CN103013921B - Establishment method of tissue-engineered human salivary gland pleomorphic adenoma living tissue model - Google Patents
Establishment method of tissue-engineered human salivary gland pleomorphic adenoma living tissue model Download PDFInfo
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Abstract
The invention discloses an establishment method for a living tissue model of tissue-engineered human salivary gland pleomorphic adenoma. The method comprises the following steps of performing in-vitro co-culture on a scaffold tissue in the same medium after primary culture and subculture of a salivary gland pleomorphic adenoma tumor tissue and normal subcutaneous fibrous tissue cells which are taken from the same patient, and then transplanting into a dorsal subcutaneous tissue of nude mice to establish the living tissue model of the tumor. The living tissue model of tissue-engineered human salivary gland pleomorphic adenoma is established by using a tissue engineering method and technology. The establishment method provides research basis for studying pathogenesis of human salivary gland pleomorphic adenoma and investigating various innovative treatment methods, and simultaneously provides a method and an idea that can be used as reference for other living tissue models of benign tumors.
Description
Technical field
The present invention relates to a kind of establishment method of tumor model, specifically a kind of establishment method of engineered people's pleomorphic adenoma of salivary gland biological tissue model.
Background technology
Common part innocent tumour has easy recurrence or multiple characteristic clinically, and the careless operation in surgical procedure tends to cause postoperative tumour to be the growth of beading implanting along operative incision.The implanting growth of postoperative tumour and recurrence are to patient's physiology and bring psychologically great wound and misery; Multiple relapse also may cause malignant proliferation of tumor, especially betides some innocent tumour of oromaxillo-facial region, as people's pleomorphic adenoma of salivary gland, WHO is defined as critical knurl, due to the singularity of its happening part, bring difficulty also to operation again, have a strong impact on patient's life quality.
The research that Chinese scholars is carried out people's pleomorphic adenoma of salivary gland at present, its research object is confined to isolated preparation more, is mainly the improvement of clinical epidemiology, nosetiology, pathology, molecular biology and modus operandi to tumour, the research and discovery of gene therapy aspect.
There is scholar subcutaneous by the tissue block of people's pleomorphic adenoma of salivary gland is migrated to nude mice back, to attempt setting up transplanted tumor animal model, but the shortcoming that the animal model of setting up exists tissue block to disappear and degenerate, thereby fail to widely apply under study for action.Also there is scholar to prepare people's pleomorphic adenoma PLAG1 transgene mouse model by transgenic technology, due to its animal model of preparing for transgenic technology, although transgenic mice tumor of submaxillary gland has been simulated mankind's pleomorphic adenoma, but occur there is larger difference with people's pleomorphic adenoma at the tissue of tumour, in histology performance, also there are differences, even in the tumour generation late stage tendency that occurs cancerating.Owing to existing at present pleomorphic adenoma of salivary gland cell cultures difficulty, biological tissue model to set up more difficult problem, cause the tissue of tumour and form genesis mechanism, plantation or recurrence mechanism to be difficult to make a breakthrough.
Summary of the invention
The object of this invention is to provide a kind of establishment method of engineered people's pleomorphic adenoma of salivary gland biological tissue model, can not set up the problem of biological tissue model to solve current people's pleomorphic adenoma of salivary gland, for the innocent tumour research that is difficult to carry out viviperception provides a kind of biological tissue model.
