阅读说明：本技术 通过分子标志物辨识激素性股骨头坏死的中医证型的系统 (System for identifying traditional Chinese medicine syndrome type of hormonal femoral head necrosis through molecular marker ) 是由 陈卫衡 林娜 张彦琼 王荣田 李泰贤 于 2019-02-03 设计创作，主要内容包括：本公开涉及一种通过分子标志物辨识激素性股骨头坏死的中医证型的系统,其中,所述分子标志物包括CD28、CD4、PLCG1、PRKCA、PTGS2、SOS2、STAT6、TLR4、IFIT1、IRF7、ISG15、MAPK14和RHOU。本公开可以应用于通过检测分子标志物的表达量对激素性股骨头坏死的不同中医证型进行区分,并协助作出较早期诊断,为指导临床诊疗提供有效依据。(The present disclosure relates to a system for identifying traditional Chinese medicine evidence of hormonal femoral head necrosis by molecular markers, wherein the molecular markers include CD28, CD4, PLCG1, PRKCA, PTGS2, SOS2, STAT6, TLR4, IFIT1, IRF7, ISG15, MAPK14 and RHOU. The method can be applied to distinguishing different traditional Chinese medicine syndrome types of the hormonal femoral head necrosis by detecting the expression quantity of the molecular marker, assist in making early diagnosis and provide an effective basis for guiding clinical diagnosis and treatment.)

1. A system for identifying traditional Chinese medicine syndrome type of 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 the hormonal femoral head necrosis, and the output device is used for outputting the traditional Chinese medicine syndrome type of the hormonal femoral head necrosis; wherein the molecular markers comprise CD28, CD4, PLCG1, PRKCA, PTGS2, SOS2, STAT6, TLR4, IFIT1, IRF7, ISG15, MAPK14 and RHOU, and the traditional Chinese medicine symptoms of the hormonal femoral head necrosis comprise phlegm stasis channel blocking syndrome, channel obstruction syndrome and liver and kidney deficiency syndrome; the computing device comprises a memory and a processor; the memory is stored with a computer program to realize a modeling algorithm and algorithms of discriminant functions shown as formulas (1), (2) and (3); the modeling algorithm is a minimum deviation two-times algorithm;

F(c-1)＝sgn[f₁(c₁)+f₂(c₂)+f₃(c₃)+f₄(c₄)-b₁]the compound of the formula (1),

F(c-2)＝sgn[f₅(c₅)+f₆(c₆)+f₇(c₇)+f₈(c₈)-b₂]the compound of the formula (2),

F(c-3)＝sgn[f₉(c₉)+f₁₀(c₁₀)+f₁₁(c₁₁)+f₁₂(c₁₂)+f₁₃(c₁₃)-b₃]the compound of the formula (3),

wherein F (c-1) represents the diagnosis result of the phlegm-blood stasis channel blocking syndrome, the return value of F (c-1) is 1, the phlegm-blood stasis channel blocking syndrome is established, and the return value of F (c-1) is-1, the phlegm-blood stasis channel blocking syndrome is not established; f (c-2) represents the diagnosis result of the channel blockage syndrome, the return value of F (c-2) is 1, which represents that the channel blockage syndrome is established, and the return value is-1, which represents that the channel blockage syndrome is not established; f (c-3) represents the diagnosis result of the liver and kidney deficiency, the return value of F (c-3) is 1, the establishment of the liver and kidney deficiency is shown, and the return value of F (c-3) is-1, the establishment of the liver and kidney deficiency is shown; c. C₁～c₁₃Sequentially and respectively representing relative expression amounts of CD28, CD4, PLCG1, PRKCA, PTGS2, SOS2, STAT6, TLR4, IFIT1, IRF7, ISG15, MAPK14 and RHOU; the relative expression amount refers to the ratio of the expression amount relative to an internal reference; f. of₁(c₁)～f₁₃(c₁₃) Are respectively based onKernel function obtained by model algorithm training, b₁～b₃The critical score value obtained by training according to the modeling algorithm.

2. The system according to claim 1, wherein the system further comprises a means 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 CD28, CD4, PLCG1, PRKCA, PTGS2, SOS2, STAT6, TLR4, IFIT1, IRF7, ISG15, MAPK14 and RHOU.

