BACKGROUND:Numerous studies have demonstrated that mild hypothermia has a better protective effect on neurons after cerebral infarction.
OBJECTIVE:To investigate theeffect of mild hypothermia on nerve regeneration microenvironment of infarcted area in rat models of cerebral infarction and analyze its possible effects on neural functional recovery after cerebral infarction.
METHODS:Twenty out of 65 adult femaleSprague-Dawleyrats were randomly selected as the sham group. The remaining 45 rats were subjected to carotid artery ligation to establish rat models of cerebral infarction. Five rats were rejected because of modeling failureor death, the remaining 40 rats were randomly and evenly divided into cerebralinfarction and mild hypothermia groups. The head temperature of rats in the cerebral infarction group was downregulated to (37±1)℃ using a semiconductor refrigeration instrument. The rats were transferred to the room with the temperature of 25℃ after the operation. Brain hypothermia was also induced in rats from the mild hypothermia group. At 13.0-14.0 minutes after establishing rat models of cerebral ischemia, the head on the side of cerebral ischemia was tightly connected with the probe of the semiconductor refrigeration instrument. The refrigerator temperature was adjusted to 6-8℃, so as to make the temperature of brain tissue on the lesion side at 32.0-33.0℃ for 4 hours.
RESULTS AND CONCLUSION:Compared with the cerebral infarction group, the BBB scores of rats inthe mild hypothermia group were distinctly increased, and the volume of infarcted area decreased. At 1 day after modeling, the expression level of growth associated protein 43 mRNA in brain tissue of rats in the mild hypothermia group was close to that in the cerebral infarction group. At 2 weeks after modeling, the expression level of growthassociated protein 43 mRNAin brain tissue of rats in the mild hypothermia group was significantly increased compared with that in the cerebral infarction group. These results suggest that mild hypothermia therapy can protect nerve cels against injury caused by cerebral infarction and promote the recovery of neurological function. Its underlying mechanism may be related to the up-regulation of the expression of growth associated protein 43 in ischemic penumbra .

Objective To test the drug susceptibilities in Mycobacterium tuberculosis by microscopic observation drug susceptibility(MODS)and evaluate the method for the detection susceptibility in second-line drugs.Methods To set up the MODS method.the drug susceptibilities of 4 second-line drugs in Mycobacterium tuberculosis were tested in 24 well plates.The test conditions were discussed.The resistance to protionamide(PTH),amikacin(AMK),capreomycin(CPM)and levofloxacin(LVF)of 60 MTB isolated from 2007 to 2008 in Shanghai Pulmonary Hospital were detected and compared with that of L-J method.The isolates were tested by minimum inhibitory concentration(MIC)method when the drug susceptibilities were not consistent.Results Among the 60 Mycobacterium tuberculosis clinical isolates.the results of drug susceptibility were confirmed by MODS,absolute concentration method and the accordance rate of PTH,AMK,CPM and LVF were 96.7%(58/60),98.3%(59/60),91.7%(55/60)and 96.7%(58/60),respectively.If the result of absolute concentration method was as the standard.the sensitivity.specificity.positive and negative predictive value as well as accuracy were 100%,87.5%,87.5%,100%and 96.7%in PTH;100%,90.0%,90.0%,100%and 98.3% in AMK;76.9%,95.7%,83.3%,93.8%,91.7% in CPM;100%,96.0%,83.3%,100% and 96.7% in LVF respectively detected by MODS assay.Conclusion MODS is a rapid and simple method for susceptibility testing of second-line drug in Mycobacterium tuberculosis.

