Effects of the Mycobacterium tuberculosis prokaryotic ubiquitin-like protein-proteasome system on the mono-resistant to rifampin resistance to Mycobacterium tuberculosis / 中国人兽共患病学报

We studied the effect of the Mycobacterium tuberculosis prokaryotic ubiquitin-like protein-proteasome system on mono-resistant to rifampin resistance to M.tuberculosis.A resazurin-based assay was employed to evaluate minimum inhibitory concentration (MIC) and comparative research on mono-resistant to rifampin MTB with Pup,Dop,PafA,Mpa genes expression and deletion of the difference.Above testing strains,respectively,carbonyl cyanide chlorobenzene hydrazone (CCCP),reserpine (RP),verapamil (VP)and chlorpromazine (CPZ) were tested.We compared and analyzed the change of rifampicin MICs on the various strains.Compared with rifampin resistant MTB,overexpression of Pup,Dop,PafA and Mpa genes were able to make monorifampicin of M.tuberculosis to enhance resistance to rifampin.Deletion of Pup gene,Mpa gene,Dop gene,PafA gene significantly decreased the resistance to rifampicin alone MTB,and the P value was ＜0.05.Results indicated that 4 kinds of efflux pump inhibitors can reduce the degree of rifampin MIC in different strains.Through the factorial analysis,there were some interactions between MTB and PPS efflux pump inhibitors,and the P value was ＜0.05.MTB PPS has influence on mono-rifampin resistance to MTB and it may regulate the efflux pathway related protein to influence its resistance.

HLA-DPB1 gene polymorphism and susceptibility to tuberculosis in Xinjiang Shihezi Han population:a case-control study / 中国免疫学杂志

Objective:In this study,we examined the HLA-DPB1 alleles in patients with tuberculosis,and health individuals attempt to investigate the association between the polymorphism of HLA-DPB1 gene and pulmonary tuberculosis in Han population in Shihezi area in Xinjiang uygur autonomous region of China.Methods:High-resolution typing of DPB1 was performed by the sequence-based typing ( SBT) method using the SBT-HLA-DPB1 generic DNA typing kit.Results:In the controls ,17 HLA-DPB1 alleles were ob-served,the HLA DPB1*0501 (28.1%) and HLA DPB1*0201(27.6%) frequency was significantly higher than other sites ,the first and second respectively.The frequency of HLA-DPB1* 0201 was observed significantly increased in patient group compared with control group ( P<0.05 );the frequency of HLA-DPB1* 0501 was observed significantly decreased in patient group compared with control group ( P<0.05 ).Conclusion: The gene frequencies of HLA-DPB1 in Xinjiang Shihezi Han population are roughly in consistence with other northern Chinese Han population;the HLA-DPB1*0201 may be the protective factor to pulmonary tuberculosis , and HLA-DPB1*0501 may be susceptible to pulmonary tuberculosis in Han population from the Xinjiang uygur autonomous region of China.

Expression of prokaryotic ubiquitin-like protein (Pup)-proteasome system in drug-resistant Mycobacterium tuberculosis / 中国免疫学杂志

Objective:To study the different gene expressions of Pup-proteasome system between the original drug resistance Mtb and the cultured Mtb( INH-Mtb,RFP-Mtb,SM-Mtb,EB-Mtb,MDR) which were at the different concentrations of the Mtb drug,and to explore whether the different Pup-proteasome system gene expression was relevent to the clinical isolates of Mtb-resistant which was widely spreaded in Xinjiang region. Methods:Culturing INH-Mtb,RFP-Mtb,SM-Mtb,EB-Mtb,MDR strains at Original drug resistance Medium,low concentrations of drug Medium and high concentrations of drug Medium to the logarithmic phase,and extract total RNA from each group of Mtb. Using SYBR GreenⅠreal-time PCR to detect the Pup-proteasome system expression level in different concen-trations of drug Mtb in each group of Mtb. Results: Compared with the original state of drug-resistant strains,Pup gene in INH-Mtb, RFP-Mtb,SM-Mtb and MDR strains group were down-regulated 0. 74,0. 23,0. 28,0. 57 times;Dop gene were up-regulated 1. 33,1. 63, 1. 14,2. 88 times;PafA gene were up-regulated 1. 69,1. 30,1. 58,1. 32 times;Mpa gene were up-regulated 3. 05,1. 79,1. 31,2. 27 times in low concentrations of Anti-Mtb drugs condition, the difference was statistically significant ( P<0. 05 ) . Compared with the original state of drug-resistant strains,Pup gene in INH-Mtb,RFP-Mtb,SM-Mtb,MDR strains group were down-regulate 0. 58,0. 37, 0. 43,0. 78 times;Dop gene were up-regulated 2. 62,2. 49,1. 69,2. 95 times;PafA gene were up-regulated 2. 16,1. 48,2. 02,2. 21 times;Mpa gene were up-regulated 1. 63,3. 22,1. 13,3. 94 times in the high concentrations of Anti-Mtb drugs condition,the difference was statistically significant(P<0. 05). Conclusion:Through the different concentrations of antibiotic selection pressure,these groups of Mtb strains expressions in the Pup-proteasome system of Pup gene,Dop gene,Mpa gene and PafA gene were different,The results reveal that Pup-proteasome system is associated with the drug resistance in Mtb which was spreaded in Xinjiang region.

