| As the most mature organ transplantation, kidney transplantation has become the preferred renal replacement therapy in the last15years, which is better than dialysis treatment in the survival time and survival quality and economic efficiency ratio. However, the shortage of donor organs severely limited further expanding the size of China’s kidney transplant. So to expand the sources of donor kidney is the first topic for further development of China and the global renal transplantation. The brain-dead donor organ donation has tremendous significance for the development of kidney transplantation in China. Currently, a large number of organs of brain-dead patients is not utilized. About13million people died in a car accident each year, a much larger number of patients in a state of brain death in the hospital due to a variety of diseases. Therefore, the kidney transplant from brain-dead donor is important research directions in the field of organ transplantation in China, which has a broad application prospects.As complex pathological process, the injury mechanism of kidney from brain-dead donor has not been fully understood. Brain death can cause some change in kidney tissue, such as microcirculation and energy metabolism, aggravation of the inflammatory response, enhance immune status of the complement system activation and oxidative stress injury, leading to apoptosis, renal ultrastructure and histopathology of structural damage and change its function. Brain death systemic inflammatory response, ischemia and reperfusion for hemodynamic instability, both of which are important mechanism of kidney damage. It has important significance for assessment for kidney quality to explore the mechanism of the pathological process of inflammatory response and ischemia-reperfusion injury of the kidney in the state of brain death, which could predict the efficacy of kidney transplantation as well as guidance targeting protection treatment. In this study, through the establishment of rat brain death model assessment of brain death on renal injury, and dynamic observation of the process of brain death in the oxidative stress injury, inflammatory response and its related signal pathways, to make sure the treatment research experimental basis of brain death donor kidney transplantation of relevant clinical diagnosis. On the basis of this study, this research is also the first Chinese herbal medicine plant extracts-icariin and specificity of MAPKs signal pathway inhibitors SB203580treatment of brain death donor kidney transplant recipients.The absence of the national organ donation system has led to a serious shortage of organs for transplantation. On the basis of animal studies, it is imperative to establish a national level organ donation system. Jointly by the Ministry of Health in2010, the Red Cross Society of China decided to provincial Red Cross units to carry out cardiac death organ donation. Henan Province Red Cross blood donation has extensive experience in hematopoietic stem cell donation and body donation to benefit tens of thousands of people over the years. It has branches in18cities in Henan, with hundreds of staff and thousands of volunteers. As a populous province, Henan, more than100million people, it is estimated that hundreds of thousands of end-stage organ failure patients need organ transplants. In order to alleviate the huge organ gap in the support of the Health Department of Henan Province, HRC began the exploration of the work of the organ donor pilot. The second part of the study summarizes the first batch of23cases of brain heart double death donation in Henan province.Part OneThe mechanism of kidney damage in brain dead ratObjective:TO explore the mechanism of kidney damage in brain dead state in rat Method:1. According to the literature, to establish the stable brain death model in the rat. Established according to our method a rat orthotopic kidney transplantation model. Fisher344rats were used as donor. Establish a rat model of brain death, to maintain brain death3h after following the above steps, cut the left kidney as donor organs. Lewis rats as kidney transplant recipients, Fisher344rats were used as donor orthotopic kidney transplantation.2. Before the induction of brain death (0h) and the induction of brain death after2h,4h,6h killed SD rats through the aorta peripheral blood at room temperature for30min and centrifuged (10OOOg,20min), draw the upper serum frozen for biochemical tests. After induction of brain death, the the rats were continuous monitored of blood pressure and heart rate. To collect all serum samples, determination of blood urea nitrogen and creatinine. Measured by ELISA of TNF-α, IL-1β, IL-6and other cell factor levels. CDNA prepared specimens using real-time PCR determination of renal tissue inflammatory cytokines (TNF-α,IL-1β,IL-6, IL-10), chemokines (MCP-1,MIP-2,the IP mRNA expression level of-10), transcription factors (NF-κB of ATF3,), and apoptosis-related genes (Bcl-2, Bax),3. Kit were used to measur tissue myeloperoxidase (MPO) activity (reflecting neutrophil infiltration), and malondialdehyde content. With hematoxylin-eosin (H&E) staining for histopathological examination, using an ordinary optical microscope at400times the light microscope, select10per field tubular scoring with light microscope, the degree of renal disease Paller’s standard score.The malondialdehyde Determination:thiobarbituric acid method.Results:1. After induction of brain death in SD rats, serum urea nitrogen and creatinine levels significantly increased in2h,4h,6h, with a statistically significant difference (p<0.05) compared with0h. And extend as the time of brain death, blood urea nitrogen and serum creatinine levels gradually increased, compared with a statistically significant difference (p<0.05) between the point in time.2. ATF3mRNA in rat kidney tissue with the time of brain death gradually increased from2.6to4.2times, the highest in6h; of ATF3protein expression levels showed similar trends, increased to3.4times in6h; ERK, JNK and p38MAPK increase in the level of protein phosphorylation; p-ERK and reached the peak at4h,6h decreased; p-JNK at4h and6h level similar; p-p38highest at6h. STAT3protein phosphorylation level increases with the time of brain death in6h pSTAT3/STAT3ratio increased to6.5times; rat kidney tissue