CN103789267B - A kind of improved primary hippocampal neurons method - Google Patents
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Abstract
The invention belongs to cell biology field, relate to a kind of neuron separation and cultural method and reagent. The object of the invention is to the problem existing for prior art, improve the method for current hippocampal neuron in-vitro separation and cultivation, solved proliferative and the active problem that cannot keep of primary hippocampal neurons. The present invention has set up the method for the Cultured Hippocampal Neurons of a set of comparative maturity, the nerve cell quantity abundance that the present invention obtains, growth conditions is better, can from mammiferous hippocampal tissue, isolate the hippocampal neurons of high activity, high quantity, meet the requirement that primary hippocampal cell is cultivated, can meet the demand of Cell Biology Experiment in Neuroscience Research.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of hippocampal neurons separation and primary culture method and reagent.
Background technology
Hippocampus (hippocampal) is the important component part of central nervous system, as the region of neuron high concentration, toolThere is the typical characteristics of central nervous system, play a significant role at aspects such as study, memory, emotional reactions and autonomic nervous functions.Neuronal cell culture model is the important experiments such as the mechanism of the differentiation of research neuronal development, nerve regneration, sacred diseaseModel, Cultured Hippocampal Neurons has become the important skill of the nerve degenerative diseases such as research Alzheimer disease, Parkinson'sArt means. Former culture (primaryculture) refers to from active somatic cell and obtains cell, tissue or organ, in vitro under conditionThe cultivation for the first time of carrying out. It is by the portion of tissue of embryo's mammalian central nervous system that culture of primary neurons is supported, as hippocampus groupKnit, cerebral cortex, cerebellum, hypothalamus, hippocampus, spinal cord and neuropile directly take out from body, inoculates the method for cultivation.The method that at present the relevant neuron in home and abroad is cultivated is more, but aspect neuronic purity and output, also exists in former cultureThe problem that some are anxious to be resolved. Because neuron is a kind of well differentiated cell, seldom division after animal birth, with respect to itIts cell, neuron is more difficult to survival and growth in vitro. Therefore cultural method and battalion that, in vitro culture neuron requiresFoster condition is all comparatively special.
At present, cause being difficult to obtaining sufficient amount and vigor primary hippocampal neurons because have following several respects: (1) hippocampus godIn organizing separation process, majority use D-hank ' s or hank ' s as rinsing liquid, in vitro mammal hippocampal tissue, removeThe impurity such as red blood cell, tunicle and connective tissue, the separation process time is longer, sometimes needs more than 2 hours, in rinsing liquidTime can be longer, the i.e. Mortality of hippocampal neuron, thus there is false-negative cultivation results. Experimentally cannot carry out hippocampusNeuronic cellar culture may be mainly because do not have the suitable neuron for hippocampal tissue sample to cultivate by rinsing liquid/solutionCut open the event of liquid. Organizing in separation process, even ice bath, neuron is still carrying out largely metabolism, the nothing of hank ' s liquidIt is very unfavorable that sugar environment separates neuron, adds DMEM or high sugar and supply with the metabolism of brain in hank ' s liquid, but grapeSugar concentration is too high, easily increases the chance of germ contamination; Add DMEM nutrient solution meeting alkalization, be unfavorable for neuronal cell survival.Chinese patent CN102978162A " a kind of neuron separation and cultural method and reagent ", Chinese patent CN102994452A "Kind efficiently separate and cultivate neuronic method " with the Chinese patent CN102994451A " improvement that a kind of neuron separates and cultivatesType method ", get brain tissue and put into the culture dish that fills 1 × PBS, the cleaning fluid that adopts is PBS liquid, the high sugar of DMEM-orDMEM-F12 and horse serum soak the brain in dissection process, supply with the metabolism of brain, have considered neuron energy generationThank to needs, but do not add any antibacterial material, concentration of glucose is too high, just easily increases the chance of cell contamination. (2) adoptAfter heavy dose of Trypsin Induced, hippocampal neurons certainly will be subject to raw ratio and the mechanical damage of certain degree; Gluing of cell surfaceAttached molecule or memebrane protein are very easily damaged, and have stereochemical structure in order to maintain the cell of cell proliferation and self for this class, be difficult to recover rapidly cell state, be often accompanied by large-scale cell death/apoptosis, cell " aging " is serious, cellVigor will obviously weaken, and in tissue, can not obtain a large amount of living cells; (3) cell seeding liquid extremely closes cultured primitive hippocampal neuronImportant, cell seeding liquid is different from ensuing cell maintenance medium, need to give nerve cell special growth factor, soCan ensure to obtain the hippocampal neuron of sufficient amount and vigor.
