【作者】 潘玲；

【导师】 马跃荣；

【作者基本信息】 成都中医药大学 ， 病理学与病理生理学， 2017， 硕士

【摘要】 研究目的:糖尿病肾病是糖尿病严重的慢性并发症,属于糖尿病全身性微血管病变表现之一。其发病率高,起病隐匿,是引起糖尿病患者过早死亡的主要原因,已成为严重威胁人类健康的重大疾病。糖尿病肾病的病机十分复杂,涉及到糖、脂类代谢紊乱,肾脏血流动力学异常,细胞因子活化等多种机制,遗传易感性也被证实参与其中。蛋白尿是糖尿病肾病最常见的早期临床症状,同时也是糖尿病造成肾脏损伤的重要原因。长期以来,肾小球基底膜的增厚以及系膜扩张被认为是糖尿病肾病蛋白尿出现的病理关键,但近年的许多研究证实糖尿病肾病实质上是一种足细胞病,足细胞损伤贯穿了糖尿病肾病发生发展的全过程,稳定足细胞的形态与功能成为控制糖尿病肾病蛋白尿的关键。通过结合中医学阴阳学说的基本理论以及临床实践经验,叶传蕙教授提出糖尿病肾病的关键病机在于脾肾亏虚、痰瘀互结、脉络阻滞。在此基础上,本课题组创立了中药复方固本化瘀通络合剂,其功效为健脾固肾、化痰祛瘀、活血通络、调和阴阳。在前期的临床观察中,我们以固本化瘀通络法作为糖尿病肾病的治疗大法,业已证实其可明显降低糖尿病肾病患者的尿白蛋白排泄率,且疗效优于血管紧张素Ⅱ受体拮抗剂（ARB）厄贝沙坦（安博维）。本研究拟以高糖干预的肾小球足细胞作为细胞模型,运用血清药理学方法制备固本化瘀通络合剂含药血清并将其作用于细胞模型上,以足细胞形态的异常改变以及细胞凋亡作为足细胞损伤的标志,观察固本化瘀通络合剂对高糖环境下足细胞损伤的影响,进一步验证其临床疗效,从而为固本化瘀通络合剂治疗糖尿病肾病蛋白尿提供细胞层面的理论依据。研究方法:1.固本化瘀通络合剂含药血清及正常大鼠血清的制备参照血清药理学的原则,选取6⁸周龄,体重为200±20g的健康雄性SPF级SD大鼠,共计20只。适应性喂养1周后将其随机分为固本化瘀通络合剂组（中药组）和空白对照组（10只/组）。中药组大鼠给予相当于临床等效剂量1倍的固本化瘀通络合剂进行灌胃,空白对照组则给予同等体积的生理盐水。灌胃剂量以1ml/100g大鼠体重进行换算,每天1次,连续7天。末次灌胃后1h（给药前禁食不禁水12h）两组同时经腹主动脉采集血液样本,离心后分离血清,经0.22μm滤过膜过滤除菌,56℃水浴灭活30min,分装,-80℃冷藏备用。2.足细胞的培养及鉴定培养条件性永生化足细胞,未分化足细胞用内含10%胎牛血清（FBS）、10U/ml小鼠重组IFN-γ、1%（青霉素和链霉素）双抗溶液的RMPI-1640培养基在33℃、5%CO₂培养箱中进行传代培养。传代后将细胞用含10%FBS、1%双抗溶液但不添加小鼠重组IFN-γ的RMPI-1640培养基于37℃、5%CO₂培养箱中诱导分化2周。利用免疫荧光技术对细胞进行鉴定,选取足细胞特异性蛋白nephrin、podocin和podocalyxin,鉴定结果显示我们所获得的细胞为符合实验要求的肾小球足细胞。实验前将分化成熟的足细胞以1×10⁵¹×10⁶个/ml左右的的密度接种于6孔板内,待其贴壁生长24h后,使用不含血清成分的RMPI-1640培养基同步化处理24h,细胞方可用于后续实验。3.不同浓度的固本化瘀通络合剂含药血清对高糖环境下足细胞损伤影响的研究将同步化处理后的成熟足细胞随机分为以下四组进行药物干预:（1）高糖对照组（Control）:高糖培养基（内含30mmol/L D-葡萄糖的RMPI-1640培养基）+10%正常大鼠血清;（2）低浓度固本化瘀通络合剂含药血清组（Lowconcentration）:高糖培养基（内含30mmol/L D-葡萄糖的RMPI-1640培养基）+2.5%固本化瘀通络合剂含药血清+7.5%正常大鼠血清;（3）中浓度固本化瘀通络合剂含药血清组（Moderate concentration）:高糖培养基（内含30mmol/L D-葡萄糖的RMPI-1640培养基）+5%固本化瘀通络合剂含药血清+5%正常大鼠血清;（4）高浓度固本化瘀通络合剂含药血清组（High concentration）:高糖培养基（内含30mmol/L D-葡萄糖的RMPI-1640培养基）+10%固本化瘀通络合剂含药血清。37℃培养48h后,分别采用倒置相差显微镜下直接观察各组足细胞的形态变化,CFSE/PI双染进行细胞毒性检测,Annexin V-FITC/PI双标染色结合流式细胞术检测足细胞凋亡率,来观察固本化瘀通络合剂含药血清对高糖环境下足细胞损伤的影响。结果:1.倒置相差显微镜下各组足细胞的形态学观察:高糖刺激48h后,高糖对照组的足细胞显示出较明显的细胞损伤形态,如细胞皱缩变形,体积变小,胞浆中空泡形成,足突变短、融合或消失,细胞数目减少等。固本化瘀通络合剂含药血清处理的三个组别的细胞形态较高糖对照组有所改善,足细胞突起增多,胞体无皱缩,胞浆内空泡减少,足细胞间排列较为紧密。其中,又以高浓度固本化瘀通络合剂含药血清组细胞的形态最佳。2.CFSE/PI双染细胞毒性检测:高糖刺激48h后,各组细胞的增殖情况无明显差异,但是高糖对照组死细胞的数量较固本化瘀通络合剂中药处理组明显增加,其中,高浓度固本化瘀通络合剂含药血清组的细胞仅见少量死细胞。3.流式细胞术（Annexin V-FITC/PI双标记）凋亡检测:高糖刺激48h后,低、中、高浓度固本化瘀通络合剂含药血清组足细胞凋亡率较高糖组均见明显降低,差异有统计学意义（P<0.05）。此外,中、高浓度固本化瘀通络合剂含药血清组正常足细胞的比例较高糖组明显增加,差异有统计学意义（P<0.05）,低浓度固本化瘀通络合剂含药血清组正常足细胞的比例与高糖组间差异无统计学意义（P>0.05）。结论:1、固本化瘀通络合剂含药血清可明显改善高糖刺激引起的足细胞损伤的形态改变,且呈一定的浓度梯度依赖。2、高糖干预可成功诱导足细胞发生凋亡,固本化瘀通络合剂含药血清通过减轻高糖诱导的足细胞凋亡,并提高正常细胞的比率,具有一定的足细胞保护作用,其具体机制有待进一步探讨。更多还原

【Abstract】 Objective: Diabetic kidney diseases is a serious chronic complication of diabetes mellitus,which is the main systemic microvascular disease in diabetes mellitus.With a high incidence and insidious onset,it be the main causes of premature death in patients with diabetes,has been a serious threat to human health.The pathogenesis of diabetic kidney diseases（DKD）is very complex.It involves many factors,such as the metabolism disorder of glucose and lipid,abnormal renal hemodynamics,cytokines activation and so on.Genetic predisposition has also been shown to be involved.Proteinuria is the most common early clinical symptoms of diabetic kidney diseases,which is also an important reason of kidney damage cause by diabetes mellitus.Pathophysiologic changes of the thickening of the glomerular basement membrane and expansion of the mesangial area have long been considered at the heart of presence of proteinuria in diabetic kidney diseases,but many studies in recent years have confirmed that diabetic kidney diseases is essentially a podocytopathy,podocyte injury occurred throughout the whole process of the occurrence and development of diabetic kidney diseases,stabilizing the morphology and function of podocyte become a key to control proteinuria in diabetic kidney diseases.Combining