CN100344764C - Engineered tissue tracing method and construction method - Google Patents
Engineered tissue tracing method and construction method Download PDFInfo
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- CN100344764C CN100344764C CNB2005101033272A CN200510103327A CN100344764C CN 100344764 C CN100344764 C CN 100344764C CN B2005101033272 A CNB2005101033272 A CN B2005101033272A CN 200510103327 A CN200510103327 A CN 200510103327A CN 100344764 C CN100344764 C CN 100344764C
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Abstract
The present invention discloses an engineered tissue cell tracing method which respectively inducts fluorescent protein genes into a seed cell required for building an engineered tissue in vitro by using a gene carrier, a cell for expressing fluorescent protein is obtained and is inoculated on an engineered tissue support frame, and the cell is observed by a fluorescence microscope. The engineered tissue cell tracing method can clearly display cells, particularly distribution and multiplication situations of cells with different varieties and all batches and distribution and function situations of cells planted in vivo.
Description
Technical field
The present invention relates to the engineering tissue technical field.
Background technology
The serious damaged or handicapped effective measure of clinical treatment histoorgan are that histoorgan is transplanted at present, but this methods of treatment has many defectives, and is limited as: donor tissue organ origin, transplanted tissue's organ is produced immunological rejection and increases virus infection probability etc.
The eighties in 20th century, scientist began to probe boldly the novel method of this world medicine difficult problem of reasonable solution along with the deep development of basic subjects such as cytobiology and engineering material.The notion of " organizational project " produces thus, and it is the principle and the method for application project and life science, and the surrogate of preparation biologically active is to recover, to keep or improve the function of damaged tissue.Adopt the learn a skill histoorgan of repair deficiency of organizational project, having graft materials wide material sources, graft can be in external shaping, avoid or reduced advantage such as sending out of immunological rejection and infectious diseases.
Adopt tissue engineering technique successfully to make up various engineering tissues such as skin, bone, cartilage, tendon, liver, cardiac muscle, bladder at present.The ultimate principle and the method for organizational project are: with the normal tissue cell of cultured and amplified in vitro as seed cell, be adsorbed on the good and biologic bracket material that can be absorbed by body of a kind of biocompatibility and form mixture, the disease of cell-scaffold material composite implanting to human body tissue, organ is decreased the position, seed cell biomaterial form in gradually by the process of body degraded and absorbed new at form and function aspects and corresponding organ, organize corresponding to tissue, and reach the purpose of repairing wound and reconstruction function.
After engineering tissue makes up and implants, need that the observation cell is grown on support, fusion situation and growth in vivo, fusion situation.At present, when these tissues carry out external structure and implant analysis, all there is certain limitation in observation technology commonly used at present, during as the tactophily of external structure observation of cell in timbering material, often use the inverted phase contrast microscope direct viewing, but because timbering material has certain thickness, cell and material contrast are little, therefore are difficult to the clearly situation of observation of cell in material; Scanning electron microscopic observation is another kind of method commonly used, but can not realize the viable cell observation, and sample process is time-consuming.In the research that cell implants, also there are open defect in cell isotopic labeling, fluorochrome label and genetic material mark etc. at present commonly used, exist radioactive radiation, fluorochrome label to exist the mark time to be shorter than for 2 weeks during as the cell isotopic labeling and the genetic material mark often exist detect loaded down with trivial details etc.
In addition, when engineering tissue makes up, according to the needs of organizing that make up, sometimes in same tissue, to inoculate different cells or cell and want the gradation inoculation, for example will be with the cell that can produce different tissues in the complex engineering tissue, and when making up bigger organizing, need inoculation repeatedly.Cell how to distinguish different cells or gradation inoculation is a more thorny problem so that understand its growth and the fusion situation, and present Observations Means also is difficult for accomplishing.
Summary of the invention
Make up and implant the above-mentioned defective that the back observation procedure exists at present engineering tissue, the object of the present invention is to provide a kind of tracing method of engineering tissue, solving the different types of cell of inoculation and to the identification problem of the cell of different batches inoculation,
Another object of the present invention also is to provide the external structure method of engineering tissue.
For realizing first purpose of the present invention, the present invention adopts following technical scheme: utilize genophore with the fluorescence protein gene of different colours external import respectively will make up the required different sorts of engineering tissue or batch seed cell in, obtain the cell of different colours express fluorescent protein, described cell is inoculated on the sub-engineering tissue support in batches, and utilized the fluorescent microscope pair cell to observe.
Described genophore can adopt retroviral vector or common carrier for expression of eukaryon.