Summary of the invention
The object of the invention is to overcome defect and the deficiency of prior art, the invention provides a kind of mammiferous hippocampal neuronThe method of primary culture in vitro, object is to set up a kind of method of simple, highly purified hippocampal neuron primary culture in vitroAnd reagent, meet the demand of Neuroscience Research.
One of technical scheme provided by the invention is:
Bifidobacterium adolescentis of the present invention (Bifidobacteriumadolescentis) LJM-001, in 2010 08 month 17Day, it was referred to as CGMCC (address: court of Beijing at China Committee for Culture Collection of Microorganisms's common micro-organisms centerNo. 3 Institute of Microorganism, Academia Sinica of No. 1, North Star West Road institute of sun district, postcode 100101) preservation, Classification And Nomenclature is the youthBifidobacterium (Bifidobacteriumadolescentis), deposit number is CGMCCNo.4090.
Bifidobacterium adolescentis of the present invention (Bifidobacteriumadolescentis) LJM-001 is located away from Zhejiang Province's healthIn young people's ight soil.
Bifidobacterium adolescentis LJM-001 bacterial strain of the present invention has following microbial characteristic:
(1) colonial morphology: bifidobacterium adolescentis LJM-001 bacterial strain bacterium colony on flat board is canescence or milky, opaque,Glossy, smooth surface, projection, quality are soft, neat in edge, diameter 1～1.5mm.
(2) individual morphology: G+Sporeless bacterium, shaft-like.
(3) physiological and biochemical property: D-ribose (+); Arabinose (+); Lactose (+); Cellobiose (-); PineTrisaccharide (+); Raffinose (+); Sorbierite (-); Starch (-); Gluconic acid sodium salt (-); Wood sugar (+); Sweet dewSugar (-); Fructose (+); Galactolipin (+); Sucrose (+); Maltose (+); Trehalose (-); Melibiose (+);Sweet mellow wine (+); Synanthrin (-); Salicin (-); F6PPK enzyme (+); Reaction to oxygen is (at aerobic solid mediumOn do not grow); Nitrate reduction (-); Catalase (-); Indole reaction (-).
Well-grown under anaerobism, does not grow in aerobic environment. 37～41 DEG C of optimum growth temperatures; 25～28 DEG C of minimum growth temperatures;The highest 43～45 DEG C; Growth optimal pH 6.5～7.0; In pH4.5～5.0 or 8.0～8.5 do not grow.
Described bifidobacterium adolescentis LJM-001, CGMCCNo.4090 is applied in the cell seeding liquid of hippocampal neuron.
The favorite outer discovery of our experimentation: the active fermentation filtrate of bifidobacterium adolescentis LJM-001 bacterial strain can provide nutrition to becomePoint, can promote again neure growth, obtain neuronic quantity and can meet the needs of experiment.
Two of technical scheme provided by the invention is: the separation of hippocampal neuron, primary culture method, comprise the following steps:
1. rinsing: get in vitro mammiferous hippocampal tissue, put in the rinsing liquid of ice bath, remove red blood cell, tunicle and knot and formTissue, with rinsing liquid flushing 2～5 times;
2. digestion: the hippocampal tissue after step 1 rinsing is cut into diameter 1mm3Fritter, with 37 DEG C of the digestive juices of 5 times of tissue volumeAct on 5～10 minutes, be organized into the rotten shape of congee, stop digestion with cell seeding liquid, blow and beat gently to tissue block 10 times withCell dispersion.
