Intractable Constipation: Clinical Diagnosis, Treatment And Study Of Aquaporin Gene Expression

Constipation is a very common disorder or disease of digestive tract. Nowadays following the changes of diet habits and the more psychosocial press on modern people the prevalence of constipation is as high as 15-20%. What s more, the intractable constipation(IC) have dramatically cut down the quality of the patients life. Great interests have been focused on the diagnosis and therapy of this disease. From the functionl changes to substantiality lesions of constipation; from laxative abuse to the Practice Guidelines for Chronic Constipation of China published; from conservative therapy to operative intervention for the treatment of constipation; from gastrointestinal hormones, neural transmitters, enteric nervous system to the study of cajal cells; the level of diagnosis and treatment and basic research for constipation in China is guadually increasing. But so far there are no reports about the etiopathogenisis and progression of constipation from the view about the changes of water absortion and mucus secretion in large intestine. There is also no systemic and relative study about aquaporins (AQPs) in large intestine. The knowledge about etiology, clinical classification and treatment of IC is limited. So we analyzed the clinical data of the patients with IC treated in our hospital in resent years and summaried the features and then present our opion and experiences about this disease. At the same time we studied the expression of AQPs in colon and rectum and explored their roles in normal condition and in pathogenesis of constipation. Part 1 Diagnosis and treatment of intractable constipation and resultsof follow-upsIC is characterized by sustainned constipation and in some casescompanied by abdominal distention, pain, anorexia, emaciation, insomnia and morbid forgetfulness, ect which can't be improved by diet and life modification, and the patients defecate depending on laxatives and/or enema which even can not help sometimes. Patients are suffering and their lives and jobs are greatly destroyed. Knowing the clinical features and control methods of IC is important for improving the management of chronic constipation. In 2002, Chinese medical association digestive disease sub-association gastrointestinal dynamics team proposed Practice guidelines for chronic constipation of China (draft) which defined IC and pointed that surgery should be considered to this disease when having abnormal examination results and no relief after strict conservative therapy. In 2003 Practice guidelines for chronic constipation showed the same idea. But IC is an aggregation of several different kinds of chronic constipative conditions, it's classification and corresponding therapies are absent in the Draft and Guidelines. We studied the clinical data(including clinical features, diagnostic methods, operation and follow-up) of IC patients having received surgery in 2002~2004 combined with the review data of 1999~2002 in our hospital to explore the proper diagnostic and therapeutic regimen to IC. We found that: 1. Besides 00C, STC and MIX types, there is another type named idiopathetic chronic colonic pseudo—obstruction (ICCPO) in IC group; ICCPO is characterized by younger onset age, shorter disease course and frequently occurrence of intestinal obstruction, and operation (total colectomy) is the the best choice for the treatment of it; 2. Some STC can be cured by conservative therapy, the reason is the existence of colonic HAPC; 3. STC with pelvic floor hernia should receive subtotal colecomy together with pelvic floor restitution; 4.00C should resort to conservative therapy in more situations.Part 2 Expression of aquaporin mRNA in large colon mucus membraneAquaporin (AQP) exists widely in the biological world. With the main function as water channel it takes a great role in the transmembrane transportation of water. As to water secretion and absorption digestive tract is the most active place in human body, but the study about AQP in which especially in colon and rectum is rare. The main function of colon and rectum is to absorb the water and salinity in the residues of chyme and then put the stool in shape. At the same time colon and rectum can secret some mucus to protect mucous membrane and to push the stool out. So we suppose that there must be abundant expression of several AQPs in large intestine and maybe the changes of expressive mode takes part in the onset and development of chronic constipation.In our study , firstly we used semi-quantity RT-PCR to examine the mRNA level of AQP1, 2, 3, 4, 5, 6, 7, 8, 9 expression in the mucous membrane and submembrane tissues from normal colon and proximal part of large intestinal adenocarcinoma surgical specimen (both divided into right colon and left colon); then we compared the AQPs expression of IC patients to this and examine whether there is a difference between them. If there 1 - then we used fluorescence real-time RT-PCR to quantify the difference. Results showed that abundant expression of AQP1, 2, 3,4,5,7,8, 9mRNA( AQP2, 9:left colon more than right colon. AQP1, 3, 4, 8: right colon more than left colon) exists in the epithelium of large intestine. AQP9 dramaticly decreased in constipation. Fluorescence real-time RT-PCR technique confirmed the significant difference.Part 3 Expression of AQP-2^ AQP-9 protein in normal, constipation andcancer of colonic tissuesIn this part we analyzed the expression of AQP^ AQP-9 protein inthe normal, constipation and adenocarcimoma of colonic tissues using S-P immunohistochemistry. The results showed that AQP-2 expressed dorainantly in cublet cells (both the luminal wall and cryptae part)and little in absorptive cells of normal colon epithelium, mildly expressed in constipation and cancer tissues but the difference is not significant; no expression in adenocarcinoma; AQP9 strongly expressed in the basement of all the luminal cublet cells and part crypt cublet cells in normal tissues (strong positive), mildly expressed in constipation and the positive proportion of cublet cells decreased , no expression in adenocarcinoma. The results suggests that these two genes especially AQP-9 play a big role in synthesis and secretion of certain kind of mucus that deriving from cublet cells, which function maybe is necessary to maintain the normal situation of colon and rectum. Decreased expression of AQP9 affects the mucus secretion, which may participate in the pathogenesis and (or)development of constipation.Part 4 Expression of mRNA of MUC2 gene in normal and cancer of colonictissuesTo know the relationship between AQP9 and cublet cells , we selected Muc2 gene, the marker of mature cublet cells, as our object of further study. We used RT-PCR to examine mRNA expression level of AQP9 and Muc2 in the adenocarcinoma and proximal part of adenocarcimoma surgical specimen. The results showed there is certain relation between them. Preliminary analysis suggests: 1)AQP9 maybe the mucus secretion pathway of colonic cublet cells, which dose not express in adenocarcinoma since there is no normal mucus secretion; 2)besides mucus synthesis and secretion, AQP9 may also take a role in maintaining the normal differentiation of colonic epithelium.