Small non-coding RNA (sRNA) is a kind of newly discovered 50 nt~500 nt small RNAs that do not encode proteins. To date, more than 150 sRNA have been found in bacteria. The small RNA acting by base-pairing with target mRNAs, resulting in post-transcriptionally regulating gene expression, is important regulators in the bacterial response to stress, virulence and metabolism. At present, researches of sRNA mainly based on bioinformatical prediction and molecular biological experiments. The sRNA that obtained through these methods needs confirmation in laboratory, and then study of its functions through a variety of experimental methods.

Objective To investigate the fluorescence quantitative PCR (FQ-PCR) based on TaqMan-MGB( Minor Groove Binder) technique for quantification of fetal DNA in maternal plasma and its variation during pregnancy. Methods Maternal DNA extracted from 237 plasma samples obtained from 30 pregnant women (5-40 gestational weeks and post delivery). The TaqMan-MGB probe and SRY primers were designed to amplify the SRY gene sequence of Y chromosome in maternal plasma by FQ-PCR. Results This system was sensitive enough to detect a male DNA among 20 000 female DNA. Fetal DNA can be detected in maternal plasma as early as 6+6 weeks of gestation and increased with the pregnant progress with the peak level at the third trimester. Between 24- 48 h after delivery, the SRY gene was negative in maternal plasma. The percentage of fetal DNA concentration in maternal total plasma DNA was 4. 88% in the first trimester, 6. 10% in the second and 4. 77% in the third trimester. The SRY positive signal was obtained from samples of 13 women bearing male fetuses and no signal was detected for all of the 17 women bearing female fetuses. Conclusions The FQ-PCR for quantification of fetal DNA in maternal plasma is highly sensitive, specific and reliable. Fetal DNA does present in maternal plasma at a higher concentraton. FQ-PCR may be useful in nonin-vasive prenatal diagnosis.

Objective:To study the effect of HLA-Gon proliferation in peripheral blood lymphocytes of childbearing age women and Treg cell subsets,and investigate the mechanisms of immune tolerance in pregnancy.Methods:The high expression of HLA-G cho-riocarcinoma cell lines JEG-3 cells with peripheral blood lymphocytes( PBLC) of healthy childbearing age women co-culture,using the HLA-G neutralizing antibodies(87G)and recombinant human tumor necrosis factor receptor typeⅡ-antibody fusion protein(rhTNFR:Fc)to intervene.Experiments were divided into seven groups:①JEG-3+PBLC culture group;②JEG-3+PBLC+87G culture group;③JEG-3+PBLC non-contact culture group;④JEG-3+PBLC+87G non-contact culture group;⑤The control group(PBLC group);⑥JEG-3+PBLC+rhTNFR:Fc culture group;⑦JEG-3+PBLC+87G+rhTNFR:Fc culture group.Detected the PBLC proliferation inhibition by CCK-8 method and the expression of TNF-αmRNA by RT-PCR in①-⑤groups.The proportion of Treg cells were detected by flow cytometry in①-⑦groups.Results:The assay of CCK-8 showed that the PBLC proliferation inhibition rate of JEG-3+PBLC culture group,JEG-3 +PBLC+87G culture group,JEG-3+PBLC non-contact culture group,and JEG-3+PBLC+87G non-contact culture group were(48.00±5.56)%,(14.67±4.04)%,(37.67±2.31)% and(8.33±3.21)%,there was a statistically significant difference on each group ( P<0.05 ).The result of RT-PCR showed that the expression of TNF-αmRNA in JEG-3+PBLC culture group and JEG-3+PBLC non-contact culture group were significantly lower than the control group.Compared with the corresponding non-87G group,the expression of TNF-αmRNA increased significantly after the intervention of 87G,P<0.05.Compared with the control group, the proportion of Treg cells of JEG-3+PBLC culture group detected by flow cytometry was significantly increased(P<0.05).There was no significant difference between JEG-3+PBLC non-contact culture group and control group ( P>0.05 ).Compared with the JEG-3+PBLC culture group,the proportion of Treg cells of JEG-3+PBLC+87G culture group was significantly decreased(P<0.05).Compared with JEG-3+PBLC culture group,the proportion of Treg cells of JEG-3+PBLC+rhTNFR:Fc culture group was significantly increased( P<0.05).Set JEG-3+PBLC+rhTNFR:Fc culture group as control,the proportion of Treg cells of JEG-3+PBLC+rh TNF:FC+87G culture was significantly decreased,but obviously higher than JEG-3+PBLC+87G culture group,there was a statistically significant difference on each group(P<0.05).Conclusion:HLA-G can inhibit peripheral blood lymphocyte proliferation of childbearing age women and inhibit the expression of TNF-α,and up-regulate the proportion of Treg cells.