the basic theory of Chinese medicine of yin-Yang and the clinical practice experiences,Professor Ye proposed the primary pathogenesis of diabetic kidney diseases is deficiency of spleen and kidney,intermingled phlem and blood stasis,channels stasis.On the basis of this,our research group has established a traditional Chinese medicine compound that named Compound Guben Huayu Tongluo,which effect is invigorating spleen and tonifying kidney,resolving phlegm and removing stasis,promoting blood circulation to remove meridian obstruction,harmonizing yin and yang.In the preliminary clinical observation,we used the method of Guben Huayu Tongluo as the treatment of diabetic kidney diseases,it has been proved that the compound can significantly reduce the urinary protein excretion rate in patients with diabetic kidney diseases,and the effect is better than that of Angiotensin II receptor antagonist（ARB）Irbesartan.In this study,we intend to establish a cell model of podocyte with hyperglycemia intervention,prepared serum containing Compound Guben Huayu Tongluo by serum pharmacological methods and apply it to the cell model,The abnormal changes of podocyte morphology and cell apoptosis were regarded as markers of podocyte injury,to observe the effects of Compound Guben Huayu Tongluo on podocyte injury under high glucose environment,to further verify its clinical efficacy,so as to provide a theoretical basis for Compound Guben Huayu Tongluo applying to the treatment of proteinuria in diabetic kidney disease.Methods: 1.Preparation of Compound Guben Huayu Tongluo containing serum and normal rat serum.Referring to the principle of serum pharmacology,20 healthy male SD rats of 6⁸ weeks old,SPF grade with body weight of 200±20g were selected.After a week of adaptive feeding,they were randomly divided into Compound Guben Huayu Tongluo group（Chinese medicine group）and blank control group（10 per group）.The rats in the Chinese medicine group was intragastric injection administration with Compound Guben Huayu Tongluo,which dose equivalent to 1 times human clinical equivalent dose.While the blank control group was given the same volume of normal saline.The dosage of gastric lavage was converted to 1ml/100 g weight,once a day,for 7 days in arow.1hour after the last intragastric administration（before gavage fasting for 12 hours but do not restricting water）,blood samples of each group were collected from abdominal aorta at the same time.Isolating serum separat after centrifugation,then filtration sterilization by micro filtration membrane with pore size of 0.22μm,56 ℃ water bath inactivated 30 min,packaged into small copies,stored at-80 ℃ standby.2.Culture and identification of podocyte Cultivating conditional immortalized mouse podocytes,The undifferentiated podocytes were cultured in RMPI-1640 medium containing 10% fetal bovine serum（FBS）,10 U / ml mouse recombinant IFN-γ,1%（penicillin and streptomycin）double solution（which consist of 100 U / ml penicillin,100 μg / ml streptomycin）at 33 ℃,5% CO₂ incubator.After subculture,the cells were induced to differentiate in RMPI-1640 medium containing 10% FBS,1% double solution,but without added mouse recombinant IFN-γ for 2 weeks at 37 ℃,5% CO₂ incubator.The cells were identified by immunofluorescence technique,and the podocyte specific proteins nephrin,podocin and podocalyxin were selected,the identification results showed that the cells obtained were in accordance with the experimental requirements of podocyte.When podocytes become differentiation and maturation,the cells were inoculated in 6 holes with a density of about 1×10⁵¹×10⁶ cells /ml.Adherent for 24 hours,the serum-free medium of RMPI-1640 was used for synchronization for a period of 24 hours,cells can be used for subsequent experiments.3.the effects of Compound Guben Huayu Tongluo containing