[Analysis on the effect of secondary mutations on Leber's hereditary optic neuropathy].

OBJECTIVE: To investigate the effect of secondary mutations on Leber's hereditary optic neuropathy (LHON). METHODS: Three primary and 24 secondary mutations were identified in 4 Chinese families which included male offspring. RESULTS: All of the four pedigrees carried classic LHON mutations at nucleotide (nt) 11778, and did not carry any point of 24 secondary mutations. Nevertheless many polymorphic points were found in the nearby fragments of these pedigrees, such as 5178, 5108, 3705, 3721, 13734, etc. CONCLUSION: Male offspring sequences should be analyzed in pedigrees with LHON to avoid the influence of familial inheritance characteristic which mitochondrial DNA polymorphism carried. Existence of the "repair genes" may affect the development of LHON.

[Detection of mtDNA*LHON G11778A mutation by real-time polymerase chain reaction using TaqMan-MGB probe technology].

OBJECTIVE: To develop a simple, rapid and reliable real-time PCR assay based on TaqMan technology using a new MGB probe for detecting mtDNA(*)LHON G11778A mutation and heteroplasmy directly. METHODS: Twenty patients with suspicion of Leber hereditary optic neuropathy (LHON) and their maternal relatives had undergone molecular genetic evaluation. Seventeen normal individuals were used as the controls. A real-time PCR involved two MGB probes (wild-type and mutation-type) in a single tube on the iCycler IQ real-time detection system was used to detect the mtDNA(*)LHON G11778A mutation. The results were then compared with the DNA sequence analysis of the PCR products. A linear standard curve was obtained by pUCm LHON-G and pUCm LHON-A clone. RESULTS: In the controls (wild type), the reaction of VIC-labeled MGB probe was positive and the channel of FAM reaction was negative, the DNA sequence was 100% matched to previously published data. In 20 LHON patients and their maternal relatives, 12 cases showed mutations in DNA sequence analysis, all of them were LHON mtDNA mutation. While 5 other cases showed the combination of LHON mtDNA mutation and wide type gene phenotype, the rate of Ct value in wild type versus gene mutation was over 25%. DNA sequence analysis showed 8 of LHON mtDNA belonged to wild types and 3 cases were heteroplasmy, and the rate of Ct value in gene mutation versus wild type was lower than 25%. CONCLUSION: This real-time PCR assay is a simple, rapid and reliable method for the detection of genotyping mtDNA mutations as well as for quantifying heteroplasmy.

Inhibition of hepatitis B virus surface antigen expression by small hairpin RNA in vitro.

AIM: To explore the anti-hepatitis B virus effect of RNA interference (RNAi) using small hairpin RNA (shRNA) expression vector. METHODS: Hepatitis B virus surface antigen green fluorescent protein (HBs-GFP) fusion vector and shRNA expression vectors were constructed and cotransfected transiently into HepG2 cells. mRNAs extracted from HepG2 cells were detected by real-time PCR. Fluorescence of HBs-GFP protein was detected by fluorescence-activated cell sorting (FACS). The effective shRNA expression vector was transfected into HepG2.2.15 cells. HBsAg and HBeAg in HepG2.2.15 cells were analyzed by radioimmunoassay (RIA) method. RESULTS: FACS revealed that shRNA targeting at HBsAg reduced the GFP signal by 56% compared to the control. Real-time PCR showed that HBs-GFP mRNA extracted from HepG2 cells cotransfected with pAVU6+27 and HBs-GFP expression plasmids decreased by 90% compared to the empty vector control. The expressions of HBsAg and HBeAg were also inhibited by 43% and 64%, respectively. CONCLUSION: RNAi using shRNA expression vector can inhibit the expression of HBsAg, providing a fresh approach to screening the efficient small interfering RNAs (siRNAs).

[Preparation of cationic liposomes and its role in enhancing cellular uptake of antisense oligonucleotides].

AIM: To prepare the liposomes which protect antisense oligodeoxynucleotides (ASON) against nuclease degradation and delivery ASON into cytoplasmic efficiently. METHODS: A cationic derivative of cholesterol, 3 beta-[N-(N',N'-dimethylaminoethan)-carbamoyl] cholesterol (DC-Chol) was synthesized and used to prepare cationic liposome. The characteristics of liposomes/ASON complexes including size, drug loaded efficiency and structure were investigated. Cellular uptake of fluorescence labled ASON (FAM-ASON) under different condition was determined by flow cytometric analysis. Denatured polyacryamide gel electrophoresis (DPGE) was used to analyze the role of liposomes in protecting ASON. RESULTS: The mean values of preliposomes and liposomes/ASON complexes size were 185.7 and 228.2 nm, respectively. Cationic liposomes showed a high adsorption capacity for ASON. When the +/- charge ratio exceeded 2:1, more than 90% of the ASON was loaded into liposomes. Agarose gel electrophoresis showed three different existence of ASON in liposomes formulation: free, absorbed and encapsulated types. Concerning cellular uptake, DC-Chol liposomes indicated high efficient effect of increasing cellular uptake of ASON. Compared with free ASON, the total fluorescence intensity in cytoplasma was significantly enhanced. The level of increasing was largely depended on +/- charge ratio. The cellular uptake of FAM-ASON decreased in the presence of serum. The cellular total fluorescence intensity in 10% and 30% fetal bovine serum of cultured medium were only 22.3% and 15.5% as that of serum-free media, respectively. DPGE confirmed that free ASON was rapidly degraded by DNase I while ASON encapsulated into liposomes was efficiently protected. CONCLUSION: The cationic DC-Chol liposomes are shown to be promising carriers to deliver ASON into cytoplasma.

[Expression of MAGE, GAGE, and BAGE genes in human hepatocellular carcinoma].

OBJECTIVE: To observe the expression of MAGE, GAGE and BAGE genes in human hepatocellular carcinoma (HCC) cell lines. METHODS: The expression levels of MAGE-1, MAGE-3, GAGE1-8, GAGE1-2 and BAGE mRNAs in HCC cell lines SMMC-7721, QQY-7701, BEL-7402 were studied by reverse transcription polymerase chain reaction and were compared with those in biopsied liver tissues. RESULTS: MAGE-1 and BAGE mRNAs were expressed in SMMC-7721 cells. MAGE-3 and BAGE mRNAs were expressed in QQY-7701 cells. MAGE-1 and GAGE1-2 mRNAs were expressed in BEL-7402 cells. None of these genes was expressed in biopsied liver tissues. CONCLUSIONS: MAGE-1, MAGE-3, GAGE1-8, GAGE1-2 and BAGE are expressed in hepatocellular carcinoma cell lines. These tumor-specific antigens can be used as molecular markers for early diagnosis and possible targets for immunotherapy of human HCC.