Specificity of screening of short hairpin RNA targeting Mcl-1 gene in Raw264.7 cells / 中国免疫学杂志

Objective:To transfect Mcl-1shRNA into Raw264.7 cells,and screen out specific shRNA eukaryotic expression plasmids with the most significant effect of silent Mcl-1 gene to figure out the effect of shRNA on Mcl-1 expression in murine macrophage cell line Raw 264.7.Methods: Specific shRNA was transfected into murine macrophage cell line Raw 264.7 via lipofectamine.Semi-quantitative RT-PCR and Western blot were respectively employed to test the changes in Mcl-1 mRNA level and Mcl-1 protein expressions 24 h and 48 h after the transfection ,and the silencing effects of the three pairs of specific shRNA fragments corresponding to different sites were analyzed.Results: Specific shRNA fragments at 24 h and 48 h could effectively reduce Mcl-1 mRNA and protein level ,with higher silencing effects than those of the normal group ,the lipofectamine group and the negative control group.There were statistically significant differences among them ( P<0.05 ).Among the three pairs of specific shRNA fragments corre-sponding to different sites ,Mcl-1 shRNA3 showed the most significant inhibiting effect on Mcl-1 mRNA and proteins.Conclusion:RNA interference can downregulate the level of Mcl-1 mRNA in murine macrophage cell line Raw 264.7 and greatly downregulate the expression of Mcl-1protein.Specific shRNA eukaryotic expression plasmids with the most significant effect of silent Mcl-1 gene have been screened out successfully.

Effects of Mcl-1 silencing on apoptosis of mouse peritoneal macrophages infected with different virulence of Mycobacterium tuberculosis / 中国病理生理杂志

AIM:To investigate the effect of inhibiting Mcl-1 gene expression on apoptosis of mouse peritoneal macrophages infected with different virulence of Mycobacterium tuberculosis using a technique of RNA interference .METH-ODS:The BALB/c mice were infected with prepared bacterium of the virulence strains of Xinjiang , H37Rv, H37Ra and BCG.Mcl-1-shRNA was applied to the mouse model of infection , and the control groups were set up .On 1 d, 3 d, 5 d and 7 d, the mouse peritoneal macrophages were collected .The expression of Mcl-1 at mRNA and protein levels was deter-mined by real-time PCR and Western blot .The apoptotic rate of peritoneal macrophages was analyzed by flow cytometry . RESULTS:The expression of Mcl-1 at mRNA and protein levels was up-regulated in the peritoneal macrophages from the mice infected with different virulence of Mycobacterium tuberculosis, and the cells from the mice infected with virulence strains of Xinjiang and H37Rv expressed higher level of Mcl-1 than the uninfected control cells (P<0.05).The expres-sion of Mcl-1 at mRNA and protein levels was reduced by RNA interference as compared with control group ( P<0.05 ) . Inhibition of Mcl-1 expression induced apoptosis of peritoneal macrophages in the mice .CONCLUSION: The Mcl-1 ex-pression at mRNA and protein levels in mouse peritoneal macrophages infected with different virulence of Mycobacterium tu-berculosis was effectively suppressed by Mcl-1-shRNA, which can induce macrophage apoptosis .

