CN111518881B - System for diagnosing hormonal femoral head necrosis through molecular markers - Google Patents

System for diagnosing hormonal femoral head necrosis through molecular markers Download PDF

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CN111518881B
CN111518881B CN201910108767.9A CN201910108767A CN111518881B CN 111518881 B CN111518881 B CN 111518881B CN 201910108767 A CN201910108767 A CN 201910108767A CN 111518881 B CN111518881 B CN 111518881B
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陈卫衡
张彦琼
林娜
李泰贤
王荣田
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Abstract

The present disclosure relates to a system for diagnosing hormonal femoral head necrosis by molecular markers, wherein the molecular markers are BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR 4. The system and the application provided by the disclosure can be applied to assessment and diagnosis of SONFH high risk groups by detecting the expression quantity of the molecular marker, and assist in making early diagnosis, so as to provide an effective basis for guiding clinical diagnosis and treatment.

Description

System for diagnosing hormonal femoral head necrosis through molecular markers
Technical Field
The disclosure relates to the technical field of clinical detection, in particular to a system for diagnosing hormonal femoral head necrosis through a molecular marker.
Background
Femoral Head necrosis (Osteoneprosis of the Femoral Head, ONFH) is a common disease of orthopedics, the number of the disease attack in China currently exceeds ten million, and 15-30 ten thousand new patients are newly added every year. The Femoral Head necrosis with different causes can be divided into traumatic Femoral Head necrosis (which is mostly caused by traumatic factors such as Femoral neck fracture and Femoral Head dislocation) and non-traumatic Femoral Head necrosis (NONFH) which is mostly caused by receiving glucocorticoid medicaments, drinking and the like.
Hormone-induced femoral head necrosis (SONFH) is a non-traumatic femoral head necrosis caused by long-term hormone use, and the incidence rate of the SONFH exceeds that of femoral head necrosis caused by trauma at present.
At present, the most effective method for diagnosing the femoral head necrosis is MRI examination, the early diagnosis rate reaches more than 90 percent, but the further development of the necrosis is difficult to prevent after the diagnosis is confirmed. Therefore, for the high risk population of SONFH, it is very important to predict the occurrence of osteonecrosis and to prevent it in advance.
Disclosure of Invention
The purpose of the present disclosure is to provide a kit and a system for diagnosing hormonal femoral head necrosis by using a molecular marker, and the use of the molecular marker in diagnosing the hormonal femoral head necrosis.
To achieve the above object, a first aspect of the present disclosure: the system comprises a computing device, an input device and an output device, wherein the input device is used for inputting the expression quantity of the molecular marker of an individual patient with femoral head necrosis, and the output device is used for outputting the diagnosis result of the femoral head necrosis; wherein the molecular marker is BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR 4; the computing device comprises a memory and a processor; the memory has a computer program stored therein, and the processor is configured to execute the computer program stored in the memory to implement a modeling algorithm and an algorithm of a discriminant function as shown in equation (1); the modeling algorithm is a minimum deviation two-times algorithm;
F(c)＝sgn[f1(c1)+f2(c2)+f3(c3)+f4(c4)+f5(c5)+f6(c6)+f7(c7)+f8(c8)-b]In the formula (1), F (c) represents the diagnosis result of the hormonal femoral head necrosis, and the return value of F (c) is 1When the return value is-1, the femoral head necrosis is induced; c. C1～c8Sequentially and respectively representing the relative expression amounts of BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR 4; the relative expression amount refers to the ratio of the expression amount relative to an internal reference; f. of1(c1)～f8(c8) The kernel functions are obtained by training according to the modeling algorithm, and b is the critical score values obtained by training according to the modeling algorithm.
Optionally, the system further comprises a device for detecting the expression level of the molecular marker; the detection device comprises a molecular marker expression quantity detection chip and a chip signal reader, wherein the molecular marker expression quantity detection chip comprises probes for respectively detecting the expression quantities of BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR 4.
