Therapeutic effects and perspective of stem cell extracellular vesicles in aging and cancer.

Senescent cells can secrete a plethora of cytokines which induce senescent phenotype of neighboring cells and was called senescence-associated secretory phenotype. Previously, it was believed that cancer was caused by the infinite division and uncontrolled proliferation of cells. Based on this, anticancer treatments were all aimed at killing cancer cells. Cancer is now considered an age-related disease. Cancer cells are not exogenous, but one of the worst results of injuries which initially induce cell senescence. Therefore, reversing cell senescence can fundamentally prevent and treat cancer. Though current anticancer treatments induce the cancer cells apoptosis, they induce senescence of normal cells at the same time, thus promoting the occurrence and development of cancer and forming a vicious circle. Extracellular vesicles (EVs) are nano-sized vesicles which partially mirror their parent cells. In the tumor microenvironment, EVs of senescent cells can change the expression profile of cancer cells, contributing to their resistance to chemotherapy. There is growing evidence indicates that stem cell EVs exert effective antiaging and anticancer actions by transferring functional microRNAs and proteins. This review will summarize the therapeutic role of stem cell EVs in reversing aging and cancer, which suggests the broad clinical application perspective.

Modifying the tumour microenvironment and reverting tumour cells: New strategies for treating malignant tumours.

The tumour microenvironment (TME) plays a pivotal role in tumour fate determination. The TME acts together with the genetic material of tumour cells to determine their initiation, metastasis and drug resistance. Stromal cells in the TME promote the growth and metastasis of tumour cells by secreting soluble molecules or exosomes. The abnormal microenvironment reduces immune surveillance and tumour killing. The TME causes low anti-tumour drug penetration and reactivity and high drug resistance. Tumour angiogenesis and microenvironmental hypoxia limit the drug concentration within the TME and enhance the stemness of tumour cells. Therefore, modifying the TME to effectively attack tumour cells could represent a comprehensive and effective anti-tumour strategy. Normal cells, such as stem cells and immune cells, can penetrate and disrupt the abnormal TME. Reconstruction of the TME with healthy cells is an exciting new direction for tumour treatment. We will elaborate on the mechanism of the TME to support tumours and the current cell therapies for targeting tumours and the TME-such as immune cell therapies, haematopoietic stem cell (HSC) transplantation therapies, mesenchymal stem cell (MSC) transfer and embryonic stem cell-based microenvironment therapies-to provide novel ideas for producing breakthroughs in tumour therapy strategies.

Lipid layer thickness and tear meniscus height measurements for the differential diagnosis of evaporative dry eye subtypes.

AIM: To explore a new diagnostic index for differentiating the evaporative dry eye (EDE) subtypes by analysis of their respective clinical characteristics. METHODS: A cross-sectional study of 139 patients (139 eyes) with EDE who were enrolled and classified as obstructive meibomian gland dysfunction (MGD) (n=81) and non-obstructive MGD (n=58) EDE. All patients completed a Standard Patient Evaluation of Eye Dryness (SPEED) questionnaire and were evaluated for average lipid layer thickness (LLT), tear meniscus height measurements (TMH), tear break-up time (TBUT), ocular surface staining score, Schirmer I test (SIT), lid margin abnormalities, and meibomian gland function and morphology. RESULTS: Age, average LLT, TMH, scores of lid margin abnormalities, meibum quality, meibomian gland loss (MGL) (all P≤0.001), and TBUT (P=0.03) were all significantly different between obstructive MGD EDE patients and non-obstructive MGD EDE patients. Average LLT in obstructive MGD EDE was correlated with meibomian expressibility (r=-0.541, P≤0.001), lid margin abnormalities were marginally not significant (r=0.197, P=0.077), and TMH was correlated with MGL (total MGL: r=0.552, P≤0.001; upper MGL: r=0.438, P≤0.001; lower MGL: r=0.407, P≤0.001). Average LLT in non-obstructive MGD EDE, was correlated with meibomian expressibility and Oxford staining (r=-0.396, P=0.002; r=-0.461, P≤0.001). The efficiency of combining average LLT and TMH was optimal, with a sensitivity of 80.2% and a specificity of 74.1%. Obstructive MGD EDE patients had an average LLT≥69 nm and TMH≥0.25 mm, while non-obstructive MGD EDE patients had an average LLT<69 nm and TMH<0.25 mm. CONCLUSION: Obstructive MGD EDE and non-obstructive MGD EDE have significantly different clinical characteristics. Combining average LLT and TMH measurements enhanced their reliability for differentiating these two subtypes and provided guidance for offering more precise treatments for EDE subtypes.