Construction and identification of Mcl-1 gene shRNA expression plasmid in targeted silenced macrophages / 西安交通大学学报(医学版)

Objective To study the expressions of myeloid cell leukemia-1 (Mcl-1 ) gene in mouse macrophages Raw264.7 and human macrophages THP-1,to screen out the cell lines with high levels of expression as the experimental cells,and based on the screening results to construct the short hairpin RNA(shRNA)eukaryotic expression plasmid targeting mice Mcl-1 gene for transfection and further screen out the shRNA expression plasmid with the most obvious effect in silencing Mcl-1 gene.Methods Semi-quantitative PCR method was used to detect the expression of Mcl-1 mRNA in the two kinds of macrophages.Western blotting was adopted to detect the expressions of Mcl-1 proteins in the two kinds of macrophages.Three different gene loci for Mcl-1 shRNA fragments were designed with small molecules interfering RNA (siRNA)software.Eukaryotic expression plasmid Mcl-1 shRNA 1-3 carrying the shRNA fragments was constructed by a company.And the eukaryotic expression plasmid vector was transfected into scavenger cells Raw264.7 mice via through the liposome.The transfection results 24 h and 48 h after transfection were observed under the inverted fluorescence microscope;Mcl-1 mRNA and protein expression were detected by real time quantitative PCR and Western blot,respectively.Results The relative expression levels of Mcl-1 mRNA and protein in mouse macrophages Raw264.7 were significantly higher than those of human macrophages (P <0.05).The shRNA expression vector constructed within 24 h and 48 h could decrease Mcl-1 mRNA and protein levels in Raw264.7 cells,especially with the most obvious silencing effect at 48 h.The 48-h transfection group differed significantly from normal group,liposome group and negative control group (P <0.05).Compared with Mcl-1shRNA1and Mcl-1shRNA2,Mcl-1shRNA3 had the strongest effect in silencing Mcl-1mRNA and protein.Conclusion We have successfully screened out experimental Raw264.7 cells and Mcl-1 shRNA eukaryotic expression plasmid which has an obvious silencing effect targeting on Mcl-1 in mice macrophages Raw264.7.

Correlation study between prokaryotic ubiquitin-like protein (Pup)-proteasome system of Mycobacterium tuberculosis and persistence of Mycobacterium tuber-culosis / 中国免疫学杂志

Objective:To explore the regulation of prokaryotic ubiquitin-like protein ( Pup )-proteasome system on the persistence of Mycobacterium tuberculosis by inducing Mycobacterium tuberculosis to being persistence state under hypoxia conditions and then analyzing the difference on the expression levels of Pup,Dop,PafA and Mpa gene at various time and different conditions. Methods:The total mRNA of the international standard virulent strains of Mycobacterium tuberculosis(H37Rv),which were cultured under hypoxia and aerobic conditions, were extracted from each group at various time and purity of the mRNA were identified.The expression of Pup,Dop,PafA and Mpa genes of M.tuberculosis strains in each group were quantified by SYBR Green I qRT-PCR,which aimed at finding the difference among the expression of Pup,Dop,PafA and Mpa genes at various time and different conditions.Results:The expression levels of Pup, Dop, PafA and Mpa genes in Mycobacterium tuberculosis under hypoxia conditions were measured at various times.The expression levels of Pup,Dop,PafA and Mpa genes:compared with the 0 d,the expression of the Pup gene was up-regulated 1.66,2.43 and 2.76-fold at 4 d,7 d,10 d respectively(P<0.05);the expression of the Dop gene was up-regulated 1.38, 1.91,2.54 and 3.28-fold at 2 d,4 d,7 d,10 d respectively(P<0.05);the expression of the PafA gene was up-regulated 1.22,1.75, 2.37 and 2.67-fold at 2 d,4 d,7 d,10 d respectively( P<0.05);the expression of the Mpa gene was up-regulated 1.66,2.21 and 2.63-fold at 4 d,7 d,10 d respectively(P<0.05).Take the aerobic conditions as control,under hypoxic conditions with the same culture time,the expression of the Pup gene was up-regulated 1.85,2.81 and 2.93-fold in 4 d,7 d,10 d respectively(P<0.05);the ex-pression of the Dop gene was up-regulated 1.20,1.76,2.01 and 3.01-fold in 2 d,4 d,7 d,10 d,respectively( P<0.05);the expression of the PafA gene was up-regulated 1.22,1.57,2.29 and 2.29-fold in 2 d,4 d,7 d,10 d,respectively(P<0.05);the expression of the Mpa gene was up-regulated 1.16,1.58,2.16 and 2.69-fold in 2 d,4 d,7 d,10 d,respectively(P<0.05).Conclusion:Under hypoxic conditions,there were significant differences on the expressions of Pup, Dop, PafA and Mpa genes at various times;what′s more, significant differences on the expressions of Pup,Dop,PafA and Mpa genes exist between hypoxic and aerobic conditions at the same time,Prokaryotic ubiquitin-like protein( Pup)-proteasome system plays a regulatory role on M.tuberculosis′s persistence.