The present invention is achieved in that a kind of establishment method of engineered people's pleomorphic adenoma of salivary gland biological tissue model, comprises the following steps:
(1) preparation of material and reagent:
Nutrient solution I: the RPMI-1640 nutrient solution that takes a morsel, add 15-20ml foetal calf serum, 1000U penicillin, 1000U Streptomycin sulphate, mix, add RPMI-1640 and mend to 100ml;
Nutrient solution II: the RPMI-1640 nutrient solution that takes a morsel, add 5-10ml foetal calf serum, 1000U penicillin, 1000U Streptomycin sulphate, mix, add RPMI-1640 and mend to 100ml;
Nutrient solution III: the RPMI-1640 nutrient solution that takes a morsel, add 15-20ml foetal calf serum, 1000U penicillin, 1000U Streptomycin sulphate, 0.25g hyaluronic acid, mix, add RPMI-1640 and mend to 100ml;
Treatment solution I: the RPMI-1640 nutrient solution that takes a morsel, add 10-15ml foetal calf serum, 10000U penicillin, 5000U Streptomycin sulphate, mix, add RPMI-1640 and complement to 100ml;
Treatment solution II: the RPMI-1640 nutrient solution that takes a morsel, add 15-20ml foetal calf serum, 1000U penicillin, 1000U Streptomycin sulphate, 1g hyaluronic acid, mix, add RPMI-1640 and mend to 100ml;
One in selection acellular dermal matrix, collagen gel, collagen sponge or poly(lactic acid) is as timbering material, described timbering material is with buffer solution for cleaning 2-4 time, in described treatment solution II, soak after 7-14 days, at 0-4 ℃ of temperature, preserve, obtain treated timbering material;
(2) former culture:
(2-1) the former culture of pleomorphic adenoma of salivary gland cell: the pleomorphic adenoma of salivary gland tumor tissues that corrective surgery takes off, immersion treatment in described treatment solution I, get tumor tissue, clean after 2-3 time, in described nutrient solution I, be cut into the tissue block that is suitable for carrying out cell cultures, be placed in culturing bottle bottom, culturing bottle is in CO
2in incubator, be inverted, make tissue block be attached at culturing bottle bottom, after 2 hours, culturing bottle is just put, add described nutrient solution I, at CO
2in incubator, continue to cultivate, after 24 hours, change described nutrient solution I, after this, every once described nutrient solution I of periodic replacement in 1 day, after 18-22 days, obtain the tumour cell of former culture;
(2-2) the former culture of subcutaneous fibrohistiocytic: from same patient's normal subcutaneous fibrous tissue, immersion treatment in described treatment solution I, then cleans, be cut into the tissue block that is suitable for carrying out cell cultures, be placed in culturing bottle bottom, add after described nutrient solution II, in CO
2in incubator, cultivate, after 24 hours, change described nutrient solution II, after this, every a nutrient solution II of periodic replacement in 3 days, after 15-19 days, obtain the inoblast of former culture;
(3) cultivation of going down to posterity:
(3-1) pleomorphic adenoma of salivary gland passage is cultivated: the tumour cell of the former culture obtaining, discard nutrient solution, with buffer solution for cleaning 2-3 time, add Digestive system and digest, under inverted microscope, observe most cells and become bowlder, add described nutrient solution I to stop digestion, collecting cell suspension after piping and druming, centrifugal abandoning after supernatant, adds described nutrient solution I dilution, then in 1: add nutrient solution I after the ratio sub-bottle of 3-4, be placed in CO
2in incubator, cultivate, changed described nutrient solution I once every 1 day, carry out the tumour cell cultivation of going down to posterity; Going down to posterity in culturing process, adopt repeatedly adherent method to remove the inoblast in tumour cell;
(3-2) the inoblast cultivation of going down to posterity: the inoblast of the former culture obtaining, discard nutrient solution, with buffer solution for cleaning 2-3 time, add Digestive system and digest, under inverted microscope, observe most cells and become bowlder, add described nutrient solution II to stop digestion, collecting cell suspension after piping and druming, centrifugal abandoning after supernatant, adds described nutrient solution II dilution, then in 1: add described nutrient solution II after the ratio sub-bottle of 3-4, be placed in CO
2in incubator, cultivate, changed described nutrient solution II once every 2 days, carry out the inoblast cultivation of going down to posterity;
