Bone marrow stromal cells reverse the microglia type from pro-inflammatory tumour necrosis factor a microglia to anti-inflammatory CD206 microglia of middle cerebral artery occlusion rats through triggering secretion of CX3CL1.

The middle cerebral artery occlusion (MCAO) model has been extensively applied to study ischaemic stroke. This study attempted to clarify effect of bone marrow stromal cells (BMSCs) on infarct injury of MCAO rats. BMSCs were isolated and identified by staining CD29/CD44 and CD31/CD45. CX3CL1 silencing vector (pLVX-shRNA-CX3CL1) was generated and infected to BMSCs. pLVX-shRNA-CX3CL1 infected BMSCs were transplanted into brain tissue of MCAO rats. Real-time PCR was used to determine CX3CL1 expression. Infarct areas were stained with TTC to evaluate infarct size. Double-staining immunofluorescence was conducted to determine anti-inflammatory type CD206 and pro-inflammatory type tumour necrosis factor a (TNF-a) microglia. Isolated BMSCs were positively presented for CD29/CD44, and negatively for CD31/CD45. CX3CL1 was significantly lower in the BMSC + pLVX-shRNA2-CX3-CL1 group compared to the BMSCs + pLVX group (p < 0.05). According to TTC and neurological scores, MCAO rats were successfully generated. BMSCs transplantation significantly increased CD206 microglia and decreased TNF-a microglia. However, shRNA-CX3CL1-infected BMSCs remarkably reduced CD206 microglia and enhanced TNF-a microglia compared to the MCAO + BMSCs group. In conclusion, BMSCs reverse microglia from pro-inflammatory type TNF-a microglia to anti-inflammatory type CD206 microglia in the infarct region of MCAO rats (3rd to 7th days post BMSC transplantation), through triggering of CX3CL1 secretion. Therefore, the potential effects of CX3CL1 secreted by BMSCs would provide an insight for stem cell-dependent therapeutic strategies in treating ischaemic stroke-associated disorders.

Evaluation of Reference Genes for Quantitative PCR in Four Tissues from Rabbits with Hypercholesterolaemia

Abstract Rabbit with hypercholesterolaemia is an important model for studying cholesterol metabolism disease. This study aimed to evaluate the expression stability of nine reference genes for quantitative PCR (qPCR) analysis in adrenal gland, liver, spleen, and kidney tissue from rabbits with hypercholesterolaemia. In total, 30 male Harbin Large White (HLW) rabbits were fed a normal feed (n = 15) or a high cholesterol feed (n = 15) for 8 weeks to induce hypercholesterolaemia. Nine reference genes were verified by qPCR using cDNA extracted from rabbit tissue samples. For qPCR analysis, reference genes were evaluated using the RefFinder and GeNorm algorithms. Overall, seven rabbits with hypercholesterolaemia were identified based on body weight and total cholesterol measurements. Combining the results of the RefFinder and GeNorm algorithms, the most stable reference genes were hypoxanthine phosphoribosyltransferase 1 (Hprt1) and eukaryotic translation elongation factor 1 alpha 1 (Eef1a1) in the adrenal gland, β-2-microglobulin (B2m) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) in the liver, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (Ywhaz) and Gapdh in the spleen, and peptidylprolyl isomerase (Ppia), β-actin (Actb), succinate dehydrogenase complex subunit A flavoprotein (Sdha), and B2m in the kidney. Taken together, our results confirmed that Hprt1 and Eef1a1, B2m and Gapdh, Ywhaz and Gapdh, and Ppia, Actb, Sdha, and B2m were the best reference genes for qPCR analyses in adrenal gland, liver, spleen, and kidney tissue, respectively, of rabbits with hypercholesterolaemia.

[A follow-up study on cognitive changes of mild cognitive impairment in the elderly].