Effect of Mcl-1 signaling pathway blockers on apoptosis of mouse macro-phages infected with Mycobacterium tuberculosis H37Rv / 中国病理生理杂志

[ ABSTRACT] AIM: To explore the effects of Mcl-1 signal pathway blockers on Mcl-1 expression, macrophage apoptosis and Mycobacterium tuberculosis in the model of mice infected with Mycobacterium tuberculosis H37Rv.METH-ODS:A mouse infection model was established by intraperitoneal injection of H37Rv suspension.The signaling pathway blockers AG490, PD98059 and LY294002 for JAK/STAT, MAPK and PI3K, respectively, were intraperitoneally injected into the mice infected with H37Rv.Cell acid-fast staining was used to observe whether the mouse peritoneal macrophages infected with H37Rv were successfully established.Immunocytochemical method was employed to detect Mcl-1 expression in the mouse peritoneal macrophages infected with H37Rv.The apoptotic rate in each group was measured by flow cytomer-ty.The scavenging capacity of apoptotic macrophages against H37Rv was determined by Mycobacterium tuberculosis colony counting.RESULTS:The result of cell acid-fast staining revealed the existence of dispersive arrangement of red short anti-acid Mycobacterium tuberculosis within infected macrophages.The result of cell immunocytochemistry showed strongly posi-tive expression of Mcl-1 protein in H37Rv infection group, AG490 treatment group and LY294002 treatment group, weakly positive expression of Mcl-1 protein in PD98059 treatment group, and negative expression of Mcl-1 protein in control group. The result of flow cytometry found that the macrophage apoptotic rate in H37Rv infection group was higher than that in con-trol group, while that in PD98059 treatment group was high than that in other groups with statistically significant differences (P<0.05).The result of Mycobacterium tuberculosis colony counting showed that PD98059 treatment had the most signifi-cant inhibitory effect on H37Rv strain.CONCLUSION: Mcl-1 signaling pathway blockers increase the apoptotic rate of macrophages infected with Mycobacterium tuberculosis H37Rv and inhibit the growth of Mycobacterium tuberculosis by inhibi-ting the signaling pathways of JAK/STAT, MAPK and PI3K, among which the MAPK has the most obvious interfering effect on Mcl-1, and leads to the highest apoptotic rate of infected macrophages and the strongest bacteriostasis.

Correlation study between Mycobacterium tuberculosis PhoPR two-component system and pathogenicity of different virulent Mycobacterium tuberculosis / 中国免疫学杂志

Objective:To explore the correlation between Mycobacterium tuberculosis PhoPR two-component system and the pathogenicity of different virulent MTB by analysing the expression levels difference of PhoP gene and PhoR gene in BCG ,H37Ra, H37Rv and XJ-MTB respectively.Methods:Total RNA extracted from four different virulent MTB strains and the integrity of total RNA were identified by using agarose gel electrophoresis.The expression of PhoP gene and PhoR gene were quantified by using SYBR Green I FQ-PCR.The expression levels difference of these genes were compared in different virulent MTB strains .Results: The relative expression levels of PhoP gene in between four different virulent MTB strains from high to low were XJ -MTB(9.05),H37Rv(1.00), H37Ra(0.25),BCG(0.08) respectively ,and the expression levels difference of PhoP gene were statistically significant in different virulent MTB strains ( P＜0.05 );the relative expression levels of PhoR gene in four different virulent MTB strains from high to low were XJ-MTB(5.72),H37Rv(1.00),H37Ra(0.18),BCG(0.07) respectively,and the expression levels difference of PhoR gene were sta-tistically significant in different virulent MTB strains ( P＜0.05 ).The expression levels of PhoP gene and PhoR gene at XJ-MTB were statistically significant difference compared with BCG ,H37Rv,H37Ra respectively (P＜0.05);the expression levels of PhoP gene and PhoR gene at H37Rv were statistically significant difference compared with BCG ,H37Ra respectively (P＜0.05);the expression levels of PhoP and PhoR gene at BCG were not statistically significant difference compared with H 37Ra (P>0.05).Conclusion:Significant expression levels difference of PhoP gene and PhoR gene are confirmed in different virulent MTB strains .Therefore,the Mycobacterium tuberculosis PhoPR two-component system is correlated with the pathogenicity in different virulent MTB strains.

