CN105823791A - Detection and analysis method for thermodynamic parameters of medulloblastoma cells and purpose thereof - Google Patents
Detection and analysis method for thermodynamic parameters of medulloblastoma cells and purpose thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of clinical examination, and relates to a detection and analysis method for thermodynamic parameters of medulloblastoma cells and purpose thereof. According to the method, through a differential scanning calorimetry (DSC), an in-vitro patient cerebrospinal fluid (CSF) specimen is heated, thermodynamic parameter variation generated during a heating process is analyzed, and then a parameter critical value is determined. The method optimizes a purification treatment process of cerebrospinal fluid protein, experiment operation is simplified, the CSF specimen Tm can be directly and rapidly detected, when Tm is 73.76 DEG C, the specificity is 69.6%, sensitivity is 86.7%, and the value is the detection threshold value which is an auxiliary determination for dissemination and transfer of the medulloblastoma cells. No expensive experiment equipment is required in the method, and the method has the advantages of simple and easy operation, and provides favorable tools for researches of dissemination and transfer behaviors of the medulloblastoma cells.
Description
Technical field
The invention belongs to clinical testing techniques field, relate to the detection of medulloblastoma cell, particularly relate to a kind of determination method to medulloblastoma cell thermodynamic parameter and application thereof.By differential scanning calorimetry (differentialscanningcalorimetry in the inventive method, DSC), to the in vitro Cerebrospinal Fluid in Patients (Cerebrospinalfluid obtained, CSF) specimen heating, thermodynamic parameter change to producing in this heating process is analyzed, determine critical parameter value, the inventive method and acquisition parameter contribute to medulloblastoma and send out the judgement of transfer.
Background technology
Document is reported, medulloblastoma (M) sends out child well, and this kind of tumor originates from embryonal rest cell, and life span is short, is one of Neuro-epithelial tumor that central nervous system's grade malignancy is the highest.Research display, the MB overwhelming majority is grown in vermis cerebelli, it can to send out transfer be its important biomolecule characteristic (BrandesAA, CritRevOncolHematol, 2004) along with cerebrospinal fluid, in usual clinical practice, MB is divided into M0-M4 type by state of sending out and scope according to medulloblastoma, wherein M0 is not for finding transfer, and M1 is discovery tumor cell in CSF, and M2-4 is to find metastatic lesion (ChangCH outside intracranial or cranium, Radiology, 1969).Having research to think, the transfer (M1-M4) of tumor is one of important symbol of tumor prognosis, which determines therapeutic modality and strategy (ZeltzerPM, JClinOncol, 1999 of tumor；PackerRJ,PediatrNeurosurg,2003).Therefore, whether identification MB shifts to send out and has very important clinical implications.
nullGenerally，Mainly by iconography and the existence of exfoliated tumor cells tumor cell in clinical practice，Wherein，Imaging diagnosis remains main standard，Especially when detecting the existence of medulloblastoma cell，The existence of the more objective display medulloblastoma cell of energy，I.e. can be provided with the detection foundation of reference significance for the outer metastatic lesion of clinical discovery MB intracranial or cranium by Imaging Method，But it need nonetheless remain for experienced neurosurgeon or iconography doctor carries out comprehensive descision to it，But it can not find the M1 state of medulloblastoma，And M1 state is also the important indicator affecting the survival of patients time，And M1 state can only be checked by exfoliative cytology at present，But regrettably，Exfoliative cytology all Shortcomings on special or in sensitivity，As: on specificity，By lumbar puncture, exfoliative cytology is checked that condition and time are strictly limited，The Exfoliated tumor cells of the most postoperative discovery might not imply that the concrete existence of tumor cell，And in sensitivity，Exfoliative cytology checks that sensitivity is only at about 35% (TerterovS，JNeurosurgPediatr，2010).In relevant Prospective Clinical development test, M1 state MB patient is not found by the detection of exfoliative cytology CSF, being confirmed that the patient's MB exfoliative cytology recall rate occurring to shift (M2-M4) state outside intracranial is only 26.7% by iconography, therefore the method about the existence of detection medulloblastoma cell is beneficial to improve and supplement to pith mother cells tumor metastasis and the judgement of sending out state.