Described fluorescin can be green fluorescent protein, red fluorescent protein, blue fluorescent protein and yellow fluorescence protein.
In order to realize second purpose of the present invention, the present invention adopts following technical scheme: a kind of external structure method of engineering tissue, and this method comprises following process: the seed cell that (1) will be to be inoculated carries out amplification cultivation; (2) according to cell category and inoculation batch, prepare the fluorescin and the genophore of corresponding different colours; (3) genophore is increased; (4) utilize genophore with the fluorescin of different colours import after the amplification corresponding kind or batch seed cell in; (5) seed cell that will be marked with the different colours fluorescin is inoculated on the engineering tissue support in batches, forms cell-material engineeringization and organizes the complex body tissue; (6) with the engineering tissue complex body in in-vitro cultivation, the seed cell form and the number change of direct viewing different zones under inverted fluorescence microscope, and the cell migration of different zones or situation such as come off.
The present invention is applied to the fluorescent protein labeling technology in the molecular biology research by utilizing fluorescin the seed cell in the engineering tissue to be carried out mark and spike in the engineering tissue structure, with respect to existing observational technique, has following advantage:
(1) utilize expression vector, pair cell mark fluorescent albumen utilizes inverted fluorescence microscope, can realize the viable cell observation;
(2) utilize the inverted fluorescence microscope direct viewing, do not need to add reaction substrate and other materials etc., have the characteristics of Real Time Observation;
(3) utilize the fluorescin of different colours that different sorts and/or batch cell are carried out mark, can distinguish the different treatment measure after, can clearly observe the distribution of different phenotype cells in tissue etc.;
(4) when utilizing retroviral vector to carry out gene transfection, cell can surpass June at the vivoexpression fluorescin, has the characteristics of long-term expression;
(5) cell still has propagation and function preferably behind different fluorescent protein labelings.
Embodiment
Below in conjunction with specific embodiment the tracing method of engineering tissue of the present invention and the construction process of engineering tissue are described further.
Embodiment 1
The external structure tissue engineering bone/cartilage need have bigger chondrocyte or stem cell concentration [(1～5) * 10 in timbering material
7/ ml], could produce near normal cartilage matrix.At present when the external structure tissue engineering bone/cartilage, the cell that is inoculated in the timbering material often is easy to come off, make seed cell not high at partial concn, therefore, sometimes cell inoculation repeatedly occurs in a timbering material, carrying out repeatedly, attempted increase seed cell concentration by a relatively large margin.Using present technique can be to the different fluorescin of seed cell mark of difference plantation batch, thereby is presented at cell quantity in the same timbering material and changes in distribution etc.
Step:
1. according to the needs of engineering tissue cartilage to be made up, prepare required seed cell, with chondrocyte or stem cell, and a large amount of amplification.
2. oneself make up or buy needed retrovirus as genophore, increase in a large number then from biotech firm.
3. the fluorescent protein labeling of cell is handled
(1) seed cell of preparing the 1st inoculation is adopted the gene transfection mode, the mark enhanced green fluorescence protein, its process is: 1. the pLEGFP-N1 plasmid DNA is through phenol-chloroform extracting, ethanol sedimentation.Cultivate PT67 packing cell to 75% and merge area, preparation A liquid (gene DNA 2 μ g+HEPES damping fluids 25 μ l), B liquid (DOTAP20 μ l+HEPES damping fluid 30 μ l) mix under the room temperature of back and leave standstill 15min, add 925 μ l and do not contain serum DMEM nutrient solution and leave standstill 30min.After the PT67 cell is used the serum-free medium rinsing, add the transfection mixed solution, put and cultivate 10h in the incubator, change complete culture solution then and cultivate 24h, change again and contain G418 (800 μ g/ml) nutrient solution, keep G418 (400 μ g/ml) behind the 4d, treat the cultivation of going down to posterity after resistance clone forms.After at the bottom of cell covers with bottle, collect the 24h nutrient solution, aperture 0.45 μ m membrane filtration can place 4 ℃ of refrigerators when using this virus liquid in 2 weeks, adds 10% glycerine in-70 ℃ of preservations otherwise should concentrate the back.
2. retroviral infection: treat that stem cell grows to 35cm
2During 50% area, change and infect liquid 6ml (containing pLEGFP-N1 virus liquid 2ml, polybrene final concentration 8 μ g/ml) at the bottom of the culturing bottle bottle, cultivate 48h for 37 ℃.
3. selection markers cell: adopt the G418 diminishing method to screen for 3 weeks.At first digest cell to be screened, adjusting inoculum density is 3 * 10
4/ cm
2, changing behind the 24h and contain G418 (800 μ g/ml) nutrient solution and cultivate 4d, G418 (500 μ g/ml) cultivates 4d then, keeps forming resistance clone and ordinary method goes down to posterity at last with G418 (300 μ g/ml).