3. The system of claim 2, wherein the chip for detecting the expression level of the molecular marker further comprises an internal reference probe, and the internal reference probe is a probe for detecting the expression level of GAPDH or β -Actin.

4. The system of claim 2, wherein the detection means comprises a real-time quantitative PCR instrument and real-time quantitative PCR primers for molecular markers including real-time quantitative PCR primers that detect the expression levels of CD28, CD4, PLCG1, PRKCA, PTGS2, SOS2, STAT6, TLR4, IFIT1, IRF7, ISG15, MAPK14 and RHOU, respectively.

5. The system of claim 4, 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.

6. The system of claim 1, wherein the internal reference is GAPDH, f₁(c₁)＝-0.6986×c₁，f₂(c₂)＝-0.6991×c₂，f₃(c₃)＝-0.1527×c₃，f₄(c₄)＝-0.1148×c₄，f₅(c₅)＝0.7766×c₅，f₆(c₆)＝0.41×c₆，f₇(c₇)＝-0.4784×c₇，f₈(c₈)＝-0.1515×c₈，f₉(c₉)＝0.5584×c₉，f₁₀(c₁₀)＝0.4779×c₁₀，f₁₁(c₁₁)＝0.433×c₁₁，f₁₂(c₁₂)＝0.3835×c₁₂，f₁₃(c₁₃)＝0.3539×c₁₃，b₁＝0.226，b₂＝0.0246，b₃0.1587; alternatively, the first and second electrodes may be,

the internal reference is β -Actin, f₁(c₁)＝-0.6192×c₁，f₂(c₂)＝-0.7702×c₂，f₃(c₃)＝0.1533×c₃，f₄(c₄)＝0.7519×c₄，f₅(c₅)＝0.5037×c₅，f₆(c₆)＝0.4773×c₆，f₇(c₇)＝-0.7201×c₇，f₈(c₈)＝-0.6149×c₈，f₉(c₉)＝0.554×c₉，f₁₀(c₁₀)＝0.4149×c₁₀，f₁₁(c₁₁)＝0.4048×c₁₁，f₁₂(c₁₂)＝0.5222×c₁₂，f₁₃(c₁₃)＝0.2906×c₁₃，b₁＝0.0091，b₂＝0.0218，b₃＝0.0547。

7. Use of a reagent for quantitatively detecting molecular markers in the preparation of a product for identifying traditional Chinese medicine evidence of hormonal femoral head necrosis, wherein the molecular markers comprise CD28, CD4, PLCG1, PRKCA, PTGS2, SOS2, STAT6, TLR4, IFIT1, IRF7, ISG15, MAPK14 and RHOU.

8. Use according to claim 7, wherein the quantitative detection of molecular markers is carried out by:

1) obtaining a serum sample of a patient with hormonal femoral head necrosis;

2) determining the expression level of the molecular marker in the serum sample.

9. A kit for identifying traditional Chinese medicine syndrome types of hormonal femoral head necrosis, wherein the kit comprises a reagent for quantitatively detecting molecular markers, and the molecular markers comprise CD28, CD4, PLCG1, PRKCA, PTGS2, SOS2, STAT6, TLR4, IFIT1, IRF7, ISG15, MAPK14 and RHOU.

10. A molecular marker combination for identifying traditional Chinese medicine syndrome of hormonal femoral head necrosis, wherein the molecular marker combination comprises CD28, CD4, PLCG1, PRKCA, PTGS2, SOS2, STAT6, TLR4, IFIT1, IRF7, ISG15, MAPK14 and RHOU.

Technical Field

The disclosure relates to the technical field of clinical detection, in particular to a system for identifying traditional Chinese medicine syndrome type of 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 (non-traumatic Osteonecrosis of the bone of the femur neck, Femoral Head dislocation and other traumatic factors) and non-traumatic Femoral Head necrosis (NONFH), the former is caused by the traumatic factors such as Femoral neck fracture and Femoral Head dislocation, and the latter is caused by the reasons of glucocorticoid drug treatment, alcohol addiction 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 disclosure is to provide a use of a molecular marker in identifying traditional Chinese medicine syndrome of hormonal femoral head necrosis, and a kit and a system for identifying traditional Chinese medicine syndrome of hormonal femoral head necrosis by the molecular marker.

