Objective To construct and screen siRNA targeting a proliferation-inducing ligand (APRIL) gene in a mouse colorectal cancer celline, CT-26. To investigate the effects to the cell growth and migrant capacity of CT-26 after knockdown APRIL gene, lay the foundation for molecular targeted therapy to colorectal cancer. Methods Four pairs of APRIL siRNA were designed and chemically synthesized. And disorder sequences were synthesized as a negative control. These sequences were transfected with LipofectAMINE 2000 into CT-26 cells, which high-expressed APRIL gene. The transfection efficency rate of 6-FAM labelled control siRNA was detected by fluorescence microscope. The inhibition effectiveness of APRIL mRNA and protein was analyzed by FQ-RT-PCR and Western blot, respectively. Cell proliferation activity was analyzed by cell counting kit-8, cell migration capacity was detected by the repair of cell damage, and MMP-2 together with TIMP-1, two important regulatory genes in cell metastasis, were measured by RT-PCR.Results The different kinds of APRIL siRNA effectively suppressed the level of APRIL mRNA and the protein expression in CT-26 (P ＜ 0.05 ). Cell proliferation and metastasis ability were repressed after APRIL siRNA transfection( P ＜ 0.05 ), compared with random siRNA control and nontransfected control. The mRNA levels of MMP-2 and TIMP-1 genes wre significantly altered among APRIL siRNA groups and two control groups ( P ＜ 0.05). Conclusion We have constructed and screened a kind of siRNA (APsi737) targeting APRIL gene in a mouse colorectal cancer cell line, CT-26. APRIL siRNA can effectively inhibit the cell growth and migration capacity, maybe be regulated by MMP-2 and TIMP-1.

Objective To analyze the distribution of HLA-C alleles in Shandong Han population of China.Methods One hundred and fifty unrelated potential donors,self-claimed as Han population from Shandong province,were selected from China Marrow Donor Program.Genotypes of HLA-C with the donors were identified by PCR-SBT.The frequencies of allele were calculated with direct counting method and the differences with other populations were analyzed with SPSS16.0 x2 software.Results A total of 25 alleles of HLA-C were observed and the most common alleles were C * 06:02 and C * 07:02 with the frequency of more than 10.00％.Moreover,there were 16 kinds of alleles with the frequency of more than 1.00％ accounting for 95.33％ of the total alleles.The distribution of HLA-C alleles in Shandong Han population was similar to that in northern Han population,but had some differences with that in southern Han population.In addition,the distribution of HLA-C alleles in Shandong Han population significantly differed from that of German/African American.Conclusion This study on the distribution of HLA-C alleles in Shandong Han population provides valuable references for further studies on the genetics of HLA,cross-match for organ transplantation and other genetic-associated diseases in this population.

Objective To study the impact of human leukocyte antigen (HLA)-B specific Bw4 epitope on disease progression of hepatitis C virus (HCV) infection.Methods Eighty-six cases of HCV infection through paid blood donation were enrolled in the study.Enzyme-linked immunosorbent assay (ELISA) to detect HCV IgG and IgM,real-time reverse transcriptation polymerase chain reaction (RT-PCR) to detect HCV RNA,and sequence specific primer-polymerase chain reaction (SSP-PCR) to analyze HLA type was performed.Categorical data were analyzed by chi-square test,and measurement data were compared by independent sample t test.Results Among the 86 HCVinfected individuals,there were 29 (33.7 ％) cases of Bw4/4 homozygote,38 cases (44.2 ％) of Bw4/6 heterozygote and 19 (22.1％) cases of Bw6/6 homozygote.The HCV RNA levels in Bw4/4 group,Bw4/6 group and Bw6/6 group were (3.98±0.32),(5.22±0.29),(5.04±0.38) lg IU/mL,respectively.The HCV RNA level in Bw4/4 group was significantly lower than those in the other two groups (t=2.821,P=0.0063 ; t =2.106,P =0.0407,respectively).The spontaneous clearance rates of Bw4/4 group,Bw4/6 group and BW6/6 group were 58.6％,26.3％ and 21.0％,respectively.The spontaneous clearance rate of Bw4/4 group was significantly higher than those of Bw4/6 group and Bw6/6 group (x2 =7.135,P =0.008; x2 =6.583,P =0.010,respectively).HCV infected individuals with homozygous epitopes of Bw4/4 had 4.351 times higher probability of spontaneous viral clearance than that of Bw4/6 heterozygote or Bw6/6 homozygote (OR=4.351,95％CI:1.676-11.294).Conclusions Homozygosity for HLA-Bw4-bearing B alleles is associated with significant lower HCV viral load and higher spontaneous clearance rate.The HLA-Bw4 epitopes have a protective effect against HCV infection.

