Objective To explore the effect of bionic urine bags on dysuria in stroke patients and improve the effect of bladder function training. Methods Forty patients with urinary incontinence were selected and randomly divided into control group (n=46) and observation group (n=44) by random number table. The patients in the control group were treated with Kangwei anti-reflux urine bags and the urine was discharged every 3 hours at daytime, once every 4 hours at night. The patients in the experimental group were treated with OT-U bionic urine bags for drainage urine, the threshold of urine bag pressure set:when the bladder pressure reached more than 35cm H2O, the bladder urine flowed into the urine bag. The two groups were compared in view of catheter catheterization time, urination after urination time, self-urination, re-intubation rate, urine overflow, urinary tract infection. Results The duration of indwelling catheters in the observation group was significantly higher than that of the control group (P <0.05). The rates of re-intubation and urinary tract infection were significantly lower as well (P<0.05). Conclusion Biomimetic urine bag can effectively promote the training of bladder function, shorten the time of indwelling catheter and reduce the occurrence of urinary tract infection after extubation.

Objective To establish a single nucleotide polymorphisms genotyping (SNP) method for a convenient, accurate, and routine analysis of clinical samples. Methods Based on the design of oligonucleotide probe, the assay was performed through three steps:the conjunction of the detection probe, universal amplification, labeling and ELISA reaction. The genotype of each SNP was revealed by reading signals of each set of reaction tubes. This assay was applied to detect sixty-two plasma samples of lung cancer for circulating DNA for three SNPs of EGFR, c.2573T＞G(L858R), EGFR, c.2582T＞G＞T(G719C). Results were compared with those obtained by direct sequencing. Results The heterozygote mutation was identified for L858R by both methods, although no mutation was detected for L861Q and G719C. Six samples were identified as heterozygotes with the new method, and only two samples were unambiguously identified as heterozygotes by the direct sequencing. Two additional samples could not be identified as heterozygotes because the peak of mutant allele was very low compared with that of wild allele. Conclusion The developed method enabled accurate identification of SNP in a convenient manner, and which is adapted to routine analysis from heterogeneous samples unambiguously.

Background Oxidative stress is a major mechanism underlying the pathogenesis of cardiovascular disease. It can trigger inflammatory cascades which are primarily mediated via nuclear factor-κB (NF-κB). The NF-κB transcription factor family includes several subunits (p50, p52, p65, c-Rel, and Rel B) that respond to myocardial ischemia. It has been proved that persistent myocyte NF-κB p65 activation in heart failure exacerbates cardiac remodeling. Mechods A recombinant adeno-associated virus serotype 9 carrying enhanced green fluorescent protein and anti-NF-κB p65 ribozyme (AAV9-R65-CMV-eGFP) was constructed. The cells were assessed by MTT assay, Annexin V–propidium iodide dual staining to study apoptosis. The expression of P65 and P50 were assessed by Western blot to investigate the under-lying molecular mechanisms. Results After stimulation with H2O2 for 6 h, H9c2 cells viability decreased significantly, a large fraction of cells underwent apoptosis. We observed a rescue of H9c2 cells from H2O2-induced apoptosis in pretreatment with AAV9-R65-CMV-eGFP. Moreover, AAV9-R65-CMV-eGFP decreased H2O2-induced P65 expression. Conclusions AAV9-R65-CMV-eGFP protects H9c2 cells from oxidative stress induced apoptosis through down-regulation of P65 expression. These observations indicate that AAV9-R65-CMV-eGFP has the potential to exert cardioprotective effects against oxidative stress, which might be of great importance to clinical efficacy for cardiovascular disease.

Objective To investigate the expression and distribution of aquaporin 3 (AQP3) in mouse early embryos at different stages.Methods Controlled ovarian hyperstimulation model of Kunming mouse was used to collect four-cell embryos,eight-cell embryos,morula stage,and early blastocysts.Immunofluorescence microscopy and laser confocal microscopy were used to detect expression and distribution of AQP3 channels in these stages.Results Fluorescence signal of AQP3 was found in four embryonic stages of mice.Distribution within embryo was different at different embryonic stages.AQP3 was mainly expressed on the karyotheca of blastomeres at four-cell and eight-cell stage.In morula stage,AQP3 was mainly expressed on cell membrane of each blastomere.In early blastocysts,AQP3 was predominantly expressed on the cell membrane and cytoplasm of trophoblastic cell.Conclusions AQP3 trans-membrane channel might have potential regulation function on mouse embryonic development.

