CN1611597A - Rat marrow MSC invivo transplanting directional differentiation into retina structure - Google Patents
Rat marrow MSC invivo transplanting directional differentiation into retina structure Download PDFInfo
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- CN1611597A CN1611597A CNA2003101033911A CN200310103391A CN1611597A CN 1611597 A CN1611597 A CN 1611597A CN A2003101033911 A CNA2003101033911 A CN A2003101033911A CN 200310103391 A CN200310103391 A CN 200310103391A CN 1611597 A CN1611597 A CN 1611597A
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Abstract
This invention provides one method of rat marrow grafting in vivo and directional differentiation into retina structure. The purpose of this invention is to provide one method of grafting mesenchyme stem cell among the marrows below the retina and differentiating into retina structure in situ. In order to fulfill this purpose, this invention assumes the following technique strategy: the strategy that separated mesenchyme stem cell of rats are directionaly induced differentiated in vivo, and its characteristic lies in making use of particular cell density and injection manners to make the mesenchyme stem cell among marrows to differentiate in situ into retina structure (including epidermal cell of retinochrome , collimation cone, rod cell, bipolar cell and ganglion cell). This invention offers complementary source to nerve cell deficiency and lays the foundation for clinic research of rehabilitation donator for retina damage.
Description
Technical field
The present invention relates to a kind of method of utilizing mesenchymal stem cells MSCs body interior orientation to be divided into retinal structure.
Background technology
Retinal diseases accounts for the over half of severe illness in eye, thereby self repairing effect is often bad, mainly take the treatment of anti-inflammatory treatment or nutritional factor at present for lighter retina injury, and severe injury is not still had good methods of treatment so far, mainly be the absorption of vitreum hemostasis, promotion blood coagulation.Many cases are owing to failing in time to treat or do not have medicine just to cause visual deterioration even blind, and it is most important for treating retinal diseases therefore to seek better methods of treatment.
The research of Stem Cell Engineering provides a kind of new approach for the treatment of retina injury.(Mesenchymal stem cells MSCs), is a similar fibroblastic class cell in marrow to mesenchymal stem cells MSCs, has multi-direction differentiation potential, can transform to multiple histocyte.Autologous bone marrow mesenchymal stem cells also has the convenience of drawing materials, non-immunogenicity, has multidirectional differentiation potential, conforms with the ethics requirement and does not have tumorigenicity, has the incomparable advantage of other stem cell.
Because the retinal structure complexity, to form cell category many, be the immune privilege district in addition herein, interior stem cell is difficult to reach blood.Even the stem cell in the marrow, have and be divided into amphiblestroid ability and also be difficult to reach damage location.Therefore, inquiring into MSCs induces differentiation condition and Transplanted cells mode to have far reaching significance to damaging amphiblestroid repairing and treating to the retina cell.
Summary of the invention
The objective of the invention is that to transplant directed differentiation be the method for retinal structure in order to provide in a kind of mesenchymal stem cells MSCs body.By the following technical solutions: the 1) scheme that isolated mesenchymal stem cells MSCs carries out the directional induction differentiation in vivo from rat marrow is characterized in that utilizing specific cell density (2 * 10
5/ mL) carry out transplanting in the body; 2) MSCs is transplanted in the rat body, the volume that uses when it is characterized in that transplanting is 10 μ L/ eyes, adopts the single-point injection; 3) MSCs is transplanted in the rat body, it is characterized in that injection system is to transplant under the retina; 4) MSCs is transplanted in the rat body, it is characterized in that the retinal structure that mesenchymal stem cells MSCs is divided in position comprises retinal pigment epithelium, the cone, rod photoreceptor cell, Beale's ganglion cells and ganglion cell.
Content of the present invention is unexposed to be delivered, and those skilled in the art can not obtain separation method of the present invention according to existing technology deduction as not spending creative work at all.
Embodiment
1. (whole process is noted aseptic technique to the vitro culture of rat MSCs, wear antimicrobial gloves): the disconnected neck of 6 male rats in age in week is put to death, soak 1min in 75% alcohol, in super clean bench, peel off the rat skin in the sterilization waist dish, do the separation and Culture of MSCs, concrete steps are: 1. remove the bilateral femur, peel top muscle off, use the flushing of D-Hank ' s liquid; 2. with the careful cross-section incision of the articulum at two, draw the low sugar DMEM insertion medullary space flushing that 2mL contains 10% foetal calf serum (Hyclone) with No. 9 syringe needles of syringe band; 3. wash so repeatedly 4-5 time, 1500rpm * 5min centrifugal collecting cell is with the nutrient solution washing once, centrifugal; 4. now join Percoll medullary cell is resuspended in the 2-3mL nutrient solution, 3000rpm * 30min is centrifugal, draws the cellular layer (wherein contain red corpuscle, the interface is light red) on the interface, is diluted to 5mL with nutrient solution, 1500rpm * 5min centrifugal collecting cell; 5. with 2.3 * 10
5Cell/well is inoculated in the plastics plate of Φ 30mm.
