TSP-1和TGF-β1表达与糖尿病大鼠多脏器损伤的研究
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摘要
研究背景：
糖尿病(diabetes Mellitus, DM)已成为世界上发病率最高、对人类健康威胁最严重的疾病之一。糖尿病所引起的糖代谢紊乱,导致微血管病变,以及高血糖本身对组织细胞的毒害作用,继而引起一系列并发症,包括糖尿病阳痿、糖尿病心肌病和糖尿病性痴呆等。对于糖尿病阳痿,有资料表明,大约一半患者在诊断为糖尿病10年后有可能合并勃起功能障碍(erectile disfunction, ED),其中约有12%的糖尿病患者ED是其首发表现。高血糖本身对心肌亦有毒害作用,流行病学研究发现,糖尿病患者70%以上死于心血管系统疾病,其中,糖尿病心肌病(diabetic cardiomyopathy)是糖尿病患者的主要并发症之一,其发病率高,危害性大,与糖尿病患者心血管疾病的高发生率和高病死率密切相关。现已公认,糖尿病心肌病是一个独立的原发病,其发病不依赖于高血压、冠状动脉疾病和其他已知心脏疾病。此外糖尿病与认知障碍和痴呆亦有密切相关性：如在欧洲,糖尿病发病率在年龄大于65的痴呆患者中是6.4%。Leibson等报道了1455例成年发病的糖尿病病人中981人经一年观察后,101人产生了痴呆,其中77例为老年性痴呆。
糖尿病上述多器官损伤的机制尚未完全明确,目前认为非酶性糖基化终末产物形成、蛋白激酶C信号通路的激活、氧化应激反应和己糖胺通路活性增高等具有重要作用。随着研究的深入,我们发现上述糖尿病上述脏器损伤均存在间质纤维化、相关细胞凋亡或突触可塑性改变等病理变化,因此高血糖状态下TSP-1和TGF-β1表达的改变和上述糖尿病患者多脏器损伤之间的关系引起了我们关注。血小板反应素(Thrombospondin, TSP),尤其是TSP-1,是重要的生理激活物之一作为一种大分子的细胞外糖蛋白,最初是在被凝血酶刺激的血小板a颗粒释放的产物中被发现,最新研究证实TSP并非血小板特有,可被肾小球系膜细胞、成纤维细胞等多种细胞分泌,许多组织如肾脏、心脏、软骨和脑中都有TSP基因产物的表达,是许多不同组织细胞外间质的重要成分。研究发现TSP-1由3条相同肽链构成的同源三聚体基质糖蛋白,每条肽链由N端、C端球状结构域、原胶原同源区、type1重复序列、type2重复序列、type3重复序列组成。其中type1重复序列(thrombospondin typel repeats,TSRs)由3个备解素样基序(TSR1、TSR2、TSR3)组成。转化生长因子β(transforming growth factor-β,TGF-β)是一个分泌型的多肽信号分子超家族,在哺乳动物组织中存在3种形式的TGF-β,分别是TGF-β1、TGF-β2和TGF-β3,其中TGF-β1所占比例最高,活性最强。激活的TGF-β可使组织纤维化,引起纤维母细胞迁移,抑制胶原酶和其它金属蛋白酶的活性,增加胶原产生,降低胶原降解,促进基质沉积。研究表明,TSP-1分子KRFK序列可与L-TGF-β1的LAP区域结合,从而改变LAP的空间构型,使TGF-β1上与受体结合的位点暴露,成为活化的TSP-1/L-TGF-β1。同时新近研究发现海马区的血小板反应素与突触的建立活化和形态功能可塑性改变均有密切关系。因此,我们提出如下假设：糖尿病状态(如高糖等刺激)条件下引起体内各类细胞TSP-1和TGF-β1表达的改变,从而促进上述细胞纤维和胶原增生,最后导致纤维化,进而引起糖尿病勃起功能障碍、糖尿病心肌病,此外TSP-1表达改变在海马区亦可引起突触可塑性的改变,因此上述通路改变亦可引起糖尿病学习记忆能力的下降。
研究已经表明,在哺乳动物中小干扰RNA(siRNA)能通过对生物学上保守结构的RNA进行干扰,从而抑制相应靶基因的表达。RNA干扰具有高度的序列特异性,能使靶蛋白的表达关闭。许多研究已经证实RNA干扰能阻断多种类型哺乳动物病毒的复制,抑制癌肿的生长。RNA干扰已经被开发成为反向遗传学的一个强有力的工具,并且已显示出广阔的治疗学上的应用前景。因此,我们有理由假设针对性开发TSP-1的小干扰RNA无疑对高血糖状态下神经元的损伤有一定的保护作用。
我们通过研究高血糖状态下对海马区神经元TSP-1和TGF-β1的表达改变,同时设计siRNA对TSP-1的表达进行了基因沉默,并评估TSP-1表达下调对神经元的形态及突触形成的影响。期望能靶向干扰TSP-1表达,为糖尿病学习记忆能力下降的基因治疗提供一定的体外实验依据。
