CN110468102A - A kind of method of in vitro culture tuberculosis infection T cell - Google Patents

A kind of method of in vitro culture tuberculosis infection T cell Download PDF

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CN110468102A
CN110468102A CN201910755331.9A CN201910755331A CN110468102A CN 110468102 A CN110468102 A CN 110468102A CN 201910755331 A CN201910755331 A CN 201910755331A CN 110468102 A CN110468102 A CN 110468102A
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culture
base fluid
tuberculosis infection
free
endotoxin
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谭玉华
江燚
刘灿
邢晓敏
冯淑怡
冯健明
孙勇
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Guangzhou Fenghua Bioengineering Co Ltd
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Abstract

The invention discloses a kind of methods of in vitro culture tuberculosis infection T cell to be mixed by inversion with anti-coagulants at once after acquiring peripheral vein fresh whole blood, dissolve anti-coagulants sufficiently.It is centrifuged removal upper plasma with centrifuge again, the isometric endotoxin-free free serum culture base fluid 1 of blood plasma is added in haemocyte, mixes well.After being centrifuged with centrifuge, upper layer liquid medium 1 is siphoned away.Then the isometric endotoxin-free free serum culture base fluid 2 of blood plasma is added in haemocyte after isolation, mixes well.Blank control pipe N is pressed again, and the sequence of testing tube T and positive control pipe P are separately added into the above blood cell sample containing liquid medium 2 of 1mL in 3 kinds of culture tubes, are mixed by inversion at once, mix well substance in culture tube.Above 3 kinds of culture tubes are uprightly put into constant incubator and are cultivated.After culture, effective centrifuge centrifugation will be cultivated, upper layer liquid medium 2 is taken to detect the concentration of gamma interferon in each culture tube.

