Objective To establish the heterologous recombinant embryonic stem cell （ES cell） with ghrelin receptor (GHS-R) gene deletion in order to study the function of the GHS-R gene. Methods PGK-neo cassette was replaced by TK-neo in X-pPNT agent. The target region was located in exon 1 and exon 2 of GHS-R. Two homologous arms were amplified from mouse ES cells genomic DNA and constructed into X-pPNT with SalⅠ/NotⅠand EcoRⅠ/BamHⅠ, respectively. ES cells were electrotransfected with the linearized targeting vector and screened with G418 and Gancyclovir. Finally, the positive ES cell clones were identified by PCR and sequencing. Results The X-pPNT-TK-neo vector was obtained. And two homologous arms were inserted correctly. Finally, 328 positive clones were obtained by G418 and Gancyclovir screening, and 3 clones were confirmed as GHS-R gene homologous recombination. Conclusion This study provides the necessary basis for the establishment of the GHS-R knock out mouse model and the further study on GHS-R gene function in vivo.
Objective To find and evaluate the existence of distant peritoneal micrometastasis of gastric cancer in rectovesical pouch or Douglas pouch by using immunohistochemist ry method. Methods Forty cases of gastric cancer were collected f rom June 2004 to March 2006 in Nanjing Gulou hospital . None of them showed obvious distant peritoneal metastasis in preoperative physical and imaging examinations and laparotomy inspection or palpation. Tissues were taken f rom rectovesical pouch or Douglas pouch during the operations , and HE and CEA/ CK220 immunohistochemistry staining were then performed on the tissues. Results Distant peritoneal micrometastasis in rectovesical pouch or Douglas pouch were found in 10 cases out of the 40 cases , all of which were found to have full-thickness invasion or invasion out side gast ric serous tunic 〔27. 8 % (10/ 36) 〕. Their occurrence rates of peritoneal micrometastasis were significantly higher than those without full-thickness invasion〔0 (0/ 4) 〕, Plt;0. 05. The number of metastatic lymph nodes was more than six in 8 cases , was only one in 2 case , the occurrence rate of peritoneal micrometastasis of the number of metastatic lymph nodes was more than seven 〔44. 4 %(8/ 18) 〕which was significantly higher than that the number was less than seven〔16. 7 % (2/ 12) 〕, Plt;0. 05. In 10 cases , 8 cases were poorly differentiated adenocarcinoma , and the other two were moderately differentiated. Conclusion When gast ric carcinoma invaded serous tunic or outside , though peritoneal metastasis may not be found by preoperational inspection or intraoperative palpation , peritoneal biopsy in rectovesical pouch or Douglas pouch may be necessary to perform as a routine procedure to detect distant peritoneal micrometastasis. It may be useful for staging , adjuvant chemotherapy and prognosis forecast.