3. prepare cell suspension: collect the rear first cell suspension of step 2 digestion, after 200 order cells sieves filter,800～1000rpm4 DEG C centrifugal 5～10 minutes, abandoning supernatant, adds cell seeding liquid, re-suspended cell, makes5×105～10×105The single cell suspension of individual/mL;
4. inoculation, cultivation: step 3 cell suspension is planted in completing in advance the culture dish and blake bottle of poly-D-lysine, putIn 37 DEG C, 5%CO2In constant incubator, after plantation, within 24～72 hours, change cell maintenance medium into, afterwards every three daysChange liquid with cell maintenance medium, each amount of changing is original volume 1/2.
Reagent is bought:
The neural basal medium of DMEM/F12, Neurobasal culture medium and B27Be purchased from Gibco company; Poly-D-lysine, tire oxSerum (FBS), trehalose and Glu are purchased from Sigma company of the U.S.; Mycillin mixed liquor (dual anti-), is purchased from the U.S.HyClone company.
The configuration of reagent:
(1) described D-Hank ' s liquid, obtains through following steps: NaCl8.0g, KCl0.4g, Na2HPO4·12H2O0.12g，KH2PO40.06g，NaHCO30.35g; Successively each composition is dissolved in about 500mL tri-distilled water and is mixed, add tri-distilled water constant volumeTo 1000mL, adjust pH value to 7.2～7.4, packing, autoclaving, packing, 4 DEG C save backup.
(2) described rinsing liquid, obtains through following steps: 2g trehalose, 3g glucose and the dual anti-100mL of being dissolved in of 10mLIn D-Hank ' s liquid, mix, add D-Hank ' s liquid and be settled to 1000mL, under 0.4MPa atmospheric pressure, dissolve in hydrogen 4～6 hours, hydrogenFinal concentration is reached for 0.5mmol/L, gamma-rays sterilization, and packing, 4 DEG C save backup.
(3) described digestive juice, obtains through following steps: 1.0g trypsase and 0.1gEDTA are dissolved in 100mLD-Hank ' sIn liquid, mix, add D-Hank ' s liquid and be settled to 1000mL, 0.22 μ m membrane filtration degerming, dissolves in hydrogen under 0.4MPa atmospheric pressure4～6 hours, hydrogen final concentration was reached for 0.5mmol/L, gamma-rays sterilization, and packing, 4 DEG C save backup.
(4) described active fermentation filtrate, obtains through following steps:
1. bifidobacterium adolescentis (Bifidobacteriumadolescentis) LJM-001, this bacterial strain was protected on 08 17th, 2010Be stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCCNo.4090;
2. bifidobacterium adolescentis LJM-001 adopts modified MRS culture medium to cultivate, and culture medium prescription is: by weight, get albumenPeptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, sodium acetate 5g, K2HPO42g，MgSO4·7H2O0.5g，MnSO4·4H2O0.2g, FOS 3g, dibasic ammonium citrate 2g, Tween-80 1mL, distilled water 1L, heating for dissolving is proofreaied and correct pHValue is to 6.5,115 DEG C of autoclaving 15-20 minute.
3. bifidobacterium adolescentis LJM-001 is inoculated in and is cooled to the step of 40 ± 2 DEG C 2. in modified MRS culture medium, bacterial classification connectsThe amount of kind is that 0.2～0.5%, 35 ± 2 DEG C of anaerobism are cultivated 24 hours, with the above-mentioned nutrient solution A of spectrophotometric determination580nmValue is 1.2 o'clock,Above-mentioned nutrient solution after centrifugal 10 minutes, is got to supernatant under revolution 5000～8500rpm, 0.22 μ m membrane filtration degerming,Obtain active-fermented broth filtrate, 4 DEG C save backup.
(5) described poly-D-lysine, obtains through following steps: 10mg poly-D-lysine is dissolved in 10mL tri-distilled water, mixedEven, add tri-distilled water and be settled to 100mL, 0.22 μ m filtering with microporous membrane degerming, packing, freezing for subsequent use.
(6) described cell seeding liquid, obtains through following steps: DMEM/F12 culture medium adds hyclone, activityFerment filtrate and dual anti-, makes that hyclone final concentration is 10%, active fermentation filtrate final concentration is 0.2%, and dual anti-final concentration is1%, with 0.22 μ m membrane filtration degerming, packing, 4 DEG C save backup.