(4) external co-cultivation:
(4-1) tumour cell of cultivating that goes down to posterity of logarithmic phase is made to single cell suspension, adjusting tumour cell concentration is 1 × 10
6individual/ml, gets 1ml single cell suspension, abandons supernatant after centrifugal, draws centrifuge tube floor cells agglomerate and is inoculated in treated timbering material central authorities, in CO
2in incubator, hatch, after 1 hour, add described nutrient solution III to carry out vitro culture, within every 8 hours, change described nutrient solution III once, carry out continuously 3-5 days vitro culture, obtain tumour cell framework;
(4-2) inoblast of cultivating of going down to posterity of logarithmic phase is made to single cell suspension, being adjusted to fibrocyte concentration is 1 × 10
6individual/ml, gets 0.5ml single cell suspension, abandons supernatant after centrifugal, draws centrifuge tube floor cells agglomerate and is inoculated in central authorities of described tumour cell framework, adds described nutrient solution III, at CO
2in incubator, carry out external co-cultivation, within every 8 hours, change described nutrient solution III once, carry out continuously 7-10 days external co-cultivation, obtain the framework of co-cultivation;
(5) transplant: the framework of co-cultivation is being carried out after histology evaluation confirmation, enter nude mice back using the framework of co-cultivation as graft transplantation subcutaneous, raise continuously after 3-6 month, set up engineered people's pleomorphic adenoma of salivary gland biological tissue model.
Described CO
2the culture condition of incubator is: culture temperature is 37 ℃, CO
2concentration is 5% of duty gas total amount, and under saturated humidity environment.
Described centrifugal operational condition is: centrifugal rotational speed is 1200r/min, and the centrifugally operated time is 5 minutes.
The operation steps of described cleaning is: first clean with 0.01M PBS damping fluid, then clean with the mixed solution of foetal calf serum and RPMI-1640 nutrient solution; The volume ratio of the foetal calf serum in described mixed solution and RPMI-1640 nutrient solution is 1: 5.
Described damping fluid is the one in PBS damping fluid, hank ' s damping fluid or D-hank ' s damping fluid.
Maximum feature of the present invention is to use method and the technology of organizational engineering, set up the biological tissue model of people's pleomorphic adenoma of salivary gland, this biological tissue model does not have the disappearance of tissue block or the problem of degeneration in model, on occurring, tumor tissues there is no difference with people's pleomorphic adenoma, in histology performance, there is no difference yet, therefore, it is a kind of useful that the research of behaviour pleomorphic adenoma of salivary gland provides, believable and feasible biological tissue research model, provide Research foundation for studying the pathogenesis of this class tumour and inquiring into various innovation methods for the treatment of, also be the foundation of other carcinoid biological tissue models simultaneously, a kind of thinking for using for reference is provided.
Accompanying drawing explanation
Fig. 1 is schema of the present invention.
Fig. 2 is the histological observation result of set up engineered people's pleomorphic adenoma of salivary gland biological tissue model.
Fig. 3 is the immunohistochemical staining observations of set up engineered people's pleomorphic adenoma of salivary gland biological tissue model.
Embodiment
With reference to figure 1, the establishment method of inventor's pleomorphic adenoma of salivary gland biological tissue model, comprises the following steps:
(1) preparation of material and reagent:
The preparation of nutrient solution I: the RPMI-1640 nutrient solution that takes a morsel, add 20ml foetal calf serum, 1000U penicillin, 1000U Streptomycin sulphate, mix, add RPMI-1640 and complement to 100ml.
The preparation of nutrient solution II: the RPMI-1640 nutrient solution that takes a morsel, add 10ml foetal calf serum, 1000U penicillin, 1000U Streptomycin sulphate, mix, add RPMI-1640 and complement to 100ml.
The preparation of nutrient solution III: the RPMI-1640 nutrient solution that takes a morsel, add 20ml foetal calf serum, 1000U penicillin, 1000U Streptomycin sulphate, 0.25g hyaluronic acid, mix, add RPMI-1640 and complement to 100ml.