Optionally, the chip for detecting the expression level of the molecular marker further comprises an internal reference probe, wherein the internal reference probe is a probe for detecting the expression level of GAPDH or β -Actin.
Optionally, the detection device comprises a real-time quantitative PCR instrument and real-time quantitative PCR primers of the molecular marker, wherein the real-time quantitative PCR primers of the molecular marker comprise real-time quantitative PCR primers for respectively detecting the expression levels of BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR 4.
Optionally, the real-time quantitative PCR primers of the molecular marker further include an internal reference primer, and the internal reference primer is a real-time quantitative PCR primer for detecting GAPDH or β -Actin.
Optionally, in formula (1), the internal reference is GAPDH, f1(c1)＝-0.097×c1，f2(c2)＝0.019×c2，f3(c3)＝-0.316×c3，f4(c4)＝0.292×c4，f5(c5)＝0.681×c5，f6(c6)＝0.375×c6，f7(c7)＝0.428×c7，f8(c8)＝0.129×c8B is 0.040; alternatively, the first and second electrodes may be,
the internal reference is beta-Actin, f1(c1)＝-0.012×c1，f2(c2)＝-0.329×c2，f3(c3)＝-0.254×c3，f4(c4)＝0.332×c4，f5(c5)＝0.561×c5，f6(c6)＝0.422×c6，f7(c7)＝0.469×c7，f8(c8)＝-0.052×c8，b＝0.024。
In a second aspect of the present disclosure: the application of a reagent for quantitatively detecting a molecular marker in preparing a product for diagnosing the hormonal femoral head necrosis is provided, wherein the molecular marker is BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR 4.
Alternatively, the quantitative detection of the molecular marker is performed by:
1) obtaining a serum sample of a patient with femoral head necrosis;
2) determining the expression level of the molecular marker in the serum sample.
A third aspect of the disclosure: a kit for diagnosing hormonal femoral head necrosis is provided, wherein the kit comprises a reagent for quantitatively detecting molecular markers, and the molecular markers are BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR 4.
A fourth aspect of the present disclosure: a molecular marker combination for diagnosing hormonal femoral head necrosis is provided, wherein the molecular marker combination comprises BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR 4.
Through the technical scheme, the system and the application provided by the disclosure can be applied to assessment and diagnosis of SONFH high-risk people by detecting the expression quantity of the molecular marker, and assist in making early diagnosis, so that an effective basis is provided for guiding clinical diagnosis and treatment.
Additional features and advantages of the present disclosure will be set forth in the detailed description which follows.
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The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure without limiting the disclosure. In the drawings:
fig. 1-2 are ROC graphs obtained by diagnosing hormonal femoral head necrosis using the molecular markers of the present disclosure in examples, wherein fig. 1 is internally referenced as GAPDH, and fig. 2 is internally referenced as β -Actin.
Detailed Description
The following detailed description of specific embodiments of the present disclosure is provided in connection with the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.
The first aspect of the disclosure: the system comprises a computing device, an input device and an output device, wherein the input device is used for inputting the expression quantity of the molecular marker of an individual patient with femoral head necrosis, and the output device is used for outputting the diagnosis result of the femoral head necrosis; wherein the molecular marker is BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR 4; the computing device comprises a memory and a processor; the memory has a computer program stored therein, and the processor is configured to execute the computer program stored in the memory to implement a modeling algorithm and an algorithm of a discriminant function as shown in equation (1); the modeling algorithm is a minimum deviation two-times algorithm;
F(c)＝sgn[f1(c1)+f2(c2)+f3(c3)+f4(c4)+f5(c5)+f6(c6)+f7(c7)+f8(c8)-b]In the formula (1), f (c) represents a diagnosis result of the hormonal femoral head necrosis, f (c) has a return value of 1 indicating that the hormonal femoral head necrosis is established, and f (c) has a return value of-1 indicating that the hormonal femoral head necrosis is not established; c. C1～c8Sequentially and respectively representing the relative expression amounts of BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR 4; the relative expression amount refers to the ratio of the expression amount relative to an internal reference; f. of1(c1)～f8(c8) Respectively, kernel functions obtained by training according to the modeling algorithm, and b is the basis of the modelingAnd training a module algorithm to obtain a critical score value. The least-squares multiplication algorithm and the operation and training method thereof are known conventional methods.
