CN101824399B - Method for inducing and differentiating into dopaminergic neuron from human amniotic sepithelial cell and application for separating obtained dopaminergic neuron - Google Patents
Method for inducing and differentiating into dopaminergic neuron from human amniotic sepithelial cell and application for separating obtained dopaminergic neuron Download PDFInfo
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- CN101824399B CN101824399B CN200910079088XA CN200910079088A CN101824399B CN 101824399 B CN101824399 B CN 101824399B CN 200910079088X A CN200910079088X A CN 200910079088XA CN 200910079088 A CN200910079088 A CN 200910079088A CN 101824399 B CN101824399 B CN 101824399B
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Abstract
The invention relates to a method for inducing and differentiating into dopaminergic neurons from human amniotic sepithelial cells, comprising the following steps of: taking a fresh human placenta; stripping a human amnion, repeatedly washing by using normal saline to remove blood clots, then soaking by using asepsis D-hank's liquor, and then rinsing by using the normal saline; placing the human amnion into 0.25 percent of a trypsin solution, and digesting at the temperature of 36.5-37.5 DEG C; stopping the digestion of the human amnion in the trypsin solution by using DMEM/F12 (Dulbecco's Modified Eagle Medium/F12) containing 10 percent of fetal calf serums, and then filtering the solution by using a cell sieve, wherein collected cells are the human amniotic sepithelial cells; separating the obtained human amniotic sepithelial cells, and carrying out in-vitro subculture on the separated human amniotic sepithelial cells by using 10 percent of culture mediums without the fetal calf serums; selecting 3 or 4 or 5 or 6 generation human amniotic sepithelial cells with good growth state, and then continuously culturing by using a neural stem cell differential medium; and harvesting dopaminergic neuron like cells.
Description
Technical field
The present invention relates to a kind of purposes of from people's amniotic epithelial cells, inducing the method that differentiates dopaminergic neuron and treating parkinsonism with the dopaminergic neuron that this method obtains.
Background technology
Parkinsonism is a kind of spontaneous, slow disease development, the cns degenerative, its pathogeneticing characteristic for motion slowly, muscle rigidity, static tremor and posture be unstable.Have now found that: it is to be continued to worsen the symptom that the back is produced by neurone painted in black, locus coeruleus and other brain stem dopaminergic cell, causes the disappearance of neurotransmitter dopamine.In elderly crowd, parkinsonism is harm the 4th kind of healthy common neurodegenerative diseases of people, it influenced 0.4% over 40 years old people and 1% over 65 years old people.No matter the size of patient age receives the patient that this disease torments for those, usually cause destructive result.The reason that causes this result is that the destruction to dopaminergic neuron is irreversible.Mainly hope it is the cell colony of development neural network for this treatment of diseases, and neural functional rehabilitation is coordinated.Some evidence has shown that the fetus dopaminergic neuron can recover the chemical abnormality of parkinsonism.But do not find suitable tissue so far.
Summary of the invention
The application's goal of the invention is: find behind healthy parturient childbirth, to have many dopaminergic neurons that are used to treat parkinsonism the in-house people's amniotic epithelial cells of the human amnion tissue on the waste people placenta, and a kind of purposes of from people's amniotic epithelial cells, inducing the method that differentiates dopaminergic neuron and treating parkinsonism with the dopaminergic neuron that this method obtains is provided.
