTEN WB represents diagram, B: vs 0P < 0.01; #, vs 0.01, P <0.05. C: PAKT represents a graph; d: p <0.01, P < 0.001.

Detailed Description

Example 1

[ Experimental techniques ]:

experimental method and materials

1. Collecting human serum and follicular fluid: PCOS and control patient sera and follicular fluid were collected.

The above cases were obtained from the reproductive medicine center of the affiliated renji hospital of Shanghai university of transportation medical school, and were reviewed by the ethical Committee of the renji hospital.

The PCOS diagnosis standard is the LUTAN standard, and the control is the patient with single oviduct factor infertility or male factor infertility. Ovulation is promoted by a conventional method, and blood and follicular fluid are collected on the day of ovum collection. Serum is obtained after blood centrifugation and is frozen, dominant follicular fluid in the first tube is frozen after centrifugation, and granulosa cells are obtained after density gradient centrifugation and enzyme digestion of the residual follicular fluid and are used for freezing and RNA extraction.

2. Granulosa cells SAA1 expression: non-PCOS ovary granular cells are collected, total RNA is extracted, after reverse transcription is carried out, SAA1 is amplified by adopting a common PCR method, an amplification product is subjected to agarose electrophoresis gel electrophoresis, and a solar imaging system is adopted for photographing after the electrophoresis is finished. Performing SAA1 protein expression detection by immunofluorescence, culturing granular cells in vitro for 1 day, adding 1% paraformaldehyde for fixation, performing membrane permeation by 0.1% PBST, performing blocking by 5% goat serum for 2 hours, incubating SAA1 antibody (1:100) overnight, washing with PBS for 3 times, incubating with Alexa Fluor 488 (green) labeled secondary antibody for 2 hours, washing with PBS for 3 times in the dark, and taking pictures by a Zeiss microscope.

3. Human granulosa cell in vitro culture: collecting follicular fluid obtained after ovum retrieval of normal non-PCOS, centrifuging, adding hyaluronidase for digestion, inoculating the isolated granulosa cells into 6-well, 12-well or 24-well plate coated with rat tail collagen, and placing in CO with volume percentage concentration of 5%₂After three days of incubation in an incubator at 37 ℃, the next step was carried out by changing to DMEM/F12 medium (GIBCO, Saimer fly) without serum and phenol red.

4. Human granular cell in-vitro culture treatment: after the granulosa cells obtained in section 1) above were treated with IL-1. beta. (1ng/ml) for 24 hours, cell culture supernatants and RNAs were collected, and the RNA and protein expression levels of SAA1 were measured. After 24 hours of treatment with SAA1(0, 0.01, 0.1, 1. mu.g/ml), the medium was replaced with serum-free medium and treated with 100nM insulin for 15 minutes to detect the expression of PTEN and Akt phosphorylation.

5. Total RNA extraction and reverse transcription: total RNA was isolated from endometrial tissue and cells using TRIzol reagent (Invitrogen) according to the instructions. RNA concentration and quality were determined by a Nanodrop ND-2000 spectrophotometer (Thermo Fisher scientific). cDNA was synthesized using PrimeScript RT kit (Takara, Dalian, China) according to the instructions, taking 500ng of total RNA.

6. Detecting the expression level of SAA1 by real-time fluorescent quantitative PCR: real-time fluorescent quantitative PCR was performed using SYBR Green Master Mix (Takara, Dalian, China) and ABI Prism System (Applied Biosystems, Thermo Fisher Scientific)

Sequence detection of SAA 1:

forward 5'-tttctgctccttggtcctgg-3'; shown as SEQ ID NO. 1.

reverse 5'-ctctggcatcgctgatcact-3'; shown as SEQ ID NO. 2.

Sequence detection for ACTB:

forward 5'-gggaaatcgtgcgtgacattaag-3'; shown as SEQ ID NO. 3.

reverse 5'-tgtgttggcgtacaggtctttg-3'; shown as SEQ ID NO. 4.

All quantitative RT-PCR reactions were performed in triplicate. Relative mRNA levels were calculated using ACTB as the internal control and using a comparative cycle threshold method.

7. Detection of serum and follicular fluid SAA 1: serum and follicular fluid of PCOS and control population were collected and assayed for SAA1 protein levels (R & D) by enzyme-linked immunosorbent assay (ELISA).

8. The statistical method comprises the following steps: adopting single-factor variance analysis to test whether the mean difference of each group has statistical significance; the checking level is p <0.05. SPSS 22.0 statistical software is used for data statistics and analysis.