In accordance with the present disclosure, BIRC3 is referenced 330 in the NCBI database; CBL reference number 867 in NCBI database; CCR5 reference number 1234 in the NCBI database; LYN is referenced 4067 in NCBI database; PAK1 reference number 5058 in NCBI database; reference number of PTEN in NCBI database is 5728; reference number 5894 of RAF1 in NCBI database; TLR4 is referenced 7099 in the NCBI database.
According to the present disclosure, the system may further comprise a device for detecting the expression level of the molecular marker. The detection device comprises a molecular marker expression quantity detection chip and a chip signal reader, wherein the molecular marker expression quantity detection chip comprises probes for respectively detecting the expression quantities of BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR 4. Or the detection device comprises a real-time quantitative PCR instrument and real-time quantitative PCR primers of the molecular marker, wherein the real-time quantitative PCR primers of the molecular marker comprise real-time quantitative PCR primers for respectively detecting the expression levels of BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR 4.
According to the disclosure, the chip for detecting the expression level of the molecular marker further comprises an internal reference probe, wherein the internal reference probe is a probe for detecting the expression level of GAPDH or beta-Actin. The real-time quantitative PCR primer of the molecular marker also comprises an internal reference primer, and the internal reference primer is a real-time quantitative PCR primer for detecting GAPDH or beta-Actin.
According to the present disclosure, when the internal reference is GAPDH, as a set of reference values obtained by training, in formula (1), f1(c1)＝-0.097×c1，f2(c2)＝0.019×c2，f3(c3)＝-0.316×c3，f4(c4)＝0.292×c4，f5(c5)＝0.681×c5，f6(c6)＝0.375×c6，f7(c7)＝0.428×c7，f8(c8)＝0.129×c8，b＝0.040; that is, the discriminant function can be simplified as shown in equation (2):
F(c)＝sgn[-0.097×c1+0.019×c2-0.316×c3+0.292×c4+0.681×c5+0.375×c6+0.428×c7+0.129×c8-0.040]formula (2).
Or, when the internal parameter is beta-Actin, the internal parameter is used as a group of reference values obtained by training, and in the formula (1), f1(c1)＝-0.012×c1，f2(c2)＝-0.329×c2，f3(c3)＝-0.254×c3，f4(c4)＝0.332×c4，f5(c5)＝0.561×c5，f6(c6)＝0.422×c6，f7(c7)＝0.469×c7，f8(c8)＝-0.052×c8B is 0.024; that is, the discriminant function can be simplified as shown in equation (3):
F(c)＝sgn[-0.012×c1-0.329×c2-0.254×c3+0.332×c4+0.561×c5+0.422×c6+0.469×c7-0.052×c8-0.024]formula (3).
In addition, f is1(c1)～f8(c8) And b may vary depending on the bias of the means for detecting the expression level of the molecular marker, or may vary depending on factors such as the size of the data scale of the training data set. The discriminant functions shown in equations (2) and (3) are obtained by training the inventors of the present disclosure with a modeling algorithm of least-squares multiplication according to the data in the examples, and do not limit the scope of the present disclosure.
In a second aspect of the present disclosure: the application of a reagent for quantitatively detecting a molecular marker in preparing a product for diagnosing the hormonal femoral head necrosis is provided, wherein the molecular marker is BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR 4.
Further, the quantitative detection of the molecular marker is performed by the following steps:
1) obtaining a serum sample of a patient with femoral head necrosis;
2) determining the expression level of the molecular marker in the serum sample.
Wherein, the method of step 2) may be a qPCR method.
The system and the application provided by the disclosure can be applied to assessment and diagnosis of SONFH high risk groups by detecting the expression quantity of the molecular marker, and assist in making early diagnosis, so as to provide an effective basis for guiding clinical diagnosis and treatment.