Description

A kind of method of in vitro culture tuberculosis infection T cell
Technical field
The invention belongs to fields of biomedicine, and in particular to a kind of method of in vitro culture tuberculosis infection T cell.
Background technique
The method of detection tuberculosis infection T cell has two major classes at present, and one kind is immune spot-ing；Another kind is release in vitro Immunization.After immune spot-ing uses in-vitro separation to T lymphocyte, with corresponding specific antigen while cultivating proliferative T cell It is stimulated.Tuberculosis infection T cell in whole blood or in the T cell that is separated to is activated by corresponding specific antigen to be remembered；There is note The cell recalled can secrete interferon；By detecting the amount of interferon, to judge whether the cell has tuberculosis infection to be formed Memory, has further reflected whether tuberculosis infection.The cell cultivation requirement of immune spot-ing is higher, needs carbon dioxide culture Case, operating procedure is complicated, is as a result judged with spot count, the degree of automation is low, and the single core of the peripheral blood in experimental system is thin The whole-course quality controls such as active somatic cell quantity, cell incubation condition, result judgement are added in born of the same parents' separation, are to determine experiment success or failure Key point.Whole blood is added in the culture tube of corresponding specific antigen by traditional release in vitro method, using whole blood cell culture, because Itself contains higher interferon in certain subject's blood, to will lead to background raising, generates mistake to the interpretation of result Sentence.
Summary of the invention
For the deficiency in the presence of the prior art, the present invention provides the sides that a kind of in vitro culture combines infection T cell Method.The interference for overcoming itself existing gamma interferon in subject's whole blood in traditional whole blood cell culture method, leads to background liter Height, the technical issues of causing result to judge by accident.
To achieve the above object, present invention employs the following technical solutions:
A kind of method of in vitro culture tuberculosis infection T cell, comprising the following steps:
Step 1: acquisition peripheral vein fresh whole blood, the whole blood of acquisition is mixed by inversion with anti-coagulants, keeps anti-coagulants abundant Dissolution；
Step 2: centrifugation, removes upper plasma, retain cellular layer；
The isometric endotoxin-free free serum culture base fluid 1 of blood plasma is added in haemocyte, mixes well for third step, from The heart siphons away the endotoxin-free free serum culture base fluid 1 on upper layer；
Step 4: the isometric endotoxin-free free serum culture base fluid 2 of blood plasma is added in haemocyte after isolation, sufficiently It mixes；
Step 5: pressing the sequence of blank control pipe N, testing tube T and positive control pipe P, it is separately added into 3 kinds of culture tubes 1mL contains 2 blood cell sample of endotoxin-free free serum culture base fluid, mixes well substance in culture tube；
Step 6: by constant temperature is uprightly put into added with 3 kinds of culture tubes of 2 blood cell sample of endotoxin-free free serum culture base fluid It is cultivated in incubator；
Step 7: culture tube is centrifuged, upper layer endotoxin-free free serum culture base fluid 2 is taken to detect each training after culture Support the concentration of gamma interferon in pipe.
Further, the anti-coagulants is heparin sodium or heparin lithium.
Further, the main component of the endotoxin-free free serum culture base fluid 1 is inorganic salts, amino acid, vitamin It is one or more.
Further, the main component of the endotoxin-free free serum culture base fluid 2 is inorganic salts, amino acid, vitamin And cell factor.
Further, the cultivation temperature of the constant incubator is 37 (± 1) DEG C.
Further, the culture tube is 16~24 hours in the incubation time of constant incubator.
Further, the inorganic salts are including but not limited to following several: calcium nitrate, anhydrous magnesium sulfate, anhydrous phosphoric acid two Hydrogen sodium, potassium chloride, sodium chloride, sodium bicarbonate.
Further, the amino acid is including but not limited to following several: L-arginine, L- asparagine, L- winter ammonia Acid, l-cysteine dihydrochloride, Pidolidone, glycine, L-Histidine, L- hydroxyproline, l-Isoleucine, L-Leu, L lysine HCL, l-methionine, L-phenylalanine, L-PROLINE, Serine, L-threonine, L-Trp, L- junket Propylhomoserin, Valine, L-Glutamine.
