CN105823791B - A kind of determination method and application thereof of pair of medulloblastoma cell thermodynamic parameter - Google Patents
A kind of determination method and application thereof of pair of medulloblastoma cell thermodynamic parameter Download PDFInfo
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Abstract
The invention belongs to clinical testing techniques fields, are related to the determination method and application thereof of a kind of pair of medulloblastoma cell thermodynamic parameter.By differential scanning calorimetry (DSC) in the method for the present invention, in vitro Cerebrospinal Fluid in Patients (CSF) sample of acquisition is heated, the thermodynamic parameter variation generated in the heating process is analyzed, determines critical parameter value；This method optimizes cerebrospinal fluid protein purification processes program, simplify experimental implementation, it can directly, rapidly check CSF sample Tm, when Tm=73.76 DEG C, specificity is 69.6%, sensibility is 86.7%, using the value as detection threshold value, facilitate the auxiliary judgment that medulloblastoma sends out transfer, this method is without expensive experimental facilities, simplicity, it is easy；There is provided the further research for shifting and sending out biobehavioral for pith mother cells to advantageous tool.
Description
Technical field
The invention belongs to clinical testing techniques field, be related to medulloblastoma cell detection more particularly to a kind of couple of marrow mother
The determination method and application thereof of cell carcinoma cells thermodynamic parameter.Pass through differential scanning calorimetry in the method for the present invention
(differential scanning calorimetry, DSC), to the in vitro Cerebrospinal Fluid in Patients (Cerebro of acquisition
Spinal fluid, CSF) sample heating, the thermodynamic parameter variation generated in the heating process is analyzed, determines parameter
The parameter of critical value, the method for the present invention and acquisition facilitates the judgement that medulloblastoma sends out transfer.
Background technique
Document report, medulloblastoma (M) send out children well, this kind of tumour originates from embryonal rest cell, and life span is short,
It is one of highest Neuro-epithelial tumor of central nervous system grade malignancy.The studies have shown that MB overwhelming majority is grown in cerebellum
Vermis, it is its important biomolecule characteristic (Brandes AA, Crit Rev Oncol that it can send out transfer with cerebrospinal fluid
Hematol, 2004), MB is divided for M0-M4 type according to send out state and the range of medulloblastoma in usual clinical practice,
Middle M0 is not find to shift, and M1 is to find tumour cell in CSF, M2-4 be found outside encephalic or cranium metastatic lesion (Chang CH,
Radiology,1969).There is research to think, the transfer (M1-M4) of tumour is the one of the important signs that of tumor prognosis, it is determined
The therapeutic modality of tumour and strategy (Zeltzer PM, J Clin Oncol, 1999；Packer RJ,Pediatr
Neurosurg,2003).Therefore, whether identification MB, which shifts, is sent out with very important clinical implications.
In general, mainly pass through the existence of iconography and exfoliated tumor cells tumor cell in clinical practice,
Wherein, imaging diagnosis is still main standard, can be more objective especially when detecting the existence of medulloblastoma cell
Display medulloblastoma cell existence, i.e., can be to shift disease outside clinical discovery MB encephalic or cranium by Imaging Method
Stove is provided with the detection foundation of reference significance, but it still needs experienced neurosurgeon or iconography doctor to it
Comprehensive descision is carried out, but it cannot find the M1 state of medulloblastoma, and M1 state is also the weight for influencing the survival of patients time
Index is wanted, and M1 state can only be checked by exfoliative cytology at present, but regrettably, exfoliative cytology is no matter on special
Or all Shortcomings in sensibility, such as: in specificity, exfoliative cytology is checked condition and time by lumbar puncture
Stringent limitation, the Exfoliated tumor cells of early stage postoperative discovery might not imply the specific existence of tumour cell, and
In sensibility, exfoliative cytology check sensibility only 35% or so (Terterov S, J Neurosurg Pediatr,
2010).In related Prospective Clinical development test, M1 state patient MB is not had found by exfoliative cytology CSF detection,
It is confirmed to occur to shift patient's MB exfoliative cytology recall rate of (M2-M4) state outside encephalic to be also 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
With the judgement for state of sending out.