To achieve the above object, a first aspect of the present disclosure: the system comprises a computing device, an input device for inputting the expression quantity of the molecular marker of an individual patient with the hormonal femoral head necrosis and an output device for outputting the traditional Chinese medicine syndrome type of the hormonal femoral head necrosis; wherein the molecular markers comprise CD28, CD4, PLCG1, PRKCA, PTGS2, SOS2, STAT6, TLR4, IFIT1, IRF7, ISG15, MAPK14 and RHOU, and the traditional Chinese medicine symptoms of the hormonal femoral head necrosis comprise phlegm stasis channel blocking syndrome, channel obstruction syndrome and liver and kidney deficiency syndrome; the computing device comprises a memory and a processor; the memory is stored with a computer program to realize a modeling algorithm and algorithms of discriminant functions shown as formulas (1), (2) and (3); the modeling algorithm is a minimum deviation two-times algorithm;

F(c-1)＝sgn[f₁(c₁)+f₂(c₂)+f₃(c₃)+f₄(c₄)-b₁]the compound of the formula (1),

F(c-2)＝sgn[f₅(c₅)+f₆(c₆)+f₇(c₇)+f₈(c₈)-b₂]the compound of the formula (2),

F(c-3)＝sgn[f₉(c₉)+f₁₀(c₁₀)+f₁₁(c₁₁)+f₁₂(c₁₂)+f₁₃(c₁₃)-b₃]the compound of the formula (3),

f (c-1) represents the diagnosis result of the phlegm-blood stasis channel blocking syndrome, the return value of F (c-1) is 1, the phlegm-blood stasis channel blocking syndrome is established, and the return value is-1, the phlegm-blood stasis channel blocking syndrome is not established; f (c-2) represents the diagnosis result of the channel blockage syndrome, the return value of F (c-2) is 1, which represents that the channel blockage syndrome is established, and the return value is-1, which represents that the channel blockage syndrome is not established; f (c-3) represents the diagnosis result of the liver and kidney deficiency, the return value of F (c-3) is 1, the establishment of the liver and kidney deficiency is shown, and the return value of F (c-3) is-1, the establishment of the liver and kidney deficiency is shown; c. C₁～c₁₃Sequentially and respectively representing relative expression amounts of CD28, CD4, PLCG1, PRKCA, PTGS2, SOS2, STAT6, TLR4, IFIT1, IRF7, ISG15, MAPK14 and RHOU; the relative expression amount refers to the ratio of the expression amount relative to an internal reference; f. of₁(c₁)～f₁₃(c₁₃) Respectively kernel functions obtained by training according to the modeling algorithm, b₁～b₃The critical score value 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 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 CD28, CD4, PLCG1, PRKCA, PTGS2, SOS2, STAT6, TLR4, IFIT1, IRF7, ISG15, MAPK14 and RHOU.

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 markers, wherein the real-time quantitative PCR primers of the molecular markers comprise real-time quantitative PCR primers for detecting expression amounts of CD28, CD4, PLCG1, PRKCA, PTGS2, SOS2, STAT6, TLR4, IFIT1, IRF7, ISG15, MAPK14 and RHOU, respectively.

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, the internal reference is GAPDH, f₁(c₁)＝-0.6986×c₁，f₂(c₂)＝-0.6991×c₂，f₃(c₃)＝-0.1527×c₃，f₄(c₄)＝-0.1148×c₄，f₅(c₅)＝0.7766×c₅，f₆(c₆)＝0.41×c₆，f₇(c₇)＝-0.4784×c₇，f₈(c₈)＝-0.1515×c₈，f₉(c₉)＝0.5584×c₉，f₁₀(c₁₀)＝0.4779×c₁₀，f₁₁(c₁₁)＝0.433×c₁₁，f₁₂(c₁₂)＝0.3835×c₁₂，f₁₃(c₁₃)＝0.3539×c₁₃，b₁＝0.226，b₂＝0.0246，b₃0.1587; alternatively, the first and second electrodes may be,

the internal reference is β -Actin, f₁(c₁)＝-0.6192×c₁，f₂(c₂)＝-0.7702×c₂，f₃(c₃)＝0.1533×c₃，f₄(c₄)＝0.7519×c₄，f₅(c₅)＝0.5037×c₅，f₆(c₆)＝0.4773×c₆，f₇(c₇)＝-0.7201×c₇，f₈(c₈)＝-0.6149×c₈，f₉(c₉)＝0.554×c₉，f₁₀(c₁₀)＝0.4149×c₁₀，f₁₁(c₁₁)＝0.4048×c₁₁，f₁₂(c₁₂)＝0.5222×c₁₂，f₁₃(c₁₃)＝0.2906×c₁₃，b₁＝0.0091，b₂＝0.0218，b₃＝0.0547。