A third aspect of the disclosure: a kit for diagnosing hormonal femoral head necrosis is provided, wherein the kit comprises a reagent for quantitatively detecting molecular markers, and the molecular markers are BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR 4.
Further, the reagents may be qPCR reagents.
A fourth aspect of the present disclosure: a molecular marker combination for diagnosing hormonal femoral head necrosis is provided, wherein the molecular marker combination comprises BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR 4.
In a particularly preferred embodiment of the present disclosure, the molecular marker combination consists of BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR 4.
The kit and the molecular marker combination provided by the disclosure can be applied to assessment and diagnosis of SONFH high-risk people by detecting the expression quantity of the molecular marker, assist in making early diagnosis and provide effective basis for guiding clinical diagnosis and treatment.
The present disclosure will be described in detail below with reference to examples. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.
In the examples, the analysis was performed using SPSS19.0 statistical software. The metrology data is expressed as mean ± standard deviation. The comparison between the counting data and the metering data adopts X2 test, the comparison between the metering data and the metering data adopts t test, the ROC curve is used for analyzing the diagnostic efficiency of the molecular marker, the prediction accuracy is calculated by the area under the ROC curve, and the difference p <0.05 has statistical significance.
Examples
This example serves to illustrate the discovery of biomarkers and the establishment and validation of predictive models of the present disclosure.
60 patients (including 20 patients with phlegm stasis collateral blocking syndrome, meridian obstruction syndrome and liver and kidney deficiency syndrome) diagnosed as SONFH at the WangJing Hospital of Chinese medical academy of sciences and the Zhengzhou bone fracture Hospital from 1 month to 2017 months in 2014 are collected as disease groups; at the same time, 20 patients who did not suffer from femoral head necrosis after receiving hormone therapy in the hematological hospital of the Chinese academy of medicine were taken as a control group. 80 patients were randomly divided into a training set (n-40, phlegm-stasis blocking collaterals, meridian obstruction, liver and kidney deficiency, 10 cases of control group) and a verification set (n-40, disease group and control group are the same as verification set).
The diagnosis of femoral head necrosis refers to the diagnosis standard provided in the literature, "Mont MA, Hungerford DS.non-particulate avascular necrosis of the femoral head [ J ] J Bone Joint Surg (Am),1995,77(3): 459-.
Inclusion criteria were: patients who meet the non-traumatic femoral head necrosis diagnosis standard; patients who do not have femoral head necrosis after 1 year follow-up of hormone treatment. Exclusion criteria: the primary disease is serious, and other treatments are needed to replace hormone.
Designing a questionnaire according to DME requirements, and investigating age, sex, height, weight, disease course, hormone using time, clinical manifestations and physical signs, and primary disease history.
The general clinical data such as sex, body mass index, age, hormone use time and the like of patients in the training set and the verification set are compared in a table 1, and the differences have no statistical significance (P >0.05) and are comparable.
TABLE 1
Figure BDA0001967300580000081
The patients in the training set and the verification set both draw 2.5ml venous Blood on an empty stomach in early morning before treatment, and then put into PAXgene Blood RNA Tubes, after being turned upside down and mixed evenly, the mixture is stored in a refrigerator at 20 ℃.
Serum: extracting with coagulant and separating gel test tube, centrifuging at 3500r/m for 10min to separate serum, and storing at-80 deg.C.
RNA extraction, quality inspection and chip detection are carried out by Shanghai Bohao Biotechnology limited.
The chip was scanned and raw data read using GeneChip Scanner 3000 image Analysis Software and Command sample Software 4.0 (Affymetrix, usa), and microarray Significance Analysis Software (SAM) was used to screen out genes with statistical Significance between different syndromes of sonofh and the control group. The percentage range of these genes was identified based on FDR (false Discovery Rate) values.
