CN110244053A - For diagnosing the molecular marker and application thereof of lupus nephritis and pulmonary hypertension disease - Google Patents
For diagnosing the molecular marker and application thereof of lupus nephritis and pulmonary hypertension disease Download PDFInfo
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Abstract
The object of the present invention is to provide the molecular markers and application thereof for diagnosing lupus nephritis and pulmonary hypertension disease, using complement factor H, complement Bb segment are as diagnosis or prediction lupus nephritis and the molecular marker of pulmonary hypertension disease can easy, accurately diagnose or whether prediction patients with lupus nephritis merges pulmonary hypertension or its onset risk.Compared to the invasive detection methods such as right cardiac catheterization, it is more suitable clinical expansion.
Description
Technical field
The present invention relates to disease molecules markers, in particular to for diagnosing point of lupus nephritis and pulmonary hypertension disease
Sub- marker and application thereof.
Background technique
Systemic loupus erythematosus (SLE) is one of most common autoimmune disease in China, and kidney is systemic erythema
Lupus is easiest to one of organ involved, and lupus nephritis (1upus nephritis, LN) is that systemic loupus erythematosus involves kidney
A kind of caused immune complex nephritis is the most common secondary glomerulopathy.And pulmonary hypertension (PAH) refer to by
Pulmonary vascular function caused by a variety of causes and/or structure change, common as SLE with the characteristics of the raising of pulmonary vascular resistance progressive
And serious complication, it is one of the principal element of SLE patient's prognosis mala.
The dysfunction of the mainly blood vessel endothelium of SLE-PAH pathogenic mechanism, the endothelium of blood vessel not only have as physics
The function of barrier, the diastole that can also adjust blood vessel are shunk, and in the function and intercellular interaction for promoting blood coagulation
Also it plays an important role.The initial stage of pulmonary hypertension vascular lesion can relate to the dysfunction of endothelial cell
And following factor: 1) as inflammatory reaction caused by pulmonary vasculitis, with hypocomplementemia.2) is mainly due to pulmonary vascular endothelial cell
With the disease of Pulmonary Vascular caused by smooth muscle cell proliferation, protopathy active level is generally lower, and related to vascular lesion, protopathy is controlled
It is lower to treat degree of reversibility.3) is mainly caused by lung thin vessels thrombus, such as anti-phospholipid syndrome, is also related to complement system function
Many-sided reason such as imbalance.
In the prior art in order to diagnose patient with the presence or absence of pulmonary hypertension, need to carry out invasive inspection, right heart catheter
Inspection is the goldstandard for making a definite diagnosis pulmonary hypertension, although right cardiac catheterization can accurately obtain pulmonary circulation and the right blood for feeling concerned about system
Hydromechanics feature, but due to its it is invasive its with intrinsic defect.
Complement system belongs to first " defence line " of natural immune system, co-exists in three kinds of activated pathway: classical pathway, side
Approach and lectin pathway are related to the participation of known more than 30 plasma proteins altogether.Complement system overactivity in order to prevent
It is damaged to caused by body itself, there is also the inhibitor of numerous complement components in vivo activates the fine adjusting of progress to it.Cause
This, complement system plays an important role in the adjustment process of body immune system.
In the pathogenic course of systemic loupus erythematosus, traditional theory thinks the immune complex activation classical pathway of complement
It is most important factor, but still believable explanation cannot be obtained there are many Clinical symptoms.And then there is scholar's proposition: immune complex
After the target organ Local activation classical pathway of complement, the further activation of alternative pathway just makes the inflammation such as C3a and C5a
Ingredient is further discharged, and then leads to the formation of " membrane attack complex ", ultimately causes target organ damage, therefore, is mended
The alternative pathway of system system has important clinical meaning in the pathogenic course of systemic loupus erythematosus.