DSC is the research tool for being widely used in inorganic and organic compound and Pharmaceutical Analysis.Its essence is one
Kind calorimetry, it is under programed temperature, and the difference power of substance and reference substance and the relationship of temperature are defeated by observation, and according to heat
Amount and temperature correlation draw DSC curve.The method can measure a variety of thermodynamics and kinetics parameters, such as specific heat capacity, enthalpy change, anti-
Answer (as shown in Figure 1) such as heat, phasor, reaction rate, crystalline rate, superpolymer crystal degree, sample dimensions.And in biological sample
In, the dependent interaction of complicated molecule can change the Thermal properties of biological sample, therefore it can be used in the list of research purifying
A biopolymer and the interaction between them, the technology are gradually applied to the body fluid such as human plasma, serum
Protein group specifically sexually revising under morbid state (Garbett NC, Plos One, 2014；Engh JA,
Neurosurgery, 2011).
Summary of the invention
The object of the present invention is to provide the determination method of a kind of pair of medulloblastoma cell thermodynamic parameter, the present invention
By differential scanning calorimetry (differential scanning calorimetry, DSC) in method, to the in vitro of acquisition
Cerebrospinal Fluid in Patients (Cerebro spinal fluid, CSF) sample heating, to the thermodynamic parameter generated in the heating process
Variation is analyzed, and determines critical parameter value.
The further object of the present invention is to provide the above method in sending out transfer for auxiliary judgment medulloblastoma
Purposes.
Based on medulloblastoma cell there is the characteristic sent out of cerebrospinal fluid and its cerebrospinal fluid can generate specificity in the present invention
Biomarker, combine, can change these biomarkers as ligand and the original high abundance receptor protein of cerebrospinal fluid
Medulloblastoma forms characteristic thermodynamic parameter in the characteristic of cerebrospinal fluid DSC Thermogram；By DSC to it in the present invention
Relevant parameter is detected, and result assists in the identification that state is sent out in MB transfer；The DSC that this method uses will be to be existing
Imaging diagnosis means provide strong supplement, are further provided with to judge the prognosis of medulloblastoma and carrying out individualized treatment
The auxiliary tool of power.
Specifically, combining DSC and CSF in the present invention purifies analytical technology, purification CSF egg is analyzed by improving technology
It is white, using poor scanning calorimetry (differential scanning calorimetry, DSC), optimize DSC operating parameter simultaneously
DSC result parameter is screened, the results show that wherein solution temperature (Tm) value is that transfer is sent out medulloblastoma patient and significantly sexually revised
Index, further by Receiver operating curve (receiver operating characteristic curve,
ROC) this method is analyzed, area is 0.835 under ROC curve, prompts have good reference to the judgement of medulloblastoma
It is worth (as shown in the figure), when Tm=73.76 DEG C, specificity is 69.6%, sensibility 86.7%, shows this detection and analysis side
The more traditional iconography of method and exfoliative cytology detection method have a clear superiority, meanwhile, this method is simple and easy to do, cost is relatively low,
Quality is stablized, and provides useful auxiliary tool for the transfering state judgement of medulloblastoma and personalized treatment.
More specifically, the determination method of a kind of pair of medulloblastoma cell thermodynamic parameter of the invention, feature
It is comprising step:
1, sample process is carried out to the cerebrospinal fluid (Cerebro spinal fluid, CSF) of in vitro patient:
1) the CSF sample under the conditions of being stored in -80 DEG C is taken out, at room temperature natural thaw；
2) it is loaded onto Amicon Ultra-0.5ml ultracentrifugation pipe, each sample cell capping, and is carried out according to sample number
Label；
3) 4 DEG C of 14000 × g ultrafiltration are centrifuged 30 minutes；
4) liquid in bottom collecting pipe is outwelled, adds the PBS (PH=7.4) of 450ul4 DEG C of pre-cooling to filter core, 14000 × g
Centrifugation 30 minutes；
5) liquid in bottom collecting pipe is outwelled, adds the PBS (PH=7.4) of 450ul4 DEG C of pre-cooling to filter core, 14000 × g
Centrifugation 30 minutes；
6) complete last wheel centrifugation after, take out filter core, upside down on another clean collecting pipe, 1000 ×
G is centrifuged 2 minutes recycling concentrates；
7) concentrate is diluted to the final volume of 600ul with PBS (4 DEG C)；
8) 20ul is respectively taken to carry out protein concentration measurement, the inspection at 4 DEG C of remaining Sample preservation with BCA method in each sample
DSC detection；
2, differential scanning calorimetry (differential scanning calorimetry, DSC) analyzes sample:
1) 400ulPBS buffer or sample are splined on dedicated 96 orifice plate of microcal VP-capillary DSC；
2) Sample Scan recording interval sets heating rate as 1.0 DEG C/min from 10 DEG C to 110 DEG C；
3) initial data is first corrected with buffer-buffer scanning data, and according to the total protein concentration of sample
It is normalized；
4) Thermogram is varied with temperature and is drawn according to specific heat capacity (Cal/ (DEG C g)), utilizes included origin software
It is drawn, and calculates Tm value.