(7) described cell maintenance medium, obtains through following steps: Neurobasal culture medium adds B27And glutamine, makeB27Final concentration is 2%, glutamine final concentration 1%, and with 0.22 μ m membrane filtration degerming, packing, 4 DEG C save backup.
(8) described dual anti-, be penicillin-streptomysin solution (100 ×), Penicillin Content is 10000U/mL, streptomysin containsAmount is 10mg/mL. 1% is dual anti-: Penicillin Content is 100U/mL, and content of streptomycin is 0.1mg/mL.
The present invention has the following advantages and effect:
1. in rinse step, hippocampal tissue infiltrates under hydrogen environment, maximizes the lock out operation that reduces tissue to hippocampus nerveThe damage of cell, and hydrogen has extremely strong BA, protects to greatest extent hippocampal neurons not to be subject toInjure, can improve survival rate and the biologically active of primary hippocampal neurons.
2. rinsing liquid contains trehalose, replaces the conventional sucrose using, and sucrose viscosity is larger, is unfavorable for organizing lock out operation.Trehalose has magical protective effect to organism, can form unique diaphragm at cell surface, effectively protectsProtect nerve cell, the life process of the body that sustains life and biological characteristic, and occurring in nature as sucrose, glucose etc. itsIts carbohydrate, does not all possess this function.
3. in digestion step, adopt the digestive juice that is rich in hydrogen, made trypsase fully be contacted and do with digestion tissueWith, Gas Stirring is even, and Trypsin Induced efficiency improves greatly, the time of trypsase using dosage and experienceShorten to some extent (shortened to and be no more than 10 minutes from original 10～20 minutes), increased again hippocampal neuron simultaneouslyPhysiological respiration, hydrogen itself is again health factor, makes Hippocampal Neuron Cells keep more original biological livingProperty. Short time, low concentration pancreatin reach necessary digestion effect, less to neure damage, have also improved godThrough first Motility rate.
In a word, the present invention has set up the method for the cultured primitive hippocampal neuron of a set of comparative maturity, i.e. rinsing under hydrogen environmentAnd enzymic digestion, the trehalose that rinsing liquid contains defencive function, the enzyme isolated cell of low concentration, short time and containing active fermentation filtrateSpecial planted medium; The nerve cell quantity abundance that the present invention obtains, growth conditions is better, can be from mammiferous seaIn horse tissue, isolate the hippocampal neurons of high activity, high quantity, meet the foster requirement of hippocampal cell culture of primary neurons.
Detailed description of the invention
Below in conjunction with specific embodiment, experiment material is got the hippocampal tissue of newborn SD rat, and the present invention is described in detail.
Embodiment 1
Bifidobacterium adolescentis of the present invention (Bifidobacteriumadolescentis) LJM-001, in 2010 08 month 17Day, it was referred to as CGMCC (address: court of Beijing at China Committee for Culture Collection of Microorganisms's common micro-organisms centerNo. 3 Institute of Microorganism, Academia Sinica of No. 1, North Star West Road institute of sun district, postcode 100101) preservation, Classification And Nomenclature is the youthBifidobacterium (Bifidobacteriumadolescentis), deposit number is CGMCCNo.4090.
Bifidobacterium adolescentis of the present invention (Bifidobacteriumadolescentis) LJM-001 is located away from Zhejiang Province's healthIn young people's ight soil.
Bifidobacterium adolescentis LJM-001 bacterial strain of the present invention has following microbial characteristic:
(1) colonial morphology: LJM-001 bacterial strain bacterium colony on flat board is canescence or milky, opaque, glossy, surperficialSmooth, protruding, quality is soft, neat in edge, diameter 1-1.5mm.
(2) individual morphology: G+Sporeless bacterium, shaft-like.
