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 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 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 develop and evaluate an aptamer based biosensor (aptasensor) for rapid colorimetric detection of enteropathogenic Escherichia coli (EPEC). Method The aptasensor was fabricated by modifying the truncated LPS-binding aptamer on the surface of nanoscale polydiacetylene vesicles using peptide bonding between the carboxyl group of the vesicle and the amine group of the aptamer. Molecular recognition between EPEC and aptamer at the interface of the vesicle led to blue-red transition of polydiacetylene which was readily visible to the naked eyes and could be quantified by colorimetric responses (CR). Transmission electron microscopy (TEM) was used to confirm the specific interactions between EPEC and polydiacetylene vesicles. Result Truncated aptamer showed the similar LPS-binding activity. The aptasensor could detect the target bacteria in a range of 105-108 colony-forming units (CFU)/ml within less than 30 minutes and its specificity was 100% for detection of EPEC O111. The sensor reproducibiliry obtained at 106 CFU/ml was 6. 08% R. S. D. The results of TEM confirmed that the specific interactions between EPEC and polydiacetylene vesicles. Conclusion A new aptasensor was developed successfully for rapid colorimetric detection of EPEC.

BACKGROUND:The reported time of bone marrow mesenchymal stem cel s induced to differentiate into chondrocytes is different. Few studies have observed and compared the cel s’ dynamic transformation during the induction process.
OBJECTIVE:To observe the dynamic differentiation and the mature time of rabbit bone marrow mesenchymal stem cel s which were directional y induced to chondroblasts for 8, 11, 14, 17, 20 days.
METHODS:Bone marrow was aspirated from the femur of New Zeal rabbits, and bone marrow mesenchymal stem cel s were isolated by gradient centrifugation. After cultivation and amplification, bone marrow mesenchymal stem cel s at passage 3 were directional y induced to chondrocytes by the serum-free medium containing transforming growth factor beta-1. The experiments were divided into five groups according to different induction time points:8 days, 11 days, 14 days, 17 days, 20 days. Then cel ular morphology, toluidine blue staining, typeⅡ col agen immunohistochemistry, aggrecan content in induction medium, and chondrogenic differentiation in each group were observed and compared.
RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cel s had apparently transformed in morphology at 8 days of induction, and presented obvious chondrocytes’ morphology at 14 days. The aggrecan in induction medium could be detected at a low level at 4 days, significantly increased at 8 days, and maintained slow increasing at 20 days. At 14 days, the metachromatic particles could be found by toluidine blue staining, and the col agen type Ⅱimmunohistochemistry was significantly positive in cel climbing slice. Experimental findings indicate that, bone marrow mesenchymal stem cel s that are monolayer cultured in a high density can be induced into chondroblasts at the effect of transforming growth factor beta-1 and other factors. There are a few chondroblasts in the early induction process, then cel s begin to have chondrocytes morphology and function after induced for 8 days, and may differentiate to mature chondrocytes at 14 days. In addition, they can keep a high biological activity in the induction process.

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.

Background The anti-proliferative effect of matrine has been demonstrated and its relevance to prevention and treatment of proliferative retinovitreopathy is concerned.Howeverthe intravitreous injection of free-matrine reiteratively may raise the risk of ocular infection.ObjectiveThe goal of the present study is to investigate the sustained releasing ability and safety of matrine polyactic acid microsphere(MAT-PLA-MSintravitreal injection.MethodsMAT-PLA-MS was prepared by Hebei Medical University and examined under the transmission electron microscope.The release of MAT-PLA-MS was monitored by ultraviolet spectrophotometry.Free-matrine with the dose of 1,2,4mg was intravitreally injected respectively in 12 eyes of New Zealand albino rabbits in free-matrine group and MAT-PLA-MS with matrine(2,4,6mg respectively was administered in 16 eyes separately in matrine microsphere group.The blank microsphere was injected in 6 right eyes as blank control group and normal saline solution was injected in 6 fellow eyes as control group.The retinal function change was evaluated by electroretinogram(ERG),and the morphological and histological change of retina following drug injection were assessed under the slit lamp biomicroscope,indirect ophthalmoscope,light microscope and transmission electron microscope.The decomposed process of MAT-PLA-MS in vitreous was recorded with ocular anterior segment and fundus color camera.Results MAT-PLA-MS containing matrine showed the spherical shape with the mean diameter of 2.28±47μm under the transmission electron microscope and the drug-loading rate 6.17% and drug-release rate 87.93% in vitro for 672 hours,presenting the controllable release characteristics.After implantation into the vitreous,the MAT-PLA-MS containing matrine decomposed gradually with the prolong of time.The b amplitudes of ERG maximum response were significantly declined in 4mg free-matrine injection group in comparison with before injection in various time points(P<0.01).However,no considerably differences were found in MAT-PLA-MS with matrine groups and control groups in various time points following the intravitreal injection(P>0.05).No obvious abnormal was seen under the slim lamp and ophthalmoscope through the study period.The changes of retinal ultrastructure were found from 1 through 28 days after injection of 4mg free-matrine,and slight retinal structural damage was seen from 7 through 28 days after injection of 6mg MAT-PLA-MS containing matrine.ConclusionThese results suggest that MAT-PLA-MS possesses good sustained release feature.MAT-PLA-MS containing matrine has less toxicity to retina than free-matrine after intravitreal injection.MAT-PLA-MS is an excellent drug delivery system.

AIM: To explore the molecular mechanism of asthenospermia(AST) by preliminary screening of nucleotide sequences from the ND3 and ND4L genes of mitochondrial DNA(mtDNA). METHODS: Samples from 50 AST patients and 42 age-matched normal controls were collected according to the WHO criteria. Density gradient centrifugation was applied to separate spermatozoa with different vigor. The ND3 and ND4L genes of mtDNA were amplified and sequenced directly from the extracted genomic DNA from AST patients and normal controls. The sequences were compared with revised Cambridge Reference Sequence(rCRS) to analyze the variants. RESULTS: A total of 22 nucleotide variations were found in ND3 and ND4L genes of mtDNA in asthenospermia group and control group. G10320A, A10398G and T10609C were missense mutations, while A10157G and A10313C were the reported for the first time in this study. Haplotype N in patients with AST(33/50) was higher than that in control group(14/42, P<0.05), and haplotype R9 in patients with AST(15/50) was also higher than that in control group(4/42, P<0.05) through genetic testing of ND3 gene. Rates of sperm progressive motility of haplotype F1, F2 and R9 were significantly lower than those of haplotype M and M rest. Two haplotype differences, haplotype M and N, were found in the same AST patient's spermatozoas which had different vigor. Haplotype M had stronger vigor, while haplotype N had lower vigor. By sequencing ND3 gene of mtDNA from 50 AST patients, we detected G10310A heteroplasmic mutation in 2 specimens of asthenospermia with poor and moderate motility spermatozoa, respectively. No mutation occurred in good motility spermatozoa. CONCLUSION: Haplotype of mitochondrial may have some correlation with sperm motility. The nt10398G-10400T polymorphisms may have benefit for sperm motility, whereas the mutation in nt10310A may impair sperm motility.