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Background Corneal blindness caused by ocular surface disease is one of the main reasons for the global blinding corneal diseases.With the development and progress of tissue engineering technology,tissueengineered cornea offers a new approach to the treatment of ocular surface disease.Objective This study was to obscrve the growth and differentiation of human umbilical cord mesenchymal stem cclls (UC-MSCs) on thc corneal stroma of receipts and investigate the feasibility of human UC-MSCs differentiated into corneal epithelium-like cells and the reparation of injury cornea.Methods Human UC-MSCs were isolated from human umbilical cord using collagenase Ⅳ digestion and passaged in DMEM/F12 containing fetal bovine serum in vitro.The immunophenotype of cultured human UC-MSCs was evaluated by flow cytometry.The differentiated osteoblasts from the human UC-MSCs by directional induce was identified.Twenty-four New Zealand albino rabbits were randomly divided into 2 groups.The human UC-MSCs were cultured on porcine corneal matrix without corneal epithelium for 4 days and then transplanted onto the 12 left eyes of 12 New Zealand albino rabbits,and porcine corneal matrix without corneal epithelium was transplanted onto the left eyes of other 12 New Zealand albino rabbits as control group.The rabbits received keratoplasty were examined using in vivo confocal microscope through focusing(CMTF).The eyeballs were taken off after 2,4 and 8 weeks,the growth and differentiation,expression of cytokeratin 3 (CK3),CK12 and ATP-binding cassette superfamily G memben 2 (ABCG2)of human UC-MSCs were observed by histopathology and immunofluorescence staining.This use of the experimental animals complied with ARVO Statement.Results Digestive human UCMSCs formed round in shape and was large in size.The attached cells displayed long-fusiform shape like fibroblasts.The cultured human UC-MSCs phenotype was CD105+/CD29+/CD44+/CD34-/CD45-and could be induced toward osteoblast differentiation under the appropriate experimental conditions.Human UC-MSCs grew well on the porcine corneal matrix.The corneal grafts survived wcll without rejection till the experiment end in experimental eyes,but the rejection of corneal graft occurred in control eyes.Confocal microscope could observe corneal epithelium-like cells.The corneal epithelium cells showed the positive response for CK3 and CK12 and absent response for ABCG2.Conclusions Human UC-MSCs with porcine corneal matrix can survive,proliferate and differentiate into corneal epithelium-like cells after transplanting onto the corneal stroma of rabbits.This result suggests that human UC-MSCs is able to repair and reconstruct the injured corneal surfaces.
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Background Clinical studies indicated that the pathogenesis of most corneal dystrophy is associated with the mutation of the transforming growth factor beta-induced (TGFBI) gene.However,the molecular mechanism of mutated TGFBI gene in corneal dystrophy is unclear. Objective The present study was to investigate the expression of the TGFBI gene in human corneal tissue and cells in vitro.MethodsHuman corneal epithelial cells and keratocytes were cultured and passaged,and donor corneal tissue was obtained for the section preparation.RT-PCR was used to detect the expression of TGFBI mRNA in human corneal tissue and cells.Immunofluorescence was used to test the expression of the TGFBI protein in the human corneal tissue,and immunohistochemistry was used to test the expression of the TGFBI protein in human corneal epithelial cells and corneal stromal cells.ResultsRT-PCR analysis showed that TGFBI mRNA could be detected as a 1274 bp band in human corneal tissue and corneal stromal cells,but no TGFBI mRNA was observed in corneal epithelial cells.Immunofluorescence assay revealed that corneal stromal cells were positive ly expressed for the TGFBI protein,but the corneal epithelial cells did not express the TGFBI protein.Immunohistochemistry indicated that the expression of TGFBI was detected the red fluoressence in the cytoplasm of corneal stromal cells;however,no positive response was found in corneal epithelial cells.ConclusionsThe expression of the TGFBI gene occurs in human corneal stromal cells but not in the corneal epithelial cells.This result might be of helpful for studying the function and role of TGFBI gene in pathogenesis of corneal dystrophy.