Sequential immune’s effects of traditional BCG primary immunization and IL-12 combined with Ag85A DNA booster immunization in mice / 中国人兽共患病学报

The aim of this study is to investigate the immune effects of BCG primary immunization and IL-12 combined with Mycobacterium tuberculosis Ag85A DNA vaccine booster immunization on mice .We randomly divided the mice into 7 groups ,namely PBS negative controls ,BCG controls ,pcAg85A controls ,BCG primary immunization combined with Ag85A booster immunization controls ,BCG primary immunization combined with Ag85A and IL-12 booster immunization controls , BCG primary immunization combined with IL-12 booster immunization controls ,and BCG primary immunization combined with pcDNA3 .1 booster immunization controls .Implementing the immune in procedure of BCG primary immunization and cytokine IL-12 booster immunization combined with mycobacterium tuberculosis Ag85A DNA ,we observed the immune effect on mice by detecting the mice serum total IgG , specific lymphocyte proliferation and cytokine levels in 4 ,6 ,8 weeks after the last immunization .Comparing the mice immunized in the strategy of BCG primary immunization and cytokine IL-12 combined with Mycobacterium tuberculosis Ag85A DNA vaccine strengthening immunization to the mice in other groups by other immune ways ,we found that ,in BCG/Ag85A+ IL-12 groups ,IgG in-creased significantly (P＜ 0 .05) ,specific lymphocyte prolifera-ted significantly ,and after strengthening immunization IFN-γlevels ,IL-2 levels and IL-4 levels in the three periods were 128 .2 ±20.4,190.2±16.51,244.2±39.14 ;146.2±17.29,271.6±16.36and16.36±28.12 ;68.6±6.62,96.6±5.5and5.5± 10 .71 ,respectively ,which were higher than those in other groups (P＜0 .05) .It’s suggested that the immunization way of BCG primary immunization and cytokine IL-12 combined with Mycobacterium tuberculosis Ag85A DNA vaccine booster immu-nization could significantly enhance the humoral and cellular immunity of the bodies ,and provide the basis for further study on protective effect test in animals .

Correlation study between prokaryotic ubiquitin-like protein (Pup)-proteasome and drug resistance of clinical isolates of Mycobacterium tuberculosis / 中国免疫学杂志

Objective:To study the correlation different drug-resistant mycobacterium tuberculosis clinical isolates of prokaryotic ubiquitin-like protein ( Pup )-proteasome of Pup, Dop, PafA, Mpa gene expression level and mycobacterium tuberculosis Pup-proteasome system with Xinjiang region widely popular drug-resistant mycobacterium tuberculosis in clinical isolates resistance.Methods:Total RNA of Mtb was extracted from cultured Mtb during the logarithmic phase in drug-susceptible strains in Xinjiang region,the clinical strains drug sensitive to INH,RFP,SM and EB respectively,and multidrug-resistant(MDR) strains.And then the purity of total RNA was identified.The expressing of Pup-proteasome relevant gene( Pup,Dop,Mpa,PafA) were quantified using SYBR GreenⅠqRT-PCR which aimed at finding the correlation between Mtb Pup-proteasome system and drug resistance of Mtb clinical isolates widespread in Xingjiang region by analyzing the expression of Pup, Dop, PafA, Mpa gene among different isolates.Results:Compared with the drug-senstive clinical isolates, mRNA expression level of Pup, Mpa gene was down-regulated in resistant M.tuberculosis clinical isolates INH ( INH-MTB) ,RFP ( RFP-MTB) ,SM ( SM-MTB) and EB ( EB-MTB) ,mRNA expression levels of genes in Dop and PafA was higher in resistant M.tuberculosis clinical isolates,the difference was statistically significant(P<0.05).Compared with MDR strain, the expression of Pup, Dop, Mpa gene were up-regulated different in the resistant M.tuberculosis clinical isolates isolates ,the expression of PafA gene was down-regulated different,the difference was statistically significant( P<0.05).Conclusion:The differentially expressed gene of Pup、Dop、PafA、Mpa gene in sensitive strains,INH-MTB,RFP-MTB,SM-MTB,EB-MTB and MDR strains.The Mycobacterium tuberculosis Pup-proteasome system.Therefore,the Pup-proteasome system have association with the drug resistance of Mtb strains widespread in Xinjiang region.

