miR145 Regulates the Proliferation and Apoptosis of Rat Vascular Endothelial Cells under Hyperglycemia by Targeting the ANGPT2 Gene and Involving the NFκB Signaling Pathway.

PURPOSE: A majority of diabetes mellitus patients with disturbances of glucose metabolism present with vascular complications. This study aimed to explore regulatory mechanisms of miR145 and its potential target gene ANGPT2 on diabetic vasculopathy under hyperglycemia. METHODS: Based on the fact that miR145 is detected in rat aortic endothelial cells (RAECs) under hyperglycemia, RAECs were transfected with miR145 mimics/inhibitor for further confirmation. RAEC proliferation was detected with CCK8 assays, and cell apoptosis and CD34+-cell population with annexinV-PI staining and anti-CD34FITC on flow cytometry, respectively. Then, qPCR and Western blot were applied to detect mRNA and protein expression of ANGPT2 and involved pathway factor NFκB p65. Subsequently, dual luciferase-reporter gene analysis was utilized to verify whether miR145 acted directly upon the 3'UTR of ANGPT2 mRNA. RESULTS: The ANGPT2 gene was confirmed to be a direct target of miR145. miR145 mimics markedly downregulated the expression of ANGPT2 and NFκB p65, boosted the percentage of the CD34+ phenotype, and promoted proliferation and suppressed apoptosis of RAECs under hyperglycemia. CONCLUSION: miR145 might regulate the viability of RAECs via targeting ANGPT2 and involving NFκB signaling to exert a protective effect on diabetic vasculature.

[Association of copy number of SMN1 and SMN2 with clinical phenotypes in children with spinal muscular atrophy].

OBJECTIVE: To study the association of copy number of SMN1 and SMN2 with clinical phenotypes in children with spinal muscular atrophy (SMA). METHODS: A total of 45 children with SMA were enrolled. Multiplex ligation-dependent probe amplification was used to measure the gene copy numbers of SMN1 and SMN2. The association of copy number of SMN1 and SMN2 with clinical phenotypes was analyzed. RESULTS: Of the 45 children with SMA, 42 (93%) had a homozygous deletion of SMN1 exons 7 and 8, and 3 (7%) had a deletion of SMN1 exon 7 alone. No association was found between SMA clinical types and the deletion types of SMN1 exons 7 and 8 (P>0.05). There was a significant difference in the distribution of SMN2 gene copy numbers between the children with SMA and the healthy children (P<0.05). The children with SMA usually had two or three copies of SMN2 gene, while the healthy children usually had one or two copies of SMN2 gene. There was a significant difference in the distribution of SMN2 copy numbers among the children with different SMA clinical types (P<0.05). The children with two copies of SMN2 gene had a significantly lower age of onset than those with three or four copies. Most of the children with type I SMA had two or three copies of SMN2 gene. Most of the children with type II SMA had three copies of SMN2 gene. Most of the children with type III SMA had three or four copies of SMN2 gene. Children with a higher copy number of SMN2 gene tended to have an older age of onset and better motor function and clinical outcome, and there was a significant association between SMN2 gene copy number and clinical outcome (P<0.05). CONCLUSIONS: The SMN2 gene can reduce the severity of SMA via the dosage compensation effect. SMN2 copy number is associated with the phenotype of SMA, and therefore, it can be used to predict disease severity.

Left iliac vein compression is not associated with infrainguinal deep venous thrombosis but is associated with iliac vein involvement.