(3) physiological and biochemical property: D-ribose (+); Arabinose (+); Lactose (+); Cellobiose (-); PineTrisaccharide (+); Raffinose (+); Sorbierite (-); Starch (-); Gluconic acid sodium salt (-); Wood sugar (+); Sweet dewSugar (-); Fructose (+); Galactolipin (+); Sucrose (+); Maltose (+); Trehalose (-); Melibiose (+);Sweet mellow wine (+); Synanthrin (-); Salicin (-); F6PPK enzyme (+); Reaction to oxygen is (at aerobic solid mediumOn do not grow); Nitrate reduction (-); Catalase (-); Indole reaction (-).
Well-grown under anaerobism, does not grow in aerobic environment. Optimum growth temperature 37-41 DEG C; Minimum growth temperature 25-28 DEG C;The highest 43-45 DEG C; Growth optimal pH 6.5-7.0; Do not grow at pH4.5-5.0 or 8.0-8.5.
Described bifidobacterium adolescentis (Bifidobacteriumadolescentis) LJM-001, CGMCCNo.4090 is applied to seaIn the neuronic cell seeding liquid of horse.
The favorite outer discovery of our experimentation: the active fermentation filtrate of bifidobacterium adolescentis LJM-001 bacterial strain can provide nutrition to becomePoint, can promote again neure growth, obtain neuronic quantity and can meet the needs of experiment.
Embodiment 2
Reagent is bought:
The neural basal medium of DMEM/F12, Neurobasal culture medium and B27Be purchased from Gibco company; Poly-D-lysine, tire oxSerum (FBS), trehalose and Glu are purchased from Sigma company of the U.S.; Mycillin mixed liquor (dual anti-), is purchased from the U.S.HyClone company.
The configuration of reagent:
(1) described D-Hank ' s liquid, obtains through following steps: NaCl8.0g, KCl0.4g, Na2HPO4·12H2O0.12g， KH2PO40.06g，NaHCO30.35g; Successively each composition is dissolved in about 500mL tri-distilled water and is mixed, add tri-distilled water constant volumeTo 1000mL, adjust pH value to 7.2～7.4, packing, autoclaving, packing, 4 DEG C save backup.
(2) described rinsing liquid, obtains through following steps: 2g trehalose, 3g glucose and the dual anti-100mL of being dissolved in of 10mLIn D-Hank ' s liquid, mix, add D-Hank ' s liquid and be settled to 1000mL, under 0.4MPa atmospheric pressure, dissolve in hydrogen 4～6 hours, hydrogenFinal concentration is reached for 0.5mmol/L, gamma-rays sterilization, and packing, 4 DEG C save backup.
(3) described digestive juice, obtains through following steps: 1.0g trypsase and 0.1gEDTA are dissolved in 100mLD-Hank ' sIn liquid, mix, add D-Hank ' s liquid and be settled to 1000mL, 0.22 μ m membrane filtration degerming, dissolves in hydrogen under 0.4MPa atmospheric pressure4～6 hours, hydrogen final concentration was reached for 0.5mmol/L, gamma-rays sterilization, and packing, 4 DEG C save backup.
(4) described active fermentation filtrate, obtains through following steps:
1. bifidobacterium adolescentis (Bifidobacteriumadolescentis) LJM-001, this bacterial strain was protected on 08 17th, 2010Be stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCCNo.4090;
2. bifidobacterium adolescentis LJM-001 adopts modified MRS culture medium to cultivate, and culture medium prescription is: by weight, get albumenPeptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, sodium acetate 5g, K2HPO42g，MgSO4·7H2O0.5g，MnSO4·4H2O0.2g, FOS 3g, dibasic ammonium citrate 2g, Tween-80 1mL, distilled water 1L, heating for dissolving is proofreaied and correct pHValue is to 6.5,115 DEG C of autoclaving 15-20 minute.
3. bifidobacterium adolescentis LJM-001 is inoculated in and is cooled to the step of 40 ± 2 DEG C 2. in modified MRS culture medium, bacterial classification connectsThe amount of kind is that 0.2～0.5%, 35 ± 2 DEG C of anaerobism are cultivated 24 hours, with the above-mentioned nutrient solution A of spectrophotometric determination580nmValue is 1.2 o'clock,Above-mentioned nutrient solution after centrifugal 10 minutes, is got to supernatant under revolution 5000～8500rpm, 0.22 μ m membrane filtration degerming,Obtain active-fermented broth filtrate, 4 DEG C save backup.