PhoP-PhoR two-component signal transduction systems in pathogenesis of Mycobacterium tuberculosis / 中国人兽共患病学报

Two-component signal transduction system (TCS) exists extensively in prokaryotic cell ,which plays a key role of regulation in the growth ,differentiation ,metabolism ,virulence ,persistence ,and pathogenicity .PhoPR two-component signal transduction system ,as one of two-component systems ,is the most basic and the most importantly sensitive to the envi-ronmental changes and makes corresponding certain reaction system adapt to changes in the host microenvironment .Therefore , PhoPR TCS which is an important regulatory system of Mycobacterium tuberculosis (MTB) to adapt to environmental change has been increasing concern ,even PhoPR TCS is becoming the new front-burner issue in the pathogenesis of M TB regulation of research .

Study progress of prokaryotic ubiquitin-like protein (Pup)-proteasome system of My cobacterium tuberculosis / 中国人兽共患病学报

Proteasome pathway is another major pathway of protein degradation in addition to lysosome in eukaryotic cell ,which involved a number of physiological functions regulation in cell .Prokaryotic ubiquitin-like protein was found in My-cobacterium tuberculosis in 2008 .With the effect of co-factor Dop ,PafA and Mpa ,Pup can mark a variety of protein ubiquitina-tion followed by importing them into proteasomal degradation .The target protein of Pup-proteasome system like FabD ,PanB , Ino1 ,Icl ,SodA ,and MtrA are involved with metabolism ,signal transduction pathways ,virulence factors ,pathogenicity and the persistence of bacteria in the host cell .Proteasome inhibitor make the function of proteasome restricted and the accumula-tion of Pup’s labeled substrate result in changes in the expression of gene indirectly ,which impacted the ability of resistance to outside pressure and the pathogenicity of Mycobacterium tuberculosis . The finding Pup-proteasome system reveals a novel mechanism of protein degradation in prokaryotes ,which is expected to become a new target of treatment of anti-TB drugs . Here ,we summarize the progress on the Pup-proteasome system in Mycobacterium tuberculosis .

Effect on expression of macrophages ferroportin and ferritin in mouse alveolar macrophages by mycobacterium tuberculosis / 中国免疫学杂志

Objective:To discuss the change of ferritin ( Fn) and ferroportin expression quantity and time-related feature in the alveolar macrophages of mice , infected with different virulence of Mycobacterium Tuberculosis infected .Methods:The prepared bacte-ria of H37Rv or BCG were injected intravenously into the mice tails .On the day 1, 3, 5, 7, 9, 11, 13 and 15, the lavage fluids were collected and the alveolar macrophages were obtained from each group of mice .The expression of FPN and Fn were detected with ELISA and /or Western blot analysis .Results:The expression of Fn in the group of either H 37Rv or BCG infected mice was decreased on the day 7, 9 and 11, and was lowest on the day 7, which showed significantly statistical difference compared to that on the other days (P<0.05).The expression of FNP in the infected mouse macrophage was decreased gradually , which was obvious on the day 5. The expression levels reached to the lowest on the day 7 and 9.The expression was much lower than that in the negative control group (P<0.05).Conclusion:The expression of Fn and FPN in macrophages isolated from lungs of mice infected with Mycobacterium tu -berculosis H37Rv or BCG become decreased , and there is no difference between these two infected mouse groups .

Relationship between expression of Mycobacteriumtuberculosis Hsp16.3 and apoptosis of infected mouse alveolar macrophages / 西安交通大学学报(医学版)