Further, the vitamin is including but not limited to following several: biotin, D-VB5 calcium, folic acid, i- inositol, Niacinamide, puridoxine hydrochloride, riboflavin, choline chloride, thiamine hydrochloride, vitamin B12, p-aminobenzoic acid.
Further, the growth factor includes following one or more: fibroblast growth factor, para-insulin are raw The long factor, epidermal growth factor, nerve growth factor, platelet derived growth factor and transforming growth factor.
Compared with the prior art, the invention has the following beneficial effects:
1) interference of existing gamma interferon in subject's whole blood in traditional whole blood cell culture method itself is overcome, effectively Background is reduced, resolution ratio and sensitivity are improved；
2) operating procedure when avoiding in-vitro separation T lymphocyte in immune spot-ing is complicated, and condition of culture requires high Limitation；
3) the method for the present invention has easy to operate, reproducible, and cultural method is easy, and resolution ratio and sensitivity height etc. are special Point.
Specific embodiment
A kind of method of in vitro culture tuberculosis infection T cell, comprising the following steps:
Step 1: acquisition peripheral vein fresh whole blood, the whole blood of acquisition is mixed by inversion with anti-coagulants, keeps anti-coagulants abundant Dissolution；
Step 2: centrifugation, removes upper plasma, retain cellular layer；
The isometric endotoxin-free free serum culture base fluid 1 of blood plasma is added in haemocyte, mixes well for third step, from The heart siphons away the endotoxin-free free serum culture base fluid 1 on upper layer；
Step 4: the isometric endotoxin-free free serum culture base fluid 2 of blood plasma is added in haemocyte after isolation, sufficiently It mixes；
Step 5: pressing the sequence of blank control pipe N, testing tube T and positive control pipe P, it is separately added into 3 kinds of culture tubes The above of 1mL is mixed by inversion containing 2 blood cell sample of endotoxin-free free serum culture base fluid, keeps substance in culture tube sufficiently mixed It is even；
Step 6: by constant temperature is uprightly put into added with 3 kinds of culture tubes of 2 blood cell sample of endotoxin-free free serum culture base fluid It is cultivated in incubator；
Step 7: culture tube is centrifuged, upper layer endotoxin-free free serum culture base fluid 2 is taken to detect each training after culture Support the concentration of gamma interferon in pipe.
Further, the anti-coagulants is heparin sodium or heparin lithium.
Further, the main component of the endotoxin-free free serum culture base fluid 1 is inorganic salts, amino acid, vitamin It is one or more.
Further, the main component of the endotoxin-free free serum culture base fluid 2 is inorganic salts, amino acid, vitamin And cell factor.
Further, the cultivation temperature of the constant incubator is 37 (± 1) DEG C.
Further, the culture tube is 16~24 hours in the incubation time of constant incubator.
Further, the inorganic salts are including but not limited to following several: calcium nitrate, anhydrous magnesium sulfate, anhydrous phosphoric acid two Hydrogen sodium, potassium chloride, sodium chloride, sodium bicarbonate.
Further, the amino acid is including but not limited to following several: L-arginine, L- asparagine, L- winter ammonia Acid, l-cysteine dihydrochloride, Pidolidone, glycine, L-Histidine, L- hydroxyproline, l-Isoleucine, L-Leu, L lysine HCL, l-methionine, L-phenylalanine, L-PROLINE, Serine, L-threonine, L-Trp, L- junket Propylhomoserin, Valine, L-Glutamine.
Further, the vitamin is including but not limited to following several: biotin, D-VB5 calcium, folic acid, i- inositol, Niacinamide, puridoxine hydrochloride, riboflavin, choline chloride, thiamine hydrochloride, vitamin B12, p-aminobenzoic acid.
Further, the growth factor includes following one or more: fibroblast growth factor, para-insulin are raw The long factor, epidermal growth factor, nerve growth factor, platelet derived growth factor and transforming growth factor.
The method of 1 in vitro culture tuberculosis infection T cell of embodiment
Peripheral vein fresh whole blood is acquired, is then mixed by inversion at once with anticoagulant sodium heparin or heparin lithium, makes anti-coagulants Sufficiently dissolution, the revolving speed with centrifuge in 3000~5000r/min are centrifuged 5~10min, remove upper plasma, retain cellular layer. The isometric endotoxin-free free serum culture base fluid 1 of blood plasma is added in haemocyte later, mixes well, with centrifuge 3000 The revolving speed of~5000r/min is centrifuged 5~10min, gently siphons away upper layer endotoxin-free free serum culture base fluid 1.
