阅读说明：本技术 基于子宫颈脱落细胞基因检测的***分期方法与检测试剂盒 (Cervical cancer staging method and detection kit based on cervical exfoliated cell gene detection ) 是由 曾新 柳宇 许争峰 李萍 胡平 毛鹏远 张根 芮璨 栾婷 于 2019-11-15 设计创作，主要内容包括：本发明公开了基于子宫颈脱落细胞基因检测的宫颈癌分期方法,采集子宫颈脱落细胞和活体组织、提取全基因组DNA、基因检测以及免疫组化检查；本发明还公开了基于子宫颈脱落细胞基因检测的宫颈癌分期检测试剂盒,包括含有特异性引物的PCR反应液；本发明对正常子宫颈、癌前病变子宫颈、子宫颈癌的脱落细胞进行DNA定量检测,从脱落细胞的角度阐明了P53、P16与子宫颈癌发生发展的相关性,为宫颈癌的分期诊断提供了新的依据；提取宫颈脱落细胞中的DNA进行检测,具有采样量低的优势；取材简便无创且患者无不适感,可在妇产科常规检查生殖系统疾病时获得待检样本,本发明还具有取材无创、副作用小、患者依从性好的优势。(The invention discloses a cervical cancer staging method based on cervical exfoliated cell gene detection, which comprises the steps of collecting cervical exfoliated cells and living tissues, extracting whole genome DNA, performing gene detection and performing immunohistochemical examination; the invention also discloses a cervical cancer stage detection kit based on cervical exfoliated cell gene detection, which comprises PCR reaction liquid containing specific primers; the invention carries out DNA quantitative detection on the cast-off cells of normal cervix, precancerous lesion cervix and cervical cancer, clarifies the correlation between P53 and P16 and the occurrence and development of cervical cancer from the angle of the cast-off cells, and provides a new basis for the staged diagnosis of cervical cancer; the DNA in the exfoliated cervical cells is extracted for detection, so that the method has the advantage of low sampling amount; the invention has the advantages of convenient material taking, no wound, no discomfort of patients, capability of obtaining a sample to be detected in the routine examination of reproductive system diseases in obstetrics and gynecology department, no wound of material taking, small side effect and good compliance of patients.)

1. The cervical cancer stage classification method based on cervical exfoliated cell gene detection is characterized by comprising the following steps:

s1: collecting cervical exfoliated cells and living tissues from three groups of people including normal cervix, cervical lesion and cervical cancer;

s2: respectively extracting whole genome DNA in cervical exfoliated cells and living tissues;

s3: carrying out quantitative detection on the P53 and P16 genes so as to determine whether the gene expression in the exfoliated cells is consistent with the detection result of the living tissues;

s4: immunohistochemical examination is carried out on tissue sections of three groups of people with normal cervix, cervix lesion and cervical cancer, so as to determine the relation between the expression of P53 and P16 genes in the exfoliated cells and the stage of the cervical cancer.

2. The cervical cancer staging detection kit based on cervical exfoliated cell gene detection according to claim 1, comprising a PCR reaction solution containing specific primers, the PCR reaction solution including: p53 forward primer, P53 reverse primer, P16 forward primer, P16 reverse primer, GAPDH forward primer, GAPDH reverse primer, 10 × PCR Buffer, ROX and nuclease-free water.

3. The cervical cancer stage detection kit based on cervical exfoliated cell gene detection according to claim 2, wherein: the final concentration of each component of the PCR reaction solution is as follows: 1 XPCR Buffer, 0.5. mu. M P53 forward primer, 0.5. mu. MP53 reverse primer, 0.5. mu. M P16 forward primer, 0.5. mu. M P16 reverse primer, 0.5. mu.M GAPDH forward primer, 0.5. mu. MGAPDH reverse primer, 1 XROX.

Technical Field

The invention relates to the technical field of in-vitro diagnosis, in particular to a cervical cancer staging method and a cervical cancer staging kit based on cervical exfoliated cell gene detection.