(2) seed cell of preparing the 2nd inoculation is adopted the gene transfection mode, the mark red fluorescent protein, its process is: the pDsRed2-C1 plasmid DNA is through phenol-chloroform extracting, ethanol sedimentation.Culturing stem cells to 50% merges area, and preparation A liquid (gene DNA 2 μ g+HEPES damping fluids 25 μ l), B liquid (DOTAP20 μ l+HEPES damping fluid 30 μ l) mix under the room temperature of back and leaves standstill 15min, adds 925 μ l and does not contain serum DMEM nutrient solution and leave standstill 30min.After cell is used the serum-free medium rinsing, add the transfection mixed solution, put and cultivate 10h in the incubator, change complete culture solution then and cultivate 48h, change again and contain G418 (800 μ g/ml) nutrient solution, keep G418 (300 μ g/ml) behind the 4d, treat the cultivation of going down to posterity after resistance clone forms.
4. press the constructing technology of engineering tissue, with the seed cell of mark enhanced green fluorescence protein with 2 * 10
7Individual/ml concentration is inoculated in the timbering material, forms cell-composite body, after 4～48 hours cultivation, carries out the 2nd inoculation of respective concentration again.The 2nd inoculation used the seed cell of red fluorescent protein that has been mark.
5. analyzed in vitro: cell-composite body of forming of inoculation back repeatedly, can be in the 2nd distribution of inoculating cell in timbering material of direct viewing under the inverted fluorescence microscope, and then can count after with whole cell dissociations, thereby draw the actual adhesion quantity of the 2nd inoculating cell by enzyme digestion.
Spike is carried out in the structure and the regeneration of 2 pairs of through engineering approaches bones of embodiment and cartilage complex tissue
Through engineering approaches bone and cartilage complex tissue are to adopt engineering tissue to learn a skill at the complex tissue of external structure through engineering approaches cartilaginous tissue and through engineering approaches osseous tissue, and the bone and the cartilage complex tissue that are used to repair the joint end are damaged.Use the fluorescin that present technique can be different to the seed cell mark of different zones, thereby study the migratory behaviour and the complex tissue characteristics such as function in vivo of different cells.
Step:
1. according to the needs of engineering tissue to be made up, prepare required seed cell, with chondrocyte, scleroblast or stem cell, and a large amount of amplification.
2. oneself make up or buy needed retrovirus as genophore, increase in a large number then from biotech firm.
3. the fluorescent protein labeling of cell is handled
(1) to preparing to be inoculated into the seed cell employing gene transfection mode in cartilage zone, mark enhanced green fluorescence protein.Its process is: 1. the pLEGFP-N1 plasmid DNA is through phenol-chloroform extracting, ethanol sedimentation.Cultivate PT67 packing cell to 75% and merge area, preparation A liquid (gene DNA 2 μ g+HEPES damping fluids 25 μ l), B liquid (DOTAP20 μ l+HEPES damping fluid 30 μ l) mix under the room temperature of back and leave standstill 15min, add 925 μ l and do not contain serum DMEM nutrient solution and leave standstill 30min.After the PT67 cell is used the serum-free medium rinsing, add the transfection mixed solution, put and cultivate 10h in the incubator, change complete culture solution then and cultivate 24h, change again and contain G418 (800 μ g/ml) nutrient solution, keep G418 (400 μ g/ml) behind the 4d, treat the cultivation of going down to posterity after resistance clone forms.After at the bottom of cell covers with bottle, collect the 24h nutrient solution, aperture 0.45 μ m membrane filtration can place 4 ℃ of refrigerators when using this virus liquid in 2 weeks, adds 10% glycerine in-70 ℃ of preservations otherwise should concentrate the back.
2. retroviral infection: treat that stem cell grows to 35cm
2During 50% area, change and infect liquid 6ml (containing pLEGFP-N1 virus liquid 2ml, polybrene final concentration 8 μ g/ml) at the bottom of the culturing bottle bottle, cultivate 48h for 37 ℃.
3. selection markers cell: adopt the G418 diminishing method to screen for 3 weeks.At first digest cell to be screened, adjusting inoculum density is 3 * 10
4/ cm
2, changing behind the 24h and contain G418 (800 μ g/ml) nutrient solution and cultivate 4d, G418 (500 μ g/ml) cultivates 4d then, keeps forming resistance clone and ordinary method goes down to posterity at last with G418 (300 μ g/ml).