DSC is a research tool being widely used in inorganic and organic compound and pharmaceutical analysis.Its essence is a kind of calorimetry, and it is under programed temperature, observes the difference power of material and reference substance and the relation of temperature of being defeated by, and draws DSC curve according to heat and temperature mutual relation.This method can measure multiple thermodynamics and kinetics parameter, such as (as shown in Figure 1) such as specific heat capacity, enthalpy change, reaction heat, phasor, reaction rate, crystalline rate, superpolymer crystal degree, sample dimensions.And in biological specimen, the dependent interaction of complicated molecule can change the Thermal properties of biological specimen, therefore it can be used in studying single biopolymer and the interaction between them of purification, this technology has gradually been applied to the body fluid protein group such as human plasma, serum specificity under morbid state and has been changed (GarbettNC, PlosOne, 2014；EnghJA, Neurosurgery, 2011).
Summary of the invention
It is an object of the invention to provide a kind of determination method to medulloblastoma cell thermodynamic parameter, by differential scanning calorimetry (differentialscanningcalorimetry in the inventive method, DSC), to the in vitro Cerebrospinal Fluid in Patients (Cerebrospinalfluid obtained, CSF) specimen heating, thermodynamic parameter change to producing in this heating process is analyzed, and determines critical parameter value.
A further object of the present invention is to provide said method in the purposes sending out in transfer for auxiliary judgment medulloblastoma.
The present invention has, based on medulloblastoma cell, the characteristic that cerebrospinal fluid is sent out, and cerebrospinal fluid can produce specific biomarker, these biomarkers are combined as part and cerebrospinal fluid original high abundance receptor protein, the medulloblastoma characteristic at cerebrospinal fluid DSC Thermogram can be changed, form distinctive thermodynamic parameter；Being detected its relevant parameter by DSC in the present invention, its result assists in MB transfer and sends out the identification of state；The DSC that this method uses will provide strong for existing imaging diagnosis means and supplement, further for judging the prognosis of medulloblastoma and carrying out the aid that individualized treatment provides strong.
nullConcrete，The present invention combines DSC Yu CSF and purifies analytical technology，Csf protein is purified by improving technology analysis，Use poor scanning calorimetry (differentialscanningcalorimetry，DSC)，Optimize DSC operating parameter and screen DSC result parameter，Result shows，Wherein solution temperature (Tm) value is that medulloblastoma patient's significance change index is sent out in transfer，Further by Receiver operating curve (receiveroperatingcharacteristiccurve,ROC) the method is analyzed，Under ROC curve, area is 0.835，The judgement to medulloblastoma is pointed out to have good reference value (as shown in the figure)，When Tm=73.76 DEG C，Specificity is 69.6%,Sensitivity is 86.7%，Show that the more traditional iconography of this determination method and exfoliative cytology detection method have clear superiority，Simultaneously，This method is simple and easy to do、Cost is relatively low、Steady quality，Transfering state for medulloblastoma judges and personalized treatment provides useful aid.
