Objective Insulin autoantibody (IAA) is known to exist in sera of type 1 diabetes mellitus (T1DM) patients and pre-T1DM individuals. The aim of this study was to establish a novel microtiter plate radioimmunoassay (RIA) for IAA and evaluate its clinical value. Methods Diluted 125Ⅰ-insulin was mixed with 5 ul serum samples in a 96-well microtiter plate and then incubated for 72 h on an orbital plate shaker (4℃). The immunocomplexes were transferred to another protein a coated Millipore plate, and then the plate was washed with Tri-Buffered Saline Tween-20 (TBT) buffer. Counts per minute (CPM) was measured with liquid scintillation and luminescence counter. The positive cut-off point of IAA index was defined as ≥0.06 based on the 99-percentile of the distribution in 317 healthy individuals. The specificity and sensitivity of the assay were calculated from the samples provided by the fourth Diabetes Autoantibodies Standardization Program (DASP 2005). The IAA levels were determined in 71 T1 DM and 551 newly diagnosed type 2 diabetes (T2DM) patients, and 317 healthy controls. The t test, non-parametric test, x2 test and linear correlation analysis were performed on the data using SPSS 11.5 software. The concordance rate was estimated with Kappa value. Results (1) The optimized testing condition was described as 2×104 CPM of 125Ⅰ-insulin, 5 ul serum sample and slowly horizontal shaking for 72 h. (2) The intra-assay CV was 4.8%-8.9% and inter-assay CV was 6.4%-10.5%. Based on DASP 2005 samples, the specificity and sensitivity of the assay were 97% (97/100) and 50% (25/50), respectively. Ninety-six serum samples with different IAA levels were selected and tested to compare between our new method and a domestic IAA RIA kit. The results showed that the IAA indices from the two methods were positively correlated (r= 0.678, P<0.001). The concordance rate was 72.9 %(Kappa value=0.402). There were 25 samples with discordant results, which were positive for IAA titer using the corresponding microtiter plate RIA but negative using the novel RIA kit. (3) In TIDM group the positive rate of IAA was 19.7% (16/71), higher than the healthy controls (0.9%, x2=54.36, P<0.001). The subgroup of T1DM children (with 0-9 years) showed the highest IAA positive rate (55.6% ,x2=4.85, P<0.05). In T2DM group the frequency of IAA was 1.5% (8/551), which had no significant difference comparing with that of healthy controls (x2= 0.95, P >0.05). Conclusions Our proposed microtiter plate RIA method for IAA is highly sensitive and specific, likely to be feasible for clinical application. The frequency of IAA is high in children with T1DM.

Objective To investigate the distribution of related single nucleotide polymorphisms (SNPs) of drug transporters in healthy Chinese Han population.Methods 10 specific SNPs of 6 drug transporters,including organic anion transporting polypeptide 1B1 (OATP1B1),OATP1B3,organic cation transporter 1 (OCT1),P-glycoprotein (P-gp),multidrug resistance protein 2 (MRF2) and breast cancer resistance protein (BCRP),were determined in healthy Chinese Han volunteers by the established rapid polymerase chain reaction (PCR) methods and sequencing.Gene frequencies were calculated and compared with other populations in the 1000 genomes project.Results The minor allele frequencies of OATP1B1 388A ＞ G,OATP1B1 521T ＞ C,OATP1B3 699G ＞ A,OCT1 181C ＞ T,OCT1 1393G ＞ A,OCT1 1258delA,MDR1 3435T ＞ C,MRP2-24C ＞ T,BCRP 421C ＞ A and BCRP 376C ＞ T were 26.97％,6.25％,23.91％,0,0,0,38.47％,18.52％,31.97％ and 1.48％,respectively.By comparing the distribution of allele frequencies between test group and Mrican,the frequencies of the OATP1 B3 699G ＞ A variant alleles were 76.09％ and 35.63％,respectively;the frequencies of the MDR1 3435T ＞ C variant alleles were 61.53％ and 85.02％,respectively;the frequencies of the MRP2-24C ＞ T variant alleles were 18.52％ and 3.10％,respectively;the frequencies of the BCRP 421C ＞ A variant alleles were 31.97％ and 1.29％,respectively.And these differences were statistically significant (P ＜ 0.05).By comparing the distribution of allele frequencies between test group and American,the frequencies of the OATP1B1 388A ＞ G variant alleles were 26.97％ and 47.26％,respectively;the frequencies of the BCRP 421C ＞A variant alleles were 31.97％ and 14.12％,respectively.And these differences were statistically significant (P ＜ 0.05).By comparing the distribution of allele frequencies between test group and European,the frequencies of the OATP1B1 388A ＞ G variant alleles were 26.97％and 40.26％,respectively;the frequencies of the OATP1B1 521T ＞ C variant alleles were 6.25％ and 16.10％,respectively;the frequencies of the OCT1 181C ＞ T variant alleles were 0 and 6.26％,respectively;the frequencies of the BCRP 421C ＞ A variant alleles were 31.97％ and 9.44％,respectively.And these differences were statistically significant (P ＜ 0.05).There was no significant difference in the distribution of allele frequencies between test group and Japanese (P ＞ 0.05).Conclusion Significant differences were observed in the distributions of some SNPs between Chinese and other populations.It is important to analyze the distribution of the related genetic polymorphisms of drug transporters in Chinese people to guide rational drug use in clinical.