(2) to preparing to be inoculated into the seed cell employing gene transfection mode in bone zone, mark red fluorescent protein.Its process is: the pDsRed2-N1 plasmid DNA is through phenol-chloroform extracting, ethanol sedimentation.Cultured osteoblast-like cells in vitro to 50% merges area, preparation A liquid (gene DNA 2 μ g+HEPES damping fluids 25 μ l), B liquid (DOTAP20 μ l+HEPES damping fluid 30 μ l) mix under the room temperature of back and leave standstill 15min, add 925 μ l and do not contain serum DMEM nutrient solution and leave standstill 30min.After cell is used the serum-free medium rinsing, add the transfection mixed solution, put and cultivate 10h in the incubator, change complete culture solution then and cultivate 48h, change again and contain G418 (800 μ g/ml) nutrient solution, keep G418 (300 μ g/ml) behind the 4d, treat the cultivation of going down to posterity after resistance clone forms.
4. press the constructing technology of engineering tissue, in the cartilage frame material, carry out the seed cell inoculation of mark enhanced green fluorescence protein, cell concn 2 * 10
7Individual/ml, form cell-composite body, in bone holder material, carry out the seed cell inoculation of mark red fluorescent protein, cell concn 5 * 10
6Individual/ml, form cell-composite body, two kinds of tissues are being assembled formation complete through engineering approaches bone and cartilage complex tissue.
5. analyzed in vitro: with through engineering approaches bone and cartilage complex tissue in in-vitro cultivation, can be in the seed cell form and the number change of direct viewing different zones under the inverted fluorescence microscope, and the cell migration of different zones or situation such as come off.
6. the analysis after the tissue transplantation: after through engineering approaches bone and cartilage complex tissue implant, can draw materials after after a while and carry out sample analysis.Sample directly places the cell that can observe different marks processing under the fluorescent microscope to be different fluorescence performances after frozen section, and should not have the fluorescence performance by the unmarked cell in zone; In conjunction with other respective detection, can carry out detailed cell function analysis to implanting tissue.
Spike is carried out in the structure and the regeneration of 3 pairs of engineering tissue skins of embodiment
For the good damaged surface of a wound of covering skin histology, during external structure through engineering approaches skin, 2 layer tissues that carry out that can bio-imitability make up, wherein one deck is inoculated keratinocyte, simulation face tissue function, another layer inoculation inoblast, simulation part dermal tissue function.Use the fluorescin that present technique can be different to the seed cell mark of different zones, thus study the migratory behaviour of different cells and organize in different seed cells characteristics such as function in vivo.
Step:
1. a large amount of amplification cultivation of seed cell to be marked are as keratinocyte, inoblast or stem cell.
2. oneself make up or buy needed retrovirus as genophore, increase in a large number then from biotech firm.
3. the fluorescent protein labeling of cell is handled
(1) to preparing to be inoculated into the seed cell employing gene transfection mode of cuticle region, mark enhanced green fluorescence protein.Its process is: the pEGFP-N1 plasmid DNA is through phenol-chloroform extracting, ethanol sedimentation.Cultivate keratinocyte to 50% and merge area, preparation A liquid (gene DNA 2 μ g+HEPES damping fluids 25 μ l), B liquid (DOTAP20 μ l+HEPES damping fluid 30 μ l) mix under the room temperature of back and leave standstill 15min, add 925 μ l and do not contain serum DMEM nutrient solution and leave standstill 30min.After cell is used the serum-free medium rinsing, add the transfection mixed solution, put and cultivate 10h in the incubator, change complete culture solution then and cultivate 48h, change again and contain G418 (800 μ g/ml) nutrient solution, keep G418 (300 μ g/ml) behind the 4d, treat the cultivation of going down to posterity after resistance clone forms.
(2) to preparing to be inoculated into the seed cell employing gene transfection mode of dermal zone, mark red fluorescent protein.Its process is: 1. the pLRFP-N1 plasmid DNA is through phenol-chloroform extracting, ethanol sedimentation.Cultivate PT67 packing cell to 75% and merge area, preparation A liquid (gene DNA 2 μ g+HEPES damping fluids 25 μ l), B liquid (DOTAP20 μ l+HEPES damping fluid 30 μ l) mix under the room temperature of back and leave standstill 15min, add 925 μ l and do not contain serum DMEM nutrient solution and leave standstill 30min.After the PT67 cell is used the serum-free medium rinsing, add the transfection mixed solution, put and cultivate 10h in the incubator, change complete culture solution then and cultivate 24h, change again and contain G418 (800 μ g/ml) nutrient solution, keep G418 (400 μ g/ml) behind the 4d, treat the cultivation of going down to posterity after resistance clone forms.After at the bottom of cell covers with bottle, collect the 24h nutrient solution, aperture 0.45 μ m membrane filtration can place 4 ℃ of refrigerators when using this virus liquid in 2 weeks, adds 10% glycerine in-70 ℃ of preservations otherwise should concentrate the back.