Objective To study the influence of Bw4 broad specific motif on hepatitis C virus (HCV) specific T cell response.Methods The 86 patients with HCV infection were enrolled in this study,who had history of non-standard paid blood donation.The sequence specific primer SSP-PCR and specific primer Amplification Refractory Mutation system was used to analyze HLA type.The T cell response,using PBMCs stimulated by HCV nonstructural protein NS3,NS4 and NS5 was tested by ELISPOT assay.Results There were 29 (33.7％) cases with homozygosity Bw4/4,38 cases (44.2％) with heterozygosity Bw4/6 and 19(22.1％) cases with homozygosity Bw6/6 in 86 patients with HCV infection.HCV viral loads in Bw4/4group,Bw4/6 group and Bw6/6 group were (3.98±0.32) Log (copy/ml),(5.22± 0.29) Log (copy/ml),(5.04±0.38) Log(copy/ml),respectively and there was a significant difference in HCV load among three groups(P=0.0153).The 24 cases with HCV infection were test HCV specific T cell response divided homozygosity Bw4/4 group and non-homozygosity Bw4/4 group.The HCV specific T cell response frequency in homozygosity Bw4/4 group and non-homozygosity Bw4/4 group was 50％ (5/10) and 14.28％ (2/14,respectively.The HCV specific T cell response magnitude in homozygosity Bw4/4 group and non-homozygosity Bw4/4 group was 70 SFU/106 PBMC (0-2020 SFU/106 PBMC)and 0 SFU/106 PBMC (0-200 SFU/106 PBMC).The response magnitude and frequency in homozygosity Bw4/4 group was significantly higher than that of non-homozygosity Bw4/4 group,P value was 0.0450 and 0.069,respectively.In homozygosity Bw4/4 group NS5 induced T cell response was dominant.The NS5 specific T cell response frequency in Bw4/4 group and non-Bw4/4 group was 50％ and 7.14％,respectively,and the difference was nearly significant(P=0.050).Conclusion The HCV-infected blood donors with homozygosity for HLA-Bw4 alleles is associated with a significant lower HCV virus load and stronger HCV specific T cell response,compared with non-homozygosity Bw4/4 group.

10.3969/j.issn.1000-3606.2013.06.005

Objective To analyze the differentiation status of CTL and to evaluate its clinical val-ue in patients with HIV/HCV coinfection .Methods Twenty-eight patients with HIV/HCV coinfection and twelve patients with single HCV infection were enrolled in this study .The technique of Fibro-Scan was used to evaluate liver fibrosis .The viral load of HCV was detected by real-time quantitative PCR .Flow cytometry analysis was performed to measure the differentiation status of CTL .Results Both of the levels of alanine transaminase ( ALT) and alkaline phosphatase ( ALP) in patients with HIV/HCV coinfection were signifi-cantly higher than those in patients with single HCV infection [(53.7464±48.1180) U/L vs (27.4750± 13.9850) U/L, P=0.012;(24.5071±8.1940) g/L vs (16.9667±7.1890) g/L, P=0.009].The liver stiffness of patients with HIV/HCV coinfection was higher than that of patients with single HIV infection [(5.9500, 5.8250) Kpa vs (5.1500, 1.0500) Kpa, P=0.117].Compared with the patients with single HCV infection, the patients with HIV/HCV coinfection showed higher viral loads of HCV [( 6.4768, 5.3434) lg copy/ml vs (2.6815, 1.6990) lg copy/ml, P=0.012], but lower clearance rate of HCV [32.14%vs 75%, P=0.032].Compared with the patients with single HCV infection , the patients with HIV/HCV coinfection showed lower percentages of CD 27+CD28+CTL [(28.265±15.095)%vs (18.068±10.263)%, P=0.017), but higher percentages of CD27+/-CD28+CTL [(62.449 ±14.561)% vs (71.111±12.681)%, P=0.066].A trend toward negative correlation was observed between the percent -age of CD27+CD28+CTL and the degree of liver stiffness (r=-0.310, P=0.058).Conclusion HIV in-fection could accelerate the progression of liver disease in patients with HIV /HCV coinfection by affecting the differentiation of CTL .

Objective: To identify a novel human leukocyte antigen (HLA) B allele in a Chinese Han individual and construct its three-dimensional structure. Methods: The initial HLA genotyping was performed by PCR-sequence-based typing (PCR-SBT). The ambiguous allele was confirmed with single-strand DNA sequencing. The DNA sequence was analyzed to identify the difference between the novel allele and its closest matching allele. Finally, the three-dimensional molecular structure of the novel allele was constructed using a Swiss-Model. Results: One allele of the subject at the HLA-B locus was B*44: 03: 01, whilst the other was a novel allele which differed from the closest matching allele B*51: 01: 01: 01 by nucleotide (nt) 329 A>C in exon 2, resulting in an amino acid change at codon 86 (p.Asn86Thr). Conclusion A novel HLA-B allele has been identified and officially named as HLA-B*51: 159 by the WHO Nomenclature Committee for Factors of the HLA System. The three-dimensional structure of B*51: 159 was simulated.