BACKGROUND:Studies concerning bone marrow mesenchymal stem cells (BMSCs) cultured by umbilical cord serum in vitro were hot topic to avoid the heterogenous serum rejection during BMSC culture and improve culture efficiency of BMSCs.OBJECTIVE:To compare biological feature of BMSCs by the umbilical cord serum and adult autoserum in vitro.DESIGN,TIME AND SETTING:The opening experiment was performed at the Laboratory of Cell Biology,Xiangya Second Hospital,Central South University,China from October 2006 to June 2008.MATERIALS:Sixteen bone marrow samples were provided by Xiangya Second Hospital,Central South University and Xiangtan Central Hospital of Hunan Province.METHODS:BMSCs were obtained from 16 fresh adult bone marrow by gradient centrifugation with Ficoll Paque,cultured with DMEM/F12 with 10% umbilical cord blood serum and 10% adult autoserum.The fourth passage was used in this study.The surface antigens of BMSCs were detected by flow cytometry.BMSCs could differentiate into ostenblasts and adipocytes under culture in conditioned medium for osteogeneais and adipogenesis and the differentiated BMSCs were identified by morphological observation,immunophenotype and immunochemical staining.MAIN OUTCOME MEASURES:The following parameters were measured:morphological observation;cell cycle and cell count;identification of surface antigen;observation of osteoblasts and adipocytea induced from BMSCs by immunochemical staining.RESULTS:The quantity and velocity of BMSCs in umbilical curd blood serum was better than those in adult autoserum (P ＜ 0.05)Only few cells were proliferating in both medium,BMSCs in S phase in umbilical cord blood serum was more than those in adult autoserum (P ＜ 0.05).The positive rate of CD29,CD73 and CD105 on BMSCs in umbilical cord serum (above 90%) was higher than those in adult autoserum (P ＜ 0.05),and the positive rates of CD31,CD34 and CD45 were lower than 2%,and the positive rate of CD31 was lower than those in adult autoserum (P ＜ 0.05).The positive rate of BMSCs differentiated into osteoblasts and adipocytes in umbilical cord blood serum was also higher than these in adult autoserum (P ＜ 0.05).CONCLUSION:Purity and differentiated potency of BMSCs in umbilical cord blood serum are better than those in the adult autoserum in vitro.The umbilical cord serum is more adapt to clinical application.

Objective To evaluate the accuracy of pyrosequencing for the mutation detection of katG gene in isoniazid resistance in Mycobacterium tuberculosis using Meta-analysis.Methods Searching PubMed,Web of Science,Elsevier,and China National Knowledge Infrastructure (CNKI),Weipu and WANFANG DATA to obtain the relevant English and Chinese-language articles,respectively.A written protocol and explicit study selection criteria was followed.Quality of included trials was assessed by QUADAS (quality assessment of diagnostic accuracy studies).Subsequently,the characteristics of the included articles were appraised and extracted.Heterogeneity of the included articles was tested by using STATA 10.0,which was used to select proper effect model.The fixed effects model was adopted using Meta-Disc software.Finally,sensitivity analysis was performed.Results Totally 114 research papers were collected and 9 articles were selected.The accordance between pyrosequencing and conventional sequencing result was 100％.Eight studies were involved including 945 specimens when katG gene was detected.The overall sensitivity and specificity were 0.77 (0.73,0.80) and 1.00 (0.99,1.00),respectively.The area under the SROC was 0.9882.As inhA gene was detected,the overall sensitivity and specificity were 0.19 (0.15,0.24) and 1.00 (0.98,1.00).The test was stable.Conclusions Our meta-analysis reveals that pyrosequencing is a highly specific tool for detection mutation of katG gene of isoniazid resistance.This result suggests that it is useful for screening of isoniazid resistance in diagnostic test.(Chin J Lab Med,2013,36:329-332)

