BACKGROUND:Studies have shown that human leukocyte antigen (HLA)-A*0206 subtype is related to the abscess of nasopharyngeal carcinoma, but there is no corresponding transgenic animal models that could used to judge the relationship between HLA-A*0206 and nasopharyngeal carcinoma on the overal level and further research of immunotherapy and gene therapy. OBJECTIVE:To construct lentiviral vectors carrying pLVX-CMV-HLA-A*0206-HA-mCMV-ZsGreen and establish HLA-A*0206 transgenic mice. METHODS:The HLA-A*0206 sequence was synthesized. EcoRI recognition site was introduced in the 5’ end by polymerase chain reaction, and influenza virus hemagglutinin labels and BamHI recognition site were introduced in the 3’ end. Eco RI and Bam HI double enzyme digestion target fragments and the pLVX-CMV-mCMV-ZsGreen plasmids were connected to the digested productions and transfected JM109 competent cel s. The positive clones were selected and identified by double enzyme digestion and sequencing. The positive plasmid and packaging plasmids were transfected into 293T cel s, which were human renal epithelial cel line that can express SV40 large T antigen. The lentivirus containing target sequence was produced. RESULTS AND CONCLUSION:Gel electrophoresis and sequencing results showed that, HLA-A*0206 was successful y inserted into pLVX-CMV-mCMV-ZsGreen frame plasmids. Transfection efficiency was 92%after 48 hours of transfecting 293T cel s. The viral suspension titer was 5 × 108 measured by fluorescence method. Experimental findings indicate that, the lentivirus containing cytomegalovirus promoter, HLA-A*0206, influenza virus hemagglutinin label and Zsgreen report gene was successful y constructed.

Objective To accurately determine the biological characteristics , especially the disease-related indexes of SPF rabbits , and their gender differences .Methods A total of thirty 70-80 day old SPF New Zealand rabbits (male :female=1:1) were fed for a week, and then we weighed the body weight and main organs , determined the blood physiological and biochemical indexes , blood gas and arterial pressure , and measured the ventricular pressure by thoracotomy under respiratory support .Results Comparison of the male and female SPF New Zealand rabbits showed that there were significant differences in the mass of thyroid , adrenal gland, and liver (P<0.05 or P<0.01); the brain, pituitary gland, thyroid gland organ coefficients ( P<0.05 or P<0.01); the thyroid/brain, adrenal/brain, and liver/brain ratios (P<0.05 or P<0.01);the mean erythrocyte volume and mean hemoglobin content (P<0.05 or P<0.01);and in glutamyl transpetidase and amylase ( P<0.05 or P<0.01 ) , but no significant differences in blood gas analysis ,heart rate and carotid artery systolic and diastolic blood pressure , left ventricular systolic and diastolic blood pressure , and right ventricular systolic and diastolic blood pressure .Conclusions The gender has an impact on organ weight and blood physiological and biochemical indexes in New Zealand rabbits , but no significant influence on blood gas , blood pressure , heart rate and ventricular pressure in the rabbits .