The preparation for the treatment of solution I: the RPMI-1640 nutrient solution that takes a morsel, add 10ml foetal calf serum, 10000U penicillin, 5000U Streptomycin sulphate, mix, add RPMI-1640 and complement to 100ml.
The preparation for the treatment of solution II: the RPMI-1640 nutrient solution that takes a morsel, add 20ml foetal calf serum, 1000U penicillin, 1000U Streptomycin sulphate, 1g hyaluronic acid, mix, add RPMI-1640 and complement to 100ml.
The preparation of 0.01M PBS damping fluid: take
KH
2PO
4·H
2O 0.27g，
Na
2HPO
4·12H
2O 2.85g，
NaCl 8.50g，
KCl 0.20g，
Add after a small amount of distilled water dissolving, continue to add distilled water to be settled to 1000ml, autoclaving, 4 ℃ of preservations.
The processing of timbering material: acellular dermal matrix (acellular dermal matrix, is abbreviated as ADM) in Biohazard Safety Equipment, is cut to 5 × 5 × 2mm under aseptic condition
3size, embathes after 3 times with 0.01M PBS damping fluid, is soaked in 7-14 days in described treatment solution II under 4 ° of C temperature condition.
Laboratory animal: the BALB/C-nu nude mice in 4 week age, female, 16-17 gram, is provided by Institute of Experimental Animals, Chinese Academy of Medical Sciences.
Tissue-derived: people's pleomorphic adenoma of salivary gland tumor tissues is taken from the sample of surgical resection, and be diagnosed as pleomorphic adenoma of salivary gland through two Pathology Doctors 's, get again same patient's normal subcutaneous fibrous tissue, under aseptic, 0-4 ℃ condition, be stored in the RPMI-1640 nutrient solution containing 100U/ml penicillin, 100U/ml Streptomycin sulphate for subsequent use.
(2) former culture:
(2-1) the former culture of pleomorphic adenoma of salivary gland cell:
The pleomorphic adenoma of salivary gland sample of preserving is taken out, being placed in treatment solution I soaks 15 minutes, remove gland tissue, tumour coating, blood clot and necrotic tissue etc. in adenoma sample, obtain needed tumor tissue, tumor tissue is first cleaned with 0.01MPBS damping fluid, use again the mixed solution (volume ratio of foetal calf serum and RPMI-1640 nutrient solution is 1: 5) of foetal calf serum and RPMI-1640 nutrient solution to clean, after repeated washing 3 times, be placed in nutrient solution I, tumor tissue be cut into 1 × 1 × 1mm that is suitable for cell cultures
3the tissue block of size, is placed in the bottom of 25ml culturing bottle, and culturing bottle is inverted in to CO
2in incubator, leave standstill 2 hours (CO
2the culture condition that incubator is cultivated is: temperature is 37 ℃, CO
2concentration is 5% of duty gas total amount, and under saturated humidity environment, lower same), make tissue block be attached at the bottom of culturing bottle, then culturing bottle is just put, add nutrient solution I, at CO
2in incubator, continue to cultivate, after 24 hours, change nutrient solution I once, after this every 1 day periodic replacement nutrient solution I once, after 18-22 days, obtain the tumour cell of former culture.
After tissue block is cultivated 7-9 days, under inverted microscope, observe, can see that tissue block grows a small pieces cell around, be referred to as " cell is dizzy ", with the continuous merisis of tumour cell, the area of " cell is dizzy " expands gradually, merges gradually with adjacent " cell is dizzy "; Cultivate 18-22 days, " cell is dizzy " fusion rate can reach 80-90%, micro-Microscopic observation demonstration, and most of tumour cell is Polygons or short shuttle shape, small portion tumour cell growth shuttle shape.
