1.26 polymorphic microsatellite markers screened from the genome of guinea pigs
Diwen LIU ; Weiwei YANG ; Baojin WU
Acta Laboratorium Animalis Scientia Sinica 2014;(3):78-83
Objective To screen microsatellite DNA markers from genome of guinea pigs for further genetic quality control and gene-mapping of this species .Methods Microsatellite sequences were obtained by magnetic bead enrichment and genome database screening , and candidate loci were chosen to design primers .Thereafter , genomic DNA of 5 different guinea pig strains were employed to select polymorphic microsatellite DNA markers based on PCR amplification results .Re-sults A total of 304 microsatellite sequences were analyzed by magnetic bead enrichment and 125 primers were designed . One polymorphic microsatellite DNA marker and 17 specific sites ( no polymorphic was found ) were determined .By gene-mapping , 292 microsatellite sequences were obtained and 178 primers were analyzed , totally 25 polymorphic microsatellite DNA markers and 28 specific sites ( without polymorphics ) were discovered .Conclusions We obtained 26 polymorphic microsatellite DNA markers and 45 potential markers in guinea pigs , and these may lay a foundation for application of mic-rosatellite DNA markers in genetic quality control and gene-mapping of guinea pigs .
2.Breeding of Zmu-1:DHP inbred strain guinea pig and preliminary analysis of molecular genetic structure of the strain
Diwen LIU ; Min XIE ; Yanhong CHEN ; Zhen WEI
Acta Laboratorium Animalis Scientia Sinica 2017;25(1):90-96
Objective To breed a guinea pig inbred strain and set up a method for detection of the microsatellite markers of genetic structure in guinea pigs. Method Using inbreeding methods we try to breed the Zmu-1:DHP inbred strain. With 15 pairs of polymorphism microsatellite primers, the genetic homozygosity of Zmu?1:DHP inbred strain,Zmu?1:DHP outbred strain and Zmu?2:DHP inbred strain ( as control) were examined by PCR. Results After breeding for 13 years, 8 sublines of Zmu?1:DHP inbred strain ( >20 generations) were bred. After identification, the gene frequency of the second subline of Zmu?1:DHP inbred strain was 86. 7%,higher than Zmu?1:DHP outbred strain (6. 7%) and Zmu?2:DHP inbred strain (66. 7%). The average number of loci of Zmu?1:DHP inbred strain was 1. 13,lower than that of Zmu?1:DHP outbred strain (2. 47%) and Zmu?2:DHP inbred strain (1. 33%). The genotypic frequency of Zmu?1:DHP in?bred strain was also higher than that of the other strains. The gene types of Zmu?1:DHP inbred strain were included in the genes of Zmu?1:DHP outbred strain, but Zmu?1:DHP inbred strain was short of 2 characteristic genes. The gene homozy?gous rates of 8 sublines of Zmu?1:DHP inbred strain were different with each other,among them, those of the 2nd and 8th sublines were higher than others. Conclusions There are both homozygosity and specificity in the Zum?1:DHP inbred strain and Zum?1:DHP outbred strain. The second Zum?1:DHP subline becomes a new inbred strain guinea pig. It is es?sential that the subline with the characteristic property is screened from these sublines. The guinea pigs of black Zmu?2:DHP inbred strain carrying microsatellite markers not present in the white strains, may carry optimal genes related with hair color properties.
