Objective To express a mutated insulin gene in HepG-2 cell line to further research of insulin gene therapy. Methods Native human insulin cDNA was obtained from fetus pancreas with RT-PCR. Furin consensus cleavage sequence was introduced into proinsulin cDNA with site-directed mutagenesis (overlap extension PCR), and the new sequence was named as INS/furin. Subsequently, INS/furin was subcloned into the multiple clone sites of plasmid p(G1RE)3BP-1Luc. The new plasmid p(G1RE)3BP-11?furin was identified with the method of enzyme digestion by Hind Ⅲ and EcoR V. HepG-2 cells were transfected with the plasmid p(G1RE)3BP-11?furin by liposome-mediated method. The transfected HepG-2 cells were incubated for 48h in a glucose-containing medium (25mmol/L), and then the conditioned media were collected and HepG-2 cells were harvested respectively. The expression of INS/furin mRNA in transfected HepG-2 cells was examined by RT-PCR, the regained DNA was sequenced and insulin in conditioned media was investigated by radioimmunoassay. Results Two enzymes, Hind Ⅲ and EcoR V, digested p(G1RE)3BP-11?furin, and 2 fragments with length of 260 bp and 4 700bp, were obtained. The 260bp fragment was identified as insulin/furin, indicating that the target gene had been successfully inserted in specific sites. RT-PCR showed that insulin/furin mRNA was expressed in transfected HepG-2 cell, and the regained DNA was confirmed as insulin/furin by sequencing; while insulin was detected by radioimmunoassay in conditioned media. Conclusion The recombinant mammalian expression plasmid p(G1RE)3BP-11?furin has been successfully constructed, and transfected into HepG-2 cells, which therefore may efficiently secrete bioactive insulin.

AIM: To study the effect of selectin on lung injury in rabbits with diabetic ketoacidosis(DKA). METHODS: 12 male New Zealand rabbits were divided into experimental group (DK groups, intravenous injection of streptozocin and alloxan monohydroate, n =6) and normal control group (received same dose of saline, n =6). After 72 hours, all the animals were killed and blood and lung tissue were collected. Blood gases, expressions of selectin-P and selectin-L were detected. RESULTS: The expression of selectin in DK group were higher than that in NS group. CONCLUSION: These results indicate that selectin expression may play an important role in lung injury during diabetic ketoacidosis.

Objective:To analyze the job preference of doctors at township health center ( THC) and provide references for formulating effective policy. Methods:238 cases of doctors were sampled from Shandong, Anhui and Shaanxi provinces. Using a discrete choice experiment, we analyzed the data with a conditional logit model. Results:The most important job attribution was salary, followed by education for children, permanent post, work location, training opportunity, and years before promotion. The OR value of the monthly income of 8 000 yuan is 7. 0 compared to 2 000 yuan. Doctors will pay 3 857 yuan and 3 294 yuan respectively for better education for children and perma-nent posts. Monthly income increasing to 8 000 yuan will reduce the quitting rate from 35% to 8%, and providing better education for children and permanent posts will reduce 5%. The permanent posts at township health center and non-permanent posts in county hospital have the same appeal to the doctor. Conclusion:When choosing a job, doc-tors at THCs valued salary the most. Improving the education level for children can be beneficial for attracting and re-taining doctors at THCs. A permanent post is more attractive to doctors than non-permanent posts at THCs.