Objective To make sure whether there is any association between genetic polymorphism of tumor necrosis factor (TNF) ? and systemic lupus erythematosus (SLE).Method PCR RFLP was used.A population based and family based study was carried out in 99 SLE patients,116 health controls and 12 families.Results The TNF ?2 allele frequency of SLE patients was significantly different from that of controls ( P

Objective To investigate the role of bacterial flagellin and CD98 in ulcerative colitis (UC).Methods A total of 60 first episode patients with active UC were recruited,including 30 mild and 30 moderate to severe UC cases.The serum of 30 healthy volunteers and normal intestinal tissues surgically removed from 15 colon cancer patients (more than 5 cm away from surgical margins) were collected as control.The content of bacterial flagellin antibodies in peripheral blood were measured with enzyme-linked immunosorbent assay (ELISA).The expression of CD98 in peripheral blood T lymphocyte was measured by flow cytometry (FACS).The expression of bacterial flagellin protein in intestinal mucosa and CD98 in intestinal epithelial basement membrane was tested by immunohistochemistry (IHC).The comparison between two groups was performed with the SNK-q method,the R×C table x2 test was used to analyze the counted data,and the Spearman correlation was used to analyze the rank materials.Results The peripheral blood concentration of bacteria flagella protein antibody of control group,mild UC group and moderate to severe group showed an upward trend,which was (7.603±2.118) pg/ml,(13.702±3.131) pg/ml and (20.813±3.004) pg/ml respectively,and the differences among groups were statistically significant (F=13.57,P＜0.01).The expression percentage of bacteria flagella protein in intestinal mucosa of the three groups also showed an upward trend,which was 3/15,56.67％ and 73.33％ respectively,and the differences among groups were statistically significant (x2 =11.553,P=0.003).The positive rate of CD98 expression in peripheral blood T lymphocytes of the three groups showed an upward trend,which was (28.42±4.31)％,(32.45±6.71)％ and (43.40±5.09) ％ respectively,and the differences among groups were statistically significant (x2 =10.110,P=0.007).The positive rate of CD98 expression in intestinal epithelial cells of the three groups also showed an upward trend,which was 1/15,36.67 ％ and 66.67％ respectively,and the differences among groups were statistically significant (x2 =5.400,P＜0.05).There was positive correlation between the peripheral blood concentration of bacteria flagella protein antibody and the expression of CD98 in peripheral blood T lymphocytes (r=0.548,P＜0.05).Conclusion Bacterial flagellin and CD98 may be important factors causing inflammatory reaction activity in UC.

