Relationship between overexpression of NK-1R,NK-2R and intestinal mucosal damage in acute necrotizing pancreatitis

AlM: To study the expression of neurokinin-1 receptor (NK-1R) and neurokinin-2 receptor (NK-2R) in distal ileum ofacute necrotizing pancreatitis (ANP) and to evaluate therelationship between expression of these two receptors andintestinal mucosal damage.METHODS: A total of 130 adult Sprague-Dawley rats wererandomly divided into two groups: the rats in ANP group(n＝80) were induced by the retrograde intraductal infusionof 30 g@L-1 sodium taurocholate. And the rats in normal controlgroup (n＝50) received laparotomy only. Sacrifices weremade 6 h, 12 h, 24 h and 48 h later in ANP and normalcontrol group after induction respectively. Intestinal mucosalpermeability was studied by intrajejunal injection of 1.SmCiradioactive isotope 99mTc-diethlene triamine pentacetic acid(DTPA) and the radioactivity of 99mTc-DTPA content in urinewas measured 6 h, 12 h, 24 h and 48 h after induction.Then the pancreas and intestine were prepared forpathology. Reverse transcription polymerase chain reaction(RT-PCR) was used to determine the mRNA expression ofNK-1R and NK-2R, and Western blot was used to investigatethe protein level of NK-1R and NK-2R.RESULTS: In ANP rats, serious histologic damages inintestinal mucosa were observed, and the radioactivity of99mTc-DTPA in urine increased significantly in the ANP group.RT-PCR revealed that NK-1R and NK-2R mRNA level wasoverexpressed in the distal ileum of ANP as compared withthe normal control group. Western blot discovered strongerNK-1R (14-fold increase) and NK-2R (9-fold increase)immunoreactivity in the intestinal mucosa of ANP rats.Moreover, the overexpression of NK-1R was associated withmucosal pathological score (r＝0.77,P＜0.01) and intestinalpermeability (r＝0.68, P＜0.01) in ANP rats.CONCLUSION: NK-1R and NK-2R contribute to disruptedneuropeptides loop balance, deteriorate intestinal damage,and are involved in pathophysiological changes in ANP.

Gene-expression analysis of single cells-nested polymerase chain reaction after laser microdissection

AIM: The structural and functional characteristics of cells are dependent on the specific gene expression profile. The ability to study and compare gene expression at the cellular level will therefore provide valuable insights into cell physiology and pathophysiology.METHODS: Individual cells were isolated from frozen colon tissue sections using laser microdissection. DNA as well as RNA were extracted, and total RNA was reversely transcribed to complementary DNA (cDNA). Both DNA and cDNA were analyzed by nested polymerase chain reaction (PCR). The quality of isolated DNA and RNA was satisfactory.RESULTS: Single cells were successfully microdissected using an ultraviolet laser micromanipulator. Nested PCR amplification products of DNA and cDNA of single cells could clearly be visualized by agarose gel electrophoresis.CONCLUSION: The combined use of laser microdissection and nested-PCR provides an opportunity to analyze gene expression in single cells. This method allows the analysis and identification of specific genes which are in volved inphysiological and pathophysiological processes in a complex of variable cell phenotypes.