CN1390950A - Process for measuring bioactivity of nerve growth factor - Google Patents
Process for measuring bioactivity of nerve growth factor Download PDFInfo
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- CN1390950A CN1390950A CN 02134339 CN02134339A CN1390950A CN 1390950 A CN1390950 A CN 1390950A CN 02134339 CN02134339 CN 02134339 CN 02134339 A CN02134339 A CN 02134339A CN 1390950 A CN1390950 A CN 1390950A
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Abstract
A method for measuring the bioactivity of nerve growth factor includes such steps as using the factor to stimulate particular cell system, reproducing it, adding a chromogenic substance to its culture medium, chromometry by enzyme linking instrument, and comparing the result with standard curve. Its advantage are high speed and high precision.
Description
Technical field:
The invention belongs to the biological activity measuring method in the biomedicine field, refer in particular to the bioactive method that method that a kind of usefulness stimulates cellular proliferation is measured nerve growth factor.
Background technology:
Nerve growth factor (nerve growth factor, be called for short NGF) be a kind of to the nutritious effect of normal neurocyte, injured nerve is repaired the biologically active factors that function has regulating effect, it can keep sympathetic nerve and sensorineural existence, promote the differentiation of neurocyte, the direction of extension of decision aixs cylinder.To the growth of promotion brain, neural growth, the regeneration and the functional rehabilitation of injured nerve have decisive role.
People source nerve growth factor (hNGF) can be applicable to diabetic peripheral neuropathy, senile dementia, Parkinson's disease, facial neuritis, and nerve injury (comprises Spinal injury, high paraplegia, severed finger reunion, brain injury, the damage of maincenter such as peripheral nerve injury and nervus peripheralis system) etc. nervous system disorders is at present unique clinical neural system rho factor that can be applicable to.Qualitative and measure the activity of nerve growth factor quantitatively, this point has great function for field of medicaments.The activity determination method of existing nerve growth factor is to adopt the chick embryonic dorsal root ganglion method.At first be that chick embryonic dorsal root ganglion is cultivated: separate the rat coccygeal nerve, be cut into several sections, alcohol disinfecting with 75%, be soaked in then in 1/1000 glacial acetic acid solution 12 hours, centrifuging and taking supernatant liquor, every 10ml add 1/100 phenol red 1ml, mixing, autoclaving, in the 2cm plate, add 1ml and evenly pave, cool to room temperature, put in the drying box, smoked 30 minutes with ammoniacal liquor, wash to neutrality repeatedly with sterilized water again.DMEM substratum (U.S. GIBCO company product), adding 10%, go out can foetal calf serum, an amount of penicillin, Streptomycin sulphate and L-glutaminate, mixing.Get the rhNGF work in-process of filtration sterilization, with the nutrient solution dilution different multiples for preparing.Get 7～9 days chicken embryo, take out dorsal root ganglion under the aseptic condition under dissecting microscope, being placed in scribbles in the culture dish of mouse tail glue, adds the substratum 1ml that contains different extent of dilution rhNGF, in 37 ℃, contain 5%CO
2Incubator in cultivated 24～48 hours, observe with inverted microscope.Relatively the growing state of projection around the neuroganglion is selected projection the closeest the longest person, and NGF amount contained in every ml substratum is decided to be an activity unit.The extension rate of stoste is half-finished activity, is expressed as A ten thousand u/ml.
Adopt this activity determination method step tolerance range loaded down with trivial details, time-consuming, that measure poor, be badly in need of wanting a kind of conveniently mode that the nerve growth factor activity is carried out method for measuring.The improvement of this method not only can be provided convenience for the efficient validity of measuring nerve growth factor, and for quantitative assay nerve growth factor activity, determines that the validity of medicine provides effective correlation data.
Summary of the invention:
The objective of the invention is to utilize different bioactive nerve growth factors inequality to the situation of the propagation stimulation of specific cells system, thereby increase the cell quantity of specific cells system, thereby measure the biological activity of nerve growth factor by the cell quantity of measuring the specific cells system after the propagation, a kind of easy, bioactive method of qualitative and quantitative assay nerve growth factor accurately and fast is provided.
Realize that the technical scheme that purpose of the present invention adopts is to stimulate specific cells system to breed with the active nerve growth factor of known organism, add substance that show color in the substratum after propagation, colorimetric on microplate reader is made typical curve; Stimulate specific cells system to breed the active nerve growth factor of unknown then, add substance that show color in the substratum after propagation, colorimetric on microplate reader will promptly draw the biological activity of the active nerve growth factor of unknown after colorimetric result and the typical curve contrast.
