Objective To analysis the β-globin gene mutation in β-thalassemia in the population of Wenzhou natives,and identify the major mutation in Wenzhou and further provide valuable information for genetic counseling,prenatal diagnosis and prevention programs in this region.Methods Patients with β-thalassemia were diagnosed and the genomics DNA were extracted from whole blood cells and amplified with PCR,sequenced and compared to the standard sequence.Some mutations were further identified by subcloned.Results 44 of 66 patients were diagnosed β-Thalassemia,9 mutations were found in the 44 sporadic patients with the sequence analysis,2 of which were known polymorphisms(exonl 59,IVS-2-665),3 belonged to the common mutations in Chinese(IVS-2-654,CD_(41/42)-TTCT and TATA box nt-28),2 were scarce abnormalities(CD_(47),CD_(66))and 2 novel variants(-24T→C,CD_(26A)→G,same sense mutation,unreported).Conclusion The mutations of β-globin gene in Han Chinese in Wenzhou are complex (9 mutations found in all),the rare and novel mutations are identified,which provide the valuable information for genetic counseling in Wenzhou.

Objective To investigate the correlation between the variations of mitochondrial gene ATP6 and type 2 diabetes mellitus ( T2DM ) and chronic complications. Methods Genomic DNA were extracted from 254 T2DM patients and 165 age-matched controls. After amplification of ATP6 by PCR and direct sequencing, all sequences were compared with the reference sequence (rCRS) to find out the variations. Bioinformatics and statistic method were used to analyze these variations. Results Many variations were detected respectively in T2DM patients and controls, a part of them only appeared in T2DM patients in low frequency, which has not been reported previously. Most of these variations are located in thethird and forth transmembrane helix of ATP synthase subunit 6 (ATPase6). Interestingly, these variationsalmost were detected in the non-obese T2DM patients with hypotension, including G8557A, A8563G,T8594C, C8609T, A8689G, G8998A and G9139A. Conclusions There were many variations in geneATP6 and must of them are mitochondrial SNP, while variations A8689G, T8825C, G8920A, G8998A andG9139A may be mild mutations which my increase the susceptibility of T2DM. G8557A, A8563G,T8594C, C8609T, A8689G, G8998A and G9139A may be associated with the biogenetics diseases suchdiabetes and hypertension.

Objective To assess the relationship between mitochondrial DNA (mtDNA) ND2 gene C5178A polymorphism and complications of cardio-cerebral-vascular in patients with type 2 diabetes mellitus (T2DM).Methods This is a case-control study.448 unrelated patients with T2DM were collected from Zhejiang Provincial People's Hospital from 2010 to 2011,including 274 males and 174 females.Direct nucleotide sequencing analysis was used to screen mtDNA ND2 gene C5178A genotyping in ）patients.Meanwhile,detailed clinical and laboratory information for all of study subjects were collected.Body mass index (BMI),blood pressure,blood lipid,blood glucose and incidence rate of cerebral infarction were compared between 5178C patients and 5178A patients.Furthermore,according to the genotyping results,we 2analyzed whether these differences exist in patients with different gender by using t test or x2 test.Results 348 out of 448 patients with T2DM were C carriers and the remaining patients were A carriers.There're significant differences between T2DM patients with 5178A and T2DM patients with 5178C on systolic pressure (124.6 mm Hg ± 9.0 mm Hg vs 127.8 mm Hg ± 10.7 mm Hg,t =2.700,P =0.007)and HDL (1.3 mmol/L ± 0.2 mmol/L vs 1.2 mmol/L ± 0.3 mmol/L,t =2.968,P =0.003).Moreover,the incidence of cerebral infarction in T2DM patients with 5178A (8.0％,8/100) was much lower than that with 5178C (21.0％,73/348 ; x2 =8.832,P =0.003).No statistical gender difference was found in the distribution of C5178A (P ＞ 0.05).Our results also revealed that the female T2DM patients with 5178A had a lower serum triglyceride (1.5 mmol/L ±0.8 mmol/L; t =2.601,P =0.011) and lower systolic pressure (123.6 mm Hg±6.6 mm Hg; t =2.887,P =0.004) than that with 5178C (1.8 mmol/L ± 1.0 mmol/L and 128.0 mm Hg ± 9.0 mm Hg,respectively).Furthermore,cerebral infarction was more common in female T2DM patients with 5178C (21.3％,29/136; x2 =5.232,P =0.022) than that with 5178A (5.3％,2/38).Similarly,male T2DM patients with 5178A had a much lower incidence rate of cerebral infarction (9.7％,6/62; x2 =3.946,P =0.047) than that with 5178C (20.7％,44/212).In contrary,the serum concentration of HDL was higher in male T2DM patients with 5178A (1.4 mmol/L ±0.2 mmol/L;t=3.511,P =0.001) than that with 5178C (1.2 mmol/L±0.3 mmol/L).Conclusions The polymorphism site mtDNA C5178A correlates with cerebral-cardiovascular complications in patients with type 2 diabetes mellitus.mtDNA 5178A allele may protect T2DM patients from developing cerebral-cardiovascular diseases through regulation of blood pressure and lipid metabolism.