(2-2) the former culture of subcutaneous fibrohistiocytic:
Come from same patient's subcutaneous fibrous tissue, be placed in treatment solution I and soak after 15 minutes, first clean with 0.01M PBS damping fluid, then the mixed solution of the foetal calf serum that is 1: 5 by volume ratio and RPMI-1640 nutrient solution cleans, after repeated washing 3 times, be cut into the 1 × 1mm that is suitable for cell cultures
2the tissue block of size, is placed in 25ml culturing bottle, adds nutrient solution II, in 37 ° of C, 5%CO
2, saturated humidity CO
2in incubator, cultivate, after 24 hours, change nutrient solution II once, after this every 3 days periodic replacement nutrient solution II once, after 15-19 days, obtain the inoblast of former culture.
Cultivate after 12-14 days in tissue block, under inverted microscope, observe, can see that tissue block peripheral cell swims out of, cell cell space is larger, becomes fusiformis or irregular shape; While being cultured to 15-19 days left and right, " cell is dizzy " fusion rate reaches 80-90%, and now cell is elongated, is arranged in radial or whirlpool shape.
(3) cultivation of going down to posterity:
(3-1) cultivation of going down to posterity of pleomorphic adenoma of salivary gland cell:
With the tumour cell of the former culture obtaining by step (2-1), discard nutrient solution I, after 0.01M PBS buffer solution for cleaning three times, add Digestive system digestion, under inverted microscope, observe most cells and become bowlder, add nutrient solution I to stop digestion, collecting cell suspension after piping and druming, under 1200r/min condition centrifugal 5 minutes, abandon supernatant, add nutrient solution I to dilute, then in after the ratio sub-bottle of 1: 3, add nutrient solution I, be placed in 37 ° of C, 5%CO
2, saturated humidity CO
2in incubator, cultivate, changed nutrient solution I once every 1 day, carry out the cultivation of going down to posterity of tumour cell.
This step Digestive system used is the mixed solution of the EDTA of 0.25% trypsinase and 0.02%, and the volume ratio of the trypsinase of 0.25% in mixed solution and 0.02% EDTA is 1: 1.
Go down to posterity latter about 48 hours time, cell starts adherent growth, and now cell majority is Polygons, and the short fusiform cell of part becomes circle or ellipse.
Going down to posterity in culturing process, adopt repeatedly adherent method to remove the inoblast mixing in tumour cell, its tool operating process is as follows:
Go down to posterity in culturing process at tumour cell, every 3-6 days, go after old nutrient solution I, before the nutrient solution I more renewing, first by cultivated hank ' s buffer solution for cleaning for tumour cell 3 times, then add Digestive system (0.2%EDTA that volume ratio is 1: 1 and 0.25% trypsinase mixed solution), under 37 ℃ of conditions, digest 10 minutes.Test under microscope, visible inoblast is circular, and tumour cell is remained stationary, sucking-off Digestive system after digestion, adds hank ' s damping fluid piping and druming cell, inoblast is come off after sucking-off, the rinsing 3 times gently of hank for tumour cell ' s damping fluid, adds new nutrient solution I to continue to cultivate.
(3-2) fibroblastic cultivation of going down to posterity:
With the inoblast of the former culture obtaining by step (2-2), discard nutrient solution II, after 0.01M PB S buffer solution for cleaning three times, add Digestive system digestion, under inverted microscope, observe most cells and become bowlder, add nutrient solution II to stop digestion, collecting cell suspension after piping and druming, under 1200r/min condition centrifugal 5 minutes, abandon supernatant, add nutrient solution II to dilute, then in after the ratio sub-bottle of 1: 4, add nutrient solution II, be placed in 37 ° of C, 5%CO
2, saturated humidity CO
2in incubator, cultivate, changed nutrient solution II once every 2 days, carry out fibroblastic cultivation of going down to posterity.
This step Digestive system used is the mixed solution of the EDTA of 0.25% trypsinase and 0.02%, and the volume ratio of the trypsinase of 0.25% in mixed solution and 0.02% EDTA is 1: 1.
Go down to posterity latter about 24 hours time, cell starts adherent growth, and now cell mostly is spindle shape, visible wire cell process.
