The Research On The Effect Of INOS To Graft Motility And Rejection Of Rat Small Bowel Transplantation

The aim of this study is to investigate the effect of inducible nitric oxide synthase (iNOS) to graft motility and its feature in rejection in small bowel transplantation(SBT), evaluate the blocking effects of N6-(l-iminoethyl)-1-Lysine (L-NIL) and sinomenine(SIN) to iNOS.Part 1. The establishment of small bowel transplantation model inrat.Objective: To establish stable orthotopic and heterotopic small bowel transplantation model in rat. Methods: To establish rat SBT model with microsurgical technique. Results: 47 and 55 orthotopic/heterotopic SBT were individually performed with successful rates as 85.10% (40/47) and 87.27% (48/55). Conclusion: Stable rat SBT model could be established after shorter period of microsurgical training so as to make foundation for research on immune reaction and graft functions.PART 2. The study on the iNOS expression in isograft of ratHeterotopic SBTObjective: To investigate the iNOS expressions in isografts in different periods of heterotopic rat SBT. Methods: Heterotopic rat SBTs were divided into 8 subgroups as the followings: subgroup 1 (normal intestine without SBT), subgroup 2 (intestine in cold preservation), and subgroups 3, 4, 5, 6, 7 and 8 separately standing for post-operative Oh, Id, 2d, 3d, 4d, and 7d. The intestine or grafts were collected from each subgroup to be assayed for iNOS mRNA and protein expression status by RT-PCR and immonohistochemical staining then examined pathologically. Results 1. The pathological grading of inflammation in intestine with cold preservation was higher than it in normal intestine (p<0.05) and reached the peak on 1 POD (p<0.05), then getting down to almost normal level on 7POD (p>0.05). 2. Few expression levels of iNOS mRNA and protein were noted in normal intestine, however these expressions in subgroup 2 rapidly up-regulated (p<0.05) and was highest on 1POD (p<0.05) followed by very quickly down-regulating to near normal value on 7POD (p>0.05). 3. The iNOS protein expressions were pathologically found in graft muscular layer and mucosa also. 4. In graft, the expression of iNOS mRNA was positive correlated with its protein expression (p<0.01). 5. The expression of iNOS in graft was positive correlated with the pathological grading of inflammatory injury (p<0.01). Conclusion: 1. The earlier high expressions of iNOS mRNA and protein following SBT appear the highest peak of iNOS expression and aspecific inflammatory reaction on 1 POD. 2. iNOS up regulating might be an important cause of earlier aspecific reaction post SBT. 3. The intramuscular cell nets, mucosa epithelial layer and crypt of graft may be the location to abnormally express iNOS. 4. The injuries to graft due to cold preservation, eschimic reperfusion and surgical procedure are the main reasons of high iNOS expression post SBT.PART3 The effect of iNOS to isograft motility in syngeneic OITObjective: To investigate the effect of iNOS to isograft motility in orthotopic total SBT. Methods: The investigating objects were divided into 4 subgroups as the followings: subgroup 1 (underwent duodenostomy only), subgroups 2 (underwent total OIT), subgroup 3 ( underwent total OIT and duodenostomy with L-NIL treatment) and subgroup 4 ( underwent underwent total OIT and duodenostomy with SIN treatment). Each subgroup consisted of 12 male Lewis rats in which 6 rats were randomly dedicated for pathological examination to observe the inflammatory injury gradings and iNOS mRNA/protein expressions with RT-PCR or immunohistchemistry on 1 POD (the peak period of iNOS expression), the other 6 were investigated for the intestinal transit function with dextran blue 2000 in vivo by observing this marker distribution and movement in small bowel cavity on 2 POD. Results: 1. Comparing with subgroup 1, the inflammatory injury score and iNOS expression levels were significantly higher (p<0.01), in contrast, these alterations above could be down-regulated with statistically change comparing with subgroup 2 (p<0.01), but no difference between subgroup 3 and subgroup 4 (p>0.05). 