第一部分TSP-1和TGF-β1在糖尿病大鼠阴茎、心肌和海马区表达改变的研究
1目的
研究TSP-1和TGF-β1蛋白和mRNA在糖尿病大鼠阴茎、心肌和海马区表达的改变情况,初步阐明TSP-1和TGF-β1通路参与了糖尿病大鼠高血糖状态下的上述脏器损伤。
2材料与方法
2.1糖尿病大鼠模型的制备
SD大鼠随机分为糖尿病组和正常对照组,其中糖尿病组大鼠腹腔注射链脲佐菌素(streptozotocin, STZ)65mg/kg制成糖尿病模型,定期测尿糖、血糖及体重,以确保维持高血糖状态。正常对照组腹腔注射等量生理盐水,其余条件同上,喂养6周。
2.2各脏器功能检测
2.2.1行为学检测
成模6周后各组大鼠分别进行Morris水迷宫实验(观察指标为逃避潜伏期和搜索策略),以检测糖尿病动物的学习、记忆能力的变化。
2.2.2阴茎勃起功能检测
成模6周后各组大鼠采用颈部皮下注射盐酸阿朴吗啡(APO)溶液(150ug/kg),剂量约1ml,持续观察30min,对其阴茎勃起情况进行检测。
2.2.3心功能检测
成模6周后各组大鼠充分麻醉后处死开胸,迅速取出心脏,置于4℃K-H液中除去血液,然后迅速转移并固定于Langendorff:灌流装置上,用改良K-H液行常规逆行恒压灌注(76mmHg),观察平衡灌注末的心脏作功量(rate-pressure product, RPP),即心率*左室发展压(HR*LVDP)的变化。
2.3各脏器组织的病理变化观察
成模6周后各组大鼠麻醉处死,分别取阴茎组织、心脏和脑,制作石蜡切片HE染色观察其病理改变,同时取心脏和海马区组织块制作电镜包埋和切片,电镜下观察其超微结构的变化。
2.4免疫组化和实时荧光定量PCR法检测TSP-1和TGF-β1的表达
成模6周后各组大鼠麻醉处死,分别取阴茎组织、心脏和脑,制作石蜡切片采用免疫组化ABC法观察上述各组织TSP-1和TGF-β1蛋白的表达,同时Trizol试剂盒提取细胞总RNA,随后逆转录合成cDNA,rt-qPCR法检测TSP-1和TGF-β1的基因转录情况。
2.5统计学分析
所有数值以均数±标准差(mean±D)表示,多组之间采用SPSS16.0统计软件进行单因素方差分析,两组间比较采用t检验。
3结果
3.1体重、血糖和尿糖检测结果
各组大鼠成模前,其体重、血糖和尿糖检测结果两组间无显著性差异,成模6周后,糖尿病组体重251.6±27.4g,血糖基本维持在19.8±2.8mmol/L,尿糖为++~+++。正常组大鼠体重397.4±21.3g,血糖为5.1±0.5mmol/L,尿糖阴性。两组差异有显著性意义(P<0.01)。
3.2各脏器功能
行为学Morris水迷宫实验显示,正常组大鼠第1天的逃避潜伏期为(40.1±26.0)s；第2天为(24.8±14.2)s；第3天为(13.4±4.8)s；穿台次数为(6.1±3.8)；糖尿病组大鼠第1天的逃避潜伏期为(71.6±33.7)s；第2天为(43.4±13.8)s；第3天为(26.9±19.1)s,穿台次数为(3.2±1.3),上述差异有显著性意义(P<0.01)。
检测其阴茎勃起功能发现糖尿病组大鼠单位时间勃起次数和勃起率(1.4±O.5次/30min和40%)明显低于正常对照组大鼠(2.4±0.8次/30min和100%)(P<0.01)。
心功能检测显示糖尿病组大鼠RPP值明显低于正常组大鼠,(P<0.01)。
3.3各脏器病理改变
各脏器组织HE染色观察发现,糖尿病组大鼠阴茎白膜厚度的增加,阴茎胶原纤维破坏,失去了其特有的起伏的外观,同时内皮和平滑肌细胞也退化及间质细胞增殖。糖尿病大鼠心肌细胞有不同程度的间质纤维化和心肌肥厚,心肌纤维出现了结构性的稀疏。电镜下可见糖尿病大鼠心肌微结构出现破坏,心肌细胞肌原纤维片状坏死、溶解,肌小节失去正常结构,甚至出现心肌细胞凋亡或坏死等现象。糖尿病大鼠海马区电镜观察可见神经元内部线粒体嵴消失,神经元之间突触结构蜕变,同时海马区单位面积突触数目明显少于正常组大鼠。
3.4免疫组化和rt-PCR检测结果
免疫组化检测发现,糖尿病大鼠各组织中TSP-1和TGF-β1的蛋白表达较正常对照组均明显上调,其中糖尿病组大鼠阴茎、心脏和海马区TSP-l和TGF-β1的单位面积阳性细胞数量均明显高于正常对照组(P<0.05),同时和实时荧光定量PCR法结果显示TSP-1和TGF-βmRNA表达较正常对照组亦明显上调(分别是对照组的2.8folds,1.7folds,6.6folds和6.9folds,4.3folds,1.42folds).
