CN116622632A - Application of CD4TEMRA in diagnosis of autism co-morbid epilepsy - Google Patents
Application of CD4TEMRA in diagnosis of autism co-morbid epilepsy Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, discloses application of CD4TEMRA in diagnosis of autism co-morbid epilepsy, and particularly discloses a cell subset for diagnosis or auxiliary diagnosis of autism co-morbid epilepsy, wherein the cell subset is CD4TEMRA, and the CD4TEMRA is CD3 + CD4 + CD45RA + CCR7 ‑ CD27 ‑ T cells. The invention is thatThe CD4TEMRA cell proportion in peripheral blood of the patient suffering from autism co-morbid epilepsy is found to be obviously improved for the first time, and when the patient is treated by risperidone, the CD4TEMRA cell proportion in the peripheral blood is obviously reduced, so that the CD4TEMRA can be used as one of laboratory immune related detection indexes for diagnosing or assisting in diagnosing the patient suffering from autism co-morbid epilepsy, and meanwhile, important reference data is provided for the selection of treatment strategies for the patient suffering from autism co-morbid epilepsy in clinic in the future.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an application of CD4TEMRA in diagnosis of autism co-morbid epilepsy.
Background
Autism spectrum disorder (Autism Spectrum Disorder, ASD) is a severe neurological disorder whose central features are manifested as social interactions and communication impairment, with limited and repetitive behaviors, common to male patients. Epilepsy (EP) is one of the common neurological disorders characterized by a persistent predisposition to seizures with neurological, cognitive, psychological and social consequences. ASDs are highly heterogeneous, with the extent of defects and behaviors potentially varying, which presents a great challenge for accurate treatment. Epidemiological studies in countries incorporating global disease burden (GBD) have estimated that 0.76% of children worldwide suffer from autism. Autism severely affects learning and social functions and may persist to adulthood, which is a significant impact on personal, household, and public health.
Since 1943 autism was first described by Kanner, the understanding of autism has been greatly improved, including environmental risk factors, genetic factors, neurobiology, molecular pathophysiology, etc. of autism, but the clear pathogenesis of autism is not clear. The diagnosis criteria for autism was a behavioral diagnosis based on DSM-5 (Diagnostic and Statistical Manual of Mental Disorders-fifth edition). Some structured diagnostic interviews and observational assessments in clinical practice may aid in early identification, screening and diagnosis of autism. But the expense of tools and training, the time required to complete these evaluations, and the need for extensive training to reliably use these tools limit their widespread use. Treatment of autism includes drug therapy and behavioral intervention. Approved drugs for treating ASD are mainly directed to autism-related co-diseases, such as restlessness, irritability, etc., and have limited effects in improving core symptoms thereof. In addition, there is a high degree of heterogeneity and complexity between individuals and within individuals, also resulting in the potential benefit of intervention being equally complex and heterogeneous.
ASD is often co-occurring with Epilepsy (EP), and ASD co-morbid epilepsy may be considered a subset of ASDs with significantly higher co-morbid rates relative to each other than their individual prevalence in the population, which may indicate the existence of a common neurogenic mechanism.
Disclosure of Invention
It is an object of a first aspect of the present invention to provide a cell subpopulation for diagnosis or assisted diagnosis of autism co-morbid epilepsy.
The object of the second aspect of the present invention is to provide a method for the preparation of a cell subpopulation according to the first aspect of the present invention.
The object of a third aspect of the present invention is to provide the use of a cell subpopulation according to the first aspect of the present invention for the manufacture of a product for diagnosis or assisted diagnosis of autism co-morbid epilepsy or for screening candidate drugs for treatment of autism co-morbid epilepsy.
The object of the fourth aspect of the present invention is to provide the use of a substance for detecting the content of a cell subpopulation according to the first aspect of the present invention.
The object of the fifth aspect of the present invention is to provide the use of a method for detecting a cell subpopulation according to the first aspect of the present invention for non-disease diagnostic or therapeutic purposes.
The object of a sixth aspect of the present invention is to provide the use of a substance for reducing the content of a cell subpopulation according to the first aspect of the present invention in blood for the manufacture of a medicament.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the invention, there is provided a cell subset for use in or in assisting diagnosis of autism co-morbid epilepsy, the cell subset being CD4TEMRA, the CD4TEMRA being CD3 + CD4 + CD45RA + CCR7 - CD27 - T cells.
In a first aspect of the invention there is provided a method of preparing a cell subpopulation according to the first aspect of the invention comprising the steps of: contacting a population of cells in blood with a binding agent; sorting cells bound to the binding agent to obtain; the materials include antibodies for detecting CD4TEMRA that label different fluorescence: CD3 antibodies, CD4 antibodies, and CD45RA antibodies.
