ABSTRACT
Human corneal stromal cells (HCSCs), a type of resting neural crest mesenchymal cell, are highly specialized transparent tissues responsible for the secretion of stroma and play an important role in maintaining the transparency of the cornea and the normal visual function of human eye.Normally, corneal stromal cells are quiescent, flat and dendritic.After the cornea is stimulated by trauma, the corneal stromal cells will be activated, and the activated human corneal keratocytes (HCK) will turn to a repair phenotype, undergo apoptosis or transform to corneal fibroblast phenotype and myofibroblast phenotype.The phenotype transformation of HCSCs is closely related to the formation of scar tissue and the reduction of ocular transparency caused by the repair process of human corneal injury.In this article, three phenotypes, phenotypic markers, phenotype transformation and in vitro transformation mechanisms of HCSCs were reviewed, and it was found that corneal fibrosis could be inhibited by interfering with the transformation process of HCK to fibrotic phenotype and TGF-β/Smad signaling pathways.Therefore, in-depth study of the molecular mechanism of phenotypic transformation of HCSCs and the regulatory mechanism of intervention in corneal scarring formation is helpful for prevention and treatment of corneal fibrosis and postoperative corneal opacity in patients.
ABSTRACT
@#AIM: To investigate the survival time and distribution of rabbit corneal stromal cells(CSCs)after transplantation of rabbit corneal <i>in vitro</i>. <p>METHODS: Primary rabbit CSCs was cultured <i>in vitro</i> and identified by immunohistochemical staining. using lentivirus(LV)with marker gene enhanced green fluorescent protein(EGFP)transfection rabbit CSCs, the growth status and fluorescence intensity of the transfected cells were observed under an inverted fluorescence microscope. The <i>in vitro</i> animal experiments were randomly divided into 2 groups. experimental group lines of LV-EGFP tag of rabbit CSCs suspension stromal injection, control group amount of normal saline injection corneal stroma, Frozen sections were taken 1wk and 1mo after surgery to observe the fluorescence of transplanted CSCs, and hematoxylin-eosin(HE)was used to observe the tissue morphology of paraffin sections. <p>RESULTS: LV-EGFP transfected rabbit CSCs showed a small amount of fluorescence after 24h under an inverted fluorescence microscope, with the strongest at 96h and 110h. There was no significant difference in the morphology of the transfected CSCs and normal CSCs. Green fluorescence can be seen in the stromal layer of the cornea in the experimental group at 1wk and 1mo, while there is no green fluorescence in the control group. Paraffin section for 1wk showed obvious epithelial cell hyperplasia and slight corneal edema in the experimental group, and a small amount of inflammatory cell infiltration. 1mo after surgery, the epithelial cell hyperplasia was weakened in the experimental group, and no corneal layer edema was observed. No obvious abnormality was found in the control group for 1wk and 1mo. <p>CONCLUSION: Extracorporeal corneal stroma transplantation of LV-EGFP labeled rabbit CSCs can survive at least 1mo in the corneal and is compatible with adjacent tissues.