Objective To investigate the therapeutic effect of tumor necrosis factor (TNF)-αsiRNA on typeⅡcolla-gen induced arthritis (CIA) in rats. Methods The expression vectors of siRNA against TNF-αgene were constructed suc-cessfully and were injected by tail veil into CIA rats. Twenty-four CIA rats were randomly divided into 4 groups including model group, empty vector group, TNF-α-siRNA1 group and TNF-α-siRNA2 group. CIA rats were injected with the same dose of phosphate buffered sodium (PBS) and pGFP-V-RS vector respectively in model group and empty vector group, while TNF-α-siRNA1 group and TNF-α-siRNA2 group were injected with TNF-α-siRNA1 eukaryotic expression vector and TNF-α-siRNA2 eukaryotic expression vector respectively. Another 6 rats, which were not established CIA model, were in-jected with PBS (blank control group). The serum expression levels of IL-1 were detected by ELISA on day 1, 5, 9 and 13 af-ter injection. The expression level of TNF-αmRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR) on day 13. Results The expression level of IL-1 was significantly higher on day 1, 5, 9 and 13 in model group than that of blank group (P＜0.05). The expression levels of IL-1 were significantly lower on day 1, 5 and 9 in TNF-α-siRNA1 group and TNF-α-siRNA2 group than that of model group and blank group (P ＜ 0.05). The expression level of TNF-αmRNA was significantly higher on day 13 in model group than that of blank group (P＜0.05). The expression levels of TNF-αmRNA were significantly lower in TNF-α-siRNA1 group and TNF-α-siRNA2 group than those of model group and emp-ty vector group (P＜0.05). Conclusion TNF-αspecific siRNA can suppress the levels of TNF-αmRNA and IL-1, which provides experimental basis for gene therapy of rheumatoid arthritis.

Objective To study therapeutic efficacy of Aredia in treating malignant metastatic bone tumors. Method 60～ 90 mg Aredia was administrated iv in 31 cases with malignant metastatic tumors,once each week. Results Pain in 12 cases was significantly relieved.14 cases acquired relif.Total effective rate was 83.9% .Activity ability was improved by 80.6% .No apparent toxicological and adverse effects as well as fever and cold symptoms were observed.Conclusion Aredia is a kind of ideal drugs for treatment of pain caused by malignant metastatic bone tumors.It is convenient in use and could be endured by patients.

Objective To establish a method for detection of gene mutations in β-thalassemia by high-resolution melting (HRM) and study its preliminary clinical application.Methods Two common mutations [ IVS-2-654 ( C ＞ T ) and -28 ( A ＞ G ) ]of β-thalassemia in Wenzhou city population were selected.The plasmid DNA fragments of these mutations were constructed by TA clone technology as PCR templates or genotyping controls.A method for detection of β-thalassemia gene mutations based on HRM analysis was established and its specificity,sensitivity and repeatability were methodologically evaluated.One hundred and seventeen patients with clinically suspected β-thalassemia from Second Affiliated Hospital and Yu ying Children's Hospital of Wenzhou Medical College were enrolled into this study.The genomic DNA was extracted from whole blood cells and detected by HRM method.The results were compared with the direct sequencing data.Results HRM method could detect the mutations [ IVS-2-654( C ＞ T) and -28 ( A ＞ G ) ]of β-thalassemia and the results did not show any non-specific amplified fragments.All within-run and between-run coefficients of variation for different DNA types' Tm were smaller than 0.1％.And minimum 103 copies of DNA of each assay and 10％ mutation could be determined by this method.One hundred and seventeen patients with clinically suspected β-thalassemia were detected with HRM and all the results were in accordance with direct DNA sequencing.There were 45 IVS-2-654 ( C ＞ T)heterozygous mutation and 9-28 ( A ＞ G)heterozygous mutation and none homozygous mutation.Conclusion The method of rapid identification of β-thalassemia gene mutations based on HRM analysis is successfully established,which is a convenient,rapid,specific,sensitive and accurate technique for screening gene mutations in β-thalassemia as well as a general technical platform to identify other gene mutations.

AIM:To investigate the effect of fenbendazole (FBZ) on the proliferation of human chronic myelogenous leukemia (CML) cell line K562.METHODS:The CCK-8 assay was used to detect the effect of FBZ on viability of the K562 cells and normal peripheral blood mononuclear cells (PBMC).The cell growth was measured by the method of Trypan blue exclusion.The cell cycle was analyzed by flow cytometry.The cell cycle-related proteins were detected by Western blot.RESULTS:The growth of K562 was significantly inhibited by FBZ.However, it elicited little cytotoxic effect on PBMC.Furthermore, FBZ induced G2/M phase arrest and mitotic catastrophe in the K562 cells based on the changes of nuclear morphology, DNA content, mitotic marker analysis and the number of polykaryocytes.CONCLUSION:Fenbendazole significantly inhibits the proliferation of K562 cells and induces cell cycle arrest at G2/M phase by the regulation of cell cycle-related proteins.

