OBJECTIVE: To investigate the expression efficiency of exogenous gene mediated by different serotypes of adeno-associated virus (AAV) vectors in retina, and to compare the expression efficiency of AAV vector and two kinds of promoters commonly used in ophthalmology after transfection into mouse retina, so as to provide the basis for selecting appropriate AAV vector and promoter for gene therapy of retinitis pigmentosa. METHODS: AAV2/2, AAV2/5, AAV2/8 and AAV2/9 were prepared. The C57BL/6J mice were injected subretinally with 1 μL purified AAV vectors (1.00×10¹³ mg/L). Then the mice were killed 2 or 4 weeks after treatment, and the eyes were enucleated for frozen section. The expression of green fluorescent protein (GFP) was observed under the confocal microscope. Two kinds of promoters, CMV and CAG, were selectd, and the expression of AAV2/8-GFP-CMV and AAV2/8-GFP-CAG was observed under confocal microscope. RESULTS: No bacterial infection or immune response were seen in the injected mice. 2 weeks after injection, the GFP green fluorescence of AAV2/8 and AAV2/9 in the mouse retina was obvious, which indicated that the GFP green fluorescence of AAV2/8 and AAV2/9 was high after transfection into the mouse retina. In these two serotypes, GFP green fluorescence of AAV2/8 was mainly concentrated in photoreceptor cells while AAV2/8 was expressed in the whole retina, indicating that AAV2/8 was more specific to photoreceptors. Further experiments on AAV2/8 showed that the GFP green fluorescence of the mouse retina was obvious 4 weeks after injection, indicating that the exogenous gene mediated by AAV2/8 could be stably expressed in vivo. For CMV and CAG promoters, CMV promoter was expressed stronger in retinal pigment epithelium (RPE)cells, while CAG promoter was stronger in photorecepters. In photorecepters, CAG promoter was expressed almost the same as CMV promoter, while CMV promoter was stronger in RPE cells. CONCLUSION AAV vectors could express transgene robustly in retinal cells; Among several AAV serotypes, AAV2/2 and AAV2/5 showed weaker GFP fluorescence than AAV2/8 and AAV2/9. AAV2/9 showed expression in each layer of the retina including ganglion cells. AAV2/8 was more specific for photoreceptor; CAG promoters had higher specificity for photoreceptors than CMV promoters.
Animals ; Dependovirus/genetics* ; Genetic Vectors ; Mice ; Mice, Inbred C57BL ; Retina ; Serogroup ; Transduction, Genetic

Objective To investigate the potential effect of hypoxia on adhersion and invasion of human cervical carcinoma cells HeLa. Methods Reverse transcription-polymerase chain reaction (RT-PCR) was used to investigate the level of HIF-1? mRNA and E-cadherin mRNA of HeLa cells cultured under normoxia, 5%O2, 3%O2, 1%O2. The invasiveness of HeLa cells was investigated by transwell chamber assay. Results Under the condition of normoxia, 5%O2, 3%O2 and 1%O2, the optical density ratio of HIF-1? mRNA/?-actin was (0.423?0.116), (0.764?0.111), (1.687?0.126) and (0.215?0.099), showing that the level of HIF-1? mRNA had a tendency of first inceasing and then decreasing with the decreasing oxygen content. The optical density ratio of E-cadherin/?-actin was (1.045?0.171), (0.719?0.114), (0.386?0.105) and (0.157?0.063), showing a decreasing tendency with the decreasing of oxygen content. The invasiveness of HeLa cells was (112.5?20.7), (254.3?27.5), (333.6?45.2) and (124.5?24.6), showing that the invasiveness of HeLa cells had a tendency of first inceasing and then decreasing with the decreasing oxygen content. Conclusion Moderate hypoxia up-regulates the mRNA levels of HIF-1?, and down-regulates the mRNA levels of E-cadherin, and enhances the tumor invasiveness. The possible mechanism is the increasing mRNA levels of HIF-1?.

Objective To study the acute inhalation toxicity of 50% Luowei,a plant molluscacide,in rats. Methods Twenty adult Wistar rats,half male and half female,were given of 5000 mg/m3 of 50% Luowei through a dynamic inhalation de?vice and the death and recovery of the rats were observed within 14 days,and LC50 was drawn. Results After exposure,the ac?tivities of the rats decreased and a few individual animals had scratching symptom,but the symptom disappeared after the expo?sure. No animals died during the whole observation period. Therefore,the acute inhalation LC50 was greater than 5000 mg/m3 for rats,and Luowei belonged to low toxicity level. Conclusions 50% Luowei will not cause animal death if it is inhaled into ani?mal body. However,people still need to wear protective equipment in production and use.

