[The Mechanisms of Trimetazidine Alleviating the Oxidative Stress in Adipose-derived Mesenchymal Stem Cells].

OBJECTIVES: To investigate the effects of trimetazidine (TMZ) on the oxidative stress injury in adipose-derived mesenchymal stem cells (ADSCs). METHODS: ADSCs derived from adipose tissue of SD rats were characterized by flow cytometry and multiline age differentiation. ADSCs apoptosis was induced by H2O2 in vitro , Dirrerent concentration of TMZ (250 µmol/L, 500 µmol/L) was used to protect ADSCs from apoptosis. The morphological features of apoptotic ADSCs were analyzed by Hoechst 33342, mitochondrial potential and structure was analyzed by JC-1 staining and electron microscope, respectively. The apoptotic proteins were detected by Western blot. The effect of TMZ on antioxidant capacity of ADSCs was evaluated by detecting reactive oxygen species (ROS), superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde (MDA). RESULTS: The isolated ADSCs expressed high levels of CD29 and CD90, low levels of CD34 and CD45 and no expression of CD31. ADSCs could be induced to adipocyte and osteoplastic cells. After being treated by H2O2, ADSCs displayed apoptosis characteristics with increased number of apoptotic cells, decreased mitochondrial transmembrane potential and damaged mitochondria. The expressions of apoptotic proteins, including Bax, Bad, and Caspase3, were dramatically increased compared to the controls; however, the anti-apoptotic protein Bcl2 was decreased. At the meantime, the contents of ROS and MDA were elevated, but the concentrations of SOD and GSH were reduced. The treatment of TMZ could partly reverse above negative impacts to ADSCs. CONCLUSION: TMZ could improve the survival rate of ADSCs by enhancing anti-oxidant defense systems to remove excessive ROS and regulating the expression of protective protein.

Genetic variants of CDH13 determine the susceptibility to chronic obstructive pulmonary disease in a Chinese population.

AIM: Adiponectin has been implicated in the development of chronic obstructive pulmonary disease (COPD). The CDH13 gene encodes T-cadherin that is an adiponectin receptor, and genetic variants of CDH13 determine blood adiponectin levels. The aim of this study was to investigate the effects of CDH13 variants on COPD susceptibility in a Chinese population. METHODS: Ten single-nucleotide polymorphisms (SNP) in CDH13 were screened using the SNaPshot method in 279 COPD patients and 367 control subjects. Association of genotypes or haplotypes constructed from these loci with COPD was analyzed in different genetic models. RESULTS: Among the 10 SNPs tested, rs4783244 and rs12922394 exhibited significant differences in allele or genotype frequencies between COPD patients and control subjects, whereas 8 other SNPs did not. The minor allele T was associated with decreased risk of COPD in the recessive model at rs4783244 (OR=0.42, P=0.023) and in the dominant model at rs12922394 (OR=0.70, P=0.022). The genotype TT at either rs4783244 or rs12922394 was associated with a significantly low level of plasma adiponectin when compared to genotypes GG and CC (P<0.05). Haplotypes GC in block 1 (rs4783244-rs12922394) as well as GTAC and ATGT in block 3 (rs4783266-rs11640522-rs11646849-rs11860282) significantly increased the risk of COPD, whereas haplotypes TT in block 1, TG in block 2 (rs11646011- rs11640875) and ATGC in block 3 were protective against COPD. CONCLUSION: CDH13 genetic variants determine Chinese individuals' susceptibility to COPD and thus are efficient genetic biomarkers for early detection of COPD.

Association between fibroblast growth factor 7 and the risk of chronic obstructive pulmonary disease.