Then the isometric endotoxin-free free serum culture base fluid 2 of blood plasma is added in haemocyte after isolation, it is sufficiently mixed It is even.By blank control pipe N, the sequence of testing tube T and positive control pipe P are separately added into 1mL containing whether there is or not interior in 3 kinds of culture tubes 2 blood cell sample of toxin free serum culture base fluid, is mixed by inversion at once, mixes well substance in culture tube.
Will 37 ± 1 DEG C be uprightly put into added with 3 kinds of culture tubes of 2 blood cell sample of endotoxin-free free serum culture base fluid above It is cultivated 16~24 hours in constant incubator.After culture, by culture tube with the revolving speed of 3000~5000r/min centrifugation 5~ 10min takes upper layer endotoxin-free free serum culture base fluid 2 to detect the concentration of gamma interferon in each culture tube.It overcomes traditional complete The interference of existing gamma interferon in itself in subject's whole blood, effectively reduces background, improves resolution ratio in blood culture method And sensitivity；Operating procedure when avoiding in-vitro separation T lymphocyte in immune spot-ing is complicated, and condition of culture is demanding Limitation；The method of the present invention has easy to operate, reproducible, the features such as cultural method is easy, resolution ratio and high sensitivity.
Wherein, 1 main component of endotoxin-free free serum culture base fluid is inorganic salts, amino acid, vitamin etc., is washed for cell It washs and suitable osmotic pressure and pH environment is provided.Inorganic salts are including but not limited to following several: calcium nitrate, anhydrous magnesium sulfate, nothing Water sodium dihydrogen phosphate, potassium chloride, sodium chloride, sodium bicarbonate；Amino acid is including but not limited to following several: L-arginine, L- Winter amide, L-ASPARTIC ACID, l-cysteine dihydrochloride, Pidolidone, glycine, L-Histidine, L- hydroxyproline, L- are different bright Propylhomoserin, L-Leu, L lysine HCL, l-methionine, L-phenylalanine, L-PROLINE, Serine, L- Soviet Union ammonia Acid, L-Trp, l-tyrosine, Valine, L-Glutamine；Vitamin is including but not limited to following several: biotin, D- It is calcium pantothenate, folic acid, i- inositol, niacinamide, puridoxine hydrochloride, riboflavin, choline chloride, thiamine hydrochloride, vitamin B12, right Aminobenzoic acid.
Wherein 2 main component of endotoxin-free free serum culture base fluid is inorganic salts, amino acid, vitamin and necessary thin Intracellular cytokine, environment and nutrition needed for proliferation is provided and plays biological function for cell.Inorganic salts are including but not limited to following It is several: calcium nitrate, anhydrous magnesium sulfate, anhydrous sodium dihydrogen phosphate, potassium chloride, sodium chloride, sodium bicarbonate；Amino acid includes but unlimited In following several: L-arginine, L- asparagine, L-ASPARTIC ACID, l-cysteine dihydrochloride, Pidolidone, glycine, L- Histidine, L- hydroxyproline, l-Isoleucine, L-Leu, L lysine HCL, l-methionine, L-phenylalanine, L- Proline, Serine, L-threonine, L-Trp, l-tyrosine, Valine, L-Glutamine；Vitamin includes but not It is limited to following several: biotin, D-VB5 calcium, folic acid, i- inositol, niacinamide, puridoxine hydrochloride, riboflavin, choline chloride, salt Allithiamine element, vitamin B12, p-aminobenzoic acid.Growth factor includes following one or more: fibroblast growth factor (aFGF and basic FGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), nerve growth factor (NGF), Platelet derived growth factor (PDGF) and transforming growth factor (TGF: α and β).Usual growth factor activity range 1~ In 10ng/mL.In addition also contain following inorganic microelement one or more: Mn, Cu, Zn, Mo, Va, Se, Fe, Ca, Mg, Si, Ni, Al, Ag, Ba, Br, Cd, Co, Cr, F, Ge, J, Rb and Zr.
The comparison of the distinct methods of 2 in vitro culture tuberculosis infection T cell of embodiment
The pattern detection result of the distinct methods of 3 in vitro culture tuberculosis infection T cell of embodiment compares
Result can obtain out of table, and the detection background result of the culture tube N of plasma exchange is trained significantly lower than traditional whole blood The method of supporting improves detection resolution, and does not influence the yin and yang attribute interpretation of result.
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to compared with Good embodiment describes the invention in detail, those skilled in the art should understand that, it can be to skill of the invention Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this In the scope of the claims of invention.