Preferably, the blood is mixed with lymphocyte separation liquid, lysed, and a binding agent is added to sort the cells bound to the binding agent.
Preferably, the sorting comprises sorting by fluorescence activated cell sorting, magnetic cell sorting, substrate assisted cell sorting, laser mediated cleavage, fluorometry, flow cytometry or microscopy.
Preferably, the cells bound to the binding agent are sorted by flow cytometry.
In a third aspect of the invention there is provided the use of a cell subpopulation according to the first aspect of the invention in (1) to (3);
(1) Preparing a product for diagnosing or assisting in diagnosing autism co-morbid epilepsy;
(2) Screening candidate medicines for treating autism co-morbid epilepsy;
(3) And preparing a product for prognosis evaluation of autism co-morbid epilepsy.
Preferably, said diagnosis or co-diagnosis of autism co-morbid epilepsy is achieved by detecting a quantitative proportion of said cell subpopulations.
In a fourth aspect of the invention there is provided the use of a substance for detecting a cell subpopulation according to the first aspect of the invention in (4) to (6);
(4) Preparing a product for diagnosing or assisting in diagnosing autism co-morbid epilepsy;
(5) Screening candidate medicines for treating autism co-morbid epilepsy;
(6) And preparing a product for prognosis evaluation of autism co-morbid epilepsy.
Preferably, the substance is used to detect the content of the cell subpopulation of the first aspect of the invention.
Preferably, the content of the cell subpopulation refers to the percentage of the number of the cell subpopulation in peripheral blood mononuclear cells.
Preferably, the substance comprises antibodies for detecting CD4TEMRA that label different fluorescence: CD3 antibodies, CD4 antibodies, CD45RA antibodies, CCR7 antibodies, and CD27 antibodies.
Preferably, the fluorescent label of the CD3 antibody is Pe-cy7.
Preferably, the fluorescent label of the CD4 antibody is BUV496.
Preferably, the fluorescent label of the CD45RA antibody is BV510.
Preferably, the fluorescent label of the CCR7 antibody is APC-R700.
Preferably, the fluorescent label of the CD27 antibody is BV650.
Preferably, the substance further comprises a red blood cell lysate, phosphate buffer solution, which lyses peripheral red blood cells.
Preferably, the product comprises reagents and/or kits.
In a fifth aspect of the invention there is provided the use of a method of detection of a cell subpopulation according to the first aspect of the invention for non-disease diagnostic or therapeutic purposes, said method comprising the steps of:
1) Forming single cell suspension by treating the peripheral blood sample;
2) Mixing the single cell suspension of step 1) with the addition of a different fluorescently labeled antibody: the CD3 antibody, CD4 antibody, CD45RA antibody, CCR7 antibody and CD27 antibody were incubated in a mix;
3) Sorting to obtain data of the cell subpopulations.
Preferably, the fluorescent label of the CD3 antibody is Pe-cy7.
Preferably, the fluorescent label of the CD4 antibody is BUV496.
Preferably, the fluorescent label of the CD45RA antibody is BV510.
Preferably, the fluorescent label of the CCR7 antibody is APC-R700.
Preferably, the fluorescent label of the CD27 antibody is BV650.
Preferably, the sorting comprises sorting by fluorescence activated cell sorting, magnetic cell sorting, substrate assisted cell sorting, laser mediated cleavage, fluorometry, flow cytometry or microscopy.
Preferably, the cells bound to the binding agent are sorted by flow cytometry.
Preferably, the incubation in step 2) is carried out at room temperature for 15-30 minutes in the dark.
Preferably, the step further comprises performing a statistical analysis of the data.
Preferably, the statistical analysis comprises a chi-square test analysis, a t-test analysis, a rank-sum test analysis, a paired Wilcoxon test analysis.
In a sixth aspect of the invention there is provided the use of a substance which reduces the content of a cell subpopulation according to the first aspect of the invention in blood for the manufacture of a medicament.
The beneficial effects of the invention are as follows:
according to the invention, the CD4TEMRA cell proportion in peripheral blood of an autism co-disease epileptic patient is obviously improved for the first time, and when the patient is treated by risperidone, the CD4TEMRA cell proportion in the peripheral blood is obviously reduced, so that the CD4TEMRA can be used as one of laboratory immunity related detection indexes for diagnosing or assisting in diagnosing the autism co-disease epileptic patient, and meanwhile, important reference data is provided for selecting treatment strategies for the autism co-disease epileptic patient clinically in the future.
The invention provides a preparation method of the cell subset in the first aspect, which is simple and can rapidly obtain the cell subset for diagnosing or assisting in diagnosing autism co-morbid epilepsy.
