Comparative study of three cell morphological analysis methods on in vitro micronucleus assay / 上海预防医学

Objective To investigate the feasibility of genotoxicity assessment for chemicals via flow cytometry (FCM) and high-content screening (HCS) based on high-throughput screening in vitro micronucleus assays. Methods In reference to the methodology of OECD TG487, the typical positive controls, cyclophosphamide (CP) and mitomycin C (MMC), were selected.And no serum MEM medium was treated as negative control.Dose range of CP was 5-20 mg/L and MMC was 0.25-1.0 mg/L.CHL cells were treated with three concentrations of each chemical for 4 h.High-throughput screening in vitro micronucleus assays based on FCM and HCS were established.The results of the frequency of micronuclei were compared to traditional cytokinesis blocking micronucleus assay in each group with or without metabolic activation. Results The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately 1.9%, 7.6%, 10.4% and 5.9%, 11.4%, 16.7%, which were obtained by conventional microscopic scoring.The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately 2.8%, 2.6%, 7.8% and 3.2%, 3.7%, 5.1%, which were obtained by flow cytometry screening.The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately2.8%, 6.2%, 9.1% and 7.9%, 10.1%, 10.2%, which were obtained by high-content screening.Compared with negative controls, the differences of the results were statistically significant(P < 0.05), and there was a dose-response relationship. Conclusion In this study, the results of high-throughput screening assays of FCM and HCS are in accordance to the results of traditional cytokinesis blocking micronucleus assay, indicating that high-throughput screening in vitro micronucleus assays could detect micronucleus formation automatically and improve the efficiency.Therefore, the method could provide data support for using high-throughput screening in vitro micronucleus assays into genotoxicity assessment of chemicals.

Comparative study of three cell morphological analysis methods on in vitro micronucleus assay / 上海预防医学

Objective To investigate the feasibility of genotoxicity assessment for chemicals via flow cytometry (FCM) and high-content screening (HCS) based on high-throughput screening in vitro micronucleus assays. Methods In reference to the methodology of OECD TG487, the typical positive controls, cyclophosphamide (CP) and mitomycin C (MMC), were selected.And no serum MEM medium was treated as negative control.Dose range of CP was 5-20 mg/L and MMC was 0.25-1.0 mg/L.CHL cells were treated with three concentrations of each chemical for 4 h.High-throughput screening in vitro micronucleus assays based on FCM and HCS were established.The results of the frequency of micronuclei were compared to traditional cytokinesis blocking micronucleus assay in each group with or without metabolic activation. Results The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately 1.9%, 7.6%, 10.4% and 5.9%, 11.4%, 16.7%, which were obtained by conventional microscopic scoring.The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately 2.8%, 2.6%, 7.8% and 3.2%, 3.7%, 5.1%, which were obtained by flow cytometry screening.The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately2.8%, 6.2%, 9.1% and 7.9%, 10.1%, 10.2%, which were obtained by high-content screening.Compared with negative controls, the differences of the results were statistically significant(P < 0.05), and there was a dose-response relationship. Conclusion In this study, the results of high-throughput screening assays of FCM and HCS are in accordance to the results of traditional cytokinesis blocking micronucleus assay, indicating that high-throughput screening in vitro micronucleus assays could detect micronucleus formation automatically and improve the efficiency.Therefore, the method could provide data support for using high-throughput screening in vitro micronucleus assays into genotoxicity assessment of chemicals.

Long term outcome of posterior lumbar pedicle screw fixation combined with isthmic bone graft fusion in the treatment of lumbar spondylolysis in young patients / 中国骨伤

