| ObjectiveConjunctiva is an important part of ocular surface.It maintains ocular surface integrity and tear-film stabilization. When large areas damaged, the loss of original function can lead to scar formation and symblepharon, eye movement is restricted, it also resulted in the tear film integrity destruction which isone of 'the high incidence of refractory eye diseases causeing of blindness in our country. The effective treatment is conjunctival reconstructs with normal conjunctiva or conjunctival substitute. Conjunctival reconstruction methods including autoallergic or allogenic conjunctival transplantation,amniotic membrane transplantation and lip mucosa transplantation. But all have different shortcomings. By now the most ideal method is to reconstruct the conjunctiva using tissue-engineering methods to get the normal physiological function. Human mesenchymal stem cells(hMSCs) is adult stem cells that can differentiate into the bone tissue, the cartilage organization, the fatty tissue, the muscular tissue and so on the many kinds of organization cell. HMSCs may massively multiply in vitro and has the ability to create clones. Because it has multidifferentiation potential and easily cultured in vitro, hMSCs has potential therapeutic value and has become biology and medical research hot spot, In this study, hMSCs has been differentiated into normal conjunctival epithelial cells, providing a new idea for the clinical treatment of conjunctival diseases. Methods一. Normal conjunctival cells culture and IdentificationMaterial and methodConjunctiva obtained from China Medical University Hospital eye cornea transplant donor's eyeballs. The donors are all healthy persons without eye medical history. The average age of donor was 34.5 years. Enucleated within 24 hours after death.2. immunohistochemical identification of cultured conjunctival cellsWhen the second cultured generation of conjunctival cells close to confluence, the glass coverslips were removed in situ fixation by methanol and DAB color immunohistochemical staining by CK13 monoclonal antibody in time.3. growth curve of conjunctival cellsThe 1st,3rd,5th passage cells were selectecd randomly and subcultured at the density of 5×10~4/ml,after culturing for another 1-9 days, were counted and compared the cell number every day. Each experiment was repeated 3 times and data were shown in mean±standard deviation.4. RT-PCR test of conjunctival cellsTotal RNA of cultured conjunctival cells was extracted by total RNA extract kit(sangon biological engineering technology,shanghai) and stored at-70℃. Primer of Muc4,Muc5ac were designed according to the gene order publicated by NCBI. 25μl PCR system with different primer was carried out. 10μl of each PCR products were examined by 2% agarose gel electrophoresis.二. Culturing and assessment of human bone mesenchymal stem cells1. Material and methodBone marrow were derived from the normal persons in clinical myeloid examination and the average age was 40.6. Bone mesenchymal stem cells were cultured and subpassaged by the means of total bone marrow adherence.2. Immunochemistry identification of cultured Bone mesenchymal stem cellsThe 3rd passage of bone mesenchymal stem cells were seeded on cover glass soaked in culture capsule. Monoclone antibody against CD44 was used to identify the near confluent cells and the positive stain was shown by DAB.3. growth curve of bone mesenchymal stem cellsWe selectecd the lst,4th,8th passage cells randomly and subcultured at the density of 1×10~4/ml, Continue to culture them for 1-9 days, then count and compare the cell number every day. Each experiment was repeated 3 times and data were shown in mean±standard deviation.4. Flow cytometry testThe amplified and subcultured bone mesenchymal stem cells of passage 1,3,5 were identified by monoclone antibody against CD34-PE, CD45-FITC, CD44-FITC,CD29-PE. Positive rate of 5 thousands mesenchymal stem cells were analysed by flow cytometry test. Each experiment was repeated 3 times and data were shown in mean±standard deviation.