BACKGROUND: Multipotency of hepatic stem cells is of important value in liver transplantation. Stem cells have been successfully identified and isolated from the animal livers. However, reports on whether stem cells exist in human hepatic tissue and how to isolate and identify them ars few.OBJECTIVE: This study was in attempt to isolate hepatic stem cells from human para-cancerous tissues of hepatoma and in vitro culture them, also to identify the stem cell surface marker, in order to find a new source of heptatic stem cells.DESIGN: Cell observation experiment.SETTING: Department of Common Surgery, First Hospital, Jilin University; Department of Common Surgery,Dongfeng Hospital of Traditional Chinese Medicine.PARTICIPANTS: Samples were harvested from 10 patients with hepatoma admitted to Department of hepatobiliary surgery, First Clinical College, Jilin University between October 2005 and June 2006, with age of 45 to 58 years.Hepatic tissue 2 cm away from cancer nest was cut when patients underwent hepatectomy, and it was pathologically confirmed as carcinoma-free tissue. Written informed consents were obtained from each patient. DMEM/F12 dry powder used for cell culture was provided by Hyclone Company, USA. Fresh fetal bovine serum was prepared by Lianxing Biotech Co.,Ltd, Tianjin. Various cell growth factors were the products of Cytolay Company, USA.METHODS: Para-cancerous tissues of hepatoma was cut into pieces, rinsed with Hank's solution and digested with type Ⅳ collagenase. Then the isolated cells were re-suspended in the DMEM/F12 medium supplemented with 0.1 volume fraction of fetal bovine serum, and hepatocyte growth factors, epidermal growth factors and α- fibroblast growth factors of 25 μg/L each were added in the above medium. When the cultured cells covered 2/3 of bottom,they were digested with trypsinase for passage and inoculated at 2×107 L-1. When cells propagated to the 3rd and 4th generations, 2.60×109 L-1 cell suspension prepared with trypsinase was added, and subsequently, anti-human C-kit antibody, immunomagnetic beads and Buffer solution were added in order. C-kit+ cells were preliminarily isolated by immunomagnetic bead separation. Haematoxylin-eosin staining and immunofluorescent histochemical double-staining were used for detecting the hepatic stem cells in para-cancerous tissues.MAIN OUTCOME MEASURES: ① Observation of cell morphology. ② Identification of hepatic stem cells from para-cancerous tissues. ③ Identification of C-kit+ cells by immunofluorescent histochemical double-staining.RESULTS :① After primarily cultured for 2 weeks, the adherent cells grew in colony. After one half of culture medium was renewed, mature hepatocytes were gradually broken and disappeared. Small round cells propagated, and most of them were located in the center and arranged in cluster. Most cells were found with one big nucleus in each, less cytoplasm and clear cell boundary. When cells propagated to the 1st and 2nd generations, they still grew in colony, but fast. Each C-kit+ cell isolated by immunomagnetic bead separation presented a spherical cell body with a very big nucleus and less cytoplasm. After in vitro cultured for 1 week, it presented broken pieces and apoptotic symptoms.② After para-cancerous tissue was stained by haematoxylin-eosin, atypically proliferated biliary tracts with small round cells could be seen in the portal area. After para-cancerous tissue was stained by immunofluorescent histochemical double-staining, small round cells in the biliary tracts proliferated in the portal arsa co-expressed red fluorescence AFP and green fluorescence cytokeratin (CK) 19 with yellow superposition arsa. ③ After C-kit+ cells were stained by fluorescence immunocytochemisty, cytoplasm expressed alpha-fetoprotein (AFP) red granules and CK19 green granules. The superposition area of both presented yellow fluorescence of AFP+/CK19+-positive cells.CONCLUSION: Hepatic stem cells exist in human para-cancerous tissues of hepatoma. Therefore, expressions of C-kit+/AFP+/CK19+, the surface markers of hepatic stem cells, can be used for identifying and isolating hepatic stem cells. Small round cells obtained by in vitro isolation and culture, i.e. hepatic oval cells possess bipotential differentiation of hepatocyte and hepatobiliary epithelial cells.