Based on the interaction information of the difference genes of the hormonal femoral head necrosis compared with the control group sample, an interaction network is established, and the topological characteristic values of the network nodes are calculated to include node connectivity (namely the number of interactions between a certain node i and other nodes in the network), node betweenness (namely the number of shortest paths between all node pairs through the certain node i), and node compactness (namely the centrality measure of the certain node i in the network graph, namely the average distance from the node i to other reachable nodes). And selecting the nodes with the topological characteristic values larger than the median number as key nodes of a hormonal femoral head necrosis network to obtain candidate molecular markers of the hormonal femoral head necrosis, wherein the molecular markers are BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR 4.
Based on the expression quantity information of the molecular markers in the peripheral blood of the SONFH patient, a differential model of the hormonal femoral head necrosis is established by adopting a partial least square method, and the weight value of each candidate molecular marker in the model and the score card value of the model are calculated. The model is shown as formula (1).
F(c)＝sgn[f1(c1)+f2(c2)+f3(c3)+f4(c4)+f5(c5)+f6(c6)+f7(c7)+f8(c8)-b]The compound of the formula (1),
in the formula (1), f (c) represents the diagnosis result of the hormonal femoral head necrosis, f (c) indicates that the hormonal femoral head necrosis is established when the return value is 1, and indicates that the hormonal femoral head necrosis is not established when the return value is-1; c. C1～c8Sequentially and respectively representing the relative expression levels of BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR 4; the relative expression amount refers to the ratio of the expression amount relative to an internal reference; f. of1(c1)～f8(c8) The kernel functions are obtained by training according to the modeling algorithm, and b is the critical score values obtained by training according to the modeling algorithm. Specifically, when the internal reference is GAPDH, f in formula (1) is used as a set of reference values obtained by training1(c1)＝-0.097×c1，f2(c2)＝0.019×c2，f3(c3)＝-0.316×c3，f4(c4)＝0.292×c4，f5(c5)＝0.681×c5，f6(c6)＝0.375×c6，f7(c7)＝0.428×c7，f8(c8)＝0.129×c8B is 0.040; that is, the discriminant function can be simplified as shown in equation (2):
F(c)＝sgn[-0.097×c1+0.019×c2-0.316×c3+0.292×c4+0.681×c5+0.375×c6+0.428×c7+0.129×c8-0.040]formula (2).
Or, when the internal parameter is beta-Actin, the internal parameter is used as a group of reference values obtained by training, and in the formula (1), f1(c1)＝-0.012×c1，f2(c2)＝-0.329×c2，f3(c3)＝-0.254×c3，f4(c4)＝0.332×c4，f5(c5)＝0.561×c5，f6(c6)＝0.422×c6，f7(c7)＝0.469×c7，f8(c8)＝-0.052×c8B is 0.024; that is, the discriminant function can be simplified as shown in equation (3):
F(c)＝sgn[-0.012×c1-0.329×c2-0.254×c3+0.332×c4+0.561×c5+0.422×c6+0.469×c7-0.052×c8-0.024]Formula (3).
And developing qPCR detection aiming at the training set samples. The total RNA is reversely transcribed into cDNA, and real-time quantitative PCR amplification is carried out by taking the cDNA as a template, and meanwhile, beta-Actin and GAPDH are taken as reference groups. QPCR was performed using a Power SYBR Green PCR Master Mit Kit using an ABI 7900HT real-time incinerator-optical quantitative PCR instrument (ABI, USA). Reaction conditions are as follows: incubate at 50 ℃ for 2min, then 95 ℃, 10 min: then 40 cycles were performed: at 95 ℃ for 15 seconds; at 60 ℃ for 1min, after the reaction is finished, confirming the amplification curve and the melting curve of QPCR
Based on independent sample sets, the expression levels of BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR4 in peripheral blood of patients are detected by a qPCR method (two groups of expression level data are generated based on two internal references of GAPDH and beta-Actin). The evaluation indexes comprise prediction Accuracy (ACC) and area under the operating characteristic curve (ROC curve for short) of a receiver, the results are shown in the figure 1-2, the ACC result is 91.20%, and the AUC result is 0.901, which indicates that BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR4 can be used as molecular markers for diagnosing the steroid-induced femoral head necrosis.
