Differentiation of human amniotic epithelial cells into corneal epithelial-like cells in vitro.

AIM: To explore the feasibility that human amniotic epithelial cells (hAECs) have the potential to differentiate into corneal epithelial-like cells under the microenvironment replicated by spontaneously immortalized human corneal epithelial cells (S-ihCECs). METHODS: hAECs were isolated by enzyme digestion, and flow cytometry was used to analysis the expression of CD29/90/166/73/34 and HLA-DR. Recovered and cultured S-ihCECs, immunocytochemistry was used to detect the expression of CK3/12. The proliferation of S-ihCECs handled by different concentrations of mitomycin was detected by CCK-8. The proliferation of hAECs cultured by S-ihCECs culture media collected at different time was analyzed by CCK-8. After filtered out the optimal conditions, we collected S-ihCECs culture media for 5 days, then prepared conditioned medium to incubate hAECs, inverted phase contrast microscope and scanning electron microscope were used to observe the change of morphology in hAECs. Quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR) was carried out to evaluate the expression of Oct-4, NANOG, PAX6, and CK12 in the differentiation period. Immunocytochemistry and western bloting were used to detect the expression of CK3/12. RESULTS: The culture media collected every 12h, from 20µg/mL mitomycin pretreatment S-ihCECs could significantly promote the proliferation of hAECs. In the period of differentiation, the morphology of differentiated hAECs was obviously different compared with the control group, and the distinctive CK3/12 for corneal epithelial cells was detected. CONCLUSION: This study showed that hAECs can differentiate into corneal epithelial-like cells by in vitro replication of the corneal epithelial microenvironment, using the culture media collected from S-ihCECs, and it is possible that S-ihCECs culture media could be used in corneal tissue engineering.

Implantation of Iakymenko keratoprosthesis in patients with severe ocular injury.

AIM: To present the results of implantation of Iakymenko keratoprosthesis in five patients with vascularized corneal leukoma caused by severe ocular injury. METHODS: Iakymenko keratoprosthesis was implanted into 5 eyes of 5 patients: 4 patients were suffered from chemical burns and 1 patient from explosive injury. The preoperative visual acuity ranged from light perception to hand motion. The implantation surgery was composed of two-stage procedures. The follow-up period was from 9 months to 11 years. The outcome measures were visual acuity, retention, and complications of the keratoprosthesis. RESULTS: Vision improvements were achieved in most patients. All keratoprosthesis were retained within the follow-up period. Corneal melting occurred in one patient and fibrous closure in another patient, both of which were successfully treated. Retinal detachment occurred in one patient after surgery. CONCLUSION: Iakymenko keratoprosthesis seems to be a promising alternative for the patients with severe corneal injury, but further investigation is needed to evaluate the role of Iakymenko keratoprosthesis.

Construction of eukaryotic plasmid expressing human TGFBI and its influence on human corneal epithelial cells.

AIM: To detect the expression of transforming growth factor beta-induced gene (TGFBI) protein in human corneal tissue and overexpress it in the human corneal epithelial cells in order to discuss the function of TGFBI in the pathogenesis of corneal dystrophy. METHODS: Immunohistochemistry (IHC) was used to detect the expression of TGFBI in the human cornea tissue. TGFBI cDNA was obtained by reverse transcription-PCR from human corneal total RNA extracted from cornea transplant donor and cloned into pCMV-N-HA vector. The recombinant pCMV-N-HA-TGFBI plasmid transfected human corneal epithelial cells. Forty-eight hours later, mRNA and proteins were harvested from cells for real-time PCR analysis and western blot assay respectively. RESULTS: IHC indicated TGFBI mainly exist below the human corneal epithelium layer. Transfection of recombinant pCMV-N-HA-TGFBI into human corneal epithelial cells resulted in effective expression of TGFBI, as shown by increased mRNA level detected by real-time PCR as well as increased protein level detected by Western blot. Meanwhile the result of real-time PCR and Western blot shown the expression of MMP1, MMP3 (matrix metalloproteinases MMP) increased while the expressin of TIMP1 (tissue inhibitors of matrix metalloproteinases TIMP) decreased. CONCLUSION: TGFBI mainly exists below the corneal epithelial layer, recombinant eukaryotic expression vector harboring human TGFBI cDNA was obtained and efficiently overexpressed in human corneal epithelial cells. Meanwhile the TGFBI overexpression in human corneal epithelial cells result in MMP1, MMP3 increasing and TIMP1 decreasing. The result might be helpful for studying the function and role of TGFBI in pathogenesis of corneal dystrophy.

Troglitazone induced apoptosis of human pterygium fibroblasts through a mitochondrial-dependent pathway.