In the sample process of CSF of the invention: using Amicon Ultra-0.5ml centrifuge tube 14000 × g ultracentrifugation
At least 3 times, average no less than 30 minutes every time, this method is enable to improve cerebrospinal fluid purity to greatest extent, reduce impurity interference letter
Number, improve sample homogeneity；
In the parameter setting of DSC detection of the invention: from 10 DEG C to 110 DEG C, setting heating rate as 1.0 DEG C/min, DSC
Curve ordinate is specific heat capacity (Cal/ (DEG C g)), and 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 sensibility is
86.7%.
The present invention has carried out prospective clinical trial, the results show that transfer sends out patient MB CSF compared with no transporting patient CSF
Tm value it is significantly raised；Further pass through Receiver operating curve (receiver operating characteristic
Curve, ROC) analysis, the results show that area under the curve is 0.829, with higher judgement reference value, as dividing value Tm=
73.76 DEG C, characteristic 69.6%, sensibility 86.7%.
The method of the present invention sends out transfer can be used for auxiliary judgment medulloblastoma.
The invention has the following advantages that
1, DSC and csf protein purification technique are combined, CSF sample Tm can directly, be rapidly checked, work as Tm=73.76
DEG C when, specificity be 69.6%, sensibility 86.7% facilitates medulloblastoma and sends out transfer using the value as detection threshold value
Judgement, auxiliary judgment value better than iconography detect；Compared with the detection of CSF exfoliative cytology, medulloblastoma is detected
The sensibility and specificity of the existence of cell significantly improves；Meanwhile experimental facilities of this method without valuableness, it is easy,
It is easy；
2, this method optimizes cerebrospinal fluid protein purification processes program, simplifies experimental implementation, and disturbing factor control is arrived
It is minimum；
3, after the Tm value that this method obtains is in conjunction with the significant small molecular protein of metastatic tumor in CSF and high abundance receptor protein
The change of CSF thermal stability is related, therefore this method provides the further research for shifting and sending out biobehavioral for pith mother cells
Advantageous tool.
In order to make it easy to understand, of the invention will be described in detail by specific drawings and examples below.It needs
It wants it is emphasized that specific example and attached drawing are merely to explanation, it is clear that those skilled in the art can be according to this
Text explanation, makes various modifications and variations to the present invention within the scope of the invention, these modifications and variations are also included in
In the scope of the present invention.In addition, the present invention refers to open source literature, these documents be in order to more clearly describe the present invention, it
Entire contents be included in and referred to herein, just look like that repeated description herein has been excessively for their full text.
Detailed description of the invention
Fig. 1 difference scanning calorimetry major parameter.
Non- Thermogram (n=23) (b) mean values for sending out transfer group medulloblastoma case of Fig. 2 (a), dash area
For 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 is non-to send out transfer group and sends out transfer group medulloblastoma case Thermogram average value (a) and Tm value (b) ratio
Compared with.
Fig. 5 .ROC curve, area under the curve 0.829, best Tm=73.76 DEG C of the dividing value of correspondence (specificity: 69.6%,
Sensibility: 86.7%).