三. Study on the differentiation of bone mesenchymal stem cells to conjunctiva cells1. Coculture of human bone mesenchymal stem cells and conjunctiva cellsSubcultured mesenchymal stem cells of passage 2-4th were seeded on the bottom of transwell culture plate at the density of 4～5×10~4/ml. Subcultured conjunctiva cells of passage 2-3rd were seeded on the upper tier of transwell culture plate at the density of 6×10~4/ml and cocultured for 11 days.2. Immunochemistry examination of cocultured bone mesenchymal stem cellsAfter cocultured for 11 days, nearly 70-80% confluent bone mesenchymal stem cells were tested by monoclone antibody against CK13.The positive stain was shown by DAB.3. RT-PCR test of cocultured bone mesenchymal stem cellsTotal RNA of cocultured bone mesenchymal stem cells were extracted and stord at-70℃. Primer of Muc4,Muc5ac were designed according to the gene order publicated by NCBI. 25μl PCR system with different primer was carried out. The result was checked by 2% agarose gel electrophoresis with 10μl PCR products.Results一. Culturing and identification of human conjunctiva cells1. morphous of conjunctiva cellsCells were harvested by tissue cultivation after digestion. Ponderosus cells adhered after 48 hours. Cells were shown in shape of round, ellipse and polygon. Cell body was loose and lucency with nucleus in center, cell membrane was also clearly seen. Cells were arranged in inlay and fuse in film after 12-14 days.2. Immunochemistry testNomogeneous immunochernical positive granules against CK13 were observed in endochylema of human conjunctiva cells.3. growth curve of conjunctival cellsSubcultured cells were aged and feeble after 5th passage. Subcultured cells grew slowlier than primary cells and the latent phase for subcultured was 48～60h. Logarithm proliferation phase for subcultured cells was 3～7d. Cells entered flat form and grew slowly after logarithm proliferation phase.3. RT-PCR testmRNA of Muc4, Muc5ac were all transcripted in human conjunctiva cells. 二. culturing and tabling of Bone MSCs1. morphous of bone mesenchymalstem cellsprimary MSCs adhered after cultured for 72 hours, the cultured cells were characterized by spindle-shaped, shuttle,polygon appearance and proliferated rapidly. Fusiform cells reached 80-90% confluent after 10 days and grown in clone. Subcultured cells nearly took the same shape of primary cells, and proliferated rapidly. Cells reached 80-90% confluent after 7-9 days2. Immunochemistry identification of cultured Bone mesenchymal stem cellsMSCs cytoplasm were stained positively by CD44 monoclonal antibody.3. growth curve of bone mesenchymal stem cellsSubcultured cells were aged and feeble after 8th passage. Subcultured cells grew slowlier than primary cells and reached confluence after 9 days.The latent phase for subcultured was 24～36h. Logarithm proliferation phase for subcultured cells was 4～6d. Cells entered flat form and grew slowly after logarithm proliferation phase.4. flow cytometry testData were shown that cultured bone mesenchymal stem cells had adhere molecular expression. Unimodality was shown in CD29, CD44, and negative results were shown in hematopoietic cell differentiation antigen-CD34, CD45. There was no significient difference in positive rate between subcultured MSCs (P＞0.05).三. Study on the differentiation of bone mesenchymal stem cells to conjunctiva cells1. Coculture of human bone mesenchymal stem cells and conjunctiva cellsPart MSCs were shown in ellipse,round and part were fusiform after cocultured for 72 hours. Cells were polygon and round ellipse in bulk, and only a small part were fusiform.2. Immunochemistry study of cocultured bone mesenchymal stem cellsAfter cocultured for 11 days, nearly 70-80% confluent bone mesenchymal stem cells were tested by monoclone antibody against CK13 and the positive stain was shown by DAB. Positive stain against CK13 was shown in differentiated bone MSCs.3. RT-PCR test of cocultured bone mesenchymal stem cellsMuc4 mRNA was transcripted by Differentiated bone MSCs, but Muc5ac was not.conclusion1. Conjunctiva cells can obtain by tissue cultivation after digestion and the 2-3rd passage subcultured cells were suitable for research.2. Highly purified normal mesenchumal stem cells can obtain by means of total bone marrow adherence and the most suitable passage for differentiation was within 4-5 passage.3. Bone mesenchymal stem cells can be differentiated into conjunctiva cells.4. Differentiated MSCs cells have partial function of normal conjunctiva cells. |
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