Background and purpose: High expression of telomerase and telomere stability are two common features in tumor cell. hTERT is a catalytic subunit of telomerase, TRF2 is extremely important to maintain the length and stability of telomerase. This study was to construct the recombinant adenovirus mediated shRNA to hTERT and TRF2, and to investigate the inhibitory effects of the vector by solo-inhibiting and connect-inhibiting in the MCF-7 cells, in order to present a new approach to the gene therapy for breast cancer. Methods: rAd-hTERT and rAd-TRF2 were constructed and the expression of hTERT mRNA and TRF2 mRNA were tested by FQ-PCR 48 hours after transfecting in MCF-7 cells. Apoptosis was observed by flow cytometry 1 to 6 days after transfection. Results: ①At 48 hours after transfection, the results of FQ-PCR showed that compared to PBS group, the expression of hTERT in rAd-hTERT group was obviously decreased and the inhibition ratio was about 86%, but TRF2 had not been obviously inhibited (P>0.05);the expression of TRF2 in rAd-TRF2 group was obviously decreased and the inhibition ratio was about 80%, but hTERT had not been obviously inhibited (P>0.05);in rAd-hTERT/rAd-TRF2 group, the inhibition ratio of hTERT and TRF2 were about 88% and 85%. Comparing rAd-hTERT/rAd-TRF2 group with rAd-hTERT group and rAd-TRF2 group, there were no significant differences of inhibition ratio between hTERT gene and TRF2 gene(P>0.05). Otherwise, comparing rAd-HK group, rAd-blank group with PBS group, there were no significant differences of inhibition ratio between hTERT gene and TRF2 gene(P>0.05). ②The result of flow cytometry showed that apoptosis was induced at the first day after transfecting in rAd-hTERT group and rAd-TRF2 group, the most obvious apoptosis was in the 3rd to 5th days,at the peak in the 5th day, and decreased in the 6th day after transfection. The apoptosis ratio of rAd-hTERT group was 46.2%, rAd-TRF2 group was 43.5%. The apoptosis ratio of rAd-hTERT/rAd-TRF2 group was 46.2% at first day, 68.5% at the second day, the most obvious apoptosis was in the 3rd to 6th days and was 77.6% in the 6th days in rAd-hTERT/rAd-TRF2 group. There were significant differences in apoptosis ratio in solo-inhibiting and connect-inhibiting(P<0.05). In addition, comparing rAd-HK group, rAd-blank group with PBS group, there were no significant differences in apoptosis ratio(P>0.05). Conclusion: ①Target sequence of RNAi which aimed at hTERT gene and TRF2 gene was designed efficiently, and the RNAi expression vectors were seen in vivo study efficiently and specifically inhibited the correspond gene expression and promoted cell apoptosis in MCF-7 cells. ②rAd-hTERT vector and rAd-TRF2 vector have no synergistical effect and antagoinstical effect on inhibiting hTERT gene and TRF2 gene mRNA expressing in MCF-7, but there was synergistical effect in terms of the induction of apoptosis. So association-RNAi-technique targeting to the genes of telomere length and stability can effectively promote tumor cell into apoptosis and inhibit breast cancer cell growth. RNAi technique of connecting correlation genes is a more effective gene therapy strategy.

AIM:To investigate the value of 3D-optical coherence tomography (OCT) in the diagnosis of macular disease before phacoemulsification.METHODS:Clinical records of 423 cataract patients (512 eyes) who underwent phacoemulsification combined with intraocular lens implantation in our hospital from June to December in 2015 were retrospectively analyzed.In addition to preoperative routine examination of fundus, Topcon 3D-OCT 2000 was used to examine the macula, the detection rate of macular disease was compared, risk factors of cataract combined with macular disease were analyzed.RESULTS:The OCT image results of 305 cases (384 eyes, 72.1%) were successfully obtained, 133 cases showed macular disease (146 eyes), the detection rate was 28.5% (95%CI:27.64%-29.40%);the macular disease of 35 cases (37 eyes) were detected by routine examination of fundus before operation, the detection rate was 7.2% (95%CI:6.72%-7.74%);the detection rate of 3D-OCT was significantly higher than routine examination of fundus for macular disease (χ2=79.05, P<0.01).Female, over 65 years old, surgical history of diseased eye, and high myopia were risk factors of cataract combined with macular disease, the relative risk was 1.705 (95%CI:1.091,2.664), 1.893 (95%CI:1.219,2.939), 6.593 (95%CI:2.027,21.447) and 95%CI:5.130 (2.841,9.263) respectively, the risk of cataract combined with macular disease showed an increasing trend with rising age.CONCLUSION:In preoperative examination of cataract patients, 3D-OCT has higher sensitivity in the detection of macular disease, especially for women, over 65 years old, high myopia and surgical history of diseased eye, 3D-OCT can be used as a routine preoperative examination.