OBJECTIVE: The purpose of this study was to investigate whether left iliac vein (LIV) compression had similar correlation with the risk of left iliac deep venous thrombosis (DVT; iliac vein involvement) and infrainguinal DVT (without iliac vein involvement). METHODS: A retrospective analysis of records and enhanced computed tomography images was conducted of 278 patients with left-sided DVT (iliac DVT, 228 patients; infrainguinal DVT, 50 patients) and 232 control patients without DVT on either side. The influences of LIV compression on the risk of left iliac DVT and infrainguinal DVT were investigated using logistic regression analysis. RESULTS: Mean percentage compression of the LIV in left iliac DVT (74.64% ± 0.99%) patients was significantly higher than in non-DVT patients (53.42% ± 1.49%; P < .01). However, mean percentage compression of the LIV in left infrainguinal DVT patients (45.37% ± 2.71%) was significantly lower than in non-DVT patients (53.42% ± 1.49%; P < .01). LIV compression was associated with increased odds of left iliac DVT (odds ratio, 1.88; 95% confidence interval, 1.64-2.15; P < .01) for each 10% increase in percentage compression of the LIV. However, LIV compression was not associated with increased odds of infrainguinal DVT (odds ratio, 0.89; 95% confidence interval, 0.76-1.03; P = .126). CONCLUSIONS: Left iliac DVT patients had more severe LIV compression than left infrainguinal DVT patients did. LIV compression was not associated with development of left infrainguinal DVT, but it did correlate with the presence of left-sided DVT with iliac vein involvement.

[Expression of VCAM-1 and caspase-3 in myocardium of persons who died from viral myocarditis].

OBJECTIVE: To observe the expression and distribution of vascular cell adhesion molecule-1 (VCAM-1) and caspase-3 in myocardium of persons who died from viral myocarditis and to explore its pathogenesis and death mechanism. METHODS: Twenty cases died from viral myocarditis were selected as the experimental group. Ten cases died from traumatic shock and massive hemorrhage shock after traffic accidents were selected as the control group. The sections of myocardium were stained by immunohistochemistry for VCAM-1 and caspase-3, and observed under microscope. The positive expressions of VCAM-1 and caspase-3 of the two groups were compared with each other by image analysis and statistical analysis. RESULTS: (1) The vascular endothelial cells expressed VCAM-1 with dark-brown colors in the experimental group, and weak expression was observed in the control group. The average optical density in the experimental group was higher than that in the control group (P < 0.05). (2) The caspase-3 positive cells were mostly inflammatory cells around the myocardial vessels with brown-red granules in the experimental group. The positive cell number in the experimental group was higher than that in the control group (P < 0.05). CONCLUSION: VCAM-1 may play an important role in the inflammatory cells exudation caused by viral myocarditis, and may provide the reference for diagnosis of viral myocarditis in forensic pathology. However, the myocardial apoptosis mediated by caspase-3 doesn't affect the lethal mechanism in the late stage of viral myocarditis.

[Study on the prevalence of prehypertension among residents living in the communities in Zhejiang].

OBJECTIVE: To investigate the prevalence and related risk factors of prehypertension in Hangzhou, Shaoxing,Jiaxing city, Zhejiang province. METHODS: 3200 people were selected by stratified cluster random sampling method, and statistical methods including chi-square test, and logistic regression through SAS 9.0 were used. RESULTS: The prevalence of pre- hypertension was 45.9%, higher for males and urban population, with significant differences seen between males and female(49.0% vs. 48.0%, P < 0.05), urban and rural areas(59.31% vs. 44.15%, P < 0.05). Data from the multiple factor logistic analysis showed that risk factors of prehypertension would include: older age, types of profession, under low education level, being urban residents, overweight and obesity, hyper triglyceride, and family history of hypertension, with ORs and 95% CI as 0.99 (0.98-0.99), 1.28(1.07-1.28), 1.31(1.10-1.56), 1.50(1.11-2.02), 1.33(0.98-1.81), 1.60 (1.19-2.16)and 1.18(1.00-1.39), respectively. CONCLUSION: Prehypertension prevalence was found high in the studied district, especially in urban residents with low education level. Strategies including reduction on risk factors as obesity and hyper triglyceride through health education as well as lifestyle modification should be taken to hold back the increasing trend on prehypertension in Zhejiang.

[Association of 1233A/G polymorphism of angiopoietin-2 gene with type 2 diabetes mellitus and diabetic nephropathy].