ObjectiveTo study the influence of anti-tuberculosis drugs on mitochondrial function in mice hepatocytes and to explore the mechanism of the anti-tuberculosis drugs induced liver injury.Methods A total of 150 mice were randomized into five groups:control group (C group),rifampin (RFP) group,isoniazid (INH) group,pyrazinamide (PZA) group and three antituberculosis drug combination group (MIX).The mice were administered intragastrically with 0.9 ％ NaC1 solution or RFP 135 mg · kg-1 · d-1 or INH 90 mg · kg-1 · d-1 or PZA 315 mg · kg-1 · d-1 or RFP+INH+ PZA (135±90+315) mg · kg-1 · d-1 once a day.Ten mice in each group were sacrificed at day 3,7 and 15 of administration,respectively.The following parameters in each group were monitored.the concentration of malondialdehyde (MDA),the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) in mitochondrion of hepatocytes and the concentration of 8-hydroxydeoxyguanosine (8-OH-dG) in mitochondrial DNA (mtDNA).The data were analyzed by one-way ANOVA or rank sum test.Results Along with the prolonged medication duration,the concentrations of MDA all gradually increased in RFP group (Z=6.020,P=0.049),IN H group (Z=10.220,P=0.006) and MIX group (Z=7.460,P=0.024),whereas the activity of SOD significantly decreased in RFP group (F=6.751,P =0.011 ) and MIX groups (F=4.891,P =0.041 ) compared with control group and PZA group.Meanwhile,the activity of GSH-PX was significantly lower in RFP group compared to the other groups (F=32.445,P＜0.01).The changes of other parameters didn't show meaningful trend.The concentrations of 8-OH-dG in mtDNA also increased in all treated groups,and those were all significantly increased in RPF group (F=6.602,P＜0.01 ),PZA group (F=5.927,P＜0.01) and MIX groups (F=7.974,P＜0.01).Conclusions Antituberculosis drugs can induce higher MDA concentration in mitochondrion and higher 8-OH-dG concentration in mtDNA,while result in lower activities of SOD and GSH-PX.The liver damage tends to become more severe along with the prolonged medication duration.The combination of three antituberculosis drugs could aggravate the damage of mitochondrion in mice hepatocytes.

Objective To investigate the relationship between polymorphisms of N-acetyltransferase 2(NAT2)genes and anti-tuberculosis drug induced hepatic-injury(ADIH).Methods A 1:1 matched case-control study was conducted.One hundred and six cases fulfilling the criteria of ADIH were selected as ADIH group from the patients who received anti-tuberculosis therapy.whereas those patients without any hepatic inj ury related elinical symptoms during three months of follow-up period were selected as control.The genetic polymorphisms of the loci,NAT2481C/T,NAT2-590G/A and NAT2-857G/A,were determined by polymerase chain reaction and restriction fragment length polymorphism technique(PCR-RFLP)in patients who received antituberculosis therapy.The major environmental factors and genotypes were analyzed by univariate and multivariate conditional Logistic analyses.Results The T,AA allele frequencies of NAT2-481C/T,NAT2-590G/A and NAT2-857G/A were 7.5%,28.8%and 17.9%respectively in ADIH group,and 6.6%,18.9%and 17.5%,respectively in the control group.Univariate analysis demonstrated that the frequency of NAT2 slow acetylation genotype in ADIH group was significantly higher than that in control group with a crude OR(95%CI)of 2.250(1.140-4.441).Among 6 potential risk factors,i.e.education level,occupation,body mass index(BMI),smoking,drinking and the type of tuberculosis,the low BMI and drinking were two risk factors for ADIH.In multivariate analysis,ADIH remained associated with acetylation genotype after adjusting for BMI and drinking status.The adjusted OR(95%CI)was 2.246(1.086-4.644).Conclusion NAT2 slow acetylation genotype may be associated with the occurrence of ADIH.

OBJECTlVE To study the reIationship between microRNA(miRNA)-122 and Iiver injury in-duced by anti-tubercuIosis drugs,and to discover the new biomarkers for earIy diagnosis of this type of Iiver injury. METHODS mice were given 2 mL isoniazid oraIIy at 90 mg·kg-1 . BIood and Iiver tissue sampIes were coIIected at 1,3,5,7,14,21 and 28 d after administration of isoniazid. Serum gIutamic-pyruvic transaminase( GPT)and gIutamic-oxaIoacetic transaminase( GOT)IeveIs were determined using an automatic biochemicaI anaIyzer. Cu/ Zn-superoxide dismutase( Cu/ Zn-SOD ) activity and maIondiaIdehyde( mDA)content were detected by biochemicaI method. ReaI-time qPCR was used to measure the expression of miRNA-122. RESULTS GPT and GOT IeveIs were significantIy higher at 14 and 21 d(P﹤0.05)than in the controI. Cu/ Zn-SOD began to decIine whiIe mDA began to increase after 5 d(P﹤0.05). miRNA-122,which progressiveIy decreased after administration,was reduced to the mini-mum 0.58 ±0.02 at 14 d. There were good correIations between miRNA-122 and GPT,Cu/ Zn-SOD, mDA(the correIation coefficients were -0.370,0.268,and -0.298,respectiveIy),but no correIation with GOT was observed. CONCLUSlON The tissue miRNA-122 profiIe can be used as a sensitive marker for anti-tubercuIosis drug-induced Iiver injury,which couId contribute to the earIy diagnosis of Iiver injury.

