To construct a subtractive cDNA library of genes transactivated by NS5A protein of hepatitis C virus with suppression subtractive hybridization technique, the mRNA was isolated from HepG2 cells transfected with pcDNA3 1(-) NS5A and pcDNA3 1(-) empty vector, respectively, then the cDNA was synthesized. After restriction enzyme RsaI digestion, small sized cDNAs were obtained. Then the tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After the tester cDNA was hybridized with the driver cDNA twice and underwent two times of nested PCR, it was then subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain JM109. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. The amplified library contained 121 positive clones. Colony PCR showed that 115 clones contained 200- 1 000 bp inserts. Sequence analysis was performed in 90 clones, and the full length sequences were obtained with bioinformatics method. Altogether 46 kinds of coding sequences were acquired, which consisted of 31 kinds of known and 15 kinds of unknown ones. The obtained sequences might be target genes transactivated by NS5A protein of HCV, among which some genes coding proteins involved in cell cycle regulation, cell apoptosis, signal transduction pathway and tumour development. The results indicated that the subtractive library of genes transactivated by NS5A protein of HCV was constructed successfully, which brought some new clues for studying the biological functions and pathogenesis of the viral proteins

Polymerase chain reaction was employed to amplify the HBV X gene from plasmid pCP10, and the product was cloned into pVR1012, then transfected HepG2 cells and cotransfected HepG2 cells with reporter plasmid pSV lacZ HBx protein produced by HepG2 cells was measured by ELISA method The activity of ? galactosidase was measured by a kit, which reflected the transactivating function of HBx protein The results showed that HepG2 cells transfected by pVR1012 X could express HBx protein The expression of ? galactosidase in HepG2 cells transfected by the pVR1012 X was 3 2 fold higher as that of control plasmid It is suggested that the recombinant plasmid pVR1012 X can be expressed in mammalian cell line, and has transactivating effect on SV40 early promoter

?AIM: To study the changes of choroidal thickness and hemodynamic parameters in patients with diabetic retinopathy and their influencing factors.?METHODS: From January 2013 to January 2015, 100 patients (100 eyes) with type 2 diabetes were divided into 3 groups:34 patients without diabetic retinopathy ( NDR) , 36 patients with non proliferative diabetic retinopathy ( NPDR) group, and 30 patients with proliferative diabetic retinopathy (PDR). According to the results of OCT, the patients with diabetic retinopathy were divided into 2 groups: diabetic macular edema ( DME ) group ( 28 cases) , and 38 cases without diabetic macular edema. During the same period in our hospital 35 subjects for physical examination were selected as the control group. The hemodynamic parameters of the posterior ciliary artery in different groups of patients at different distance from the center of the macular were compared, analyzing influencing factors.?RESULTS:With the aggravation of diabetic retinopathy, the choroidal thickness in different distance from the center of the macular decreased. The choroidal thickness of NPDR and PDR group were thinner than that of the control group (P<0. 05). The choroidal thickness of NDR group was not different from the control group (P>0. 05). There was no significant difference in the choroidal thickness between the DME patients and non DME patients (P>0. 05). Pearson correlation analysis showed that there was no significant correlation between choroidal thickness of patients with diabetic retinopathy and diabetic duration, fasting blood glucose, HbA1c, eye axis length, systolic blood pressure and diastolic blood pressure (P>0. 05), but there was a correlation with BCVA (logMAR) (P<0. 01). EDV and PSV in NDR group and NPDR group were significantly lower than those in control group, RI was higher than that in control group. PSV and EDV in PDR group were significantly lower than those in other three groups, RI was higher than the other three groups, and the difference was statistically significant ( P<0. 05).?CONCLUSION:With the severity of retinopathy in type 2 diabetic patients, the choroidal thickness decreased, and the thickness of the choroid is beneficial in the comprehensive analysis of 2 diabetic retinopathy.