OBJECTIVE: To investigate the association between single nucleotide polymorphisms (SNP) of angiopoietin-2 (Ang-2) gene and type 2 diabetes mellitus (T2DM) and diabetic nephropathy (DN). METHODS: Genotype and allele frequency of Ang-2 were detected by amplification refractory mutation system-PCR(ARMS-PCR) in 221 cases with T2DM and 104 normal controls. Monocyte chemoattractant protein-1(MCP-1) was measured by ELISA. DN patients were divided into three groups according to urinary albumin excretion rates (UAER), i.e. DN0: UAER < 30 mg/24 h, DN1: UAER 30-300 mg/24 h and DN2: UAER > 300 mg/24 h. RESULTS: Statistics showed: (1) Genotype frequencies and allele frequencies in Ang-2 1233A/G had significant difference but not Ang-2 759T/G and 1078A/G; (2) Comparing with those with genotype AA, the relative risk of genotype (AG+ GG) suffered from T2DM and DN were 2.265 fold (OR= 2.265, 95% CI: 1.223-1.402, P= 0.031), 1.789 fold (OR= 1.789, 95% CI: 0.889-1.021, P= 0.012), respectively; (3) The onset of DN was related to Ang-2 1233A/G allele G (r= 1.321, OR= 1.427, 95% CI: 2.324-4.177, P= 0.034). CONCLUSION: Ang-2 1233A/G polymorphism may be associated with T2DM and involved in onset and development of DN.

[Study on key techniques and intervention in reducing birth defects].

OBJECTIVE: To investigate key techniques and intervention in reducing birth defects. METHOD: Down's syndrome (DS), trisomy-18 (Edwards syndrome, ES), neural tube defects (NTD), Duchenne muscular dystrophy (DMD), spinal muscular atrophy (SMA), thalassemia, and glucose-6-phosphate dehydrogenase deficiency (G6PD) were chosen as target disease. From Jan. 2007 to Dec. 2009, the condition of intake folic acid were investigated in 5004 pregnant women in Panlong District and Wuhua District of Kunming City. All of the 27 660 pregnant women undergoing prenatal examination were enrolled into the study from the First People's Hospital of Yunnan Province, the Second People's Hospital of Yunnan Province, the First People's Hospital of Qujing City, the Second People's of Qujing City, Qujing Women and Children's Hospital, People's Hospital of Lincang City, Kunming Maria Women's Hospital, Maternal and Infant's Care Unit of Panlong District of Kunming City, Maternal and Infant's Hospital of Dali City. The screening was performed on serum of those pregnant women at 8 - 20(+6) gestational weeks. Prenatal cytogenetic analysis and fetal ultrasonograpy were performed on the high risk or indicated women after genetic counseling. DNA analysis was administered on those women with family or childbearing history of DMD, SMA, thalassemia, or G6PD. Outcome of pregnancy was followed up to evaluate the effect of intervention. RESULTS: Approximately 30.10% (1506/5004) of pregnant women were administered by oral folic acid during perinatal period. Two thousand three hundred and thirteen women with high risks of DS, ES, or NTD fetuses were observed among 27 660 undergoing maternal serum screening. Two thousand and ninety-six pregnant women including two twins pregnant women were performed cytogenetic analysis. Other 67 pregnant women at high risk of DMD, SMA, thalassemia, and G6PD accepted genetic counseling and prenatal gene analysis. Two thousand one hundred and sixty-three pregnant women (2165 fetuses) underwent prenatal examination. One hundred and two cases chromosome abnormalities, 17 cases NTD, 4 cases DMD, 1 cases α-thalassemia major were found. All of the 91 fetuses with major birth defects were terminated after genetic counseling. Another affected DS fetus in a twin pregnancy dead intrauterine at 24 gestational weeks. Thirty-two women bearing fetuses with balanced translocations or inversions continued their pregnancies. Totally 2071 normal term fetuses were born in the prenatal diagnosis group. Two fetuses with normal chromosome were lost within 1 week after amniocentesis. Four affected DS fetuses were born from their high risk mothers who refused further prenatal diagnosis service. In a random sampling follow-up cohort of 5000 mothers at low risk, none of affected child suffering target diseases was found. The DS detection rate of maternal serum screening was 84% (27/32), with the false positive rate was 6.153% (1702/27 660). CONCLUSIONS: Folic acid intake before conception and in the first trimester would reduce the risk of birth defects, only 1/3 reproductive women took folic acid actively. Maternal serum screening could effectively detect high risk of DS, ES and NTD. The genetic counseling is critical in women at high risk or who had family history of inherited disorders. The prenatal screening and diagnosis combined with routine obstetric care could reduce the incidence of major birth defects, which should become prenatal care strategy in our country.

