MG53对重度烫伤小鼠肾脏保护作用的研究

发布时间：2018-05-18 15:10

本文选题：rhMG53 + PTRF　；参考：《第三军医大学》2013年硕士论文

【摘要】：内脏器官是机体各项生命活动的参与者和维持者。严重烧伤后，急性的缺血缺氧、炎症因子等因素，都会造成机体内脏器官的损伤。内脏器官受损，特别是多脏器功能损伤是烧伤病情加重、并发症发生以及死亡率增加的重要特征。加强重要内脏损伤的防治和促进受损脏器早日修复与重建是提高救治成功率和大幅度降低死亡率的关键。严重烧伤后，由于脏器本身存在肉眼难辨的缺血性损害，加之这类损伤范围广，累计器官多，发病机制复杂、隐蔽以及缺乏有效的促修复措施，因而其损伤后的修复常不易引起人们的重视[1]。既往采取的各种措施，常常仅是针对发病机制的某一环节或减轻其损害程度，并不直接作用于修复细胞本身，故缺乏对脏器的主动修复。因此，如何加速与促进受损内脏细胞的主动修复有可能为防治多器官功能障碍综合症(multipleorgan dysfunction syndrome, MODS)、多脏器功能衰竭(multiple system organ failure,MSOF)发生以及提高救治成功率提供新的有效途径，具体重要的理论意义和临床应用价值。 MG53(Mitsugumin53)，一个肌肉特有的TRIM家族蛋白，是细胞膜损伤修复的一个必不可少的组分。Ma等的研究表明，重组的人MG53(Recombinant HumanMitsugumin53，rhMG53)能够治疗肌营养不良症以及以细胞膜损伤为发病机理的其他人类疾病。当培养肌肉和非肌肉细胞的培养液中存在rhMG53时，rhMG53能够识别细胞膜损伤信号，进而提高这些细胞的膜修复能力。小鼠动物实验研究表明，通过各种途径(肌内、静脉、皮下)给予rhMG53，都能够提高骨骼肌细胞膜修复能力，同样也能一定程度上改善肌营养不良症的组织病理变化[2]。因此我们推测，机体严重烫伤后，通过外源性的rhMG53治疗，可能对烫伤造成的各种脏器损伤起到一定的修复作用，改善脏器功能，提高生存率。本研究以30%的Ⅲ度烫伤小鼠为模型，观察静脉注射rhMG53对重度烧伤小鼠生存率和脏器组织的保护作用的影响，及其可能的机制。研究内容和方法: 1. MG53对30%Ⅲ°烫伤小鼠死亡率影响及脏器病理改变的实验研究 1)小鼠Ⅲ°烧伤模型的制作使用YLS-Q5超级台式温控烫伤仪致伤Bal b/c小鼠，通过温度和时间两个因素控制烫伤深度。使用致伤温度为90℃，自重500g，烫头直接2cm恒温金属烫头垂直接触小鼠皮肤6s，8s，10s，经HE染色观察烧伤皮肤组织学改变，确定小鼠Ⅲ°烧伤模型的制作条件。 2)MG53对30%Ⅲ°烫伤小鼠死亡率影响及脏器病理改变的研究实验前分装外源性rhMG53蛋白。16只20g左右的雄性Bal b/c小鼠，随机分两组，为MG53实验组及BSA对照组，分别制作30%Ⅲ°烫伤面积，伤后予以腹腔注射2.5ml乳酸钠林格注射液，烫伤后0.5h，烫伤后6.5h，分别通过尾静脉注射外源性rhMG53蛋白及BSA蛋白，伤后每6小时记录小鼠死亡数目至伤后48小时，统计死亡率。伤后48小时存活的小鼠心脏灌注，取小鼠的心，肝，脾，肺，肾，小肠，结肠，脑，肌肉，皮肤，胃组织，4%多聚甲醛固定，24h组织脱水，石蜡包埋、切片，行苏木精-伊红(HE)染色，显微镜下观察各组组织损伤情况。 3)重度烫伤小鼠肾脏损伤的评估 ①重度烫伤小鼠肾小管坏死程度的评分随机选取每只小鼠肾脏切片一张，并在显微镜下随机选取20个视野观察，根据肾小管的坏死程度，采用Erdogan等的半定量病理评估法评分：评分越高说明肾小管坏死越严重，0分：正常肾脏；1分：最少坏死(5%肾小管坏死)；2分：轻度坏死(5%～25%肾小管坏死)；3分：中度坏死(25%～75%肾小管坏死)；4分：重度坏死(75%的肾小管坏死)。分析的实验结果以(x±s)表示，采用Microsoft Excel软件及SPSS13.0统计软件行统计分析，P 0.05为差异有统计学意义。 ②免疫组织化学SP法检测重度烫伤小鼠肾脏中KIM-1的表达取前面实验小鼠的肾脏石蜡切片，分MG53实验组及BSA对照组，，每组选取4张切片，使用肾损伤分子-1(Kidney Injury Molecule-1，KIM-1)多克隆抗体行免疫组化(IHC)染色，显微镜下观察各组肾脏中KIM-1的表达情况；每张切片随机选取5个视野，显微镜下拍照，拍照后，IPP软件半定量分析，分析的实验结果以(x±s)表示，采用Microsoft Excel软件及SPSS13.0统计软件中的独立样本t检验进行统计分析，P 0.05为差异有统计学意义。 2．通过尾静脉注射的外源性rhMG53蛋白在体内的分布及重度烫伤后PTRF体内表达的变化 1)免疫组化方法检测外源性rhMG53蛋白在小鼠体内的分布取前面实验小鼠脏器的石蜡切片，使用MG53多克隆抗体，分别进行免疫组织化学染色，以心肌及骨骼肌表达内源性MG53组织作阳性对照，观察其他脏器中外源性rhMG53分分布，显微镜下观察，拍照。 