More specifically, a kind of determination method to medulloblastoma cell thermodynamic parameter of the present invention, it is characterised in that it includes step:
1, the cerebrospinal fluid (Cerebrospinalfluid, CSF) of in vitro patient is carried out sample process:
1) taking-up is stored in the CSF specimen under the conditions of-80 DEG C, the most naturally thaws；
2) being loaded onto AmiconUltra-0.5ml ultracentrifugation pipe, each sample cell covers, and is marked according to sample number；
3) 4 DEG C of 14000 × g ultrafiltration are centrifuged 30 minutes；
4) outwelling the liquid in bottom collecting pipe, the PBS (PH=7.4) adding 450ul4 DEG C of pre-cooling arrives filter element, and 14000 × g is centrifuged 30 minutes；
5) outwelling the liquid in bottom collecting pipe, the PBS (PH=7.4) adding 450ul4 DEG C of pre-cooling arrives filter element, and 14000 × g is centrifuged 30 minutes；
6) complete last take turns centrifugal after, take out filter element, upside down is on another clean collecting pipe, and 1000 × g is centrifuged 2 minutes and reclaims concentrated solution；
7) concentrated solution PBS (4 DEG C) is diluted to the final volume of 600ul；
8) each sample respectively takes 20ul BCA method and carries out protein concentration measurement, the censorship DSC detection at 4 DEG C of residue Sample preservation；
2, differential scanning calorimetry (differentialscanningcalorimetry, DSC) analysis sample:
1) 400ulPBS buffer or sample are splined on 96 special for microcalVP-capillaryDSC orifice plates；
2) Sample Scan recording interval is from 10 DEG C to 110 DEG C, sets programming rate as 1.0 DEG C/min；
3) initial data is first corrected by the data of buffer-buffer scanning, and is normalized according to the total protein concentration of sample；
4) Thermogram varies with temperature draw according to specific volume (Cal/ DEG C of .g), and utilization carries origin software and draws, and calculates Tm value.
In the sample process of the CSF of the present invention: use AmiconUltra-0.5ml centrifuge tube 14000rpm ultracentrifugation at least 3 times, average every time no less than 30 minutes, make this method can improve cerebrospinal fluid purity to greatest extent, reduce impurity interference signal, improve sample homogeneity；
The parameter of the DSC detection of the present invention is arranged: from 10 DEG C to 110 DEG C, sets programming rate as 1.0 DEG C/min, and DSC curve vertical coordinate is specific volume (Cal/ DEG C of .g), abscissa is temperature value；
In the present invention, Diagnostic parameters is solution temperature Tm, and when Tm=73.76 DEG C, specificity is 69.6%, and sensitivity is 86.7%.
The present invention has carried out prospective clinical trial, and result shows, it is significantly raised compared with the Tm value without transporting patient CSF that patient MB CSF is sent out in transfer；Being analyzed by Receiver operating curve (receiveroperatingcharacteristiccurve, ROC) further, result shows, area under curve is 0.829, has higher judgement reference value, when dividing value Tm=73.76 DEG C, characteristic is 69.6%, and sensitivity is 86.7%.
The inventive method sends out transfer can be used for auxiliary judgment medulloblastoma.
The invention have the advantages that
1, DSC and csf protein purification technique are combined, CSF specimen Tm can be checked directly, rapidly, when Tm=73.76 DEG C, specificity is 69.6%, sensitivity is 86.7%, with this value for detection threshold value, contributing to medulloblastoma and send out the judgement of transfer, its auxiliary judgment is worth and is better than iconography detection；Compared with the detection of CSF exfoliative cytology, the Sensitivity and Specificity of the existence of its detection medulloblastoma cell all significantly improves；Meanwhile, this method is without expensive experimental facilities, easy, easy；
2, this method optimizes cerebrospinal fluid protein purification processes program, simplifies experimental implementation, controls interference factor to minimum；
3, after this method obtains Tm value small molecular protein significant with metastatic tumor in CSF and high abundance receptor protein combine, CSF heat stability changes is relevant, and therefore this method is by for pith mother cells transfer with send out the further of Biology seed coating and study the instrument that offer is favourable.
By concrete drawings and Examples, the present invention will be described in detail in order to make it easy to understand, following.It needs to be noted, instantiation and accompanying drawing are merely to explanation, obviously the present invention can be made various correction and change according to illustrating herein by those of ordinary skill in the art within the scope of the invention, and these are revised and change and also include in the scope of the present invention.It addition, the present invention refer to open source literature, these documents are to more clearly describe the present invention, and their entire contents is all included in and carried out reference herein, just look like they full text repeated description the most in this article excessively as.