4结论
STZ诱导的糖尿病大鼠出现血糖增高、尿糖强阳性、体重增加不明显等一般情况改变,而且大鼠勃起功能、心功能和学习记忆能力均不同程度下降,同时伴阴茎、心脏和海马区不同程度的病理改变,此外糖尿病大鼠各组织中TSP-1和TGF-β1的蛋白和：mRNA表达较正常对照组大鼠亦明显上调。
第二部分siRNA靶向干扰TSP-1对高血糖培养神经元形态和TSP-1表达改变的影响
1目的
明确siRNA靶向干扰TSP-1可有效改善高血糖状态下神经元形态改变和TSP-1/TGF-β1的表达,以及保护神经元形态和突触连接。
2材料与方法：
2.1细胞培养
取孕16天SD大鼠,0.5%戊巴比妥钠麻醉,打开子宫,取出胎鼠,取大脑并分离海马。研磨组织并通过200目的滤网,收集细胞悬液,离心、重悬。接种于1×Polylysine包被的6孔培养板以及有小圆玻片的48孔培养板中,于37℃、5%CO2培养箱中培养,随机分为正常培养组、高糖培养组、高糖培养+转染组、高糖培养+阴行转染组、高糖培养+空白转染组。
2.2siRNA制备与转染
各对siRNA序列如下TSP-1sence5'-UACACUUGGCACCAGCAAAGCAGGG-3', LacZ引物序列上游引物
5'-ACCAGAAGCGGRGCCGGAAA-3'下游引物
5'-CCACAGCGGATGGTTCGGAT-3',上述siRNA由英維捷基(上海)贸易有限公司合成。上述细胞准备后,进行转染,而后轻轻振荡混匀,5%二氧化碳、37℃湿化空气中常规培养48小时。
2.3扫描电镜观察
上述细胞培养48小时后,吸干铺有小圆玻片48孔培养板中的培养液,4℃生理盐水洗1次,2.5%戊二醛固定1h,0.01M PBS洗2次,每次15min,1%锇酸固定1h,0.1M PBS溶液漂洗2次,每次15分钟。梯度酒精脱水,100%丙酮脱水20min。用50%醋酸异戊酯置换15min,100%醋酸异戊酯置换15min,临界点干燥,粘台,镀金,上机观察。
2.4免疫组化检测及Western Blot检测
上述细胞培养48小时后,0.01MPBS含0.3%的TRITON X-100(pH值7.4,PBST)分别洗净,然后沉浸含2%正常山羊血清的PBS2小时,TSP-1或TGF-β1抗体(1:100)和含有1%的牛血清白蛋白的PBS中4℃过夜,PBS(3×5min)洗净,生物素标记的羊抗兔IgG(1:200)孵育4h,PBS(3×5min)洗净,免疫标记diaminobenzdine0.05%,加0.3%H202的PBS,染色,镜下控制反应时间,然后用乙醇和二甲苯梯度脱水。其中PBS被用来代替一抗作为阴性对照。
等量提取的蛋白装载到SDS/PAGE胶上并被分离后,再转移到PVDF膜上,然后用含有5%脱脂奶粉的TBST液封闭1小时,再分别用TSP-1或TGF-β1抗体(1:100)在常温下孵育,膜用TBST液洗涤4次,然后用抗鼠IgG-HRP(DAKO)在常温下孵育1小时,最后蛋白用ECL体系进行检测。蛋白相对表达量=待测蛋白的光密度值／内参的光密度值。
2.5统计学分析
所有数值以均数士标准差(mean±SD)表示,多组之间采用SPSS16.0统计软件进行单因素方差分析,两组间比较采用t检验。
3结果
3.1扫描电镜观察
各组培养细胞扫描电镜可见,正常组神经元表面细胞微绒毛结构完整,神经元间联系广泛而紧密,高糖培养组神经元细胞表面微绒毛结构缺失,伴脱落,细胞膜可见轻微破损,神经元间联系稀疏,而RNA转染组神经元上述病变较轻,同时阴性转染组神经元显示和高糖培养组程度类似的损伤。
3.2免疫组化检测及Western Blot检测结果
通过检测神经元TSP-1和TGF-β1蛋白表达来评估TSP-1siRNA对神经元上述蛋白表达的影响,结果发现TSP-1siRNA转染组TSP-1和TGF-β1蛋白的表达与高糖培养组相比显著地减少(P<0.01)。
4结论
通过siRNA靶向干扰TSP-1能有效改善高血糖状态下神经元形态改变和TSP-1和TGF-β1蛋白的表达,同时保护神经元之间的连接和形态。
Background
Diabetes (Diabetes Mellitus, DM) has become the one of highest incidence and the most serious disease to human health. One important mechanism of type2diabetes was insulin resistance and insulin dysfunction, then leading to islet failure. Disorder of glucose metabolism, that leading to microvascular disease, and toxic effects of high blood sugar on tissue cells, could give rise to a series of complications, including diabetic erectile dysfunction, diabetic nephropathy, diabetic cardiomyopathy, and diabetic dementia, etc. The research data indicated that about half of patients diagnosed with diabetes after10years with a possible merger erectile dysfunction (ED). ED was the first manifestation in about12%of ED patients with diabetes. Hyperglycemia itself has toxic effects on cardiac, epidemiological studies have found that more than70%of patients with diabetes died of cardiovascular diseases. Diabetic cardiomyopathy was a major cardiac complications in patients with diabetes, and closely related to high incidence and high mortality of cardiovascular disease. It is well established, diabetic cardiomyopathy was an independent primary disease, and was independent on hypertension, coronary artery disease and other known heart disease. Diabetes was also closely related to the cognitive impairment and dementia, as in Europe, the incidence rate of dementia in diabetes patients older than65is6.4%. Leibson et al reported1455cases of adult onset diabetes,981patients were observed after one year later,101had dementia, of which77cases of senile dementia. The mechanism of multi-organ damage in diabetes has still unclear. The non-enzymatic formation of advanced glycation end products, protein kinase C signaling pathway, oxidative stress, polyol pathway activation and increased hexosamine pathway activity were the some roles involved in. With further research, we found