Low concentration of sodium hyaluronate temporarily elevates the tear film lipid layer thickness in dry eye patients with lipid deficiency.

AIM: To investigate the effects of different concentrations of artificial tears on lipid layer thickness (LLT) and blink rate (BR) in dry eye patients. METHODS: This study included 106 eyes of 58 patients with dry eye. The lipid deficiency type was defined as the LLT baseline <75 nm. The LLT and BR were measured at baseline and 1, 5 and 15min after the instillation of 0.1% or 0.3% sodium hyaluronate (SH) eye drops by using the LipiView ocular surface interferometer. RESULTS: In the lipid deficiency group, the LLT increased from baseline at 1min post instillation. The LLT after the instillation of 0.1% SH was significantly higher than that after the instillation of 0.3% SH (P<0.001). The LLT returned to baseline at 15min post instillation of 0.1% SH and at 5min post instillation of 0.3% SH. In the non-lipid deficiency group, the LLT decreased from baseline at 1min and returned to baseline at 5min for both treatments. The BRs were not significantly different at different time points for both treatments. CONCLUSION: SH eye drops induce a short-term increase in LLT of patients with lipid deficiency. A low concentration of artificial tears have a stronger effect than a high concentration of artificial tears on the increase in LLT. In comparison, SH eye drops induce a transient and slight decrease in LLT of patients without lipid deficiency. A low concentration of artificial tears might be better for patients with lipid deficiency.

Novel 9, 10-dihydrophenanthrene derivatives from Eria bambusifolia with cytotoxicity aganist human cancer cells in vitro.

The present study was designed to identify bioactive compounds similar to those isolated from Dendrobium genus from its relative specie Eria bambusifolia. Compounds 1-10 were isolated and purified using silica gel, MCI CHP-20 gel, Sephadex LH-20, and Lichroprep RP-18 chromatography methods. Their structures were elucidated by means of extensive spectroscopic analyses. The cytotoxicity of these compounds against five human cancer cell lines was tested. Erathrins A and B (1 and 2) were new compounds, and compound 1 represented a novel carbon framework having a phenanthrene-phenylpropane unit with a dioxane moiety. Moreover, compound 1 showed selective cytotoxic activity against HL-60 cells (IC50 = 14.50 µmol·L(-1)). These results provided a basis for future development of these agents as anticancer lead compounds.

Importance of the stem cell microenvironment for ophthalmological cell-based therapy.

Cell therapy is a promising treatment for diseases that are caused by cell degeneration or death. The cells for clinical transplantation are usually obtained by culturing healthy allogeneic or exogenous tissue in vitro. However, for diseases of the eye, obtaining the adequate number of cells for clinical transplantation is difficult due to the small size of tissue donors and the frequent needs of long-term amplification of cells in vitro, which results in low cell viability after transplantation. In addition, the transplanted cells often develop fibrosis or degrade and have very low survival. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPS) are also promising candidates for cell therapy. Unfortunately, the differentiation of ESCs can bring immune rejection, tumorigenicity and undesired differentiated cells, limiting its clinical application. Although iPS cells can avoid the risk of immune rejection caused by ES cell differentiation post-transplantation, the low conversion rate, the risk of tumor formation and the potentially unpredictable biological changes that could occur through genetic manipulation hinder its clinical application. Thus, the desired clinical effect of cell therapy is impaired by these factors. Recent research findings recognize that the reason for low survival of the implanted cells not only depends on the seeded cells, but also on the cell microenvironment, which determines the cell survival, proliferation and even reverse differentiation. When used for cell therapy, the transplanted cells need a specific three-dimensional structure to anchor and specific extra cellular matrix components in addition to relevant cytokine signaling to transfer the required information to support their growth. These structures present in the matrix in which the stem cells reside are known as the stem cell microenvironment. The microenvironment interaction with the stem cells provides the necessary homeostasis for cell maintenance and growth. A large number of studies suggest that to explore how to reconstruct the stem cell microenvironment and strengthen its combination with the transplanted cells are key steps to successful cell therapy. In this review, we will describe the interactions of the stem cell microenvironment with the stem cells, discuss the importance of the stem cell microenvironment for cell-based therapy in ocular diseases, and introduce the progress of stem cell-based therapy for ocular diseases.