Objective To investigate the changes of T cells and dendritic cells (DCs) and their related factors in children with immune thrombocytopenia (ITP) before and after therapy,and to analyze their clinical significance.Methods T-cells and DCs subsets were determined by flow cytometry both in 64 children with ITP (ITP group) before and after therapy and the control group.The serum levels of interleukin (IL)-4,interferon-γ (IFN-γ),transforming growth factor beta 1 (TGF-β1),and IL-27 were detected by enzyme linked immunosorbent assay (ELISA).Results Treatments of glucocorticoid or IVIg were effective in 41 cases of 64 ITP children.Compared to the control group,helper T cells (Th),Th/suppressor T cells (Ts),T regulatory cells (Treg),plasmacytoid DC (pDC),pDC/myeloid DC (mDC),and TGF-β1 in ITP patient group before treatment were significantly lower,while IFN-γ and IFN-γ/IL-4 were significantly higher (P ＜0.05).In ITP group,Th,Th/Ts,Treg,pDC,pDC/mDC,TGF-β1,and IL-27 were significantly increased,while IFN-γ and IFN-γ/IL-4 were decreased in children with ITP after therapy and achieved response (P ＜ 0.05).However,there was no significant difference between before and after therapy in ITP children without treatment response (P ＞ 0.05).Conclusions T cells and DCs subsets disorder and abnormal cytokine levels are observed in children with ITP,which can be corrected by immunosuppressive therapy,indicating that Th1 overactivity and the decrease of Treg and pDC both in quantity and function may be related to the pathogenesis of ITP in children.

Objective To identify the recombinant aldolase (ALD) of Plasmodium falciparum, and to develop monoclonal antibodies (McAbs) against the recombinant ALD. Methods ALD gene was amplified by PCR from genomic DNA of FCC1/HN strain, and expressed in E.coli DH5?. BALB/c mice were immunized with the recombinant ALD of P. falciparum via celiac injection for 3 times with 2 weeks interval. Three days after a booster injection, spleen cells of the immunized mice were used for producing McAbs. The immune serum was tested by IFAT and Western blotting. Results BALB/c mice immunized with purified aldolase protein developed strong immune response to the antigen, and the titer of specific antibody reached 1∶105 in all immune sera after the third immunization. Moreover, immune sera specifically recognized the cultured P. falciparum. Western blotting showed that the immune sera recognized specifically a Mr 41 000 band of crude malaria antigen. No cross-reaction with human red cells was detected. Seven positive hybridoma cell lines were obtained after 3 rows of selection. All the McAbs′subclasses belong to IgG1. IFAT showed that only 4 McAbs could recognize the cultured P.falciparum. Conclusion Plasmodial aldolase has been successfully expressed and purified, and the established hybridoma cell lines can secrete McAbs specific to the aldolase of P. falciparum.

Objective To obtain DNA oligonucleotide aptamers which can specifically bind to MPT64 protein from Mycobacterium tuberculosis by SELEX(systematic evolution of ligands by exponential enrichment)technology. Methods A random ssDNA library with in vitro synthesized 78 nucleotides in length was subiected to 12 rounds of selection by SELEX method against MPT64 protein. The binding ability of the aptamers to the protein was examined by biotin-streptavidin-horseradish peroxidase system. Results The selective system used was as follows:in PCR amplification,annealing temperature was 65℃ and the concentration of Mg2+ was 1.5 mmol/L in optimizing library, and when preparation of ssDNA with asymmetrical PCR amplification, 0.75 mmol/L of Mg2+ was used. When using the plate for ELISA as the substrate for the selection, the pattern of electrophoretic band of PCR product after the tenth round selection became unitary and denser than that of the first round. The binding assay demonstrated that A value at 450 nm of the tenth round increased by 9.18 times as compared with that of the first round. The results showed that the affinities of the aptamers were different. The highest A value at 450 nm was 1.606, and the lowest 0.572. Conclusion A set of aptamers with considerable binding affinity to MPT64 protein are successfully picked out from the initial random DNA library.

Objective To construct the recombinant plasmid of protein CFP10-MPT48-TB8.4 of Mycobacterium tuberculosis and to investigate the diagnosis potential of this fusion protein in tuberculosis serodiagnosis.Methods The recombinant fusion protein CFP10-MPT48-TB8.4 was expressed, and identified by Western blot.The ELSIA based on the purified fusion protein was done,and used for screening in 230 cases of clinical serum samples including pulmonary tuberculosis patients ( n =150 ),pulmonary disease patients other than tuberculosis (n =70) and health controls (n =103 ).The test result was analyzed by Medcale11.5 software.Results The fusion protein CFP10-MPT48-TB8.4 was successfully expressed with a purity over 95％.Specific immunogenicity of the recombinant protein was confirmed by Western blot.The overall sensitivity and specificity obtained of ELISA were 56.7％ (85/150) and 90.8％ ( 157/173 ),respectively.The specificity was 85.7 ％ (60/70) in non-tuberculosis group and 94.2％ (97/103 ) in healthy group,respectively.Conclusion The recombinant protein of CFP10-MPT48-TB8.4 has a high sensitivity and specificity and may be a potential candidate antigen in tuberculosis serodiagnosis.