OBJECTIVE@#To investigate the long-term effect of posterior lumbar pedicle screw fixation combined with isthmus bone grafting and fusion in young patients with spondylolysis.@*METHODS@#A retrospective study was carried out, consisting of 16 young patients with lumbar spondylolysis without spondylolisthesis treated by lumbar posterior pedicle screw fixation combined with isthmic bone grafting fusion from January 2006 to July 2014. There were 11 males and 5 females, aged from 18 to 21 years old, with an average age of 19.3 years old, and the course of disease ranged from 12 to 26 months, with an average of 22 months. All the patients suffered from lumbar pain and difficulty in getting out of bed. Preoperative CT confirmed 12 cases of L₅ isthmus fissure and 4 cases of L₄ isthmus fissure. Bone graft fusion was confirmed and internal fixation was removed after operation. Lumbar spondylolysis was evaluated by lumbago visual analogue scoring method at preoperative and postoperative time points. Lumbar isthmic fusion was evaluated by lumbar CT, and degeneration of fixed and adjacent segments of lumbar intervertebral disc was evaluated by lumbar MRI.@*RESULTS@#Of the 16 patients, 13 patients (26 sides) were followed up, with a mean duration of 96 months. The operation time ranged from 80 to 105 minutes, with an average of 95 minutes. The intraoperative bleeding volume ranged from 150 to 300 ml, with an average of 225 ml. All the patients were successfully operated without any complications related to the operation. VAS scores at each time point after operation were improved compared with those before operation(<0.01). Postoperative CT scans of lumbar spine showed osseous fusion at 6 to 14 months, with an average of 12 months. There were no changes of adjacent segment degeneration, fixed segment disc degeneration and protrusion on lumbar spine MRI, and no symptomatic recurrence or recurrent spondylolysis in the long term.@*CONCLUSIONS@#The posterior lumbar pedicle screw fixation combined with isthmic bone grafting and fusion is safe and effective in the treatment of young spondylolysis. The fusion rate is high and the interference of normal physiological range is reduced. The long-term effect is satisfactory.

Clinical effect of Ranibizumab combined with PPV on PDR with neovascular glaucoma at early stage / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To investigate the clinical efficacy of intravitreal injection of Ranibizumab combined with pars plana vitrectomy ( PPV ) in patients with proliferative diabetic retinopathy ( PDR ) complicated with early neovascular glaucoma.? METHODS: Totally 80 patients ( 80 eyes ) of PDR patients with early neovascular glaucoma were selected in the patients went to Ophthalmic Center from July 2014 to December 2016. According to whether the injection of ranibizumab taken, they were divided as pretreatment group ( 40 patients 40 eyes, preoperative ranibizumab injection combined with PPV treatment ) , conventional group (40 patients 40 eyes, only taken PPV surgery). The changes of visual acuity, intraocular pressure and other indicators were compared between the two groups.?RESULTS: The operation time, neovascular bleeding times, coagulation times of pretreatment groups were significantly lower than those of the conventional group, the differences were statistically significant (P<0. 05). The differences of preoperative intraocular pressure, BCVA between the two groups were not statistically significant (P>0. 05). At 1wk, 3mo after the surgery, the BCVA of pretreatment group was better than that of conventional group (P<0. 05). At 1wk after the operation, the IOP of pretreatment group were lower than that of conventional group ( P< 0. 05 ). The preoperative VEGF in aqueous humor of the two groups was not significantly different (P>0. 05 ). At 1mo after operation, VEGF in aqueous humor of the pretreatment group was significantly lower than conventional group ( P < 0. 05 ). At 3mo after operation, the fovea retinal thickness of pretreatment group was lower than that of the conventional group ( P<0. 05).?CONCLUSION: Ranibizumab combined with PPV can significantly reduce the difficulty of operation in PDR patients with early neovascular glaucoma, improve the postoperative visual acuity, and reduce the level of VEGF in aqueous humor.