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Background The human transforming growth factor beta-induced gene (TGFBI) is the first determined pathogenic gene to corneal dystrophy.But the molecular genetic mechanism is completely unknown.The study of concerning role of TGFBI is very important for us understand the physiological function of cornea,and the pathogenesis of corneal dystrophy.Objective The vector of human transforming growth factor beta-induced gene (TGFBI) in eukaryotic expression was constructed and transfected into the human corneal epithelial cells in order to explore its influence on the growth of human corneal epithelial cells.Methods Total RNA was extracted from normal donor cornea tissue and cDNA was obtained by reverse transcription.TGFBI cDNA was synthesized by reverse transcription-PCR and cloned into pCMV-N-HA vector and identified by sequencing with PCR and EcoRV,XhoI double restriction endonuclease.The cells were grouped into recombinant pCMV-N-HA-TGFBI plasmid group,pCMVN-HA plasmid group,non-transfected group and pGFP-C2 transfected group.The recombinant pCMV-N-HA-TGFBI plasmid was transfected to human corneal epithelial cells and identified by observing the expression of enhanced green fluorescence protein(EGFP) in the cells.The TGFBI mRNA and proteins were harvested from the cells for real-time PCR analysis and Western blot assay respectively in 58 hours after transfection.The growth of the transfected cells was assessed by Cell Counting Kit-8.The expressions of matrix metalloproteinase(MMP) and tissue inhibitors of matrix metalloproteinase (TIMP) proteins and their mRNA in transfected cells were detected using SYBR fluorescence realtime PCR analysis and Western blot assay.Results The sequencing result of pCMV-N-HA-TGFBI positive clone plasmid showed that amplified TGFBI eDNA inserted into the vector at the correct sequence.EGFP was expressed in transfected cells in 48 hours after transfer of pGFP-C2 with the transfer efficacy 70％.The expression intensity of TGFBI mRNA was significantly higher in recombinant pCMV-N-HA-TGFBI plasmid group compared with pCMV-N-HA plasmid group and non-transfected group,and TGFBI protein was expressed in recombinant pCMV-N-HA-TGFBI plasmid group.No significant difference was found in the A450value among recombinant pCMV-N-HA-TGFBI plasmid group,pCMV-N-HA plasmid group and non-transfected group ( F=3.34,P＞0.05 ).The mRNA level of MMP1,MMP3in the transfected cells was significant elevated but that of TIMP1 was declined in the recombinant pCMV-N-HA-TGFBI plasmid group compared with pCMV-N-HA plasmid group and non-transfected group (all P ＜ 0.05 ).Meanwhile,the expressions of MMP1,MMP3 and TIMP1 proteins appeared the same tendency( all P＜0.05).Conclusions Eukaryotic expression vector harboring human TGFBI eDNA can be successfully constructed and efficiently overexpressed in human corneal epithelial cells.TGFBI gene is involved in the physical and pathological conditions of human corneal epithelial cells by regulating the activity of MMP1,MMP3 and TIMP1.The results offer a new approach for the study of the role of TGFBI in pathogenesis of corneal transparency.