(4) external co-cultivation:
(4-1) go down to posterity cultivate growth of tumour cell to the 3-4 for time, arrive logarithmic phase.According to a conventional method the tumour cell of logarithmic phase is made to single cell suspension, adjusting tumour cell concentration is 1 × 10
6individual/ml, gets 1ml single cell suspension under 1200r/min condition centrifugal 5 minutes, abandons after supernatant, draws centrifuge tube floor cells agglomerate and is inoculated in treated timbering material central authorities, is placed in 37 ° of C, 5%CO
2, saturated humidity CO
2in incubator, hatch after 1 hour, add nutrient solution III at CO
2in incubator, continue to cultivate, within every 8 hours, change nutrient solution III once, carry out continuously 3 days vitro culture, obtain tumour cell framework.
(4-2) go down to posterity cultivate fibroblastic growth to the 3-4 for time, arrive logarithmic phase.According to a conventional method the inoblast of logarithmic phase is made to single cell suspension, being adjusted to fibrocyte concentration is 1 × 10
6individual/ml, gets 0.5ml single cell suspension and after centrifugal 5 minutes, abandon supernatant under 1200r/min condition, draws centrifuge tube floor cells agglomerate and is inoculated in central authorities of described tumour cell framework, adds nutrient solution III, at CO
2in incubator, carry out external co-cultivation, within every 8 hours, change nutrient solution III once, carry out continuously the external co-cultivation of 7 days, obtain the framework of co-cultivation.
Inoblast, in process of growth, can secrete various kinds of cell epimatrix and somatomedin, for the growth of tumour cell and for so form tissue block nutrition is provided.
(5) framework of co-cultivation is being carried out after histology evaluation confirmation, in SPF animal housing, enter nude mice back using the framework of co-cultivation as graft transplantation subcutaneous, postoperative nude mice continues to raise 3 months under SPF condition, builds up engineered people's pleomorphic adenoma of salivary gland biological tissue model.
(6) post-transplantation 3 months, under animal general anesthesia state, take out graft, set up organize models is carried out to histological examination and immunohistochemical staining observation, visible by observing: there is angiogenesis on the surface in organize models, graft relatively loose tissue block before implanting becomes reality agglomerate, the tumour of growth is similar round, around visible fibrous capsule holds, histology shows that it has the typical histologic characteristics of pleomorphic adenoma of salivary gland, tumour cell is fusiformis, Polygons or circle, the HE observation of dyeing, visible DLC structure (Fig. 2); Immunohistochemical staining is observed, the visible DLC structural outside layers cell S-100 protein staining positive (Fig. 3).
It is more than the establishment method of the engineered pleomorphic adenoma of salivary gland biological tissue model that provides as an example of people's pleomorphic adenoma of salivary gland example, pleomorphic adenoma of salivary gland belongs to carcinoid one, because it has the general characteristic that the especially critical knurl of general innocent tumour has, therefore the given method of above embodiment also for the foundation of the carcinoid biological tissue model of other types provides can be for method and the thinking used for reference.