The method that differentiates dopaminergic neuron of from people's amniotic epithelial cells, inducing of the present invention is implemented in the following manner:
A kind of method that differentiates dopaminergic neuron of from people's amniotic epithelial cells, inducing of the present invention, this method may further comprise the steps:
(1) gets the Freshman placenta tissue, human amnion tissue is peeled off, wash repeatedly with saline water and remove blood clot, use aseptic D-hank ' s liquid to soak 1.2-2 after individual hour then, use saline water rinsing 2-5 time again;
(2) the people's amnion with rinsing places 0.25% trypsin solution, and digestion is 10-20 minute under 36.5 ℃-37.5 ℃ temperature;
(3) stop the digestion of above-mentioned human amnion tissue in trypsin solution with the DMEM/F12 that contains 10% foetal calf serum, with 180-220 order cell sieve above-mentioned solution is filtered then, collect the cell behaviour amniotic epithelial cells that obtains;
(4) separate the people's amniotic epithelial cells obtained, above-mentioned people's amniotic epithelial cells of separating is carried out the subculture in vitro separately cultivation with the substratum of 10% no foetal calf serum;
(5) select 3 or 4 or 5 or 6 good generation people amniotic epithelial cells of growth conditions, continue to cultivate with the cell differentiation of nerve cord substratum then;
(6) above-mentioned people's amniotic epithelial cells is cultivated 7-14 days in the cell differentiation of nerve cord substratum after; Results part cell is identified; Confirm the existence of tyrosine hydroxylase positive cell, and carry out quantitative analysis, obtained containing the cell of dopaminergic neuron-like cell;
(7) gather in the crops all above-mentioned cells.
A kind of method that differentiates dopaminergic neuron of from people's amniotic epithelial cells, inducing of the present invention, wherein: aseptic D-hank ' the s liquid in the described step (1) contains: 1000U/ml penicillium mould, 1000U/ml Streptomycin sulphate, 100U/ml qingfengmeisu qiong and 2.5ng/ml B fungizone.
A kind of method that differentiates dopaminergic neuron of from people's amniotic epithelial cells, inducing of the present invention; Wherein: in described step (1); Human amnion tissue uses aseptic D-hank ' s liquid to soak after 1.5 hours, and using concentration again is 0.9% saline water rinsing 3 times.
A kind of method that differentiates dopaminergic neuron of from people's amniotic epithelial cells, inducing of the present invention, wherein: in described step (2), people's amnion digested 15 minutes under 37. ℃ temperature.
A kind of method that differentiates dopaminergic neuron of from people's amniotic epithelial cells, inducing of the present invention, wherein: in described step (3), above-mentioned solution is filtered with 200 order cells sieve.
A kind of method that differentiates dopaminergic neuron of from people's amniotic epithelial cells, inducing of the present invention, wherein: the NSC basic medium in the described step (5) contains for every milliliter: the vitamins C of Prostatropin 8, the 200 μ M of the Sonic hedgehogSHH of 15% serum, 200ng, the fibroblast growth factor 8 of 100ng, 10ng and the BDNF of 20ng.
A kind of method that differentiates dopaminergic neuron of from people's amniotic epithelial cells, inducing of the present invention; Wherein:: the quantitative analysis in the described step (6) is to utilize immunofluorescence cell chemistry double label method to carry out mark; Under inverted biologic microscope, select at least three low-power fields to carry out analysis of accounts then, the multiple of this low-power field is 10 times.
A kind of method that differentiates dopaminergic neuron of from people's amniotic epithelial cells, inducing of the present invention; Wherein: it is to be that 37 ℃, humidity are to carry out in the incubator of 95% and 5% carbonic acid gas in temperature that subculture in vitro separately in the described step (4) is cultivated, and the monobasic breeding time of people's amniotic epithelial cells is 10-12 days in the step (5).
A kind of method that differentiates dopaminergic neuron of from people's amniotic epithelial cells, inducing of the present invention, wherein: described aseptic D-hank ' s, DMEM/F12 and the substratum that does not have a foetal calf serum are the commercially available prod.
Purposes of the present invention is for to treat parkinsonism with the described dopaminergic neuron that obtains in the method that differentiates dopaminergic neuron of from people's amniotic epithelial cells, inducing.
The present invention utilizes the human amniotic membrane tissue-derived people's amniotic epithelial cells of Biohazard Waste behind the healthy parturient childbirth, and vitro culture is induced the positive dopaminergic neuron of differentiation tyrosine, is used for Parkinsonian treatment.(human amnioticepithelial cells, it is early stage hAECs) to come from the embryo, keeps many differentiation potentials of similar embryonic stem cell, can be divided into entoderm (pancreas, liver), mesoderm (myocardial cell) and ectoderm (neurocyte) for people's amniotic epithelial cells.The people's amniotic epithelial cells that wherein has NSC (neurocyte) characteristic is the good cell source of cell replacement treatment nervous system disorders.
