低能量冲击波和骨髓间充质干细胞对慢性环孢素肾病大鼠的治疗研究

发布时间：2018-06-30 20:24

本文选题：低能量冲击波 + 骨髓间充质干细胞　；参考：《第二军医大学》2017年博士论文

【摘要】：背景:冲击波属于机械波,做为无创物理治疗方法已经应用临床30余年。从最初的体外碎石治疗,再发展到骨不连、疼痛治疗,特别是在上世纪发现低能量冲击波对于慢性缺血性疾病的改善治疗作用,使得冲击波的应用范围越来越广阔。慢性肾脏疾病是我国的常见病多发病,我国约有1.2亿慢性肾脏疾病患者,然而对于绝大多数的肾脏疾病,特别是慢性肾功能不全目前还缺乏有效的治疗手段。肾脏局部微循环障碍、小管间质纤维化在慢性肾脏疾病发生发展中起至关重要的作用。低能量冲击波治疗作为一种新型治疗手段是否也能对慢性肾功能不全有效目前尚未有报道。干细胞治疗技术是目前医学研究的热点,近年来关于干细胞治疗慢性肾脏疾病基础及临床研究有大量报道,干细胞治疗业已被证实在急性肾损伤、肾小管损伤修复、缺血性肾脏病、肾间质纤维化防治方面有一定疗效。在体外实验中低能量冲击波曾有报道可以增强干细胞的分化和迁徙能力,那么在体内是否也能增强干细胞治疗的作用,目前尚未有相关文献报道。为了证实以上的假说,我们选取慢性环孢霉素肾病模型作为治疗对象,环孢霉素是肾脏病领域治疗的常见药物,主要用于肾移植排斥、肾病综合征、自身免疫系统疾病等的治疗,但其副作用是可能引起慢性环孢素肾病,其主要病理特征有入球小动脉透明样变性、灶状和条带状肾间质纤维化、肾小管萎缩、炎性细胞浸润。缺血性损害是其肾毒性的主要机制,而这也正是低能量冲击波和骨髓间充质干细胞治疗的长处所在。为此在本研究中我们选取环孢素肾病模型,使用低能量冲击波和骨髓间充质干细胞进行干预,观察低能量冲击波、干细胞技术是否对慢性肾脏病有治疗效果,同时观察低能量冲击波、干细胞技术间是否有协同作用,探索低能量冲击波应用于慢性肾脏病治疗的可行性以及其与干细胞共同应用的前景。研究目的:观察低能量冲击波、干细胞治疗技术是否对慢性环孢素肾病具有治疗作用,两者间能否有协同作用,并初步探讨其机制。研究方法:1.使用不同能量级(6、8、10、12KV)的冲击波干预正常SD大鼠,动态监测肾脏功能(血肌酐、尿素氮)及大鼠生长情况变化,28d切取肾脏进行组织形态学评估,同时测定部分细胞因子的表达水平,为下一步实验选取合适的冲击波能量级。2.实验大鼠分成5组:c组:正常对照组;h组:模型组;l1、l2、l5组:8KV冲击波干预组,分别于第7、14,21天给予冲击波治疗,l1组(每次左右肾各100次)、l2组(每次左右肾各冲击200次)、l5组(每次左右肾各冲击500次);,动态监测肾脏功能(血肌酐、血尿素氮)及大鼠生长情况变化,28d切取肾脏进行组织形态学评估,同时测定部分细胞因子(α-SMA、TGF-β_1、NF-κB、VEGF)的表达水平。3.实验大鼠分成5组:c组:正常对照组;h组:模型组;l组:低能冲击波干预组;b组:干细胞治疗组;bl组:低能冲击波联合干细胞治疗组,动态监测肾脏功能(血肌酐、血尿素氮)及大鼠生长情况变化,28d切取肾脏进行组织形态学评估,同时测定部分细胞因子(α-SMA、TGF-β_1、NF-κB、VEGF)的表达水平。结果:1.与正常对照组比较,低能量冲击波治疗组(6KV、8KV)在肾组织形态上无明显改变,10KV组的肾脏组织中可见到少量的肾脏淤血情况,12KV干预组大鼠皮肤可见瘀斑,肾脏表面也可见到有散在的出血点,肾组织内有较多的肾脏淤血情况,肾小管可以见到浊肿发生。肾功能和体重情况,与正常对照组比较,不同能量级冲击波干预组的血肌酐、血尿素氮、体重变化趋势无明显差别,差异不具有统计学意义。肾间质纤维化(RIF)评分,与正常对照组比较,低能量冲击波治疗组(6KV、8KV)肾间质纤维化评分无明显差别,10KV和12KV的RIF评分较正常组升高,差异具有显著性。2.与c组比较,h组与冲击波治疗组(l1、l2、l5组)在体重、血肌酐、血尿素氮水平均显著升高,差异具有统计学意义。与h组相对比,l1、l2组血肌酐、血尿素氮水平有明显下降,体重增加。但是l5血肌酐、尿素氮、体重水平与h组大鼠无显著差异。肾组织形态上,模型组与治疗组都出现了明显的肾脏损害,RIF评分均显著升高。与h组比较,l1、l2组RIF评分显著下降,但是l5组与h组之间比较RIF评分高于模型组,差异有统计学意义。在机制初步探索上,与肾脏病进展密切相关的因子α-SMA、TGF-β1、NF-κB、VEGF在模型组和干预组之中表达均明显上升。与H组比较,L1、L2组的上述因子表达程度有所下降,而L5组大鼠的肾脏上述因子表达水平比模型组大鼠显著升高,差异有统计学意义。3.与C组比较,H组与治疗组(L、B、BL组)的血肌酐、尿素氮水平均显著升高,体重显著下降。与H组比较,L、B、BL组对大鼠的血肌酐、尿素氮水平有明显下降,体重增加。肾组织形态上,模型组与治疗组都出现了明显的肾脏损害,RIF评分均显著升高;与H组比较,L、B、BL组RIF评分显著下降。细胞因子表达上α-SMA、TGF-β1、NF-κB、VEGF在模型组和治疗组之中均明显上升。但是BL组与B组比较,在血肌酐、尿素氮水平、体重、RIF评分、细胞因子表达水平上均无显著差异。结论:适量的低能量冲击波治疗和骨髓间充质干细胞治疗对慢性环孢素肾病大鼠有改善作用,两者间在体内未发现协同作用,两者改善肾功能的机理仍需进一步的研究。
[Abstract]:Background: shock wave is a mechanical wave. It has been used as a noninvasive physical therapy for more than 30 years. From the original extracorporeal lithotripsy treatment to bone nonunion and pain treatment, especially in the last century, it was found that low energy shock waves were used to improve the treatment of chronic ischemic diseases, which made the application of shock waves wider and wider. There are about 120 million chronic renal diseases in our country. There are about 120 million patients with chronic kidney disease in our country. However, most renal diseases, especially chronic renal insufficiency, are still lack of effective treatment. The local microcirculation disorder of the kidney and tubulointerstitial fibrosis are important in the development of chronic renal disease. Low energy shock wave therapy is not yet reported as a new therapy for chronic renal insufficiency. Stem cell therapy is a hot spot in medical research. In recent years, the basic and clinical research of stem cell therapy for chronic kidney disease has been reported. Stem cell therapy has been proved to be urgent. There is a certain effect on renal injury, renal tubular