Objective To investigate the iodine nutrition level of vulnerable people in Inner Mongolia after adjustment of iodized salt standard and to provide theoretical bases for scientific iodine supplementation. Methods In 2013, 3 cities were selected from eastern, central and western parts of Inner Mongolia in accordance with the random number table, 3 or 4 counties were selected from each target city, 5 units according to their sub-area position of east, south, west, north and center were selected from each county, and then 1 township was selected from each unit, 5 groups of target population including school children aged 8- 10, women of childbearing age, pregnant and lactating women and infants each at least 10 people were investigated in each township. Edible salt samples from their homes and urine samples were collected. The direct titration method among the generic methods of iodide testing for salt production industry (GB/T 13025.7-2012) was used to determine the salt iodine level, and As3+-Ce4+catalytic spectrophotometry using ammonium per sulfate digestion (WS/T 107-2009) was used to test the urinary iodine level. Results Totally 3 300 samples of edible salt from local residents had been examined and median iodine was 26.20 mg/kg. The median of urinary iodine was 190.6μg/L of 1 289 school-age children;was 183.6μg/L of 621 women of childbearing age; was 178.2 μg/L o f 876 pregnant women; was 178.6 μg/L of 664 lactating women and was 167.7μg/L of 599 infants. Conclusion After adjustment of iodized salt standard, iodine nutrition level is suitable in all vulnerable people.

Objective:To investigate the application value of superb microvascular imagingin monitoring angiogenesis in the treatment of chronic ischemic diseases of lower limbs by tibial transverse transport.Methods:From May 2019 to December 2020, 12 patients with ischemic diseases of lower limbs who received tibial transverse transport therapy were retrospectively analyzed, including 7 male patients and 5 female patients, aged 30-86 years, with an average age of 64.0±16.6 years. Among them, there were 10 patients with diabetic foot, with a disease course of 5-30 years (mean 14.4±8.3 years), and 2 patients with arteriosclerosis obliterans. The results of lower extremity superb microvascular imaging before and after treatment were recorded and compared.Results:After treatment, 12 patients were found to have obvious new vessels around the artery of lower extremity, the number of vessels 1-8, the average number of 3.25±2.73. We found that a total of 12 patients 20 inherent lower limb artery collateral established, collateral circulation was found around 12 dorsal metatarsal artery (DMA) in 11 patients, around 3 dorsal pedis artery (DPA) in 2 patients, around 3 anterior tibial artery (ATA) in 2 patients, and around 1 posterior tibial artery (PTA) in 2 patients. The lateral branches were established in ipsilateral lower limbs in 6 patients, contralateral lower limbs in 2 patients, and bilateral lower limbs in 4 patients.Conclusion:The phenomenon of angiogenesis is obvious in the clinical application of tibial transverse transport. Superb microvascular imaging can effectively reflect the degree of promoting the establishment of collateral circulation by tibial transverse transport.

This study aimed to validate the high yield and soluble expression of proteins carrying the transactivator of transcription (Tat) peptide tag, and further explored the potential mechanism by which the Tat tag increases expression. Escherichia coli superoxide dismutase (SOD) proteins, including SodA, SodB and SodC, were selected for analysis. As expected, the yields and the solubility of Tat-tagged proteins were higher than those of Tat-free proteins, and similar results were observed for the total SOD enzyme activity. Bacterial cells that overexpressed Tat-tagged proteins exhibited increased anti-paraquat activity compared with those expressing Tat-free proteins that manifested as SodA>SodC>SodB. When compared with an MG1655 wild-type strain, the growth of a ΔSodA mutant strain was found to be inhibited after paraquat treatment; the growth of ΔSodB and ΔSodC mutant strains was also slightly inhibited. The mRNA transcript level of genes encoding Tat-tagged proteins was higher than that of genes encoding Tat-free proteins. Furthermore, the α-helix and turn of Tat-tagged proteins were higher than those of Tat-free proteins, but the β-sheet and random coil content was lower. These results indicated that the incorporation of the Tat core peptide as a significant basic membrane transduction peptide in fusion proteins could increase mRNA transcripts and promote the high yield and soluble expression of heterologous proteins in E. coli.
Escherichia coli* ; Escherichia* ; HIV-1* ; Membranes ; Paraquat ; RNA, Messenger* ; Solubility ; Superoxide Dismutase ; Superoxides* ; Trans-Activators