CN104338181B - A kind of method for removing cells of people's corneal stroma - Google Patents
A kind of method for removing cells of people's corneal stroma Download PDFInfo
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- CN104338181B CN104338181B CN201410503431.XA CN201410503431A CN104338181B CN 104338181 B CN104338181 B CN 104338181B CN 201410503431 A CN201410503431 A CN 201410503431A CN 104338181 B CN104338181 B CN 104338181B
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- corneal stroma
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Abstract
The object of the invention is, in order to solve the problem that corneal stroma donor is in short supply, to improve the deficiency of traditional method for removing cells, a kind of method for removing cells of people's corneal stroma is provided, belong to organizational engineering cornea technical field. The method is specially: collects and contains histiocytic corneal stroma and be placed in cleaning solution, and clean 2-3 time in cleaning solution, and 2-5min at every turn, more above-mentioned corneal stroma is sealed in and in nitrogen, preserves 5～7d; After preservation finishes, corneal stroma is taken out from nitrogen, with cleaning solution cleaning 2-3 time, each 2-5min, then in cleaning solution, isothermal vibration processing, and change cleaning solution 1 time every 1-2h, obtain the corneal stroma of de-stroma cell. The de-cell of the method is complete, corneal Collagen not damaged, and the medicament of all applying in flow process is all non-toxic, and at utmost the biological characteristics of shielding angle membrane matrix is also removed stroma cell effectively.
Description
Technical field
The invention belongs to organizational engineering cornea technical field, be specifically related to a kind of de-cell side of people's corneal stromaMethod.
Background technology
Chinese citizen, because of the about 300-500 ten thousand of blind person simple eye and the sick blinding of cornea of both eyes, is only second to cataract, occupies eyeThe second of section's blinding disease. Cause the principal disease of corneal blindness comprise various infectious keratonosuses, corneal degeneration,Serious mechanical wound, eye table chemical injury, severe xerophthalmia, complicated pteryium, ocular pemphigoid infection etc.,All keratonosus kinds are almost contained. Wherein most of patient to obtain the unique method of useful eyesight be operative treatment,And the major way of operation is cornea holostrome or flaggy transplanting. China is owing to being subject to traditional concept and eye bank's conditionRestriction, Corneal donors quantity is very limited, treatment cost costliness, and part cornea donor is due to time and skyBetween restriction can not obtain utilizing in time fully, therefore can not meet clinical demand far away, give patient family andSociety brings immense pressure.
In the situation that cornea donor is very deficient, utilizes cytology and organizational engineering means to obtain and there is normal lifeThe cornea tissue of reason 26S Proteasome Structure and Function is the key technology that these patients are cast off illiteracy. Superficial keratectomy tissue comprises corneaEpithelial layer and part hypothallus, its structure has very maintaining the physiological function of eye table and the visual performance of eyeballImportant meaning. The at present existing multiple method for reconstructing of corneal epithelium has also been applied to clinical, has obtained objectively and has controlledTherapeutic effect. Though and that the material source of corneal stroma and de-cell processing mode have is multiple, there are many problems:(1) corneal stroma material comes from human eye or animal eyes, and people's cornea tissue compatibility is best, but holostrome cornea comesSource scarcity, and the corneal stroma that derives from animal eyes is difficult to avoid the problems such as immune response; (2) for reduce as far as possibleThere is immunoreactive risk, need shift to an earlier date the stroma cell in corneal matrix for the corneal stroma of corneal transplantationTake off cell processing. De-cell processing mode is developed to the current comparatively generally table of application by freeze-thaw method originallyAll there are some problems that are difficult to evade in surface-active agent elution method: for example multigelation may cause collagen fracture,The tension force of damage corneal stroma; And surfactant all has larger toxicity, after processing, be difficult to be applied to clinical,Stay at present laboratory stage more. Therefore, the reliable sources of searching people corneal stroma donor and safe and effectiveMethod for removing cells be the problem with great clinical practice meaning.