Molecular mechanisms of Arg77Cys missense mutation and Arg174stop nonsense mutation of factor XIII A gene causing severe factor XIII deficiency / 中国实验血液学杂志

The present study was aimed to investigate the molecular mechanisms responsible for the pathogenesis of severe factor XIII (FXIII) deficiency. Site-directed mutagenesis was conducted to obtain human FXIIIA expression plasmids bearing the mutations. Wild type FXIIIA recombinant plasmid (pcDNA3.1-FXIIIA-wt) and 2 mutant FXIIIA recombinant plasmids (pcDNA3.1/FXIIIA/77mut, pcDNA3.1/FXIIIA/174mut) were transfected into the cultured COS-7 cells using lipofectamine 2000 transfection reagent, respectively. FXIII activities were measured by the Berichrom(®) FXIII chromogenic assay. The expression levels of FXIIIA mRNA were detected by real-time RT-PCR. The recombinant FXIIIA mutants were determined by using Western blot and ELISA. The results showed that the normalized mRNA levels of 2 mutants in transfected COS-7 cells were 0.82 ± 0.21 and 0.76 ± 0.17, respectively. The relative levels of both mRNA transcripts were not significantly decreased as compared with the wild type (1.06 ± 0.51). FXIII activity and FXIIIA antigen levels in concentrated media of cell expressing the wild type protein were (24.0 ± 2.9)% and (13.2 ± 2.3)%, respectively. FXIII activity and FXIIIA antigen levels in cell lysates containing the wild type recombinant protein were (61.6 ± 30.4)% and (32.8 ± 14.5)%, respectively. However, the antigen levels and activity of 2 mutants were severely decreased as compared to the wild type. It is concluded that both mutations severely disturb the normal expression of FXIIIA protein. The reduction of expression levels and decreased activities of the 2 mutants provides a convincible explanation for the deficiency phenotype in the index case.

Identification of Arg77Cys and Arg174stop double heterozygous mutation in a Chinese family with inherited FXIII deficiency / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify the gene mutation type of an inherited coagulation factor XIII (FXIII) deficiency pedigree.</p><p><b>METHODS</b>PCR and DNA sequencing were used to identify the mutations in the 15 exons and the flank sequence of FXIII gene in the proband. The identified mutations were validated by allele specific PCR, PCR restriction fragment length polymorphism technique or DNA sequencing in the family members and 100 healthy volunteers.</p><p><b>RESULTS</b>Arg77Cys and Argl74stop double heterozygous mutations were discovered in the proband. The pedigree analysis showed that Arg77Cys missense mutation inherited from her father, and Arg174stop from her mother. The Arg77Cys missense mutation in exon 3 was not found in her husband and the other 100 healthy volunteers.</p><p><b>CONCLUSION</b>A novel Arg174stop nonsense mutation was discovered in human FXIII gene. A simple DNA assay based on PCR for detection of this mutation was developed. The congenital FXIII deficiency in the proband might be caused by the coinheritance of the Arg77Cys missense mutation in exon 3 and the Arg174stop nonsense mutation in exon 4.</p>

Clinical study on the vertebral plasty for the treatment of osteoporotic vertebral compressed fractures in elderly / 中国骨伤

<p><b>OBJECTIVE</b>To study therapeutic effects of Sky-bone expander for the treatment of osteoporotic vertebral compressed fractures.</p><p><b>METHODS</b>Fifteen patients (18 vertebrae) suffering from vertebral compression fractures were treated with Sky bone expander system which expanded and reconstructed the vertebral body with PMMA cement. The clinical effect was evaluated by observing the changing of visual analogue scale (VAS). The preoperative and postoperative mean VAS scores were compared by paired-sample t test. All the patients were followed up by telephone or clinic consulting after being discharged from our hospital.</p><p><b>RESULTS</b>The procedure was performed successfully in 15 patients. The operation time ranged from 45 to 110 minutes (65 minutes per vertebra on average). The patients were followed up and the duration ranged from 6 to 12 months (8 months on average). The mean VAS score of the patients were improved significantly at the third postoperative day compared with those before the operation (2.5 +/- 1.3, vs 7.7 +/- 1.1, all P < 0.05). The mean VAS score at the end of the follow-up was 2.2 +/- 1.2.</p><p><b>CONCLUSION</b>Sky bone expander system provides significant pain relief effect in the cases of osteoporotic vertebral compression fractures, shortens the duration of lying in bed, and its procedure is convenient with few complications.</p>