that the multiple organ injury in diabetes, that had interstitial fibrosis, apoptosis-related or pathological changes in synaptic plasticity. The high glucose activiated TSP-1and TGF-β1signaling pathway, then caused the multiple organ damage in patients with diabetes, brought to our concern. Thrombospondin (Thrombospondin, TSP), especially TSP-1, is an important physiological activator. TSP-1is an extracellular glycoprotein molecules, initially in platelets stimulated by thrombin product of a-granule release was found, after study has confirmed the TSP is not a platelet-specific, can be mesangial cells, fibroblasts and other cells, many organizations such as the kidney, heart, cartilage and brain are the product of the TSP gene expression, many different organizations, an important component of the extracellular matrix. Study found that TSP-1peptide by the three chains of the same homologous matrix glycoprotein trimer, each peptide chain from the N-terminal, C-terminal globular domain, procollagen homology region, typel repeats, type2repeats, type3repeat sequences. One typel repeats (thrombospondin typel repeats, TSRs) prepared by the three solution-like motif (TSR1, TSR2, TSR3) composition. Studies have shown that, TSP-1molecule KRFK sequence with L-TGF-β1of the LAP area, thereby changing the spatial configuration of the LAP, and exposure TGF-β1receptor binding site, then become activated TSP-1/L-TGF-β1. The research also showed that thrombospondin in hippocampus are closely related to the establishment of synaptic activation, plasticity the morphology and function. Therefore, we propose the following hypothesis:the diabetic state (such as high sugar stimulation) conditions caused by various types of body cells TSP-1ang TGF-β1activation, thus promoting proliferation of these cells and collagen fibers, leading to fibrosis, and changes of synaptic plasticity, which leads to diabetic multiple organ injury. Recent studies have revealed that interference in the mammalian small RNA (siRNA) through the conservative structure of biological RNA interference to inhibit the expression of the corresponding target genes. RNA interference is highly sequence-specific, can shut down the expression of target proteins. Many studies have confirmed that RNA interference can block many types of mammalian virus replication, inhibition of cancer growth. RNA interference has been developed as a powerful reverse genetics tool and has shown broad prospects for therapeutic application on.
In this study, we investigated the expression of TSP-1and TGF-β1changes under high glucose on hippocampal neurons, design of siRNA on silencing expression of TSP-1gene, and to assess the reduction of TSP-1expression on neuronal morphology and synapse formation. It would provide the in vitro experimental evidence on a target gene therapy of TSP-1in diabetic learning and memory ability injury.
PART Ⅰ TSP-1and TGF-β1expression in penis, myocardium, and hippocampus of diabetic rat
1Objective
Investgating the TSP-1and TGF-(31express in penis, myocardium, and hippocampus of diabetic rat, proved TSP-1and TGF-β1was involved in diabetic above-mentioned organ injury under high glucose.
2Materials and Methods
2.1Preparation of diabetic rats
SD rats were randomly divided into two groups, diabetes rat's model were induced by intraperitoneal injection of streptozotocin (STZ)65mg/kg, regular testing urine, blood glucose and body weight in order to ensure the maintenance of high blood sugar status. While the normal control group rats were injected with normal saline, with same feeding conditions for six weeks.