Objective To predict the CTL epitopes of Tat exon 1 region in HIV-1 CRF07_BC strains, which were prevailing in China. Methods Total of 236 plasma samples were from the 3rd National HIV Molecular Epidemic Survey (NMES3). All the subjects were infected with HIV-1 CRF07_BC viruses. The tat exon 1 region was amplified by reverse transcription reaction and nested polymerase chain reaction (nested-PCR), then the PCR products were sequenced. The distribution of CTL epitopes of this region were predicted by on-line software BIMAS HLA Peptide Binding Predictions and statistics software. Results To-tal of 236 CRF07_BC strains were from 16 provinces, mainly in intravenous drug asers(58.9%)and then sex(25.0%). It was showed that there were 12 CTL epitopes of 236 Tat exon 1 region of CRF07_BC strains mainly located in proline-rich region, cysteine-rich region and core-region. Those epitopes were banded by 5 HLA presenting molecules in genotype(A * 2501 ,A * 2902, B * 15,B * 5301 and Cw * 1203) and 6 HLA presenting molecules in serotype (B53, B58 ,B57 ,A3 ,A68 and Cw12). The frequency of single amino acid substitution was more than 50% in 7 CTL epitopes. Conclusion The CTL epitopes in Tat exon 1 of CRF07 _BC strains were located in different functional regions, and there were some amino acid variations in them.

Objective To investigate the application value for predicting human cytomegalovirus(HCMV) infection with viral load in urinary epithehal cell (EC).Methods Peripheral blood and urine specimens from 82 infants with HCMV latent infection and 84 infants with HCMV active infection were collected respectively.Plasma HCMV DNA load and the levels of HCMV lgM/IgG antibody were detected by real-time fluorescence quantitative polymernse chain reaction (FQ-PCR) and chemiluminescence immunsassay.HCMV pp65 antigen in peripheral blood polymorphonuclear leukocytes (PMNLs) was detected by indirect immunofluorescence assay.The urinary EC count and HCMV DNA load were detected by UF-100 automated urine sediment analyzer and FQ-PCR,respectively.HCMV DNA load in urinary EC was calculated accordingly.At the same time,the sensitivity and specificity for diagnosis of active HCMV infection were evaluated by receiver operating characteristic curve (ROC).Results The positivity of HCMV DNA in urinary EC was 94.58% (157/166),which was the highest among the urinary EC from 166 cases of HCMV infection.HCMV DNA load ranged from 5.67×102to 1.31×107 copies/103 EC There was no significantly statistical difference among urine specimens from different periods of time(P>0.05).HCMV DNA load in active infection group [5.13±0.99(copies/103EC,lg)]is significantly higher than that in latent infection group [3.92±0.82 (copies/103 EC,lg),t = 8.52,P < 0.01];According to ROC curve analysis,its sensitivity and specificity were 71.4% and 75.2% respectively when cut-off value was 4.55.The virus load was significantly decreased in urinary EC in post-treatment infants as compared with pre-trestment(t=5.44,P<0.01).Conclusion Detection of HCMV DNA load in the urinary EC is a cost-effective method and can be used to predict HCMV active infection in infants and monitor treatment of HCMV infection.

Objective To construct quantitative standard for quantification of hepatocyte growth factor (HGF) mRNA and establish its real-time fluorescence quantitative(FQ)-PCR assay to estimate its clinical relevance in lymphoma.Methods Recombinant plasmid Was constructed with target cDNA obtained from isolated total RNA by RT-PCR After PCR products were identified and purified,recombined plasmids were quantitated and then acted as quantitative standard.A new real time FQ-PCR analysis system Was established with the second pair of primers and the probe after amplification condition and the concentrations of components were optimized.HGF mRNA expressions in 47 lymohoma cases[11 Hodgkin disease(HD) cases,36 non-Hodgkin lyphoma(NHL)cases.among these patients,36 patients in remission while 11 patients without remission ] were analyzed quantitatively,and its specificity and sensitivity for lymphoma diagnosis were evaluated by receiptor operation character(ROC)curve method.Results HGF mRNA quantitative standard was constructed successfully.and its real time FO.PCR analysis system Was established combined with hot.start PCR and down.touch PCR technique. According to slope of standard curve (-3.513)and correlation cofficient(0.999),amplification efficiency of the system was 92.6%.Coefficient variation of intra-assay,intra-day and inter-day-assay were 2.1%,4.0% and 6.8%,respectively.Sensitivity of FQ-PCR Was 2 eopies/μl.Expressions of HGF mRNA in lymphoma group Was higher than that in control group(6.425±2.172 and 0.317±0.192,respectively,t=15.883,P<0.001),and its expressions in remission group was lower than no remission group(6.157±1.712 and 7.59l ±1.184,respectively,t=2.768,P<0.05).However,there Was not difference of HGF mRNA level between group HD and group NHL(P>0.05).According to ROC analysis,its sensitivity and specificity were 93.6% and 100% when cutoff value for lymphoma clinical diagnosis Was 3.136.Conclusion HGF mRNA'8 quantitative standard and its real time F9-PCR analysis system have been successfully constructed,and it can be used for quantitative detection of its mRNA expression in lymphoma.