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Background Studies demonstrated that human amniotic epithelial cells (AECs) have some characteristics of embryonic stem cells and they were used to re-establish the surface of eyes. Human AECs may serve as new seed cells in tissue engineering for corneal epithelium reconstitution in the future. Objective The present study was to investigate the application value of human amniotic epithelium cells transfected by lentiviral vectormediated enhanced green fluorescent protein (EGFP) gene as new seed cell source for engineering the corneal surfacelayer. Methods Lentiviral vector carrying the objective gene EGFP was transfected into human amniotic epithelial cells (pLenti6/V5-DEST),and the transient expression of the transgene in the human amniotic epithelial cells was observed under the fluorescence microscope. Flow cytometry was used to detect the positive expression rates of EGFP in transfected cells. The transfected human amniotic epithelial cells were seeded onto the fresh corneal stromal surface of New Zealand white rabbit and cultured in vitro. The stem cell deficiency ( SCD ) models were established by cutting off the limbus of cornea in 20 eyes of New Zealand white rabbits, and the model rabbits were then divided into 2 groups randomly. The transplanted grafts carrying the pLenti6/V5-DEST-EGFP gene-transferred human amniotic epithelium cells were regarded as the pLenti6/V5-DEST-EGFP group, and the corneal stroma graft without any epithelial cell served as the control group. The opacity of stroma and corneal conjunctivalization and vascularization were observed daily. The rabbits' eyes were extracted one month after operation. The expression of EGFP in the cornea was detected under the fluorescence microscope, and the expression of CK8, CK18 and CK12 in cornea was detected by immunohistochemical staining. Results The shape of the transferred human amniotic epithelial cells resembled normal human amniotic epithelial cells. 48 hours after the transient transfection of EGFP presented with the highest expression level throughout the observation duration, with a positive expression rate of EGFP of 61.5% ,showing significant differences in comparison with that of 12 ( 5.24% ) , 24 ( 38.27% ) or 96 ( 39. 10% ) hours ( P ＜0. 05) post-transfection; but no obvious difference was found in the positive rate of transiently transfected EGFP between 48 hours and 72 hours ( 58.36% ) ( P＞0. 05 ). Six cornea grafts were clear in 1 month and two corneas were rejected during the observation period in the pLenti6/V5-DEST-EGFP group. A few new blood vessels were seen around the graft. Ten corneas of the control group became opaque and cloudy with new blood vessels growth around the grafts. Imunohistochemistry revealed the positive expressions of CK8, CK1 8 and CK12 in the corneal epithelial layer in the pLenti6/V5-DEST-EGFP group. However,the expression of CK12 was absent in the control group. Conclusion Human amniotic epithelium cells transfected with the pLenti6/V5-DEST-EGFP gene is a new and ideal feed cell type to reconstruct the corneal surface layer. Lentivirus is a relatively safe gene transfection vector.
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<p><b>OBJECTIVE</b>To investigate the injury in the retina of rats exposed to n-hexane.</p><p><b>METHODS</b>Thirty-two SD male rats were randomly divided into control group and four n-hexane groups. The rats in the four n-hexane groups inhaled 35.2 g/m3 n-hexane statically for 1, 3, 7 and 14 days respectively (6 rats in every group) while 8 rats in the control group inhaled air. Histopathology and ultrastructure changes of the retina of rats were analyzed.</p><p><b>RESULTS</b>Rats in control group had clear layers of retinal structure, stained evenly and with regular cell shape. Retinal degeneration was observed in the rats exposed to n-hexane for 7 d and 14 d, and aggravated by degrees with time exposed to n-hexane. In the rats exposed to n-hexane for 14 d, the outer segments of photoreceptor were arranged in a confusing order, and topically there appeared dissolution; in the inner segments, mitochondria were swollen or disappeared. Pyknotic chromatin and cytoplasmic edema were observed in the outer nuclear layer. There were degeneration of horizontal cells, bipolar cells and amacrine cells in the inner nuclear layer. Cytoplasmic edema and organelle dissolution were observed in ganglionic cells. In the neurofibromas layer, outer and inner plexiform layers, there was neuron cell tuber edema, and the microfilament and vacuole of synapse decreased.</p><p><b>CONCLUSION</b>The histopathology and ultrastructure of retina are damaged in the rats exposed to n-hexane, thus leading to ocular fundus disease.</p>