2.2The organ function tests
2.2.1Behavioral testing: The learning and memory changes in each group of rats were to detect by Morris water maze test (recording the escape latency and search strategy).
2.2.2Erectile function testing: Using subcutaneous injection of apomorphine hydrochloride (APO) solution (150ug/kg) about1ml, observation30min, recording its erection situation.
2.2.3Heart function testing: The rats were killed after full anesthesia thoracotomy, the heart quickly removed, placed in4°C KH solution to remove blood, then quickly transferred and fixed on the Langendorff perfusion device with a modified KH solution by conventional retrograde perfusion constant (76mmHg), observed the end of perfusion balance the amount of cardiac work changes (rate-pressure product, RPP), that is left ventricular developed pressure*heart rate (HR*LVDP).
2.3Pathological changes of organs observation
The rats were killed after anesthesia, penis, heart and brain were taken out and making paraffin slice. Pathological changes of these organs were observed by HE staining, while heart and hippocampus tissue slices were produced embedding and using electron microscope to observe the ultra-micro-structure changes.
2.4Immunohistochemistry and real-time quantitative PCR
The rats were killed after anesthesia, penis, heart and brain were taken out. TSP-1and TGF-β1protein expression were examed by immunohistochemical ABC method, while TSP-1and TGF-β1mRNA expression was detected by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR).
2.5Statistical analysis
Values are mean±SD. The differences in multiple groups were analysised by statistical software SPSS16.0one-way analysis of variance, while the differences between the two groups were analysised by/t test.
3Results
3.1The body weight, blood and urine glucose
Before diabetes model induction, the body weight, blood and urine glucose were no significant differences between the two groups (P>0.05). After6weeks, the diabetic group body weight251.6±27.4g, blood glucose were22.6±3.6mmol/L, urine for the++-+++. Normal rats weighing397.4±21.3g, blood glucose5.1±0.5mmol/L, urine glucose (-). There are significant differences between the two groups (P<0.01).
3.2The organs function
The erectile function was detected in rats with diabetes and erectile erections per unit time and rate were1.4±0.5times/30min and40%. It was significantly lower than the normal control group (2.4±0.8times/30min and100%)(P<0.01).
Heart function tests also showed that diabetic rats RPP was significantly lower than normal rats,(P<0.01).
Morris water maze behavioral experiments showed that normal rats on day1of the escape latency of (40.1±26.0s);2days (24.8±14.2s);3days (13.4±4.8s); through platform number is (6.1±3.8); diabetic rats on day1of the escape latency of (71.6±33.7s);2days (43.4±13.8s);3days (26.9±19.1s), through platform number is (3.2±1.3), the difference was statistically significant (P<0.01).
3.3Pathological changes in penis, myocardium and hippocampus
HE staining of the tissues showed that the thickness of the albuginea penis increased, penis collagen fiber damaged, and lost its characteristic undulating appearance, but also degradation of endothelial and smooth muscle cells in diabetic rats. Diabetic rats heart have varying degrees of interstitial fibrosis and myocardial hypertrophy, myocardial fiber structural appeared sparse. Micro-structure of diabetic rat heart revealed that myocardial myofibril necrosis, dissolution, loss of normal sarcomere structure, and even myocardial apoptosis under electron microscope. Diabetic rat hippocampus shows that mitochondria within neurons disappear, transformed the synaptic structure between neurons, while the number of synapses per unit area of the hippocampus was significantly less than normal rats.
3.4Immunohistochemistry and real-time quantitative PCR assay
TSP-1and TGF-β1protein expression in penis, heart and hippocampus of diabetes rats were significantly up-regulated, in which the penis, heart and hippocampus of TSP-1and TGF-β1in unit area of positive cells were respectively higher than the control group rats (P<0.01). The real-time quantitative PCR showed that TSP-1mRNA expression in penis, myocardium and hippocampus were2.8folds,1.7folds,6.6folds and TGF-β1mRNA expression were6.9folds,4.3folds,1.4folds while compared with the comtrol group rats.
4Conclusion
STZ-induced diabetic rats had increased blood sugar, urine sugar, and decreased the weight, sexual function, cardiac function and learning and memory ability, with the penis, the heart and hippocampus of different degrees of pathological changes. In addition, TSP-1and TGF-β1protein and mRNA expression in dibetes rats were significantly increased while comparing with the normal control rats.
PART Ⅱ The protection of siRNA against TSP-1on high glucose cultured neurons form TSP-1expression changes
1Objective
To explore the siRNA targeting TSP-1can effectively improve the high glucose cultured neuronal morphology and expression of TSP-1, and the protection of neurons and synapses of the contact normal.
2Materials and Methods
2.1Cell culture
SD rats pregnant16days,0.5%sodium pentobarbital anesthesia, opening the uterus, remove the fetus, and isolated hippocampal brain to take. Organization and the purpose of grinding through200mesh, cells were collected, centrifuged and resuspended. Inoculated at1×Polylysine-coated6-well plates and a small round glass slides in48-well culture plates at37℃with5%CO2and95%air.