Main regulatory protein of the H factor as complement bypass activated channel, is preventing alternative pathway of complement excessive activation side
Face is even more the very important effect that plays.The encoding gene of the H factor is located at 1q32, is by 20 short identical repeated fragments
The glycoprotein for the 150kDa that (short consensus repeats, SCRs) structural domain, 1213 amino acid residues form, liver
Dirty is its main synthetic organ.In addition, kidney mesangial cell, retinal pigment epithelium, blood platelet, the single core in periphery are thin
Born of the same parents, Deiter's cells, fibroblast and endothelial cell etc. can also express.Two main functional areas of the H factor are located at
The both ends of albumen: the SCR1-4 positioned at N-terminal is mainly the formation for inhibiting the C3 convertase (C3bBb) of alternative pathway, auxiliary i factor
Degrade C3bBb, to inhibit the overactivity of alternative pathway of complement；And the SCR19-20 for being located at C-terminal mainly can be thin with host
The mucopolysaccharide of cellular surface combines the identification for completing target, by the combination with these sites, the H factor is assisted to inhibit host cell
The excessive activation of surface alternative pathway of complement.In addition, the H factor can also be with the fragment, DNA, histone of a variety of apoptotic cells etc.
Ingredient combines, and adjusts and removes apoptotic cell.Therefore, the H factor is adjusting complement system participation innate immune defence, is inhibiting complement
Overactivity and apoptosis substance removing in play important " brake ".
To sum up, the needs of pulmonary hypertension whether are merged for easy, accurate diagnostic system patients with SLE, still
So there is demand searching that can such as diagnose and/or as target using molecular marker come treatment system by non-intrusive inspection
Property lupus erythematosus and pulmonary hypertension/lupus nephritis and pulmonary hypertension.
Summary of the invention
To solve the deficiencies in the prior art, the present invention provides a kind of lupus nephritis and the molecule marks of pulmonary hypertension disease
Will object, the marker is complement factor H, compared to lupus nephritis pulmonary arterial pressure normal patient, the expression of complement factor H
It significantly reduces.
Further, described the present invention also provides a kind of lupus nephritis and the molecular marker of pulmonary hypertension disease
Marker is complement Bb segment, and compared to lupus nephritis pulmonary arterial pressure normal patient, the expression of complement Bb segment is significantly risen
It is high.
Further, the molecular marker is complement factor H and complement Bb segment.
The present invention also provides a kind of molecular markers to diagnose or predict the use in lupus nephritis and pulmonary hypertension disease
On the way, the purposes is to prepare diagnostic reagent.
Further, the molecular marker is complement factor H, compared to lupus nephritis pulmonary arterial pressure normal patient, is mended
The expression of body factor H significantly reduces.
Further, the marker is complement Bb segment, compared to lupus nephritis pulmonary arterial pressure normal patient, complement Bb
The expression of segment significantly increases.
Further, the marker is complement factor H and complement Bb segment.
Further, the diagnostic reagent further include detection anti-RNP antibody, anticardiolipin antibodies, d-dimer and/or
The related reagent of hemoglobin.
The present invention provides a kind of diagnostic kit, and the kit includes measurement diagnosis or prediction lupus nephritis and pulmonary artery
The related reagent of the molecular marker of high pressure disease.
Further, the molecular marker is complement factor H.
Further, the molecular marker is complement Bb segment.
Further, the molecular marker is complement factor H and complement Bb segment.
Further, the kit further includes detection anti-RNP antibody, anticardiolipin antibodies, d-dimer and/or blood
The related reagent of Lactoferrin.
The present invention also provides it is a kind of diagnosis or prediction lupus nephritis and pulmonary hypertension disease method, the method includes
Detect the expression of the molecular marker of patient to be diagnosed, and the molecular marker with lupus nephritis pulmonary arterial pressure normal patient
Expression compare, it is diagnosable or be predicted as lupus nephritis and lung when there were significant differences for the expression of molecular marker
Arterial hypertension.
Further, the molecular marker is complement factor H, compared to lupus nephritis pulmonary arterial pressure normal patient, is mended
The expression of body factor H significantly reduces.
Further, the marker is complement Bb segment, compared to lupus nephritis pulmonary arterial pressure normal patient, complement Bb
The expression of segment significantly increases.
Further, the marker is complement factor H and complement Bb segment.
Further, the method also includes detection anti-RNP antibodies, anticardiolipin antibodies, d-dimer and/or blood red
The expression of albumen and compared with the expression of lupus nephritis pulmonary arterial pressure normal patient.
Further, the expression of the Complement H factor shows that patients with lupus nephritis is closed less than or equal to 241 μ g/ml
And pulmonary hypertension or with merge pulmonary hypertension high risk.