Objective To study the relationship between the expression of Mycobacterium tuberculosis small heat shock protein Hsp16.3 and the apoptosis of infected mouse alveolar macrophages.Methods The laboratory mice were infected with bacterial suspension of the international standard virulent strain of Mycobacterium tuberculosis H37Rv strains (H37Rv),Hsp16.3 gene deletion mutants of the international standard virulent of Mycobacterium tuberculosis H37Rv strains(△H37Rv),or sterile saline solution (normal control)by the tail vein. After successful replication of mouse infection model in each group,we cleaved the alveolus of each group of mice and collected lavage fluid to obtain alveolar macrophages of the infected mice at days 1 ,3 ,5 ,7 ,9 ,1 1 ,1 3 and 1 5 .Then the infection status of macrophages was observed with confocal laser scanning microscopy;flow cytometry was used to detect the apoptosis rate of alveolar macrophages of the infected mice;Caspase-3 and Bcl-2 expressions were examined by Western blot.Results The apoptosis rate of Hsp16.3 gene was higher in deletion strain (△H37Rv)group and H37Rv strains (H37Rv)group than in control group.The apoptosis rate of alveolar macrophages in △ H37Rv group gradually increased,peaked at day 7 ,and then gradually decreased.It was significantly higher in H3 7 Rv group than in H3 7 Rv strain group from day 1 to 7 and from day 1 3 to 1 5 (P<0 .0 5 ).Caspase-3 and Bcl-2 protein expressions in the macrophages of△H37Rv group and H37Rv group were higher than those of control group.Caspase-3 expression in the microphages of △H3 7 Rv group and H3 7 Rv group gradually increased from day 1 to 7 and peaked at day 7;it peaked again at day 13 in H37Rv group.However,Caspase-3 expression remained significantly higher in△H37Rv group than in H3 7 Rv group (P<0 .0 5 ).Bcl-2 expression in △H3 7 Rv group did not change much at the early stage of infection (P<0 .0 5 ),but gradually increased after day 9 .Bcl-2 expression in H3 7 Rv group did did not change much from day 1 to 7 (P<0.05),but gradually increased after day 7.However,it remained lower in△H37Rv group than in H37Rv group,especially after 7 days(P<0.05).Conclusion Mycobacterium tuberculosis small heat shock protein Hsp16.3 can inhibit the apoptosis of macrophages during the early and late stages of infection,and this inhibition may be achieved by inhibiting the expression of apoptotic protease Caspase-3 and promoting the expression of Bcl-2 protein.

Correlation study between Mycobacterium tuberculosis PhoPR two-component system and drug resist-ance of widespread clinical isolates of Mycobacterium tuberculosis / 中华微生物学和免疫学杂志

Objective To explore the correlation between Mycobacterium tuberculosis ( MTB ) PhoPR two-component system and drug resistance of MTB clinical isolates widespread in Xinjiang region by analyzing the expression of PhoP gene and PhoR gene among different isolates .Methods Total RNA of MTB was extracted from drug-susceptible strains , the strains only resistant to a single first-line anti-TB drugs (INH, RFP, SM and EB) and multidrug-resistant (MDR) strains, respectively.The purity of total RNA was checked by agarose gel electrophoresis .The expressions of PhoP gene and PhoR gene were quantified by using SYBR Green I qRT-PCR and the differences of their gene expression in different isolates were ana-lyzed.Results Compared with the drug-susceptible strains of MTB, the expression of PhoP gene was up-regulated for about 1.48 times in MTB strains resistant to RFP (RFP-MTB) and 2.74 times in MDR strain (P<0.05).Compared with MDR strain, the expressions of PhoP gene in the isolates resistant to INH (IN-HMTB), RFP (RFP-MTB), SM (SM-MTB) and EB (EB-MTB) were down-regulated for 0.70, 0.50, 0.25 and 0.21 times respectively.The expressions of PhoR gene were down-regulated for 0.36, 0.54, 0.35 and 0.19 times, respectively (P<0.05).The expressions of PhoR gene in the isolates of INH-MTB, RFP-MTB, SM-MTB and EB-MTB were up-regulated for 6.33, 4.56, 2.34, 1.85 and 9.06 times, respectively as compared with the drug-susceptible strains (P<0.05).Conclusion Significant differences of PhoR gene and PhoP gene expressions were observed among drug-susceptible strains , INH-MTB, RFP-MTB, SM-MTB, EB-MTB and MDR strains.Therefore, the Mycobacterium tuberculosis PhoPR two-component system is asso-ciated with the drug resistance of MTB strains prevalent in Xinjiang region .