Drawings
FIG. 1 shows a general separation of various components of the blood.
FIG. 2 is a graph of flow cytometry analysis results.
FIG. 3 is a graph showing the analysis results of the ratio of CD4TEMRA in peripheral blood of the control group and the ASD-EP group, wherein P <0.05 is represented by the graph, and no statistical difference is represented by ns.
FIG. 4 is a graph showing the analysis of the proportion of CD4TEMRA in peripheral blood of ASD-EP patients treated with risperidone, wherein P <0.05 is represented.
Fig. 5 is a ROC graph of CD4TEMRA in peripheral blood of control and ASD-EP groups (auc=0.8261, p=0.0406).
Detailed Description
The invention will now be described in detail with reference to specific examples, without limiting the scope of the invention.
The materials, reagents and the like used in this example are commercially available materials and reagents unless otherwise specified.
Example 1
Male infants with ASD co-morbid epilepsy, who were diagnosed in the women child medical center autism intervention center in guangzhou, in 2021 to 2022, were selected as ASD-EP group. Meanwhile, the children who visit the medical center of women in Guangzhou city in 2021 to 2022 and regularly participate in normal physical examination are selected as a control group (TD). The infant completes the clinical history collection, the comprehensive physical examination and the nervous system examination during the primary visit, and extracts 2mL of peripheral blood circulation cell detection before the administration and after 1-3 months of the administration respectively. Both ASD-EP group and control group children signed informed consent.
ASD-EP group inclusion criteria: 1) The age is between 3 and 9 years old; 2) Diagnosis meets the diagnosis standard of ASD in the manual for diagnosis and statistics of mental disorders, 5 th edition (Diagnostic and Statistical Manual of Mental Disorders-fifth edition, DSM-5); 3) Epilepsy diagnosis meets the diagnosis and classification standards of the international antiepileptic consortium (International League Against Epilepsy, ILAE) in 2017; 4) Parents of the children agreed to participate in the study and signed informed consent.
ASD-EP group exclusion criteria: 1) Other known etiologies of neurodevelopmental disorders (e.g., fragile X syndrome, etc.); 2) Acute or chronic infection conditions exist.
Control group inclusion criteria: 1) Male children with ages matched to the experimental group; 2) Children who participated in normal physical examination.
Control group exclusion criteria: 1) Children with defined neurological abnormalities or developmental disorders, such as ASD, epilepsy, and developmental disorders; 2) Children with acute and chronic infections exist.
2mL of peripheral blood of the ASD-EP group children and the control group children (when the participants have common diseases such as fever or diarrhea, the children can draw blood after the health condition of the children is stable) are respectively collected, the peripheral blood is placed in an EDTA anticoagulation tube, then the tube is immediately inverted for 6-7 times, so that the anticoagulation agent is mixed with the blood, the blood is sent to a laboratory within 12 hours after collection, and the laboratory immediately carries out cell analysis on the blood sample after the blood sample is received. The method comprises the following specific steps:
(1) Centrifuging the blood sample at 2000rpm for 5 minutes, extracting plasma, and preserving at-80deg.C;
(2) The remaining blood was mixed with a 1 x balanced salt solution (PBS) containing no calcium and no magnesium ions at 1:1, mixing;
(3) Mixing the diluted blood with lymphocyte separation solution Ficoll-hypaque (polysucrose-diatrizoic amine) (TBD, LTS 1077), and centrifuging at 800G for 18 min at room temperature;
(4) Peripheral blood mononuclear cells (Peripheral Blood Mononuclear Cell, PBMC) were then collected from the buffy coat (see fig. 1), washed once with 1×pbs buffer, centrifuged at 2000rpm for 5 minutes, and the supernatant was discarded;
(5) 3mL of erythrocyte lysate (TIANGEN, RT 122) was added for 5 min, then lysis was stopped by adding 2 volumes of PBS, centrifugation was performed at 2000rpm for 5 min, and the supernatant was discarded;
(6) The cell suspension concentration was adjusted to 0.5X10 6 cells/mL；
(7) Adding 50 μl of the prepared antibody solution (Table 1) into 50 μl of cell suspension, mixing, and incubating at normal temperature under dark condition for 20 min;
(8) Adding 1mLPBS, washing once, centrifuging at 2000rpm for 5 minutes, and discarding the supernatant;
(9) 200-300 mu L (according to cell quantity) of PBS is added to resuspend the cells;
(10) Cells were analyzed on a flow cytometer (BD company, USA LSRFortessa X-20) gated at FSC/SSC to obtain 30X 10 5 Individual cells are the end point and the target population is mononuclear cells (PBMCs). The cell surface markers are shown in Table 2, and the gating is shown in detail in FIG. 2.