2)免疫组化方法检测正常小鼠及重度烫伤小鼠体内的PTRF的表达取正常小鼠及重度烫伤小鼠肾脏的石蜡切片，使用PTRF多克隆抗体，分别进行免疫组化，观察重度烫伤后，小鼠体内PTRF表达的变化，显微镜下拍照，拍照后，IPP软件半定量分析，分析的实验结果以(x±s)表示，采用Microsoft Excel软件及SPSS13.0统计软件中的独立样本t检验进行统计分析，P 0.05为差异有统计学意义；继续取重度烫伤小鼠小肠及皮肤石蜡切片，性免疫组化染色，观察PTRF的表达情况。实验结果: 1. MG53对30%Ⅲ°烫伤小鼠死亡率影响及脏器病理改变的实验研究 1)小鼠Ⅲ°烧伤模型的制作在自然状态下，使用烫头直接接触小鼠皮肤8s，压力为500g，烫头温度为90℃时，组织学显示皮肤各层、皮下附件及皮下脂肪组织均发生凝固性坏死，无完整的细胞结构，真皮内胶原肿胀变形，符合Ⅲ°烧伤组织学改变。 2)MG53对30%Ⅲ°烫伤小鼠死亡率影响及脏器病理改变的研究 MG53给药组伤后48h的死亡率为25%，BSA组伤后48h的死亡率为37.5%，MG53给药组死亡率低于BSA对照组，但统计学Kaplan-Meier分析，P值＞0.05，无统计学差异；HE染色结果显示，严重烧伤后小鼠的心、肝、脾、肺、胃、小肠、结肠、皮肤、肌肉、脑组织等脏器均有不同程度的组织学变化，但MG53给药组和BSA对照组在上述多数组织中无明显的差别，有明显差别的脏器是肾脏。组织切片显示MG53}f药组的肾脏近曲小管和远曲小管上皮细胞浊肿变性、坏死脱落以及肾间质炎症细胞浸润方面较对照组有明显改善，肾小球无明显变化。 3)重度烫伤小鼠肾脏损伤的评估 ①重度烫伤小鼠肾小管坏死程度的评分 MG53给药组的Erdogan半定量病理评分显著低于BSA对照组肾小管病理损伤评分(P0.0001)。 ②免疫组织化学SP法检测重度烫伤小鼠肾脏中KIM-1的表达各组肾脏KIM-1免疫组化结果显示：肾皮质髓质的损伤部位均可见KIM-1的表达，且表达量随肾脏损伤范围及程度的增加而成明显升高趋势。显微镜下观察结果显示，MG53给药组小鼠肾脏的KIM-1表达明显弱于BSA对照组，而在用PBS代替一抗的损伤肾脏中，没有发现阳性染色；通过IPP软件分析各组小鼠KIM-1表达的平均光密度显示，MG53给药组平均光密度OD值显著低于BSA对照组，差异有明显的统计学意义(P0.0001)。 2．通过尾静脉注射的外源性rhMG53蛋白在体内的分布及重度烫伤后PTRF体内表达的变化 1)免疫组化方法检测外源性rhMG53蛋白在小鼠体内的分布免疫组化结果显示，没有给予rhMG53的小鼠体内，内源性MG53主要分布在心肌和骨骼肌，可见骨骼肌细胞胞浆和心肌细胞呈强阳性染色。除心肌和骨骼肌外，其它组织均为阴性。在rhMG53给药组，小鼠的rhMG53主要分布于部分肾皮质肾小管上皮细胞和部分肾髓质肾小管上皮细胞，并且强阳性染色主要集中于损伤严重的区域，而对照组在这些部位未见阳性细胞。另外MG53实验组小鼠的肺脏中，发现大量rhMG53的分布，且rhMG53主要分布于肺脏部分小静脉内皮细胞胞浆中，而其他组织中未见明显阳性细胞。 2)免疫组化方法检测正常小鼠及重度烫伤小鼠体内的PTRF的表达肾脏PTRF免疫组化结果显示：正常小鼠肾脏和重度烫伤小鼠肾脏，皆可见大量的强阳性染色，且在肾脏的皮质和髓质都存在大量的强阳性区域。显微镜下初步观察结果显示，两组肾脏PTRF的表达无明显差别，而在用PBS代替一抗的肾脏中，没有发现阳性染色；通过IPP软件分析各组小鼠PTRF表达的平均光密度OD值，结果显示，正常小鼠肾脏平均光密度OD值与重度烫伤小鼠相比，无明显的统计学差异(P=0.890)；另外小鼠的小肠及皮肤中，皆有PTRF的弱阳性表达，较肾脏相比较，阳性区域的面积及染色强度明显较弱。实验结论：动物实验研究发现，尾静脉注射rhMG53蛋白能够选择性到达PTRF的高表达的肾脏。尾静脉注射rhMG53蛋白能够降低30%Ⅲ°烫伤小鼠早期死亡的发生，并改善肾脏的病理损伤情况。上述研究提示，严重烧伤后外源性rhMG53蛋白能够通过肾脏局部的PTRF早期对肾脏起到保护作用。
[Abstract]:Visceral organs are the participants and maintainers of various life activities. After severe burns, acute ischemic anoxia, inflammatory factors and other factors will cause the injury of the internal organs of the body. The damage of the viscera, especially the injury of multiple organs, is the important feature of the aggravation of the burn condition, the complication and the increase of mortality. The prevention and treatment of visceral injury and the promotion of early repair and reconstruction of damaged organs are the key to improve the success rate and reduce the