BACKGROUND:The technique of vacuum sealing drainage, initialy used for better wound healing, has been widely applied to al kinds of refractory wounds. OBJECTIVE:To summarize the research progress in vacuum sealing drainage for treatment of severe soft tissue injury. METHODS: An electronic retrieve was performed in Wanfang database, VIP database and PubMed database from January 1989 to August 2014 was performed for papers regarding the vacuum sealing drainage technology with the key words of “vacuum sealing drainage technology; injury; gentamicin; hyperbaric oxygen; nerve growth factor; chymotrypsin” in Chinese or English. Finaly 48 articles were involved in the final analysis according to the inclusion criteria. RESULTS AND CONCLUSION:Vacuum sealing drainage is a highly effective treatment technique to promote wound healing which can promote wound blood circulation, reduce sweling, inhibit bacterial growth, stimulate growth of granulation tissue by mechanical stress, inhibit cel apoptosis, thereby helping wound healing. Compared to conventional open-dressings, vacuum sealing drainage has a faster wound healing speed, lower infection rate, and fewer dressing change frequency. With recent advances in medicine, vacuum sealing drainagetechnology cannot be simply used for treatment of skin soft tissue damage, but in combination with gentamicin, hyperbaric oxygen, nerve growth factor and chymotrypsin to achieve better therapeutic effects.

Objective To study the relationship between activity and virulence of secretory acid proteinase and extracellular phosphlipase of different Candida albicans isolated from various specimens.Methods 190 strains of Candida albicans were isolated from various specimens(sputum,blood,thrush,wound,secretion of vaginitis) of patients with Candida albicans infection in hospital.Milk-plate medium and egg-yolk medium were used respectively to test the activities of secretory acid proteinase and extracellular phosphlipase of Candida albicans.The suspension of two forms of Candida albicans(5?106CFU?mL-1) were injected to vein of tail in mice respectively in virulence test.The mortality and mean survival time of mice were observed in 1 month.The virulence was appreciated with the mean survival time of mice.Results All of 190 strains,the positive detectable rates of secretory acid proteinase and extracellular phosphlipase were 83.68% and 85.26%,respectively.Animal experimental results showed the activities of secretory acid proteinase and extracellular phospolipase in hyphal forms were significantly higher than that in spore form(P

Objective: All-trans retinoic acid(ATRA) is a classic drug that can induce tumor differentiation,and its influence on the aberrant methylation of cancer cells has been studied insufficiently.The objective of this study was to observe the influence of ATRA on the methylation of the RARa gene promoter in ovarian cancer cell line COC2 and its relationship with the RARa expression.Methods: The ovarian cancer cell line COC2 was treated with different concentrations of ATRA for different times.Methylation specific PCR(MSP) and bisulfate sequencing methods were used to detect the changes in the methylation of the RARa promoter after ATRA treatment.RT-PCR was employed to observe the changes in the expression level of RARa mRNA.Results: Aberrant methylation of the RARa gene promoter was found in the ovarian cancer cell line COC2.ATRA treatment decreased the number of RARa promoter methylation sites in COC2 within a certain scope in a concentrationand time-dependent manner,and increased the expression of RARa mRNA.Conclusion: Aberrantly high methylation of the RARa promoter exists in the ovarian cancer cell line COC2;ATRA can partially reverse the methylation and increase the RARa mRNA expression.

Recent studies demonstrate that the long non-coding RNAs (lncRNAs), pseudogenes, circular RNAs (circRNAs) and so on can be used as competitive endogenous RNAs (ceRNAs), binding to microRNAs (miRNAs), to regulate the expression level of their targets.This novel interplay optimizes traditional liner miRNA→RNA pattern and has become a research hotspot in the scientific community.Dysregulation of ceRNA interplay will influence the body′s normal life activities, leading to the occurrence of diseases, and even tumor formation.This article briefly introduces the key components of ceRNA crosstalk and its research progress in gastric cancer pathogenesis, so as to provide a new thought for cancer research, clinical diagnosis and therapeutics.

