Objective To isolate and identify the photoreceptor precursor cells isolated from newborn mouse retina. Methods Fourteen newborn littermate mice were randomly divided into seven groups (P1-P7,2 each). The retinal cells were isolated from the newborn mice on postnatal 1-7d respectively,and then cultured in vitro. The cellular growth state was observed under inverted phase contrast microscope. After in vitro expansion,5-bromo-2-deoxy-uridine (BrdU),neuroepithelial stem protein (Nestin),neural retina leucine zipper (Nrl) and Opsin in cultured cells were detected by immunohistochemistry and immunofluorescence techniques. Results Most of retinal cells isolated from the seven groups (P1-P7) of newborn mice proliferated and adhered to the plate in the particular medium. Most of the growing cells were BrdU-positive cells. A part of the cells could express neural stem cell specific antigen-Nestin,the positive expression rate of Nestin in the seven groups (P1-P7) were 31.0%,31.6%,32.3%,30.2%,31.2%,30.9% and 29.5%,respectively,with no significant difference. Some growing cells expressed Nrl,and the expression of Nrl was increased significantly on the third day after birth,and then increased in small range,the positive expression rate of Nrl in the seven groups (P1-P7) were 20.6%,35.2%,65.5%,68.6%,71.6%,73.0% and 73.3%,respectively. The positive expression of Opsin was found only in a few cells of P5-P7,while the cells in groups P1-P4 did not express Opsin. Conclusions The photoreceptor precursor cells are presented in neonatal mouse retina. They have the ability of proliferation and may differentiate into retinal photoreceptor cells,having a potentiality of expressing Opsin. The photoreceptor precursor cells are increased significantly on the third day after birth,and then differentiate into mature retinal photoreceptor cells gradually.

Objective:To develop an economical,simple and portable device for imitation of ship shock motion produced by non-contacting under water explosion.Methods:The device was developed based on aerodynamical principles and sensor detection technology,and was tested with animal experiments to verify its performance.The injuries caused by 4 grades of shock motions with different accelerations,duration and displacement were observed in 40 black rabbits(10 for each grade).Results:The device was safe and functionally stable,with a peak acceleration of 1 000 m/s~2,a shock duration of about 2 ms and a device displacement of 100 mm.The accelerations of 4 shock motion grades were significantly different and with good reproducibility.The damage to the rabbits were mainly haemorrhage of different extents in organs of pleuroperitoneal cavity,especially in the lungs,but no rupture of organs was found.The degree and involvement of damage had an increasing tendency with the increase of acceleration of shock motion.Conclusion:The device we developed can imitate the effects of ship shock motion.

The purpose of this study was to investigate the function of stereo visual acuity in patients after phacoemulsifications and intraocular lens implantation. Mensuration of stereopsis with laser holographic stereovision test chart was done in 80 cases who had undergone phacoemulsifications and intraocular lens implantation. In this group, 43 patients were male, 37 were female .Ages of the patients were 52 to 81 years. Single eye operation was done in 18 cases, while both eye operation in 62 cases. The corrected vision was all over 0 4 in both eyes.In the 80 cases 67 (83 7%) had near stereopsis and 13 cases (16 3%)did not have; 71 cases (88 8%) had distance stereopsis and 9 cases (11 2%)did not have. Function of stereopsis weakens gradually following aging and is closely related with vision. The function of sterovision would recover to varying degrees after phacoemulsifications and intraocular lens implantation. Recovery of distance stereopsis was better than that of near stereopsis.The results of this study show that the status of stereopsis is an important index to evaluate the curative effect in patients after phacoemulsifications and intraocular lens implantation.

Objiective To observe the morphological changes of rabbits' retina at different time points at which seawater was injected into the vitreous of rabbits. Methods Twenty-five New-Zealand white rabbits were randomly assigned to five groups, as 30 minutes, 2 hours, 6 hours, 12 hours and 24 hours group following the seawater injection in the vitreous. The right eyeball served as experimental eyeball and the left one as control in each group. 0.5ml artificial seawater was injected into the intermedius of rabbits right vitreous. 0.5ml balanced salt sdution was injected into the left vitreous in teh same way. At different time points following the eyeball injury, the morphological changes of retina were observed with light microscope and transmission electron microscope. Results The retina of control eyeballs had clear layers and regular cell shape in retinal structures with conventional staining. For the experimental eyeballs, the ganglion cell layer began to swelling after 30 minutes of seawater injection. The nerve fiber swelled obviously and the mitochondria vesiculated and swelled in the inner segment at 2 hours of seawater injection. The cell population of the inner nuclear layer began to reduce in 6 hours. At 12 hours of seawater injection, the retinal damages included disorganized cell nuclei, decreased cell population in ganglion cells and inner nuclear layer. Mitochondria vesiculated in photoreceptor cells, nuclear membrane shrank and sank inward. The vesiculation on inner and outer segments increased obviously. Conclusions Seawater may cause the morphological changes of rabbits' retina at early stage, and the histological damages become aggravation following the time prolongation. The damages would be unrecoverable 6 to 12 hours after seawater injury.