mortality of the injured organs. After severe burns, the viscera itself has an ischemic damage to the naked eye. In addition, the scope of this kind of injury is wide, the accumulative organs are many, the pathogenesis is complex, and the effective measures for promoting the repair are lack. Therefore, the repair after the injury is often not easy to cause people to pay attention to the various measures taken by [1]., often only for a certain link of the pathogenesis or to reduce the degree of damage, it does not directly affect the repair of the cell itself, so it lacks the active repair of the viscera. Therefore, how to accelerate and promote the active repair of the damaged viscera cells. It is possible to provide a new effective way to prevent multiple organ dysfunction syndrome (Multipleorgan dysfunction syndrome, MODS), multiple organ failure (multiple system organ failure, MSOF) and to improve the success rate of treatment. It is of important theoretical significance and clinical application value.
MG53 (Mitsugumin53), a muscle specific TRIM family protein, is an essential component of cell membrane damage repair,.Ma and other studies suggest that the recombinant human MG53 (Recombinant HumanMitsugumin53, rhMG53) can treat muscular dystrophy and other human diseases with cell membrane damage as the pathogenesis. When cultured and not, the muscle and other human diseases are developed. When rhMG53 is present in the culture fluid of the muscle cells, rhMG53 can identify the signal of cell membrane damage and improve the membrane repair ability of these cells. Experimental study in mice shows that rhMG53 can be improved by various pathways (intramuscular, venous, subcutaneous), and it can also improve muscle nutrition to some extent. Histopathological changes in adverse events [2].
Therefore, we speculate that after severe scald, exogenous rhMG53 therapy may play a certain role in repairing the various organs damage caused by scald, improving the function of the organs and improving the survival rate. In this study, a model of 30% degree scald mice was used to observe the survival rate and organs of severely burned mice by intravenous injection of rhMG53. The effect of protection and its possible mechanisms.