Specific embodiment
Embodiment 1 tests the detection and analysis of medulloblastoma cell thermodynamic parameter
1, sample process is carried out to the cerebrospinal fluid of in vitro patient (Cerebro spinal fluid, CSF):
1) the CSF sample under the conditions of being stored in -80 DEG C is taken, at room temperature natural thaw；
2) it is loaded onto Amicon Ultra-0.5ml ultracentrifugation pipe, each sample cell capping, and is carried out according to sample number
Label；
3) 4 DEG C of 14000 × g ultrafiltration are centrifuged 30 minutes；
4) liquid in bottom collecting pipe is outwelled, adds the PBS (PH=7.4) of 450ul4 DEG C of pre-cooling to filter core, 14000 × g
Centrifugation 30 minutes；
5) liquid in bottom collecting pipe is outwelled, adds the PBS (PH=7.4) of 450ul4 DEG C of pre-cooling to filter core, 14000 × g
Centrifugation 30 minutes；
6) complete last wheel centrifugation after, take out filter core, upside down on another clean collecting pipe, 1000 ×
G is centrifuged 2 minutes recycling concentrates；
7) concentrate is diluted to the final volume of 600ul with PBS (4 DEG C)；
8) 20ul is respectively taken to carry out protein concentration measurement, the inspection at 4 DEG C of remaining Sample preservation with BCA method in each sample
DSC detection；
2, differential scanning calorimetry (differential scanning calorimetry, DSC) analyzes sample:
1) 400ulPBS buffer or sample are splined on dedicated 96 orifice plate of microcal VP-capillary DSC；
2) Sample Scan recording interval sets heating rate as 1.0 DEG C/min from 10 DEG C to 110 DEG C；
3) initial data is first corrected with buffer-buffer scanning data, and according to the total protein concentration of sample
It is normalized；
4) Thermogram is varied with temperature and is drawn according to specific heat capacity (Cal/ (DEG C g)), utilizes included origin software
It is drawn, and calculates Tm value.
For the present invention from sending out in the numerous parameters of transporting patient CSF DSC, it is significant for filtering out wherein solution temperature Tm
Change parameter, verified through prospective clinical trial, wherein to send out patient MB CSF obvious compared with the Tm value of no transporting patient CSF for transfer
Increase, further by Receiver operating curve (receiver operating characteristic curve,
ROC it) analyzes, area under the curve 0.829, judgement with higher auxiliary is worth, and when Tm=73.76 DEG C of dividing value, characteristic is
69.6%, sensibility 86.7%.
Claims (4)
1. for being used for auxiliary judgment in preparation to the reagent in the determination method of medulloblastoma cell thermodynamic parameter
The purposes of medulloblastoma sent out in the reagent of transfer；The determination method comprising step:
1) sample process is carried out to the cerebrospinal fluid CSF of in vitro patient:
(1) the CSF sample under the conditions of being stored in -80 DEG C is taken out, at room temperature natural thaw；
(2) it is loaded onto Amicon Ultra-0.5mL ultracentrifugation pipe, each sample cell capping, and is marked；
(3) 4 DEG C of 14000 × g ultrafiltration are centrifuged 30 minutes；
(4) outwell the liquid in bottom collecting pipe, add 450 μ L, 4 DEG C of pre-coolings PH=7.4 PBS to filter core, 14000 × g from
The heart 30 minutes；
(5) outwell the liquid in bottom collecting pipe, add 450 μ L, 4 DEG C of pre-coolings PH=7.4 PBS to filter core, 14000 × g from
The heart 30 minutes；
(6) after completing centrifugation, filter core is taken out, is upside down on another collecting pipe, 1000 × g is centrifuged 2 minutes recycling concentrates；
(7) concentrate is diluted to the final volume of 600 μ L with 4 DEG C of PBS；
(8) 20 μ L are respectively taken to carry out protein concentration measurement with BCA method in each sample, remaining Sample preservation send DSC to examine at 4 DEG C
It surveys；
2) differential scanning calorimetry dsc analysis sample:
(1) 400 μ LPBS buffers or sample are splined on to 96 orifice plates of microcal VP-capillary DSC；
(2) Sample Scan recording interval sets heating rate as 1.0 DEG C/min from 10 DEG C to 110 DEG C；
(3) initial data is first corrected with buffer-buffer scanning data, and is carried out according to the total protein concentration of sample
Normalized；
(4) Thermogram is varied with temperature according to specific heat capacity and is drawn, and is drawn using origin software, and calculates dissolution
Temperature Tm value；Wherein, specific heat capacity unit be Cal/(DEG C g).
2. purposes according to claim 1, which is characterized in that in the determination method, in the sample process of CSF:
It is centrifuged at least 3 times using Amicon Ultra-0.5ml centrifuge tube 14000 × g ultrafiltration, average no less than 30 minutes every time.
3. purposes according to claim 1, which is characterized in that in the determination method, the parameter setting of DSC detection
In: from 10 DEG C to 110 DEG C, heating rate is set as 1.0 DEG C/min, DSC curve ordinate is specific heat capacity, and abscissa is temperature
Value.
4. purposes according to claim 1, which is characterized in that in the determination method, solution temperature Tm is judgement
Parameter, when Tm=73.76 DEG C, specificity is 69.6%, sensibility 86.7%.
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