AIM: Fibroblast growth factor 7 (FGF7) is involved in a number of physiological and pathological processes, including lung disease. However, relatively little is known about the effect of FGF7 gene polymorphisms on chronic obstructive pulmonary disease (COPD) susceptibility. This study aimed to investigate the association between FGF7 polymorphisms with COPD susceptibility in a Chinese Han population. METHODS: We conducted a case-control study of 279 COPD patients and 367 age- and gender-distribution-matched control subjects. The tagging SNPs rs10519225 and rs7170426 in FGF7 were genotyped by SNaPshot. The associations of each SNP genotype and haplotype constructed by these loci with COPD were analyzed. RESULTS: A multivariate analysis showed that rs10519225 was significantly associated with an increased risk of COPD (P=0.011, OR=1.535, FDR q=0.022), whereas no association was found for rs7170426. Linkage disequilibrium (LD) analysis showed that these loci were in weak LD, with an r(2) of 0.033 and a D' of 0.232 (95% CI: 0.150-0.520). The haplotype constructed by allele G at rs10519225 and allele A at rs7170426 was associated with a decreased susceptibility to COPD (P=0.012, OR=0.751, FDR q=0.048). CONCLUSION: These findings suggest that FGF7 may be one susceptibility factor for COPD.

[A study on the haplotype of the solute carrier family 11 member 1 gene in Tibetan patients with pulmonary tuberculosis in China].

OBJECTIVE: To investigate the association of the haplotype of the solute carrier family 11 member 1 (SLC11A1) gene with susceptibility to pulmonary tuberculosis in Tibetans. METHODS: Four polymorphisms of the SLC11A1 gene [5' (GT)n, INT4, D53N, and 3' UTR] were investigated by denaturalization high performance liquid chromatography and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 140 patients (the patient group) and 139 PPD-positive healthy controls (the control group) of the Tibetan nationality from June 2004 until January 2005. The relationship between the haplotype and susceptibility to pulmonary tuberculosis in these patients was studied by chi2 test, and the linkage disequilibrium as well as the haplotype were analyzed by the SHESIS software. RESULTS: The haplotype frequencies of 5'(GT)9/INT4 G, 3'UTR TGTG/D543N G, 3'UTR TGTG del/D543N A were 64.8% (181/280), 76.6% (215/280), 12.0% (34/280) among the patients and 78.1% (217/276), 84.4% (235/276), 6.4% (18/276) among the controls. 5' (GT)9/INT4 G, 3'UTR TGTG/D543N G haplotypes rendered a lower risk (chi2 = 11.026, P<0.01, chi2 = 6.547, P<0.05, respectively), but 3'UTR TGTG del/D543N A haplotype a higher risk (chi2 = 6.547, P<0.05) for tuberculosis. CONCLUSIONS: 5' (GT)9/INT4 G, 3'UTR TGTG/D543N G and 3'UTR TGTG del/D543N A haplotypes of the SLC11A1 gene may be associated with the susceptibility of the Tibetan population to pulmonary tuberculosis.

[Study on the association of two polymorphisms of the vitamin D receptor (VDR) gene with the susceptibility to pulmonary tuberculosis (PTB) in Chinese Tibetans].

OBJECTIVE: To investigate the relationship between Fok I and Taq I polymorphisms of the VDR gene and the susceptibility of PTB in Chinese Tibetans. METHODS: Two polymorphisms of the VDR gene were typed by PCR-RFLP among 140 patients and 139 healthy controls who had been sensitized to tuberculosis. The relationship among two polymorphisms and the susceptibility of PTB were studied, and cases were grouped according to the genotypes. RESULTS: The genotype FF, Ff, ff were observed in 60, 56 and 24 cases among tuberculosis group while in 70, 60 and 9 cases among control group. A significant increase of VDR genotype ff (mutant homozygotes) was observed in the PTB patients when compared to controls (P < 0.05, OR 3.111, 95% confidence interval 1.343-7.207). The genotype of TT were in 137 cases, Tt in 3 cases among patients while TT in 134 cases , Tt in 5 cases among controls. No significant difference in these genotype frequencies was observed between two groups (P > 0.05, OR 1.704, 95% confidence interval 0.399-7.272). Two polymorphisms didn't show linkage disequilibrium and D' is 0.475. CONCLUSION: The Fok I polymorphism of VDR gene might be associated with PTB in Chinese Tibetan population, but no evidences supported that the Taq I polymorphism were associated with PTB in Chinese Tibetan. The two polymorphisms didn't show linkage disequilibrium.

[A method of haplotype analysis for multiple single-nucleotide polymorphisms].