Claims (10)

1. a kind of method of in vitro culture tuberculosis infection T cell, it is characterised in that: the following steps are included:
Step 1: acquisition peripheral vein fresh whole blood, the whole blood of acquisition is mixed by inversion with anti-coagulants, keeps anti-coagulants sufficiently molten Solution；
Step 2: centrifugation, removes upper plasma, retain cellular layer；
The isometric endotoxin-free free serum culture base fluid 1 of blood plasma is added in haemocyte, mixes well, is centrifuged, inhales for third step Walk the endotoxin-free free serum culture base fluid 1 on upper layer；
Step 4: the isometric endotoxin-free free serum culture base fluid 2 of blood plasma is added in haemocyte after isolation, it is sufficiently mixed It is even；
Step 5: pressing the sequence of blank control pipe N, testing tube T and positive control pipe P, 1mL is separately added into 3 kinds of culture tubes Containing 2 blood cell sample of endotoxin-free free serum culture base fluid, mix well substance in culture tube；
Step 6: by constant temperature incubation is uprightly put into added with 3 kinds of culture tubes of 2 blood cell sample of endotoxin-free free serum culture base fluid It is cultivated in case；
Step 7: culture tube is centrifuged, upper layer endotoxin-free free serum culture base fluid 2 is taken to detect each culture tube after culture The concentration of middle gamma interferon.
2. a kind of method of in vitro culture tuberculosis infection T cell according to claim 1, which is characterized in that described anticoagulant Agent is heparin sodium or heparin lithium.
3. a kind of method of in vitro culture tuberculosis infection T cell according to claim 1, which is characterized in that in the nothing The main component of toxin free serum culture base fluid 1 be inorganic salts, amino acid, vitamin it is one or more.
4. a kind of method of in vitro culture tuberculosis infection T cell according to claim 1, which is characterized in that in the nothing The main component of toxin free serum culture base fluid 2 is inorganic salts, amino acid, vitamin and cell factor.
5. a kind of method of in vitro culture tuberculosis infection T cell according to claim 1, which is characterized in that the constant temperature The cultivation temperature of incubator is 37 ± 1 DEG C.
6. a kind of method of in vitro culture tuberculosis infection T cell according to claim 1, which is characterized in that the culture Pipe is 16~24 hours in the incubation time of constant incubator.
7. a kind of method of in vitro culture tuberculosis infection T cell according to claim 3 or 4, which is characterized in that the nothing Machine salt is including but not limited to following several: calcium nitrate, anhydrous magnesium sulfate, anhydrous sodium dihydrogen phosphate, potassium chloride, sodium chloride, carbonic acid Hydrogen sodium.
8. a kind of method of in vitro culture tuberculosis infection T cell according to claim 3 or 4, which is characterized in that the ammonia Base acid is including but not limited to following several: L-arginine, L- asparagine, L-ASPARTIC ACID, l-cysteine dihydrochloride, L- paddy Propylhomoserin, glycine, L-Histidine, L- hydroxyproline, l-Isoleucine, L-Leu, L lysine HCL, L- first sulphur ammonia Acid, L-phenylalanine, L-PROLINE, Serine, L-threonine, L-Trp, l-tyrosine, Valine, L- glutamy Amine.
9. a kind of method of in vitro culture tuberculosis infection T cell according to claim 3 or 4, which is characterized in that the dimension Raw element is including but not limited to following several: biotin, D-VB5 calcium, folic acid, i- inositol, niacinamide, puridoxine hydrochloride, core yellow Element, choline chloride, thiamine hydrochloride, vitamin B12, p-aminobenzoic acid.
10. a kind of method of in vitro culture tuberculosis infection T cell according to claim 4, which is characterized in that the growth The factor includes following one or more: fibroblast growth factor, insulin-like growth factor, epidermal growth factor, nerve Growth factor, platelet derived growth factor and transforming growth factor.
CN201910755331.9A 2019-08-15 2019-08-15 A kind of method of in vitro culture tuberculosis infection T cell Pending CN110468102A (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103364545A (en) * 2013-05-21 2013-10-23 佛山迪安医学检验所有限公司 Method for detecting tuberculosis effector T lymphocytes by using enzyme-linked immunospot assay
CN105259354A (en) * 2015-11-13 2016-01-20 夏晶 Kit for detecting tuberculosis T cell release gamma-interferon and use method of kit
CN106248934A (en) * 2016-08-25 2016-12-21 中国疾病预防控制中心传染病预防控制所 Antigen of mycobacterium tuberculosis albumen Rv0446c and the application of t cell epitope peptide thereof
CN106645729A (en) * 2016-12-07 2017-05-10 上海芯超生物科技有限公司 Chemiluminescent kit for quantitatively detecting mycobacterium tuberculosis r interferon as well as preparation method, detection method and evaluation method of chemiluminescent kit
CN108490189A (en) * 2018-03-14 2018-09-04 江苏瑞安生物技术有限公司 Detect the chromatography method and test strips preparation method of mycobacterium tuberculosis interferon

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103364545A (en) * 2013-05-21 2013-10-23 佛山迪安医学检验所有限公司 Method for detecting tuberculosis effector T lymphocytes by using enzyme-linked immunospot assay
CN105259354A (en) * 2015-11-13 2016-01-20 夏晶 Kit for detecting tuberculosis T cell release gamma-interferon and use method of kit
CN106248934A (en) * 2016-08-25 2016-12-21 中国疾病预防控制中心传染病预防控制所 Antigen of mycobacterium tuberculosis albumen Rv0446c and the application of t cell epitope peptide thereof
CN106645729A (en) * 2016-12-07 2017-05-10 上海芯超生物科技有限公司 Chemiluminescent kit for quantitatively detecting mycobacterium tuberculosis r interferon as well as preparation method, detection method and evaluation method of chemiluminescent kit
CN108490189A (en) * 2018-03-14 2018-09-04 江苏瑞安生物技术有限公司 Detect the chromatography method and test strips preparation method of mycobacterium tuberculosis interferon

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
赵有利: "《淋巴结结核中西医诊治学》", 30 November 2018 *

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