The preferred embodiments of the present disclosure are described in detail above with reference to the accompanying drawings, however, the present disclosure is not limited to the specific details in the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.
It should be noted that, in the above embodiments, the various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations will not be further described in the present disclosure.
In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure as long as it does not depart from the gist of the present disclosure.

Claims (6)

1. A system for diagnosing hormonal femoral head necrosis is characterized by comprising a computing device, an input device and an output device, wherein the input device is used for inputting the expression quantity of a molecular marker of an individual patient with femoral head necrosis; wherein the molecular marker is BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR 4; the computing device comprises a memory and a processor; the memory has a computer program stored therein, and the processor is configured to execute the computer program stored in the memory to implement a modeling algorithm and an algorithm of a discriminant function as shown in equation (1); the modeling algorithm is a minimum deviation two-times algorithm;
F (c) = sgn [ f1(c1) + f2(c2) + f3(c3) + f4(c4) + f5(c5) + f6(c6) + f7(c7) + f8(c8) -b ] formula (1),
in the formula (1), f (c) represents the diagnosis result of the hormonal femoral head necrosis, f (c) has a return value of 1 indicating that the hormonal femoral head necrosis is established, and has a return value of-1 indicating that the hormonal femoral head necrosis is not established; c 1-c 8 respectively represent the relative expression amounts of BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR4 in sequence; the relative expression amount refers to the ratio of the expression amount relative to an internal reference; f1(c1) -f 8(c8) are kernel functions obtained through training according to the modeling algorithm respectively, and b is a critical score value obtained through training according to the modeling algorithm;
the system also comprises a device for detecting the expression level of the molecular marker; the detection device comprises a molecular marker expression level detection chip and a chip signal reader, wherein the molecular marker expression level detection chip comprises probes for respectively detecting the expression levels of BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR 4;
the molecular marker expression level detection chip also comprises an internal reference probe, wherein the internal reference probe is a probe for detecting the expression level of GAPDH or beta-Actin;
in formula (1), the internal parameter is GAPDH, f1(c1) = -0.097 × c1, f2(c2) =0.019 × c2, f3(c3) = -0.316 × c3, f4(c4) =0.292 × c4, f5(c5) =0.681 × c5, f6(c6) =0.375 × c6, f7(c7) =0.428 × c7, f8(c8) =0.129 × c8, and b = 0.040; alternatively, the first and second electrodes may be,
The internal parameters are β -Actin, f1(c1) = -0.012 × c1, f2(c2) = -0.329 × c2, f3(c3) = -0.254 × c3, f4(c4) =0.332 × c4, f5(c5) =0.561 × c5, f6(c6) =0.422 × c6, f7(c7) =0.469 × c7, f8(c8) = -0.052 × c8, b = 0.024.
2. The system according to claim 1, wherein the detection device comprises a real-time quantitative PCR instrument and real-time quantitative PCR primers of the molecular markers, and the real-time quantitative PCR primers of the molecular markers comprise real-time quantitative PCR primers for respectively detecting the expression levels of BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR 4.
3. The system of claim 2, wherein the real-time quantitative PCR primers of the molecular marker further comprise an internal reference primer, and the internal reference primer is a real-time quantitative PCR primer for detecting GAPDH or beta-Actin.
4. Use of a reagent for quantitatively detecting molecular markers in the preparation of a product for diagnosing hormonal femoral head necrosis, wherein the molecular markers are BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR 4.
5. Use according to claim 4, wherein the quantitative detection of the molecular marker is carried out by:
1) obtaining a serum sample of a patient with femoral head necrosis;
2) Determining the expression level of the molecular marker in the serum sample.
6. A kit for diagnosing hormonal femoral head necrosis, wherein the kit comprises a reagent for quantitatively detecting molecular markers, and the molecular markers are BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1 and TLR 4.
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