serum on podocyte injury under high glucose environment After synchronization treatment,the mature podocytes were randomly divided into the following four groups for drug intervention:（1）high glucose control group（Control）: high glucose medium（RMPI-1640 medium containing 30mmol/L Dglucose）+ 10% normal rat serum;（2）low concentration of Compound Guben Huayu Tongluo containing serum（low concentration）: high glucose medium（RMPI-1640 medium containing 30mmol/L D-glucose）+ 2.5% Compound Guben Huayu Tongluo containing serum;（3）moderate concentration of Compound Guben Huayu Tongluo containing serum group（moderate concentration）: high-sugar medium（RMPI-1640 medium containing 30mmol/L D-glucose）+ 5% Compound Guben Huayu Tongluo containing serum;（4）high concentration of Guben Huayu Tongluo ricipe containing serum group（high concentration）: high glucose medium（RMPI-1640 medium containing 30mmol/L D-glucose）+ 10% Compound Guben Huayu Tongluo containing serum.After 48 hours of intervention at 37 ℃,the morphological changes of podocytes were observed under the inverted phase contrast microscope,CFSE/PI double staining was used to measure the viability of podocytes.Annexin V-FITC/PI double staining combined with flow cytometry were used to detect the apoptosis of podocytes.to observe the effect of Compound Guben Huayu Tongluo containing serum on podocytes injury under high glucose environment.Results: 1.Morphological observation of podocytes in inverted phase contrast microscope:After 48 h of high glucose stimulated,podocytes in high glucose group showed more obvious cell damage,sunch as cells shrunken and deformed,the volume became smaller,the vacuoles formed in the cytoplasm,the FPs were short,fused or disappeared,and the number of cells decreased.The cell morphology of the three groups treated with Compound Guben Huayu Tongluo containing serum was higher than that of the sugar group,The number of podocytes increased,the cell body did not shrink,the vacuoles in the cytoplasm decreased,and the arrangement of the podocytes was more compact.Among them,the high concentration of traditional Chinese medicine containing serum cells of the best form.2.CFSE/PI double staining cytotoxicity test:After 48 h of high glucose stimulated,The number of dead cells in the high glucose control group was significantly higher than that in the traditional Chinese medicine treatment group.Among them,there is only a few dead cells in the high concentration of Compound Guben Huayu Tongluo containing serum group.3.Flow cytometry（Annexin V-FITC/PI double staining）apoptosis detection: After 48 h of high glucose stimulation,The apoptosis of podocytes in the low concentration 、moderate concentration and high concentration of Compound Guben Huayu Tongluo containing serum group was significantly lower than that in high glucose group,the difference was statistically significant（P < 0.05）.In addition,the percentage of normal podocytes in the moderate concentration and high concentration of Compound Guben Huayu Tongluo containing serum group was significantly higher than that in the high glucose group,the difference was statistically significant（P < 0.05）,there was no significant difference in the percentage of normal podocytes between the low concentration of Compound Guben Huayu Tongluo containing serum group and the high glucose group（P>0.05）.Conclusion: 1、The serum contained Compound Guben Huayu Tongluo can significantly improve the morphological changes of podocyte injury induced by high glucose stimulation,and has a certain concentration gradient dependence.2、 High glucose intervention can induce podocyte apoptosis,the serum contained Compound Guben Huayu Tongluo can reduce the apoptosis of podocytes induced by high glucose and improve the ratio of normal cells,the specific mechanism remains to be further explored.更多还原