Abstract: Objective　To analyze the emergency response and long-term intervention effects after the detection of infectious snails epidemic by loop-mediated isothermal amplification (LAMP) assays in Hannan District, Wuhan City, and to explore the application of LAMP in early surveillance and early-warning of schistosomiasis transmission. Methods Snails picked up by the risk monitoring system in Hannan District were examined by anatomical microscopy and LAMP technology to identify the schistosomiasis infection. Emergency response and intensive intervention were initiated in the environment where positive snails appeared, and the long-term effects were evaluated. Results In May 2018, the infectious snails were detected by LAMP technology in Hannan District, and the positive snails were located in Zhujiacha, Dongzhuang Village, Obstacles and weeds were removed and buried by machine in Zhujiacha. 12 700 m2 of snails were killed by drugs, and the mortality rate of snails was more than 80%; no new seropositive persons were found in the emergency examination within 500 m of the positive snail sites. 506 people were examined in Dong Zhuang Village at the end of the year, and 30 positive IHA cases were detected with a blood positive rate of 5.93%, no positive fecal test was found, and all positive blood test patients took preventive medication. The monitoring results of sentinel rats and wild feces were all negative. Health education was carried out, 7 warning signs were deployed and refreshed, and 500 publicity brochures were distributed. After nearly three years of intensified intervention and monitoring in the villages where the positive environment is located, and the density of snails on the stubborn snail has dropped from 0.094/frame to 0.027/frame, and the positive rate of blood test in Dongzhuang Village has steadily dropped from 5.93% to 3.74%. Conclusions The infected snails missed by microscopy were detected by LAMP in Hannan District, which created conditions for the rapid emergency treatment of environment and elimination of positive snail and improved the sensitivity of the surveillance and early warning system in transmission-interrupted areas.

Adult ; Factor VII Deficiency ; complications ; Female ; Humans ; Liver Cirrhosis, Biliary ; complications

Objective To discuss a real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) wether if can be used to detect Brucella. Methods According to the BCSP31 gene sequences specific for Brucella, one pair of primers and one TaqMan probe were designed. A real-time PCR was developed with the BCSP31 fragments cloned into PMD18-T vector. The standard cure was established and the sensitivity, the species specificity and the stability of the assay were evaluated. The clinical blood specimens were detected by QT-PCR and compared with clinical diagnosis. Results The standard curve was established with the standard template and the relationship between the Ct and the DNA copy number was linear(r=0.999). The sensitivity of the real-time PCR was 5 copies/μl. The sensitivity of the common PCR was 5×102 copies/μl. The sensitivity was about 100 times higher than common PCR. Species specificity of this FQ-PCR assay evaluated using genomic DNA from 6 Bmcella strains and 5 non-Brucella strains and strong fluorescence was detected in all Brucella strains. The CV of intra-assay and inter-assay reproducibility were 0.71%,7.23%, reprectively. Twenty-four specimens from clinical brucellosis cases, 19 showed positive, the positive coincident rate was 79%(19/24). The negative results were obtained for all 31 negative control, and the negative coincident rate was 100%(31/31). Two were positive from all 30 specimens clinically suspected. Conclusions Highly specific, sensitive, repeatable and coincidental with clinic, this FQ-PCR is quite useful for rapid detection of tiny DNA of Brucella in various samples and laboratory diagnosis.

OBJECTIVETo explore the effect of combined gene therapy with interference hTERT and TRF2 gene on the treatment of breast cancer.

METHODSRecombinant adenovirus rAd-hTERT and rAd-TRF2 expressing siRNA-hTERT and siRNA-TRF2 was constructed, and the vectors were transfected into MCF-7 cells. Than the expressions of hTERT and TRF2 proteins were detected by Western blot, the inhibition of MCF-7 cell proliferation by MTT colorimetry, and the changes of MCF-7 cell cycle by flow cytometry and the colony forming ability of MCF-7 cells by clone form test.