Second, experimental results

1. Serum SAA1 was significantly elevated in the PCOS population and correlated with the clinical profile of PCOS

We tested serum SAA1 in PCOS and control populations using ELISA and found that serum SAA1 levels were significantly elevated in PCOS populations (P <0.001, N ═ 32/group), as shown in figure 1. Correlation analysis is carried out with clinical indexes, and the serum SAA1 level is found to be in significant positive correlation with BMI, basic LH, LH/FSH, T and AMH levels, which indicates that the serum SAA1 level is closely related with the clinical characteristics of PCOS (Table 1).

BMI, baseline LH, LH/FSH, T and AMH level indicators are important clinical indicators of PCOS in order to show that SAA1 is closely related to the clinical features of PCOS.

TABLE 1 correlation of serum SAA1 with clinical indicators of PCOS

2. The follicular fluid SAA1 is significantly increased in ovary local part of PCOS patient

When the SAA1 level in follicular fluid is detected by ELISA method by collecting follicular fluid of PCOS and control, the SAA1 level of follicular fluid of PCOS patient is remarkably increased (P)<0.001, N ═ 32/group), see fig. 2. Further correlation analysis, we found that the level of SAA1 in follicular fluid was positively correlated with the level of SAA1 in serum (R)²＝0.18,P<0.001), see fig. 3.

3. Ovary granular cell SAA1 expression and synthesis

We found that ovarian granulosa cells express both SAA1mRNA and SAA1 protein using immunofluorescence and PCR techniques, as shown in FIG. 4. We collected cell culture supernatant by using a granulosa cell in vitro culture model, and found that the granulosa cells can secrete SAA1 protein, the basal expression level of the protein is about 30ng/ml, and the addition of inflammatory factor interleukin-1 beta can significantly increase the ability of the granulosa cells to secrete SAA1, as shown in FIG. 5. This suggests that granulosa cells have the ability to de novo synthesize SAA 1.

SAA1 causing insulin resistance in ovarian granulosa cells

We used an in vitro granular cell culture model, and added SAA1 in vitro, and found that SAA1 can promote PTEN expression with a concentration gradient. But significantly down-regulated insulin-stimulated Akt phosphorylation, leading to granular cell insulin resistance, see figure 6. This suggests that too much SAA1 may lead to granulocytic insulin resistance.

The results show that the SAA1 is remarkably increased in the serum and the ovary of the PCOS patient, and the serum SAA1 level is closely related to the clinical characteristics of PCOS high LH blood disease, high androgen blood disease and the like. At the same time, locally increased SAA1 of the ovary can cause the insulin resistance of the granulosa cell to cause the dysfunction of the granulosa cell, and further influences the development of the PCOS follicle. These studies suggest that SAA1 plays an important role in the development of PCOS, and SAA1 can be used as a new biomarker to indicate the occurrence and progression of PCOS diseases.

Sequence listing

<110> Shanghai university of traffic medical college affiliated renji hospital

Application of <120> serum amyloid A1 in preparation of biomarker for diagnosing polycystic ovarian syndrome

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tttctgctcc ttggtcctgg 20

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ctctggcatc gctgatcact 20

<210> 3

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gggaaatcgt gcgtgacatt aag 23

<210> 4

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tgtgttggcg tacaggtctt tg 22

<210> 5

<211> 122

<212> PRT

<213> Homo sapiens

<400> 5

Met Lys Leu Leu Thr Gly Leu Val Phe Cys Ser Leu Val Leu Gly Val

1 5 10 15

Ser Ser Arg Ser Phe Phe Ser Phe Leu Gly Glu Ala Phe Asp Gly Ala

20 25 30

Arg Asp Met Trp Arg Ala Tyr Ser Asp Met Arg Glu Ala Asn Tyr Ile

35 40 45

Gly Ser Asp Lys Tyr Phe His Ala Arg Gly Asn Tyr Asp Ala Ala Lys

50 55 60

Arg Gly Pro Gly Gly Ala Trp Ala Ala Glu Val Ile Thr Asp Ala Arg

65 70 75 80

Glu Asn Ile Gln Arg Phe Phe Gly His Gly Ala Glu Asp Ser Leu Ala

85 90 95

Asp Gln Ala Ala Asn Glu Trp Gly Arg Ser Gly Lys Asp Pro Asn His

100 105 110

Phe Arg Pro Ala Gly Leu Pro Glu Lys Tyr

115 120

Claims

1. Use of serum amyloid A1 in preparation as a biomarker for diagnosing polycystic ovary syndrome.

2. The purposes of the reagent for detecting serum amyloid protein A1 in the preparation of the diagnostic composition in the biomarker for in vitro diagnosis of polycystic ovary syndrome, the upstream primer sequence for the detection of serum amyloid protein A1 is such as SEQ ID As shown in NO: 1, the downstream primer sequence for detecting serum amyloid A1 is shown in SEQ ID NO: 2, and the diagnosis comprises: detecting the level of serum amyloid A1 biomarkers from patients.