Objective To investigate the gestation-specific reference intervals (GRIs) and dynamic changes of thyroid function at different gestational ages in Chongqing .Methods Combining self-sequential longtitudinal with cross-sectional study, the serum samples from 640 pregnant women with different gestational age were collected from June 2014 to September 2015 in the Third Military Medical University. The free triiodothyronine (FT3), free thyroxine (FT4), thyroglobulin (TG), thyroid-stimulating hormone (TSH), thyroid peroxidase antibody (TPOAb), and antithyroglobulin antibody (TGAb) were detected by the direct chemiluminescence method.According to China Guideline for the diagnosis and treatment of thyroid disease in pregnancy and postpartum in 2012, the reference interval of the thyroid function was calculated.The data were analyzed by Chi square test .Results Established GRIs of thyroid function during pregnancy in Chongqing:The GRIs was 3.68-5.59 pmol/L for FT3, 9.34-17.02 pmol /L for FT4, 0.18-5.26 mIU/L for TSH in 6-9+6 weeks of pregnancy; the GRIs was 3.69-6.03 pmol /L for FT3, 8.42-15.75 pmol/L for FT4、0.09-4.85 mIU/L for TSH in 10-13+6 weeks of pregnancy; the GRIs was 3.24-5.46 pmol /L for FT3, 6.50-14.24 pmol/L for FT4, 0.11-5.13 mIU/L for TSH in 14-27+6 weeks of pregnancy;the GRIs was 3.06-5.05 pmol /L for FT3, 6.12-11.69 pmol/L for FT4, 0.75-3.67 mIU/L for TSH in 30-34 weeks of pregnancy; the GRIs was 2.96-5.00 pmol/L for FT3, 6.26-11.36 pmol /L for FT4, 0.84-5.54 mIU/L for TSH in 36-40 weeks of pregnancy.Screening by GRIs, the prevalence of thyroid dysfunction was 8.75% (46), however, the prevalence was 37.07% (195) in according with the guidelines,χ2 =120.5,P =0.000.The overdiagnosis rate was 28.32%(149 /526).Using the guidelines of thyroid disease and our GRIs, the thyroid disease was found 116 (22.05%) and 30 (5.70%) in the first screening. Moreover, the thyroid disease was found 79(19.27%) and 10(3.23%) during the repeat screening in the normal population.Conclusions Using the GRIs for thyroid function tests in normal singleton pregnant women could reduce the risk of over diagnosis .The detection rate of repeat screening of TPOAb negative patients was close to the first screening detection rate , and repeated screening could reduce the risk of missed diagnosis for thyroid dysfunction in pregnancy women .(Chin J Lab Med, 2016, 39:511-515 )

Objective To explore the significance of detecting miR-145 hypermethylation in non-small cell lung cancer (NSCLC). Methods Seventy-five NSCLC tissues and adjacent tissues was collected from May 2012 to January 2015. The methylation status of miR-145 promoter was tested by bisulfite sequencing PCR (BSP) and the expression of miR-145 evaluated by ISH. The correlation between miR-145 methylation and clinical parameters , and the relationship between miR-145 methylation and expression were statistically analyzed. Results The hypermethy-lation ratio of miR-145 in NSCLC tissues was higher than that in the adjacent tissues. Hypermethylation of miR-145 was correlated with NSCLC differentiation stage, but not with gender and age. In NSCLC, miR-145 hypermethyla-tion was negatively related with its expression. Conclusion MiR-145 hypermethylation is closely related with dif-ferentiation stage in NSCLC tissues. MiR-145 hypermethylation may be a potential biomarker for estimating NSCLC differentiation stage.

Objective To explore the influence of G4 cyberknife treatment of large hepatocellular car-cinoma on liver function,and to evaluate its treatment safety.Methods Sixty-three large liver cancer patients treated with routine G4 cyberknife treatment were retrospectively analyzed,and then statistical analysis of the difference in liver function before and after treatment was conducted.Results After G4 cyberknife treatment of 1 2 months,the levels of ALT,ALP,TBIL,PA were respectively 23.00 U /L,1 1 1 .00 U /L,1 3.70 μmol/L, (81 .87 ±1 3.94)%.Compared with the levels before treatment [28.00 U /L,32.00 U /L,1 1 .30 μmol/L, (86.07 ±1 4.07)%],there were no signi-ficant differences found (Z =-1 .677,P =0.094;Z =-0.504, P =0.61 4;Z =-1 .945,P =0.053;t =1 .271 ,P =0.21 3).The level of ambumin was (34.84 ±4.75)g/L at 1 2 months after treatment,which decreased and the difference compared with the level before treatment [(37.45 ±4.1 4)g/L]was significant (t =3.357,P =0.002).The Child-Pugh grade was 5.80 ±1 .1 7 respectively at the time points of 1 2 months after treatment,and no significant difference was found compared with the Child-Pugh grade before treatment (5.48 ±0.81 ,t =-1 .668,P =0.1 06).Conclusion G4 cyberknife treatment does not cause liver injury.It is safe and reliable in large liver cancer treatment.So,it is worth widely clinical popularizing.

0.05) .The total survival rate of N group and H I group was significantly higher than H II group(P0.05) .The abdominal stinking leak was more severe and death rate was higher in N group and H I group than H II group.Conclusion:Dry heat environment in the desert could obviously impair the total survival rate of pigs.