Background

Cervical cancer is one of the most common malignancies in women, with about 50 million new cases per year worldwide and 25 million deaths; only in China, about 13.5 million new cases account for 1/4 of the total cases of the world each year on average, and the death cases are more than 5 ten thousands of the year; therefore, the prevention and treatment of cervical cancer is not slow. According to histopathological typing, cervical cancer is mainly classified into squamous cell carcinoma (abbreviated as squamous carcinoma) and adenocarcinoma, wherein squamous carcinoma accounts for about 75-80% and adenocarcinoma accounts for about 20-25%. Early detection of cervical cancer is usually achieved by regular health examinations. In the course of cervical cancer, cells are transformed from normal to abnormal before canceration, and a series of precancerous lesions exist. Therefore, cervical lesions are discovered in time through screening, corresponding treatment schemes are formulated according to the grades and the stages of the cervical lesions, the trend of the cervical lesions towards the development of the cervical cancer is stopped, and the method has important significance for improving the prevention and treatment effect of the cervical cancer.

At present, cervical cancer screening generally adopts cervical lesion three-step diagnosis (liquid-based cytology, colposcope and histopathological diagnosis) combined HPV detection, but early diagnosis of cervical cancer is still difficult, and the method mainly comprises the following four points that ① liquid-based cytology examination has no obvious distinction on cervical cell lesion levels, and is greatly influenced by subjective factors, such as overlapping of blood and mucous cells influences identification of abnormal cells, reduces accuracy, causes high false negative rate and is easy to miss diagnosis and misdiagnosis, ② colposcope and histopathological diagnosis are invasive examination methods, and simultaneously have the problems that materials are not easy to obtain, typical and sufficient pathological specimens are difficult to obtain, ③ DNA detection sensitivity is high, but specificity is low, positive results only indicate whether transient infection exists, and the value of prediction of occurrence and progress of cervical cancer needs to be further confirmed.

In view of the limitations of the existing methods for screening cervical cancer at present, researchers have been trying to find new methods for early diagnosis from the aspects of protein, gene and the like; except for more conventional technical methods such as immunohistochemistry, Polymerase Chain Reaction (PCR), in-situ hybridization and the like, the research on screening cervical cancer markers by using new technologies such as DNA ploidy analysis, gene sequencing, circulating free DNA detection and the like is reported, but most of the new technologies still stay in the scientific research stage, cannot be really used for clinical early screening and cannot be used for the staging of cervical cancer; therefore, the development of noninvasive detection kits for early and staged diagnosis of cervical cancer is still urgent.

The history of cervical cancer has been studied more often, and its onset and progression is typically a quantitative to qualitative process that takes years to decades. Among them, the continuous and repeated infection of HPV virus is the important reason for the generation of cervical cancer, mainly the combination of HPV virus oncogene protein and cell gene product (P53, P16, etc.) to change cell cycle and DNA repair, resulting in genomic instability and cell cycle abnormality, thereby making cervical cells undergo malignant transformation and develop into cervical cancer; therefore, the detection of some cell gene products (P53, P16 and the like) except HPV virus is necessary for early screening of cervical cancer, and the change of the cell gene products can also provide a basis for the stage of cervical cancer.

Disclosure of Invention

The invention aims to provide a cervical cancer staging method and a cervical cancer staging kit based on cervical exfoliated cell gene detection, so as to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme: the cervical cancer stage classification method based on cervical exfoliated cell gene detection comprises the following steps:

s1: collecting cervical exfoliated cells and living tissues from three groups of people including normal cervix, cervical lesion and cervical cancer;

s2: respectively extracting whole genome DNA in cervical exfoliated cells and living tissues;

s3: carrying out quantitative detection on the P53 and P16 genes so as to determine whether the gene expression in the exfoliated cells is consistent with the detection result of the living tissues;

s4: immunohistochemical examination is carried out on tissue sections of three groups of people with normal cervix, cervix lesion and cervical cancer, so as to determine the relation between the expression of P53 and P16 genes in the exfoliated cells and the stage of the cervical cancer.