Objective To investigate the pathological features of rabbit' retina at the early stage of sunlight-induced injury.Methods Twenty New Zealand rabbits without any eye diseases were randomly allocated to 4 groups(5 each): animals in group A were fed under normal illumination environment,the animals in group B were fed under normal illumination environment and exposed to constant simulated sunlight,the animals in group C were fed under low illumination environment,and the animals in group D were fed under low illumination environment and exposed to constant simulated sunlight.The pathological changes in rabbits' retina were observed with light and transmission electron microscope.Results The structure of retina in group A and group C were histologically well-defined,uniform in staining,and with regular cell shape.In the animals in group B,vesiculation occurred in the ganglion cell layer,inner nuclear layer and outer nuclear layer,and some of the mitochondria showed swelling.The intramembranous structure of external segments became disorganized.In the animals in group D,vesiculation and swelling occurred in the ganglion cell layer and inner nuclear layer,with disruption of the cell membrane.Vesiculation and swelling were also found in the outer nuclear layer,with reduction in cell quantity.The structure was disorganized,with disorganized cell membrane,obvious swelling of mitochondria,disorganized intramembranous structure of external segments,and loss and vesiculation of the laminal structure.Conclusion The rabbit' retina will be injured when it is exposed to 30 000 lux constant simulated sunlight for 30 minutes.Rabbits fed in a low illumination environment and exposed to constant simulated sunlight may suffer severer injury than those fed in a normal environment and exposed to constant simulated sunlight.

Objective To evaluate the differences of the thinnest-point corneal thickness (TCT) decrease after three different corneal crosslinking (CXL) protocols for progressive keratoconus.Methyds Retrospective clinical case study.From August 2010 to November 2015,consecutive 85 patients (110 eyes) with progressive keratoconus were enrolled and treated with CXL in Department of Opthalmology,Navy General Hospital.21 patients of 25 eyes underwent standard epithelium-off corneal crosslinking (S-CXL),14 patients of 22 eyes underwent 1 g · L-1 riboflavin-sodium lactate Ringer's solution iontophoresis-assisted CXL (I-CXLa),and 50 patients of 63 eyes underwent 0.1％ riboflavin-distilled water solution I-CXLb.Preoperative and postoperative TCT were measured by ALLEGRO oculyzer.The differences of TCT decrease after treatment were compared among the three CXL protocols.Results The differences of TCT from baseline after 3 months,6 months and 12 months in the S-CXL group were (-14.93 ±27.16) μm,(-31.94 ±22.89) μm,(-27.71 ±26.01) μm,respectively,the I-CXLa group were (-20.14 ± 19.09) μm,(-10.10 ± 24.28) μm,(-7.11 ± 22.26)μm,respectively,the I-CXLb group were (-28.08 ± 26.14) μm,(-21.08 ± 25.62) μm,(-15.91 ± 19.19)μm,respectively.Three months after treatment,the differences of TCT decrease in the three groups was not statistically significant (P =0.188);Six and 12 months after treatment,the differences between S-CXL and I-CXLa were statistically significant (all P ＜0.05),but the differences between S-CXL and I-CXLb,between I-CXLb and I-CXLa showed no significant difference (all P ＞ 0.05).Conclusion Six and 12 months after treatment,TCT decrease is related to the CXL protocol.TCT decrease degree may reflect the intensity of crossinking.TCT decrease in I-CXLb is smaller than that in S-CXL,but there is no statistical difference.

Objectives:To establish animal model of anterior proliferative vitreoretinopathy (aPVR) with cultured homologous dermal fibroblasts of rabbit, and to provide evidence why hypotony was caused by aPVR. Methods:Animal models of aPVR were established with cultured homologous dermal fibroblasts on pigmented rabbits. Rabbits were sacrificed on the 14th, 28th and 56th day after the operation to prepare naked eyes and to receive histological examinations. Results:Naked eye examination showed that the peripheral retina was detached by dragging in the experimental group 28 and 56 days postoperatively. Microscopic examination showed atrophy or absence of the non-pigmented ciliary epithelium on the 28th and 56th postoperative day in the experimental group. Conclusions:The epiciliary membrane in aPVR dragged the ciliary body, made atrophy of non-pigmented epithelium, which perhaps was the main cause of hypotony.

Objective To investigate the effect of all-trans retinoic acid (ATRA) on expression of gap junction genes 26, 32 and 43 in two human hepatocellular carcinoma cell lines. Method Human hepatocellular carcinoma cell lines SMMC-7721 and BEL-7404 were cultured and inoculated in culture plate. Cells were divided into 2 groups and cultured respectively with normal medium and medium contains ATRA at a concentration of 10-mol/L. After 24, 48 and 72 hours, cells were collected and total mRNA were extracted. RT-PCR was used to detect the transcription of gap junction genes 26, 32 and 43. Result SMMC-7721 and BEL-7404 cultured under condition of normal medium do not express CX26 mRNA and CX32 mRNA. With the effect of ATRA for 48 and 72 hours, SMMC-7721 cell expressed CX26 mRNA and CX32 mRNA. BEL-7404 expressed CX26 mRNA and CX32 mRNA while cultured with ATRA for 72 hours. CX43 mRNA expression of the two cell lines was not altered by ATRA. Conclusion ATRA affects the expression of CX26 and CX32 in HCC cell lines SMMC-7721 and BEL-7404 at the transcription level.

