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 determine the content of gallic acid and evaluate the quality of Galla chinensis of different habitats in Guizhou Province. Methods HPLC method was used to determine the content of Gallic acid on Alltima C18 column (4.6 mm?250 mm, 5 ?m) with the mobile phase of methanol-water-phosphoric acid (15∶85∶0.085). The flow rate was 1.0 mL/min and the detective wave length was 270 nm. Results The contents of Gallic acid in Galla chinensis of 10 different habitats in Guizhou Province were 51.43%～ 60.25%. Conclusion The quality of Galla Chinensis in different habitats in Guizhou Province are up to the Chinese Pharmacopoeia, and the individual varibility is significant.

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.

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?.

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

Data were analyzed from self-filled questionnaires for the students who took the elective course of general practice in China Medical University. 95.5% of students thought it was necessary to study general practice in the college. 0-10 was used to score the degree of how they want to choose general practice as a career, the median was 0 before they had the class and the median was 5 after the class (Wilcoxon rank test, P<0.01). But there were still some places need to be improved in terms of curriculum design, teaching methods and community practice. The General Practice teaching need to be innovated and the teaching mode should be different from the traditional one.

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 assess the clinical significance of glucose-6-phosphate isomerase in RA patients. Methods The level of serum GPI in 100 patients with RA, 98 patients with other rheumatic diseases and 108 normal controls were assessed by sandwich ELISA methods. The level of RF, CRP, anti-CCP antibodies were also assessed in RA patients. Results The level of GPI was higher in RA patients [(2.4?5.0) ?g/ml] than that of normal control group [(0.12?0.14) ?g/ml (P

BacKground:RectaI carcinoid is a rareIy seen neuroendocrine tumor. TiII now,there is controversiaI for the treatment modaIities of rectaI carcinoids with diameter between 1-2 cm. Aims:To study the expression of Ki-67 in rectaI carcinoids and the efficacy and safety of endoscopic resection. Methods:A totaI of 83 rectaI carcinoid patients with tumor diameter Iess than 1. 5 cm were enroIIed. AII patients were pathoIogicaIIy diagnosed and underwent endoscopic mucosaI resection from Jan. 2008 to Dec. 2013 at Chongqing Three Gorges CentraI HospitaI and Wuhan Tongji HospitaI. The medicaI records were retrospectiveIy reviewed and expression of Ki-67 in tumor tissue was assessed by immunohistochemistry. Results:AII patients underwent preoperative endoscopic uItrasonography. Tumors were Iimited to mucosa or submucosa, and no muscuIaris propria invoIvement or metastasis was seen. The mean foIIow-up duration was 38 months,and no recurrence or metastasis occurred. Ki-67 was IowIy expressed in aII rectaI carcinoids(0. 84% ± 0. 67%). When tumors were grouped by size,no significant differences in gender,age,tumor site and Ki-67 expression IeveI were seen between <1. 0 cm group and 1. 0-1. 5 cm group(P >0. 05). Furthermore,when tumors were grouped by a cutoff vaIue of mean Ki-67 index 0. 84%,differences in cIinicopathoIogicaI parameters between the two groups were not significant either( P >0. 05 ). Conclusions:Ki-67 is IowIy expressed in rectaI carcinoids Iess than 1. 5 cm in diameter enroIIed in this study,which denoted an inactive proIiferation. For rectaI carcinoids with diameter Iess than 1. 5 cm,with Iow Ki-67 expression and without muscuIaris propria invoIvement and metastasis, IocaI endoscopic excision is an effective and safe treatment modaIity.

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