Realize that a concrete technical scheme of the present invention is that the specific cells that somatomedin stimulates proliferation can be TF-1 clone (a human body medullary cell system).Because TF-1 clone is a clone that depends on GM-CSF (CM-CSF), its cell surface has NGF high-affinity receptor TrkA albumen, high-affinity receptor TrkA albumen with can induce TrkA albumen autophosphorylation after NGF combines, thereby cause the propagation of TF-1 cell.The NGF of known different concns is added in the TF-1 cell culture, observe the growing state of TF-1 cell, find that 0.5～50ng/ml NGF can obviously promote its propagation, to reach 50ng/ml saturated when above when the concentration of NGF.During concrete the measurement, in the NGF adding TF-1 cell culture with known different concns, behind the adding substance that show color, colorimetric on microplate reader is made typical curve in the substratum after propagation.The NGF that need are measured unknown concentration adds in the TF-1 cell culture and cultivates, in the culture after cultivation, add substance that show color after, colorimetric on microplate reader with the typical curve contrast, draws the occurrence of unknown concentration.
Employed cell substance that show color is MTT or XTT in the technical scheme of the present invention.
Disclosed concrete technical scheme of the present invention can be in the TF-1 cell suspension of cultivating with GM-CSF, continue behind the medicine of the NGF of adding concentration known and biological activity situation to cultivate, because the proliferate situation of the NGF pair cell of different concns is inequality, after adding cell substance that show color MTT, culture is carried out colorimetric on microplate reader, measure its wavelength, make typical curve.Then in the TF-1 cell suspension of cultivating with GM-CSF, continue to cultivate after adding the medicine that needs measurement nerve growth factor biological activity situation, because the proliferate situation of the NGF pair cell of different concns is inequality, after adding cell substance that show color MTT, culture is carried out colorimetric on microplate reader, measure its wavelength, with the reference as a comparison of the active NGF typical curve of known organism, by the reference standard curve, calculate the biological activity numerical value of NGF sample to be measured.
A preferable scheme of the present invention is that the active rhNGF reference substance of a plurality of known organisms is made gradient by certain numerical value, and adding TF-1 cell suspension that GM-CSF cultivates respectively, to be adjusted to concentration be 1 * 10
5～2 * 10
5/ ml adds 96 well culture plates, and every hole 100 μ l contain 1-50IU/ml GM-CSF.NGF dilutes every hole, back according to a certain percentage and adds 10 μ l, makes blank with 10 μ l serum-free RPM1,1640 substratum.Culture plate is put 37 ℃, 5%CO
2Cultivate 48h in the incubator.Every then hole adds 5mg/ml MTT 20 μ l and continue to cultivate 4～6h, cultivates when finishing, and every hole adds 15%SDS 100 μ l, fully behind the mixing on microplate reader colorimetric, the measurement wavelength is 570nm.The average result of 3 experiments, drawing standard curve are got in each experiment.It is 1 * 10 that the TF-1 cell suspension that adds the GM-CSF cultivation behind the NGF diluted sample to be measured is adjusted to concentration
5～2 * 10
5/ ml adds 96 well culture plates, and every hole 100 μ l contain 1-50IU/mlGM-CSF.NGF dilutes every hole, back according to a certain percentage and adds 10 μ l, makes blank with 10 μ l serum-free RPM11640 substratum.Culture plate is put 37 ℃, 5%CO
2Cultivate 48h in the incubator.Every then hole adds 5mg/ml MTT 20 μ l and continue to cultivate 4～6h, cultivates when finishing, and every hole adds SDS 100 μ l, fully behind the mixing on microplate reader colorimetric, draw numerical value and typical curve and contrast, draw the biological activity numerical value of NGF.
Adopt the measuring method of nerve growth factor of the present invention, can be qualitative and the biological activity of quantitative measurment NGF, have easy, advantage accurately and fast.
Description of drawings:
Fig. 1 is the FB(flow block) that typical curve is obtained.
The canonical plotting that Fig. 2 is among the embodiment to be drawn.
Fig. 3 is the FB(flow block) of measuring method.