2. transplantation treatment and observe under the retina: MSCs transplants the following method of all taking: 1. cell is through 0.25% trysinization, to final concentration be 2 * 10
5/ mL, it is standby to draw 10 μ L with 50 μ L microsyringes; 2. rat (♀) is cut off approximately 1/2 after anesthesia at once along conjunctiva temporo lateral edges 1mm place, is as the criterion can expose temporo branch hole ball; 3. use the oblique insertion temporo of syringe needle side sclera No. 6.5, the syringe needle that will draw the microsyringe of cell again inserts this hole along circumscribed direction, and dial is aimed at retina, gently cell liquid is injected under the retina, drips the antibiotics medicament for the eyes and prevents trauma infection contamination.
3. the acquisition of rat Y chromosome sry gene and sry gene probe mark:
1. design amplimer: determine that according to document sry gene two ends primer is at rat sry gene:
primer?1：5′-AGA?TCT?TGA?TTT?TTA?GTG?TTC-3′
primer?2：5′-TGC?AGC?TCT?ACT?CCA?GTC?TTG-3′
2. extract genomic dna from the male rat kidney, utilize pcr amplification sry gene fragment;
3. the DIG mark of sry probe: use PCR DIG probc synthesis kit (Rochc, cat#1 636 090) probe is carried out the DIG mark of PCR method, utilize Roche High Pure PCR product purification kit to reclaim;
4. the detection of tissue slice: with go ahead of the rest HE dyeing of tissue slice, observe its structure, the section of carrying out scan-type is carried out original position DNA hybridization and is detected.
The result: organize each layer at normal rat injection MSCs and all find sry hybridization positive cell, cellular form conforms to each confluent monolayer cells.
Claims (4)
1. the method for an enrichment rat bone marrow mesenchymal stem cells is characterized in that the mesenchymal stem cells MSCs of cultivating is former generation in generation to 2 cell.
2. carry out the scheme of directional induction differentiation by the isolated cell of the separation method of claim 1 in vivo, it is characterized in that transplanting with specific cell density and volume injected.
3. by the method for claim 2 isolated cell is carried out the scheme that directional induction is broken up in vivo, it is characterized in that injection system is a subretinal injection.
4. by the method for claim 3 isolated cell is carried out the scheme that directional induction is broken up in vivo, it is characterized in that making mesenchymal stem cells MSCs to be divided into retinal structure (comprising the cone, rod photoreceptor cell, Beale's ganglion cells and ganglion cell) in position.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2003101033911A CN1611597A (en) | 2003-10-31 | 2003-10-31 | Rat marrow MSC invivo transplanting directional differentiation into retina structure |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2003101033911A CN1611597A (en) | 2003-10-31 | 2003-10-31 | Rat marrow MSC invivo transplanting directional differentiation into retina structure |
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| CN1611597A true CN1611597A (en) | 2005-05-04 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNA2003101033911A Pending CN1611597A (en) | 2003-10-31 | 2003-10-31 | Rat marrow MSC invivo transplanting directional differentiation into retina structure |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101875914B (en) * | 2009-04-30 | 2012-07-04 | 四川大学华西医院 | Inducing liquid for differentiation of bone marrow mesenchymal stem cells into epithelial cells and preparation and application thereof |
| CN102618497A (en) * | 2012-03-15 | 2012-08-01 | 中国人民解放军第三军医大学第一附属医院 | Method for preparing retinal pigment epithelia by utilizing human marrow mesenchymal stem cells |
-
2003
- 2003-10-31 CN CNA2003101033911A patent/CN1611597A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101875914B (en) * | 2009-04-30 | 2012-07-04 | 四川大学华西医院 | Inducing liquid for differentiation of bone marrow mesenchymal stem cells into epithelial cells and preparation and application thereof |
| CN102618497A (en) * | 2012-03-15 | 2012-08-01 | 中国人民解放军第三军医大学第一附属医院 | Method for preparing retinal pigment epithelia by utilizing human marrow mesenchymal stem cells |
| CN102618497B (en) * | 2012-03-15 | 2013-10-02 | 中国人民解放军第三军医大学第一附属医院 | Method for preparing retinal pigment epithelia by utilizing human marrow mesenchymal stem cells |
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