Further, the expression of the complement Bb segment shows patients with lupus nephritis more than or equal to 1.26 μ g/ml
Merge pulmonary hypertension or with the high risk for merging pulmonary hypertension.
Beneficial effect
The present invention specifies that alternative pathway of complement merges in pulmonary hypertension disease in patients with lupus nephritis with important
Effect.
Present invention finds factors H, Bb to compare as lupus nephritis and the molecular marker of pulmonary hypertension disease
In invasive inspection methods such as right cardiac catheterizations, capable of accurately and efficiently diagnosing patients with lupus nephritis, whether to merge lung dynamic
Arteries and veins high pressure or prediction merge pulmonary hypertension risk.
Detailed description of the invention
Fig. 1: patient registers selected flow chart.
The immunofluorescence dyeing of complement pathway activation related component is as a result, described group in Fig. 2: LN-PAH patient lungs tissue
Dividing includes Bb, C3d and C5b-9, carries out total dyeing, Fig. 2A, Fig. 2 B, Fig. 2 C point to main component laminin outside blood vessel
It is not related to Bb, C3d and C5b-9, Fig. 2 D is the colocalization area Results of Pearson correlation coefficient assessment.
Specific embodiment
The present invention is described below in more detail to facilitate the understanding of the present invention.
It should be understood that the term or word used in the specification and in the claims is not construed as having
The meaning limited in dictionary, and be interpreted as having on the basis of following principle and its meaning one in the context of the present invention
The meaning of cause: the concept of term can suitably limit best illustration of the invention by inventor.
In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition such as Sambrook et al.,
Molecular cloning: condition described in laboratory manual, or according to the normal condition proposed by manufacturer.
The differential expression of 1 lupus nephritis of embodiment and pulmonary hypertension complement pathway GAP-associated protein GAP
Data and method
Research object:
Review evaluation in January, 2002 in May, 2009 is medical in Peking University First Hospital and through Renal biospy first
The case of 389 patients of lupus nephritis is suffered from confirmation, and the patient carries out during being hospitalized through chest echocardiography
Diagnosis, and it is hard by all types of Renal vascular lesions of Pathological observation assessment, including blood vessel immune complex deposit, artery
Change, thrombotic microvascular disease, non-inflammatory gangrenosum acne angiosis, true property vasculitis.In turn, according to the standard of Fig. 1, lupus kidney is chosen
24 patients that inflammation merges pulmonary hypertension (LN-PAH) are studied.Wherein the diagnosis of lupus nephritis is according to U.S.'s rheumatism
What the standard of sick association carried out, PAH excludes the congenital heart using standard as defined in European Society of Cardiology (ESC) 2015
Popular name for, portal hypertension, pulmonary vein occlusion, pulmonary embolism, chronic obstructive pulmonary disease, left heart failure medical history.
Method:
Research object is divided into LN-PAH group (24) and LN-non-PAH group (328) and normal person group (100).
The clinical indices of LN-PAH group and LN-non-PAH group are detected, clinical indices include: age, gender, fever, cheekbone portion
Erythema, photosensitization, alopecia, canker sore, arthritis, pleurisy, Raynaud's phenomenon, nervous system damage, anaemia, leucocyte subtract
Low, decrease of platelet, the scoring of Lupus activity degree.The above diagnosis, assessment are operated according to standard method.
The lab index of LN-PAH group and LN-non-PAH group is detected, lab index includes: hemoglobin, blood flesh
Acid anhydride, D dimer, autoantibody repertoire index of correlation.The index analysis establishing criteria method is carried out according to kit specification
Operation.
The expression of complement pathway main component in LN-PAH group and LN-non-PAH group blood plasma is detected, in specific blood plasma
The concentration of main complement component is determined by ELISA, including complement fragment C5a (Quidel
Corporation, San Diego, CA), C3a (Quidel Corporation, San Diego, CA), Bb (Quidel
Corporation, San Diego, CA), soluble C5b-9 (SC5b-9, Quidel Corporation, San Diego,
CA), properdin properdin (Uscnk life science Inc, Wuhan, China) and C3 (Quidel
Corporation,San Diego,CA).All complement components are measured all in accordance with manufacturer's explanation.In addition, in blood plasma
Also establishing criteria method is measured for the measurement of C1q, MBL, C4BP.