In a second aspect of the present disclosure: the application of a reagent for quantitatively detecting molecular markers in preparing a product for identifying traditional Chinese medicine evidence of hormonal femoral head necrosis is provided, wherein the molecular markers comprise CD28, CD4, PLCG1, PRKCA, PTGS2, SOS2, STAT6, TLR4, IFIT1, IRF7, ISG15, MAPK14 and RHOU.

Alternatively, the quantitative detection of the molecular marker is performed by:

1) obtaining a serum sample of a patient with hormonal femoral head necrosis;

2) determining the expression level of the molecular marker in the serum sample.

A third aspect of the disclosure: provided is a kit for identifying traditional Chinese medicine evidence of hormonal femoral head necrosis, wherein the kit comprises a reagent for quantitatively detecting molecular markers, and the molecular markers comprise CD28, CD4, PLCG1, PRKCA, PTGS2, SOS2, STAT6, TLR4, IFIT1, IRF7, ISG15, MAPK14 and RHOU.

A fourth aspect of the present disclosure: the molecular marker combination comprises CD28, CD4, PLCG1, PRKCA, PTGS2, SOS2, STAT6, TLR4, IFIT1, IRF7, ISG15, MAPK14 and RHOU.

By the technical scheme, the method can be applied to distinguishing different traditional Chinese medicine syndrome types of the hormonal femoral head necrosis by detecting the expression quantity of the molecular marker, assist in making early diagnosis and provide an effective basis for guiding clinical diagnosis and treatment.

Additional features and advantages of the disclosure will be set forth in the detailed description which follows.

Drawings

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 is a ROC graph obtained by diagnosing hormonal femoral head necrosis (phlegm-blood stasis collateral obstruction) using the molecular markers of the present disclosure in examples.

FIG. 2 is a ROC graph obtained by diagnosing hormonal femoral head necrosis (meridian obstruction syndrome) using the molecular markers of the present disclosure in examples.

Fig. 3 is a ROC graph obtained by diagnosing hormonal femoral head necrosis (liver and kidney deficiency) using the molecular markers of the present disclosure in examples.

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 for inputting the expression quantity of the molecular marker of an individual patient with the hormonal femoral head necrosis and an output device for outputting the traditional Chinese medicine syndrome type of the hormonal femoral head necrosis; wherein the molecular markers comprise CD28, CD4, PLCG1, PRKCA, PTGS2, SOS2, STAT6, TLR4, IFIT1, IRF7, ISG15, MAPK14 and RHOU, and the traditional Chinese medicine symptoms of the hormonal femoral head necrosis comprise phlegm stasis channel blocking syndrome, channel obstruction syndrome and liver and kidney deficiency syndrome; the computing device comprises a memory and a processor; the memory is stored with a computer program to realize a modeling algorithm and algorithms of discriminant functions shown as formulas (1), (2) and (3); the modeling algorithm is a minimum deviation two-times algorithm;

F(c-1)＝sgn[f₁(c₁)+f₂(c₂)+f₃(c₃)+f₄(c₄)-b₁]the compound of the formula (1),

F(c-2)＝sgn[f₅(c₅)+f₆(c₆)+f₇(c₇)+f₈(c₈)-b₂]the compound of the formula (2),

F(c-3)＝sgn[f₉(c₉)+f₁₀(c₁₀)+f₁₁(c₁₁)+f₁₂(c₁₂)+f₁₃(c₁₃)-b₃]the compound of the formula (3),

f (c-1) represents the diagnosis result of the phlegm-blood stasis channel blocking syndrome, the return value of F (c-1) is 1, the phlegm-blood stasis channel blocking syndrome is established, and the return value is-1, the phlegm-blood stasis channel blocking syndrome is not established; f (c-2) represents the diagnosis result of the channel blockage syndrome, the return value of F (c-2) is 1, which represents that the channel blockage syndrome is established, and the return value is-1, which represents that the channel blockage syndrome is not established; f (c-3) represents the diagnosis result of the liver and kidney deficiency, the return value of F (c-3) is 1, the establishment of the liver and kidney deficiency is shown, and the return value of F (c-3) is-1, the establishment of the liver and kidney deficiency is shown; c. C₁～c₁₃Sequentially and respectively representing relative expression amounts of CD28, CD4, PLCG1, PRKCA, PTGS2, SOS2, STAT6, TLR4, IFIT1, IRF7, ISG15, MAPK14 and RHOU; the relative expression amount refers to the ratio of the expression amount relative to an internal reference; f. of₁(c₁)～f₁₃(c₁₃) Respectively kernel functions obtained by training according to the modeling algorithm, b₁～b₃The critical score value obtained by training according to the modeling algorithm.