3.Comparison of biological characteristics related to eyeballs between two strains of guinea pigs and the retinal mechanism of myopia
Yanhong CHEN ; Zhen WEI ; Zhipeng AI ; Diwen LIU
Acta Laboratorium Animalis Scientia Sinica 2018;26(2):201-206
Objective To compare the biological characteristics of eyeballs between Zmu-1:DHP and DHP guinea pigs,and to explore the retinal mechanism of myopia in Zmu-1:DHP guinea pigs. Methods To measure the refraction, corneal curvature and axial length of the two guinea pig strains at age of 4-12 weeks. Those spontaneous myopic Zmu-1:DHP guinea pigs were chosen to take the retina for pathological examination. The pathological changes in the retina were determined and compared with the DHP guinea pigs. The expression of RALDH, ALDHTH, TH, TK, iNOS, nNOS, bFGF and TGFβ mRNA in the retina were detected by real time-PCR. Results The myopic rate of 3-week old Zmu-1:DHP guinea pigs was 90.21%,while of the DHP guinea pig was only 18.00%. From 4 to 12 weeks, compared with the DHP guinea pigs,myopia and axial length of the Zmu-1:DHP guinea pigs were significantly increased(P<0.01),and the corneal curvature of Zmu-1:DHP guinea pigs was significantly less than the DHP guinea pigs(P<0.01). The retina outer nuclear layer of Zmu-1:DHP guinea pigs was reduced in thickness,the cell volume was smaller,and the cell number was less compared with the DHP guinea pigs. The choroid of Zmu-1:DHP guinea pigs showed atrophy and became thinner. There were few pigment granules in the pigment epithelium of Zmu-1:DHP guinea pigs,while there were plenty of pigment granules in the DHP guinea pigs. Compared with the DHP guinea pigs,the expression of TH mRNA was significantly down-regulated in the retina of Zmu-1:DHP guinea pigs(P<0.01),and the expression of TK,iNOS,nNOS,bFGF and TGFβ was significantly down-regulated(P<0.01, P <0.05, P <0.05, P <0.05, P <0.05). Conclusions Zmu-1:DHP strain guinea pig has a high rate of spontaneous axial myopia. The retinal mechanism of myopia has a relationship with the regulation of several myopia factors.
4.Uncontrolled preliminary study on the clinical efficacy of fecal microbiota transplantation in irritable bowel syndrome and its influence on gut microbiota
Diwen SHOU ; Haoming XU ; Hongli HUANG ; Bailing LIU ; Wenjuan TANG ; Huiting CHEN ; Youlian ZHOU ; Yongqiang LI ; Qingling LUO ; Jie HE ; Yuqiang NIE ; Yongjian ZHOU
Chinese Journal of Digestion 2021;41(1):23-28
Objective:To investigate the efficacy and safety of fecal microbiota transplantation (FMT) in the treatment of irritable bowel syndrome (IBS), and to explore the effects of FMT on the gut microbiota of IBS patients.Methods:From September 2016 to August 2017, at Guangzhou First People′s Hospital, 28 hospitalized IBS patients who underwent FMT treatment were enrolled. Before FMT, four and 12 weeks after FMT, all the IBS patients completed the irritable bowel syndrome quality of life scale (IBS-QOL), irritable bowel syndrome severity scoring system (IBS-SSS) and gastrointestinal symptom rating scale (GSRS). 16S rDNA sequencing was performed before FMT and four weeks after FMT. The effects of FMT on gut microbiota diversity and microbiota structure of IBS patients were analyzed respectively from the level of phylum, family and genus, and linear discriminant analysis effect size (LEfSe) was further used to screen the different bacteria. Paired t test and paired rank sum test were used for statistical analysis. Results:Twelve weeks after FMT, the scores of the six dimensions of IBS-QOL including dysthymia, behavioral disorder, auto imagery, health concerns, eating avoidance, and relationship expansion were all lower than those before FMT (43.750, 22.656 to 56.250 vs. 48.438, 32.031 to 60.938; 37.500, 18.750 to 56.250 vs. 46.429, 21.429 to 62.500; 31.250, 14.063 to 42.188 vs. 31.250, 18.750 to 50.000; 41.667, 27.083 to 56.250 vs. 50.000, 41.667 to 66.667; 54.167, 43.750 to 72.917 vs. 66.667, 58.333 to 83.333; 8.333, 0.000 to 33.333 vs. 16.667, 8.333 to 33.333, respectively), and the differences were statistically significant ( Z=-2.157, -3.429, -2.274, -3.197, -3.042 and -2.329, all P<0.05). Twelve weeks after FMT, the scores of the two dimensions of IBS-QOL including behavioral disorder and relationship