Objective To investigate the expression and effect of miR-22-3p in non-small cell lung cancer (NSCLC). Methods The miR-22-3p expression level in seventy-six NSCLC tissues and para-cancer tissues was detected by qRT-PCR. The relationship between the expression of miR-22-3p and gender,age,tumor size,histolo-gy grade,pathological type and lymph node metastasis was analyzed. The function of miR-22-3p on the prolifera-tion of NSCLC cells was tested by growth curve assay. Target genes of miR-22-3p were predicted by online software Targetscan. Luciferase reporter assay and qRT-PCR was used to certificate the prediction. Results The expression of miR-22-3p was increased in NSCLC tissues than the para-cancer tissues and was correlated to lymph node metas-tasis. Overexpression of miR-22-3p could suppress the proliferation of A549 cells. Astrocyte-Elevated Gene-1(AEG-1) was predicted to be a target of miR-22-3p. MiR-22-3p was revealed to bind to AEG-13′UTR by luciferase report-er assay. Overexpression of miR-22-3p could inhibit the expression of AEG-1 in A549 cells. Suppression of miR-22-3p could increase AEG-1 expression. Conclusion MiR-22-3p could inhibit the proliferation of NSCLC by tar-geting AEG-1.

Objective To observe the changes of serum and fecal taurine-conjugated bile acid levels and its association with glucose metabolism during the spontaneous development of type 2 diabetes in OLETO rats.Methods Twenty male OLETF rats(4 weeks old)were included and 10 male LETO rats of the same age were used as the normal control group.OLETF rats were fed with high fat diet whereas LETO rats were fed with normal diet.Serum and fecal taurine-conjugated bile acid levels of OLETF rats were tested at different stage of diabetes including baseline, normal glucose tolerance, impaired glucose tolerance and diabetes periods, and the association of taurine-conjugated bile acid level with body weight, blood glucose, and glucose-regulating hormones were also investigated.Results Compared with LETO rats, the baseline serum levels of taurine-conjugated bile acid in OLETF rats did not change, but the levels of fecal taurine-conjugated bile acid including taurine-conjugated chenodeoxycholic acid(TCDCA), taurocholic acid(TCA)and taurine-conjugated deoxycholic acid(TDCA)were significantly decreased [(14.25±7.18 vs 0.90±0.31)mg/kg,(7.12±4.14 vs 1.30±0.35)mg/kg,(4.30±1.78 vs 1.02±0.14)mg/kg, all P<0.01].During the development of diabetes, the fecal levels of TCDCA, TCA and TDCA were still lower than those in the control rats.TDCA was negatively associated with the level of fasting blood glucose(r=-0.470, P=0.032),but positively associated with the serum level of glucagon-like peptide(GLP)-l(r=0.406, P=0.044).Conclusion The decrease of intestinal taurine-conjugated bile acid level is involved in the development of diabetes in OLETF rats.Intestinal TDCA may regulate the secretion of GLP-1 by paracrine pathway.

[Summary] A total of 128 individuals with type 2 diabetes underwent continuous glucose monitoring for 3 consecutive days.The dawn phenomenon was defined by three different parameters according to the previous research:(1)the absolute increase of glucose level from nocturnal nadir to prebreakfast value(?G) above 20 mg/dl;(2)?G above 10 mg/dl;( 3 ) insulin requirement increased at least 20%.The participants were secondarily separated by presence/absence of a dawn phenomenon based on the definitions above.The impact on blood glucose fluctuation of different groups was assessed according to the standard deviation of blood glucose( SDBG) , the area under curve above 10 mmol/L ( AUC ) , and the mean amplitude of glycemic excursions ( MAGE ) , etc.The frequencies of dawn phenomenon were 64.8%(?G≥20mg/dl), 85.2%(?G≥10 mg/dl), and 59.4%(rise in insulin requirement≥20%)respectively.The impacts on SDBG, AUC, MAGE, and MODD were without statistical difference(P>0.05) between the presence and absence of the dawn phenomenon patients when?G≥10 mg/dl.However, the differences reached statistical significance(P<0.05) when ?G≥20 mg/dl and the increase in insulin requirement≥20%. Besides, the incidence of dawn phenomenon was positively correlated with HOMA-IR, HbA1C , and free C-peptide.Dawn phenomenon is a very frequent event in type 2 diabetes and not only impacts the overall glycemic control but also exaggerates glucose fluctuation.To be clinically relevant, ?G≥20mg/dl should be taken as the quantitative criterion of the dawn phenomenon.