2. In subgroup 2 the value of geometry center (GC) of the marker distribution was higher companying with a shorter transit distance in grafts (p<0.01). The value of GC increased and transit distance of marker in grafts was lower after treatment either by L-NIL or SIN (p<0.01), however no difference between L-NIL and SIN was found.(p>0.05). 3. iNOS expressions were positively correlated with pathological grading of inflammatory injury (pO.Ol), and negative correlated with GC value of marker in graft ((p<0.01). Conclusion: 1. The high expressions of iNOS mRNA and protein earlier post SBT pathologically appear aspecific inflammatory reaction in graft and result in earlier dysfunction of motility in graft. 2. iNOS plays a important role in graft inflammatory reaction7and dysfunction lead by inflammation of graft. 3. L-NIL and SIN are able to inhibit the expression of iNOS mRNA and protein, alleviate inflammatory injury and reduce the earlier dysfunction of motility in graft after transplantation.PART4 The effect of iNOS to acute rejection in SBTObjective: To study the effect of iNOS to acute rejection in SBT. Methods: Establishing heterotopic rat SBT according to the divisions as the followings: subgroup 1 (isograft), subgroup 2 (allograft), subgroup 3 (allograft with L-NIL treatment) and subgroup 4 ( allograft with CsA treatment, 4mg/kg.d"1) . Except isograft (Lewis to Lewis) in subgroup 1, allografts (BN to Lewis) were performed in the other 3 subgroups. Grafts were collected for pathological examination to observe the inflammatory injury gradings and iNOS mRNA/protein expressions with RT-PCR or immunohistchemistry on 7 POD (the period of strongest rejection). Results: 1. iNOS expression levels significantly up-regulated in subgroup 2 after SBT and very low expression in subgroup 1 (p<0.01). 2. iNOS expressions were considerably lower in subgroups 3 and 4 than in subgroup 2 (p<0.01), but no difference between subgroups 3 and 4 (p>0.05). 3.On 7 POD, pathological evidence of severe rejection presented in subgroup 2, in contrast slight infiltration of inflammatory cells in graft were dedected in subgroup 1 on the same time. 4. Moderate rejection in subgroup 3 and mild rejection in subgroup 4 were found on 7 POD, and statistic differences were showed either between subgroups 3 and 2 or 3 and 4 (p<0.05). 5. The histological foundings in each subgroup were positively correlated with iNOS mRNA and protein expressions (p<0.01). Conclusion: 1. iNOS has promotion effect to rejection in SBT. 2. iNOS might be a earlier monitoring index in SBT rejection and reference of mediation or dosage adjustment of immune suppressions. 3. As an inhibitor ofiNOS, L-NIL can inhibit the producing of iNOS, its inhibiting efficiency to iNOS is similar to CsA, and may alleviate the rejection.Part5. The preliminary research on anti-rejection effect ofSinomenine in SBTObjective: Through observing SIN effect to inhibit iNOS expression to explore its effect to anti-rejection in SBT. Methods: Establishing heterotopic rat SBT according to the divisions as the folio wings: subgroup 1 ( iso graft), subgroup 2 (allograft), subgroup 3 (full dosage CsA treatment, 4mmg/kg/d"1), subgroup 4 (half dosage CsA treatment, 2mg/kg/d"'), subgroup 5 ( SIN treatment), and subgroup 6 ( SIN and half dosage CsA treatment). Except isograft (Lewis to Lewis) in subgroup 1, allografts (BN to Lewis) were performed in the other 5 subgroups. Grafts were collected for pathological examination to observe the inflammatory injury gradings and iNOS mRNA/protein expressions with RT-PCR or immunohistchemistry on 7 POD (the period of strongest rejection). Results: 1. Severe rejection occurred in subgroup 2 on 7 PODwith iNOS significantly up-regulated, however slight infiltration of inflammatory cells in the graft was found with very few expression of iNOS in subgroup 1 on the same time (p<0.01). 2. The rejections were obviously inhibited with significantly lower levels of iNOS expression in subgroup 3 and 6 (p<0.01), but no difference between these two subgroups (p>0.05). 3. Although iNOS expressions in subgroups 4 and 5 were lower than that in subgroup 2 companying with some abatement of rejection, significant difference was found comparing with subgroup 3 (p<0.05). Conclusion: 1. Sinomenine may inhibit the iNOS expressions and partially alleviate rejection in SBT. 2. CsA can not be completely replaced by Single...