Accompanying drawing explanation
Fig. 1. difference scanning calorimetry major parameter.
Fig. 2. (a) non-Thermogram (n=23) (b) mean values sending out transfer group medulloblastoma case, dash area is standard deviation.
Fig. 3. (a) sends out Thermogram (n=15) (b) mean values of transfer group medulloblastoma case, and dash area is standard deviation.
Fig. 4. non-send out transfer group and send out transfer group medulloblastoma case Thermogram meansigma methods (a) and Tm value (b) compares.
Fig. 5 .ROC curve, area under curve is 0.829, corresponding optimal dividing value Tm=73.76 DEG C (specificity: 69.6%, sensitivity: 86.7%).
Detailed description of the invention
The detection of medulloblastoma cell thermodynamic parameter is analyzed experiment by embodiment 1
1, the cerebrospinal fluid (Cerebrospinalfluid, CSF) of in vitro patient is carried out sample process:
1) take the CSF specimen being stored under the conditions of-80 DEG C, the most naturally thaw；
2) being loaded onto AmiconUltra-0.5ml ultracentrifugation pipe, each sample cell covers, and is marked according to sample number；
3) 4 DEG C of 14000 × g ultrafiltration are centrifuged 30 minutes；
4) outwelling the liquid in bottom collecting pipe, the PBS (PH=7.4) adding 450ul4 DEG C of pre-cooling arrives filter element, and 14000 × g is centrifuged 30 minutes；
5) outwelling the liquid in bottom collecting pipe, the PBS (PH=7.4) adding 450ul4 DEG C of pre-cooling arrives filter element, and 14000 × g is centrifuged 30 minutes；
6) complete last take turns centrifugal after, take out filter element, upside down is on another clean collecting pipe, and 1000 × g is centrifuged 2 minutes and reclaims concentrated solution；
7) concentrated solution PBS (4 DEG C) is diluted to the final volume of 600ul；
8) each sample respectively takes 20ul BCA method and carries out protein concentration measurement, the censorship DSC detection at 4 DEG C of residue Sample preservation；
2, differential scanning calorimetry (differentialscanningcalorimetry, DSC) analysis sample:
1) 400ulPBS buffer or sample are splined on 96 special for microcalVP-capillaryDSC orifice plates；
2) Sample Scan recording interval is from 10 DEG C to 110 DEG C, sets programming rate as 1.0 DEG C/min；
3) initial data is first corrected by the data of buffer-buffer scanning, and is normalized according to the total protein concentration of sample；
4) Thermogram varies with temperature draw according to specific volume (Cal/ DEG C of .g), and utilization carries origin software and draws, and calculates Tm value.
The present invention is from sending out the numerous parameter of transporting patient CSFDSC, filter out wherein solution temperature Tm for significantly changing parameter, verify through prospective clinical trial, wherein to send out patient MB CSF significantly raised compared with the Tm value without transporting patient CSF in transfer, further by Receiver operating curve (receiveroperatingcharacteristiccurve, ROC) analyze, area under curve is 0.829, there is higher judgement auxiliary be worth, when dividing value Tm=73.76 DEG C, characteristic is 69.6%, and sensitivity is 86.7%.