Specific embodiment:
Needing measure sample is recombinant human nerve growth factor and mouse source nerve growth factor.As shown in Figure 1, the TF-1 cell suspension that adds the GM-CSF cultivation being adjusted to concentration is 1 * 10
5～2 * 10
5/ ml adds 96 well culture plates, and every hole 100 μ l contain 1-50IU/ml GM-CSF.NGF dilutes every hole, back according to a certain percentage and adds 10 μ l, makes blank with 10 μ l serum-free RPM1,1640 substratum, and by 0.025,0.1,0.4,1.6,6.4,25.6U/ml makes gradient with the active rhNGF reference substance of known organism.Culture plate is put 37 ℃, 5%CO
2Cultivate 48h in the incubator.Every then hole adds 5mg/ml MTT 20 μ l and continue to cultivate 4～6h, cultivates when finishing, and every hole adds SDS 100 μ l, fully behind the mixing on microplate reader colorimetric, the measurement wavelength is 570nm, reference wavelength is 630nm.The average result of 3 experiments is got in each experiment, observed value such as following table:
| The 1st observed value | The 2nd observed value | The 3rd observed value | Equal observed value | |
| ????0.025 | ????0.170 | ????0.172 | ????0.159 | ????0.167 |
| ????0.1 | ????0.165 | ????0.080 | ????0.162 | ????0.169 |
| ????0.4 | ????0.168 | ????0.181 | ????0.155 | ????0.168 |
| ????1.6 | ????0.214 | ????0.217 | ????0.196 | ????0.209 |
| ????4.8 | ????0.306 | ????0.340 | ????0.314 | ????0.320 |
| ????9.6 | ????0.358 | ????0.365 | ????0.345 | ????0.356 |
| ????25.6 | ????0.368 | ????0.375 | ????0.364 | ????0.369 |
According to the numerical value drawing standard curve of average measurement as shown in Figure 2.
As shown in Figure 3, measure the active reference substance A570 of unknown, it is 1 * 10 that the TF-1 cell suspension that adding GM-CSF cultivates is adjusted to concentration
5～2 * 10
5/ ml adds 96 well culture plates, and every hole 100 μ l contain 1-50IU/ml GM-CSF.With reference substance A
570Every hole, dilution back adds 10 μ l in proportion, makes blank with 10 μ l serum-free RPM1,1640 substratum, and culture plate is put 37 ℃, 5%CO
2Cultivate 48h in the incubator.Every then hole adds 5mg/ml MTT 20 μ l and continue to cultivate 4～6h, cultivates when finishing, and every hole adds SDS 100 μ l, fully behind the mixing on microplate reader colorimetric, measured wavelength value contrast known organism typical curve that active gradient is done shown in Figure 2.Institute's test sample product activity can be found from typical curve, is expressed as A ten thousand u/ml.
Claims (9)
1. measure the bioactive method of nerve growth factor for one kind, it is characterized in that stimulating specific cells system to breed the active nerve growth factor of unknown, add substance that show color in the substratum after propagation, colorimetric on microplate reader will promptly draw the biological activity of the active nerve growth factor of unknown after colorimetric result and the typical curve contrast.
2. the method for claim 1 is characterized in that the specific cells that nerve growth factor stimulates proliferation can be a TF-1 clone; Cultivate in the NGF adding TF-1 cell culture with unknown concentration, in the culture after cultivation, behind the adding substance that show color, colorimetric on microplate reader with the typical curve contrast, draws the occurrence of unknown concentration.
3. arbitrary method as claimed in claim 1 or 2 is characterized in that employed cell substance that show color is any among MTT or the XTT.
4. method as claimed in claim 3, it is characterized in that it being in the TF-1 cell suspension of cultivating with GM-CSF, continue to cultivate after adding the medicine that needs measurement nerve growth factor biological activity situation, after adding cell substance that show color MTT continuation cultivation, culture is carried out colorimetric on microplate reader, with the reference as a comparison of the active NGF typical curve of known organism,, calculate the biological activity numerical value of NGF sample to be measured by the reference standard curve.
5. method as claimed in claim 4 is characterized in that it being NGF to be measured to be added TF-1 cell suspension that GM-CSF cultivates to be adjusted to concentration be 1 * 10
5～2 * 10
5/ ml adds 96 well culture plates, and every hole 100 μ l contain 1-50IU/ml GM-CSF.NGF dilutes every hole, back according to a certain percentage and adds 10 μ l, makes blank with 10 μ l serum-free RPM11640 substratum; Culture plate is put 37 ℃, 5%CO
2Cultivate 48h in the incubator; Every then hole adds 5mg/ml MTT 20 μ l and continue to cultivate 4～6h, cultivates when finishing, and every hole adds SDS 100 μ l, fully behind the mixing on microplate reader colorimetric, measure the wavelength absorption value and contrast with typical curve, draw the biological activity numerical value of NGF.