OBJECTIVE: To observe and assess the cognitive changes of mild cognitive impairment (MCI) in the elderly. METHODS: A cohort study design was conducted among 47 patients with MCI and 21 control selected from the same convalescent camp, Montreal cognitive assessment (MoCA), mini mental state examination (MMSE) and clock drawing test (CDT) were performed to all subjects at the onset of study and 12 months later. RESULTS: The score of MMSE, CDT, MoCA and its subitems including visuospatial skill and delayed recall of MCI group were lower than the baseline after 12 months, with significantly decline in the score of MoCA (P = 0.041) and delay recall (P = 0.003). There was no obvious difference in the score of control between the baseline and that after 12 months. CONCLUSION: The decline of delayed recall occurred early and significantly, which may be a predictor in the conversion of mild cognitive impairment to dementia.

[Association analysis between SNPs of the growth hormone receptor gene and growth traits in arctic fox].

Using single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing, single nucleotide polymorphisms (SNPs) of growth hormone receptor (GHR) gene were detected in an arctic fox population. Correlation analysis between GHR polymorphisms and growth traits were carried out using the appropriate model. Four SNPs, G3A in the 5'UTR, C99T in the first exon, T59C and G65A in the fifth exon were identified on the arctic fox GHR gene. The G3A and C99T polymorphisms of GHR were associated with female fox body weight (Pamp;0.05) and the T59C and G65A polymorphisms of GHR were associated with male fox body weight (Pamp;0.05) and the skin length of the female fox (Pamp;0.01). Therefore, marker assistant selection on body weight and skin length of arctic foxes using these SNPs can be applied to get big and high quality arctic foxes.

Establishment and characterization of a fibroblast cell line from the Mongolian horse.

A fibroblast line was successfully established from Mongolian horse ear marginal tissue by using a primary explant technique and cell cryogenic preservation technology. Biological analysis showed the following: The cells were adherent and exhibited density-dependent inhibition of proliferation; assays of microbial contamination from bacteria, fungi, and mycoplasma were negative; the population doubling time of the cells was 33.9 h; and a 2n chromosome number of 64 at a frequency higher than 80%. A lack of cross-contamination of this cell line with other species was confirmed by isoenzyme analysis of lactic and malic dehydrogenases. In order to study exogenous gene expression, four fluorescent proteins, pEGFP-N3, pEGFP-C1, pDsRed1-N1, and pEYFP-N1, were transfected into the cells. The corresponding fluorescence was distributed throughout the cytoplasm and nucleus 12 h after transfection. This cell line not only preserves the genetic resources of the Mongolian horse at the cellular level but also provides valuable materials for genomic, postgenomic, and somacloning research in this species.

[Correlation analysis between MC3R and MC4R gene polymorphism and growth traits in pigeon.].

The SNPs in partial coding sequence of MC3R and MC4R genes were identified by polymerase chain reaction followed by single strand conformation polymorphism (PCR-SSCP) analysis and DNA sequencing in shack-Kee and Columba domestica from Harbin area. Correlation analysis between MC3R and MC4R polymorphism and growth and body composition traits was carried by the least square analysis. The genotypes of T91G mutation in MC3R gene and A903G mutation in MC4R gene proved to have significant association with body weight, carcass weight, and holo-carcass weight in shack-Kee (P<0.05). The interaction of MC3R-T91G and MC4R-A903G was discussed through combination genotype analysis. The least square analysis showed that the combined genotype had significant association with holo-carcass weight (P<0.05). Multiple comparisons revealed that BBAA genotype birds had a higher holo-carcass weight than AABB genotype birds and BBAA genotype was the beneficial genotype for the growth of body weight.

[An analysis on the relationship between susceptibility to cirrhosis and polymorphisms at C-1350T and G-944C sites of CIITA gene promoter IV in chronic HBV carriers].