People's amniotic epithelial cells is through 10%FCS (FCS foetal calf serum) substratum vitro culture; Can obtain purity at the positive dopaminergic neuron-like cell of 85% tyrosine hydroxylase; Striatum is transplanted in the animal model for parkinsonism brain; Not only can improve the behavior disorder of animal model for parkinsonism, brain inner tissue morphological abnormalities also improves, and our research confirms to transplant the interior substantia nigra dopaminergic neuron of animal brain and obviously is superior to normal control.
Description of drawings
Figure 1A-Fig. 1 C proves with method of the present invention and can turn out dopaminergic neuron-like cell for the cell of turning out with method of the present invention demonstration at microscopically;
Fig. 2 A-Fig. 2 D is dopaminergic phenotypic differentiation and the neural genes involved that takes place;
Fig. 3 A-Fig. 3 C is for after being transplanted to cultured cells of the present invention in the brain of mouse, to the mouse analysis of cutting into slices, the tyrosine hydroxylase positive cell that obtains, people's amniotic epithelial cells and the expression of exogenous tyrosine hydroxylase positive cell in mouse brain;
Fig. 4 A-Fig. 4 C is for after being transplanted to cultured cells of the present invention in the brain of mouse, to the mouse analysis of cutting into slices, the tyrosine hydroxylase positive cell that obtains, anti-people's NA and the expression of exogenous tyrosine hydroxylase positive cell in mouse brain.
Embodiment
A kind of method that differentiates dopaminergic neuron of from people's amniotic epithelial cells, inducing of the present invention, this method may further comprise the steps:
(1) gets the Freshman placenta tissue; Human amnion tissue is peeled off; Wash repeatedly with saline water and to remove blood clot; Use commercially available aseptic D-hank ' s liquid to soak after 1.5 hours then, use 0.9% saline water rinsing again 3 times, aseptic D-hank ' s liquid contains: 1000U/ml penicillium mould, 1000U/ml Streptomycin sulphate, 100U/ml qingfengmeisu qiong and 2.5ng/ml B fungizone;
(2) the people's amnion with rinsing places 0.25% trypsin solution, and digestion is 15 minutes under 37 ℃ temperature;
(3) with the digestion of the above-mentioned people's amnion of DMEM/F12 (commercially available) termination in trypsin solution that contains 10% foetal calf serum, with 200 order cells sieve above-mentioned solution is filtered then, collect the cell behaviour amniotic epithelial cells that obtains;
(4) separate the people's amniotic epithelial cells that is obtained; With the substratum of 10% commercially available no foetal calf serum above-mentioned people's amniotic epithelial cells of separating is carried out subculture in vitro separately and cultivate, the environment that this subculture in vitro separately is cultivated is to be that 37 ℃, humidity are to carry out in the incubator of 95% and 5% carbonic acid gas in temperature;
(5) select 3 or 4 or 5 or 6 good generation people amniotic epithelial cells of growth conditions; The monobasic breeding time of people's amniotic epithelial cells is 10-12 days; Continue to cultivate with the cell differentiation of nerve cord substratum then, the cell differentiation of nerve cord substratum contains for every milliliter: the vitamins C of Prostatropin 8, the 200 μ M of the Sonic hedgehog SHH of 15% serum, 200ng, the fibroblast growth factor 8 of 100ng, 10ng and the BDNF of 20ng;
(6) above-mentioned people's amniotic epithelial cells is cultivated 7-14 days in the cell differentiation of nerve cord substratum after; Results part cell is identified; Confirm the existence of tyrosine hydroxylase positive cell; Because dopaminergic neuron-like cell has tyrosine hydroxylase, so, just can confirm the existence of dopaminergic neuron-like cell through confirming the existence of tyrosine hydroxylase.And utilize immunofluorescence cell chemistry double label method to carry out the quantitative mark analysis; Under inverted biologic microscope, select at least three low-power fields to carry out analysis of accounts then; The multiple of this low-power field is 10 times, has obtained containing the cell of dopaminergic neuron-like cell;
(7) gather in the crops above-mentioned all cells.