injury repair, ischemic kidney disease, and renal interstitial fibrosis. In vitro, low energy shock waves have been reported to enhance the differentiation and migration of stem cells, and whether the role of stem cell therapy can be enhanced in the body. The above hypothesis, we choose chronic cyclosporin nephropathy model as an object of treatment, cyclosporin is a common drug in the field of kidney disease, mainly used in the treatment of renal transplant rejection, nephrotic syndrome, and autoimmune disease, but its side effect is the possible cause of slow cyclosporin nephropathy, its main pathological feature is a small ball movement. Hyaline degeneration, focal and banded renal interstitial fibrosis, renal tubules atrophy, and inflammatory cell infiltration. Ischemic damage is the main mechanism of its renal toxicity, and this is the advantage of the low energy shock wave and bone marrow mesenchymal stem cells. In this study we selected cyclosporine nephropathy model and use low energy shock wave. Intervention with bone marrow mesenchymal stem cells to observe the effects of low energy shock wave, stem cell technology on chronic kidney disease, and whether there is a synergistic effect between low energy shock wave and stem cell technology, explore the feasibility of low energy shock wave for chronic kidney disease and the prospect of CO application with stem cells. The purpose of this study is to observe the therapeutic effect of low energy shock wave and stem cell therapy on chronic cyclosporin nephropathy, whether there is a synergistic effect between them and explore its mechanism. 1. the methods of study: 1. the normal SD rats were interfered with different energy levels (6,8,10,12KV), and the dynamic monitoring of renal function (blood creatinine, urea nitrogen) and large amount of renal function (blood creatinine, urea nitrogen) The rat growth was changed. 28d was cut into the kidney for histomorphological evaluation, and the expression level of some cytokines was measured. The next experiment was divided into 5 groups: the C group: the normal control group, the H group, the model group, the L1, L2, L5 group: the 8KV shock wave intervention group, which were given the impact wave treatment at day 7,14,21, respectively. Treatment, group L1 (100 times of kidney each time), group L2 (200 times of renal impact each time), group L5 (500 times each kidney), and the dynamic monitoring of renal function (blood creatinine, blood urea nitrogen) and rats' growth changes. 28d was taken for histomorphological evaluation of kidney, and a table of some cytokines (alpha -SMA, TGF- beta _1, NF- kappa B, VEGF) was measured. .3. rats were divided into 5 groups: Group C: normal control group; group H: model group; group L: low-energy shock wave intervention group; B group: stem cell therapy group; BL group: low-energy shock wave combined with stem cell therapy group, dynamic monitoring of renal function (blood creatinine, blood urea nitrogen) and rat growth, 28d cut kidney for histomorphological assessment, simultaneous measurement of kidney, simultaneous measurement of kidney, simultaneous measurement and measurement of kidney, simultaneous measurement and measurement of kidney, simultaneous measurement of kidney The expression level of partial cytokine (alpha -SMA, TGF- beta _1, NF- kappa B, VEGF). Results: 1. compared with the normal control group, there was no significant change in the renal tissue morphology of the low