6. the method for claim 1 is characterized in that stimulating specific cells system to breed with the active nerve growth factor of known organism, adds substance that show color in the substratum after propagation, and colorimetric on microplate reader is made typical curve; Stimulate specific cells system to breed the active nerve growth factor of unknown then, add substance that show color in the substratum after propagation, colorimetric on microplate reader will promptly draw the biological activity of the active nerve growth factor of unknown after colorimetric result and the typical curve contrast.
7. method as claimed in claim 6 is characterized in that the specific cells that nerve growth factor stimulates proliferation is a TF-1 clone; Cultivate in the nerve growth factor adding TF-1 cell culture with concentration known, in the culture after cultivation, behind the adding substance that show color, colorimetric on microplate reader, drawing standard curve; Cultivate in the NGF adding TF-1 cell culture with unknown concentration, in the culture after cultivation, behind the adding substance that show color, colorimetric on microplate reader with the typical curve contrast, draws the occurrence of unknown concentration.
8. as claim 3 or 7 described arbitrary methods, it is characterized in that it being in the TF-1 cell suspension of cultivating with GM-CSF, continue to cultivate after adding the medicine of known nerve growth factor biological activity situation, after adding cell substance that show color MTT continuation cultivation, culture is carried out colorimetric, drawing standard curve on microplate reader; In the TF-1 cell suspension with the GM-CSF cultivation, continue to cultivate after adding the medicine that needs measurement nerve growth factor biological activity situation, after adding cell substance that show color MTT continuation cultivation, culture is carried out colorimetric on microplate reader, with the reference as a comparison of the active NGF typical curve of known organism, by the reference standard curve, calculate the biological activity numerical value of NGF sample to be measured.
9. method as claimed in claim 8 is characterized in that it being known recombinant human nerve growth factor and mouse source nerve growth factor to be added TF-1 cell suspension that GM-CSF cultivates to be adjusted to concentration be 1 * 10
5～2 * 10
5/ ml adds 96 well culture plates, and every hole 100 μ l contain 1-50IU/ml GM-CSF; RhNGF dilutes every hole, back according to a certain percentage and adds 10 μ l, makes blank with 10 μ l serum-free RPM11640 substratum, and presses 0.025,0.1,0.4,1.6,6.4 with the rhNGF reference substance, and 25.6/ml makes gradient, and culture plate is put 37 ℃, 5%CO
2Cultivate 48h in the incubator; Every then hole adds 5mg/ml MTT 20 μ l and continue to cultivate 4～6h, cultivates when finishing, and every hole adds SDS 100 μ l, fully behind the mixing on microplate reader colorimetric, measure wavelength, draw numerical value drawing standard curve; NGF to be measured is added TF-1 cell suspension that GM-CSF cultivates, and to be adjusted to concentration be 1 * 10
5～2 * 10
5/ ml adds 96 well culture plates, and every hole 100 μ l contain 1-50IU/ml GM-CSF.NGF dilutes every hole, back according to a certain percentage and adds 10 μ l, makes blank with 10 μ l serum-free RPM11640 substratum; Culture plate is put 37 ℃, 5%CO
2Cultivate 48h in the incubator; Every then hole adds 5mg/ml MTT 20 μ l and continue to cultivate 4～6h, cultivates when finishing, and every hole adds SDS 100 μ l, fully behind the mixing on microplate reader colorimetric, measure wavelength and draw numerical value and contrast with typical curve, draw the biological activity numerical value of NGF.
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| CN 02134339 CN1390950A (en) | 2002-07-11 | 2002-07-11 | Process for measuring bioactivity of nerve growth factor |
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| CN 02134339 CN1390950A (en) | 2002-07-11 | 2002-07-11 | Process for measuring bioactivity of nerve growth factor |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103376248A (en) * | 2012-04-26 | 2013-10-30 | 舒泰神（北京）生物制药股份有限公司 | Quantitative determination method for activity of nerve growth factor |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103376248A (en) * | 2012-04-26 | 2013-10-30 | 舒泰神（北京）生物制药股份有限公司 | Quantitative determination method for activity of nerve growth factor |
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