2.2siRNA preparation and transfection
Each of the siRNA sequence is as follows. TSP-1sence5'-UACACUUGGCACCAGCAAAGCAGGG-3', LacZ primers upstream primer5'-ACCAGAAGCGGRGCCGGAAA-3'primer5'-CCACAGCGGATGGTTCGGAT-3', cell preparation, transfection, and then gently Shakers,5%carbon dioxide,37℃humidified air in the conventional culture for48hours. After transfected for48h, cells were harvested for the next experiments.
2.3Scanning electron microscopy observation
The cell culture, the dry glass slides covered with small round48-well culture plates in culture medium,4℃saline wash1,2.5%glutaraldehyde1h,0.01M PBS washed2timesx15min,1%osmium tetroxide fixed1h,0.1M PBS solution, rinsed twice for15minutes. Gradient alcohol dehydration,100%acetone dehydration20min. With50%isoamyl acetate replacement of15min,100%amyl acetate replacement of15min, the critical point drying, sticky table, gold-plated, observed on the machine.
2.4Immunohistochemistry and Western Blot assay
Immunohistochemistry:0.01M PBS containing0.3%of the rest were washed TRITON X-100(pH value7.4, PBS-T), then immersed in PBS2%normal goat serum for2hours at37℃4℃overnight, TSP-1antibody (1:100) and PBS containing1%bovine serum albumin, washed in PBS (3×5minutes), biotinylated goat anti-rabbit IgG (1:200) were incubated in PBS wash, PBS-T (3x5minutes), at room temperature for2hours, the immune markers diaminobenzdine0.05%, plus0.3%H2O2in PBS, staining, endoscopic control of reaction time, and then dehydrated with graded ethanol and xylene. Which PBS was used instead of primary antibody as negative control.
Western Blot asay:the same amount of protein extract loaded onto SDS/PAGE gel and was separated, then transferred to PVDF membrane, and then with5%skim milk TBST solution closed1hour, then using the antibody at room temperature incubated for2hours, then TBST membranes were washed4times, then use the anti-mouse IgG-HRP (DAKO) incubated for1hour at room temperature, the final protein with the ECL detection system. Protein relative expression=optical density of test protein/internal reference optical density value.
2.5Statistical analysis
Values are mean±SD. The differences in multiple groups were analysised by statistical software SPSS16.0one-way analysis of variance, while the differences between the two groups were analysised by ttest.
3Results
3.1SEM observation
