發(fā)布時間：2018-06-30 20:24

  本文選題：低能量沖擊波 + 骨髓間充質干細胞��； 參考：《第二軍醫(yī)大學》2017年博士論文

【摘要】：背景:沖擊波屬于機械波,做為無創(chuàng)物理治療方法已經應用臨床30余年。從最初的體外碎石治療,再發(fā)展到骨不連、疼痛治療,特別是在上世紀發(fā)現(xiàn)低能量沖擊波對于慢性缺血性疾病的改善治療作用,使得沖擊波的應用范圍越來越廣闊。慢性腎臟疾病是我國的常見病多發(fā)病,我國約有1.2億慢性腎臟疾病患者,然而對于絕大多數(shù)的腎臟疾病,特別是慢性腎功能不全目前還缺乏有效的治療手段。腎臟局部微循環(huán)障礙、小管間質纖維化在慢性腎臟疾病發(fā)生發(fā)展中起至關重要的作用。低能量沖擊波治療作為一種新型治療手段是否也能對慢性腎功能不全有效目前尚未有報道。干細胞治療技術是目前醫(yī)學研究的熱點,近年來關于干細胞治療慢性腎臟疾病基礎及臨床研究有大量報道,干細胞治療業(yè)已被證實在急性腎損傷、腎小管損傷修復、缺血性腎臟病、腎間質纖維化防治方面有一定療效。在體外實驗中低能量沖擊波曾有報道可以增強干細胞的分化和遷徙能力,那么在體內是否也能增強干細胞治療的作用,目前尚未有相關文獻報道。為了證實以上的假說,我們選取慢性環(huán)孢霉素腎病模型作為治療對象,環(huán)孢霉素是腎臟病領域治療的常見藥物,主要用于腎移植排斥、腎病綜合征、自身免疫系統(tǒng)疾病等的治療,但其副作用是可能引起慢性環(huán)孢素腎病,其主要病理特征有入球小動脈透明樣變性、灶狀和條帶狀腎間質纖維化、腎小管萎縮、炎性細胞浸潤。缺血性損害是其腎毒性的主要機制,而這也正是低能量沖擊波和骨髓間充質干細胞治療的長處所在。為此在本研究中我們選取環(huán)孢素腎病模型,使用低能量沖擊波和骨髓間充質干細胞進行干預,觀察低能量沖擊波、干細胞技術是否對慢性腎臟病有治療效果,同時觀察低能量沖擊波、干細胞技術間是否有協(xié)同作用,探索低能量沖擊波應用于慢性腎臟病治療的可行性以及其與干細胞共同應用的前景。研究目的:觀察低能量沖擊波、干細胞治療技術是否對慢性環(huán)孢素腎病具有治療作用,兩者間能否有協(xié)同作用,并初步探討其機制。研究方法:1.使用不同能量級(6、8、10、12KV)的沖擊波干預正常SD大鼠,動態(tài)監(jiān)測腎臟功能(血肌酐、尿素氮)及大鼠生長情況變化,28d切取腎臟進行組織形態(tài)學評估,同時測定部分細胞因子的表達水平,為下一步實驗選取合適的沖擊波能量級。2.實驗大鼠分成5組:c組:正常對照組;h組:模型組;l1、l2、l5組:8KV沖擊波干預組,分別于第7、14,21天給予沖擊波治療,l1組(每次左右腎各100次)、l2組(每次左右腎各沖擊200次)、l5組(每次左右腎各沖擊500次);,動態(tài)監(jiān)測腎臟功能(血肌酐、血尿素氮)及大鼠生長情況變化,28d切取腎臟進行組織形態(tài)學評估,同時測定部分細胞因子(α-SMA、TGF-β_1、NF-κB、VEGF)的表達水平。3.實驗大鼠分成5組:c組:正常對照組;h組:模型組;l組:低能沖擊波干預組;b組:干細胞治療組;bl組:低能沖擊波聯(lián)合干細胞治療組,動態(tài)監(jiān)測腎臟功能(血肌酐、血尿素氮)及大鼠生長情況變化,28d切取腎臟進行組織形態(tài)學評估,同時測定部分細胞因子(α-SMA、TGF-β_1、NF-κB、VEGF)的表達水平。結果:1.與正常對照組比較,低能量沖擊波治療組(6KV、8KV)在腎組織形態(tài)上無明顯改變,10KV組的腎臟組織中可見到少量的腎臟淤血情況,12KV干預組大鼠皮膚可見瘀斑,腎臟表面也可見到有散在的出血點,腎組織內有較多的腎臟淤血情況,腎小管可以見到濁腫發(fā)生。腎功能和體重情況,與正常對照組比較,不同能量級沖擊波干預組的血肌酐、血尿素氮、體重變化趨勢無明顯差別,差異不具有統(tǒng)計學意義。腎間質纖維化(RIF)評分,與正常對照組比較,低能量沖擊波治療組(6KV、8KV)腎間質纖維化評分無明顯差別,10KV和12KV的RIF評分較正常組升高,差異具有顯著性。2.與c組比較,h組與沖擊波治療組(l1、l2、l5組)在體重、血肌酐、血尿素氮水平均顯著升高,差異具有統(tǒng)計學意義。與h組相對比,l1、l2組血肌酐、血尿素氮水平有明顯下降,體重增加。但是l5血肌酐、尿素氮、體重水平與h組大鼠無顯著差異。腎組織形態(tài)上,模型組與治療組都出現(xiàn)了明顯的腎臟損害,RIF評分均顯著升高。與h組比較,l1、l2組RIF評分顯著下降,但是l5組與h組之間比較RIF評分高于模型組,差異有統(tǒng)計學意義。在機制初步探索上,與腎臟病進展密切相關的因子α-SMA、TGF-β1、NF-κB、VEGF在模型組和干預組之中表達均明顯上升。與H組比較,L1、L2組的上述因子表達程度有所下降,而L5組大鼠的腎臟上述因子表達水平比模型組大鼠顯著升高,差異有統(tǒng)計學意義。3.與C組比較,H組與治療組(L、B、BL組)的血肌酐、尿素氮水平均顯著升高,體重顯著下降。與H組比較,L、B、BL組對大鼠的血肌酐、尿素氮水平有明顯下降,體重增加。腎組織形態(tài)上,模型組與治療組都出現(xiàn)了明顯的腎臟損害,RIF評分均顯著升高;與H組比較,L、B、BL組RIF評分顯著下降。細胞因子表達上α-SMA、TGF-β1、NF-κB、VEGF在模型組和治療組之中均明顯上升。但是BL組與B組比較,在血肌酐、尿素氮水平、體重、RIF評分、細胞因子表達水平上均無顯著差異。結論:適量的低能量沖擊波治療和骨髓間充質干細胞治療對慢性環(huán)孢素腎病大鼠有改善作用,兩者間在體內未發(fā)現(xiàn)協(xié)同作用,兩者改善腎功能的機理仍需進一步的研究。
[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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R692

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2 葉，

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