Objective To investigate the relation between homeostasis model assessment of insulin resistance(HOMA-IR) and the results of coronary angiography in coronary artery disease(CAD) patients complicated with hypertension.Methods One hundred and two CAD patients complicated with hypertension were enrolled into the investigation group and another 80 CAD patients without hypertension were considered as the control group.The HOMA-IR and the results of coronary angiography were compared between the 2 groups and their correlation was further analyzed.Results The HOMA-IR of the investigation group was higher than that of the control group(8.10?1.25 vs 4.70?2.13,P

BACKGROUND:Recombinant adeno-associated virus serotype 9 has a high affinity in myocardial tissue, and the expression of recombinant adeno-associated virus serotype 9-enhanced green fluorescent protein (rAAV9-eGFP) in the aorta of atherosclerosis mice is not clear. OBJECTIVE:To explore the optimal time point of rAAV9-eGFP expression in the aorta of atherosclerosis mice. METHODS:Atherosclerosis model was established with high-fat diet in 30 ApoE-/-mice for 16 weeks. Among them, 25 mice were injected with 5.0×1011 vg (virus genomes) rAAV9-eGFP through the tail vein, while the remaining 5 mice were injected with saline, serving as the control group. The virus-transfected mice were kil ed at 14, 21, 28, 35 and 60 days after transfection, and aortic tissue was harvested. The expression of enhanced green fluorescent protein was detected with laser scanning confocal microscope. Western blot assays were used to detect the expression of enhanced green fluorescent protein in aorta. The expression of enhanced green fluorescent protein in vivo was observed and the optimal expression time point was determined. RESULTS AND CONCLUSION:rAAV9-eGFP effectively transfected the aorta of atherosclerosis mice, enhanced green fluorescent protein was expressed in aortic tissue, and the expression intensity increased gradual y with the increasing transfection time. The highest expression level was found at 35 days after transfection and then maintained stable at 60 days. There were significant differences at different time points after transfection (P<0.001). These data indicate that rAAV9-eGFP can be effectively expressed in the aorta of atherosclerosis ApoE-/-mice and rAAV9-eGFP can be regarded as the optimal vector in the treatment of atherosclerosis.

BACKGROUND:A lot of work has been carried out on the development of the primary cultured rat myocardial cel s at home and abroad. The primary culture technology of rat myocardial cel s becomes more mature, but myocardial cel s from neonatal mice are not easy to be obtained under the same experimental conditions. The mouse genome has more similarities with the human genome, which has a higher research value. OBJECTIVE:To improve the primary culture method of neonatal mouse myocardial cel s, and to obtain myocardial cel s with high purity, vitality and original structure and function. METHODS:The mouse cardiac tissues were treated using an enzyme digestion method to isolate isolated single myocardial cel s:first, the cardiac tissues were digested using trypsin, and then col agenous fibers were treated with col agenase to isolate single myocardial cel s. The concentration and action time of trypsin and type II col agenase were adjusted, and the pH values of reagents and temperature of each step were strictly control ed. RESULTS AND CONCLUSION:At 24 hours after inoculation, the myocardial cel s began to be adherent;at 48 hours, independent pulsation of myocardial cel s could be observed;at 72 hours, myocardial cel s were cross-linked;and at 96 hours, myocardial cel s formed cel clusters and presented with consistent beating. The survival rate and purity of myocardial cel s were both over 95%. This modified method could successful y culture myocardial cel s with high purity and viablility from neonatal mice, and the structure and function of myocardial cel s could be retained. Therefore, it is a feasible culture method.