This study was purposed to construct a vector containing human suppressor gene p53 and p16, and to investigate their expression and effect on K562 and HL-60 cells. pBudCE4.1-53-16 is a vector designed for simultaneous expression of human suppressor gene p53 and p16 in mammalian cell line. After transfection into K562 cells with lipofectamine(TM) 2000, the expression of p53 and p16 genes was detected by Western blot and immunocytochemical method. The growth curve, apoptosis, cell cycle were assayed by CCK-8 and flow cytometry. The results showed that the recombinant plasmid pBudCE4.1-53-16 was constructed successfully and were verified by PCR and restriction analysis. The expression of P53 and P16 protein could be detected after transfection into leukemia cells (K562 and HL-60) for 48 hours. As compared with control group, the cell proliferation in experimental group was inhibited, the cells were arrested in G0 phase and apoptotic cells increased (p<0.001). It is concluded that the recombinant plasmid pBudCE4.1-53-16 has been established. p16 and p53 in the recombinant plasmid pBudCE4.1-53-16 synchronously express in leukemic cells after transfection in vitro for 2 days and results in reduced proliferation, G0 arrest and apoptosis increase.
Apoptosis ; genetics ; Cell Cycle ; genetics ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; Gene Expression ; Genes, p53 ; Genetic Vectors ; HL-60 Cells ; Humans ; K562 Cells ; Plasmids ; Transfection

Objective To observe effects of Heyutai Fuzhu Jiangtang Tablets combined with metformin in insulin resistance (IR); To discuss its mechanism of action. Methods 6–7 week old male ZDF (fa/fa) rats were randomly divided into model group,metformin group,Heyutai Fuzhu Jiangtang Tablets group(Jiangtang Tablets group),and metformin combined with Heyutai Fuzhu Jiangtang Tablets group.ZDF(fa/+)rats were chosen as normal group.Each medication group was given relevant medicine for gavage for 6 weeks. Body weight, FBG, TG, TC, FFA, FINS, HOMA-IR, OGTT and HE staining were tested. HE staining was used to observe the pathological changes of skeletal muscle. RT-PCR and Western blot were used to detect skeletal muscle corresponding gene and protein expression. Results Compared with Jiangtang Tablets group and metformin group, TC, FFA, FBG, and HOMA-IR in metformin combined with Heyutai Fuzhu Jiangtang Tablets group decreased significantly (P<0.05, P<0.01). Blood glucose level and AUC significantly decreased at each time point in OGTT. HE staining of skeletal muscle fibers arranged in order; nucleus increased and internal movement was not significant, without obvious infiltration of inflammatory cells. Expressions of skeletal muscle InsR, Akt, and Glut4 mRNA expression increased (P<0.05, P<0.01). Expressions of skeletal muscle p-InsR, p-Akt, and Glut4 protein expression increased (P<0.05, P<0.01). Conclusion Heyutai Fuzhu Jiangtang Tablets combined with metformin can improve IR in type 2 diabetic rats, and the effect is better than single-application.

OBJECTIVETo investigate serum IL-18 levels in mice with collagen-induced arthritis treated by recombinant adenoviral vector containing mIL-18BP and mIL-4 fusion gene (AdmIL-18BP/mIL-4).

METHODSArthritis was induced by injection of collagen in male DBA-1/BOM mice. Mice with collagen-induced arthritis (CIA) were intra-articularly injected with 10(7)pfu/6μL of AdmIL-18BP/mIL-4; and in mice of control groups AdLacZ or PBS were used. The animals were sacrificed at week 1, 2 and 4 after treatment. Serum IL-18 levels were determined by ELISA at the different time points.

RESULTThe mean serum levels of IL-18 at weeks 1, 2, and 4 after injection of AdmIL-18BP/mIL-4 were (36.5±5.4)ng/L, (32.5 ± 3.2) ng/L and (28.7 ±2.9)ng/L, respectively, which were significantly lower than those at the same time point of AdLacZ group [(66.2 ±5.1)ng/L, (69.2 ±4.2)ng/L and (77.7 ±3.9)ng/L] and PBS group [(67.3 ±7.1)ng/L, (71.9 ±1.8)ng/L and (78.7±4.1)ng/L] (P<0.01 at all time points). In the therapy group, there were no significant differences in the mean serum concentrations of IL-18 at all time points.

CONCLUSIONThe serum IL-18 levels in CIA mice are down-regulated by treatment of recombinant adenovirus containing mIL-18BP and mIL-4 fuse gene, which might be a promising therapeutic strategy for rheumatoid arthritis.

Adenoviridae ; genetics ; Animals ; Arthritis, Experimental ; blood ; therapy ; Gene Fusion ; Genetic Therapy ; Genetic Vectors ; Interleukin-18 ; blood ; genetics ; Interleukin-4 ; genetics ; Male ; Mice ; Mice, Inbred DBA

Objective:To analyze the contractile properties of the lumbar erector spinae in patients with chronic nonspecific low back pain (CNLBP), and to explore their correlation with pain and dysfunction. Methods:From January to June, 2020, 24 patients with CNLBP in the outpatient and the ward of geriatric rehabilitation medicine department and 26 asymptomatic volunteers were included. Their contractile properties of the lumbar erector spinae were measured with tensiomyography, including maximum radial muscle displacement (Dm), contraction time (Tc), delay time (Td), sustain time (Ts), half-time relaxation (Tr) and lateral symmetry (LS). The contraction velocity (V_C) was calculated. Potential associations of tensiomyography parameters to Visual Analogue Scale (VAS) and Oswestry Disability Index (ODI) were assessed using correlation analysis. Results:No significant differences were found in Td, Ts, Tc, Tr and LS between two groups (P > 0.05). Dm and Vc were significantly lower in both sides of CNLBP group than in the control group (t > 2.058, P < 0.01). Dm or Vc were not correlated with VAS and ODI (P > 0.05). Conclusion:Erector spinae are stiff and fatiguable in patients with CNLBP, however, they are not associated with pain and dysfunction. Tensiomyography could be used for accurate diagnosis and treatment of CNLBP.