Objective To establish a method of competitive polymerase chain reaction combined with restrictive endonucleases to measure the methylation quantitatively in MDR1 promoter region.Methods One sense primer and two anti-sense primers were designed to amplify a fragment of MDR1 gene promoter region, which was located between -102 and +186 bp containing a methylation site. The internal reference DNA fragment, witch was less 30bp than target fragment was made by three times of polymerase chain reaction(PCR)using different anti-sense primer and then subcloned into the Pbluescriptsk+ plasmid. The genomic DNA digested by Hpa, a methylation-sensitive restrictive endonuclease and competitive internal reference DNA were competitively amplified for MDR1 promoter in the same tube by PCR. The PCR products were electrophoresed on agarose gel, stained by ethidium bromide and subjected to image analysis scanner. The amount of target fragment was calculated as following, the optical density ratio of target fragment to competitive internal reference fragment multiplied the amount of competitive internal reference DNA. The ratio of PCR products amplified from HpaⅡ digested DNA and undigested DNA was named the methylation rate.Results The genomic DNA serially diluted with optimal amount of competitive internal reference DNA were co-amplified for MDR1 promoter. The significant positive correlation between the ratio of two products and the amount of genomic DNA was demonstrated. The correlation coefficient was 0.992, P

Objective:To investigate if the combined use of CDAⅡ and sodium butyrate can induce demethylation and re-expression of retinoic acid receptor?2(RAR?2)gene in cultured human breast cancer cells MCF7.To explore if the two drugs can inhibit cell growth and induce cell apoptosis synergetically.Methods:MCF7 cell line was treated with CDAⅡ,sodium butyrate,combination of the two drugs respectively.Methylation was assessed by methylation-specific polymerase chain reaction(MSP)for RAR?2 gene.Gene expression was evaluated by reverse transcription polymerase chain reaction(RT-PCR).Apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling(TUNEL)and Hoechst33342/propidiumiodide(PI)staining.Cell growth inhibition was measured by MTT assay.Results:Neither CDAⅡ nor sodium butyrate induced demethylation and re-expression of RAR?2 gene,Combination of the two drugs partially demethylated gene promoter accompanied by re-expression of RAR?2.The apoptotic cells in the double-drug group were obvious following Hoechst33342/PI staining.The percentage of apoptotic cells in the double-drug group was significantly higher than that of the two single-drug group(39.5% vs 5.2%,8.1%)(P

Objectives To report a generalized atrophic benign epidermolysis bullosa family,identify the deficient protein and related pathogenic gene mutation. Methods The diagnosis was confirmed based on clinical manifestations and necessary examinations. Electron microscopy and immunofluorescent staining were used to detect the deficient protein. The pathogenic gene mutation was identified by PCR amplification of ge-nomic DNA with primers targeting the flanking introns, followed by direct automated sequencing. Results In the family, the affected individuals were homozygous for a novel 4-bp deletion in COL17A1, 3897delATCT, which resulted in a downstream premature termination codon. Conclusions 3897delATCT of COL17A1 is the pathogenic gene mutation in the patients and probably results in nonsense-mediated mRNA decay and abrogation of type XⅦ collagen synthesis, as documented in the literature.

Objective To explore brain activity features during the resting state in alcohol dependent individuals,and study the relationship between the brain activity features and alcohol dependent individuals' clinical symptoms.Methods Twenty-four alcohol dependent individuals and 22 healthy control subjects,well matched in gender,age,education and handedness,were enrolled as the alcohol dependent group and control group respectively.AGE 3.0 T MR scanner was used to acquire all the subjects' resting state data. DPARSF software was used to process resting functional MRI data,and then the whole brain fractional amplitudes of low frequency fluctuation (fALFF) data were acquired.Two-sample t test statistical analysis was made to access fALFF difference between the two groups. Results In comparison with the control group,the alcohol dependent group showed reduced fALFF in bilateral medial prefrontal gyrus,right inferior occipital gyrus,left precuneus,left inferior temporal gyrus,and left posterior lobe of cerebellum (0.64-1.69 vs.0.87-1.78,t =- 4.23- - 2.79,P ＜ 0.05 ). fALFF was increased in the alcohol dependent group at the anterior cingulate,bilateral inferior frontal gyrus,right middle frontal gyrus,bilateral insular lobe,bilateral dorsal thalamus ( 0.86-1.82 vs. 0.76-1.58,t =3.56-3.96,P ＜ 0.05 ).Conclusion Alcohol dependent individuals had abnormal activity at the bilateral prefrontal lobe,anterior cingulate,bilateral dorsal thalamus,bilateral insular lobe,left posterior lobe of cerebellum et al,during the resting state,and these abnormal activities might be related with clinical manifestation and pathophysiology.

Objective To map the specific gene responsible for primary erythromelalgia and identify gene mutations in a Chinese family and one sporadic patient with primary erythromelalgia. Methods Geno-mic DNA was extracted from peripheral lymphocytes of the family members of the pedigree and the sporadic patient. Scanning the genes on chromosome 2q that had been identified was performed by using 6 microsatellite markers for the family members with primary erythromelalgia. Then linkage analysis and haplotype analysis were conducted. All exons of SCN9A gene were analyzed by PCR-DNA sequencing. The mutation identification was also confirmed by restriction fragment length polymorphism(RFLP). Results A maximum 2-point LOD score of 2.11 was found at a recombination fraction (? = 0.00) with markers D2S2370 and D2S2330. Recombination events were detected by markers D2S1353 and D2S2345 in this family by the haplotype analysis. There were two missense heterozygous point mutations in the 15th exon of SCN9A gene both in the family(T2573A) and the sporadic patient(T2543C), leading to the substitution of the amino acid leucine to histidine(L858H) and isoleucine to threonine(I848T), respectively. The above mutations were not found in 400 normal alleles. Conclusion It is proved that primary erythromelalgia is caused by mutations in SCN9A gene.

We report the diagnosis and treatment of a pregnant woman with acute Stanford type B aortic dissection in the second trimester who underwent thoracic endovascular aortic repair under local anesthesia and later gave birth to a live neonate. The patient was admitted due to acute upper back pain at 27 weeks of gestation, who was diagnosed as acute Stanford type B aortic dissection. Thoracic endovascular aneurysm repair was performed with low radiation dose under local anesthesia. A live neonate was born through cesarean section at 33 +6 gestational weeks due to the flat baseline of the fetal heart monitor, with a birth weight of 1 840 g and Apgar score of 9 at 1 min. The neonate was discharged after a 20-day treatment. During the follow-up of 12 months, the infant grew and developed well, and covered stent was well placed in the mother without leakage in the distal or proximal ends of the stent or any other complications.