Genetic diagnosis for female carriers of glucose-6-phosphate dehydrogenase deficiency by RT-PCR-DGGE / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the feasibility of genetic diagnosis for female carriers of human glucose-6-phosphate dehydrogenase (G6PD) deficiency by reverse transcriptase-PCR-denaturing gradient gel electrophoresis (RT-PCR-DGGE).</p><p><b>METHODS</b>Blood samples were collected from suspected 54 female carriers of G6PD deficiency. Total RNAs of peripheral blood were prepared and reverse-transcripted into cDNA. Design of 6 primer pairs for DGGE was based on 17 mutation sites of G6PD cDNA described in the Chinese population. Mutations in the coding region of G6PD gene were screened and genotyped by combination of PCR-DGGE and DNA sequencing.</p><p><b>RESULTS</b>One case of 1024C/T, 20 cases of 1376G/T and 12 cases of 1388G/A were detected in the 54 samples. The total detection rate was 66.1% (33/54).</p><p><b>CONCLUSIONS</b>Heterozygous mutation rate in female carriers of G6PD deficiency detected by RT-PCR-DGGE is high. RT-PCR-DGGE is value of clinical diagnosis for G6PD-deficiency female carriers.</p>

[Chromosomal aberration in vulvar squamous cell carcinoma analyzed by comparative genomic hybridization].

BACKGROUND & OBJECTIVE: Vulvar squamous cell carcinoma (VSCC) accounts for about 80%-90% of female vulvar malignant tumors, but the etiology is still unclear. This study was to identify genetic alteration in VSCC by comparative genomic hybridization (CGH). METHODS: The genomic imbalance, that is, gains or losses of chromosomes, in 21 cases of vulvar squamous cell carcinoma was detected by CGH. RESULTS: The common chromosomal alterations in VSCC included gains of chromosomes 3q, 8q and 12q, and losses of chromosomes 4p and 3p. CONCLUSIONS: There are multiple chromosomal aberrations in VSCC. The amplification of the tumor suppressor genes and loss of the oncogenes on these regions may be involved in the development and progression of VSCC.

Effects of antisense human telomerase reverse-transcript protein subunit (hTERT) gene on biological characteristics of hepatoblastoma cell line in vitro / 中华儿科杂志

<p><b>OBJECTIVE</b>Telomerase, a complex of ribose and nucleoprotein, is a specific marker of tumor, which expresses in 98% infinite cell lines and 90% malignant tumor organizations and whose function is to maintain the length of telomere. Human telomerase reverse-transcript protein subunit (hTERT) is the key element and rate-limiting factor of telomerase activity. Our study was to investigate the effects of antisense hTERT gene on biological characteristics of hepatoblastoma cell line in vitro.</p><p><b>METHODS</b>The sense and antisense hTERT eukaryotic expression vectors that we had constructed before were transfected into hepatoblastoma cell line HepG2 by using the SuperFect transfection reagent (Qiagen) according to the manufacturer's instructions, then the HepG2-s and HepG2-as of G418-resistant colonies were obtained with G418 and identified for the presence of hTERT insert by PCR with T7 and pcDNA3.1/BGH reverse primers. After that, we have detected the endogenous hTERT mRNA expression and telomerase activity by quantitative real-time RT-PCR and TRAP-silver staining assay in cells from each group. Meanwhile, MTT cellular proliferation assay, soft agar colony formation assay and flow cytometry were employed to analyze if the proliferation capacity of liver cancer cells was affected in vitro and the tumor cells could be induced to apoptosis by antisense hTERT.</p><p><b>RESULTS</b>Antisense hTERT significantly down-regulated the endogenous hTERT mRNA expression (15.35 +/- 1.72/HepG2-as, 43.8 +/- 2.89/HepG2-s, 45.2 +/- 3.46/HepG2) (n = 10, t = 7.61, P < 0.01) and telomerase activity in HepG2, compared to blank control and sense hTERT. After 20 passages of three group cells, a 7-day cell growth curve and the numbers (size) of soft agar colony formation showed the proliferation and the anchorage-independent growth in HepG2-as were significantly suppressed (50.6 +/- 4.8/HepG2-as, 113.52 +/- 8.15/HepG2-s, 119.12 +/- 10.82/HepG2) (n = 10, t = 4.54, P < 0.01 and n = 10, t = 3.96, P < 0.01), compared to HepG2 and HepG2-s. However there was a significant increase in apoptosis percentage of HepG2-as by flow cytometry (n = 10, t = 9.24, P < 0.01 and n = 10, t = 8.37, P < 0.01), compared to control group.</p><p><b>CONCLUSIONS</b>Antisense hTERT could significantly suppress the hepatoblastoma cell growth and reverse its malignant phenotypes in vitro and cause the increase in apoptosis percentage of HepG2, thus it might be applied in malignant tumor gene therapy through the telomerase-targeted molecular mechanism.</p>