[Study on the treatment of dry eye with Loteprednol Etabonate].

OBJECTIVE: To observe the efficacy and safety of 0.5% Loteprednol Etabonate ophthalmic suspension in the treatment of moderate dry eye. METHODS: Totally 34 dry eye patients (68 eyes) in grade 2 or grade 3 (DEWS standard) enrolled in our hospital from March 2009 to September 2010 were randomly divided into two groups: the experimental group (Loteprednol Etabonate Group) and the control group (Cyclosporine A, CsA group). 0.5% Loteprednol Etabonate ophthalmic suspension or 1% CsA eye drops was applied 2 times a day respectively together with 0.2% Liposic eye drops (4 - 6 times/day). Questionnaire was used in these patients before the treatment and repeated every 2 weeks during the treatment till 8 weeks. Slit lamp microscope examination, fluorescent staining, tear break-up time (BUT), Schirmer I test (SIt) and intraocular pressure measurement were carried out at the same time point. The conjunctival impression cytology (IC) was performed before the treatment and 8 weeks after the treatment. The mean of the results were compared by t-tests and χ(2) test. RESULTS: After 2 weeks of the treatment, the mean score of the questionnaire was significantly lower than that before the treatment in each group (t = 5.36, 3.63, P < 0.01). After 4 weeks of the treatment, the inflammation of the ocular surface was relieved obviously in both group and the mean score of the corneal fluorescein staining (FL) was lower than that before the treatment in each group. The average density of the goblet cells before the treatment was (181.2 ± 16.1)/mm(2) and (179.4 ± 17.5)/mm(2) in each group respectively. After 8 weeks of the treatment, this increased to (348.6 ± 22.5)/mm(2) and (360.4 ± 27.8)/mm(2) significantly (t = 16.9, 16.3, P < 0.05). BUT was significantly prolonged in each group after the treatment (P < 0.01). There was no significant change in ST I or NCT in each group (P > 0.05). CONCLUSIONS: Topical 0.5% Loteprednol Etabonate ophthalmic suspension is safe and effective for the treatment of moderate dry eye.

[The effects of transplantation of compound keratoprosthesis in clinical practice and managements of complications after operation].

OBJECTIVE: To explore the clinical value and management of complications of the transplantation of Titanium skirt compounded keratoprosthesis for severe corneal blindness eyes. METHODS: It was a retrospective case series study. Nine eyes from 9 male patients, aged 28 to 52 years old, accepted permanent keratoprosthesis transplantation in Zhongshan Ophthalmic Center from March 2002 to June 2005. All patients had corneal lesion in both eyes for 1.5 to 5.0 years. Among the 9 treated eyes, 6 eyes was severe vascularization after alkali burns, 3 eyes explosive injuries. Light perception was remained in all patients before surgery, however, 2 eyes only had a questionable orientation of light perception among them. Surgical management was divided into two stages. In the first stage, transplantation of Titanium skirt compound keratoprosthesis was performed, and the explant was reinforced by the self auricular cartilage and Tendons capsule. The second stage of surgery was performed in 5 to 6 months later, in which the membrane in the front of keratoprosthesis was cut. After the surgery, visual acuity, visual field, intraocular pressure and retina were examined. The complications were noticed and managed. RESULTS: All treated eyes were followed up for 1 to 3 years. After the treatment, 7 eyes divorced from blindness with uncorrected visual acuity 20/200 (0.1), and 2 eyes among them got corrected visual acuity 20/30 (0.6). Two eyes with the questionable orientation of light perception before treatment gained uncorrected visual acuity 4/200 (0.02) and 8/200 (0.04) after treatment respectively. Complications were found to include 5 recurrent frontal membrane of keratoprosthesis, one back membrane of keratoprosthesis, and one limited corneal melting. Complications were controlled by the corresponding treatments, such as membrane resection for the recurrent frontal membrane of keratoprosthesis, courage under microscope for back membrane of keratoprosthesis, and reinforcement of acellular dermis for corneal melting. All keratoprosthesis were maintained in situ, and no rejection and leakage of aqueous humor happened. CONCLUSIONS: It is effective to use transplantation of keratoprosthesis for the severe corneal blindness eyes. Combination with self auricular cartilage and Tendons capsular reinforcement may reduce the complications and improve the biocompatibility of keratoprosthesis.

[Keratoplasty in the management of Terrien's marginal degeneration].

