Protective Effects Brought By Transplantations Of Bone Marrow Stromal Stem Cells Based On Ischemic Postconditioning Upon Ischemia Reperfusion Injury Of Pulmonary Allograft In Rats

Part1.The Improvements in Establishment of Rat Orthotopic Left Lung Transplantation Model.Objective To establish rat orthotopic left lung allograft transplantation model by improved techniques. Methods This study improved the traditional cuff technique in many aspects, including graft retrieval, cuff self-making, recipient pneumonoresection,"muff-like"vessel and trachea anastomosis techniques. In a group with 30 pairs of clean-grade SD rats, the left lung allograft was stored at 4 oC LPDG solution for 3 h, and then the left lung was transplanted into the recipient rat without microscope. When the transplanted lung had been reperfused for 4 h. Airway pressure, blood gas analysis were detected before and after the right hilus of lung was block up, then left lung was removed, and W/D, histological changes under microscopy were determined. The reproducible ability of this model about ischemic- reperfusion injury of donor lung was evaluated. Results 30 pairs of rats receiving transplantation were performed the anastomosis of pulmonary artery, pulmonary vein and bronchus by single person without microscope. The time consuming was (5±1)min, (9±4)min(,2±0.5)minutes respectively. The total operational time consuming was (59±10)min. The operation successful rate was 90%, survival rate arrived 100%. The experimental outcome demonstrated that the model could duplicated the change of the ischemia-reperfusion injury. Conclusion The merit of this model corresponds with the construction of rat lung transplantation model. All the manipulations were performed by single person without microscope. The harvesting of donor lung was so fast that ischemic time was nearly 0 min. The cuff structure and the anastomosis technique were simpler than before. The improved"muff-like"technique increased the successful rate and shortened the total operational time consuming. This model duplicated the changes of ischemia reperfusion injury on lung allograft, and was demonstrated to be an ideal and suitable model for some researches just like IRI of donor lung .Part2.BMSCs Were Isolated, Cultivated, Identified and Marked,and Its Protective Effects on Ischemia-reperfusion Injury of Pulmonary Allograft in RatsObjective Bone marrow stromal stem cells were isolated, cultivated, identified and marked,and its protective effects on ischemia-reperfusion injury of pulmonary allograft in rats and explore the mechanisms. Methods A left lung transplantation formwork was constructed by using SD rat both donor and recipient by improved muff-like technique. Three groups of rats were examined. The donors in group 1 were sham operated,the donors in group 2 sufferd from ischemia-reperfusion injury,the donors in group 3 were received BMSCs .Each group have seven recipient. The donor lungs were all ex vivo reperfused by 4oC LPDG through pulmonary vein . After the left lung allograft was reperfused for 4 hours, we blocked the hilum of right lung, then measured airway pressure and made arterial blood gas analysis to measure PaO2 and PaCO2 .At last, rat was executed. Lung allograft tissue was harvested to be detected. W/D ratio, superoxide dismutase(SOD) activity, malondialdehyde (MDA) contents and myeloperoxidase(MPO) activity were detected. Graft pathologic histology was examined under light microscope. The message of expression of VEGF in donor lung tissue was measured by Real-time PCR and Western-blot. The content of TNF- a and IL-10 were quantified by ELISA. The expression of TNF-a,and bcl-2 were detected by immunohistochemical staining. Apoptotic cell death was determined by TUNEL. Results Compared with the sham operation group , the airway pressure, PaO2 ,the activity of SOD decreased and W/D ratios, content of MDA, activity of MPO increased(P<0.01)in the ischemia-reperfusion group,which demonstrated ischemia-reperfusion injury do occurred in the ischemia-reperfusion group. Compared with the ischemia-reperfusion group, the airway pressure, PaO2 ,the activity of SOD increased and W/D ratios, content of MDA, activity of MPO decreased(P<0.01)in the ischemia-reperfusion group. In the group of BMSCs, tissue edema, interstitial inflammation and exudation alleviated under light microscope, lower expression of