ABSTRACT
<p><b>Background</b>Corneal stromal cells (CSCs) are components of the corneal endothelial microenvironment that can be induced to form a functional tissue-engineered corneal endothelium. Adipose-derived mesenchymal stem cells (ADSCs) have been reported as an important component of regenerative medicine and cell therapy for corneal stromal damage. We have demonstrated that the treatment with ADSCs leads to phenotypic changes in CSCs in vitro. However, the underlying mechanisms of such ADSC-induced changes in CSCs remain unclear.</p><p><b>Methods</b>ADSCs and CSCs were isolated from New Zealand white rabbits and cultured in vitro. An Exosome Isolation Kit, Western blotting, and nanoparticle tracking analysis (NTA) were used to isolate and confirm the exosomes from ADSC culture medium. Meanwhile, the optimal exosome concentration and treatment time were selected. Cell Counting Kit-8 and annexin V-fluorescein isothiocyanate/propidium iodide assays were used to assess the effect of ADSC- derived exosomes on the proliferation and apoptosis of CSCs. To evaluate the effects of ADSC- derived exosomes on CSC invasion activity, Western blotting was used to detect the expression of matrix metalloproteinases (MMPs) and collagens.</p><p><b>Results:</b>ADSCs and CSCs were successfully isolated from New Zealand rabbits. The optimal concentration and treatment time of exosomes for the following study were 100 μg/ml and 96 h, respectively. NTA revealed that the ADSC-derived exosomes appeared as nanoparticles (40-200 nm), and Western blotting confirmed positive expression of CD9, CD81, flotillin-1, and HSP70 versus ADSC cytoplasmic proteins (all P < 0.01). ADSC-derived exosomes (50 μg/ml and 100 μg/ml) significantly promoted proliferation and inhibited apoptosis (mainly early apoptosis) of CSCs versus non-exosome-treated CSCs (all P < 0.05). Interestingly, MMPs were downregulated and extracellular matrix (ECM)-related proteins including collagens and fibronectin were upregulated in the exosome-treated CSCs versus non-exosome-treated CSCs (MMP1: t = 80.103, P < 0.01; MMP2: t = 114.778, P < 0.01; MMP3: t = 56.208, P < 0.01; and MMP9: t = 60.617, P < 0.01; collagen I: t = -82.742, P < 0.01; collagen II: t = -72.818, P < 0.01; collagen III: t = -104.452, P < 0.01; collagen IV: t = -133.426, P < 0.01, and collagen V: t = -294.019, P < 0.01; and fibronectin: t = -92.491, P < 0.01, respectively).</p><p><b>Conclusion:</b>The findings indicate that ADSCs might play an important role in CSC viability regulation and ECM remodeling, partially through the secretion of exosomes.</p>
Subject(s)
Animals , Rabbits , Adipose Tissue , Cell Biology , Cell Proliferation , Physiology , Cell Survival , Physiology , Cells, Cultured , Exosomes , Metabolism , Extracellular Matrix , Metabolism , Fibroblasts , Cell Biology , Metabolism , Matrix Metalloproteinases , Metabolism , Mesenchymal Stem Cells , Cell Biology , MetabolismABSTRACT
Background Corneal chemical burn is one of blinding eye diseases.Previous therapies for corneal chemical burn is limited to certain extent.However,transplantation of adipose-derived mesenchymal stem cells (ADSCs) for corneal diseases is drawing more and more attention.Objective This study was to observe the effect of rabbit ADSCs transplantation for ocular alkali burns and explore its mechanism.Methods Rabbit corneal stromal cells (CSCs) were isolated and cultured by suspended matrix method,and rabbit ADSCs were obtained and digested from inguinal fat tissue with enzyme digestion method (0.25％ trypsin) and identified by flow cytometry.CSCs cocultured with ADSCs,and CSCs-induced ADSCs were identified by double-label of with immunofluorescence and reverse transcription PCR (RT-PCR).Then induced or uninduced ADSCs were inoculated on amniotic membrane to prepared ADSCs-amnion patch.Corneal alkali burn models were established in the right eyes of 60 New Zealand rabbits by placing a filter paper with 1％ NaOH solution at the central cornea for 50 seconds.The models were randomized into the induced ADSCs+ amnion implanted group,the uninduced ADSCs + amnion implanting group,amnion implanted group and model group.Corneal opacification and neovascular area were examined and corneal inflammation was graded by slit lamp microscope 1 week,2 weeks and 1 month after surgery.The contents of CD45,interferon-γ (IFN-γ) and interleukin-10 (IL-10) in corneal homogenate as well as vascular endothelial growth factor (VEGF) in aqueous humor were detected by ELISA assay.The use and care of experiment animals followed the Statement of ARVO.Results ADSCs showed the positive responses for CD105,CD29,CD44 with the positive rate 90.23 ％,88.56％ and 98.88％,respectively.CSCs was positively reactive for vimentin.The double-label staining was positive after coculture of CSCs with ADSCs.Hematoxylin-eosin stain exhibited that ADSCs grew well