OBJECTIVE: To evaluate the efficacy and safety of keratoplasty combined with corneal foci resection in the treatment of Terrien's marginal degeneration (TMD). METHODS: In this nonrandomized retrospective case series, the records of 48 eyes from 40 patients with TMD who received keratoplasty from January 1995 to December 2005 in Zhongshan Ophthalmic Center were reviewed retrospectively. Orbscan topography examination was undertaken in 8 eyes of 8 patients and the refractive error of 9 eyes from 9 patients was tested before and after the operation. RESULTS: The mean age of the patients was (30 +/- 6) years old. The mean follow up period was (7 +/- 6) years. It took (3 +/- 1) months postoperatively to obtain a stable visual acuity. Before operation, the naked eye and best corrected visual acuity (VA) (Q25,Q75) were (0.05,0.4), and (0.1,0.5), respectively, while improved to (0.2,0.6) and (0.4,0.7) after operation, respectively (Z = 4.63, 3.85, both P<0.01). VA was improved in 39 eyes (81.3%), remained at the same level in 4 eyes (8.3%), decreased 1-2 lines in 3 eyes (6.3%), and decreased more than 2 lines in 2 eyes (4.1%) after the operation. The median spherical diopter and cylinder diopter were (-2.00 D, -8.50 D) and (2.50 D,12.00 D) before operation, while decreased to ( -1.25 D, -4.75 D) and (0.75 D, 4.25 D) after operation (Z= 2. 49, 2.54, P = 0.01, 0.01). The improvement in Sim K's astigmatism, astigmatism in 3 mm zone and mean power in 3 mm and 5 mm zone were reduced statistically significant after the operation (P <0.05); with the exception of astigmatism in the 5 mm zone, which was not reduced significantly after the operation (Z = 1.86, P = 0.06) . The operative complications included corneal perforation during operation in 5 eyes (10.4%), hydrops between graft and recipient interface in 8 eyes (16.7%), epithelial in-growth in 4 eyes (8.3%), choroidal detachment in 1 eye (2.1%), graft rejection in 7 eyes (14.6%), and recurrence in 3 eyes (6.3%). Secondary surgery was required in 5 eyes (10.4%) for interface hydrops, epithelial in-growth and recurrence of TMD. CONCLUSIONS: Keratoplasty combined with foci resection is effective and safe in the treatment of TMD. This procedure can preserve and improve the visual activity.

Sutureless fixation of amniotic membrane patch as a therapeutic contact lens by using a polymethyl methacrylate ring and fibrin sealant in a rabbit model.

PURPOSE: To introduce and evaluate a sutureless technique by using a polymethyl methacrylate (PMMA) ring and fibrin sealant to fix an amniotic membrane (AM) patch on the ocular surface as a therapeutic contact lens in a rabbit model. METHODS: PMMA rings were fabricated by duplicating an impression of a rabbit conjunctival fornix. The central cornea of the left eye in 16 rabbits was deepithelialized (diameter = 10 mm). A human AM patch was fixed to the ocular surface by using either a PMMA ring and fibrin sealant or interrupted 10-0 nylon sutures. The fibrin sealant was used to create the PMMA ring-AM complex but not to attach the AM/PMMA ring to the ocular surface. The rabbits were followed up with slit-lamp examination and fluorescein staining for 7 days. Reepithelialization and complications were recorded. RESULTS: The corneal epithelial defect was recovered in each rabbit of both groups after 5 days. In the sutureless group, all membranes remained in place and intact during the follow-up period. One eye was noted to have a partial conjunctival epithelial defect caused by exposure to the PMMA ring. In contrast, >50% of rabbits in the interrupted suture group exhibited complications including conjunctival edema, suture loosening, patch detachment, bleeding, and conjunctival epithelial defects. CONCLUSIONS: The sutureless technique that uses a PMMA ring and fibrin sealant for AM patch placement has a lower incidence of complications than the interrupted suture method. This sutureless technique may promote increased clinical use of AM patch by alleviating patients' pain and shortening surgical time.

[Implantation of modified polyhydroxyethyl methacrylate-polymethyl methacrylate keratoprostheses in rabbit and monkey corneas].