Use the dopaminergic neuron-like cell that from aforesaid method, obtains to treat parkinsonism, the dopaminergic neuron cell that promptly obtains with aforesaid method is transplanted in the brain of suffering from the gloomy comprehensive patient of gold.
Utilize immunofluorescence cell chemistry double label method to carry out quantitative mark and analysis to resulting part cell of the present invention, can see examining and (representing) dopaminergic neuron-like cells of above-mentioned two kinds of common marks of fluorescence of usefulness shown in Fig. 1 C shown in Figure 1A at microscopically by bright spot with the tyrosine hydroxylase of (the expressing) of red fluorescence mark, (representing) all cells shown in Figure 1B by bright spot with the blue-fluorescence mark by luminous point.Demonstrate to people through above-mentioned three figure: have the Dopamine HCL appearance neurocyte of some amount with the resulting cell of method of the present invention, can be used as and be used to treat Parkinsonian cell source.
Carry out corresponding gene test with the resulting part cell of method of the present invention, obtain dopaminergic phenotypic differentiation shown in Figure 2 and the neural genes involved that takes place; Fig. 2 A-Fig. 2 B representes dopaminergic phenotypic differentiation genes involved respectively: Math1, NGN2; Fig. 2 C-Fig. 2 D representes neural genes involved: the Mash1 of generation, Noth1 respectively; Can prove with the resulting cell of method of the present invention and contain the basis that generates dopaminergic neuron-like cell.
The resulting cell of the present invention is transplanted in the mouse brain, and to the brain of mouse the analysis of cutting into slices, Fig. 3 A demonstrates the tyrosine hydroxylase of useful FITC (green fluorescence) mark in mouse brain at microscopically.Behind the plain PKH26 mark of red fluorescence, be transplanted in the mouse brain external with cell on the resulting people's amnion of method of the present invention; Bright spot among Fig. 3 B shows: striatum is distributed with the expression with PKH26 mark positive human amniotic epithelial cells in the mouse brain; Fig. 3 C demonstrates useful FITC in the mouse brain (mark green fluorescence) and PKH26 double labeling cells; Confirm to have people's amniotic epithelial cells in the mouse brain, and this people's amniotic epithelial cells contains the dopaminergic neuron cell from exogenous transplanted cells.
To mouse intracerebral transplantation the application's the cell of from people's amniotic epithelial cells, separating, Fig. 4 A demonstrates the tyrosine hydroxylase of useful FITC (green fluorescence) mark in mouse brain; The specific marker thing that anti-people's NA is a human archeocyte, Fig. 4 B demonstrate the people's amniotic epithelial cells with anti-people's NA immunofluorescence dyeing of red fluorescence mark that is present in the mouse brain; Fig. 4 C demonstrates wherein part tyrosine hydroxylase (FITC mark green fluorescence) and anti-people's NA (Teasered mark red fluorescence) double labeling cells (yellow); The people's amniotic epithelial cells that exists in the mouse brain from exogenous transplanted cells is described, and this people's amniotic epithelial cells contains the dopaminergic neuron cell.
From above test, can draw: after the cell that will from people's amniotic epithelial cells, induce differentiation to come out is transplanted to mouse brain; In mouse brain, found people's amniotic epithelial cells, and in this people's amniotic epithelial cells the dopaminergic neuron cell has been arranged from exogenous transplanted cells.
Above method is not that institute of the present invention restricted portion is referring to claim to the qualification of invention just to explanation of the present invention, and under the situation of spirit of the present invention, the present invention can do any type of modification.
