Objective: To investigate the effects of miR-200a on the proliferation of lung cancer cells and to identify its direct target genes. Methods:Real-time PCR was performed to analyze the miR-200a expression in 15 paired clinical specimens of non-small cell lung cancer and adjacent noncancerous tissues, human lung cancer cell lines (A549, NCI-H520, and SK-MES-1), and one human normal lung bronchial epithelial cell line (16HBE). The effects of miR-200a on the proliferation of A549 lung cancer cells were detected through CCK-8 method. The candidate target genes of miR-200a were identified by bioinformatics screening and then verified by dual luciferase reporter gene assay, real-time PCR, and Western blot. The effects of YAP1 downregulation on the proliferation of A549 lung cancer cell line were also observed through CCK-8 method. Results:The miR-200a expression in non-small cell lung cancer tissues and lung cancer cell lines was significantly decreased (P<0.01). The upregulation of miR-200a expression could significantly inhibit the pro-liferation of A549 lung cancer cells (P<0.01). Dual luciferase reporter gene indicated that miR-200a could directly affect the 3′-untrans-lated region of the YAP1 gene to inhibit luciferase activity (P<0.01). Real-time PCR and Western blot revealed that the upregulation of miR-200a expression could significantly reduce the mRNA and protein expression levels of YAP1 in A549 lung cancer cells (P<0.01). CCK-8 method indicated that the downregulation of YAP1 could significantly prevent the proliferation of A549 lung cancer cells (P<0.01). Conclusion:MiR-200a inhibits the proliferation of lung cancer cells by targeting YAP1. Thus, miR-200a elicits tumor suppression effects.

AimTo study the effect of interferon gamma( IFN-γ) and tumor necrosis factor alpha(TNF-α) in the immune pathogenesis of dengue virus infection. Method The serum levels of IFN-γ and TNF-α were measured with emzyme linked immunosorbent assay (ELISA) method in 30 cases of the patients with the dengue virus infection in Guangzhou district. The results were treated with t-test of two sample mean. ResultsThe serum levels of IFN-γ and TNF-α in patients with the dengue virus infection were much higher than healthy controls( P ＜ 0.05、 P ＜ 0.01 ). IFN-γ was detectable on the first day of postinfection. Level of IFN-γ reached their peaks on the second day, then declined . The level of TNF-α had an obvious rise from the second day and reached their peaks on the third day, then declined. ConclusionThe data suggest that the IFN-γ and TNF-α may play an important role in the dengue virus pathogenicity and immunity.

Objective:To establish a method of multi-PCR to amplify the complete DNA sequence (CDS) of TCR ? and ? chain of the antigen-specific T lymphocytes in local pathologic specimen of active pulmonary tuberculosis patients, and to analyze ?/? T cell receptor gene rearrangement and CDR3 repertoire.Methods:The lymphocytes in bronchoalveolar lavage (BAL) of active pulmonary tuberculosis patients were separated. Following total RNA extraction, cDNA synthesis, Multi-PCR, recombinant clones construction, and sequencing, the CDS of TCR ? and ? chains from these lymphocytes were analyzed by using software of DNAstar and internet TCR resources.Results:24 of ? chain CDS and 13 of ? chain CDS from 3 samples of BAL were obtained. As for TCR ? chain, AV1S2 (54%), AV12S3 (41%), and AV12S2(5%) appeared frequently. BV2(38%), BV29S1(46%), BV14(3%), and BV4S2(3%) in TCR ? chain appeared more often. There were CDR3 diversities between samples and even in the same sample by amino acid sequence analysis, but there were a few identical or similar amino acid sequences. There was the same amino acid sequence of SVGTGTLHQETQY in CDR3 region of ? chain of BAL sample No.1 and No.2; The sequence of AVRDWAGNMLT appeared in two ? chains of BAL sample No.2 and No.3; Moreover, the sequence of AV…DNN…RLM appeared in ? chains of BAL sample No.2 and No.3.Conclusion:A method of Multi-PCR is used to amplify TCR ? and ? chain CDS of tuberculosis patients. There are characteristic T cell clones to proliferate,with TCR ? and ? chain repertiore skewing in local infective focus. The sequences of CDR3 in different TCR clones are mostly different but there are a few identical or similar sequences in the same patient or even between different patients. The identical amino acid sequences of CDR3 are possibly specific for recognizing MTB polypeptide.

