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Objective To study the relation between stroke and both plasma leptin level and polymorphism of leptin receptor gene. Methods Ninty-nine patients with stroke and 97 patients with other diseases (controls), admitted to our hospital from January 2007 to December 2008, were chosen in the experiment. Polymerase chain reaction-restriction fragment length polymorphism (LEPR) examination was performed to detect the leptin receptor gene (Gln223A rg gene) polymorphism in these patients. The level of serum leptin was analyzed by enzyme linked immunosorbent assay. Results The genotype frequencies of GG, GA and AA in Gln223Arg gene in patients with stroke were 66.67%, 20.20% and 13.13%, respectively, while those in controls were 78.35%, 15.46% and 6.19%, respectively; those in these 2 groups were not significantly different (P＞0.05). The allele frequencies of A and G in patients with stroke were 76.77% and 25.23%, while those in controls were 86.08% and 13.92%; those in the 2 groups were significantly different (P＜0.05). The level ofleptin in patients with stroke was markedly higher that that in controls (P＜0.05). Binary regression analyses showed that history of hypertension, elevated fasting blood glucose and cholesterol, increased plasma leptin level were associated with stroke. Conclusion The allele frequencies of A in Gln223A rg gene, hypertension, elevated fasting blood glucose and cholesterol and increased plasma leptin level might be correlated with the increased risk of stroke.
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Objective To observed hepatolenticular degeneration gene (ATP7B gene) expressed in Me32aT22/2L cell and detected whether A TP7B could transport redundant copper and reduce copper-induced cell apoptosis, which will make the basement for future gene therapy. Methods Piasmid pRc/CMV-WD containing A TP7B cDNA was transfected into Me32aT22/2L cell using lipofectamin2000 transfection methods. Intracellular distribution of ATP7B was detected by immunofluorescence histochemistry method and copper transportation function was studied using high-concentration copper incubation experiments, meanwhile, cell apoptosis induced by high-concentration copper incubation was observed. Results Expression of A TB7B gene could be detected and located around nuclei within the Me32aT22/2L cell. After incubation of high- concentration copper solution after 24, 48 and 72 hours, in empty vector group intracellular copper content was (600.60±69.71) ng/mg, (890.72±65.74) ng/mg and (1189.20±85.71) ng/mg respectively, however, intracellular copper content was (351.33±49.86) ng/mg, (427.38±30.95) ng/mg and (539.10±34.91) ng/mg in A TP7B group. Comparison of two groups has a statistical significance (P<0.01). Meanwhile, cell apoptotic rate was markedly decreased in A TPTB group compared with empty vector group (P<0.01). Conclusion Exogenous A TP7B gene could be expressed in Me32aT22/2L cell and A TPTB could transport intracellular redundant copper, subsequently reduced copper-induced cell apoptosis.
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Objectives To detect RHBDF1 gene expression in normal glial and glioma cells and prove the apoptosis inducement and growth inhibition in C6 cells after its RHBDFI gene being silenced in vitro by small interfering RNA (siRNA), so as to define the new target for gene therapy of glioma. Methods RHBDF1 gene expression was detected by Western blotting in normal glial cells and glioma cells including C6, U251 and MGR2. C6 cells transfeetion was performed using lipofection of RHBDF1 siRNA and control siRNA, and the change of RHBDF1 gene and protein levels was detected by RT-PCR and Western blotting. After the RHBDF1 gene silencing, cell apoptosis rate was determined by TUNEL methods and cell proliferation rate was measured by Ki-67 immunostaining. Results As compared with the normal glial cells, RHBDF1 gene was over-expressed in the glioma cells. After siRNA transfection, the cell apoptosis rate was 2.96%±1.25%, 36.35%±4.85% and 33.58%±4.08% in the control siRNA, RHBDF1 siRNA1 and RHBDF1 siRNA2 groups, respectively. Proliferation rate is 95.96%± 3.25%, 24.57%±5.53% and 25.68%±4.08% in the control siRNA, RHBDF1 siRNA1 and RHBDF1 siRNA2 groups, respectively. Compared with the control siRNA group, significantly high apoptosis rate and low proliferation rate in RHBDF 1 siRNA groups were showed (P<0.05). Conclusion RHBDF1 gene was over-expressed in glioma cells and RHBDF1 gene silencing can induce cell apoptosis and inhibit cell growth, indicating that RHBDF1 gene is a new potential target in gene therapy of glioma.
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<p><b>OBJECTIVE</b>To study the exons deletion mechanisms for dystrophin gene, the molecular characters of breakpoints of junction fragments for deletion-type Duchenne muscular dystrophy (DMD) patients with 46 and 51 exons deletion were compared and analyzed.</p><p><b>METHODS</b>Deletion-type DMD patients were detected by multiplex polymerase chain reaction(mPCR). The breakpoints of junction fragments with 46 and 51 exons deletions were cloned and sequenced respectively.</p><p><b>RESULTS</b>Analysis of sequences of deletion-junction fragment of exon 46 showed that the 5'breakpoint was located in AT-rich region of intron 45 and the 3' breakpoint was in medium reiteration repeats (MER1) sequence. There existed 2 bp(ta) junction homology between two breakages. No small insertion, small deletion or point mutation was located near the junction point. Similarly, analysis of sequences of deletion-junction fragment of exon 51 showed that the 5 breakpoint was located in transposon-like human elements (THE1) of intron 50 and the 3' breakpoint was in L2 sequence. There existed 3 bp(cta) junction homology between two breakages. No small insertion, small deletion or point mutation was located near the junction point. By analyzing the secondary structure of junction fragments with 46 and 51 exons deletions, it was demonstrated that all breakpoints of junction fragments were located at the non-matching regions of single-strand hairpin.</p><p><b>CONCLUSION</b>By comparing the junction fragments with 46 or 51 exons deletion, it was found that all of breakpoints were located in repeat sequences and the repeat sequences formed the single-strand hairpin which could make the introns instable and result in exon deletion.</p>