on the amnion.Corneal porcelain opacity and a lot of new blood vessels were seen in the model group,and corneal was clear in the induced ADSCs+ amnion implanted group 1 month after surgery.The inflammatory scores were 1.65 ±0.18,2.05 ± 0.17,2.68±0.25,2.90 ±0.18,and the areas of neovasculization were (10.59 ± 1.78),(22.58 ± 1.63),(37.98 ± 1.90),(45.37±1.65)mm2 respectively in the induced ADSCs+ amnion implanted group,uninduced ADSCs+ amnion implanted group,amnion implanted group and the model group.The inflammatory scores of 1 week,2 weeks,1 month after operation among the four groups had statistically significant differences (F =280.826,330.172,465.707,all at P =0.000),and the areas of neovasculization of 1 week,2 weeks,1 month after operation among the four groups had statistically significant differences (F=60.020,670.811,1 510.231,all at P =0.000),the inflammatory scores in the induced ADSCs + amnion implanted group were remarkably lower than those of the other groups,the areas of neovasculization in the induced ADSCs+ amnion inplanted group were smaller than those of the other groups (all at P＜0.01).In 1 month after surgery,the contents of CD45,IL-10,IFN-γ in cornea and VEGF in aqueous humor were statistically different among the groups(F =916.545,1 739.358,462.134,129.126,all at P =0.000).Compared with the uninduced ADSCs+ amnion implanted group,amnion implanted group and the model group,CD45 and IFN-γ contents were declined,and IL-10 content was elavated in the induced ADSCs+ amnion implanted group (all at P＜ 0.01).In addition,VEGF contents in aqueous humor were significantly lower than those in the other groups (all at P＜0.01).Conclusions Rabbit CSCs-induced ADSCs amnion patch transplantation is effective for the reconstruction of ocular surface after alkali damage probably by differentiation of ADSCs into epithelial-like cell after CSCs induced.Moreover,amnion can alleviate immuno-inflammatory response and suppress neovascularization.
ABSTRACT
AlM:To investigate the effects of pirfenidone ( PFD) on the proliferation and transfomring growth factor-β1 ( TGF-β1 ) expression in vitro culture rat corneal stromal cells.METHODS: Corneal stromal cells from 8 to 10wk SD rats were isolated, cultured and treated with different concentrations of PFD 0mg/mL (control group), 0. 15mg/mL (experimental group▏), 0. 3mg/mL (experimental group‖), 1mg/mL (experimental group Ⅲ) for 48h. CCK-8 assay was performed to assess cell proliferation, while immunocytochemistry and Western Blot were used to detect the expression of ki-67 and TGF-β1 expression, respectively. RESULTS: Compared with control group, PFD significantly inhibited the proliferation in a dose -dependent manner ( all P < 0. 05 ), so was protein expression of ki-67. PFD significantly down-regulated the expression of TGF-β1 in a dose-dependent manner (P<0. 05).CONCLUSlON: Pirfenidone can significantly inhibit the proliferation of rat corneal stromal cell by down regulating TGF-β1 expression, therefore, it has potential prospect in lightening the corneal wound healing reaction.
ABSTRACT
AIM:To determine whether acellular porcine cornea stroma (APCS) could support the growth of the rabbit corneal cells in vitro.METHODS: APCS was prepared. The rabbit's corneal epithelium and stromal cells were cultured and seeded on, APCS in vitro.The observation of phase contrast photograph and histological examination were performed.RESULTS: Histological examination showed the epithe- lium grew on the scaffold of APCS in 2-3 layers at 10th day. The stromal cells adhered to the surface of the scaffold after 24 hours and invaded into the interlaminar of the material at 5th day.CONCLUSION: These results indicate that APCS can support the growth and proliferation of the corneal epithelium and stromal cells in vitro.
ABSTRACT
Objective To investigate the construction of compound membrane with corneal stromal cells and collagen-chitosan by tissue engineering technique and its biocompatibility.Methods Rabbit and human corneal stromal cells were separated and seeded into collagen-chitosan membrane.The compound membrane was transplanted into rabbit corneal stroma.Then the growth condition of keratocytes,the effect on normal keratocytes and degradation of compound membrane were detected by corneal confocal microscope,anterior OCT and histological and immunohistochemical methods ex vivo 1,2,4 weeks after grafting.Results The rabbit and human corneal stromal cells grown well in collagen-chitosan scaffold.The compound membrane degradated gradually after grafting.There was no necrosis and dissolvation.Corneal epithelium,stroma and endothelial cells were all normal.Conclusion Collagen-chitosan can be used as a biological scaffold for construction of corneal stroma.Corneal confocal microscopy and anterior OCT are new methods to observe the biological activity of constructed corneal stroma.