of proinflammatory cytokines IL-1β, TNF-α, IL-6, IL-10and other cytokines mRNA expression levels were significantly increased, and was a time-dependent trend within6h after brain death. Macrophage chemokine MCP-1, MIP-2, the IP-10mRNA expression in brain death4h after the more obvious increase peaked6h.3. Inflammatory transcription factor NF-κ B in the kidney tissue mRNA expression gradually increased, peaked around4h,6h decreased slightly. Detected by ELISA in serum levels of TNF-α,IL-1β, IL-6and other pro-inflammatory cytokines levels. IL-6levels were significantly increased. TNF-α levels showed no elevated; elevated levels of IL-1β is not obvious. The Bax/Bcl-2ratio renal tissue after brain death was significantly elevated,. Kidney tissue in the rat brain death state content of MPO and MDA level:Compared with the control group, after the death of the rat brain renal tissue MPO content was significantly higher; renal tissue MDA increased obviously.4. Histopathological examination showed6h after brain death the kidney presents significant pathological damage., Conclusion:1. A successful establishment of the the rats stabilized brain death model, and the model of rat orthotopic kidney transplantation.2. SD rats after induction of brain death and the induction of brain death for2h,4h,6h, blood urea nitrogen, creatinine levels significantly increased. With the extension of time of brain death, blood urea nitrogen and serum creatinine levels increased gradually.3. records oxidative stress injury in the process of brain death, inflammation and its associated signaling pathway data to provide experimental evidence for the brain-dead donor kidney transplantation clinical diagnosis and treatment.Part TwoThe protective effect of icariin and specific inhibitor of p38-MAPK in kidney injury from brain-dead donorObjective:To explore the protective effect oficariin and specific inhibitor of p38-MAPK in brain-dead donor kidney injury.Method:1. Using Fisher344rats to establish brain death model.2. Induction of brain death after intraperitoneal injection of icariin (100mg/kg/day) or p38-MAPK specific inhibitor-SB203580(1mg/kg/day), or both combined.3. Maintain brain death after3h, the rats were sacrificed in accordance with the donor kidney preparation procedure to remove the kidney as a donor.4. Build the kidney transplantation model in rat.5. The day of surgery (day0) and after a week give icariin daily (100mg/kg/day) or SB203580(1mg/kg/day). Experimental groups and dosing regimens. Observed in each group graft survival time detection laboratory indicators. Results:1. After treatment of Icariin and SB203580, the brain-dead donor kidney transplant graft survival time (median) from31.5days,36days to53.5days,52.5days; after combination treatment, graft survival time (median) extended from35days to97.5days,50%of renal allograft survival over100days.2. Ten days after surgery, icariin group, SB203580group and combined treatment group graft function decreased compared with the control group, but no significant difference (p>0.05).3. Organization in accordance with the the Banff grading criteria for brain death kidney pathological changes were scored in each group from each of three rats. Compared with the control group, treated with icariin or SB203580, vasculitis alleviate the most obvious, tubular atrophy also improved. Vasculitis and tubular atrophy is the most obvious improvement in the combined treatment group.Conclusion:1. Icariin can significantly prolong the brain-dead donor kidney transplant graft survival time, and SB203580can also significantly extend the brain-dead donor kidney transplant graft survival time.2. graft survival time was significantly longer in joint application of icariin and SB203580in brain-dead donor kidney transplant.Part ThreeThe23Cases of organ donation after brain and heart death in Henan Province,Objective:To summary of donation after brain death and cardiac death (DBCD) in Henan Province,, explore Chinese organ standardized donation mode.Method:Uniting the Red Cross and the Health Department to establish the Office of organ donation, using Chinese type3death standard (brain-heart double death), the organization was established with reference to the relevant national organizational structure. Red Cross staff coordination members were the team as the core, and cardiac death organ donation guidelines was for action standards. We carry out human organ donation in Henan Province.Results:1.Since July1,2010to2012, the office received more than1,000copies of Application for organ donation. Received a total of95cases of the application of the standards-compliant. The potential for primary disease mainly traffic accident trauma (51/95,53.7%), and other accidental trauma (17/95,17.9%), spontaneous intracerebral hemorrhage (15/95,15.8%), and cranial tumors (5/95,5.3%) and other (6/95,6.3%); informed of the main ways of organ donation coordinator informed (69/95,72.6%) and by the media or publicity materials (26/95,27.4%). Success Stories72cases, the reasons include the families go back (32/72,44.4%), coordination problems (15/72,20.8%), family members of opposition (9/72,12.5%), does not meet the brain death (7/72,9.7%), organ failure (6/72,8.3%) and for the death of (3/72,4.2%).2.Successful implementation of DCD organs donated23cases (23/95,24.2%) and the remains of the cornea donated two cases, a total of18donated liver,44kidney, a pancreas, cornea,16diverted to six transplant centers, allThe operations were successfully completed. Primary disease was23cases successfully donated cases of traumatic brain injury (60%);3.Our hospital completed the DCD kidneys transplanted23cases, surgery is successfully completed, the immunosuppressive regimen after all he tacrolimus, MMF combined with prednisone, patients transplanted kidney rupture bleeding, kidney transplant surgery again,15days after the patient died in DIC; kidney transplant recipients with acute rejection reaction rate of4/23, a kidney transplant the delayed reply incidence of7/23.Conclusion:In accordance with the relevant provisions in China, we explore the establishment of a complete system of organ donation in Henan, which is fair, transparent, harmless and feasibility. More organ failure patients will benefit from this system. |
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