energy shock wave group (6KV, 8KV), and a small amount of renal congestion in the renal tissue of the 10KV group. The skin of the 12KV intervention group was seen in the skin of the rats and the surface of the kidney was also visible. There were scattered bleeding points, more renal blood stasis in renal tissue, renal tubules can be seen cloudy swelling, renal function and body weight, compared with the normal control group, there was no significant difference in blood creatinine, blood urea nitrogen and body weight in different energy level shock wave intervention group, and the difference was not statistically significant. RIF evaluation was not significant. Compared with the normal control group, there was no significant difference in the renal interstitial fibrosis score of the low energy shock wave therapy group (6KV, 8KV). The RIF score of 10KV and 12KV was higher than that of the normal group. The difference was significant between the.2. and the C group. The difference was statistically significant between the H group and the shock wave group (L1, L2, L5 group) in the body weight, the blood creatinine and the blood urea nitrogen water. Significance. Compared with group H, L1, L2 group blood creatinine, blood urea nitrogen level obviously decreased, weight increased. But L5 blood creatinine, urea nitrogen, body weight level had no significant difference with the H group. The renal tissue morphology, both the model group and the treatment group had obvious renal damage, and the RIF score increased significantly. Compared with the H group, the RIF score of L1 and L2 group was significantly lower. But the RIF score of L5 group and H group was higher than that of the model group, and the difference was statistically significant. In the preliminary exploration of the mechanism, the expression of factor alpha -SMA, TGF- beta 1, NF- kappa B, VEGF in the model group and the intervention group were obviously increased. Compared with the H group, the expression degree of the above-mentioned factors in L1 and L2 group decreased, while L5 group was larger. The expression level of the above factors in the rat kidney was significantly higher than that in the model group. The difference was statistically significant between the.3. and the C group. The blood creatinine in the H group and the treatment group (L, B, BL group) increased significantly and the body weight decreased significantly. Compared with the H group, the serum creatinine and the urea nitrogen level of the rats were significantly decreased, and the body weight was increased. The body weight was increased. The renal tissue shape was increased. In the model group, both the model group and the treatment group had obvious renal damage, and the RIF score increased significantly. Compared with the H group, the RIF scores in the L, B and BL groups decreased significantly. The expression of alpha, TGF- beta 1, NF- kappa B, VEGF in the model group and the treatment group were significantly higher than that in the H group. There is no significant difference in the expression level of cytokine. Conclusion: a moderate amount of low energy shock wave therapy and bone marrow mesenchymal stem cell therapy can improve the chronic cyclosporin nephropathy, and there is no synergistic effect between the two. The mechanism of improving renal function still needs further study.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R692

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