Claims (5)
1. an establishment method for engineered people's pleomorphic adenoma of salivary gland biological tissue model, is characterized in that, comprises the following steps:
(1) preparation of material and reagent:
Nutrient solution I: the RPMI-1640 nutrient solution that takes a morsel, add 15-20ml foetal calf serum, 1000U penicillin, 1000U Streptomycin sulphate, mix, add RPMI-1640 and mend to 100ml;
Nutrient solution II: the RPMI-1640 nutrient solution that takes a morsel, add 5-10ml foetal calf serum, 1000U penicillin, 1000U Streptomycin sulphate, mix, add RPMI-1640 and mend to 100ml;
Nutrient solution III: the RPMI-1640 nutrient solution that takes a morsel, add 15-20ml foetal calf serum, 1000U penicillin, 1000U Streptomycin sulphate, 0.25g hyaluronic acid, mix, add RPMI-1640 and mend to 100ml;
Treatment solution I: the RPMI-1640 nutrient solution that takes a morsel, add 10-15ml foetal calf serum, 10000U penicillin, 5000U Streptomycin sulphate, mix, add RPMI-1640 and complement to 100ml;
Treatment solution II: the RPMI-1640 nutrient solution that takes a morsel, add 15-20ml foetal calf serum, 1000U penicillin, 1000U Streptomycin sulphate, 1g hyaluronic acid, mix, add RPMI-1640 and mend to 100ml;
One in selection acellular dermal matrix, collagen gel, collagen sponge or poly(lactic acid) is as timbering material, described timbering material is with buffer solution for cleaning 2-4 time, in described treatment solution II, soak after 7-14 days, at 0-4 ℃ of temperature, preserve, obtain treated timbering material;
(2) former culture:
(2-1) the former culture of pleomorphic adenoma of salivary gland cell: the pleomorphic adenoma of salivary gland tumor tissues that corrective surgery takes off, immersion treatment in described treatment solution I, get tumor tissue, clean after 2-3 time, in described nutrient solution I, be cut into the tissue block that is suitable for carrying out cell cultures, be placed in culturing bottle bottom, culturing bottle is in CO
2in incubator, be inverted, make tissue block be attached at culturing bottle bottom, after 2 hours, culturing bottle is just put, add described nutrient solution I, at CO
2in incubator, continue to cultivate, after 24 hours, change described nutrient solution I, after this, every once described nutrient solution I of periodic replacement in 1 day, after 18-22 days, obtain the tumour cell of former culture;
(2-2) the former culture of subcutaneous fibrohistiocytic: from same patient's normal subcutaneous fibrous tissue, immersion treatment in described treatment solution I, then cleans, be cut into the tissue block that is suitable for carrying out cell cultures, be placed in culturing bottle bottom, add after described nutrient solution II, in CO
2in incubator, cultivate, after 24 hours, change described nutrient solution II, after this, every a nutrient solution II of periodic replacement in 3 days, after 15-19 days, obtain the inoblast of former culture;
(3) cultivation of going down to posterity:
(3-1) pleomorphic adenoma of salivary gland passage is cultivated: the tumour cell of the former culture obtaining, discard nutrient solution, with buffer solution for cleaning 2-3 time, add Digestive system and digest, under inverted microscope, observe most cells and become bowlder, add described nutrient solution I to stop digestion, collecting cell suspension after piping and druming, centrifugal abandoning after supernatant, adds described nutrient solution I dilution, then in 1: add nutrient solution I after the ratio sub-bottle of 3-4, be placed in CO
2in incubator, cultivate, changed described nutrient solution I once every 1 day, carry out the tumour cell cultivation of going down to posterity; Going down to posterity in culturing process, adopt repeatedly adherent method to remove the inoblast in tumour cell;
(3-2) the inoblast cultivation of going down to posterity: the inoblast of the former culture obtaining, discard nutrient solution, with buffer solution for cleaning 2-3 time, add Digestive system and digest, under inverted microscope, observe most cells and become bowlder, add described nutrient solution II to stop digestion, collecting cell suspension after piping and druming, centrifugal abandoning after supernatant, adds described nutrient solution II dilution, then in 1: add described nutrient solution II after the ratio sub-bottle of 3-4, be placed in CO
2in incubator, cultivate, changed described nutrient solution II once every 2 days, carry out the inoblast cultivation of going down to posterity;
(4) external co-cultivation:
(4-1) tumour cell of cultivating that goes down to posterity of logarithmic phase is made to single cell suspension, adjusting tumour cell concentration is 1 × 10
6individual/ml, gets 1ml single cell suspension, abandons supernatant after centrifugal, draws centrifuge tube floor cells agglomerate and is inoculated in treated timbering material central authorities, in CO
2in incubator, hatch, after 1 hour, add described nutrient solution III, at CO
2in incubator, carry out vitro culture, within every 8 hours, change described nutrient solution III once, carry out continuously 3-5 days vitro culture, obtain tumour cell framework;
(4-2) inoblast of cultivating of going down to posterity of logarithmic phase is made to single cell suspension, being adjusted to fibrocyte concentration is 1 × 10
6individual/ml, gets 0.5ml single cell suspension, abandons supernatant after centrifugal, draws centrifuge tube floor cells agglomerate and is inoculated in central authorities of described tumour cell framework, adds described nutrient solution III, at CO
2in incubator, carry out external co-cultivation, within every 8 hours, change described nutrient solution III once, carry out continuously 7-10 days external co-cultivation, obtain the framework of co-cultivation;
(5) transplant: the framework of co-cultivation is being carried out after histology evaluation confirmation, enter nude mice back using the framework of co-cultivation as graft transplantation subcutaneous, raise continuously after 3-6 month, set up engineered people's pleomorphic adenoma of salivary gland biological tissue model.