[Objects]To isolate and identify the pathogen of the large outbreak of dengue in Guangdong province in 2014. To understand the origin and the phylogenetic characteristics of the isolates ,and provide scientific foundation for the surveillance and prevention of dengue fever.[Methods]Collected the patient serum samples over all the Guangdong province during the 2014 outbreakperiod,isolated and identified the virus from these samples. Amplified complete E gene and complete genome with certain primers and sequenced all the products. Then the Phylogenetic ,Bayesian phylogeography and mutations analysis were carried.[Results]40 DENV-1 strains were isolated and identified. 40 complete E gene sequences and 6 complete genome sequences of DENV-1 were obtained. Phylogenetic analysis with E gene sequences revealed that the 40 isolates were classified into two genotypes including 16 genotypeⅠ(Asia)and 24 genotypeⅤ(America/Africa). 14 genotypeⅠisolates were clustered closest with isolates from Guangdong province(2013)and Sigapore(2013)which share the nucletide identities of 99.6% ~ 99.9%,other two genotypeⅠisolates were clustered with strains from Malaysia (2013) and both share the nucletide identities of 99.7%;24 genotypeⅤisolates were all classified in one clade with striains from Bangladesh(2009),China(2009)and Bhutan(2013)which share nucletide identities of 99.0%-99.9%. Further analysis with six complete genome sequences showed that five isolates were clustered closest with strains isolated from Guangdong province(2013)share the nucletide identities of 99.6%-99.8% while the sixth stains closest with strains isolated from Myanmar(2002)share the nucletide identities of 98.8%. The isolates have five amino acid mutations compared with strains epidemic in Guangdong province in 2013,three mutations(S88V,E203G,T275R)are in the EⅡdomain and one mutation (S305P)is in the EⅢdomain which associated with virulence.[Conclusions]During the outbreak in Guangdong province in 2014, DENV-1 is the predominant causative serotype,and there are at least two different kinds of genotypes of DENV-1 largely epidemiced in the whole province. Evolution analysis reveals the multiple origins of the isolates which may origin from Guangdong province , Sigapore,Malaysia,Myanmar so that we should enhance the study and surveillance of autochthonous and vectors in order to understand the epidemic way of dengue in Guangdong province. The isolates have had four mutations in the domain associated with virulence which remain further study to know their biological effects.

Objective To detect dengue virus infection by serological method and to determine the sequences of E gene of dengue virus isolated from Guangzhou in 2006.so as to clarify the possible origin of dengue fever.Methods IgM and IgG antibodies to dengue virus were detected by immunochromatographic test(ICT);NSI antigen and IgM antibody were detected by enzyme-linked immunosorbent assay(ELISA).The virus was cultured and isolated from the serum samples within 2 days using C6/36 cell lines and was identified by immuno-fluorescence assay(IFA)and RT-PCR.The E gene of isolated virus DV1-GZ42/06 was sequenced;homological analysis and phylogenetic tree analysis were performed by comparing with the reference strains and epidemic virus strains.Results The positive rates of IgM and IgG of dengue virus in patients were 89.5%(433/484)and 38.0%(184/484)by ICT,respectively.The positive rates of NS1 antigen were 92.7%(38/41)in day 1 to day 2,83.3%(70/84)in day 3 to day 5,and 10.9%(5/46)in day 6 to day 10;and the IgM detection rates were 2.4%(1/41),51.2%(43/84)and 97.8%(45/46)at the same period by ELISA.Twenty-five strains of dengue virus were isolated from 41 serum samples(6 1.O%)and were identified as type 1 dengue virus by IFA and RT-PCR.The sequencing and phylogenetic analysis of the E gene showed that the homology between the isolated Guangzhou/42/06 strain and standard strain Hawaii/45 was 94.6%.and it had a high homology with the Thailand/NI09V104,Vietnam/06.and Vietnam/07 isolates(99.0%,98.6%and 98.6%,respectively)and belonged to the same cladogram,but had low homology with the isolated strain from Guangdong before 2006.Conclusions The detection of NS1 antigen is important in the early diagnosis of dengue fever.The outbreak of dengue fever in Guangzhou in 2006 was possibly caused by the cases from neighboring countries.

Peak Torque(PT),Total Work(TW),Average Power(AP),Torque Acceleration Energy(TAE),Set Total Work(STW),Endurance Ratio (ER)and DOrsal/plantar ratio of ankle dorsal and plantarflexion were tested in 101 patients with lumbar disc protrusion using a Cybex一6000 isokinetic muscle test-ing instrument.The results showed the most tested of the affected side was signficantly lower than that onthe opposite side. The PT,TW and STW of the affected side was lower 70一80%than that on the oppo-site side. The rest was lower 20一30%than another side. No significant difference of endurance ratios be-tween the affected and unaffected sides was noted.