About people's corneal stroma donor, laser refractive surgery may be ideal source. Modern Laser dioptric handThe corneal stroma lens that take off in art process, thickness is 40 μ m-140 μ m left and right, diameter 8mm, optics spyProperty, size and operability all meet the requirement of corneal lamellar transfer operation, was used as medical rubbish after operation in the pastRubbish processing. According to rough Statistics, China annual laser refractive surgery amount, more than 500,000 examples, and is increase year after yearTrend, if make full use of corneal stroma lens treatments corneal blindness patient, a bright lamp showing the way for them beyond doubt.
The existing many decades of research of corneal matrix method for removing cells, as previously mentioned, previously multiple method for removing cells allBecause its limitation is difficult at clinical expansion. The scholar in recent domestic keratonosus field payes attention to these graduallyLimitation, starts to explore the method that the damage of corneal Collagen is less, toxicity is lower.
Summary of the invention
The object of the invention is, in order to solve the problem that corneal stroma donor is in short supply, to improve traditional method for removing cells notFoot, provides a kind of method for removing cells of people's corneal stroma, and the de-cell of the method is complete, corneal Collagen withoutDamage, the medicament of all applying in flow process is all non-toxic, the at utmost biological characteristics of shielding angle membrane matrixAnd effectively remove stroma cell.
For achieving the above object, the present invention includes following main contents:
A method for removing cells for people's corneal stroma, comprises the steps:
1, under aseptic condition, collect the corneal stroma that contains stroma cell and be placed in cleaning solution, and at cleaning solutionMiddle cleaning 2-3 time, each 2-5min, more above-mentioned corneal stroma is sealed in nitrogen to normal temperature or 4 DEG C of preservations5～7d；
2, after preserving and finishing, under aseptic condition, corneal stroma is taken out from nitrogen, clean 2-3 with cleaning solutionInferior, each 2-5min, then be placed in cleaning solution, and under rotating speed 150～250rpm condition, 15～25 DEG C of constant temperature shakesSwing and process 24h-36h, and change cleaning solution 1 time every 1-2h, obtain the corneal stroma of de-stroma cell;
Wherein, the described corneal stroma that contains stroma cell is postoperative people's holostrome corneal stroma or people of eyeball exciseEye cuts through laser refractive surgery the corneal stroma lobe of removing;
Described cleaning solution is to contain the physiological saline of 0.3wt% mycillin or the balanced salt containing 0.3wt% mycillinSolution.
The present invention compared with prior art, it is advantageous that:
1, corneal matrix of the present invention is rinsed the physiological saline or the balanced salt solution that adopt containing 0.3wt% mycillin,To the effect of human eye totally nontoxic, and can prevent to infect. Isothermal vibration is sloughed cell fragment, and environment temperature is15～25 DEG C, can not damage corneal stroma collagen, the medicament of all applying in flow process, can all without obvious toxicityAt utmost the biological characteristics of shielding angle membrane matrix is also removed stroma cell effectively.
2,, because nitrogen environment can make Cell hypoxia apoptosis, the present invention adopts gaseous nitrogen in normal temperature or 4 DEG C of environmentThe de-cell of mode that makes the stroma cell apoptosis on corneal stroma, easy operating, has avoided multigelation to cause glueFormer fracture and damaged tissue tension force, and tissue is made while having avoided the de-cell of the chemical reagent such as use surfactantThe toxic action becoming.
3, while sloughing cell fragment in the present invention, be in SPSS or balanced salt solution, to carry out isothermal vibrationMode, can keep the electrolyte level in people's corneal stroma.
Detailed description of the invention
Isothermal vibration incubator: Labnet company, model 211DS;
Physiological saline, balanced salt solution (ringer's solution, containing the ringer's solution of sodium lactate), blue or green strepto-Element is market and buys product;
Solution, container and the instrument using in this method is product aseptic or that sterilized.