OBJECTIVETo study the genetic polymorphisms of UDP-glucuronosyltransferase 1F(UGT1F) and the relationship between polymorphisms and susceptibility to hepatocellular carcinoma (HCC).

METHODSThe polymorphisms of UGT1F of 84 patients with HCC and 144 healthy controls were detected by PCR-denaturation gradient gel electrophoresis-sequencing or PCR-single strain conformation polymorphsim-sequencing.

RESULTSThree new single nucleotide polymorphisms(SNP) were found: the first one was a transversion of TrarrG at nucleotide 232; the second one was the transition of ArarrG at nucleotide 528 in exon 1; the last one was the transition of ArarrG at nucleotide 376 in intron 2. Additionally, the polymorphism at nucleotide 754 was proved in this study. The frequencies of genotype and allele of 4 loci in cases and controls were analyzed. Both frequencies of genotype G/G(13.10%) and allele G (29.17%) of position 754 of UGT1F in cases were sig nificantly greater than those in controls (2.78% and 19.44% ) respectively. For other loci, the difference between the two groups were not significant.

CONCLUSIONExons 2-5 of UGT1F are highly conservative, but exon 1 emerges highly polymorphic. And the polymorphism at locus 754 may be related with HCC.

Alleles ; Amino Acid Substitution ; Base Sequence ; Carcinoma, Hepatocellular ; enzymology ; genetics ; DNA Mutational Analysis ; DNA, Neoplasm ; chemistry ; genetics ; Gene Frequency ; Genetic Predisposition to Disease ; genetics ; Genotype ; Glucuronosyltransferase ; genetics ; Humans ; Liver Neoplasms ; enzymology ; genetics ; Odds Ratio ; Polymorphism, Single Nucleotide ; Polymorphism, Single-Stranded Conformational

Objective To investigate whether the gene polymorphisms of cytochrome P450(CYP) 2E1 are associated with the risk of anti-tuberculosis drug induced hepatotoxity (ADIH).Methods In this case control study, 339 patients who matched the diagnosis criteria of tuberculosis were included. The gcneral healthy status and liver biochemical parameters were checked in all these patients. Polymerase chain reaction-restriction fragment length polymorphism (PCR RFLP) technique was used to determine CYP 2Et polymorphisms. The statistic analysis were performed by using both univariate and multivariate Logistic regression analysis. Results The allele frequencies of CYP 2E1 7632T/A, 1019C/T and 1259G/C in 103 tuberculosis patients of ADIH group were 17.5%, 26.2%and 27.2 % respectively, while those in 236 tuberculosis patients of control group were 29.7 % ,39.4 %and 40.7%, respectively (x2 =5.539, P＜0.05; x2 =5.458, P＜0.05; x2 =5.628, P＜0.05). The results of univariate analysis demonstrated that the risk of concurrent ADIH was significantly higher in patients with wild genotypes of CYP 2E1-7632T/A, CYP 2E1-1259G/C, CYP 2E1-1019C/T than in patients with other genotypes. After adjusted for sex, occupation and alcohol consumption status, the results of multivariate Logistic regression analysis also showed that wild genotypes of CYP 2E1-7632T/A, CYP 2El-1259G/C, CYP 2E1-1019C/T were significantly associated with higher risk of ADIH. The results of interaction analysis indicated that the wild genotypes of CYP 2E1-7632T/A and CYP 2E1-1259G/C or CYP 2E1-1019C/T had synergetic effects on the development of ADIH.Conclusions The risk of concurrent ADIH is significantly higher in patients with wild genotypes of CYP 2E1-7632T/A, CYP 2E1-1259G/C, CYP 2E1-1019C/T compared to patients with othergenotypes. Wild genotypes of CYP 2E1-7632T/A and CYP 2E1-1259G/C or CYP 2El-1019C/T have synergetic effects on the development of ADIH.