Objective: To investigate the bacterial species, characteristics and differences of oral bacteria flora of saliva in the longevous elderly between in Bama county and in Debao county in Guangxi, in order to explore the relationship between longevity and oral salivary bacteria flora in the elderly. Methods: The saliva was taken from the longevous elderly in Bama county(BM group)and people aged over 60 years in Debao county(BS group)separately, and the total DNA was extracted.The 16S rDNA-V4 region was amplified by PCR and analyzed by sequencing.The main species and diversity of bacterial colonies were recorded for difference analysis. Results: A total of 14 saliva samples were collected from 7 cases in BM group and 7 cases in BS group.A total of 369 OTUs were generated by cluster analysis of 14 samples.At the genus level, the dominant salivary bacteria flora were Ctinomyces, Capnocytophaga, Chryseobacterium, Fusobacterium, Haemophilus, Lactobacillus, Leptotrichia, Neisseria, Porphyromonas, Prevotella, Rothia, Streptococcus, Veillonella in both BM group and BS group.The OTU PCA analysis showed that some evidence for indeterminate differences was found, but statistically significant differences did not exist in the dominant components of oral flora between the two groups(P>0.05). Also, the same tendency toward the diversity(P>0.05)was presented.Similarly, the species annotation analysis and the heat map showed that there were no significant differences(P>0.05)in oral salivary flora composition between the two groups.Lactobacillu was always the prevailing flora in the Phylume, Class, Order, Family and Genus, but the abundance ratio was different between the two groups as following: Lactobacillus abundance in salivary bacteria flora was higher in BM Group than in the BS group, while Mycoplasma abundance was lower in BM Group than in the BS group(P<0.05). Conclusions The dominant salivary bacteria flora is Lactobacillus in both BM and BS group, while, the abundance of Lactobacillus is higher in the BM group than in the BS group, which indicates that the longevity of population in Bama county may be related to Lactobacillus.

Objective To study the effect of flumazenil on hypnotic mice induced by diazepam and zolpidem ,and to eval-uate the possibility of flumazenil oral administration .Methods First ,Kunming mice were injected intraperitoneally with nor-mal saline and sodium pentobarbital (S + W) ,diazepam and pentobarbital sodium (D + W) ,zolpidem and pentobarbital sodi-um (Z + W) .The hypnotic effect of diazepam and zolpidem on prolonging the sleep time of pentobarbital sodium would be ver-ified by (D + W) group and (Z + W) group .Then the mice were injected intraperitoneally with flumazenil .The sleep time was used as the evaluation index to evaluate the effect of flumazenil against hypnosis . Finally , the oral administration of flumazenil was observed against hypnosis ,which was evaluated by using sleep time as an index .Results Compared with the control group (S+W) ,the diazepam group (D+W) and the zolpidem group (Z+W) significantly prolonged the sleep time in-duced by pentobarbital sodium (P<0 .001 ,P<0 .05);After Intraperitoneal injection of flumazenil ,compared with the diazepam group (D+W) and the zolpidem group (Z+W) ,the sleep time of the diazepam group [F(ip)+D+W] and the zolpidem group [F(ip)+Z+W] were significantly shorter (P<0 .001 ,P<0 .05);After oral administration of flumazenil ,the sleep time of the diazepam group [F(ig)+ D+ W] and the zolpidem group [F(ig)+ Z+ W] were also significantly shorter (P< 0 .001 ,P<0.05) .Conclusion Flumazenil ,whether intraperitoneal injection or intragastric administration ,could antagonize the hypnotic effect of diazepam and zolpidem .It was proved that oral administration of flumazenil had the same effect compared with intrap-eritoneal injection of flumazenil ,which provided the possibility of preparation of oral administration of flumazenil .

Objective To explore the risk factors of osteoporosis in elderly patients with ischemic stroke by intracranial atherosclerotic stenosis(ICAS). Methods 342 elderly patients(≥ 65 year)with ischemic stroke by ICAS were divided into osteoporosis group and non-osteoporosis group,multivariate unconditional Logistic re-gression analysis was used to analyze the risk factors for elderly patients with ischemic stroke by ICAS. Results Univariate analysis shows that female,unilateral anterior circulation infarct,involvement of vessels of more than two branches,complete paralysis,delayed paralysis,BI index were significantly associated with osteoporosis in el-derly patients with ischemic stroke by ICAS between the two groups(all P < 0.05). Multiple Logistic regression analysis revealed that gender,disease duration,involvement of cerebral circulation,paralysis degree,muscle ten-sion state were independent risk factors for the development of osteoporosis in elderly patients with ischemic stroke by ICAS(OR = 3.459,2.486,4.540,3.819,3.699,P < 0.05). Conclusions Gender,disease duration,in-volvement of cerebral circulation,paralysis degree,muscle tension state are independent risk factors of osteoporo-sis in elderly patients with ischemic stroke by ICAS,provides a theoretical basis for the prevention of osteoporosis in elderly patients with ischemic stroke by ICAS.