Claims (8)
1. from people's amniotic epithelial cells, induce the method that differentiates dopaminergic neuron for one kind, this method may further comprise the steps:
(1) gets the Freshman placenta tissue, human amnion tissue is peeled off, wash repeatedly with saline water and remove blood clot, use aseptic D-hank ' s liquid to soak 1.2-2 after individual hour then, use saline water rinsing 2-5 time again;
(2) human amnion tissue with rinsing places 0.25% trypsin solution, and digestion is 10-20 minute under 36.5 ℃-37.5 ℃ temperature;
(3) stop the digestion of above-mentioned people's amnion in trypsin solution with the DMEM/F12 that contains 10% foetal calf serum, with 180-220 order cell sieve above-mentioned solution is filtered then, collect the cell behaviour amniotic epithelial cells that obtains;
(4) separate the people's amniotic epithelial cells obtained, above-mentioned people's amniotic epithelial cells of separating is carried out the subculture in vitro separately cultivation with the substratum of 10% no foetal calf serum;
(5) select 3 or 4 or 5 or 6 good generation people amniotic epithelial cells of growth conditions; Continue to cultivate with the cell differentiation of nerve cord substratum then, wherein: the cell differentiation of nerve cord substratum contains for every milliliter: the vitamins C of Prostatropin 8, the 200 μ M of the Sonic hedgehog SHH of 15% serum, 200ng, the fibroblast growth factor 8 of 100ng, 10ng and the BDNF of 20ng;
(6) above-mentioned people's amniotic epithelial cells is cultivated 7-14 days in the cell differentiation of nerve cord substratum after; Part cell to results is identified; Confirm the existence of tyrosine hydroxylase positive cell, and carry out quantitative analysis, obtained comprising the cell of dopaminergic neuron-like cell;
(7) results steps (6) obtain containing the cell of dopaminergic neuron-like cell.
2. the method that differentiates dopaminergic neuron of from people's amniotic epithelial cells, inducing as claimed in claim 1 is characterized in that: aseptic D-hank ' the s liquid in the described step (1) contains: 1000U/ml penicillium mould, 1000U/ml Streptomycin sulphate, 100U/ml qingfengmeisu qiong and 2.5ng/ml B fungizone.
3. the method that differentiates dopaminergic neuron of from people's amniotic epithelial cells, inducing as claimed in claim 2; It is characterized in that: in described step (1); Human amnion tissue uses aseptic D-hank ' s liquid to soak after 1.5 hours, and using concentration again is 0.9% saline water rinsing 3 times.
4. the method that differentiates dopaminergic neuron of from people's amniotic epithelial cells, inducing as claimed in claim 1 is characterized in that: in described step (2), people's amnion digested 15 minutes under 37. ℃ temperature.
5. the method that differentiates dopaminergic neuron of from people's amniotic epithelial cells, inducing as claimed in claim 1 is characterized in that: in described step (3), with 200 order cells sieve above-mentioned solution is filtered.
6. the method that differentiates dopaminergic neuron of from people's amniotic epithelial cells, inducing as claimed in claim 1; It is characterized in that: the quantitative analysis in the described step (6) is to utilize immunofluorescence cell chemistry double label method to carry out mark; Under inverted biologic microscope, select at least three low-power fields to carry out analysis of accounts then, the multiple of this low-power field is 10 times.
7. the method that differentiates dopaminergic neuron of from people's amniotic epithelial cells, inducing as claimed in claim 1; It is characterized in that: the environment that subculture in vitro separately in the described step (4) is cultivated is to be that 37 ℃, humidity are to carry out in the incubator of 95% and 5% carbonic acid gas in temperature, and the monobasic breeding time of people's amniotic epithelial cells is 10-12 days in the step (5).
8. the method that differentiates dopaminergic neuron of from people's amniotic epithelial cells, inducing as claimed in claim 1 is characterized in that: described aseptic D-hank ' s, DMEM/F12 and the substratum that does not have a foetal calf serum are the commercially available prod.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1894401A (en) * | 2003-03-12 | 2007-01-10 | 睿良思生命科学私人有限公司 | Derivation of terminally differentiated dopaminergic neurons from human embryonic stem cells |
| CN101092608A (en) * | 2006-06-23 | 2007-12-26 | 中日友好医院 | Method for separating and purifying nerve stem of human amnion tissue |
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| CN1894401A (en) * | 2003-03-12 | 2007-01-10 | 睿良思生命科学私人有限公司 | Derivation of terminally differentiated dopaminergic neurons from human embryonic stem cells |
| CN101092608A (en) * | 2006-06-23 | 2007-12-26 | 中日友好医院 | Method for separating and purifying nerve stem of human amnion tissue |
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