Research contents and methods:
Effect of 1. MG53 on mortality and visceral pathological changes in 30% ~ 3 degree scalded mice
1) the production of mouse model of third degree burn
The Bal b/c mice were injured by the YLS-Q5 Super Table temperature control scald instrument, and the scald depth was controlled by two factors of temperature and time. The injury temperature was 90 C, the weight of self weight was 500g, the mouse skin was directly exposed to 6S, 8s, 10s with the scald 2cm constant temperature metal perm. The histological changes of burn skin skin were observed by HE staining, and the production of the model of the mice was determined. Conditions.
2) effect of MG53 on mortality and visceral pathological changes in 30% ~ 3 degree scalded mice
Before the experiment, the male Bal b/c mice were divided into two groups of exogenous rhMG53 protein.16 only about 20g, which were randomly divided into two groups: MG53 experimental group and BSA control group. The area of 30% degree scald was made, 2.5ml Sodium Lactate Ringer's Injection was injected intraperitoneally after injury, 0.5h after scald, 6.5h after scald, and exogenous rhMG53 protein and BSA protein were injected through the tail vein. Every 6 hours after injury, the death number of mice was recorded to 48 hours after injury, and the death rate was recorded. The heart, liver, spleen, liver, spleen, lung, kidney, intestine, colon, brain, muscle, skin, stomach tissue, 4% polyformaldehyde fixed, 24h tissue dehydration, paraffin embedding, HE staining and microscopic observation under microscope were observed. Injury in the group.
3) evaluation of renal injury in severely scalded mice
Score of renal tubular necrosis in severely scalded mice
Randomly selected one slice of each mouse kidney and randomly selected 20 visual fields under the microscope. According to the necrosis degree of renal tubules, Erdogan and other semi quantitative pathological evaluation were used. The higher the score, the more serious the renal tubular necrosis was, the 0 points: the normal kidney; 1 points: the least necrosis (5% renal tubular necrosis); 2 points: mild necrosis. (5% ~ 25% renal tubular necrosis); 3: moderate necrosis (25% to 75% renal tubular necrosis); 4 points: severe necrosis (75% of renal tubular necrosis). The results of the analysis were (x + s), and the statistical analysis was performed by Microsoft Excel software and SPSS13.0 statistical software, and P 0.05 was statistically significant.
Immunohistochemical SP method was used to detect the expression of KIM-1 in kidney of severely scalded mice.
The paraffin section of the kidney of the experimental mice was taken, divided into 4 sections of MG53 experimental group and BSA control group, each group selected 4 slices, using Kidney Injury Molecule-1, KIM-1 polyclonal antibody and immunohistochemistry (IHC) staining. The expression of KIM-1 in the kidneys of each group was observed under microscope; each slice was randomly selected for 5 fields of vision and under microscope. After taking pictures and taking pictures, the IPP software is semi quantitative analysis, the results of the analysis are (x + s), and the statistical analysis is carried out by the t test of independent samples in Microsoft Excel software and SPSS13.0 statistics software. P 0.05 has statistical significance.
2. the distribution of exogenous rhMG53 protein through tail vein in vivo and the changes of PTRF expression after severe scald.
1) immunohistochemical method was used to detect the distribution of exogenous rhMG53 protein in mice.
The paraffin section of the viscera of the previous experimental mice was taken with MG53 polyclonal antibody and immunohistochemical staining was performed respectively. The expression of endogenous MG53 tissues expressed in myocardium and skeletal muscle was used as positive control, and the source rhMG53 distribution in other organs was observed and observed under microscope, and photographed.
2) immunohistochemical method was used to detect the expression of PTRF in normal mice and severely scalded mice.
The paraffin section of normal mice and severely scalded mice was taken by using PTRF polyclonal antibody and immunohistochemistry. The changes of PTRF expression in the body of the mice were observed after severe scald. Under the microscope, the IPP software was semi quantitative analysis. The results of the analysis were (x + s), and the Microsoft Excel software and SPSS13.0 statistics were used. The independent sample t test in the software was statistically analyzed, and the difference was statistically significant in P 0.05, and the paraffin section of small intestine and skin in severe scalded mice was continued, and the expression of PTRF was observed by sexual immunohistochemical staining.