Objective To investigate the features of risk factors of minor stroke with CISS classification in Guangdong Province. Methods We retrospectively investigated the patients admitted within 3 days of the occurrence of a minor stroke, and were classified by CISS criteria as large artery atherosclerosis (LAA), cardiogenic stroke (CS), penetrating artery disease (PAD), other etiology (OE), undetermined etiology (UE). Results In this study, 303 pa-tients met the inclusion criteria of minor stroke. The highest percentage of the risk factors included hypertension (72.3%), hyperlipidemia (58.3%), and diabetes mellitus (39.9%). Among different subtypes, 41.9% were diagnosed with LAA, and 50.8% with PAD. Plasma triglyceride (TG)(1.765 ±1.18)mg/L vs.(2.19 ±1.84)mg/L,P=0.03], apolipoproteinsB (ApoB) [(0.95±0.29)mg/L vs.(1.11±0.46)mg/L,P=0.009]C-reactive protein (CRP) [(6.63±11.30) mg/L vs.(3.42 ±5.02)mg/L,P=0.042] and ApoB/ApoA1 ratio [(0.754 ±0.25)mg/L vs.(0.875 ±0.49)mg/L,P=0.019], differed significantly between group LAA and PAD. Conclusion Hypertension, hyperlipidemia and diabetes mellitus are the major risk factors of minor stroke. The most common subtypes of the minor stroke patients in Guangdong Province are LAA and PAD, and detecting their TG, apoB, CRP level and apoB/apoA1 ratio might help subclassify minor stroke according to CISS.

Objective To establish methods for the determination of doxorubicin and elacridar, and prepare PLGA nanoparticles for the co-delivery of doxorubicin and elacridar.Methods Ultraviolet spectrophotometry (UV) and high performance liquid chromatography (HPLC) were used to establish the determination method of doxorubicin and elacridar, respectively;co-delivery nanoparticles system was prepared by nanoprecipitation method, and optimizing the prescription was by adjusting the dosage ratio of the two drugs to investigate the particle size,morphology, encapsulation efficiency (EE), drug loading (DL) and in vitro release.Results The linearity of doxorubicin was better in the rang of 1 to 40 μg/ml, A=0.021C+0.002,r=0.999 5;the linearity of elacridar was better in the rang of 0.5 to 100 μg/ml,A=120 742.462 6C+1 974.570 4,r=1.000 0;the particle size was about 50 nm;transmission electron microscope (TEM) showed that nanoparticles were round in shape and had a good dispersion;EE of doxorubicin and elacridar were 56.58%、51.66%,respectively, DL of doxorubicin and elacridar were 1.48%、1.85%,respectively,the molar ratio of two drugs was about 1∶1;the nanoparticles released slowly in vitro.Conclusion The established methods of doxorubicin and elacridar were convenient and efficient, accurate and repeatable.The Co-delivery nanoparticles system was well dispersionand smaller size, which could be used for further studies.

Objective To investigate the effects of Bucinnazine Hydrochloride on the pain behavior and the expression of caveolin-1 (Cav-1) in the anterior cingulate cortex of neuropathic pain mice.Methods 64 adult male Kunming mice (20-25g) were divided randomly into 4 groups with 16 in each group:Sham+BH(Bucinnazine Hydrochloride) group,Sham+NS (Normal Saline) group,CCI+ BH group and CCI+ NS group.The corresponding drugs were administered by intraperitoneal injectionfrom the forth day after CCI once a day for three days.Paw thermal withdrawal latency was measured by Hargreaves methods.Mechanicalwithdrawal threshold was assayed by electronic dolorimeter.c-Fos protein in anterior cingulate cortex was detected by immunohistochemistry staining and the expression of t-Cav-1,p-Cav-1was detected by Western blot.Results Bucinnazine Hydrochloride administered by intraperitoneal injection(0.1 mg/10 g,mice) alleviated thermal hyperalgesia and mechanical allodynia of CCI mice.Compared with the forth day (4.92±0.41) s of CCI+BH group,paw withdrawal latency on the fifth day(5.92±0.61) s was increased(P＜0.05),and on the sixth day(7.93±0.91) s and seventh day (9.12±0.69)s were increased more(P＜0.01,P＜0.01).The paw withdrawal mechanical threshold on the sixth and seventh day of CCI+BH group mice((2.54 ±0.41)g,(3.68±0.61)g) were increased significantly (P＜0.01,P＜0.01)compared with the forth day(1.55± 0.31)g.Immunohistochenistry results showed that the expression of c-Fos decreased after treated with Bucinnazine Hydrochloride in the anterior cingulate cortex of CCI mice(P＜0.001).Western Blotting showed that the expression of t-Cav-1 (1.97±0.31) and p-Cav-1 (0.11 ±0.09) in the anterior cingulate cortex of CCI +BH group mice decreased compared with that of in CCI+NS group mice(t-Cav-1:2.87±0.15,p-Cav-1:0.48± 0.09) (P＜0.01,P＜0.01).Conclusion Bucinnazine Hydrochloride can alleviate both thermal hyperalgesia and mechanical allodynia of neuropathic pain of mice,and reduce the expression of c-Fos,t-Cav-1,p-Cav-1 in the anterior cingulate cortex of neuropathic pain mice.