Haplotypes from multiple single nucleotide polymorphisms(SNPs) spaced in longer DNA were constructed by multi-step PCR and DNA sequencing methods. Two allele-specific primers were synthesized and used for long DNA fragments (approximately 10 kb) amplification from human genome DNA. Fragments within these long DNA fragments were amplified by using these PCR products as templates in the second round PCR. The second round PCR products were subsequently sequenced. Haplotype construction was performed based on the character of nucleotides at the 3'-end of the allele-specific primers and the sequencing results from the second round PCR products. The DNA fragment (approximately 16 kb) from promoter to exon 4 of lipoprotein lipase (LPL) gene was amplified by allele-specific primers,and the DNA fragments including exon 2 or exon 3 of LPL gene were amplified and sequenced. A SNP of +13,557G-->A within intron 2 was identified. Four haplotypes including -421 G/ +13557G/ + 15222A, - 421A/ +13557G/ +15222A, -421G/ +13557G/ + 15222G, - 421G/+ 13557A/ +15222A were detected. The method is effective and relatively simple for construction of haplotypes from multiple single nucleotide polymorphisms.

[Progress in study of ADAM33 gene].

A disintegin and metalloprotease-encoding gene (ADAM) 33, located on the short arm of chromosome 20 (20p13), was recently identified through a genome-wide scan for asthma. It is a new member of a family of genes encoding a group of the metalloproteinase superfamily called the ADAM family. The proteolytic activity of the ADAMs plays important role in the healthy and disease states. The identification of ADAM33 as a candidate gene for asthma may provide a newly therapeutic target for this disease.

A novel polymorphism A(+884)-->G in the hepatic lipase gene and its association with coronary artery disease.

Hepatic lipase (HL) activity may influence susceptibility to coronary artery disease (CAD). Association between the single nucleotide polymorphisms (SNPs) in the HL gene with the occurrence of CAD has been investigated thoroughly, but to date most studies focused on the base variation in the promoter of HL gene, little is known about the variation in the coding region. In present study, the SNP in all exons of the HL gene were analyzed. All 9 exons with their flanking sequences of the HL gene were amplified from the Chinese patients with CAD and normal controls by PCR technique, and the PCR products were detected by denaturing high performance liquid chromatography (DHPLC) and sequenced with a dideoxy terminal termination method. As the result, a novel SNP A(+884)-->G within the sixth exon of HL gene was found, the 276 codon AAA was changed into AGA and resulted in the substitution of arginine for lysine. Compared with the control group, more CAD patients carried the G+884 allele (AG+GG) (54.9% vs. 41.5%, chi(2)=6.164, df=2, P=0.046). The prevalence of the G+884 allele was significantly higher in the CAD patients than that in control subjects (31.4% vs. 21.3%, chi(2) =4.652, df=1, P=0.031). Data from the linkage disequilibrium analysis showed that the A(+884)-->G polymorphism was strong in linkage disequilibrium with the T(-2)-->C variation we identified previously(D'=0.699, 0.742 in CAD patients and controls, respectively), and the frequency of the C(-2)/G(+884) haplotype (mutation) is significantly higher in CAD patients than that in controls (0.253 vs. 0.172, P<0.05).

Relationship between a novel polymorphism of hepatic lipase gene and coronary artery disease.

Hepatic lipase (HL) is a lipolytic enzyme involved in the catabolism of plasma lipoproteins, and is an important determinant of high density lipoproteins(HDL) concentration and low density lipoproteins(LDL) subclass distribution. Accordingly, HL activity may influence body's susceptibility to coronary artery disease (CAD). Association on the single nucleotide polymorphisms (SNPs) in the HL gene to post-heparin plasma HL activity and the plasma HDL-cholesterol concentration have been investigated thoroughly, but to date, little is known about th is in Chinese. In present study, the SNPs of the HL gene were analyzed. The promoter region and all the 9 exons with their flanking sequences of the HL gene were amplified from the Chinese patients with CAD and normal controls by PCR technique, and the PCR products were detected by denaturing high performance liquid chromatography (DHPLC) and sequenced with a dideoxy terminal termination method. As the result, a novel SNP-2T right curved arrow C in the promoter of HL gene was found. Compared with the control group, more CAD patients carried the -2C allele(TC+CC) (57.9% versus 42.7%, chi(2) =4.181, df=2, =0.041). The prevalence of the -2C allele was significantly higher in the CAD patients than in control subjects (chi(2)=3.988, df=1, P=0.046) and the odds ratio(OR) of -2C allele associated with the risk of CAD is 1.58 [95% confidence interval(CI): 1.01-2.47]. The -2C allele homozygous carriers in the CAD patients had a significantly higher HDL-cholesterol level than the noncarriers [(1.13-/+0.24) mmol/L versus (0.91-/+0.14) mmol/L, P<0.05]. These suggest that a T right curved arrow C substitution at -2 of the HL promoter may be associated with th e variation of HDL-cholesterol concentration and therefore affect the risk of CAD in Chinese.