(5) described poly-D-lysine, obtains through following steps: 10mg poly-D-lysine is dissolved in 10mL tri-distilled water, mixedEven, add tri-distilled water and be settled to 100mL, 0.22 μ m filtering with microporous membrane degerming, packing, freezing for subsequent use.
(6) described cell seeding liquid, obtains through following steps: DMEM/F12 culture medium adds hyclone, activityFerment filtrate and dual anti-, makes that hyclone final concentration is 10%, active fermentation filtrate final concentration is 0.2%, and dual anti-final concentration is1%, with 0.22 μ m membrane filtration degerming, packing, 4 DEG C save backup.
(7) described cell maintenance medium, obtains through following steps: Neurobasal culture medium adds B27And glutamine, makeB27Final concentration is 2%, glutamine final concentration 1%, and with 0.22 μ m membrane filtration degerming, packing, 4 DEG C save backup.
(8) described dual anti-, be penicillin-streptomysin solution (100 ×), Penicillin Content is 10000U/mL, streptomysin containsAmount is 10mg/mL. 1% is dual anti-: Penicillin Content is 100U/mL, and content of streptomycin is 0.1mg/mL.
Embodiment 3
Separation, primary culture method with SD hippocampus of rats comprise the following steps:
The reagent of being prepared with embodiment 1 and embodiment 2 and configuration reagent.
(1) rinsing: get in vitro mammiferous hippocampal tissue, put in the rinsing liquid of ice bath, remove red blood cell, tunicle and knotForm tissue, with rinsing liquid flushing 2～5 times;
(2) digestion: the hippocampal tissue after step 1 rinsing is cut into diameter 1mm3Fritter, with the digestive juice of 5 times of tissue volume37 DEG C act on 5～10 minutes, are organized into the rotten shape of congee, stop digesting with cell seeding liquid, blow and beat gently to tissue block10 times with cell dispersion.
(3) prepare cell suspension: collect the rear first cell suspension of step 2 digestion, after 200 order cells sieves filter,800～1000rpm4 DEG C centrifugal 5～10 minutes, abandoning supernatant, adds cell seeding liquid, re-suspended cell, systemBecome 5 × 105～10×105The single cell suspension of individual/mL;
(4) inoculation, cultivation: step 3 cell suspension is planted in completing in advance the culture dish and blake bottle of poly-D-lysine,Be placed in 37 DEG C, 5%CO2In constant incubator, after plantation, within 24～72 hours, change cell maintenance medium into, afterwards everyWithin two days, change liquid with cell maintenance medium, each amount of changing is original volume 1/2.
(5) microscopy is judged: nerve cell is planted after latter 12 hours, and inverted microscope is observed, show: most cells is adherent,Form is rounded, and wherein minority nerve cell outward appearance is long shuttle type, and starts to stretch out 1～2 projection. The 2nd dayChange cell maintenance medium, cultivate after 24 hours, cellular morphology is not various, as fusiformis, triangle, cone shape or notRule shape, the nerve cell that stretches out projection increases different in size gradually. Cultivate the 72nd hour, pericaryon increasesGreatly, full; After 3 days, neuron shape is very typical: cell space is full, is fusiformis, taper more, and it is many that minority isLimit shape, endochylema is abundant, and core is large, and kernel mays be seen indistinctly, and projection rises appreciably, visible only a few still in backgroundThe polygonal Deiter's cells place mat of flat.
Cultivating 3rd～5 days, carry out sediments microscope inspection, cultured cell occurs that volume becomes large and most cells appearance is typicalNeuron morphology, does not have heteroproteose cell propagation, and explanation is cultivated successfully.
The primary hippocampal neurons cell that the inventive method is set up can be survived 5～9 days, can meet and carry out neuronal cell meritThe needs that can study.
(6) cell purification
Exist if observed heteroproteose cell, carry out cell purification processing.