(11) Data analysis and mapping between groups was performed using GraphPad Prism 9.3. Classification variables (gender) were checked using chi-square. The measurement data conforming to normal distribution is expressed by mean ± standard deviation (x±s), the comparison between two experimental groups adopts t test, and the paired samples adopt paired sample t test; the measurement data which do not accord with the normal distribution are expressed by median (quarter bit interval) [ M (P25, P75) ] and the comparison between groups adopts a rank sum test and the paired samples adopt a paired Wilcoxon test. P <0.05 is considered statistically significant, representing P <0.05, P <0.01, P <0.001, ns no statistical difference.
TABLE 1 antibody solution Components
TABLE 2 cell surface markers
As can be seen from Table 3, 4 selected children with autism co-morbid epileptic children peripheral blood matched with the sex of the control group (TD) were compared by flow cytometry, and found CD4 of the children of ASD-EP group + Terminally differentiated effector memory T cells (CD 4 TEMRA) were significantly higher than in the control group, P<0.05 (FIG. 3). To verify the diagnostic value of CD4 EMRA in ASD-EP, the inventors predicted the status of autism co-morbid epileptic patients using ROC curves, and the results are shown in fig. 5, with AUC of CD4TEMRA of peripheral blood of the control group and ASD-EP group of 0.8261, p=0.0406.
Further, 4 ASD-EP infants taking risperidone were followed for 1-3 months, and peripheral blood was collected for flow cytometry analysis, and it was found that CD4TEMRA was significantly decreased after treatment compared to before treatment (Table 4, FIG. 4).
TABLE 3 comparison of control group with ASD-EP group children
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. Be used for diagnosis or supplementary diagnosis autismA cell subset of symptomatic epileptic disease, said cell subset being CD4TEMRA, said CD4TEMRA being CD3 + CD4 + CD45RA + CCR7 - CD27 - T cells.
2. A method of preparing a cell subpopulation according to claim 1, comprising the steps of: contacting a population of cells in blood with a binding agent; sorting cells bound to the binding agent to obtain; the binding agents include antibodies that label different fluorescence: CD3 antibodies, CD4 antibodies, and CD45RA antibodies.
3. The method of claim 2, wherein the sorting comprises sorting by fluorescence activated cell sorting, magnetic cell sorting, substrate assisted cell sorting, laser mediated cleavage, fluorometry, flow cytometry, or microscopy.
4. The use of the cell subpopulation of claim 1 in (1) to (3);
(1) Preparing a product for diagnosing or assisting in diagnosing autism co-morbid epilepsy;
(2) Screening candidate medicines for treating autism co-morbid epilepsy;
(3) And preparing a product for prognosis evaluation of autism co-morbid epilepsy.
5. The use according to claim 4, wherein said diagnosis or co-diagnosis of autism co-morbid epilepsy is achieved by detecting the quantitative proportion of said cell subsets.
6. Detecting the use of a substance of the cell subpopulation according to claim 1 in (4) to (6);
(4) Preparing a product for diagnosing or assisting in diagnosing autism co-morbid epilepsy;
(5) Screening candidate medicines for treating autism co-morbid epilepsy;
(6) And preparing a product for prognosis evaluation of autism co-morbid epilepsy.
7. The use according to claim 6, wherein the substance is used to detect the content of the cell subpopulation according to claim 1; preferably, the content of the cell subpopulation refers to the percentage of the number of the cell subpopulation in peripheral blood mononuclear cells; preferably, the substance comprises antibodies for detecting CD4TEMRA that label different fluorescence: CD3 antibodies, CD4 antibodies, CD45RA antibodies, CCR7 antibodies, and CD27 antibodies.
8. Use of a method of detection of a cell subpopulation according to claim 1 for non-disease diagnostic or therapeutic purposes, said method of detection comprising the steps of:
1) Forming single cell suspension by treating the peripheral blood sample;
2) Mixing the single cell suspension of step 1) with the addition of a different fluorescently labeled antibody: the CD3 antibody, CD4 antibody, CD45RA antibody, CCR7 antibody and CD27 antibody were incubated in a mix;
3) Sorting to obtain data of the cell subpopulations.
9. The use according to claim 8 for studying the mechanism of T cell immune abnormality of the cell subpopulation according to claim 1 in diagnosis or co-diagnosis of autism co-morbid epileptic patients.
10. Use of a substance that reduces the content of a cell subpopulation according to claim 1 in blood for the manufacture of a medicament for the prevention or treatment of autism co-morbid epilepsy.
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