Objective To investigate motor nerve function status in rats with diabetes mellitus by motor unit number estimation (MUNE), and discuss it′s early diagnostic value in diabetic peripheral neuropathy (DPN). Methods Diabetic rat model (DM group) was induced by streptozotocin. The MUNE of gastrocnemius muscle and motor nerve conduction (MCV, CMAP) of the sciatic nerve were measured at the 4th, 8th and 12th week after onset of hyperglycemia in the DM group and the control group (normal SD rats). The ultrastructure of sciatic nerve was observed by electron microscope. Results At the 4th week, MUNE of gastrocnemius muscle was significantly decreased in DM group compared to that of the control group (275.88 ± 87.87 vs 369.71 ± 75.64,P<0.05). There were no significant differences in MCV and CMAP of sciatic nerve be?tween two groups. The electron microscopy observation showed that most nerve fibers were normal;a small amount of axonal atrophy, and myelin lamellar structure was separated in DM group. At the 8th week, compared with the control group, MUNE were reduced in gastrocnemius muscle in DM group (357.49±72.68 vs 221.26±92.41, P<0.01). There were no significant dif?ferences in MCV and CMAP of the sciatic nerve between DM group and control group. The electron microscope observation showed that part of nerve fibers were normal, the myelin focal plate layer was loose and separated, axonal atrophy, the axonal membrane and myelin sheath inner layer was separated with big gap. At the 12th week, MUNE of gastrocnemius muscle (127.87±19.80 vs 366.85±51.25), sciatic nerve MCV [(35.06±4.43) m/s vs (50.47±6.07) m/s] and CMAP [(2.91±1.37) mV vs (5.98±2.14) mV] were significantly decreased in DM group than those of control group (P<0.01). The electron microscopy observation showed severely damaged myelin flex and axonal squeeze. Conclusion MUNE is much earlier in detecting ear?ly motor nerve dysfunction in DM than conventional motor nerve conduction test.

Objective To assess the function of motor nerve fiber in patients with diabetic peripheral neuropathy (DPN) by single-fiber conduction studies.Methods According to the diagnostic standard of DPN issued in Toronto meeting in 2009,on the basis of the result of peroneal nerve conventional conduction study,a total of 65 patients with DPN in the Department of Endocrinology and the Department of Neurology of Tianjin Third Central Hospital from October 2012 to October 2013 were enrolled into the study,from whom 33 had abnormal sensory conduction (sensory-diabetic peripheral neuropathy group,S-DPN group),32 had abnormal sensory motor conduction (sensory motor-diabetic peripheral neuropathy group,SM-DPN group).Single-fiber conduction velocity (SF-CV) and single-fiber distal motor latency (SF-DML)were detected in all subjects.The obtained results were compared with the data from 34 healthy volunteers (control group).The relationship of SF-CV,SF-DML and the duration of diabetes mellitus,fasting glucose,HbA1 c was also studied in DPN patients.Results The SF-CV ((43.1 ± 3.6) m/s) was decreased in S-DPN group compared with control group ((47.5 ± 3.3) m/s,t =5.077,P ＜ 0.01).There were no significant differences in SF-DML ((3.6 ± 0.7) ms),motor nerve conduction velocity (MCV (49.5 ± 2.6)m/s) and DML ((3.4 ± 0.6) ms) in S-DPN group compared with that of control group ((3.4 ± 0.5) ms,(50.9 ± 3.5) m/s,(3.2 ± 0.5) ms,respectively).SM-DPN group had lower SF-CV ((35.2 ± 3.6)m/s,t =9.119,14.219),MCV ((40.9 ± 3.2) m/s,t =11.131,13.025) and increased SF-DML ((4.5±0.7) ms,t=5.692,7.231),DML ((4.2 ±0.7) ms,t=5.561,6.975) compared with the other two groups (P ＜0.01).SF-CV in DPN patients was negatively related to the diabetic duration (r =-0.340,P =0.006),while SF-DML had no correlation with duration of DM,fasting blood glucose and HbAlc.Conclusions Detection of SF-CV is easy to find early motor nerve dysfunction in DPN patients.SF-CV is decreased with the increasing duration of diabetes.

Breast cancer is one of the most common cancers in China. With increasing of the incidence and mortality, the harm to women is becoming much serious. Estrogen positive patients need to take 5-10 years′ endocrine drugs to prolong survival and prevent recurrence. Endocrine therapy has become one of the most common and effective methods in the treatment of breast cancer. In order to achieve the desired effect of cancer treatment, reduce cancer metastasis and recurrence rate, good compliance is essential. In conclusion, this review summarizes the evaluation of compliance, various evaluation tools and their use.