OBJECTIVE: To investigate the biocolonization of polyhydroxyethyl methacrylate (PHEMA) sponge with cornea tissue and evaluate the therapeutic effects of modified porous PHEMA-PMMA (polymethyl methacrylate) Keratoprostheses (KPro) on rabbit and monkey corneas. METHODS: The KPro were made using two-stage polymerization combined with mechanical cutting. The experiment was divided into two groups. In the control group (A group), ten normal rabbit eyes received lamellar implantation of PHEMA sponges. The sponges were obtained 2 weeks, 1, 2, 3 and 4 months after operation. The cell proliferation and neovascularization inside the sponges were observed using light and transmission electron microscopy (TEM) and immunohistochemistry. In the experimental group (B group), the porous PHEMA-PMMA KPros were inserted into the lamellar pockets of ten rabbit corneas and two monkey corneas (stage I operation). The healing process was investigated by slit-lamp microscopy. The anterior lamellar cornea tissues were removed 3 months after surgery, exposing the underneath transparent core (stage II operation). The operated eyes were then followed up for 3 - 6 months. RESULTS: No complication was observed in A group. Under the light microscope, fibroblasts started to grow into the cornea 2 weeks after operation; lots of cells, accompanied with new blood vessels, invaded into the cornea 2 - 3 months after surgery. Invading cells of sponge, as well as keratocyte, were positive for vimentin. Under the electron microscope, the invading cells looked healthy and were surrounded by extracellular matrix and collagen. In B group, eight rabbit eyes which have received KPro implantation, anterior lamellar cornea melting happened in two eyes after the stage I operation. The remaining six corneas retained their central cores during observation after the stage II operation. Two monkey operated eyes were found no complication throughout the whole follow-up. CONCLUSIONS: The PHEMA sponge can obtain a tight fusion with the host cornea. The modified PHEMA-PMMA KPros have obtained a relatively stable therapeutic results after implantation into animal corneas.

[Preliminary clinical study of sutureless, small-incision deep lamellar endothelial keratoplasty surgery].

OBJECTIVE: To investigate the surgical procedure, clinical efficacy, and the prevention and management of complications of sutureless, small-incision deep lamellar endothelial keratoplasty (DLEK). METHODS: Nine patients (nine eyes) with bullous keratopathy underwent sutureless, small-incision DLEK surgery, six of them was combined with anterior vitrectomy. Visual acuity, graft clearance, corneal curvature, astigmatism and endothelial cell density (ECD) were observed over a 3 - 5 month follow-up period. RESULTS: All grafts remained transparent, and six eyes had improved visual acuity. After the surgery, mean corneal curvature was (43.96 +/- 3.38) D. Mean corneal astigmatism was (3.32 +/- 1.20) diopter (D). Mean ECD was (2124 +/- 278) cells/mm(2). No severe complications occurred. CONCLUSION: Sutureless, small-incision DLEK, as compared with penetrating keratoplasty (PKP) and microkeratome-associated deep lamellar endothelial keratoplasty, has more advantages and is expected to be the initial surgical treatment for bullous keratopathy.

[The transfection and expression of IL-1ra gene to the rabbit cornea in situ via cation polymer mediation].

OBJECTIVE: To investigate the efficiency and safety of transfection of PEGFP-IL-1ra plasmid via cation polymer mediation (poly-ethylenimine, PEI) by injection into the corneal stroma. METHODS: Human IL-1ra cDNA fragments were cloned by RT-PCR. Plasmid PEGFP-hIL-1ra recombinants were constructed and transferred into corneal endothelial cells (CEC) via cation polymer mediation. Expression of IL-1ra mRNA and IL-1ra was detected by green fluorescent protein (GFP) and Western-blotting. In the experiment group, 20 microl preparation containing 10 microg plasmid PEGFP-hIL-1ra recombinants and PEI-in-vivo was injected into the corneal stroma of Wistar rats (n = 30). Equivalent PEI-in-vivo solution was injected into another 15 corneas as the controls. Corneas were harvested at different time points (day 1, 3, 6, 14 and 21) after injection. The changes of tissue structure and function after IL-1ra in situ transfection were studied by HE staining, transmission electron microscopy, trypan blue-alizarin red staining and immunohistochemistry. The location and intensity of IL-1ra-GFP fusion protein expression were monitored by fluorescence microscopy. RESULTS: The size of the RT-PCR product of hIL-1ra fragments was approximately 500 bp in agarose gel electrophoresis. Restrictive enzyme digestion analysis of PstI, BamHI and DNA sequence analysis showed that expression of plasmid PEGFP-hIL-1ra recombinants had been constructed successfully. Twelve hours after the transfection of PEGFP-hIL-1ra, GFP fluorescence was detected in 10% - 15% endothelial cells. IL-1ra protein (RMW: 44,000) was detected by Western-blotting. In PEGFP-hIL-1ra treated group, fluorescence was appeared at day 1 in cornea basal epithelial cells, peaked at day 6 in whole cornea, began to weaken at day 14, and only weak fluorescence remained in cornea epithelial cells at day 21. No fluorescence appeared in the control group. No significant pathologic changes could be found in HE stained cornea tissues in both transfected group and the controls. p63 immunocytochemical staining in cornea epithelium was positive in both groups. Trypan blue-alizarin red staining confirmed that there was no damage in cornea endothelial cells. IL-1ra-GFP granules could be found by transmission electron microscope in every layer of cornea in the transfected group, but none in the controls. There was no impairment in the ultrastructure of cells in both groups. CONCLUSIONS: By direct injection of PEGFP-hIL-1ra into corneal stroma and mediated by cation polymer, IL-1ra genes could be transferred and expressed in corneal tissue efficiently and safely, and might provide a novel technique of gene transfection to cornea in situ.