2. retroviral infection: treat that stem cell grows to 35cm
2During 50% area, change and infect liquid 6ml (containing pLRFP-N1 virus liquid 2ml, polybrene final concentration 8 μ g/ml) at the bottom of the culturing bottle bottle, cultivate 48h for 37 ℃.
3. selection markers cell: adopt the G418 diminishing method to screen for 3 weeks.At first digest cell to be screened, adjusting inoculum density is 3 * 10
4/ cm
2, changing behind the 24h and contain G418 (800 μ g/ml) nutrient solution and cultivate 4d, G418 (500 μ g/ml) cultivates 4d then, keeps forming resistance clone and ordinary method goes down to posterity at last with G418 (300 μ g/ml).
4. press the constructing technology of engineering tissue, in the dermal scaffold material, carry out the stem cell inoculation of mark red fluorescent protein, cell concn 1 * 10
7Individual/ml, form cell-composite body, cultivate after 2 days, carry out the keratinocyte inoculation of mark enhanced green fluorescence protein, cell concn 5 * 10 in the surface of cell-composite body one side
6Individual/ml, form complex body tissue with two confluent monolayer cells.
5. analyzed in vitro: the through engineering approaches skin histology that will have 2 layers of structure can be in the seed cell form and the number change of direct viewing different zones under the inverted fluorescence microscope in in-vitro cultivation.
6. the analysis after the tissue transplantation: after the through engineering approaches skin histology is implanted the skin injury place, can draw materials after after a while and carry out sample analysis.Sample directly places the cell that can observe different marks processing under the fluorescent microscope to be different fluorescence performances after frozen section, and should not have the fluorescence performance by the unmarked cell in zone; In conjunction with other respective detection, can carry out detailed cell function analysis to implanting tissue.
Claims (6)
1, a kind of tracing method of engineering tissue, it is characterized in that: utilize genophore with the fluorescence protein gene of different colours external import respectively will make up the required different sorts of engineering tissue or batch seed cell in, obtain the cell of different colours express fluorescent protein, described cell is inoculated on the engineering tissue support in batches, and utilizes the fluorescent microscope pair cell to observe.
2, the tracing method of engineering tissue as claimed in claim 1 is characterized in that: described genophore adopts retroviral vector or common carrier for expression of eukaryon.
3, the tracing method of engineering tissue as claimed in claim 1 or 2 is characterized in that: described fluorescin can be green fluorescent protein, red fluorescent protein, blue fluorescent protein and yellow fluorescence protein.
4, a kind of external structure method of engineering tissue, this method comprises following process: the seed cell that (1) will be to be inoculated carries out amplification cultivation; (2) according to cell category and inoculation batch, prepare the fluorescin and the genophore of corresponding different colours; (3) genophore is increased; (4) utilize genophore with the fluorescin of different colours import after the amplification corresponding kind or batch seed cell in; (5) seed cell that will be marked with the different colours fluorescin is inoculated on the engineering tissue support in batches, forms cell-material engineeringization and organizes the complex body tissue; (6) with the engineering tissue complex body in in-vitro cultivation, the seed cell form and the number change of direct viewing different zones under inverted fluorescence microscope, and the cell migration of different zones or dropping situations.
5, the external structure method of engineering tissue as claimed in claim 4 is characterized in that: described genophore adopts retroviral vector or common carrier for expression of eukaryon.
6, as the external structure method of claim 4 or 5 described engineering tissues, it is characterized in that: described fluorescin can be green fluorescent protein, red fluorescent protein, blue fluorescent protein and yellow fluorescence protein.
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Non-Patent Citations (4)
| Title |
|---|
| 动态监测组织工程软骨的体外构建及三维静态培养 段小军等，中华实验外科杂志，第22卷第3期 2005 * |
| 增强型绿色荧光蛋白在人软骨细胞中的表达及修复重建人软骨示踪方法的研究 江逊等，中国临床康复，第8卷第20期 2004 * |
| 应用荧光蛋白标记技术对体外构建组织工程软骨的监测 段小军 等，中国矫形外科杂志，第13卷第2期 2005 * |
| 绿色荧光蛋白体外转染与体内示踪成骨细胞的研究 任高宏等，中华整形外科杂志，第20卷第6期 2004 * |
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