expansion were both lower than those of four weeks after FMT (37.500, 18.750 to 56.250 vs. 39.286, 19.643 to 62.500 and 8.333, 0.000 to 33.333 vs. 16.670, 2.083 to 41.667, respectively), and the differences were statistically significant ( Z=-1.998 and -2.110, both P<0.05). Four and 12 weeks after FMT, the scores of IBS-SSS and GSRS were both lower than those before FMT ((190.32±106.51), (201.43±102.48) vs. (245.93±86.10) and 5.50, 4.00 to 9.00 and 5.50, 4.00 to 8.75 vs. 7.00, 6.00 to 9.75), and the differences were statistically significant ( t=4.402 and 3.848, Z=-3.081 and -3.609; all P<0.01). No serious adverse reactions occurred in the patients after FMT. At the phylum level, after FMT the abundance of Verrucomicrobia in the feces of IBS patients was richer than that before FMT (6.74% vs. 0.37%); at the family level, after FMT the abundance of Verrucomicrobiaceae in the feces of IBS patients was richer than that before FMT (6.74% vs. 0.37%); at the genus level, after FMT the abundance of Akkermansia was richer than that before FMT (6.74% vs. 0.37%); and the differences were statistically significant (all Z=-2.589, all P=0.010). The results of LEfSe method indicated that four weeks after FMT the abundance of Akkermansia in the gut microbiota of IBS patients was richer than that before FMT (6.74% vs. 0.37%), and the difference was statistically significant (linear discriminant analysis value=4.5, P=0.049). Conclusions:FMT is safe and effective in the treatment of IBS. The mechanism may be through upregulating the diversity of gut microbiota and changing the structure of gut microbiota of IBS patients.
5.Polymorphism and Tissue Expression Analysis of TYR and MC1R Genes in Guinea Pigs with Different Coat-Color Phenotypes
Yingen TANG ; Yaxian FENG ; Min ZHONG ; Zhen WEI ; Lie WANG ; Diwen LIU
Laboratory Animal and Comparative Medicine 2025;45(1):21-29
Objective To explore the polymorphism of tyrosinase (TYR) and melanocortin 1 receptor (MC1R) genes and their mRNA expression levels in relation to coat-color phenotypes in guinea pigs, providing genetic markers for locating dominant traits in guinea pigs. Methods A total of 57 self-bred ordinary-level guinea pigs were selected and divided into three groups based on coat color: white (n=22), variegated (n=22) and black (n=13). The guinea pigs were euthanized with an overdose of pentobarbital sodium via intraperitoneal injection. DNA was then extracted from the dorsal skin tissue. Polymorphism in the coding sequence (CDS) of the exons of the TYR and MC1R genes in each group was detected by cloning and sequencing. The mRNA expression of the two genes in skin tissues was detected by real-time fluorescent quantitative PCR to investigate the relationship between these genes and guinea pig coat color. Results A single nucleotide polymorphism (SNP) site was found in the CDS region of TYR exon Ⅰ, where the base A was replaced by G. All white guinea pigs had the G/G genotype for TYR, while no deep-colored (variegated and black) guinea pigs exhibited the G/G genotype for TYR. Most deep-colored guinea pigs had the A/A genotype, and a few had A/G genotype. The A/A genotype frequency in black guinea pigs was higher than in variegated guinea pigs. A 2 760 bp sequence deletion was identified in the exon of the MC1R gene, marked as the - gene, with non-deleted samples marked as N gene. Most white guinea pigs had the -/- genotype for MC1R, variegated guinea pigs mainly had the -/N genotype, and black guinea pigs mainly had the N/N genotype, with a few showing the -/N. The TYR gene expression level was higher in white guinea pigs, lower in variegated guinea pigs, and intermediate in black guinea pigs, but there was no significant difference among the three groups (P>0.05). The MC1R gene expression level in white guinea pigs was extremely low, while both variegated and black guinea pigs showed significantly higher levels than white guinea pigs (P<0.01). Black guinea pigs showed significantly higher levels than variegated guinea pigs (P<0.05). ConclusionThe TYR and MC1R genes synergistically regulate coat color of guinea pigs. The G-site mutation in the TYR gene may lead to albinism, and the change of N-site in the MC1R gene affects the depth of the coat color.