Neural differentiation of embryonic stem cells induced by conditioned medium from neural stem cell.

Embryonic stem cells can proliferate indefinitely and are capable of differentiating into derivatives of all three embryonic germ layers in vitro, including the neural lineage. The main objective of this study is to test the effects of neural stem cell conditioned medium on the neural differentiation of mouse embryonic stem cells. When cultured in neural stem cell conditioned medium, mouse embryonic stem cells can form floating cell spheres composed of many nestin-positive cells. After trypsinization and growth on gelatin, these embryonic stem cell-derived neural progenitor cells can be expanded for more than 3 months without loss of neural progenitor characteristics. Both neuronal and glial cells can be readily generated from these cells under differentiation conditions. Thus, neural stem cell conditioned medium is a highly potent reagent for inducing the development of mouse embryonic stem cells into the neural lineage, especially neural progenitor cells.

[Induction of epidermal stem cells of rhesus monkey into human conjunctival epithelial cells: an in vitro experiment].

OBJECTIVE: To observe the possibility of inducing the epidermal stem cells of rhesus monkey into human conjunctival epithelial cells and to investigate the plasticity of epidermal stem cells. METHODS: Epidermal stem cells of rhesus monkey were cultured in vitro and separated with type IV collagen. The cells were analyzed before and after isolation with beta1 integrin and keratin 15 by flow cytometry. The separated stem cells were characterized with markers, beta1 integrin and keratin 15 by immunohistochemistry and RT-PCR. The stem cells were transfected by green fluorescent protein (GFP) gene. 24 hours later, the fluorescent cells were selected out and co-cultured with human conjunctival epithelial cells in Transwell for 10 days. Then the cells were characterized with the markers of beta1 integrin, keratin 15, mucin 4 and keratin 4 by immunohistochemistry and RT-PCR. RESULTS: The purity of the separated epidermal stem cells was almost 90%, and all the cells were positive of beta1 integrin and keratin 15. After being transfected with GFP gene for 24 hours, the cells expressed green fluorescence. 10 days later, after co-culture with human conjunctival epithelial cells, the epidermal stem cells were positive of mucin 4 and keratin 4. CONCLUSION: Epidermal stem cells of rhesus monkey can be induced into conjunctival epithelial cells in vitro by co-culturing with primary passage conjunctival epithelial cells.

[The effect of protein kinase C alpha on expression of developmental genes during differentiation of mouse embryonic stem cells into neuron-like cells in vitro].

OBJECTIVE: To study the effect of protein kinase C alpha on the expression of developmental genes pax-6, slit-2 and netrin-1 during differentiation of mouse embryonic stem cells into neuron-like cells in vitro, in an attempt to elucidate their roles in signaling. METHODS: ES-BALB/c cells were induced to form embryoid bodies in the ES conditioned medium for 4 days, and were plated separately on coated glass coverslip into 6-well culture dishes for immunohistochemical study and into 100 mm dishes for RT-PCR assay. These cultures were collected after 1, 3, 5, 7 and 14 days in the presence of 5 x 10(-7) mol/L retinoic acid (RA). The neuron-like cells were stained with antibody to NSE and NF-200. mRNA level of the development related genes (pax-6, slit-2 and netrin-1) in undifferentiated and differentiated ES cells was assessed by RT-PCR assay. Effects of PMA and D-sphingosine on the developmental genes were also observed. RESULTS: Most of the neuron-like cells stained with the antibodies to NSE and NF-200. RT-PCR assay showed that levels of PKC alpha, pax-6 and netrin-1, but not slit-2 transcripts decreased dramatically upon induction on the 1st day, but then raised slowly and resumed to normal level on 14th day. PMA upregulated the levels of PKC alpha, pax-6 and netrin-1; while D-sphingosine downregulated their levels. CONCLUSIONS: The results of the present experiments demonstrate that the developmental genes pax-6 and netrin-1 play a very important role during differentiation of mouse ES cells into neuron-like cells through PKC alpha signaling pathway.