Study on the association of 3'UTR polymorphisms of NRAMP1 gene with susceptibility to tuberculosis in Uighurs / 中华微生物学和免疫学杂志

Objective To determine whether 3'UTR polymorphisms of the NRAMP1 gene are as-sociated with tuberculosis in Uighurs. Methods 3'UTR polymorphisms of NRAMP1 gene were typed by PCR-RFLP among 224 patients with active tuberculosis and 225 healthy individuals. The relationship be-tween 3'UTR polymorphisms and susceptibility to tuberculosis was studied, and cases were grouped accord-ing to genotypes. Results In the tuberculosis patients, genotype TGTG/TGTG,TGTG/TGTG deleted, and TGTG deleted/TGTG deleted were observed in 159,56 and 9 cases respectively, while the genotypes of the healthy controls were TGTG/TGTG in 185, TGTG/TGTG deleted in 36 and TGTG deleted/TGTG deleted in 4 case. The frequency of the genotype TGTG/TGTG was found more often among controls than that in pa-tients (X2=7.94 ,P <0.01). The frequency of allele TGTG and the frequency of variant allele were 0.87 and 0.13 respectively. Conclusion 3'UTR polymorphisms of NRAMP1 gene are associated with suscepti-bility to tuberculosis in Uighurs.

Screening of Mycobacterium tuberculosis distinctive genes by suppression subtractive hybridization technique / 中华微生物学和免疫学杂志

Objective To screen differential Mycobacterium tuberculosis genes between Xinjiang clinical strains and H37Rv by suppression subtractive hybridization( SSH), and to analyze the function of these specifically pathopoiesis genes. Methods Both M. tuberculosis Xinjiang clinical strains and H37Rv as tester and driver each other, most identical genome was drived whereas some distinctive genes was re-mained and enriched by utilization SSH technique. Meanwhile through inserting differential genes to E. coli all of sequences that we have cloned were determined by BLAST in GenBank. The function of differential genes between M. tuberculosis H37Rv and Xinjiang clinical strains were analyzed. Results We cloned and analyzed six different DNA fragments that only existed in Xinjiang clinical strains. One is the fragment of a gene ceding monooxygenase, flavin-binding family identified by Glimmer2. One fragment belongs to acyl-transferase family protein. One for aminotransferase, class Ⅱ, acyl carrier protein. One fragment belongs to chromosomal replication initiator protein DNA and one for M. tuberculosis paralogous family 11-pyridoxamine 5'-phosphate oxidase-related. Meanwhile, we cloned ten DNA fragments only in H37Rv. Conclusion SSH technique can efficiently screen differential genes in M. tuberculosis in Xinjiang clinical strains. They are possible key genes that M. tuberculosis survive and fortify virulence in mal-environment as same as their ho-mogenic genes, such as enhanced adsorbability in wall-held protein, counteracted digestion by nitro-oxygen-ase, elevated composition capability in the acyhransferase, control chromosomal replication initiator protein, synthesized aminotransferase acyl cartier protein and pyridoxamine 5'-phosphate oxidase.

Effects of transforming growth factor 1 and hypoxia on the expression of vascular endothelial growth factor for Hepatoma cells / 基础医学与临床

Objective To observe the effect of Vascular Endothelial Growth Factor(VEGF) to the expression in hepatoma cells affected by TGF-?1 and hopxia. Methods HepG2 cells were cultured in vitro, and treated with different doses of TGF-?1 and Cobalt chloride hexahydrate(CoCl2), or with TGF-?1 and CoCl2. The change of VEGF protein and mRNA was detected by immunohistochemistry and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Results Immunohistochemistry and RT-PCR showed that the expression of VEGF protein and mRNA were significantly higher in TGF-?1 groups or CoCl2 groups than that of control group(P

Preliminary study on detecting drug-resistance mycobacterium tuberculosis by using DNA microarray hybridization / 中国病理生理杂志

AIM:To detect the drug-resistance mycobacterium tuberculosis by using DNA microarray hybridization.METHODS: DNA microarray for detecting drug-resistance of mycobacterium tuberculosis was prepared; Clinical isolated strains were cultivated and their drug-resistance sensitivity was detected. The genome DNA of mycobacterium tuberculosis was prepared and the drug-resistance genes of the mycobacterium tuberculosis were amplified by PCR. Then the gene chip was hybridized, washed, detected and analyzed. RESULTS: Results of cultivating mycobacterium tuberculosis and detecting the drug-resistance sensitivity: one strain was drug-sensitive; four strains were multi-drug-resistant; The detecting results of the drug-resistance was consistent with the results of diagnosis therapy of the 5 clinical patients. The detecting results of gene chip confirmed the above facts. CONCLUSION: Detecting drug-resistance mycobacterium tuberculosis by the gene chip is precise, fast and highly-efficient.