Experimental results:
Effect of 1. MG53 on mortality and visceral pathological changes in 30% ~ 3 degree scalded mice
1) the production of mouse model of third degree burn
Under natural condition, the skin 8s was directly exposed to the skin of mice with a perm head, the pressure was 500g, and the perm temperature was 90 C. The histology showed that all layers of skin, subcutaneous appendages and subcutaneous adipose tissue were all coagulated necrosis, without complete cell structure, the swelling and deformation of the collagen in the dermis, and the histological change of the third degree burn.
2) effect of MG53 on mortality and visceral pathological changes in 30% ~ 3 degree scalded mice
The mortality of 48h after injury in MG53 group was 25%, the mortality of 48h after injury in group BSA was 37.5%, and the mortality of MG53 administration group was lower than that of BSA control group, but statistically Kaplan-Meier analysis, P value was > 0.05, no statistical difference, and HE staining results showed that the heart, liver, spleen, lung, stomach, intestines, colonic, skin, muscle and brain tissues of the mice after severe burns were found. There was no obvious difference between the MG53 administration group and the BSA control group, but the visceral organs with distinct differences were renal. The tissue section showed that the renal proximal convoluted tubule and the distal tubule epithelial cells in the MG53}f drug group were cloudy and swollen, necrosis and exfoliation and the infiltration of renal interstitial inflammatory cells were more obvious than those in the control group. There was no significant change in the glomeruli.
3) evaluation of renal injury in severely scalded mice
Score of renal tubular necrosis in severely scalded mice
The Erdogan semi quantitative pathological score in the MG53 administration group was significantly lower than that in the BSA control group (P0.0001).
Immunohistochemical SP method was used to detect the expression of KIM-1 in kidney of severely scalded mice.
The results of KIM-1 immunohistochemistry in each group showed that the expression of KIM-1 was visible in the injured parts of the medulla of the renal cortex, and the expression was obviously increased with the increase of renal damage range and degree. Under the microscope, the expression of KIM-1 in the kidney of the MG53 administration group was significantly weaker than that of the BSA control group, and the PBS was used instead of the one resistance. No positive staining was found in the injured kidneys, and the average optical density of KIM-1 expression in each group was analyzed by IPP software. The average optical density of MG53 in the group was significantly lower than that of the BSA control group, and the difference was statistically significant (P0.0001).
2. the distribution of exogenous rhMG53 protein through tail vein in vivo and the changes of PTRF expression after severe scald.
1) immunohistochemical method was used to detect the distribution of exogenous rhMG53 protein in mice.
The results of immunohistochemistry showed that the endogenous MG53 was mainly distributed in the myocardium and skeletal muscle in the mice without rhMG53, and the cytoplasm and myocardial cells of the skeletal muscle cells were strongly positive. All the other tissues were negative except for the myocardium and the skeletal muscle. In the rhMG53 administration group, the rhMG53 of the mice was mainly distributed in the epithelium of some renal cortex and renal tubules. Cell and partial renal medullary renal tubular epithelial cells, and strong positive staining mainly concentrated in the area of severe injury, but in the control group, no positive cells were found in these areas. In addition, a large number of rhMG53 were found in the lungs of the MG53 experimental group, and rhMG53 was mainly distributed in the small venous endothelial cell cytoplasm of the lungs and in other tissues. No obvious positive cells were found.
2) immunohistochemical method was used to detect the expression of PTRF in normal mice and severely scalded mice.
The renal PTRF immunohistochemical results showed that a large number of strong positive staining was found in kidney of normal mice and severely scalded mice, and a large number of strong positive regions were found in the cortex and medulla of the kidneys. Under the microscope, the results showed that the expression of PTRF in the two groups of kidneys had no obvious difference, but in the use of PBS instead of the kidney, no kidney was used. The average optical density o value of PTRF expression in each group was analyzed by IPP software. The results showed that the average optical density o value of normal mice kidney had no significant difference compared with that of severe scalded mice (P=0.890). In addition, there was a weak positive expression of PTRF in the small intestine and skin of mice, compared with the kidney. The area and dyeing strength of the region are obviously weak.
Experimental results: the experimental study found that the injection of rhMG53 protein in the tail vein can selectively reach the high expression of PTRF in the kidney. The injection of rhMG53 protein in the tail vein can reduce the occurrence of early death in 30% degree scald mice and improve the pathological damage of the kidney. The above study suggests that exogenous rhMG53 protein can pass after severe burns. Early renal PTRF plays a protective role in kidney.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R644

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