Identification of two novel mutations of human blood coagulation factor V gene in a Chinese family with congenital factor V deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To discover the mutations of human blood coagulation factor V (FV) gene in a Chinese family with congenital factor V deficiency, and to explore the molecular mechanism associated with the congenital factor V deficiency.</p><p><b>METHODS</b>PCR and DNA sequencing were used to look for the FV gene mutations in the proband. And the novel mutation were testified by PCR restriction fragment length polymorphism technique or reverse DNA sequencing. One hundred healthy volunteers were chosen as controls at random.</p><p><b>RESULTS</b>Two novel mutations were discovered in the FV gene of proband, which were the A1763C missense mutation in exon 11 and the splicing site mutation in the 3' terminal of intron 16 (G-->T). The pedigree analysis showed that the two mutations inherited from his parents respectively: the A1763C came from his father, and the G-->T from his mother. The A1763C missense mutation in exon 11 was not found in each of 100 healthy volunteers.</p><p><b>CONCLUSION</b>The congenital deficiency of FV in the proband might be caused by the A1763C missense mutation in exon 11 and the splicing site mutation in the 3' terminal of intron 16, which jointly caused the proband to be a double heterozygote.</p>

Investigation of dietary fiber intakes and varies in 53 patients with diabetes / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the intakes of total dietary fiber (TDF), soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) in Subjects With Type 2 Diabetes so as to provide the base for making the adequate intakes of dietary fiber.</p><p><b>METHODS</b>The enzymatic-gravimetric methods for dietary fiber were established on basis of a collaborative study. Dietary intake was measured by means of 3-day food records through weighting and using food pictures. TDF, SDF and IDF were analyzed by enzymatic-gravimetric methods.</p><p><b>RESULTS</b>The reproducibility relative standard deviations for DF ranged from 2.63% to 9.67%. Vegetable foods were the mainly sources of DF. The total dietary intakes, insoluble and soluble fibers were 26.5 +/- 9.8, 14.6 +/- 5.8, 10.4 +/- 4.4 (g/d) respectively.</p><p><b>CONCLUSION</b>The dietary fiber intake of the diabetes remains in the range of intakes recommended by American Diabetes Association.</p>

Hemoglobin H disease combining hemoglobin Q-thailand and hemoglobin E disease:a clinical and laboratory genealogical research / 中华检验医学杂志

Objective To investigate clinically and in laboratory a genealogical tree with hemoglobin H disease Combining Hemoglobin Q-Thailand and Hemoglobin E Disease.Methods Genealogical laboratory studies were carried out with the following methods:hemoglobin electrophoresis, various biochemical determinations,and DNA analysis.Results Father's genotype of ?-THAL:??/?~Q ?~(4.2); genotype of ?-THAL:?E/N;phenotype:minor ?-THAL carrier combining Hb Q and Hb E multiple heterozygote;mother' s genotype of ct-THAL:--~(SEA)/??;genotype of ?-THAL:?n/?n.According to comprehensive analysis,mother's phenotype:minor ?-THAL,complex minor ?-THAL carrier combining Hb F ? Initial sign of ?-THAL genotype:--~(SEA)/?~Q ?~(4.2);phenotype:deletion type Hb H genotype disease;?- THAL genotype:?E/?E;phenotype:? E homozygote.According to comprehensive analysis:deletion type Hb H combining HbE multiple heterozygote.Youger brother's ?-THAL genotype:--~(SEA)/?~Q ?~(4.2);?-THAL genotype:?n/?n;phenotype:deletion type Hb H disease.Both mother and her youngest son have G6PD deficiency.Conclusions Guangdong Province is an area with high morbidity of ?-THAL and ?-THAL,Hb E and Hb Q as well as G6PD deficiency.There may be some correlation between Hb E and Fib Q in term's of the high morbidity of regional Hb,but the two types of Hb combining Hb H disease are rare in China and the world in point of nonhomologous chromosome.Attention should be paid to the problems of double heterozygote of ?-THAL complex ?-THAL,and THAL complex G6PD deficiency.Data from the study have enriched the scientific information of molecular genetics of erythroeyte thalassemia and of molecular pathology with important significance in genetics guidance and clinical treatment for patients.