OBJECTIVE: To investigate the relationship between the susceptibility to cirrhosis and the single nucleotide polymorphisms (SNPs) at C-1350T and G-944C loci of class II transactivator (CIITA) gene promoter IV in chronic HBV carriers. METHODS: C-1350T and G-944C loci of CIITA gene promoter IV were analyzed by sequence-specific primer PCR (PCR-SSP) in 544 chronic HBV carriers and 125 non-HBV infected healthy blood donors. RESULTS: Among the chronic viral hepatitis B patients, there were significantly decreased frequencies of CC and TG haplotypes, and significantly increased frequency of CG haplotype among patients with liver cirrhosis (CG vs. CC: chi2=8.274, df=1, P < 0.01; CG vs. TG: chi2 = 15.027, df =1, P <0.01). There were no significant differences in the frequencies of CC and TG haplotypes between chronic hepatitis B and liver cirrhosis patients (chi2 = 1.231, df =1, P < 0.05). There were significantly increased frequencies of CC/CC (Group 1) genotype and genotypes contained CG haplotype (Group 3), and significantly decreased frequencies chi2= 7.176, df = 1, P < 0.01; Group1 vs Group 4, chi2 = 19.818, df = 1, P < 0.01; Group 3 vs Group 2, chi2 = 11.423, df = 1, P < 0.01; Group 3 vs Group 4, chi2 = 34.226, df = 1, P < 0.01; Group 1 vs Group 3, chi2 = 0.009, df = 1; Group 2 vs Group 4, chi2 = 2.176, df = 1). CONCLUSION: Polymorphisms at -1350 and -944 loci of CIITA gene promoter IV are associated with susceptibility to liver cirrhosis in chronic HBV carriers. The chronic HBV carriers bearing CC/CC genotype or genotypes containing CG haplotype progress into liver cirrhosis with more probability.

[Construction of eukaryotic expression vectors containing different haplotype cDNA of human CIITA gene].

OBJECTIVE: To construct eukaryotic expression vectors containing three different haplotype cDNAs of human CIITA gene. METHOD: cDNA fragments of three different CIITA haplotypes were obtained by inducing one or two single nucleotide mutations of wild type recombinant plasmid EBS-NPL-CIITA cDNA, which correspond to two non-homonymy single nucleotide polymorphism (SNP) sites in the coding region of human CIITA gene, using overlap extension PCR site-directed mutagenesis technology. The above-mentioned three haplotype cDNAs were respectively cloned to EBS-NPL-CIITA linearized vectors. Positive clones were identified by colonial PCR and restriction endonuclease digestion and were sent to be sequenced. Then eukaryotic expression vectors containing four different haplotypes and an empty vector EBS-NPL were transfected into HepG2 cells respectively. HLA-DR was detected by indirect cell immunofluorescence technique. RESULTS: The cDNA fragments of three different human CIITA haplotypes were successfully constructed, and the eukaryotic expression vectors containing three different haplotype cDNAs of human CIITA gene were obtained. No expression of HLA-DR was observed in the original HepG2 cells and empty vector transfected HepG2 cells and the expression of HLA-DR emerged in the HepG2 cells transfected with four eukaryotic expression vectors. CONCLUSION: The eukaryotic expression vectors containing three different haplotype cDNAs of human CIITA gene were successfully constructed, and they are essential for our further study of the functional differences of them.

[Association analysis between SNPs of the growth hormone gene and antler production in spotted deer].

We report the effects of candidate gene growth hormone (GH) gene on antler production in the current study. Single nucleotide polymorphisms (SNPs) of the GH gene were identified and genotyped by polymerase chain reaction followed by single strand conformation polymorphism (PCR-SSCP) analysis and DNA sequencing in a deer population from the farm of Jilin Agricultural University. Correlation analysis between GH polymorphisms and antler production was carried out using the appropriate mixed model. Results showed an effect of GH gene on antler production. Deer with the SNP genotypes G-->A had a significant difference in antler production of the fifth saw (P<0.2). BB deer had a higher antler pro-duction than AA ones (P<0.2).

[SSSR fingerprinting in broiler selected for very low density lipoprotein (VLDL)].

SSR fingerprints were analyzed in three generations of fat line (FL) and lean line (LL) of broiler chickens. Changes in gene frequencies of every locus were evaluated. Thus the relationship between SSR markers and VLDL (a trait representing fat mass of broiler), which is the basis for early selection of LL broiler, was examined. Fourteen microsatellite locus were successfully amplified with 4 of 5 primers used. The results of chi2 test for the gene frequencies of every locus show that one locus was significantly different in generation 1(P<0.05), two in generation 2 and 4 in generation 3.