Promoterless DNA fragments inhibiting the expression of integrated gene in human pancreatic carcinoma cells / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate whether introducing promoterless DNA containing the cDNA sequence of green fluorescent protein (GFP) induces gene-specific silencing in human pancreatic cancer cell line harboring genomically integrated GFP gene.</p><p><b>METHODS</b>Using G418 selection and fluorescent separation we established a highly purified monoclonal pancreatic cancer cell line, recombinant PANC-1, which had a steady level of GFP expression. GFP cDNA was amplified by PCR from plasmid pEGFP-C1 and ligated to the promoterless plasmid PUC19. PUC-GFP was then transfected into monoclone cells along or cotransfected with pEGFP-C1 to panc-1 cells. Each had PUC plasmid transfected group as their control to eliminate possibility of plasmid toxicity and GFP small interferent RNA (siGFP) transfected group as positive control. Western blot, flow cytometry and phase contrast fluorescence microscopy were used to detect the changes of GFP expression.</p><p><b>RESULTS</b>The data showed that (1) PUC-GFP inhibited the GFP expression in monoclonal cell line in a dosage dependent manner. The inhibitory effect of 3 microg PUC-GFP did not show significant difference with siGFP. (2) The significant repression appeared on the fourth day after transfecting monoclonal cells with 3 microg PUC-GFP. By the end of the sixth day, GFP expression in PUC-GFP group and siGFP group remained at a low level. (3) Cotransfecting PUC-GFP with pEGFP-C1 plasmids into PANC-1 cells showed a decreased transfection efficiency when compared with transfecting pEGFP-C1 alone. Higher PUC-GFP vs pEGFP-C1 corresponded with lower transfection efficency. (4) When adding new pEGFP-C1 plasmid to cells after inhibition appeared, the GFP expression recovered.</p><p><b>CONCLUSION</b>Transfection of the promoterless DNA fragment containing full-length cDNA effectively induces a gene-specific silencing in mammalian cells.</p>

Tetramethylpyrazine inhibits agiontensin II-induced nuclear factor-kappaB activation and bone morphogenetic protein-2 downregulation in rat vascular smooth muscle cells / 生理学报

Tetramethylpyrazine (TMP), an effective component of traditional Chinese medicine Chuanxiong, is commonly used to resolve embolism. Its possible therapeutic effect against atherosclerosis has received considerable attention recently. Angiotensin II (Ang II) is highly implicated in the proliferation of vascular smooth muscle cells (VSMCs), resulting in atherosclerosis. The mechanisms of TMP in the proliferation of VSMCs induced by Ang II remain to be defined. The present study was aimed to study the effect of TMP on Ang II-induced VSMC proliferation through detection of nuclear factor-kappaB (NF-kappaB) activity and bone morphogenetic protein-2 (BMP-2) expression. Primary cultured rat aortic smooth muscle cells were divided into the control group, Ang II group, Ang II + TMP group and TMP group. Cells in each group were harvested at different time points (15, 30 and 60 min for detection of NF-kappaB activity; 6, 12 and 24 h for measurement of BMP-2 expression). NF-kappaB activation was identified as nuclear staining by immunohistochemistry. BMP-2 expression was observed through Western blot, immunohistochemistry and in situ hybridization. The results showed that: (1) Ang II stimulated the activation of NF-kappaB. Translocation of NF-kappaB p65 subunit from cytoplasm to nucleus appeared as early as 15 min, peaked at 30 min (P<0.01) and declined after 1 h. (2) TMP inhibited Ang II-induced NF-kappaB activation (P<0.01). (3) Ang II increased BMP-2 expression at 6 h but declined it significantly at 12 and 24 h (P<0.01). (4) BMP-2 expression was also kept at high level at 6 h in Ang II + TMP group but maintained at the normal level at 12 and 24 h. (5) There was no significant difference in NF-kappaB activation and BMP-2 expression between the control group and TMP group. These results indicate that TMP inhibits Ang II-induced VSMC proliferation through repression of NF-kappaB activation and BMP-2 reduction, and BMP-2 expression is independent of the NF-kappaB pathway. In conclusion, TMP has therapeutic potential for the treatment of atherosclerosis.

K-ras mutation analysis in ovarian serous borderline and malignant tumors / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the roles of K-ras gene in the tumorigenesis of ovarian serous borderline and malignant tumors.</p><p><b>METHODS</b>Fifty one tissue samples of ovarian serous tumors, including 18 conventional serous borderline tumors, 11 micropapillary serous borderline tumors, 12 invasive micropapillary serous carcinomas, and 10 conventional serous carcinomas were investigated for the presence of K-ras mutation. DNA was extracted after microdissection of the tumor tissue, the exon 1 of K-ras gene was amplified by PCR, and the presence of mutation at the codons 12 and 13 was evaluated by direct sequencing analysis.</p><p><b>RESULTS</b>GGT to GTT mutation at codon 12 of the K-ras gene was found in one conventional serous borderline tumors, resulting in valine to glycine substitution. All other 50 cases showed no K-ras mutation. All tumors had a wild-type codon 13.</p><p><b>CONCLUSIONS</b>Mutations of K-ras at codons 12 and 13 in ovarian serous tumors are very rare in this series of patients, suggesting a difference present between the Chinese and Caucasian populations. K-ras mutations may play a less important role in the tumorigenesis of ovarian serous tumor of the Chinese patients.</p>