The cultured cells by scanning electron microscopy showed normal group of neurons surface microvilli structural integrity, neuronal links between broad and close, high glucose group of neurons in cell surface microvilli structure loss, the membrane slightly injury, and neurons contact sparse, while the RNA interference group of neurons showed less severe injury.
3.2Immunohistochemistry and Western Blot assay
Detection of neuronal morphology and TSP-1and TGF-β1protein expression to assess the impact of siRNA on neurons. TSP-1and TGF-β1protein expression was significantly reduced in the TSP-1siRNA group while compare with the normal control group (P<0.01). The TSP-1and TGF-β1protein expression of negative transfection group and control group, the difference was not statistically significant (P>0.05).
4Conclusion
Our study implied that interference by siRNA targeting TSP-1can effectively improve the high glucose neuronal morphological and synapses changes and reduce TSP-1and TGF-β1protein expression.
糖尿病(diabetes Mellitus, DM)已成为世界上发病率最高、对人类健康威胁最严重的疾病之一。糖尿病所引起的糖代谢紊乱,导致微血管病变,以及高血糖本身对组织细胞的毒害作用,继而引起一系列并发症,包括糖尿病阳痿、糖尿病心肌病和糖尿病性痴呆等。对于糖尿病阳痿,有资料表明,大约一半患者在诊断为糖尿病10年后有可能合并勃起功能障碍(erectile disfunction, ED),其中约有12%的糖尿病患者ED是其首发表现。高血糖本身对心肌亦有毒害作用,流行病学研究发现,糖尿病患者70%以上死于心血管系统疾病,其中,糖尿病心肌病(diabetic cardiomyopathy)是糖尿病患者的主要并发症之一,其发病率高,危害性大,与糖尿病患者心血管疾病的高发生率和高病死率密切相关。现已公认,糖尿病心肌病是一个独立的原发病,其发病不依赖于高血压、冠状动脉疾病和其他已知心脏疾病。此外糖尿病与认知障碍和痴呆亦有密切相关性：如在欧洲,糖尿病发病率在年龄大于65的痴呆患者中是6.4%。Leibson等报道了1455例成年发病的糖尿病病人中981人经一年观察后,101人产生了痴呆,其中77例为老年性痴呆。
糖尿病上述多器官损伤的机制尚未完全明确,目前认为非酶性糖基化终末产物形成、蛋白激酶C信号通路的激活、氧化应激反应和己糖胺通路活性增高等具有重要作用。随着研究的深入,我们发现上述糖尿病上述脏器损伤均存在间质纤维化、相关细胞凋亡或突触可塑性改变等病理变化,因此高血糖状态下TSP-1和TGF-β1表达的改变和上述糖尿病患者多脏器损伤之间的关系引起了我们关注。血小板反应素(Thrombospondin, TSP),尤其是TSP-1,是重要的生理激活物之一作为一种大分子的细胞外糖蛋白,最初是在被凝血酶刺激的血小板a颗粒释放的产物中被发现,最新研究证实TSP并非血小板特有,可被肾小球系膜细胞、成纤维细胞等多种细胞分泌,许多组织如肾脏、心脏、软骨和脑中都有TSP基因产物的表达,是许多不同组织细胞外间质的重要成分。研究发现TSP-1由3条相同肽链构成的同源三聚体基质糖蛋白,每条肽链由N端、C端球状结构域、原胶原同源区、type1重复序列、type2重复序列、type3重复序列组成。其中type1重复序列(thrombospondin typel repeats,TSRs)由3个备解素样基序(TSR1、TSR2、TSR3)组成。转化生长因子β(transforming growth factor-β,TGF-β)是一个分泌型的多肽信号分子超家族,在哺乳动物组织中存在3种形式的TGF-β,分别是TGF-β1、TGF-β2和TGF-β3,其中TGF-β1所占比例最高,活性最强。激活的TGF-β可使组织纤维化,引起纤维母细胞迁移,抑制胶原酶和其它金属蛋白酶的活性,增加胶原产生,降低胶原降解,促进基质沉积。研究表明,TSP-1分子KRFK序列可与L-TGF-β1的LAP区域结合,从而改变LAP的空间构型,使TGF-β1上与受体结合的位点暴露,成为活化的TSP-1/L-TGF-β1。同时新近研究发现海马区的血小板反应素与突触的建立活化和形态功能可塑性改变均有密切关系。因此,我们提出如下假设：糖尿病状态(如高糖等刺激)条件下引起体内各类细胞TSP-1和TGF-β1表达的改变,从而促进上述细胞纤维和胶原增生,最后导致纤维化,进而引起糖尿病勃起功能障碍、糖尿病心肌病,此外TSP-1表达改变在海马区亦可引起突触可塑性的改变,因此上述通路改变亦可引起糖尿病学习记忆能力的下降。
研究已经表明,在哺乳动物中小干扰RNA(siRNA)能通过对生物学上保守结构的RNA进行干扰,从而抑制相应靶基因的表达。RNA干扰具有高度的序列特异性,能使靶蛋白的表达关闭。许多研究已经证实RNA干扰能阻断多种类型哺乳动物病毒的复制,抑制癌肿的生长。RNA干扰已经被开发成为反向遗传学的一个强有力的工具,并且已显示出广阔的治疗学上的应用前景。因此,我们有理由假设针对性开发TSP-1的小干扰RNA无疑对高血糖状态下神经元的损伤有一定的保护作用。