Subject(s)
Animals , Male , Rats , Hexanes , Toxicity , Rats, Sprague-Dawley , Retina , PathologyABSTRACT
<p><b>OBJECTIVE</b>To investigate the injury in the corneal nerve and cornea of rats exposed to n-hexane.</p><p><b>METHODS</b>Thirty-two SD male rats were randomly divided into one control group and four n-hexane groups. The four n-hexane groups inhaled 35.2 g/m(3) n-hexane statically for 1, 3, 7 and 14 d respectively, while the rats in the control group inhaled air. The corneal nerve damage was investigated with golden staining and transmission electron microscope. Histopathological and ultrastructure changes of cornea were analyzed also.</p><p><b>RESULTS</b>The concentration of n-hexane in blood of rats in different experimental groups was (242.91 +/- 59.68), (668.77 +/- 221.74), (1021.21 +/- 545.71) and (1140.42 +/- 468.44) microg/L, increased gradiently with time exposed to n-hexane. In the rats exposed to n-hexane for 7 and 14 d, there appeared fewer corneal nerve bundles and lower density of nerve fiber at the center of cornea, under electron microscope, the lamellar sheath of nerve fiber in the corneal epitheliums appeared intermittent, the neuroplasm of endings was partly lysed and became vacuolar, the microfilament and racuole of neuraxon decreased. In the group exposed to n-hexane for 14 d, the microvillus of cornea epithelium were decreased. In some basal cells there appeared pyknotic nucleus and vacuole, mitochondria were swollen or disappeared.</p><p><b>CONCLUSION</b>The structure of corneal nerve and cornea is damaged in the rats exposed to n-hexane, thus leading to dysfunction of cornea.</p>
Subject(s)
Animals , Rats , Cornea , Nerve TissueABSTRACT
The chromatographic fingerprint of Gastrodia elata Bl. (Tianma) was developed to compare the quality of Tianma samples from different habitats and processing methods. The above analysis method was established by HPLC-DAD technique. And an HPLC method was used to analysis the contents of gastrodin (GAS) and p-hydroxybenzyl alcohol (HBA) in Tianma from different habitats and processed methods. Experiments of chromatographic fingerprint analysis were carried out with a Zorbax XDB C18 column (250 mm x 4.6 mm, 5 microm). The mobile phase consisted of acetonitrile and 0.1% aqueous acetic acid in gradient elution mode. The column was maintained at 25 degrees C. Detection was set at 270 nm. The mass spectra were recorded using as ESI source in the negative mode with ion spray voltage at 3500 V, source temperature at 335 degrees C, gas spray at 8.3 kPa and gas flow rate at 9 L x min(-1). The HPLC methods of quantitative analysis were the same as those of chromatographic fingerprint analysis except the mobile phase, which consisted of acetonitrile and 0.1% aqueous acetic acid in isocratic elution mode with the ratio of 4.5 to 95.5 (v/v). Data of chromatographic fingerprint were analyzed by the "similarity evaluation system for chromatographic fingerprint of TCM (Version 2004 A)" software to compare the quality of Tianma. Samples from different habitats with the same processing method were of high similarity, though a few samples showed evident difference in fingerprint graphics. For Tianma samples with different processing methods, the contents of common peaks were different and the processing method of freezing to dry was better than others. With HPLC-MS technique, 8 major common peaks in the fingerprint of Tianma were identified by their MS spectra and comparison with the reference standards. The results of similarity analysis for chromatographic fingerprint were basically consistent with those of quantitative analysis. The established HPLC-DAD/MS methods can be used to evaluate the quality of Tianma.