Experimental method has made following improvement according to prior art for the detection of plasma complement H factor level: with pH9.6's
0.05M carbonate buffer solution dilutes Goat anti-Human H factor polyclonal antibody (Calbiochem company, Germany) to 10 μ g/ml, in advance
First coated elisa plate, 4 DEG C are incubated overnight.The H factor standard product diluted, patient's blood plasma sample are originally separately added into reacting hole, 37
DEG C be incubated for 1h.Mouse anti human H factor monoclonal antibodies (Usbiological company, the U.S.) 1: 500 is added after board-washing to dilute, 37
DEG C be incubated for 1h.The sheep anti-mouse igg (Sigma company) of alkali phosphatase enzyme mark, 37 DEG C of incubation 30min are added after board-washing.Finally plus
Enter alkaline phosphatase substrate colour developing, measures absorbance value under 405nm wavelength with microplate reader.By drawing standard curve, foundation
Standard items quantify plasma complement H factor level.
Immunofluorescence assay: the expression of measurement Bb, C3d and C5b-9 in the lung tissue of LN-PAH patient, specifically
Are as follows: paraffin mass is made after fixation-dehydration-embedding in the lung tissue for taking LN-PAH patient, with 8um thickness slice, dewaxed to
Water and antigen retrieval, with the activation product Bb of Immunofluorescence test complement alternative route, terminal common pathway product C3d and complement
Deposition of the terminal compound C5b-9 at tissue blood vessel is activated, Confocal is imaged and carries out gray-scale statistical.
Data analysis:
Continuous variable is described as mean+SD (SD) or median (IQR, quartile deviation), and group difference is by making
With the double factor analysis of variance test and non-parametric test.Classified variable is expressed as a percentage, and is analyzed with Chi-square Test.
Hardy-Weinberg equilibrium is analyzed using x2 degree of fitting.Single argument and multivariable are using logistic regression analysis assessment survival rate.
As a result it is indicated using the ratio of 95% confidence interval.Data statistic analysis is calculated using 12.0 software of SPSS.Double tail p values are less than
0.05 is considered to have statistical significance.
Experimental result:
In 24 patients of LN-PAH patient group, 2 be male, 22 be women, experiment carry out when average age be
35.29 ± 15.13 years old；In 328 patients of LN-non-PAN patient group, 53 be male, 275 be women, experiment carry out
When average age be 32.83 ± 11.30；LN-PAH patient group and LN-non-PAH patient group do not have in terms of age and gender
Significant difference (p value is 0.758).
Wherein the particularly relevant information of LN-PAH patient group is as shown in table 1.
Table 1: lupus merges patients with pulmonary hypertension relevant information
The clinical evaluation data and laboratory examination results of LN-PAH group patient and LN-non-PAH group patient are respectively such as table 2
With shown in table 3.
Table 2: lupus merges patients with pulmonary hypertension compared with lupus pulmonary arterial pressure normal patient clinical data
Table 3: ruthless sore merges patients with pulmonary hypertension compared with ruthless sore pulmonary arterial pressure normal patient laboratory data
As shown in Table 2, compared to LN-non-PAH patient group, LN-PAH patient group has more decrease of platelet symptoms
(p < 0.001), lower cheekbone portion erythema (p=0.01), alopecia (p=0.017) and arthritis (p=0.03) symptom, in explanation
State the generation of symptom and pulmonary hypertension and asynchronous.
Lupus merges patients with pulmonary hypertension and the level of complement comparison result of lupus pulmonary arterial pressure normal patient is shown in Table 4.
Table 4: lupus merges patients with pulmonary hypertension compared with lupus pulmonary arterial pressure normal patient level of complement
As shown in Table 4, the horizontal difference of the serum mannose agglutinin in normal population, C3a, C5a and solubility C5b-9
For 1532 ± 1020ng/ml, 100.87 ± 70.55ng/ml, 9.32 ± 7.88ng/ml and 467.41 ± 545.23ng/ml.
Compared to normal population, the serum mannose agglutinin of LN-PAH patient and LN-non-PAH patient, C3a, C5a and
The level of soluble C5b-9 all has significant raising (p value is respectively P < 0.01, P < 0.01, P < 0.01, P < 0.01).