[Genetic linkage map of Betula pendula Roth and Betula platyphylla Suk based on random amplified polymorphisms DNA markers].

Based on the genetic inheritance and segregation of random amplified polymorphism DNA (RAPDs) markers, the first mid-density linkage map for silver birch was constructed by using a pseudo-testcross mapping strategy. A segregating population including 80 progenies from the cross between Betula pendula Roth and B. platyphylla Suk was obtained. A set of 1,200 random oligonucleotide primers were screened, and 208 primers were selected to generate RAPD markers within a sample of 80 F1 progenies. A total of 364 segregating sites were identified. Among them, 307 belonged to 1 : 1 segregating site, and 36 belonged to 3 : 1 segregating site, others were found distorted from the normal 1 : 1 ratio. Altogether 307 sites segregating 1 : 1 (testcross configuration) were used to construct parent-specific linkage maps, 145 for B. pendula and 162 for B. platyphylla. The resulting linkage maps consisted of 145 marker sites in 14 groups (four or more sites per group), 6 triples and 6 pairs for B. pendula, which covered the map distance about 955.6 cM (Kosambi units). The average map distance between adjacent markers was 14.9 cM, and 162 linked marker site for B. platyphylla were mapped onto 15 groups (four or more sites per group), 4 triples and 6 pairs, which covered the map distance about 1,545.8 cM, and the average map distance between adjacent markers was 15.2 cM. Further study is warranted to integrate the two maps to one density map and to locate important genes on the maps.

[Study on genetic instability of NM23H1 gene in Chinese with the epithelial ovarian tumor].

The aim of this study was to examine microsatellite instability (MSI) and loss of heterozygosity (LOH) of locus D17S396 on chromosome 17 and their influence on the expression of nm23H1 in the epithelial ovarian tumors, which may provide experimental basis for the mechanism of nm23H1 gene and tumor metastasis. Techniques such as DNA extraction from formalin-fixed paraffin-embedded tissues, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), ordinary silver stain were used to study MSI and LOH of locus D17S396. Envision immunohistochemistry and Leica-Qwin computer imaging techniques were used to assess the expression of nm23H1 gene. In our experiments, the frequency of heredity instability of malignant ovarian tumors was 40%, which is higher than that of borderline ovarian tumors, while there were no heredity instability occurred in benign ovarian tumors and normal ovarian tissue. Among 25 epithelial ovarian carcinomas, the frequency of LOH in lymph node metastasis cases (66.67%) was significantly higher than those without metastasis (10.53%). Moreover, the frequency of LOH was higher in FIGO stage III and IV than in stage I and II. However, the frequency of MSI showed no correlation with any clinicopathologic characteristics. The positive frequency of nm23H1 protein in the ovarian epithelial carcinoma and borderline tumors were 56.00% and 57.14%, respectively. They were both higher than those of the benign tumors and normal ovarian tissue. In the epithelial ovarian carcinomas, the positive frequency of nm23H1 protein in lymph node metastasis case was significantly lower than those without metastasis. FIGO stage III and IV also exhibited lower positive frequency of nm23H1 protein compared with stage I and II. Furthermore, there was no difference in nm23H1 protein expression intensity analyzed by computer imaging. In the epithelial ovarian carcinomas, the positive frequency of nm23H1 protein in LOH positive group was 0.00%, which is lower than that of LOH negative group (P < 0.01). The results indicated that the heredity instability of nm23H1 gene might be implicated in pathogenesis and progression of epithelial ovarian tumor. The occurrence of LOH might be the molecule marker of the deteriorism of ovarian tissue. Both MSI and LOH of nm23H1 gene controlled development of the epithelial ovarian tumor independently in different paths. LOH could inhibit the expression of nm23H1 in local tissue of the epithelial ovarian carcinoma, which endowed it with high aggressive and poor prognosis. Increasing the amount of nm23H1 protein expression could effectively restrain metastasis of the ovarian epithelial carcinoma and improve prognosis of patients.

