Objective:To construct the lentiviral-vector encoding human interleukin-10 protein(LV-hIL-10) and to observe the effect of LV-hIL-10 on controlling neuropathic pain via intrathecal administration in CCI rats. Methods:hIL-10 gene fragment was isolated and amplified from pCYIL-10 plasmid by PCR, and was cloned into pWPXL. The recombinant plasmid pWPXL-IL-10,envelope plasmid pMD2.G and packaging plasmid psPAX2 were cotransfected into 293T cells, LV-hIL-10 is prepared by concentrating the collected supernatant .At the same time, the empty plasmid pWPXL-GFP,pMD2.G and psPAX2 were cotransfected into 293T cells, LV-GFP is prepared for contrast.135 sheer breed pathogen-free adult male Sprague-Dawley rats divided into 9 arrays at random: CCI models 4 arrays (C0,C1,C2,C3), sham operatived rats 4 arrays (S0,S1,S2,S3) and a normal contrast array (N), each respectively intrathecal injection LV-hIL-10 (C1,S1)、LV-GFP (C2, S2),isotonic Nachloride (C3,S3) and control (no implanted catheters and no administration, C0,S0), the pain threshold of each array and the expression of mRNA and protein of IL-10 in spinal cord,pallium and hippocampus on different time were observed after intrathecal administration LV-hIL-10 in successful CCI model rats . Results:The hIL-10 gene fragment was obtained from pCYIL-10 plasmid, pWPXL-hIL-10 was recombinated successfully. the cloned gene segment was validated by DNA sequencing .High titer(2?1010)and highly purified LV-hIL-10 particles were obtained by three plasmids were cotransfected into 293T cells. The mechanical allodynia and thermal hyperalgesia were alleviated via intrathecal injection LV-hIL-10 in CCI rats. The overexpression of IL-10 were detected in spinal cord,pallium and hippocampus , especially in the spinal cord .Conclusions:The mechanical allodynia and thermal hyperalgesia can be relieved by intrathecal injection LV-hIL-10 in CCI rats.

Acute Disease ; Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; China ; Humans ; Infant ; Inpatients ; Insecticides ; poisoning ; Middle Aged ; Organophosphorus Compounds ; Pesticides ; poisoning ; Poisoning ; etiology ; mortality ; therapy ; Risk Factors ; Survival Rate

Epigenetics comprises the modifications made in gene expressions without changing the DNA sequence itself. Significant epigenetic changes take place during spermatogenesis and fertilization and exert direct influences on embryogenesis. This article provides an overview of the latest researches on epigenetics of male germ cells and a brief discussion on the correlation of sperm with embryogenesis in four aspects: DNA methylation, histone modification, regulation of non-coding RNAs, and genomic imprinting.
Animals ; DNA Methylation ; Embryonic Development ; Epigenesis, Genetic ; Genomic Imprinting ; Histones ; metabolism ; Humans ; Male ; Spermatozoa

Background Hypertension is a multigenetic inheritable disease.Gene therapy with long-term effects and less side effects by regulating gene expression has been shown to be a potential and exciting prospect. Objective To investigate the effects of RNA interference(RNAi)targeting angiotensin-converting enzyme(ACE)on the blood pressure and ACE expression in kidney of spontaneously hypertensive rats(SHR).Methods SHR were randomly to receive placebo(n=12)or control adenovirus Ad5-EGFP)or a single injection of recombinant adenovi- ral vectors,Ad5-EGFP-ACE-shRNA(n=12,iv).Normotensive Wistar-Kyoto rats(WKY)were served as normal control group.SBP was measured before and after the intervention.Aorta,lung,myocardium and kidney were studied using fluorescence microscope to identify the sites of Ad5-EGFP-ACE-shRNA.Expressions of ACE mRNA and protein in kidney were evaluated by RT-PCR and Western blot.Results SBP of the treat group was effectively reduced by 19.0?3.2 mmHg at the 3rd day,and 22.1?3.3 mmHg at the 13th day of the experiment.The anti- hypertensive effect significant remained at least for 14 days.On the contrary,increase in BP was shown in placebo and the adenovirus control group.Compared with placebo or adenovirus control rats,ACE mRNA expression level in kidney of the treated rats was lower by 61.1% and 62.3% respectively,with ACE protein expression level lower- ing by 56.2% and 53.30% as well(ail P0.05). Conclusion RNA interference targeting ACE gene inhibits the expressions of ACE mRNA and protein.A single dose injection resulted in a prolonged decrease in BP.The evidence of strong antihypertensive effect by genetic therapy justifies efforts for further investigation.