Embodiment 1
With the micro-Smooth forceps of sterilization by the smooth surface of removing in laser refractive surgery, thickness 40-140 μ m, straightThe corneal stroma lobe of footpath 8 ± 2mm is put into and is filled containing the physiological saline of 0.3wt% mycillin on operating tableIn 1ml specification eppendorf pipe, be transferred to secondary B2 type Biohazard Safety Equipment, in culture dish, inject containing 0.3wt%The physiological saline of mycillin, takes out corneal stroma lobe, cleans each 3 minutes in culture dish 3 times; AgainCorneal stroma lobe is put into 5ml centrifuge tube, use portable nitrogen cylinder by being full of nitrogen in centrifuge tube, screwCentrifuge tube pipe lid is also used tube sealing film tube sealing, and normal temperature is preserved 5d; After 5d, take out corneal stroma lobe, be transferred to secondary B2In type Biohazard Safety Equipment, in culture dish, clean 3 times each 3 with the physiological saline containing 0.3wt% mycillinMinute; Corneal stroma lobe after cleaning is put into the reagent bottle filling containing the physiological saline of 0.3wt% mycillin,In 25 DEG C of isothermal vibration incubators, with the rotating speed concussion 24h of 150rpm, per hourly change liquid 1 time, taken offThe corneal stroma lobe of stroma cell.
Cell free corneal stroma lobe is carried out showing after HE and Hoechst33342 dyeing, and this method takes off matrixCell is complete.
Embodiment 2
With sterilization micro-Smooth forceps the holostrome corneal stroma of getting rid of its hetero-organization is put into and is filled the green grass or young crops containing 0.3wt%In the test tube of the physiological saline of streptomysin, be transferred to secondary B2 type Biohazard Safety Equipment, in culture dish, inject and containThe physiological saline of 0.3wt% mycillin cleans corneal stroma 3 times in culture dish, each 2 minutes; GetGo out corneal stroma, put into 5ml centrifuge tube, use portable nitrogen cylinder by being full of nitrogen in centrifuge tube, screwPipe covers and uses tube sealing film tube sealing, and normal temperature is preserved 7d; After 7d, take out corneal stroma lobe, in culture dish, use containing 0.3wt%The physiological saline of mycillin cleans 3 times, each 2 minutes; Corneal stroma after cleaning is put into fill and containThe reagent bottle of the physiological saline of 0.3wt% mycillin, turning with 250rpm in 20 DEG C of isothermal vibration incubatorsSpeed concussion 36h, changes liquid 1 time in every 2 hours, obtains the corneal stroma lobe of de-stroma cell.
Cell free corneal stroma is carried out showing after HE and Hoechst33342 dyeing, and the de-matrix of this method is thinBorn of the same parents are complete.
Embodiment 3
With the micro-Smooth forceps of sterilization by the smooth surface of removing in laser refractive surgery, thickness 40-140 μ m, straightThe corneal stroma lobe of footpath 8 ± 2mm is put into the balanced salt solution filling containing 0.3wt% mycillin on operating table1ml specification eppendorf pipe in, be transferred to secondary B2 type Biohazard Safety Equipment, in culture dish, inject and containThe balanced salt solution of 0.3wt% mycillin, takes out corneal stroma lobe, cleans each 5 in culture dish 2 timesMinute; Again corneal stroma lobe is put into 5ml centrifuge tube, use portable nitrogen cylinder to be full of nitrogen in centrifuge tubeGas, screws centrifuge tube pipe lid and with tube sealing film tube sealing, preserves 6d for 4 DEG C; After 6d, take out corneal stroma lobe, in trainingSupport in ware and clean 2 times with the balanced salt solution containing 0.3wt% mycillin, each 5 minutes; By the angle after cleaningMembrane matrix lobe is put into the reagent bottle filling containing the balanced salt solution of 0.3wt% mycillin, at 15 DEG C of isothermal vibrationsIn incubator, with the rotating speed concussion 30h of 200rpm, per hourly change liquid 1 time, obtain the cornea of de-stroma cellMatrix lobe.
Cell free corneal stroma lobe is carried out showing after HE and Hoechst33342 dyeing, and this method takes off matrixCell is complete.