Background The incidence of dry eye is gradually increased,and researches showed that inflammation participated in the pathogenesis and development of dry eye.The current therapy for dry eye can only relieve symptom but not achieve final cure.Stem cell therapy has been used in the treatment of limbal stem cell deficiency.However,whether it is feasible for the stem cell treating dry eye is still unclear.Objective This study attempted to investigate a new approach to treat dry eye syndrom by using human umbilical cord mesenchymal stem cells (HUCMSCs).Methods The use and care of experimental animals complied with the Tsinghua University School of Medicine Laboratory Animal Care Details.HUCMSCs were cultured and cell suspension was prepared with the cell density of 5×105/ml.Twenty 24-week-old male NOD/Ltj mice were randomized into 4 groups.0.1 ml PBSHUCMSCs suspension was injected via tail vein or lacrimal respectively in the caudal vein injection group and lacrimal injection group,and 10 μ1 PBS-HUCMSCs suspension was topically administed in the eye drops group.The NOD/Ltj mice without any treatment served as the model group.Five male ICR mice were used as the normal control group.Tear secretion was quantitatively detected with phenol red cotton thread in 1,2,3 weeks after injection,and corneal epithelial defect was scored by fluorescein staining.The serum levels of interleukin (IL)-6,IL-17a,tumor necrosis factor-α (TNF-αt) and interferon-γ (IFN-γ) were assayed by ELISA.The relative expression levels of p65,Stat3,Stat5 and extracellular signal-regulated kinases (Erk)-1 in lacrimal gland were detected by Western blot.Results The tear secretion amount was significantly different among the model group,caudal vein injection group,lacrimal injection group and eye drops group in various time points (1 week:F =3.700,P =0.040;2 weeks:F =5.150,P =0.008;3 weeks:F=10.130,P＜0.001).The tear secretion amount was increased in the caudal vein injection group and lacrimal injection group compared with the model group in different time points (all at P＜0.05),and no significant difference was seen in tear secretion amount between eye drops group and model group among various time points (all at P＞0.05).The fluorescein staining score was 3.00±0.63,9.40±1.62,5.20±1.17,4.20±1.17 and 7.20±0.98 in the ICR mouse control group,model group,caudal vein injection group,lacrimal injection group and eye drops group 1 week after injection respectively,and the scores were significantly lower in the caudal vein injection group,lacrimal injection group and eye drops group than those in the model group (P =0.001,0.000,0.033).The serum levels of IL-6,IL-17a and TNF-α in the caudal vein injection group were evidently lower than those in the model group (t =4.70,3.46,11.0,all at P＜0.01),but no significant difference was displayed in the serum IFN-γ level among the five groups (F=1.740,P=0.170).The expressions of STAT5 were significantly decreased in the mice treated with tail vein injection and lacrimal injection compared with mice without treatment (both at P＜0.05).Conclusions Administration of HUCMSCs via intravenous and lacrimal injection can alleviate the inflammatory response during progression of dry eye syndrome by down-regulating the serum level and expression of inflammation-related factors in NOD/Ltj mice.The topical administration of HUCMSCs eye drops can attenuate the symptom of dry eyes by lubricating the cornea and suppling nutrition.

Objective To construct lentiviral vectors containing peptide P1-GFP fusion genes.Umbilical cord derived mesenchymal stem cells were infected with lentivirus carrying peptide P1 and GFP fusion genes.To inject the targeted umbilical cord derived mesenchymal stem cells into mice and to detect GFP expression in the spleen.Methods Umbilical cord derived mesenchymal stem cells were cultured with adhered tissues of umbilical cord smaller than 1 mm3 . Lentiviral vector containing P1-GFP fusion genes with engineering technology was constructed and infected the umbilical cord derive mesenchymal stem cells.Targeted umbilical cord derived mesenchymal stem cells were intravenously injected in the mouse tail vein and after 18 hours GFP expression was detected with immunohistochamical staining of the spleen tissues.Results Harvested umbilical cord derived mesenchymal stem cells grew well in culture medium. Green fluorescence on umbilical cord derived mesenchymal stem cells were observed under fluorescence microscope at 18 hours after infected with lentivirus.Green fluorescence intensity of umbilical cord derived mesenchymal stem cells was increasing over time and reached a peak at 72 hours.Umbilical cord derived mesenchymal stem cells highly expressed CD105 (90.0%)/CD44 (98%) and CD73 (85.0%)/CD90 (98.5%) molecules.GFP expression was detected in the spleen after intravenous injection of targeted umbilical cord derived mesenchymal stem cells in the mice 18 hours later.GFP expressing cells intimately contacted with lymphocytes.Conclusions Targeted umbilical cord derived mesenchymal stem cells contain P1-GFP fusion genes are constructed.Targeted umbilical cord derived mesenchymal stem cells can be targeted to mouse spleen and intimately contact with lymphocytes after intravenous injection.Our results lay the groundwork for further studies.