BACKGROUND: Bone marrow-derived mesenchymal stem cells (BMSCs) can not only differentiate into multiple nonhematopoietic cell lineages, but seek out damaged tissues and repair them as well. Hence, they were largely studied for their potential clinical use. However, their biological characteristics have not been fully discovered. OBJECTIVE: To compare the biological characteristics of BMSCs of different species cultured in vitro, in order to provide basis for the clinical research of stem cell therapy.DESIGN: Randomized controlled observation was designed.SETTING: Medical Research Department of General Hospital of Guangzhou Military Area.MATERIALS: The experiment was performed in Medical Research Department of General Hospital of Guangzhou Military Area from June 2004 to July 2005. Thirty SD rats weighing (160±20) g, aged 35 to 40 days, 30 Kunming mice weighing (16.0±2.0) g, aged about 40 days, 8 New Zealand white rabbits weighing (2.0±0.2) kg, aged 80 to 90 days and 10 healthy volunteers (25-32 years old) were selected. All the animals were of clean grade, which were purchased from the Animal Center of Southern Medical University.METHODS: The BMSCs of mice and rats were prepared according to the protocol developed in the Caplan laboratory, while those of rabbits and human were isolated from bone marrow suspension obtained by iliac puncture.The morphology of BMSCs was observed by light microscope and transmission electron microscope. Cell growth curve was tested by MTT. Expression of Stro-1 was analyzed by immunofluorescence cytochemistry and flow cytometry. To evaluate the specific response of BMSCs to osteogenic supplements(10 nmol/L dexamethasone, 10 mmol/L β-glycerophosphate,and 50 mg/L ascorbic acid), the activity of alkaline phosphatase (AKP) activity was tested by a commercial kit. Expression of osteocalcin was examined by immunocytochemistry and hydroxyapatite crystals were shown by von Kossa staining. Adipogenic differentiation was evaluated by estimating percent of cells containing Oil Red-O- stained oil droplets.MAIN OUTCOME MEASURES: ① Morphological observation and growth situation of BMSCs. ② Expression of Stro-1: BMSC marker. ③ Differentiation in osteogenic medium.RESULTS: ①The morphology of adherent BMSCs ofthose four species observed by optic microscopy was obviously different. When they became mature or aged, the mouse cells turned into flat shape, irregularly polygonal, fell to pieces and deposited on the flask-bottom, while the rat, rabbit and human cells would enlarge and become polygonal, vacuoles would appear in their cytoplasm, finally, the cells were detached from the flask-bottom, floating off like cotton wool. The cultures of different species also had some commonness, such as poly-layer growing manner, without contact inhibition and consisting of two groups. Cells of one group grew into colonies from single cells and expanded quickly, while cells of the other group were sporadic and did not proliferate. Electron microscopy revealed that all of the primary cells had microvilli and that they could be divided into two subpopulations according to their ultrastructures. Some cells were rich in organelles and most chromatin was euchromatin, while the other subpopulation cells had much fewer organelles and more heterochromatin. Growth curves of BMSCs of different species were almost the same. ② The positive rate of human adherent bone marrow-derived cells for Stro-1: BMSC marker was (91.4±8.3) %, and that of mouse adherent cells was (83.5±6.2) % .③Treated with osteogenic supplements, mouse BMSCs differentiated into adipose tissue, rabbit ones died, while rat and human ones differentiated into osteocytes. BMSCs also demonstrated spontaneous differentiation in vitro.CONCLUSION: Mouse, rat, rabbit and human BMSCs can be easily expanded in vitro, although the harvest of the current method is a mixture of mesenchymal cells with various maturities, most of which are poor-differen-tiated cells. BMSCs of those species are different in morphology and response to the same inductive supplements. Therefore, in order to establish a kind of stem cell therapy, it is necessary not only for evidence from animal experiments but for that from human experiments as well.

OBJECTIVETo study the role of neuronal nitric oxide synthase (nNOS) in aged rats' hippocampal delayed neuronal death (DND) following brain ischemia.

METHODSModels of incomplete brain ischemia were induced by clipping common carotid artery. A total of 46 aged SD rats were divided into 8 groups: normal control group (Group A, n=5), sham-operation group (Group B, n=5), reperfusion 1, 6, 12, 24, 48, and 96 hours groups after brain ischemia for 30 minutes (Group C, D, E, F, G, and H, n=6/group). The expression of nNOS was examined by immunohistochemistry and neuronal ultrastructural changes were observed by the transmission electron microscopy (TEM) at different time points after reperfusion.

RESULTSImmunohistochemistry showed that nNOS expression in the hippocampal neurons was high in Group E, low expression in Group D, moderate expression in Group F and G. There was nearly no expression of nNOS in Group A, B, C, and H. Ultrastructure of hippocampal neurons was damaged more severely in reperfusion over 24 hours groups.

CONCLUSIONSNitric oxide (NO) may be one of the important factors in inducing DND after ischemia/reperfusion.

Animals ; Apoptosis ; Brain Ischemia ; enzymology ; Female ; Hippocampus ; enzymology ; pathology ; Immunohistochemistry ; Male ; Microscopy, Electron ; Neurons ; enzymology ; Nitric Oxide Synthase ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; enzymology