Objective To discuss the correlation between MTHFR gene polymorphism and unexplained recurrent spontaneous abortion.Methods A case control study was used in this study,140 patients with unexplained recurrent spontaneous abortion(UR-SA) (abortion group)and 143 cases of normal women(control group)were recruited.Genomic DNA was obtained and extracted from the oral mucosa cells.Fluorescence quantitative PCR was used to examine the MTHFR gene polymorphisms,and Taqman-MGB technology was conducted to analysis the relationship between single nucleotide polymorphism and disease.Results There was statistically significant difference in the frequencies of C677T genotype and alleles between the two groups(P＜0.05).However,no significant difference in the frequencies of A1298C genotype and alleles between the two groups(P＞0.05).Conclusion MTHFR gene C677T polymorphism might be one of the genetic risk factors of URSA.

Objective To investigate the influence of the genotype distribution of methylenetetrahydrofolate reductase (MTHFR) C677T,A1298C and methionine synthase reductase (MTRR) A66G in threatened abortion of Chinese Han gestationalage women in Sanya city,which involved in the folic acid biosynthetic pathway among.Methods One hundred and thirty-nine samples of case group and the same number of control group were recruited from Sanya region in Hainan Province.Genomic DNA was extracted from the mucosal epithelium of the subjects.The gene polyrnorphisms of MTHFR and MTRR were detected by Fluorescence quantitative PCR technology.The distribution frequencies of both case group and control group.were analyzed and compared,to investigate the effect of the gene polymorphisms on threatened abortion.Results Both the case group and the control group complied with Hardy-Weinberg law.The genotype frequency of MTHFR C677T,MTHFR A1298C and MTRR A66G were not significantly different.Conclusion This study suggests that the gene polymorphism which involved in folic metabolism was not significantly different from the group of threatened abortion and the control group,and whether the metabolism related genes are the risk factors of threatened abortion need to be further discussed.

Objective To study and optimize the preparation condition of pVAX1‐wapA‐loaded nanoparticles and deter‐mine the transfection efficiency .Methods The related effects of the crucial factors for the formation of nanoparticles:concen‐tration of chitosan and TPP ,pH value ,N/P ratio were studied by single‐factor experiment ,with nanoparticles size and zeta potential as index .Cell transfection test was carried out to indicate that enhancement of cell transfection efficiency of nano‐car‐rier .Results Nanoparticles loaded DNA vaccine were nearly spherical shape with uniform particle size chitosan nanoparticle (CS) ,(219 .2 ± 18 .2) nm ;quaternary ammonium chitosan nanoparticles(CSTM) ,(222 .5 ± 15 .6) nm .Zeta potential of CS and CSTM was (24 .7 ± 3 .5) mV ,(19 .6 ± 1 .2) mV and encapsulation efficiency was 91 .24% ,87 .66% ,respectively .CSTM nano‐particle could enhance cellular uptake of pVAX1‐wapA obviously .Conclusion CSTM nanoparticle was proved to be an efficient DNA vaccine delivery vector .

Objectives To investigate the relationship of methylenetetrahydrofolate reductase (MTHFR) and methioninesynthase reductase (MTRR) with unexplained recurrent spontaneous abortion (URSA). Methods Case control study was used to select 244 patients with URSA (miscarriage group) and 116 normal women (control group) who were admitted to Tianjin Medical University General Hospital and Tianjin Women’s and Children’s Health Center from January 2013 to March 2015. The oral mucosal epithelial cells were extracted using fluorescence quantitative PCR to detect MTHFR gene C677T, A1298C and MTRR gene loci of A66G single nucleotide polymorphisms (SNP). The relationship between folate metabolism related gene polymorphisms of MTHFR and MTRR and URSA was analysed. Results The frequency of C677T genotype MTHFR was significantly higher in URSA group than that in the control group, and the frequency of CT genotype was significantly lower than that of the control group (P<0.05). There was no significant difference in the frequencies of A1298C MTRR and A66G MTHFR between the two groups. The activity of MTHFR, red cell folate and plasma folate levels were significantly lower in URSA group than those of control group. Homocysteine levels were significantly higher in URSA group than those of control group (P<0.05). There were no significant differences in serum folic acid, red cell folate, homocysteine cysteine levels between patients <35 years old and ≥ 35 years old in URSA group. Conclusion C677TMTHFR gene polymorphism is associated with unexplained recurrent spontaneous abortion.