According to the present disclosure, a hormone-induced femoral head necrosis (SONFH) can be classified into three stages according to an early stage (phlegm stasis obstruction in collaterals), a medium stage (meridian obstruction) and a late stage (liver and kidney deficiency) according to a conventional diagnosis and treatment of common traditional Chinese medicine diseases in Beijing area and a document ' Chen Wei Heng, Liu Dai Bing, Zhanghong Mei, etc. ' three-stage four-type syndrome differentiation thought of femoral head necrosis [ J ]. a Chinese traditional medicine basis, a medical journal, 2003,9(12):51-52 '. The pattern of phlegm-blood stasis obstructing the collaterals refers to the pathological process of bone destruction in early stage of disease; the meridian obstruction syndrome is the middle stage of the disease, the pain of the patient is aggravated, and the function is limited; the liver and kidney deficiency refers to the pathological process of repairing necrotic bones in later stage of disease. Has good clinical curative effect on the femoral head necrosis according to the syndrome differentiation thought of 'three-phase four-type'.

CD28 is referenced 940 in the NCBI database according to the present disclosure; CD4 reference number 920 in NCBI database; reference number 5335 in NCBI database of PLCG 1; PRKCA reference number 5578 in NCBI database; PTGS2 reference number 5743 in NCBI database; SOS2 is reference number 6655 in NCBI database; STAT6 is referenced 6778 in the NCBI database; TLR4 reference number 7099 in NCBI database; IFIT1 reference number 3434 in NCBI database; IRF7 reference number 3665 in NCBI database; ISG15 reference number 9636 in NCBI database; the reference number of MAPK14 in NCBI database is 1432; RHOU is referenced 58480 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 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 CD28, CD4, PLCG1, PRKCA, PTGS2, SOS2, STAT6, TLR4, IFIT1, IRF7, ISG15, MAPK14 and RHOU. Or the detection device comprises a real-time quantitative PCR instrument and real-time quantitative PCR primers of the molecular markers, wherein the real-time quantitative PCR primers of the molecular markers comprise real-time quantitative PCR primers for respectively detecting the expression amounts of CD28, CD4, PLCG1, PRKCA, PTGS2, SOS2, STAT6, TLR4, IFIT1, IRF7, ISG15, MAPK14 and RHOU.

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, f₁(c₁)＝-0.6986×c₁，f₂(c₂)＝-0.6991×c₂，f₃(c₃)＝-0.1527×c₃，f₄(c₄)＝-0.1148×c₄，f₅(c₅)＝0.7766×c₅，f₆(c₆)＝0.41×c₆，f₇(c₇)＝-0.4784×c₇，f₈(c₈)＝-0.1515×c₈，f₉(c₉)＝0.5584×c₉，f₁₀(c₁₀)＝0.4779×c₁₀，f₁₁(c₁₁)＝0.433×c₁₁，f₁₂(c₁₂)＝0.3835×c₁₂，f₁₃(c₁₃)＝0.3539×c₁₃，b₁＝0.226，b₂＝0.0246，b₃0.1587; that is, the discriminant function can be simplified as shown in equations (4) to (6):

F(c-1)＝sgn[-0.6986×c₁-0.6991×c₂-0.1527×c₃-0.1148×c₄-0.226]the compound of the formula (4),

F(c-2)＝sgn[0.7766×c₅+0.41×c₆-0.4784×c₇-0.1515×c₈-0.0246]the compound of the formula (5),

F(c-3)＝sgn[0.5584×c₉+0.4779×c₁₀+0.433×c₁₁+0.3835×c₁₂+0.3539×c₁₃-0.1587]formula (6).