2. the establishment method of engineered people's pleomorphic adenoma of salivary gland biological tissue model according to claim 1, is characterized in that described CO
2the culture condition of incubator is: culture temperature is 37 ℃, contains 5% CO in air
2, and under saturated humidity environment.
3. the establishment method of engineered people's pleomorphic adenoma of salivary gland biological tissue model according to claim 1, is characterized in that, described centrifugal operational condition is: centrifugal rotational speed is 1200r/min, and the centrifugally operated time is 5 minutes.
4. the establishment method of engineered people's pleomorphic adenoma of salivary gland biological tissue model according to claim 1, it is characterized in that, the operation steps of described cleaning is: first clean with 0.01M PBS damping fluid, then clean with the mixed solution of foetal calf serum and RPMI-1640 nutrient solution; The volume ratio of the foetal calf serum in described mixed solution and RPMI-1640 nutrient solution is 1: 5.
5. the establishment method of engineered people's pleomorphic adenoma of salivary gland biological tissue model according to claim 1, is characterized in that, described damping fluid is the one in PBS damping fluid, hank ' s damping fluid or D-hank ' s damping fluid.
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Non-Patent Citations (10)
| Title |
|---|
| Fujita Y et al.Reconstruction of pleomorphic adenoma ofthe salivary glands in three-dimensional collagen gel matrix culture.《Virchows Arch》.1999,全文. |
| Reconstruction of pleomorphic adenoma ofthe salivary glands in three-dimensional collagen gel matrix culture;Fujita Y et al;《Virchows Arch》;19990228;第138页左栏材料和方法部分 * |
| 人涎腺多形性腺瘤细胞的体外培养及其生物学特性;张秀琴;《中国优秀硕士学位论文全文数据库》;20040328;正文方法部分 * |
| 体外培养人涎腺多形性肿瘤细胞及表面标记物的初步研究;孟超等;《科学技术与工程》;20120630;全文 * |
| 孟超等.体外培养人涎腺多形性肿瘤细胞及表面标记物的初步研究.《科学技术与工程》.2012,全文. |
| 张秀琴.人涎腺多形性腺瘤细胞的体外培养及其生物学特性.《中国优秀硕士学位论文全文数据库》.2004,全文. |
| 新生小鼠皮肤成纤维细胞的分离培养与鉴定;胡素贤等;《中国医药导报》;20111031;正文第1段2.3.1部分 * |
| 涎腺多形性腺瘤细胞体外培养的研究进展;肖翠翠等;《口腔颌面外科杂志》;20100831;第299页第2.3.1部分 * |
| 肖翠翠等.涎腺多形性腺瘤细胞体外培养的研究进展.《口腔颌面外科杂志》.2010,全文. |
| 胡素贤等.新生小鼠皮肤成纤维细胞的分离培养与鉴定.《中国医药导报》.2011,正文第1段. |
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