Subject(s)
Benzyl Alcohols , Chromatography, High Pressure Liquid , Gastrodia , Chemistry , Glucosides , Mass Spectrometry , Plants, Medicinal , Chemistry , Quality Control , Rhizome , ChemistryABSTRACT
<p><b>AIM</b>To develop methods for the fingerprint analysis of Rhizoma Coptidis and the determination of berberine, palmatine and jatrorrhizine in Rhizoma Coptidis, and analyze the contents of these three alkaloids in Rhizoma Coptidis under different cultivation conditions, from different areas and processed with different methods.</p><p><b>METHODS</b>Two methods (HPLC-UV and HPLC-MS) have been developed and used in fingerprint analysis of Rhizoma Coptidis. An HPLC method was used to determine the contents of three alkaloids.</p><p><b>RESULTS</b>With HPLC-MS techniques, seven major chromatographic peaks in the fingerprint analysis of Rhizoma Coptidis were identified by their MS spectra and compared with the reference standards. In different cultivation conditions, shading conditions and growing ages have obvious influence on the contents of three alkaloids in Rhizoma Coptidis, while planting density was not the major factor that influenced the contents of three alkaloids. The contents of three alkaloids of Coptidis samples were almost higher than those of Coptidis reference material. For Coptidis samples from different cultivation area, the contents of these three alkaloids were different greatly. For Coptidis samples processed with different methods, the contents of three alkaloids were not influenced obviously by processing methods.</p><p><b>CONCLUSION</b>The results showed that the ecology cultivation method to replace the traditional shading method was feasible and provided the theoretical foundation for scientifically processing Rhizoma Coptidis.</p>
Subject(s)
Berberine , Reference Standards , Berberine Alkaloids , China , Chromatography, High Pressure Liquid , Methods , Coptis , Chemistry , Ecosystem , Plants, Medicinal , Chemistry , Quality Control , Reference Standards , Reproducibility of Results , Rhizome , Chemistry , Spectrometry, Mass, Electrospray Ionization , MethodsABSTRACT
Traditional cultivation of Coptis chinensis was carried out under shield by disafforestation, which has been used for over 300 years and lead to the severe destruction of natural environment. Several ecological modes for cultivation of Coptis chinensis have been developed, which increase the yields of Coptis chinensis, protect the resources of forest, and obtain economic and ecologic benefit.
Subject(s)
Agriculture , Methods , Conservation of Natural Resources , Coptis , Ecosystem , Forestry , Methods , Plants, MedicinalABSTRACT
<p><b>OBJECTIVE</b>To establish ecologically new modes of cultivating Coptis chinensis in woods and co-cultivating it with maize.</p><p><b>METHODS</b>Based on the experience obtained from plot comparative test and production test, we used application-oriented research methods, and established new Coptis chinensis cultivation techniques that protect the natural environment.</p><p><b>RESULTS</b>Coptis chinensis was harvested 6 years after cultivation. Yield of Coptis chinensis cultivated in forest (69.5 kg per 0.067 hm2) was higher with 3.7% than that in controls which cultivated under shed (67 kg). Yield of Coptis chinensis co-cultivated together with maize (168.4 kg per 0.067 hm2) was lower with 15.8% than that in controls which cultivated under shed (200 kg).</p><p><b>CONCLUSION</b>The new cultivation modes of Coptis chinensis is an ecologically new technique that assures the good growth of medicinal plants, cereals, forest and animal husbandry.</p>
Subject(s)
Agriculture , Methods , Conservation of Natural Resources , Coptis , Ecosystem , Forestry , Methods , Plants, Medicinal , Zea maysABSTRACT
<p><b>OBJECTIVE</b>To develop technique of storing Coptis chinensis seeds in damp sand under shed and fine cultivation of seedling and to enhance the cultivation seedling rate from seeds of Coptis chinensis.</p><p><b>METHODS</b>With the old technique of seedling in woods as control, we screened the new method for storing seeds and cultivating seedlings.</p><p><b>RESULTS</b>Seeds were picked up at May and stored in damp sand under shed until November, and then planted with technique of fine cultivation of seedling. The germination rate of seeds was up to 98%. One hundred and forty thousands seedlings could be getting from per kilogram seeds.</p><p><b>CONCLUSION</b>Compared to the old method of cultivating seedling in woods, the technique of fine cultivation of seedling significantly increased the rate of cultivation seedlings by 8 times. This technique has been widely applied.</p>
Subject(s)
Coptis , Drug Storage , Germination , Humidity , Plants, Medicinal , Seedlings , Seeds , Silicon DioxideABSTRACT
To summarize the research and application of ecological planting technique for Coptis chinensis, and describe the recent development of its chemical components, pharmacological effects and clinical applications.