6.Simultaneous Determination of Eleven Active Constituents in Zhenwutang Decoction by HPLC
Ping TIAN ; Kai MA ; Wei ZHANG ; Diwen ZHANG ; Bibi LIU ; Xiaoyan GUO ; De’en HAN
China Pharmacy 2019;30(18):2491-2496
OBJECTIVE: To establish a method for simultaneous determination of eleven active constituents in Zhenwutang decoction, such as 5-hydroxymethylfurfural, (+)-cianidanol, paeoniflorin, benzoylaconitine, benzoylhypacoitine, benzoylpaeoniflorin, 6-gingerol, 8-gingerol, atractylenolide Ⅱ, 6-shogaol and pachymic acid. METHODS: HPLC method was adopted. The separation was performed on Phenomenex Kinetex C18 column with mobile phase consisted of acetonitrile-0.2 % phosphoric acid solution(gradient elution) at flow rate of 1.0 mL/min. The detection wavelength was set at 285 nm (4.4-7 min, 5-hydroxymethylfurfural), 203 nm [7-12 min,(+)-cianidanol], 233 nm (12-50 min,paeoniflorin, benzoylaconitine, benzoylhypacoitine, benzoylpaeoni- florin), 200 nm (50-62.3 min, 6-gingerol, 8-gingerol; 62.9-90 min, 6-shogaol, pachymic acid) and 222 nm (62.3-62.9 min, atractylenolide Ⅱ). The column temperature was set at 35 ℃, and the sample size was 20 μL. RESULTS: The linear ranges of 5-hydroxymethylfurfural, (+) -cianidanol, paeoniflorin, benzoylaconitine, benzoylhypacoitine, benzoylpaeoniflorin, 6-gingerol, 8-gingerol, atractylenolide Ⅱ, 6-shogaol, pachymic acid were 0.62-12.47 μg/mL (r=0.999 6),2.36-47.25 μg/mL (r=0.999 7),200.80-4 016 μg/mL (r=0.999 7),4.45-89.04 μg/mL (r=0.999 6),4.28-85.54 μg/mL (r=0.999 5),5.16-103.13 μg/mL (r=0.999 9),5.53-110.66 μg/mL (r=0.999 9),0.84-16.89 μg/mL (r=0.999 8),0.60-12.04 μg/mL (r=0.999 9),0.53-10.62 μg/mL (r=0.999 5),1.04-20.78 μg/mL (r=0.999 7), respectively. The limits of quantitation were 0.155, 0.590, 1.210, 1.112, 1.070, 0.258, 0.553, 0.421, 0.153, 0.354, 0.431 μg/mL, respectively. The limits of detection were 0.047, 0.179, 0.134, 0.337, 0.324, 0.078, 0.168, 0.128, 0.046, 0.107, 0.131 μg/mL, respectively. RSDs of precision, stability and reproducibility tests were all lower than 3%. The average recovery rates were 96.06%-103.01%(RSD=2.64%,n=6), 95.11%-101.57%(RSD=2.58%,n=6), 97.22%-102.11%(RSD=1.93%,n=6), 96.43%-102.78%(RSD=2.35%,n=6), 96.42%-101.43%(RSD=2.15%,n=6), 96.86%-102.05%(RSD=2.10%,n=6), 95.32%-100.55%(RSD=1.87%,n=6), 97.04%-103.25%(RSD=2.22%,n=6), 96.78%-103.22%(RSD=2.62%,n=6), 97.04%-103.14%(RSD=2.28%,n=6), 97.08%-103.51%(RSD=2.94%,n=6), respectively. CONCLUSIONS: The method is accurate and specific, and suitable for simultaneous determination 11 active components of Zhenwutang decoction.