[Study on the association of cholesteryl ester transfer protein gene mutations with the susceptibility to coronary atherosclerotic heart disease].

OBJECTIVE: To determine the frequencies of 4 mutations of cholesteryl ester transfer protein (CETP) gene in Chinese population and to investigate the association of the mutations with lipid metabolism and the susceptibility to coronary atherosclerotic heart disease (CHD). METHODS: The target fragments of CETP gene were amplified and analyzed by PCR-restriction fragment length polymorphism technique in 209 unrelated control individuals and 203 CHD patients. The test for Hardy-Weinberg equilibrium was performed using HWE program and statistical analysis was implemented in statistical package SPSS. RESULTS: IVS14A and 451Q mutant genes were not found in either control group or patient group. The frequencies of 405V mutant allele were 0.443 and 0.413 in controls and patients, respectively, while 442G mutant gene frequencies were 0.007 and 0.025, respectively. The observed allele frequencies of I405V and D442G mutation were in accord with Hardy-Weinberg equilibrium. The frequency of 442G mutant gene in patients was significantly higher than that in controls (P=0.043). Compared with the CHD patients without D442G mutation, the 442G heterozygous CHD patients exhibited a significant increase in plasma TC and LDL-C concentration (P=0.017; P=0.041). CONCLUSION: IVS14A and 451Q mutants of CETP gene were rare in Chinese population and 442G mutant gene was possibly one of the susceptibility factors to CHD in Chinese.

[Association of HMG-CoA reductase gene polymorphism with levels of lipids].

OBJECTIVE: To study the distribution of ScrF1 restriction polymorphism in intron 2 of the 3-hydroxy-3-methylglutaryl coenzyme A(HMG-CoA) reductase gene in Chinese Han population and the association of the polymorphism with coronary heart disease(CHD). METHODS: HMG-CoA reductase genotyping was performed using polymerase chain reaction-restriction fragment polymorphism. RESULTS: HMG-CoA reductase allelic frequencies of A, a were 0.519, 0.481; 0.440, 0.560 in CHD group and control group respectively. There was no significant difference in frequencies of allele and genotype in ScrF1 polymorphism between CHD group and control group(P>0.05). However, the levels of plasma very low density lipoprotein (VLDL) and TG in CHD patients with AA genotype were higher than those in CHD patients with other genotypes(P<0.05). The frequencies of A, a alleles at ScrF1 polymorphic site were significantly different from those reported in European Caucasians (0.44 vs 0.55, 0.56 vs 0.45, P<0.05). CONCLUSION: No direct association was found between the ScrF1 polymorphism and CHD, but there is a significant correlation between the AA genotype of the HMG-CoA reductase gene and the levels of plasma VLDL and TG in CHD group.

[Association of APOA5 gene single nucleotide polymorphism with levels of lipids and coronary heart disease in Chinese].

OBJECTIVE: To investigate the single nucleotide polymorphism 4 (SNP4) of the apolipoprotein A5 (APOA5) gene possible association with coronary heart disease(CHD) and its distribution of in Chinese Han population. METHODS: APOA5 SNP4 genotyping was performed using polymerase chain reaction and Hae III restriction fragment length polymorphism analysis. RESULTS: APOA5 allelic frequencies of T, C were 0.435, 0.565 and 0.374, 0.626 in CHD group and control group, respectively. There is significant difference in allele and genotype frequencies between CHD group and control group (P<0.05). The levels of plasma high density lipoprotein in CHD patients with CC genotype were higher than those in CHD patients with other genotypes (P<0.01). The frequencies of T allele and C allele in Chinese was significantly different from those in Caucasians (0.374 vs 0.663, 0.626 vs 0.337, P<0.01). The C allele was much more common in Chinese population. CONCLUSION: The association is found between the Hae III polymorphism and CHD, There is a significant correlation between the CC genotype of the APOA5 and the levels of plasma high density lipoprotein-cholosteal in the CHD group.