[Preimplantation genetic diagnosis for a patient with Robertsonian translocation].

OBJECTIVE: To investigate the feasibility and risk of preimplantation genetic diagnosis (PGD) for screening normal offspring of Robertsonian translocation carriers. METHODS: This case was clinically diagnosed as primary infertility for 6 years; the husband was found to have chromosome der (13;14) (q10;q10) and oligozoospermia. For the solution of the couple's problem, controlled ovarian hyperstimulation (COH) and intracytoplasmic sperm injection (ICSI) were performed to obtain embryos. The embryos were drilled in zona by acidified Tyrode's solution at 6-8 cell stage (day 3 post-fertilization) and a single blastomere was removed from each embryo. All blastomeres were analyzed by fluorescence in situ hybridization (FISH) using the double color probes LSI 13q labeled by SpectrumOrange and Tel 14q labeled by SpectrumGreen. The embryos biopsied were cultured at once and the normal ones selected were transferred the next day. Prenatal diagnostic techniques were used to detect the karyotype of fetus at 18 weeks of gestation. RESULTS: Unbalanced, normal or balanced, and unclear embryos were separated. The couple obtained 50a (4/8)normal or balanced,and 37.5a (3/8)unbalanced, and 12.5a (1/8) unclear embryos. A singleton pregnancy followed, and the karyotype of the fetus (46,XY) was detected by prenatal diagnostic techniques. CONCLUSION: PGD is useful for screening out unbalanced embryos and is very valuable for solving the reproductive problem of Robertsonian translocation carriers and for avoiding fetal beings with severe disorders.

[Lymphatic metastasis and VEGF-C expression in ovarian epithelial tumors].

The aim of the present study was to explore the role of vascular endothelial growth factor-C (VEGF-C) in the process of angiogenesis, lymphangiogenesis and lymphatic metastasis in epithelial ovarian tumors. In situ hybridization and immunohistochemical staining for VEGF-C were performed in 30 epithelial ovarian carcinomas, 9 borderline tumors and 26 benign cystadenomas. Endothelial cells were immunostained with anti-VEGFR-3 pAb and anti-CD31 mAb, and VEGFR-3 positive vessels and microvessel density (MVD) were assessed by image analysis. VEGF-C mRNA and protein expression in ovarian epithelial carcinomas were significantly higher than that in borderline tumors and benign cystadenomas (p < 0.05 or p < 0.01). In ovarian epithelial carcinomas, VEGF-C protein expression, VEGFR-3 positive vessels and MVD were significantly higher in the cases of clinical stage III-IV and with lymphatic metastasis than those of clinical stage I-II and without lymphatic metastasis respectively (p < 0.05 or p < 0.01), VEGFR-3 positive vessels and MVD was significantly higher in the VEGF-C protein positive tumors than negative tumors (p < 0.05), VEGFR-3 positive vessels was significantly correlated with MVD(p < 0.01). These data suggest that VEGF-C might play a role in lymphatic metastasis via lymphangiogenesis and angiogenesis in epithelial ovarian carcinomas, and VEGF-C could be used as a biologic marker of metastasis in ovarian epithelial carcinomas.