7.Auditory mismatch negativity in attention deficit hyperactivity disorder in children: a Meta-analysis
Lili XIAO ; Chunxia LIU ; Yuxin DU ; Diwen ZHANG ; Libo WANG ; Xin SHU
Sichuan Mental Health 2023;36(2):123-130
ObjectiveTo explore the differences existing in the auditory mismatch negativity (MMN) amplitude and latency between children with attention deficit hyperactivity disorder (ADHD) and normal children, and to probe into the significance of MMN latency and amplitude for assessing the auditory perception and attention level in ADHD children and normal children. MethodsOn December 1, 2022, a systematic search was performed in PubMed, Embase, Cochrane Library, China National knowledge Infrastructure (CNKI), Wanfang Data Knowledge Service Platform and VIP databases to identify all well qualified literature focusing on MMN of ADHD children, then the valid data relevant to MMN amplitude and latency were extracted. The Newcastle-Ottawa Scale (NOS) was used to assess the quality of the included studies, and Stata 20.0 was employed for Meta-analysis. ResultsA total of 9 qualified studies comparing ADHD children (n=170) against healthy controls (n=159) were finally included. Among the included literature, there were 18 matched pairs of MMN amplitude data and 10 matched pairs of MMN latency data at different recording sites. Meta-analysis denoted that ADHD group resulted in potentials of slightly lower MMN amplitude (WMD=-0.334, 95% CI: -1.426~0.758, P=0.549) and notably longer MMN latency (WMD=14.768, 95% CI: 4.660~24.876, P=0.004) compared to control group, and the Bgger's funnel plot did not reveal any publication bias. ConclusionCompared with healthy controls, ADHD children have longer MMN latency, suggesting that the auditory perception and attention level of ADHD children may be reduced.
8.Influencing factors of depressive symptoms in patients with vestibular neuritis
Shuangzhi WANG ; Feihu CAO ; Liu WANG ; Jiawei HE ; Xin ZOU ; Diwen ZHANG
Sichuan Mental Health 2024;37(6):557-561
BackgroundVestibular neuritis is a common clinical acute peripheral vertigo disorder. Some patients may experience negative emotions states, leading to chronic exacerbation of vestibular neuritis and a poorer prognosis. Further research is needed to understand the psychological state of patients with vestibular neuritis and its influencing factors. ObjectiveTo explore the relationship between depressive symptoms and vestibular symptoms in patients with vestibular neuritis and its influencing factors, so as to provide references for clinical intervention. MethodsA total of 86 patients with vestibular neuritis, hospitalized in the Department of Neurology of the Third Hospital of Mianyang from June 2021 to June 2023, were included in the study. Assessments were conducted using the Hamilton Depression Scale-17 item (HAMD-17), Dizziness Assessment Rating Scale (DARS) and Dizziness Handicap Inventory (DHI). Patients were divided into depression group(n=46) and non-depression group(n=40) based on HAMD-17 score. Pearson correlation analysis was used to examine the correlation among each scale score. Binary Logistic regression was used to identify influencing factors for depressive symptoms. ResultsAmong the 86 patients, 46 (53.49%) exhibited depressive symptoms. Statistically significant differences were observed between depression group and non-depression group in terms of age, disease duration, years of education, DARS score and DHI score (t=4.512, 4.921, 2.712, 3.529, 5.471, P<0.01 ). In depression group, HAMD-17 score was positively correlated with DARS score and DHI score (r=0.345, 0.335, P<0.01). Binary Logistic regression analysis showed that age (OR=4.352, 95% CI: 1.520~12.462), disease duration(OR=3.772, 95% CI: 1.339~10.630), years of education (OR=0.074, 95% CI: 0.235~0.923), DARS score (OR=1.213, 95% CI: 0.405~3.628) and DHI score (OR=3.619, 95% CI: 1.246~10.514) were the influencing factors of depressive symptoms among patients with vestibular neuritis. ConclusionDepressive symptoms in patients with vestibular neuritis are positively correlated with vestibular symptoms. Risk factors for depressive symptoms in patients with vestibular neuritis include age, disease duration, DARS score and DHI score, while years of education serve as a protective factor.