The cervical cancer stage detection kit based on cervical exfoliated cell gene detection comprises a PCR reaction solution containing specific primers, wherein the PCR reaction solution comprises: p53 forward primer, P53 reverse primer, P16 forward primer, P16 reverse primer, GAPDH forward primer, GAPDH reverse primer, 10 × PCR Buffer, ROX and nuclease-free water.

Preferably, the final concentration of each component of the PCR reaction solution is: 1 XPCR Buffer, 0.5. mu. M P53 forward primer, 0.5. mu. M P53 reverse primer, 0.5. mu. M P16 forward primer, 0.5. mu. M P16 reverse primer, 0.5. mu.M GAPDH forward primer, 0.5. mu.M GAPDH reverse primer, 1 XROX.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention carries out DNA quantitative detection on the cast-off cells of normal cervix, precancerous lesion cervix and cervical cancer, clarifies the correlation between P53 and P16 and the occurrence and development of cervical cancer from the angle of the cast-off cells, and provides a new basis for the staged diagnosis of cervical cancer;

2. the DNA in the exfoliated cervical cells is extracted for detection, so that the method has the advantage of low sampling amount;

3. based on the position and the structural characteristics of the cervix, the exfoliated cells of the cervix/vagina are collected for detection, the materials are easy to obtain, the wound is not generated, the patient does not feel uncomfortable, and the sample to be detected can be obtained when the reproductive system diseases are routinely checked in the department of obstetrics and gynecology.

Drawings

FIG. 1 Aa shows the change in expression level of P53 gene in cervical tissue of patients with cervical normality, precancerous lesion, and cervical cancer;

FIG. 1 Ab is a graph showing the change in expression level of P53 gene in exfoliated cervical cells of patients with cervical normality, precancerous lesion, and cervical cancer;

FIG. 2 Ba shows the change in expression level of P16 gene in cervical tissue of patients with normal cervix, precancerous lesion, and cervical cancer;

FIG. 2 Bb shows the expression level change of P16 gene in exfoliated cervical cells of patients with normal cervix, precancerous lesion, and cervical cancer;

FIG. 3 shows the immunohistochemical results of P53 gene in cervical tissue of patients with cervical normality, precancerous lesion, and cervical cancer;

FIG. 4 shows the immunohistochemical results of P16 gene in cervical tissue of patients with cervical normality, precancerous lesion, and cervical cancer;

FIG. 5 shows an amplification curve and a melting curve of the P53 gene;

FIG. 6 shows an amplification curve and a melting curve of the P16 gene;

FIG. 7 shows an amplification curve and a melting curve of the GAPDH gene;

FIG. 8 is an electrophoretogram of gene amplification products.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1 to 8, the present invention provides a technical solution: the cervical cancer stage classification method based on cervical exfoliated cell gene detection comprises the following steps:

s1: collecting cervical exfoliated cells and living tissues from three groups of people including normal cervix, cervical lesion and cervical cancer;

s2: respectively extracting whole genome DNA in cervical exfoliated cells and living tissues;

s3: carrying out quantitative detection on the P53 and P16 genes so as to determine whether the gene expression in the exfoliated cells is consistent with the detection result of the living tissues;

s4: immunohistochemical examination is carried out on tissue sections of three groups of people with normal cervix, cervix lesion and cervical cancer, so as to determine the relation between the expression of P53 and P16 genes in the exfoliated cells and the stage of the cervical cancer.

Cervical cancer stage detection kit based on cervical exfoliated cell gene detection comprises PCR reaction liquid containing specific primers, wherein the PCR reaction liquid comprises: p53 forward primer, P53 reverse primer, P16 forward primer, P16 reverse primer, GAPDH forward primer, GAPDH reverse primer, 10 × PCR Buffer, ROX and nuclease-free water.