First component of the C1q as classical complement activation pathway, expression and LN- in LN-PAH patient
Expression in non-PAH patient is not significantly different (p=0.206).
Mannose-binding lectin is the inducer of agglutinin complement activation pathway, equally, the expression water in LN-PAH patient
The flat expression with LN-non-PAH patient is also not significantly different (p=0.874).
C4BP is that the main liquid phase of complement activation inhibits albumen, can be turned by enhancing classics/lectin pathway C3
Change the failure of enzyme C4b2a to play inhibiting effect, moreover it is possible to play suppression by enhancing the failure of alternative pathway C3 convertase C3bBb
Production is used.Expression in LN-PAH patient in the expression of C4BP and LN-non-PAH patient is not significantly different (p
=0.308).
Above-mentioned experimental result explanation can not distinguish LN-PAH trouble by classical pathway and lectin pathway Related Component
Person and LN-non-PAH patient also can not be diagnosed or be predicted that lupus is suffered from by classical pathway and lectin pathway Related Component
The risk of person's merging pulmonary hypertension.
Properdin plays a key effect in alternative pathway C3 convertase is stablized, and Bb is the activity of factor B in alternative pathway
Segment, and complement factor H is a kind of plasma complement regulatory factor abundant, can inhibit the formation of C3 convertase and C3 is promoted to turn
Change the failure of enzyme and inactivate C3b as the common factor of Complement factor I, by above-mentioned function, complement factor H is able to suppress
The activation of alternative pathway.
It can learn whether alternative pathway is activated by the expression of Bb, properdin and factor H in measurement blood plasma.
As the result is shown (referring to table 4), the expression of Bb is compared with the expression in LN-non-PAH patient in LN-PAH patient
There is significant raising (1.26 ± 0.758 μ g/ml vs.0.74 ± 0.186 μ g/ml, p=0.049)；The factor H in LN-PAH patient
Expression have significant decrease (241.00 ± 116.502 μ g/ml compared with the expression in LN-non-PAH patient
Vs.417.13 ± 188.017 μ g/ml, p=0.024), and in LN-PAH patient properdin expression and LN-non-
Expression in PAH patient is not significantly different (p=0.102).
The above results illustrate that in LN-PAH patient, overactivity has occurred in alternative pathway of complement, mainly passes through Bb's
Be overexpressed and factor H expression reduction and realize.
The activation of classical pathway of complement, which is mainly reflected in, converts C3a and C3b for C3, and then forms C5 convertase polymolecular
C5, can be cut into C5a and C5b by enzyme, final complement complex C5b-9 due to complement system activation and make C5b with
C9 is assembled and is obtained, and therefore, is had studied C3, C3a, C5a and solubility C5b-9, is able to reflect the complement in the circulatory system
Activation.
The results show that in LN-PAH patient C3, C3a, C5a and solubility C5b-9 expression and LN-non-PAH
Expression in patient is not significantly different, and shows that activation degree of the classical pathway of complement in two groups of patients does not have significance difference
It is different.
In order to further confirm that complement pathway related component in lung tissue's positioning of LN-PAH patient and expression, leads to
It crosses immunofluorescence assay to detect the deposition of C3d, C5b-9 and Bb, as the result is shown (referring to fig. 2), as warp
The Bb segment of C3d, C5b-9 of the marker of allusion quotation complement pathway and the Specific marker as alternative com-plement pathway is in lung
Be in tissue it is positive, in conjunction with the differential expression of C3d, C5b-9, Bb in aforementioned LN-PAH patient and LN-non-PAH patient,
Further demonstrate the overactivity in LN-PAH patient there are alternative pathway of complement.
Injury of blood vessel is a common attribute of SLE, and simultaneously pulmonary hypertension is considered as being drawn by its blood vessel endothelium to lupus
The lesion risen.One of SLE vascular lesion generally acknowledges that assuming is the immune complex triggering inflammatory reaction on blood vessel endothelium, including swashs
Complement cascade reaction living forms C5b-9 film complement attack and then finally destroys basement membrane of blood vessel and inflammatory cell infiltration occurs.
