Construction of eukaryotic expression vector of short hairpin RNA targeting human xylosyltransferase-I gene / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To design and construct the plasmids expressing short hairpin RNA (shRNA) targeting human xylosyltransferase- I (XT- I) which is the initiating enzyme in the biosynthesis of proteoglycans (PC).</p><p><b>METHODS</b>Short chain oligonucleotides were designed according to the sequence of XT-I provided by GenBank. The DNA segments were gained through annealing after chemosynthesis, and were cloned into Pgenesil-1 vector. The recombinant XT- I shRNA expression vectors were identified by digestion and sequencing analysis. At last the constructed XT-I expression vectors were transfected into salivary adenoid cystic carcinoma cell line (ACC-M) by Lipofectomine 2000. The expression of green fluorescent protein (GFP) was detected by inverted fluorescent microscope and the rates of transfection were detected by flow cytometer. Semiquantitative RT-PCR was used to detect the expression of mRNA level of XT- I in transfected ACC-M cells and the protein expression of XT- I was detected by Western blot.</p><p><b>RESULTS</b>The plasmids expressing shRNA targeting XT-I gene are called WJ1, WJ2, WJ3, WJ4, WJ5 and WJ6. Successful constructions were identified by digestion and sequencing. The mean rate of transfection was 50.26%. ACC-M cells transfected with WJ1-WJ6 expressed GFP successfully. And by RT-PCR and Western blot, WJ3 showed the most powerful RNAi gene silencing of inhibitory. The inhibition rate was 72.39% of mRNA level and 70.18% of protein level respectively.</p><p><b>CONCLUSION</b>The XT-I shRNA expression vectors were constructed successfully which lays the foundation for RNAi study on the biosynthesis of PG in salivary gland tumors.</p>

wt-p53 gene repair for p53 mutations of salivary pleomorphic adenoma cells / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To analyze the original mutated codon of p53 gene of salivary pleomorphic adenoma (SPA) and to evaluate the repair effects of wt-p53 on SPA cells.</p><p><b>METHODS</b>Four cases of SPA were obtained from clinical fresh samples and SPA cells were separated and cultured, and then the cells were transduced by Ad-wt-p53. The cells and the corresponding tumor tissue DNA were extracted, PCR and single strand conformational polymorphism (SSCP) and DNA sequencing analysis were performed.</p><p><b>RESULTS</b>PCR-SSCP analysis showed 3 out of 4 SPA with abnormal exon 8 and abnormal exon 6. DNA sequencing analysis showed that exon 6 point mutation was found at codon 203 (GTG-->GCG), poly-codon mutations were found in exon 8 at codon 272 (GTG-->GT square), 275 (TGT-->T square T), 276 (GCC--> square CC) and at codon 290 (CGC-->CGCC). After transduced by Ad-wt-p53, all of the mutated codons were repaired.</p><p><b>CONCLUSIONS</b>p53 gene mutation involved many codons that occurred frequently in the tumorigenesis of SPA. Exogenous wt-p53 could compensate and repair all the mutated p53 codons of SPA cells. SPA cells transduced by Ad-wt-p53 showed the typical apoptosis.</p>

The effects of HSV-tk suicide gene and wild-type p53 gene on pleomorphic adenoma cells of salivary gland / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To study the therapeutic effects of combined gene therapy of wild type p53 (wt-p53) and herpes simplex virus thymidine kinase (HSV-tk) gene on pleomorphic adenoma cells of salivary gland.</p><p><b>METHODS</b>Wild type p53 and HSV-tk gene were transfected into human pleomorphic adenoma cells of salivary gland by using recombinant adenovirus vector. The efficiency of transfection was checked and gene was expressed by RT-PCR methods. The cell inhibition after transfected was verified by light microscope and MTT.</p><p><b>RESULTS</b>The proliferation of the pleomorphic adenoma cells transfected wt-p53 and HSV-tk gene was inhibited and the cell survival rate decreased to 54% and 38% respectively in 5 days. However, when wt-p53 gene combined with HSV-tk/GCV system, the killing effects was significantly stronger (P < 0.05) and the cell survival rate decreased to 20%.</p><p><b>CONCLUSION</b>Combining p53 gene with HSV-tk/GCV system for gene therapy in pleomorphic adenoma cells of salivary gland is a valuable method.</p>