Claims (5)
1. the determination method to medulloblastoma cell thermodynamic parameter, it is characterised in that it includes step:
1) cerebrospinal fluid (CSF) in vitro patient carries out sample process:
(1) taking-up is stored in the CSF specimen under the conditions of-80 DEG C, the most naturally thaws；
(2) being loaded onto AmiconUltra-0.5ml ultracentrifugation pipe, each sample cell covers, and labelling；
(3) 4 DEG C of 14000 × g ultrafiltration are centrifuged 30 minutes；
(4) outwelling the liquid in bottom collecting pipe, the PBS (PH=7.4) adding 450ul4 DEG C of pre-cooling arrives filter element, and 14000 × g is centrifuged 30 minutes；
(5) outwelling the liquid in bottom collecting pipe, the PBS (PH=7.4) adding 450ul4 DEG C of pre-cooling arrives filter element, and 14000 × g is centrifuged 30 minutes；
(6) after completing to be centrifuged, taking out filter element, upside down is on another collecting pipe, and 1000 × g is centrifuged 2 minutes and reclaims concentrated solution；
(7) concentrated solution PBS (4 DEG C) is diluted to the final volume of 600ul；
(8) each sample respectively takes 20ul BCA method and carries out protein concentration measurement, the censorship DSC detection at 4 DEG C of residue Sample preservation；
2) differential scanning calorimetry (DSC) analysis sample:
(1) 400ulPBS buffer or sample are splined on 96 orifice plates of microcalVP-capillaryDSC；
(2) Sample Scan recording interval is from 10 DEG C to 110 DEG C, sets programming rate as 1.0 DEG C/min；
(3) initial data is first corrected by the data of buffer-buffer scanning, and is normalized according to the total protein concentration of sample；
(4) Thermogram varies with temperature draw according to specific volume (Cal/ DEG C of .g), utilizes origin software to draw, and calculates Tm value.
2. the determination method to medulloblastoma cell thermodynamic parameter as described in claim 1, it is characterized in that, in the sample process of described CSF: use AmiconUltra-0.5ml centrifuge tube 14000rpm ultracentrifugation at least 3 times, average every time no less than 30 minutes.
3. the determination method to medulloblastoma cell thermodynamic parameter as described in claim 1, it is characterized in that, during the parameter of described DSC detection is arranged: from 10 DEG C to 110 DEG C, set programming rate as 1.0 DEG C/min, DSC curve vertical coordinate is specific volume (Cal/ DEG C of .g), and abscissa is temperature value.
4. the determination method to medulloblastoma cell thermodynamic parameter as described in claim 1, it is characterised in that in described method, solution temperature Tm is for judge parameter, and when Tm=73.76 DEG C, specificity is 69.6%, and sensitivity is 86.7%.
5. the determination method to medulloblastoma cell thermodynamic parameter described in claim 1 is in the purposes sent out in transfer for auxiliary judgment medulloblastoma.
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| CN111948398A (en) * | 2019-05-14 | 2020-11-17 | 复旦大学附属华山医院 | Cerebrospinal fluid VGF protein kit and application thereof in medulloblastoma metastasis evaluation |
| CN112083168A (en) * | 2019-06-14 | 2020-12-15 | 复旦大学附属华山医院 | Polypeptide fragment diagnosis model and application thereof in predicting medulloblastoma metastasis risk |
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| US20080172184A1 (en) * | 2007-01-12 | 2008-07-17 | University Of Louisville Research Foundation, Inc. | Proteomic profiling method useful for condition diagnosis and monitoring, composition screening, and therapeutic monitoring |
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| US20080172184A1 (en) * | 2007-01-12 | 2008-07-17 | University Of Louisville Research Foundation, Inc. | Proteomic profiling method useful for condition diagnosis and monitoring, composition screening, and therapeutic monitoring |
Non-Patent Citations (2)
| Title |
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| CHAGOVETZ, ALEXIS A. ET AL.: "Differential Scanning Calorimetry of Gliomas: A New Tool in Brain Cancer Diagnostics?", 《NEUROSURGERY》 * |
| CHAGOVETZ, ALEXIS A. ET AL.: "Preliminary use of differential scanning calorimetry of cerebrospinal fluid for the diagnosis of glioblastoma multiforme", 《JOURNAL OF NEURO-ONCOLOGY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111948398A (en) * | 2019-05-14 | 2020-11-17 | 复旦大学附属华山医院 | Cerebrospinal fluid VGF protein kit and application thereof in medulloblastoma metastasis evaluation |
| CN112083168A (en) * | 2019-06-14 | 2020-12-15 | 复旦大学附属华山医院 | Polypeptide fragment diagnosis model and application thereof in predicting medulloblastoma metastasis risk |
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