OBJECTIVETo evaluate the affection of crushing technology on quality. The dissolution of Pills of Six Herbs with Rehmunnia prepared by different crushing technology was determined by taking the dissolution of Paeonol as test marker.

METHODThe Pills was prepared with the fine powder which was crushed by normal crusher or super fine crusher. The rotatory-basket method was used, and the cumulative dissolution percentage was determined by UV.

RESULTStatistics indicated there was a significant difference in dissolution parameter (T50) between super fine crushing powder Pills and normal fine-crushing powder Pills (P < 0.01), and there was a difference in dissolution of different batches of Pills of Six Herbs with Rehmunnia prepared by the normal crush technique (P < 0.05).

CONCLUSIONThe determination of dissolution of Pills of Six Herbs with Rehmunnia is necessary. In order to improve the quality of drugs, we should adopt the technique of super fine crushing in the preparation procedure.

Acetophenones ; analysis ; chemistry ; Drug Combinations ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; Particle Size ; Plants, Medicinal ; chemistry ; Quality Control ; Rehmannia ; chemistry ; Solubility ; Technology, Pharmaceutical ; methods

Objective To investigate the possibility of inducing mesenchymal stem cells(MSCs)from human umbilical cord Wharton's Jelly to differentiate into spermatogonia.Methods To isolate,culture and purify MSCs with adherent method,the growth and proliferation of human umbilical cord-derived MSCs were observed,and their immunophenotypes were determined by flow cytometry;MSCs of the third generation were divided into 2 groups to be induced and cultured,MSCs of the control group were cultured in basal medium,while those of the experimental group with conditional medium.The morphologic and ultrastructure changes of control group and experimental group cells were compared with phase contrast microscopy,electron microscopy(EM)and transmission electron microscope(TEM)respectively ;the spermatogonial cells differentiated were then evaluated by immunohistochemistry stained for CD117and CD49f ;the method of Western-blot was used to test if the cells induced could express CD49f.Results A population of MSCs were isolated from human umbilical Wharton's Jelly;they were processed to obtain a fibroblast-like population of cells and could be maintained in vitro for extended periods with stable population doubling;After induction,the shape of MSCs changed greatly from the fibroblast to the round,even familiar to the tadpole;expressed the known molecular markers of spermatogonial cells,such as CD49f,CD117.Conclusion The induced MSCs not only undergo spfermatogonial-cell like morphologic changes,ultramicrostructure mature with increasing cell organs,but also express the spermatogonial cell markers,which show that human umbilical cord derived MSCs are capable of differentiating into spermatogonial cell.

Objective To evaluate the efficacy and risk of endovascular thrombolysis with Urokinase and Tirofiban in patients with cerebral venous sinus thrombosis (CVST).Methods Nine patients with severe CVST were performed selective intravenous sinus Urokinase and Tirofiban thrombolysis combined with mechanical thrombus maceration in our hospital from January 2009 to January 2011; their clinical data and treatment efficacy were analyzed.Results Normal cerebrospinal fluid (CSF) pressure was noted in 8 patients before discharging from the hospital; DSA indicated that 7 achieved complete recanalization of main branch of the venous sinus,cortical veins and deep vein.Only 1 achieved reeanalization of sinuses partly,and partial compensation was noted in the cortical veins.No operation-related complication was noted during and after the surgery.After thrombolysis,symptoms and signs of 8 patients improved obviously,and headache disappeared in 7 of them with only mild degree in 1; 1 patient died.Conclusion Intravenous sinus Urokinase and Tirofiban thrombolysis is an effective and safe method for patients with potentially catastrophic intracranial dural sinus thrombosis.

BACKGROUNDDiagnosis and appropriate treatment of multidrug-resistant tuberculosis (MDR-TB) remain major challenges. We sought to elucidate that persons who share a household with drug resistance tuberculosis patients are at high risk for primary drug resistance tuberculosis and how to prevent these outbreaks.

METHODSWe used 12-locus mycobacterial interspersed repetitive unit and 7-locus variable-number tandem repeat to identify household transmission of extensively drug resistant and multiple drug resistant Mycobacterium tuberculosis in three families admitted in Shanghai Pulmonary Hospital affiliated with Tongji University. Drug susceptibility tests were done by the modified proportion method in the MGIT 960 system in the same time. Clinical data were also obtained from the subjects' medical records.