[A pilot study on transdifferentiation of skin stem cell in reconstructing corneal epithelium].

OBJECTIVE: To observe the differentiation and development of skin stem cells on corneal stroma and to discuss the possibility of reconstructing corneal epithelium with skin stem cells. METHODS: Pieces of human and rabbit skin were obtained during operation. Rabbit eye balls were taken, and pieces of corneal stroma without epithelium were prepared. Skin stem cells from the rabbit skin and human skin were cultured. The human skin stem cells of the first generation to 4th generation were implanted on the rabbit corneal stroma and cultured. Three rabbits underwent autotransplantation of the rabbit skin stem cells of the first generation to 4th generation on the pieces of corneal stroma with the superficial lamina removed and then fed for 100 approximately 114 days. Another 3 rabbits underwent allotransplantation of the rabbit skin stem cells of first to 4th generation on the pieces of corneal stroma with the superficial lamina removed and then fed for 100 days. Then the rabbits were killed and their eye balls taken out. The rabbit corneas implanted with human or rabbit epithelial cells and the rabbit corneas with the autogeneous or heterogeneous epithelial cells were sliced and underwent immunohistochemistry with human AE5 antibody corresponding to the specific surface marker keratin K3/K12 common to humankind and rabbit, and human epithelial cell keratin K-19 monoclonal antibody. RESULTS: Since the 3(rd) day of transplantation the transplanted human epithelial cells formed multiplayer and were human AE5 antibody and human K19 monoclonal antibody positive. The autotransplanted corneas remained basically transparent without obvious vascular hyperplasia till the cornea specimens were taken. Histological examination showed intact multiplayer epithelium and immunohistochemistry showed human AE5 positive. The allotransplanted\rabbit corneas showed congestion since the 9(th) day. Histological examination showed that the corneas were nor so transparent as the autotransplanted ones and the epithelium was nor intact with a lot of lymphocyte infiltration. CONCLUSION: Corneal epithelium can be reconstructed from skin stem cell, which may be an alternative for constructing autogeneous bioengineered corneas.

[Long-term results of amniotic membrane transplantation for conjunctival surface reconstruction].

OBJECTIVE: To investigate the long-term results of amniotic membrane transplantation (AMT) for conjunctival surface reconstruction after symblepharon resection and analysis the related factors. METHODS: Fifty-one cases (55 eyes) with symblepharon due to eye burns (chemical or heat) or Stevens-Johnson syndrome were selected for symblepharon resection and AMT. Ten eyes were performed within 1 year and twenty-three eyes in 1.0 to 8.0 years after chemical eye burns. RESULTS: Observation time varied from 26.0 to 30.0 months [mean value (27.4 +/- 2.6) months]. No necrosis and ulceration were found in all amnion grafts at the early stage after transplantation. Deep conjunctival fornix and free of eye movement were obtained in 56.4% (31/55) of those eyes received surgery. Slight symblepharon recurrence and limited eye movement restrict were revealed in 16.4% of them (9/55), Fifteen eyes of them (27.3%) shown moderate recurrence of symblepharon. The effects of AMT for those patients with different degree of symblepharon had significant difference statistically (Pearson Chi-Square, P = 0.000). The same results were observed between those patients who were performed in different time after chemical eye burns (Likelihood Ratio, P = 0.039) and with different severity of dry eye (Pearson Chi-Square, P = 0.000). CONCLUSIONS: Amniotic membrane can be used to reconstruct ocular surface effectively, but multiple surgeries may be needed. These microenvironments such as symblepharon severity, dry eye, and whether remained partial healthy conjunctiva in the affected eyes before surgery will influence the long-term results of AMT for conjunctival surface reconstruction.