TNF- a and higher expression of IL-10 were detected by ELISA(P<0.01), lower expression of TNF- a and higher expression of bcl-2 were detected by immunohistochemistry(P<0.01),Less apoptotic cells were found by TUNEL(P<0.01). Paracrine secretion cytokine of VEGF in the group of BMSCs was obviously higher than the sham operation group and the ischemia-reperfusion group (P<0.01) . Conclusion (1)Compared with the sham operation group , ischemia-reperfusion injury do occurred in the ischemia-reperfusion group.The transplantations of BMSCs reduced ischemic-reperfusion injury of lung allograft. (2) BMSCs reduced ischemic-reperfusion injury of lung allograft through means of anti-oxidative, anti-inflammatory and anti- apoptosis, paracrine secretion effect.So the transplantations of BMSCs may become a beneficial try which can prevent and reduce the IRI of lung allografts.Part3.Protective Effects Brought by Transplantations of Bone Marrow Stromal Stem Cells Based on Ischemic Postconditioning upon Ischemia- reperfusion Injury of Pulmonary Allograft in RatsObjective To investigate protective effects brought by transplantations of bone marrow stromal stem cells based on ischemic postconditioning upon ischemia-reperfusion injury of pulmonary allograft in rats and explore the mechanisms. Methods A left lung transplantation formwork was constructed by using SD rat both donor and recipient with improved muff-like techniques. Methods A left lung transplantation formwork was constructed by using SD rat both donor and recipient with improved muff-like techniques. The experiment was divided into four groups: In group A,open pulmonary blood flow directly to replicate ischemia-reperfusion injury . In group B, the donor lung suffered ischemic postconditioning before IRI. In group C, administration of BMSCs after IRI. In group D, administration of BMSCs after ischemic postconditioning. Each group have fourteen recipient. Then the left lung orthotopic transplantation was performed. After the left lung allograft was reperfused for 4 hours and 72 hours, extract seven rats randomly .,we blocked the hilum of right lung, then measured airway pressure and made arterial blood gas analysis to measure PaO2 and PaCO2 . At last, rat was executed. Lung allograft tissue was harvested to be detected W/D ratio, superoxide dismutase (SOD) activity, malondialdehyde (MDA) contents and myeloperoxidase(MPO) activity. Graft pathologic histology was examined under light microscope. The message of expression of VEGF in donor lung tissue was measured by Real-time PCR and Western-blot. The content of TNF- a , IL-10, G-CSF and GM-CSF were quantified by ELISA. The expression of TNF-a,and bcl-2 were detected by immunohistochemical staining. Apoptotic cell death was determined by TUNEL. Localization of BMSCs was detected through immumofluorescence method. Results Compared with group A and B, paracrine secretion cytokine of VEGF which detected by Real-time PCR and Western-blot was obviously increased in group C and D,so the G-CSF,GM-CSF detected by ELISA. Compared with group A , the airway pressure, PaO2 ,the activity of SOD decreased and W/D ratios, content of MDA, activity of MPO increased(P<0.01)in group B,C and D.TNF- a decreased ,IL-10 increased(P<0.01). Lower expression of TNF- a and higher expression of bcl-2 were detected by immunohistochemistry(P<0.01),Less apoptotic cells were found by TUNEL(P<0.01). In group D, tissue edema, interstitial inflammation and exudation alleviated significantly under light microscope. Through immunofluorescence method,we detected BMSCs were located in lung tissue after 72 hours. Conclusion Protective effects brought by transplantations of bone marrow stromal stem cells based on ischemic postconditioning upon ischemia-reperfusion injury of pulmonary allograft in rats were significant.Its mechanisms may through means of anti-oxidative, anti-inflammatory and anti- apoptosis, paracrine secretion effect and homing of BMSCs.Reduce ischemic-reperfusion injury of lung allograft by ischemic postconditioning firstly,and then administration of BMSCs may improve the microenvironment of which BMSCs survived. This study demonstrated that the transplantations of bone marrow stromal stem cells based on ischemic postconditioning upon ischemia-reperfusion injury of pulmonary allograft in rats may become a new strategy, which can reduce the IRI of lung allografts.