AIM: To study the effect of troglitazone on primary culture human pterygium fibroblasts (HPF). METHODS: Cell viability loss and apoptosis were quantified by cell counting kit-8, AnnexinV-FITC/PI double staining, caspases activity test and western blotting. Flow cytometry was used to detect mitochondrial membrane potential. RESULTS: Peroxisome proliferator-activated receptor γ (PPAR-γ) was positively expressed in pterygium specimens (n=5). Troglitazone showed dose-dependent inhibition of cell survival, induced phospholipids redistribution, activated caspase-3, -9, and altered mitochondrial potential. Western blot assay demonstrated the increase of Bax/Bcl-2 protein ratio. CONCLUSION: Troglitazone induced apoptosis of HPF through a mitochondrial-dependent pathway.

Endogenous aspergillus endophthalmitis after kidney transplantation.

Endogenous aspergillus endophthalmitis(EAE) after kidney transplant is a rare but important clinical problem due to potentially devastating consequences. Early diagnosis of EAE, timely removal of affected vitreous by vitrectomy, proper anti-fungal treatment, all contributed to the successful control of the disease. Therapeutic success of EAE in post-transplant patients depends largely on prompt diagnosis. Definite diagnosis of EAE is based on positive culture results of vitreous specimen, while fundoscopy and B scan ultrasound may aid early diagnosis. In terms of anti-fungal medicine, amphotericin B has long been the first choice, but its systemic applicaiton has severe adverse reactions, especially for patients with impaired renal function. Herein, we report the treatment modality of EAE after kidney transplant with vitrectomy, systemic administration of micafungin plus voriconazole, topical application of fluconazol and amphotercin B.

Pioglitazone inhibits TGFß induced keratocyte transformation to myofibroblast and extracellular matrix production.

Phenotype transformation of corneal keratocyte to myofibroblast plays an important role in the wound healing process of cornea and TGFß is considered to be the most important mediator to induce myofibroblast trans-differentiation. Peroxisome proliferator-activated receptors-γ (PPAR-γ) activation has been proved to exert anti-fibrotic effect in many tissues. In this study, we investigated the effect of PPAR-γ agonist, pioglitazone, on myofibroblast transformation, extracellular matrix production and cell proliferation. The results showed pioglitazone inhibited the TGFß-driven myofibroblast differentiation, as determined by F-actin fluorescence staining, α-smooth muscle actin-specific immunocytochemistry and western blot analysis. Pioglitazone also potently attenuated TGFß induced type I collagen and fibronectin mRNA and protein production. Moreover, pioglitazone showed inhibitory effect on TGFß induced cell proliferation. The irreversible PPAR-γ antagonist GW9662, partially reversed the inhibition of collagen I and fibronectin expression but not myofibroblast transformation, suggesting both PPAR-γ dependent and PPAR-γ independent mechanisms were involved in the action of pioglitazone. Therefore, our study indicates pioglitazone has a potential application in therapy of corneal fibrosis and PPAR-γ might be a promising therapy target.

[Preliminary study on human bone marrow mesenchymal stem cells differentiation into epithelial-like cells].

OBJECTIVE: To investigate the differentiation potential of human bone marrow mesenchymal stem cells (hMSC) inducing into epithelial-like cells, even corneal epithelial-like cells, and to discuss the plasticity that make hMSC the seed cells used in corneal tissue engineering. METHODS: hMSC were isolated and purified by density gradient centrifugation combined with an attachment culture method and passaged in vitro. hMSC were identified by flow cytometry. The passaged hMSC were planted on fresh pig corneal Bowman's membrane. The expression of CK12, ABCG2 and CK19 in hMSC was identified by immunofluorescence staining. We used in vitro method to obtain a multilayer culture of hMSC. When hMSC formed a monolayer, the cells were inserted to Millicell culture and grew into multilayers by using the air-lifting cultivation methodology. Four weeks later, after fixed and dehydrated, the hMSC were observed under the light microscope after hemotoxylin and eosin (HE) and immunohistochemistry staining. RESULTS: hMSC could be cultured, expanded in vitro, and showed great potential of proliferation. The result of flow cytometry showed that the positive staining percentage was 0.06% for CD45, 0.41% for CD34, 86.43% for CD44, 85.72% for CD29 and 90.72% for CD105. This indicated that hMSC expressed CD44, CD29, CD105 but not CD45 and CD34. After four weeks induction, part of hMSC expressed CK12 and CK19 but not ABCG2. In the in vitro stratification, HE and immunohistochemical staining showed that there were one or two layers epithelial-like cells, even corneal epithelial-like cells after using the air-lifting cultivation. CONCLUSIONS: This study suggests that hMSC have the potential to differentiate into epithelial cells, even corneal epithelial cells. hMSC could be the option of cells used to reconstruct the corneal epithelium by tissue engineering technology.