Claims (4)
1. a method for removing cells for people's corneal stroma, is characterized in that, comprises the steps:
(1) under aseptic condition, collect the corneal stroma that contains stroma cell and be placed in cleaning solution, and in washingIn liquid, clean 2-3 time, each 2-5min, more above-mentioned corneal stroma is sealed in nitrogen and is preserved;
(2) preserve and finish, under aseptic condition, corneal stroma is taken out from nitrogen, clean 2-3 with cleaning solutionInferior, each 2-5min; Be placed in again the processing of cleaning solution isothermal vibration, obtain the corneal stroma of de-stroma cell;
Wherein, described cleaning solution is contain the physiological saline of 0.3wt% mycillin or contain 0.3wt% mycillinBalanced salt solution.
2. the method for removing cells of a kind of people's corneal stroma according to claim 1, is characterized in that, described inThe corneal stroma that contains stroma cell be that the postoperative people's holostrome corneal stroma of eyeball excise or human eye are bent through laserThe corneal stroma lobe that light operation cutting is removed.
3. the method for removing cells of a kind of people's corneal stroma according to claim 1, is characterized in that, step(1) described corneal stroma is sealed in the condition of preserving in nitrogen and is: at normal temperature or 4 DEG C, preserve 5～7d.
4. the method for removing cells of a kind of people's corneal stroma according to claim 1, is characterized in that, step(2) described isothermal vibration processing method is: under rotating speed 150～250rpm condition, and 15～25 DEG C of isothermal vibrations24h-36h, and change cleaning solution 1 time every 1-2h.
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| CN201410503431.XA CN104338181B (en) | 2014-09-25 | 2014-09-25 | A kind of method for removing cells of people's corneal stroma |
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| CN201410503431.XA CN104338181B (en) | 2014-09-25 | 2014-09-25 | A kind of method for removing cells of people's corneal stroma |
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| CN104338181A CN104338181A (en) | 2015-02-11 |
| CN104338181B true CN104338181B (en) | 2016-05-11 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104399122B (en) * | 2014-11-28 | 2017-08-25 | 武征 | A kind of acellular matrix and preparation method thereof |
| CN106730006A (en) * | 2016-12-22 | 2017-05-31 | 深圳艾尼尔角膜工程有限公司 | A kind of method for removing cells of cornea |
| CN109464704B (en) * | 2018-11-19 | 2021-12-10 | 爱尔眼科医院集团股份有限公司 | RPE cell sheet and application and preparation method thereof |
| CN109568663B (en) * | 2018-11-27 | 2021-01-15 | 中山大学中山眼科中心 | Preparation method of corneal stent material and corneal stent material |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2491528A1 (en) * | 2002-06-28 | 2004-01-08 | Cardio, Inc. | Decellularized tissue |
| CN101274106A (en) * | 2008-03-24 | 2008-10-01 | 中山大学中山眼科中心 | Method for preparing acellular matrix |
| CN101985051A (en) * | 2010-10-21 | 2011-03-16 | 暨南大学 | Acellular cornea or acellular corneal stroma, preparation method and application thereof |
| CN104001215A (en) * | 2014-06-13 | 2014-08-27 | 深圳艾尼尔角膜工程有限公司 | Acellular corneal stroma and preparing method for acellular corneal stroma |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2491528A1 (en) * | 2002-06-28 | 2004-01-08 | Cardio, Inc. | Decellularized tissue |
| CN101274106A (en) * | 2008-03-24 | 2008-10-01 | 中山大学中山眼科中心 | Method for preparing acellular matrix |
| CN101985051A (en) * | 2010-10-21 | 2011-03-16 | 暨南大学 | Acellular cornea or acellular corneal stroma, preparation method and application thereof |
| CN104001215A (en) * | 2014-06-13 | 2014-08-27 | 深圳艾尼尔角膜工程有限公司 | Acellular corneal stroma and preparing method for acellular corneal stroma |
Non-Patent Citations (1)
| Title |
|---|
| 角膜修复材料的制备及生物相容性研究;龙玉宇;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20130115(第01期);E080-32 * |
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