RESULTSAt 48 h after transfection, the relative expression amounts of hTERT protein of the PBS control group, rAd-blank group, rAd-HK control group, rAd-hTERT group, rAd-TRF2 group and rAd-hTERT and rAd-TRF2 group were 1.00, 0.94 +/- 0.02, 0.95 +/- 0.04, 0.18 +/- 0.04, 0.95 +/- 0.01 and 0.18 +/- 0.04, respectively. The relative expression amounts of TRF2 protein were 1.00, 1.01 +/- 0.08, 0.96 +/- 0.02, 0.95 +/- 0.08, 0.22 +/- 0.01 and 0.26 +/- 0.02, respectively. After transfection of rAd-hTERT or rAd-TRF2 into MCF-7 cells separately, the inhibition rate of cell proliferation was only 54.6% and 48.4%, there was 8.9% +/- 1.2% or 9.2% +/- 2.3% of MCF-7 cells into M phase, 66.4% +/- 1.5% or 64.6% +/- 1.9% of MCF-7 cells was arrested at G(0)/G(1) phase, and the cell colony forming ability was decreased significantly (cell colony number from 100 in PBS control group down to 41.3 +/- 5.1 and 43.7 +/- 6.4). But after transfection by rAd-hTERT and rAd-TRF2 simultaneously, the inhibition rate of cell proliferation was about 82.1%, and M phase cells was significantly reduced to 4.4% +/- 1.2%. Large numbers of cells were arrested at G(0)/G(1) phase (81.4% +/- 1.3%), and the cell colony forming ability was more significantly decreased (cell colony number there were only 29.2 +/- 3.9).

CONCLUSIONMore effective effect of tumor gene therapy can be achieved by combination of interference hTERT and TRF2 genes as compared with interference by either of the single gene alone.

Adenoviridae ; genetics ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Female ; Gene Expression Regulation, Neoplastic ; Genetic Therapy ; Genetic Vectors ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics ; Telomerase ; genetics ; metabolism ; Telomeric Repeat Binding Protein 2 ; genetics ; metabolism ; Transfection ; Tumor Stem Cell Assay

OBJECTIVE: To study cellular mechanism of cardiomyocytes injury in the early stage of crush injury by observing some effects of crush injury rat sera on cultured neonatal rat cardiomyocytes. METHODS: One to three days old neonatal rat cardiomyocytes were cultured in vitro and some effects of crush injury rat sera on beating rate, cell surface area, total protein content, 3H-Leu incorporation, intracellular calcium concentration ([Ca2+]i) and Fos protein expression were observed in cultured rat cardiomyocytes. RESULTS: Compared with normal rat serum group, crush injury rat sera decreased beating rate(beats/min) of cardiomyocytes from 88.3 to 26.4, cell surface area, total protein content, 3H-Leu incorporation, [Ca2+]i (nmol/L) and PI of Fos protein expression were increased. CONCLUSION Crush injury rat sera suppress cell beating, increase intracellular calcium, induce Fos protein synthesis and cause cell hypertrophy, which may cause cardiac injury in the early stage of rush injury.
Animals ; Calcium/metabolism* ; Cell Size/drug effects* ; Cells, Cultured ; Disease Models, Animal ; Extremities/injuries* ; Heart Injuries/pathology* ; Heart Rate/drug effects* ; Immune Sera/pharmacology* ; Myocytes, Cardiac/pathology* ; Proto-Oncogene Proteins c-fos/metabolism* ; Rats ; Rats, Sprague-Dawley

Adolescent ; Adult ; Female ; Humans ; Male ; Mercury ; urine ; Middle Aged ; Reference Values ; Spectrophotometry, Atomic

Objective To investigate the epidemic pattern,diagnosis and treatment of schistosomiasis after the 16-years intervention program on its transmissionin Jiaxing region of Zhejiang province.Methods Clinical data of schistosomiasis patients during the last 10 years and pathological specimens with deposited schistosomal eggs during the last 8 years were retrospectively analyzed.Results The total numbers of schistosomiasis patients admitted to hospital increased from 194 in 2001 to 960 in 2010,and from 78 to 266 with complications.Number of the ones with accompanied diseases increased from 116 to 694.All the numbers of the above said three groups showed an yearly increase.The hardest hit age of the patients was between 60 and 74.The number of specimens with deposited schistosomal eggs increased from 192 in 2003 to 298 in 2010.While the ratio of specimens with deposited schistosomal eggs to the total number of pathological specimens became slightly decreasing,the average age of patients increased,Eggs were mainly deposited on appendix,colon,rectum,stomach,liver,gallbladder and small intestine.Conclusion Schistosomiasis still hit the Jiaxing region,with the average age of patients increased.

Objective To investigate the epidemiology,diagnosis and treatment of an imported falciparum malaria patient in Xilin County,Baise City,so as to provide the reference for improving the diagnosis and treatment of falciparum malaria pa-tients in the future. Methods The epidemiological and clinical data were collected and analyzed. Results The patient had lived in Africa where the malaria was epidemic. The disease attacked him after his coming back home from abroad. The blood test for Plasmodium falciparum was positive. In Xilin County,no local Plasmodium infection was found from 2004 to 2016,and therefore,we concluded that this case was overseas imported. Conclusion The monitoring of overseas returnees in Xilin Coun-ty should be strengthened to timely diagnose and treat the imported cases of malaria.