[Histopathology and ultrastructure changes of retina in rats exposed to n-hexane].

OBJECTIVE: To investigate the injury in the retina of rats exposed to n-hexane. METHODS: 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. RESULTS: 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. CONCLUSION: The histopathology and ultrastructure of retina are damaged in the rats exposed to n-hexane, thus leading to ocular fundus disease.

[Evaluation of the quality of Gastrodia elata Bl. by HPLC-DAD/MS].

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.

[Morphological changes of corneal nerve in rats exposed to n-hexane.].

OBJECTIVE: To investigate the injury in the corneal nerve and cornea of rats exposed to n-hexane. METHODS: 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. RESULTS: 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. CONCLUSION: The structure of corneal nerve and cornea is damaged in the rats exposed to n-hexane, thus leading to dysfunction of cornea.

[Quality analysis and evaluation of Rhizoma Coptidis under different cultivation conditions].

AIM: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Variety of differential display on gene and corresponding gene's clone following corneal alkali burns in rats].

OBJECTIVE: To study the condition of differential gene caused after corneal alkali burns in rats and clarify the molecular biological foundation of corneal denatured protein. METHODS: The animals were sacrificed on the 3rd day and the 2nd week after alkali burns. Total RNA was isolated from the excised corneas and then reverse-transcribed into cDNA. The differential gene was detected by mRNA differential display reverse transcription polymerase chain reaction (DDRT-PCR) with two kinds of anchoring primer and 12 kinds of random primers after corneal alkali burns in the rats. The differential gene fragments were cloned, and their homogeneity was compared with each other in the Gene Bank. RESULTS: Compared with the normal cornea, the cornea of alkali burns on the 2nd week produced one differential gene fragment of 630 bp in the same reaction condition, and this differential gene was homologous to the rattus norvegicus mitochondrial cytochrome oxidase subunits I, II, III gene. CONCLUSIONS: It is found that there is differential gene in the cornea of alkali burns on the 2nd week in rats, and this differential gene is homologous to the rattus norvegicus mitochondrial cytochrome oxidase subunits I, II, III gene. It can be concluded that the occurrence of this differential gene is possible to be related with the action of the superoxide free radicals caused by alkali burns.

[Application and prospect on the researches of ecological cultivation of Coptis chinensis].

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.

[Ecological technique of cultivating Coptis chinensis in woods and co-cultivating it with maize].

OBJECTIVE: To establish ecologically new modes of cultivating Coptis chinensis in woods and co-cultivating it with maize. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

[Technique of storing Coptis chinensis seeds in damp sand under shed and fine cultivation of seedling].

OBJECTIVE: 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. METHODS: With the old technique of seedling in woods as control, we screened the new method for storing seeds and cultivating seedlings. RESULTS: 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. CONCLUSION: 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.

[Research development of Coptis chinensis].

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.

[The study of FK506 eye drops preventing and treating immune rejection on limbal allograft transplantation].

OBJECTIVE: To evaluate the general cause of limbal allograft transplantation rejection and the effects of the immunosuppressant FK506. METHODS: Limbal deficiency models were established in 48 New Zealand rabbits, which were randomized into three groups of 16 rabbits each. Limbal allograft transplantation was performed one month later. After transplantation, the FK506 group, the CsA group and the untreated group were treated with FK506 eye drops (0.5 g/L), 1% CsA eye drops and physiological saline, respectively. Graft survival and ocular surface were observed clinically for 10 weeks. Impression cytology was performed on the central cornea of the rabbit one month after limbal ablation, 1 day before allograft transplantation, and two and four weeks after transplantation. The expression of CD25 in peripheral T cells was identified dynamically one day before and one and eight weeks after transplantation. The expression of CD25 on the limbal allograft and lymphocyte infiltration was also identified dynamically the fourth, eighth and tenth week after transplantation. RESULTS: Conjunctivalization of corneal epithelium was found in the limbal deficiency model by impression cytology. Two weeks after limbal allograft transplantation, the central corneal epithelium regained corneal phenotype. Four weeks later, the ocular surface of FK506 group and CsA group continued to express corneal phenotype. Epithelium rejection appeared first in the untreated group (P < 0.05), which had the highest level of the lymphocyte infiltration and the expression of CD25 in peripheral blood and allografts after limbal allograft transplantation (P < 0.05). Epithelium rejection occurred next with the CsA group. FK506 group epithelium rejection occurred last and had the lowest level of the lymphocyte infiltration and the expression of CD25 in peripheral T cell and limbal allografts. CONCLUSIONS: Topically administered FK506 eye drops early after limbal allograft transplantation can delay allograft rejection by effectively inhibiting the expression of CD25 in peripheral T cells and limbal allografts. Also, FK506 is more effective than CsA.