If cell is cultivated 48～72 hours, observed heteroproteose cell and existed, every hole add cytarabine (concentration is 2～10 μ g/mL) 1mL, then add the cell seeding liquid of preparing with the isopyknic step of cytarabine solution (3), make cytarabineFinal concentration is 1～5 μ g/mL, after 24～48 hours, changes liquid completely with the cell maintenance medium of step (4) preparation.
Embodiment 4
1. cell viability test
The cell viability of the hippocampal neuron that table 1 is cultivated
Note: * represents that P < 0.05 represents that difference is extremely remarkable.
The cell viability effect of cell seeding liquid of the present invention (containing active fermentation filtrate) group is best.
2. Neuronal Survival rate test
The embodiment of the present invention 3 plays obvious facilitation to the survival of nerve cell, neuron survival rate while cultivating 4 days, withDMEM/F12+20% calf serum group (survival rate 58%), NeurobasalMedium+2%B27 group (survival rate 62%)Compare, 3 groups of Neuronal Survival rates of the embodiment of the present invention are 83%, and the present invention obviously promotes the survival of nerve cell, the difference utmost pointSignificantly (p < 0.01).
Conclusion: the primary culture in vitro very neuron of " fragile " is not difficult, the hippocampal neuron of cultivating by the present invention programComparatively a large amount of, survival rate is high, and the good and external viability of form is strong, and the neuronic survival rate of first three day of in vitro culture is far above literary compositionOffer 50% of report, can be used as Neuroscience Research.
Claims (1)
1. the separation of a hippocampal neuron and primary culture method comprise the following steps:
(1) rinsing: get in vitro mammiferous hippocampal tissue, put in the rinsing liquid of ice bath, remove red blood cell, tunicle and knot and form groupKnit, with rinsing liquid flushing 2～5 times; Described rinsing liquid, obtains through following steps: 2g trehalose, 3g glucose and 10mL are twoAnti-being dissolved in 100mLD-Hank ' s liquid, mixes, and adds D-Hank ' s liquid and is settled to 1000mL, under 0.4MPa atmospheric pressure, dissolves in hydrogen4～6 hours, hydrogen final concentration was reached for 0.5mmol/L, gamma-rays sterilization, and packing, 4 DEG C save backup; Describedly dual anti-ly be100 × penicillin-streptomysin solution: Penicillin Content is 10000U/mL, content of streptomycin is 10mg/mL; Described D-Hank ' sLiquid, obtains through following steps: NaCl8.0g, KCl0.4g, Na2HPO4·12H2O0.12g，KH2PO40.06g，NaHCO30.35g; Successively each composition is dissolved in about 500mL tri-distilled water and is mixed, add tri-distilled water and be settled to 1000mL, adjust pH value extremely7.2～7.4, packing, autoclaving, packing, 4 DEG C save backup;
(2) digestion: the hippocampal tissue after step (1) rinsing is cut into diameter 1mm3Fritter, with 37 DEG C of the digestive juices of 5 times of tissue volumeAct on 5～10 minutes, be organized into the rotten shape of congee, stop digestion with cell seeding liquid, blow and beat gently to tissue block 10 times with cell dispersion;Described digestive juice, obtains through following steps: 1.0g trypsase and 0.1gEDTA are dissolved in 100mLD-Hank ' s liquid, mixes,Add D-Hank ' s liquid and be settled to 1000mL, 0.22 μ m membrane filtration degerming, dissolves in hydrogen 4～6 hours, hydrogen under 0.4MPa atmospheric pressureGas final concentration is reached for 0.5mmol/L, gamma-rays sterilization, and packing, 4 DEG C save backup; Described D-Hank ' s liquid, preparation sideThe same step of method (1); Described cell seeding liquid, obtains through following steps: DMEM/F12 culture medium adds hyclone, workProperty ferment filtrate and dual anti-, makes that hyclone final concentration is 10%, active fermentation filtrate final concentration is 0.2%, and dual anti-final concentration is1%, with 0.22 μ m membrane filtration degerming, packing, 4 DEG C save backup; Described dual anti-, the same