Or when the internal parameter is β -Actin, f is used as a group of reference values obtained by training₁(c₁)＝-0.6192×c₁，f₂(c₂)＝-0.7702×c₂，f₃(c₃)＝0.1533×c₃，f₄(c₄)＝0.7519×c₄，f₅(c₅)＝0.5037×c₅，f₆(c₆)＝0.4773×c₆，f₇(c₇)＝-0.7201×c₇，f₈(c₈)＝-0.6149×c₈，f₉(c₉)＝0.554×c₉，f₁₀(c₁₀)＝0.4149×c₁₀，f₁₁(c₁₁)＝0.4048×c₁₁，f₁₂(c₁₂)＝0.5222×c₁₂，f₁₃(c₁₃)＝0.2906×c₁₃，b₁＝0.0091，b₂＝0.0218，b₃0.0547; that is, the discriminant function can be simplified as shown in equations (7) to (9):

F(c-1)＝sgn[-0.6192×c₁-0.7702×c₂+0.1533×c₃₃+0.7519×c₄-0.0091]the compound of the formula (7),

F(c-2)＝sgn[0.5037×c₅+0.4773×c₆-0.7201×c₇-0.6149×c₈-0.0218]the compound of the formula (8),

F(c-3)＝sgn[0.554×c₉+0.4149×c₁₀+0.4048×c₁₁+0.5222×c₁₂+0.2906×c₁₃-0.0547]formula (9).

In addition, f is₁(c₁)～f₁₃(c₁₃) 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 (4) to (9) are obtained by training the inventors of the present disclosure with a modeling algorithm of a minimum-deviation-two 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 molecular markers in preparing a product for identifying traditional Chinese medicine evidence of hormonal femoral head necrosis is provided, wherein the molecular markers comprise CD28, CD4, PLCG1, PRKCA, PTGS2, SOS2, STAT6, TLR4, IFIT1, IRF7, ISG15, MAPK14 and RHOU.

According to the disclosure, the CD28, CD4, PLCG1 and PRKCA are molecular markers for identifying patients with hormonal femoral head necrosis caused by phlegm-stasis blocking collaterals.

According to the present disclosure, the PTGS2, SOS2, STAT6 and TLR4 are molecular markers for identifying patients with vasoocclusive, hormonal femoral head necrosis.

According to the present disclosure, the IFIT1, IRF7, ISG15, MAPK14 and RHOU are molecular markers for identifying patients with hormonal femoral head necrosis due to liver and kidney deficiency.

Further, the quantitative detection of the molecular marker is performed by the following steps:

1) obtaining a serum sample of a patient with hormonal 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 distinguishing different traditional Chinese medicine syndrome types of the hormonal femoral head necrosis by detecting the expression quantity of the molecular marker, help to make early diagnosis, have high accuracy and good stability of the diagnosis result, provide effective basis for guiding clinical diagnosis and treatment, and provide objective basis for scientific connotation of differential typing of the hormonal femoral head necrosis.

A third aspect of the disclosure: provided is a kit for identifying traditional Chinese medicine evidence of hormonal femoral head necrosis, wherein the kit comprises a reagent for quantitatively detecting molecular markers, and the molecular markers comprise CD28, CD4, PLCG1, PRKCA, PTGS2, SOS2, STAT6, TLR4, IFIT1, IRF7, ISG15, MAPK14 and RHOU.

Further, the reagents may be qPCR reagents.

A fourth aspect of the present disclosure: the molecular marker combination comprises CD28, CD4, PLCG1, PRKCA, PTGS2, SOS2, STAT6, TLR4, IFIT1, IRF7, ISG15, MAPK14 and RHOU.

Wherein, CD28, CD4, PLCG1 and PRKCA can be used for judging the syndrome of phlegm-blood stasis blocking collaterals;

PTGS2, SOS2, STAT6 and TLR4 can be used for judging the channel blockage syndrome;

IFIT1, IRF7, ISG15, MAPK14 and RHOU can be used to determine liver and kidney deficiency.

The kit and the molecular marker combination provided by the disclosure can be applied to distinguishing different traditional Chinese medicine syndrome types of the hormonal femoral head necrosis by detecting the expression quantity of the molecular marker, help to make early diagnosis, have high accuracy and good stability of the diagnosis result, provide effective basis for guiding clinical diagnosis and treatment, and provide objective basis for scientific connotation of differential typing of the hormonal femoral head necrosis.

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.