| Stem cell belongs to the cell with multi-directional differentiation and is capable of self-renewal. Stem cell comes from the cell line which is origin of the primitive cell and differentiate to other tissular cells in specified conditions. The limbal epithelium is located between the corneal epithelium and conjunctival epithelium, and thought to be the reservoir of stem cells for the corneal epithelium. At present, people universally accepted the corneal epithelium can be regenerated efficiently from the limbal epithelium. The limbal epithelium is vital to the cornea for repairing in trauma, self-renewal and integrating. Because of the basal cell uninterruptly substituted the cast-off epithelium. In addition to the integrate limbal epithelium prevent the conjunctival blood vessel invasion for helping the cornea of the state of transparent and the normal physiological function. At present, corneal transplant is only effective method of curing corneal disease. But there is few donor's cornea. Recently, from the establishment of technique of stem cell, artificial cornea to the development tissue engineering cornea, the treatment outlook of corneal defect is opened up. Therefore, it is importance that obtained limbal stem cells and its proliferating ability in vitro. The techniques of culture in vitro have great progresses, established the basic for the study of biologic characters. From clinical application's angle: to obtain succeedly limbal stem cells can construct the tissue engineering cornea that can heal cornea wound and transplant the patient who has the deficient of limbal stem cell, such as the treatment of Stevens-Johnson symptom-complex, Cicatricial pemphigiod, warmhearted or alkali burnt. In view of rabbit cornea is similar to human being's, we use rabbit limbal stem cells substitute human being's as object. It is expected to provide the base of technology for adult keratocyte. This experiment studied separation, culture and biological characterization of rabbit corneal limbal stems in vitro and the effect of some growth factors on proliferation of the cells. It can search for the best of cultural method and empirical study of rabbit limbal stem cell growth factor.The two facts are as follows:1.Separation, culture and biological characterization of rabbit corneal limbal stems in vitroTo digest the rabbit coneal limbal tissue makes use of 0.25% of the trypsin and mechanical method, it were cultured in medium containing 15% fetal bovine serum (FBS), and the medium was Dulecco's modified Eagle medium and F12 medium, and the morphology of the monolayer-cultured cells was observed by inverted phase contract microscope everyday. The cultured cells were identified immunochemically by using monoclonal antibody AE1/AE3 and AE5, because AE1/AE3 is recognized a group of acidic keratins and basic keratins and AE5 is highly specific for a 64KD keratin. Primary cells grew within 48hours, and formed a confluent layer in 10-14days, the shape of the primary isolated cells were similar to corneal epithelial cells with round or oval shape. Immunochemical staining showed that most cultured cells were AE1/AE3 positive and a few AE5 positive in the early ,later, these cells were AE5 positive. The method of culturing limbal stem cells of rabbit in medium containing 15% fetal bovine serum (FBS) in vitro is established in this experiment and the biological characteristics of imbal stem cells have been observed, and it is basic for further study of corneal limbus stem cells.2.The empirical study of rabbit limbal stem cell growth factorThe 100μl cell suspension containing 5×104 cells that grow at the best state—2 generation cells is added in 96-well cell culture plate, and then add 100μl medium contain different concentration of epidermal growth factor(EGF),basic fibroblast growth factor(bFGF) and nerve growth factor(NGF) in every well. There are five samples with regard to every different concentration. To search for the suitable growth factor concentration of rabbit limbal stem cells, MTT colorimetry method was applied to detect the condition of corpuscular growth. EGF is a kind of single strand polypeptide which is composed of 53 amino acid and its acceptor belongs to PTK acceptor. When the growth factor is bonding with PTK acceptor, it can stimulate the interior cell's 2-amino-3-p-hydroxyphenylpropionic acid activation. This process adjusts the cell generation and the cell differentiation. There are affluent PTK acceptor in limbus of cornea cellula epithelialis. When culturing in vitro, EGF stimulate corneal epithelium cell the condition of cell proliferation and movement. bFGF is a sort of microamount polypeptide and multi-functional cell growth factor. bFGF is role of many species of cells, which is origin of the neural ectoderm, neural crista and mesoblast. It effects the cell splitting, growth and differentiation, morphous construction and regeneration. NGF is glucoprotein, which is the weight of 140,000. It induces protein synthesis, stimulationg IgM secretion and adjusting choline acetyltransferase action. The experimental result is shown that EGF,bFGF,NGF can promote the role of cell proliferation of rabbit limbal stem cells. The concentration of EGF at 10ng/ml～80ng/ml can promote its growth,but the best suitable concentration is 10ng/ml. The concentration of bFGF at 5ng/ml～100ng/ml can promote its growth,but the best suitable concentration is 20ng/ml. The concentration of EGF at 1ng/ml～200ng/ml can promote its growth,but the best suitable concentration is 100ng/ml.The experiment provide the basis of the proliferating of rabbit limbal stem cells in vitro.
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