Objective To develop an assay to determine Mycobacterium tuberculosis resistance to rifampin, isoniazid, ofloxacin and amikacin by pyrosequencing and evaluate the value of this method in clinical application. Methods Eighty-nine clinical isolates of Mycobacterium tuberculosis from tuberculosis patients were collected from Shanghai Pulmonary Hospital during 2008 to 2009. All strains were isolated from decontaminated sputum, cultured on Lowenstein-Jensen media and identified by traditional biochemical tests with standard methods. Ten Mycobacterium tuberculosis were selected from the strain bank of Shanghai Pulmonary Hospital. The optimal conditions of detection of rpoB, katG, gyrA and rrs gene by pyroseuencing were determined, using the 10 Mycobacterium tuberculosis strains whose drug susceptibility of Bactec 960 and sequence of rpoB, katG, gyrA, rrs gene were known. Then the drug susceptibility of 89 Mycobacterium tuberculosis clinical isolate strains were detected by pyrosequencing using this conditions and the results were compared with that of the Bactec 960 methods. Results The pyrosequencing program of sequence analysis was suitable for the detection of the mutations of rpoB and gyrA genes, and the program of single nucleotide polymorphism was suitable for katG and rrs genes. Among the 89 Mycobacterium tuberculosis clinical isolates,when using the drug susceptibility of Bactec 960 as the standard, the sensitivity of rifampin, isoniazid,ofloxacin and amikacin is 98.0%, 64. 1%, 79.5%, 78. 3% respectively, the specificity is 97.5%,100. 0%, 90. 0%, 100. 0% respectively, the accuracy is 97.8%, 77. 5%, 85.4%, 94. 4% respectively,tested by pyrosequencing. The results of the detection of resistance to rifampin, isoniazid, ofloxacin and amikacin in Mycobacterium tuberculosis using pyrosequencing technique were almost the same with that of Bactec 960, and Kappa ≥0. 7 in each detection. Conclusion Pyrosequencing is thus a rapid, accurate and high throughput method for detecting Mycobacterium tuberculosis resistance to these four drugs.

OBJECTIVE To set up and evaluate the microscopic observation drug susceptibility assay(MODS)technology and use it to detect rapidly Mycobacterium tuberculosis(MTB).METHODS The 24-hole cell culture plate method of liquid culture were used to set up MODS technology.The MODS technology was used to detect MTB and non-M.tuberculosis(NMT)isolates comparing with Lowenstein-Jensen(L-J) method.RESULTS When bacterial concentration was 3?103 CFU/ml,the time of reading test-result was the seventh day by MODS;four kinds of NMT(M.phlei,M.kansasii,M.chelonaeand M.marinum) in the liquid medium in the observation was similar to the growth of cording,it was difficult to distinct from the cording of MTB in the liquid medium;When using the 4-Nitro-benzoic acid 800 ?g/ml,thiophene-2-carboxylic acid hydrazine 2.5 ?g/ml for testing conditions,the correct diagnosis can be improved;the test results of clinical isolates by MODS were highly concordance rate with the results of L-J.If the results of L-J was the golden standard,the sensitivity,specificity,positive and negative predictive value as well as accuracy by MODS was 95.7%,100%,100%,99.9% and 97% respectively.CONCLUSIONS The results of MODS in detection of MTB are highly concordance wih the results of L-J method;MODS assay can be used for rapid detection of tuberculosis,with a rapid,simple,inexpensive,and other advantages.