Establishment, Identification And Cloning Of An Untransfected Human Corneal Stromal Cell Line

Corneal layer is a transparent membrane, which is located in the anterior wall of theeyeball, provides most of the eye’s refractive power, coupled with the refractive power of thelens, human can make accurate focus light on the retina imaging. Human CornealStroma(HCS) is to constitute a major part of the cornea, accounting for90%of cornealthickness and play an important role in maintaining the transparency and allowing lightthrough. Many reasons can cause the HCS abnormal lesions and the HCS lesions havefeatures of high prevalence, blinding intensity and difficult clinical treatment. Now the onlycure is a corneal transplantation surgery, but due to the highly lack of the donated cornea, thevast majority of patients with abnormal corneal stroma cannot receive donor cornea. Inrecent years human corneal tissue engineering make it possible to reconstruct tissue-engineered human corneal stroma(TE-HCS) in vitro, bring much hope to tens of millions ofpatients with abnormal corneal stroma. The source of human corneal stromal cell (HCSC)and ideal carrier bracket is the two key issues which are much-needed to be solved in thereconstruction of TE-HCS. The source of HCS is the bottlenecks which restrict the TE-HCSscale reconstruction in vitro. It is reported that constructing HCS cell lines throughtransfection of cancer genes, but this type of cell cannot applied in clinical because of itspotential tumorigenicity. Constructing an untransfected and nontumorigenic HCS cell linesis the hope of solving the source of seed cells problem. But there is no report aboutconstructing untransfected and nontumorigenic HCS cell lines. In order to address sources ofseed cells in HCSC, this thesis uses the continuous cultivation method to launch the HCSC’sprimary culture. Through adding materials which can promote adherence, growth anddivision, we can get HCSC that can subculture and construct untransfected human cornealstroma cell line, and verify the cell properties, functional protein expression andtumorigenicity. Through clonal culture, we select monoclonal cell lines from this cell lineand verify the cell properties again after amplified. This lay the foundation for relatedtheoretical study of HCSC and the study of reconstruction of TE-HCS in vitro using normalmonoclonal cell lines. In order to succeed establishes the untransfected human corneal stroma cell line, thisthesis will rip the corneal stroma piece which obtains except the cornea endodermis and theprevious cerebral cortex to carry on the trypsin to digest moderately, then pastes the matrixorganization block in uses the gelatin diaper in24-well plates, add DMEM/F12mediumwhich contains5%fetal bovine serum（FBS）. The primary culture was carried out at37℃with5%CO2. After24h, change the medium to HCSC special medium, DMEM/F-12medium which namely to contain basic fibroblast growth factor （bFGF）, epidermalgrowth factor（EGF）, chondroitin sulfate （CS）, carboxymethyl chitosan（CM-CH）and20%FBS. During primary culture, fibroblast-like cells migrated from cornea pieces after2d’s culture,\the HCSC growing into a confluent monolayer in14d. Then we carry onsubculture successfully by using the HCSC special medium. Observations showed thatHCSC were highly transparent and spindle in shape. During subculture, the HCSCmaintained their spindle shape, and grew and proliferated at a steady rate. After frozenreservation, their shape and proliferation are not affected. After being subcultured to overpassage40, a novel continuous untransfected HCS cell line was established.Morphological observation, growth properties, chromosome analysis,immunocytochemistry analysis and tumorigenesis assay were used to identify the properties,functions of established HCS cell line. According to the results of morphological observationwith optical microscopy, light microscopic observation showed that the higher the density ofthe cultured human corneal stromal cells in vitro, more neatly arranged, showing the typicalspindle shape; The characteristics analysis showed that population doubling time of the celllines is41.44hours, indicating the strong cell growth and division of the cell lines; Thechromosome count and karyotype analysis results showed that the cell ‘s characteristicnumber of remain at46, and has a typical human chromosome characteristics despite theaneuploidy of chromosomes. The cells express vimentin positively, which is a specificmarker of HCSC. Therefore, the untransfected HCSC can be used as seed cells for in vitroreconstruction of TE-HCS. In order to indentify the potential of the cell lines, this thesiscarried on the immunocytochemistry analysis of functional protein-cell connectional proteinsand membrane transportation proteins. According to the results of, the HCSC positivelyexpress conneexin-43and integrin β1, and functional proteins Na+/K+-ATPase and Ca2+-ATPase. All these indicated that the HCSC maintain the characteristic HCS functions likecell junctions and potential of active transport of Na+, K+and Ca2+. The untransfected celllines constructed not only contains properties of HCSC, but also contains expressions of cell junction proteins and membrane transportation proteins and has the potential of performingthe normal function of HCSC.In order to obtain normal karyotype of human corneal seeds of human corneal cells invitro reconstruction, this thesis has cloning experiment on constructed untransfected HCSCcell lines using clonal culture. Using limited dilution method, cultured in DMEM/F-12containing20%FBS at37℃with5%CO2, we selected15monoclonal steains.Chromosome specimens by conventional methods, the chromosome count and karyotypeanalysis, screening with3normal karyotype of2n=46monoclonal strains. Pick onemonoclonal cell strain, C5G, to identify the growth feature and expression of mark proteins.The characteristics analysis showed that population doubling time of the cell lines is40.39hours, indicating the strong cell growth and division of the cell lines, and had the expressionof vimentin. This means that the cell strain is the monoclonal cell lines of HCSC and can becultured to be used as seed cells for reconstruction of TE-HCS and complete cornea in vitro.In conclusion, this thesis has success established the human corneal stroma cell linewhich the non-extension dyes, and screens the genome normal HCSC seed cell, has solvedthe HCSC seed cell origin and quantity limit question, is the TE-HCS formalization in vitroreconstruction and the clinical practice, as well as the organization project human entirecornea’s in vitro reconstruction has laid the foundation.