BACKGROUND:Platelet-derived growth factor-B (PDGF-B) is an effective pro-angiogenic growth factor, and adeno-associated virus type 9 (rAAV9) has a strong cardiomyocyte targeting affinity, which is an ideal vehicle for ischemic heart disease gene therapy.
OBJECTIVE:To explore the PDGF-B gene transfection of in vitro neonatal rat myocardial cells mediated by rAAV9 against ischemia and hypoxia-induced cardiomyocytes apoptosis.
METHODRat neonatal myocardial cells were isolated and cultured, and then transfected by rAAV9-PDGF-B and empty virus, rAAV9 with enhance green fluorescent protein (eGFP), under multiplicity of infection (MOI) of 105, 106 and 107, respectively. We observed the expression of eGFP under fluorescence microscopy every day, and used flow cytometry to measure transfection efficiency of vector rAAV9. Western blot and immunofluorescence were used to evaluate protein expression of PDGF-B. Myocardial ischemia and hypoxia injury model was established in vitro on the 5th day of transfection of rAAV9-eGFP and rAAV9-PDGF-B with 107 MOI. The number of myocardial apoptosis was measured by TUNEL assay. Western blot was employed to detect the protein expression of Bax and Caspase-3 which were related apoptosis, and the effect and its possible mechanism of PDGF-B gene overexpression against myocardial apoptosis were explored.
RESULTS AND CONCLUSION:rAAV9 vector can efficiently transfect neonatal rat myocardial cells. eGFP and PDGF-B protein expressed in myocardial cells correctly and efficiently, and the expression intensity increased gradual y with the increasing of time course and MOI. The expression became stable on the 5th day, and the transfection efficiency showed significant difference among these groups (P<0.01). Myocardial apoptosis rate was significantly reduced in the rAAV9-PDGF-B group than the rAAV9-eGFP group (P<0.05), and protein levels of Bax and Caspase-3 in the rAAV9-PDGF-B group were significantly lower than those of the rAAV9-eGFP group (P<0.05). These data indicate that overexpression of PDGF-B gene can effectively reduce ischemia and hypoxia-induced myocardial apoptosis, and the possible mechanism might be by inhibiting Bax and Caspase-3 protein expression, which can provide evidence of rAAV9-PDGF-B vector in the gene therapy of ischemic heart diseases.

BACKGROUND:Macrophage migration inhibitory factor (MIF) is a multi-potent cytokine that makes considerable contribution to the regulation of inflammatory response and immune response in the body. MIF rs1007888 is associated with various inflammatory diseases, but the correlation between rs1007888 and coronary heart disease in the Kazakhs of China has been rarely explored. OBJECTIVE:To investigate the relationship between rs1007888 gene polymorphisms in MIF gene and coronary heart disease in the Kazakhs from Xinjiang Uygur Autonomous Region, China. METHODS:A total of 230 Kazakh patients with coronary heart disease evidenced by coronary arteriography between December 2012 and July 2014 were recruited, and another 478 Kazak controls were free from coronary artery disease with normal angiograms. Real-time fluorescence quantitative PCR assay was used to detect the rs1007888 polymorphisms of MIF gene. Alele and genotype distributions of the rs1007888 polymorphism were compared between patients and controls. RESULTS AND CONCLUSION:Distribution of genotypes in the two groups appeared to be in Hardy-Weinberg equilibrium (P> 0.05). The alele frequencies and genotypes of MIF-rs1007888 showed no significant difference between the two groups (P > 0.05). Therefore, the genetic variation of rs1007888 in MIF gene is not associated with coronary heart disease in the Kazakhs of China.

Objective To optimize primary cultures techniques of isolating neonatal rat cardiomyocytes and to com?pare three different methods of extractingβ3-adrenergic receptor(β3-AR)membrane protein from cultured neonatal rat car?diomyocytes. Methods TypeⅡcollagen and differential velocity adhesion were used to collect primary cardiomyocytes. To?tal protein method, ultracentrifugation method, extract kit method were used to isolate cardiomyocytesβ3-AR membrane pro?teins. The BCA method was applied for protein quantification. Relative content ofβ3-AR membrane protein and GADPH in the sample were examined by western blot. Results Optimizing culture and isolation skills can produce a great quantity of cardiomyocytes in high concentration.The kit method acquired a higher level of protein concentration(8.26±0.29)g/L than to?tal protein method(5.12±0.47)g/L does than ultracentrifugation method(3.20±0.37)g/L does all of which were with signifi?cant difference(P < 0.05). The concentration of β3-AR membrane protein was higher if obtained by kit method(0.22 ± 0.05)than ultracentrifugation method(0.09 ± 0.03)than total protein method (0.01 ± 0.01) with significant difference(P <0.05). Conclusion optimizing methodology can obtain abundant myocardial cells in high concentraion. The kit method of isolating primary culturedβ3-AR membrane proteins result in improved concentration and specificity of membrane protein.