Mechanism of the complement pathway in LN-PAH is contradictory, on the one hand, classical or alternative route complement component in early days
Hereditary homozygote defect makes patient be susceptible to suffer from lupus, and on the other hand, excess complement activation leads to inflammation and tissue damage.
Previous studies only confirm the activation of alternative com-plement pathway can react SLE activity, vascular lesion especially with
Merging thrombotic microvascular disease becomes the severity of main vascular lesion.The overactivity for adjusting alternative com-plement pathway can have
The generation and progress of the inhibition SLE of effect.
And the application has found that the raising of Bb, C3d and C5b-9 expression and the reduction of factor H content show that bypass is mended
Body pathway activation plays an important role in LN-PAH, and Bb, C3d, C5b-9 are to have deposition in the lung tissue of LN-PAH patient
's.
Therefore, patients with lupus nephritis can be diagnosed by the expression of Bb in detection blood plasma, factor H, and whether to merge lung dynamic
Arteries and veins high pressure or prediction merge pulmonary hypertension risk.
Embodiment 2 utilizes complement pathway differential protein diagnosis lupus nephritis and pulmonary hypertension
Patients with lupus nephritis 7 that Peking University First Hospital newly accepts for medical treatment are chosen to make using Complement H factor, complement Bb segment
Its whether simultaneously pulmonary hypertension is diagnosed for molecular marker, while using right cardiac catheterization result as control.
Testing result is as shown in table 5:
| Patient | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| Factor H (μ g/ml) | 253.00 | 326.12 | 469.56 | 500.78 | 189.65 | 385.46 | 156.27 |
| Bb(μg/ml) | 1.20 | 0.72 | 0.69 | 0.61 | 1.29 | 0.69 | 2.11 |
| Right cardiac catheterization detects (+/-) | + | - | - | - | + | - | + |
The results show that accurately can diagnose or predict wolf using complement factor H, complement Bb segment as molecular marker
Whether sore nephritis patient merges pulmonary hypertension disease.
The above is only a preferred embodiment of the present invention, it should be pointed out that: for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (9)
1. the molecular marker of a kind of lupus nephritis and pulmonary hypertension disease, it is characterised in that: the marker be complement because
Sub- H, compared to lupus nephritis pulmonary arterial pressure normal patient, the expression of complement factor H is significantly reduced.
2. the molecular marker of a kind of lupus nephritis and pulmonary hypertension disease, it is characterised in that: the marker is complement Bb
Segment, compared to lupus nephritis pulmonary arterial pressure normal patient, the expression of complement Bb segment is significantly increased.
3. the molecular marker of a kind of lupus nephritis and pulmonary hypertension disease, it is characterised in that: the marker be complement because
Sub- H and complement Bb segment；Compared to lupus nephritis pulmonary arterial pressure normal patient, the expression of complement factor H is significantly reduced, and
The expression of complement Bb segment significantly increases.
4. the reagent or molecular marker of detection molecules marker expression level are being used to prepare diagnosis or prediction lupus nephritis simultaneously
Purposes in the reagent of pulmonary hypertension disease, it is characterised in that: the molecular marker is complement factor H, compared to lupus
The expression of ephritis pulmonary arterial pressure normal patient, complement factor H significantly reduces.
5. purposes according to claim 4, it is characterised in that: the molecular marked compound is complement Bb segment, compared to wolf
Sore ephritis pulmonary arterial pressure normal patient, the expression of complement Bb segment significantly increase.
6. purposes according to claim 4, it is characterised in that: the molecular marked compound is complement factor H and complement Bb piece
Section, compared to lupus nephritis pulmonary arterial pressure normal patient, the expression of complement factor H is significantly reduced, and complement Bb segment
Expression significantly increases.
7. according to the described in any item purposes of claim 4-6, it is characterised in that: the reagent further include detection anti-RNP antibody,
The related component of anticardiolipin antibodies, d-dimer and/or hemoglobin.
8. a kind of diagnostic kit, it is characterised in that: the kit includes measurement diagnosis or prediction lupus nephritis and pulmonary artery
The related reagent of the molecular marker of high pressure disease, the molecular marker are complement factor H and/or complement Bb segment.
9. kit according to claim 8, it is characterised in that: the kit further includes detection anti-RNP antibody, the anti-heart
The related reagent of phospholipid antibody, d-dimer and/or hemoglobin.
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