step of preparation method (1); DescribedActive fermentation filtrate, obtains through following steps: 1. bifidobacterium adolescentis (Bifidobacteriumadolescentis) LJM-001,This bacterial strain has been stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number on 08 17th, 2010For CGMCCNo.4090; 2. bifidobacterium adolescentis LJM-001 adopts modified MRS culture medium to cultivate, and culture medium prescription is: withWeighing scale, gets peptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, sodium acetate 5g, K2HPO42g，MgSO4·7H2O0.5g，MnSO4·4H2O0.2g, FOS 3g, dibasic ammonium citrate 2g, Tween-80 1mL, distilled water 1L, heatingDissolve and proofread and correct pH value to 6.5,115 DEG C of autoclaving 15-20 minute; 3. bifidobacterium adolescentis LJM-001 is inoculated in and is cooled toThe step of 40 ± 2 DEG C is 2. in modified MRS culture medium, and bacterial classification inoculum concentration is that 0.2～0.5%, 35 ± 2 DEG C of anaerobism are cultivated 24 hours, with pointThe above-mentioned nutrient solution A of light photometric determination580nmValue was 1.2 o'clock, by above-mentioned nutrient solution under revolution 5000～8500rpm centrifugal 10 minutesAfter, get supernatant, 0.22 μ m membrane filtration degerming, obtains active-fermented broth filtrate, and 4 DEG C save backup;
(3) prepare cell suspension: collect the rear first cell suspension of step (2) digestion, after 200 order cells sieves filter, 800～1000rpm4 DEG C centrifugal 5～10 minutes, abandoning supernatant, adds cell seeding liquid, re-suspended cell, makes 5 × 105～10×105The list of individual/mLCell suspension; Described cell seeding liquid, the same step of preparation method (2);
(4) inoculation, cultivation: step (3) cell suspension is planted in completing in advance the culture dish and blake bottle of poly-D-lysine,Be placed in 37 DEG C, 5%CO2In constant incubator, after plantation, within 24～72 hours, change cell maintenance medium into, tie up with cell every three days afterwardsHold liquid and change liquid, each amount of changing is original volume 1/2; Described cell maintenance medium, obtains through following steps: NeurobasalCulture medium adds B27And glutamine, make B27Final concentration is 2%, glutamine final concentration 1%, and with 0.22 μ m membrane filtration degerming,Packing, 4 DEG C save backup.
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| CN103805565B (en) * | 2014-02-21 | 2016-03-09 | 温州医科大学附属第二医院、育英儿童医院 | Hippocampal neuron is separated and primary culture method and reagent |
| CN104818251B (en) * | 2015-05-20 | 2018-08-21 | 妙顺（上海）生物科技有限公司 | Hippocampus of adult rat neuron In-vitro separation culture method |
| CN105695411B (en) * | 2016-03-22 | 2019-07-02 | 中国人民解放军第二军医大学 | A kind of naked mole cortical neuron cultural method |
| CN105754943B (en) * | 2016-03-22 | 2019-07-02 | 中国人民解放军第二军医大学 | A kind of naked mole cultured hippocampal neuron method |
| CN111979178B (en) * | 2020-08-20 | 2021-04-02 | 创芯国际生物科技(广州)有限公司 | Culture medium and culture method for animal lung bud organoid |
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| CN102286604A (en) * | 2011-06-25 | 2011-12-21 | 温州医学院 | Microbiological method for screening dimethoate residues in food |
| CN102304481A (en) * | 2011-06-25 | 2012-01-04 | 温州医学院 | Bifidobacterium adolescentis strain |
| CN102533654A (en) * | 2012-02-02 | 2012-07-04 | 温州医学院附属第二医院 | Culture solution for primary culture of newly born rat hippocampal neuron and preparation method and application thereof |
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| CN102286604A (en) * | 2011-06-25 | 2011-12-21 | 温州医学院 | Microbiological method for screening dimethoate residues in food |
| CN102304481A (en) * | 2011-06-25 | 2012-01-04 | 温州医学院 | Bifidobacterium adolescentis strain |
| CN102533654A (en) * | 2012-02-02 | 2012-07-04 | 温州医学院附属第二医院 | Culture solution for primary culture of newly born rat hippocampal neuron and preparation method and application thereof |
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