RESULTSAll of the six strains were defined as Beijing genotype by the deletion-targeted multiplex PCR (DTM-PCR) identification on the genomic deletion RD105. Strains from family-1 had the same minisatellite interspersed repetitive unit (MIRU) pattern (232225172531) and the same MIRU pattern (3677235). Strains from family-2 had the same MIRU pattern (2212261553323) and the same MIRU pattern (3685134). Strains from family-3 did not have the same MIRU pattern and they differed at only one locus (223326173533, 223325173533), and did not have the same VNTR pattern with two locus differed (3667233, 3677234).

CONCLUSIONSHousehold transmission exists in the three families. A clear chain of tuberculosis transmission within family exists. Tuberculosis susceptibility should be considered when there is more than one tuberculosis patients in a family. Household tuberculosis transmission could be prevented with adequate treatment of source patients.

Adult ; Female ; Genotype ; Humans ; Male ; Middle Aged ; Multiplex Polymerase Chain Reaction ; Mycobacterium tuberculosis ; classification ; genetics ; pathogenicity ; Radiography ; Tuberculosis, Multidrug-Resistant ; diagnostic imaging ; transmission ; Young Adult

OBJECTIVETo select the optimal culture media for the mass production of gamma delta T cells used in adoptive immunotherapy.

METHODSThree different culture media (RPMI-1640, AIM-V, and OpTmizer, with or without autologous serum) were used to culture gamma delta T cells. The survival rate, purity, proliferation efficiency, and biological functions of the expanded gamma delta T cells were examined and compared.

RESULTSThe survival rate of gamma delta T cells expanded in RPMI-1640 decreased over culture time. The purities of gamma delta T cells cultured in AIM-V or OpTmizer with or without serum were higher than those cultured in RPMI-1640. After two weeks of culture in the absence of serum, the purity and proliferation efficiency of gamma delta T cells cultured in OpTmizer were significantly higher than those cultured in RPMI-1640 (P < 0.05) and AIM-V (P < 0.05). gamma delta T cells in different culture media had similar CD107a expression and tumor necrosis factor-alpha production (P > 0.05). However, cells expanded in RPMI-1640 exhibited significantly weaker cytotoxicity against Daudi lymphoma cells than those expanded in OpTmizer (P < 0.05) and AIM-V (P < 0.05).

CONCLUSIONDue to low serum-dependence, high proliferation efficiency, and favorable biology function of expanded cells, OpTmizer is the most suitable medium for the mass production of gamma delta T cells used in adoptive immunotherapy.

Cell Culture Techniques ; Culture Media ; Humans ; Immunotherapy, Adoptive ; T-Lymphocytes ; cytology

The present study was to investigate in vitro the rat cardiomyocyte injury with targeting of home-made perfluorocarbon lipid particles with avidin-biotin interaction. Neonatal rat cardiomyocytes were cultured in vitro and divided into two groups: TNF-alpha activated group and non-activated group. Those in the TNF-alpha activated group were exposed to 200 ng/ml TNF-alpha solution for 6 hours and then cardiomyocytes in both groups were pretargeted with biotinylated ICAM-1 monoclonal antibodies, and were exposed to streptavidin, and then to homemade green fluorescently-labeled biotinylated perfluorocarbon lipid particles. Cardiomyocytes nucleus stained with Hoechst. The results were detected with fluorescence microscope. As a result, in TNF-alpha activated group, around blue fluorescent cardiomyocytes nucleus, a great amount of green fluorescent particles were found, while there were few green fluorescent particles in non-TNF activated group. It has been shown that ICAM-1 is expressed in the surface of cardiomyocytes when they are stimulated by TNF-alpha. Perfluorocarbon lipid particles associated with ICAM-1 monoclonal antibodies can be targeted to injured cardiomyocytes by avidin-biotin interaction.
Animals ; Animals, Newborn ; Antibodies, Monoclonal ; metabolism ; Cells, Cultured ; Contrast Media ; Female ; Fluorocarbons ; immunology ; metabolism ; Intercellular Adhesion Molecule-1 ; metabolism ; Lipids ; chemistry ; Male ; Microspheres ; Myocytes, Cardiac ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; pharmacology ; Ultrasonography