Objective To investigate the relationships between Cyclooxygenase(COX-2) and angiogenesis and expressions of COX-2 and vascular endothelial growth factor(VEGF) in thyroid cancer and its clinical significance.Methods Immunohistochemistry was used to detect the expressions of COX-2 and VEGF in 60 thyroid cancer,(15 thyroid)adenomas and 10 normal thyroid tissues.Results The expression rates of COX-2 and VEGF in thyroid cancer were higher than those in thyroid adenomas and normal tissues(P

Alzheimer Disease ; metabolism ; therapy ; Amyloid beta-Peptides ; immunology ; metabolism ; Antibodies ; therapeutic use ; Humans ; Immunotherapy

0.05),but that MFI and/or PPC of CAR in the 2 types cells markedly increased compared with lymphocytes in the same group(Pa

Objective To investigate the effect of clinical pathway on inpatients for rehabilitation. Methods The hospital expense, the ex-pense for medicine and length of stay in hospital were compared in patients with cerebral hemorrhage, cerebral infarction, spinal cord injury and spastic cerebral palsy before (January 1st, 2012 to April 30th, 2013) and after (May 1st, 2013 to December 31st, 2015) clinical pathway implementation, 50 inpatients for each disease, totally 200 patients. Results The length of stay in hospital reduced after of clinical pathway implementation, in all the diseases (t>5.226, P<0.001), with the decrease of hospital expense for cerebral infarction (t=3.327, P<0.001). There was no significant increase in any expense in the disease (t<1.777, P>0.05). Conclusion The implementation of rehabilitation clinical pathway can reduce the length of stay in hospital for the patients rehabilitation in hospital, without increasing their cost.

Objective To investigate influence of nano-?-linolenic acid on expression of macrophage migration inhibitory factor (MIF) in murine model with viral myocarditis (VM).Methods Eighty male Balb/c mice were randomly divided into 4 groups equally:control group,model group,low and high dose intervention group.Mice in control group were inoculated introperitoneally with Eagle's solution.Every mouse in other groups was treated with 0.1 mL Coxsackie B3 virus(CVB3) intraperitoneally.Then,mice in low and high dose intervention group were treated with 60 and 180 mg/kg nano-?-linolenic acid solution for 1 week,respectively.Mice in control group and model group were treated with 9 g/L saline for 1 week.All mice were killed on day 15.The expression levels of myocardial MIF mRNA and protein were detected by reverse transcriptase polymerase chain reaction(RT-PCR) and immunohistochemistry.Serum MIF concentration were exa-mined by enzymelinked immunosorbent assay(ELISA).Myocardial histopathology was determined with hematoxylin and eosin stain.Results The mortality rate was 0,45%,30% and 20% in control group,model group,low and high dose intervention group,respectively.The mortality rate was significantly lower in high dose intervention group compared with model group (P

Objective To study local people's habits and customs concerning the prevention of dental fluorosis in an indoor coal-combustion-type fluorosis area of Zhaotong city Yunnan province, and to provide reference value for monitoring and control of the disease. Methods In 2008, 600 people of a 12-year-old group (400 people) and a 35 - 44-year-old group(200 people) were examined for dental fluorosis and oral health status in an indoor coal-combustion-type fluorosis area of Zhaotong. A questionnaire survey of 120 students and 60 adults was carried out on their habits and customs, stove changing status, knowledge of prevention of dental fluorosis, social psychology status, oral health behavior and their need for medical treatment. Results The total prevalence of dental fluorosis was 91.0% (546/600), dental fluorosis index was 2.58. The total prevalence of dental fluorosis of 12-year-old group was 86.5%(346/400), and dental fluorosis index was 2.12. The total prevalence of dental fluorosis of rural student was 90.5% (181/200), which was obviously higher in the rural group than the urban group [82.5% (165/200), χ2 = 5.48, P < 0.05]. The total prevalence of dental fluorosis of 35-44-year-old group was 100% (200/200), which was obviously higher than that of the 12-year-old group(χ2 = 29.67, P < 0.01). The rates of using coal, baking of food, changing stoves were 66.7% (60/90), 14.4% (13/90),57.8% (52/90), respectively, and these values of the urban were much lower than those of the rural[95.6%(86/90) ,31.1%(28/90) ,76.7%(69/90),χ2 = 24.51,7.11,7.29, all P < 0.01]. Only 23.3% (42/180) of people surveied had the knowledge of prevention of dental fluorosis, and the rate in the adult group[56.7% (34/60)]was higher than that of the student group [6.7% (8/120), χ2 = 55.9, P < 0.01]. The same rate in the urban student group was 0(0/60), and the rural student group was 13.3%(8/60), there was a significant difference between them(χ2 = 5.66, P < 0.05). Seventy-three point nine per cent( 133/180) of these people thought dental fluorosis had significant effect on their life, 91.1%(164/180) of these 'people wished to be treated. Eighty-four point four per cent( 152/180) of the people had a habit of toothbrushing,and 50.7% (77/152) of these people were still using toothpaste containing fluorine. Conclusions Zhaotong is a serious indoor coal-combustion-type fluorosis area. Enhancing health education to local people especially to students, and changing people's unhealthy habits and customs are keies to control the prevalence of indoor coal-combustion-type dental fluorosis.