[A novel gene in APOA1/C3/A4/A5 cluster: apolipoprotein A5].

Using methods of comparative and functional genomics, a new gene coding for apolipoprotein A5 was identified in the vicinity of APOA1/C3/A4 cluster on human chromosome 11q23 by Pennaccio team and Vliet team. The open reading frame of human APOA5 encoded a 366-amino acid protein with high sequence homology to mouse Apoa5 and human APOA4. Mice expressing a human APOA5 transgene showed a decrease in plasma triglyceride concentrations to one-third of those in control mice; conversely, knockout mice lacking Apoa5 had four times as much plasma triglycerides as controls. Single nucleotide polymorphisms (SNPs) in APOA5 (S19W, -1131T>C) and APOA5 haplotype (APOA5*3) were independently associated with high plasma triglyceride levels. These findings indicate that APOA5 is an important determinant of plasma triglyceride levels, a major risk factor for coronary artery disease.

Advanced systems biology methods in drug discovery and translational biomedicine.

Systems biology is in an exponential development stage in recent years and has been widely utilized in biomedicine to better understand the molecular basis of human disease and the mechanism of drug action. Here, we discuss the fundamental concept of systems biology and its two computational methods that have been commonly used, that is, network analysis and dynamical modeling. The applications of systems biology in elucidating human disease are highlighted, consisting of human disease networks, treatment response prediction, investigation of disease mechanisms, and disease-associated gene prediction. In addition, important advances in drug discovery, to which systems biology makes significant contributions, are discussed, including drug-target networks, prediction of drug-target interactions, investigation of drug adverse effects, drug repositioning, and drug combination prediction. The systems biology methods and applications covered in this review provide a framework for addressing disease mechanism and approaching drug discovery, which will facilitate the translation of research findings into clinical benefits such as novel biomarkers and promising therapies.

Transforming growth factor-beta1 gene polymorphisms associated with chronic obstructive pulmonary disease in Chinese population.

AIM: To determine the frequencies of polymorphism and haplotype in the transforming growth factor-beta 1 (TGF-beta1) gene promoter in the Chinese population and to investigate the susceptibility of this population to chronic obstructive pulmonary disease (COPD). METHODS: The target fragments of the TGF-beta1 gene promoter were amplified and analyzed by polymerase chain reaction-restriction fragment length polymorphism technique in 84 COPD patients and 97 age- and sex-matched healthy controls. The test for Hardy-Weinberg equilibrium was performed using HWE program of the LINKUTIL package and statistical analysis was carried out with the SPSS statistical package. An expectation maximization algorithm was used for the pairwise linkage disequilibrium test and haplotype analysis. RESULTS: More carriers of the -800A allele, or fewer carriers of the -509T allele, were detected in the COPD patients compared with the non-symptomatic control subjects [for the -800A allele, 29.8% vs 14.4%, respectively, Chi2=6.257, degrees of freedom (df)=1, P=0.012; for the -509T allele, 27.3% vs 44.3%, respectively, Chi2=5.582, df=1, P=0.018]. The prevalence of the -800A allele was significantly higher in the COPD patients than in control subjects (P=0.009), whereas the frequency of the -509T allele was significantly higher in control subjects than in the COPD patients (P=0.008). In addition, this distribution tendency for the -800A or -509T allele was similar in heavy smokers (smoking history >20 pack years); (number of packs of cigarettes per day multiplied by the number of years of smoking) Chi2=7.235, P=0.007, and Chi2=5.636, P=0.018, respectively). The linkage disequilibrium was found between -800 G-A and -509 C-T (D>0.60, P<0.0001), and the frequency of the AC haplotype, consisting of the least common base at -800 and the most common base at -509, was significantly higher in patients with COPD than in controls (0.056 vs 0.021, P<0.05). CONCLUSIONS: The single nucleotide polymorphism (SNP) in the TGF-beta1 gene promoter might be associated with COPD, and the -800A/-509C haplotype is possibly one of the susceptibility factors for COPD.