[Objective]This study was designed to investigate the genetic evolution of the neuraminidase(NA)gene of seasonal A/H1N1 and 2009 novel A/H1N1 inflilenza virus,and discuss the genetic variation of influenza A virus.[Methods]The virus strains were separately isolated from the clinical samples collected in 2006 and 2009,and then identified as seasonal A/H1N1 and novel A/H1N1.The full length of the NA gene of these strains was amplified by RT-PCR.Then the genetic evolution and mutations of important functional sites were analyzed.[Results]The homology of NA gene between the 2009 novel A/H1N1 isolates and 2006 seasonal A/H1N1 isolates was low(77.9%～78.8%),so was the homology of NA gene between the 2009 novel A/H1N1 isolates and representative strains of different periods and 1979-2001 WHO recommended vaccine strains(78.1%～79.3%).But compared with the WHO recommended vaccine strains of 2009 novel A/H1N1,the homology reached more than 99%.The genetic evolution analysis revealed that NA gene of 2009 novel A/H1N1 had the closest genetic relationship with the swine influenza A virus(A/swine/Belgium/1/1983)from Eurasian Iineage,and some of the antigenic sites and neuraminidase active sites of NA gene of seasonal A/H1N1 were mutated after 2005.[Conclusion]The NA gene of 2009 novel A/H1N1 may originate from Eurasian Iineage of swine influenza virus.The variation of NA gene of seasonal A/H1N1 has occurred in a certain degree.Hence,it is very necessary to continuously monitor the variant of influenza A virus.

Objective To investigate the risk factors of acute kidney injury (AKI) occurring in patients with critical neurological disease, and the related factors affecting their prognosis. Methods The clinical data of 207 patients with critical neurological disease admitted to the Department of Critical Care Medicine of Anhui Provincial Hospital Affiliated to Anhui Medical University (South District) from January 2016 to March 2017 were analyzed retrospectively, they were assigned into an AKI group (40 cases) and a non-AKI group (167 cases), and according to the prognosis, the patients with AKI were subdivided into a survival subgroup (14 cases) and a death subgroup (26 cases). Clinical data of Glasgow coma score (GCS), acute physiology and chronic health evaluation Ⅱ (APACHEⅡ), blood glucose, white blood cell count (WBC), central venous pressure (CVP), blood sodium, cystatin C, urea nitrogen (BUN) etc. index levels and the proportions of patients using glycerin fructose and furosemide before occurrence of AKI were collected. The indexes with statistical significant differences found in the univariate analysis were analyzed by multivariate logistic regression analysis to screen out the risk factors influencing the occurrence of AKI and the factors related to the prognosis of the AKI patients; the receiver operating characteristic curve (ROC) was drawn to assess the predictive value of risk factors in patients with severe neurological disease to develop AKI. Results The incidence of AKI was 19.3% (40/207) in the patients with critical neurological disease. The hospital mortality in AKI group was significantly higher than that in the non-AKI group [65.0% (26/40) vs. 22.2% (37/167), P < 0.01]. Compared with non-AKI group, GCS (4.44±1.65 vs. 5.39±1.62), CVP [cmH2O (1 cmH2O = 0.098 kPa): 7.69±2.66 vs. 8.98±2.56] were obviously lower in AKI group at admission, APACHEⅡ(24.50±3.67 vs. 20.05±4.42), blood glucose (mmol/L: 12.33±6.53 vs. 9.33±3.26), serum sodium (mmol/L: 144.75±10.85 vs. 140.58±5.23), WBC (×109/L: 16.15±6.25 vs. 12.79±4.22), Cystatin C (mg/L: 1.27±0.74 vs. 0.74±0.26) and BUN (mmol/L: 7.81±3.33 vs. 5.53±3.20) and proportion of male [77.5% (31/40) vs. 59.9% (100/167)], patients with the comorbidity of hypotension [37.5% (15/40) vs. 19.8% (33/167)], use of glycerin fructose [17.5% (17/40) vs. 3.6% (6/167)], or use of furosemide [70.0% (28/40) vs. 13.8%(6/167)] were significantly increased in AKI group, there was a statistically significant difference between the above two groups (all P < 0.05). Multivariate logistic regression analysis showed that the hyperglycemia [odds ratio (OR) = 1.201, 95% confidence interval (95%CI) = 1.01-1.42, P < 0.05] and use of furosemide for treatment (OR = 24.493, 95%CI =4.92-120.36, P < 0.01) were the independent risk factors for occurrence of AKI in critical neurological patients. ROC curve analysis showed that blood sugar had certain predictive value of developing AKI in patients with critical neurological disease, the area under the ROC curve (AUC) of blood glucose was 0.733, when the optimal cut-off value of blood glucose was 9.05 mmol/L, the sensitivity was 77.5% and the specificity was 62.6%. Compared with the survival subgroup in the patients with AKI, the GCS at admission in death subgroup was significantly lower (3.77±0.87 vs. 5.50±2.03), but their levels of blood glucose (mmol/L: 16.51±9.10 vs. 10.09±2.89) and BUN (mmol/L: 10.26±3.07 vs. 6.48±2.70) were obviously higher than those in the survival subgroup (all P < 0.05). Conclusion AKI is one of the common complications in patients with critical neurological disease, hyperglycemia and the use of furosemide are the independent risk factors of occurrence of AKI in such patients; the blood glucose has moderate predictive value; and lower GCS, higher glucose and BUN levels in AKI patients may enhance their risk of death.