我们通过研究高血糖状态下对海马区神经元TSP-1和TGF-β1的表达改变,同时设计siRNA对TSP-1的表达进行了基因沉默,并评估TSP-1表达下调对神经元的形态及突触形成的影响。期望能靶向干扰TSP-1表达,为糖尿病学习记忆能力下降的基因治疗提供一定的体外实验依据。
第一部分TSP-1和TGF-β1在糖尿病大鼠阴茎、心肌和海马区表达改变的研究
1目的
研究TSP-1和TGF-β1蛋白和mRNA在糖尿病大鼠阴茎、心肌和海马区表达的改变情况,初步阐明TSP-1和TGF-β1通路参与了糖尿病大鼠高血糖状态下的上述脏器损伤。
2材料与方法
2.1糖尿病大鼠模型的制备
SD大鼠随机分为糖尿病组和正常对照组,其中糖尿病组大鼠腹腔注射链脲佐菌素(streptozotocin, STZ)65mg/kg制成糖尿病模型,定期测尿糖、血糖及体重,以确保维持高血糖状态。正常对照组腹腔注射等量生理盐水,其余条件同上,喂养6周。
2.2各脏器功能检测
2.2.1行为学检测
成模6周后各组大鼠分别进行Morris水迷宫实验(观察指标为逃避潜伏期和搜索策略),以检测糖尿病动物的学习、记忆能力的变化。
2.2.2阴茎勃起功能检测
成模6周后各组大鼠采用颈部皮下注射盐酸阿朴吗啡(APO)溶液(150ug/kg),剂量约1ml,持续观察30min,对其阴茎勃起情况进行检测。
2.2.3心功能检测
成模6周后各组大鼠充分麻醉后处死开胸,迅速取出心脏,置于4℃K-H液中除去血液,然后迅速转移并固定于Langendorff:灌流装置上,用改良K-H液行常规逆行恒压灌注(76mmHg),观察平衡灌注末的心脏作功量(rate-pressure product, RPP),即心率*左室发展压(HR*LVDP)的变化。
2.3各脏器组织的病理变化观察
成模6周后各组大鼠麻醉处死,分别取阴茎组织、心脏和脑,制作石蜡切片HE染色观察其病理改变,同时取心脏和海马区组织块制作电镜包埋和切片,电镜下观察其超微结构的变化。
2.4免疫组化和实时荧光定量PCR法检测TSP-1和TGF-β1的表达
成模6周后各组大鼠麻醉处死,分别取阴茎组织、心脏和脑,制作石蜡切片采用免疫组化ABC法观察上述各组织TSP-1和TGF-β1蛋白的表达,同时Trizol试剂盒提取细胞总RNA,随后逆转录合成cDNA,rt-qPCR法检测TSP-1和TGF-β1的基因转录情况。
2.5统计学分析
所有数值以均数±标准差(mean±D)表示,多组之间采用SPSS16.0统计软件进行单因素方差分析,两组间比较采用t检验。
3结果
3.1体重、血糖和尿糖检测结果
各组大鼠成模前,其体重、血糖和尿糖检测结果两组间无显著性差异,成模6周后,糖尿病组体重251.6±27.4g,血糖基本维持在19.8±2.8mmol/L,尿糖为++~+++。正常组大鼠体重397.4±21.3g,血糖为5.1±0.5mmol/L,尿糖阴性。两组差异有显著性意义(P<0.01)。
3.2各脏器功能
行为学Morris水迷宫实验显示,正常组大鼠第1天的逃避潜伏期为(40.1±26.0)s；第2天为(24.8±14.2)s；第3天为(13.4±4.8)s；穿台次数为(6.1±3.8)；糖尿病组大鼠第1天的逃避潜伏期为(71.6±33.7)s；第2天为(43.4±13.8)s；第3天为(26.9±19.1)s,穿台次数为(3.2±1.3),上述差异有显著性意义(P<0.01)。
检测其阴茎勃起功能发现糖尿病组大鼠单位时间勃起次数和勃起率(1.4±O.5次/30min和40%)明显低于正常对照组大鼠(2.4±0.8次/30min和100%)(P<0.01)。
心功能检测显示糖尿病组大鼠RPP值明显低于正常组大鼠,(P<0.01)。
3.3各脏器病理改变
各脏器组织HE染色观察发现,糖尿病组大鼠阴茎白膜厚度的增加,阴茎胶原纤维破坏,失去了其特有的起伏的外观,同时内皮和平滑肌细胞也退化及间质细胞增殖。糖尿病大鼠心肌细胞有不同程度的间质纤维化和心肌肥厚,心肌纤维出现了结构性的稀疏。电镜下可见糖尿病大鼠心肌微结构出现破坏,心肌细胞肌原纤维片状坏死、溶解,肌小节失去正常结构,甚至出现心肌细胞凋亡或坏死等现象。糖尿病大鼠海马区电镜观察可见神经元内部线粒体嵴消失,神经元之间突触结构蜕变,同时海马区单位面积突触数目明显少于正常组大鼠。
3.4免疫组化和rt-PCR检测结果
免疫组化检测发现,糖尿病大鼠各组织中TSP-1和TGF-β1的蛋白表达较正常对照组均明显上调,其中糖尿病组大鼠阴茎、心脏和海马区TSP-l和TGF-β1的单位面积阳性细胞数量均明显高于正常对照组(P<0.05),同时和实时荧光定量PCR法结果显示TSP-1和TGF-βmRNA表达较正常对照组亦明显上调(分别是对照组的2.8folds,1.7folds,6.6folds和6.9folds,4.3folds,1.42folds).
4结论
STZ诱导的糖尿病大鼠出现血糖增高、尿糖强阳性、体重增加不明显等一般情况改变,而且大鼠勃起功能、心功能和学习记忆能力均不同程度下降,同时伴阴茎、心脏和海马区不同程度的病理改变,此外糖尿病大鼠各组织中TSP-1和TGF-β1的蛋白和：mRNA表达较正常对照组大鼠亦明显上调。
第二部分siRNA靶向干扰TSP-1对高血糖培养神经元形态和TSP-1表达改变的影响
1目的
明确siRNA靶向干扰TSP-1可有效改善高血糖状态下神经元形态改变和TSP-1/TGF-β1的表达,以及保护神经元形态和突触连接。
2材料与方法：
2.1细胞培养
取孕16天SD大鼠,0.5%戊巴比妥钠麻醉,打开子宫,取出胎鼠,取大脑并分离海马。研磨组织并通过200目的滤网,收集细胞悬液,离心、重悬。接种于1×Polylysine包被的6孔培养板以及有小圆玻片的48孔培养板中,于37℃、5%CO2培养箱中培养,随机分为正常培养组、高糖培养组、高糖培养+转染组、高糖培养+阴行转染组、高糖培养+空白转染组。
2.2siRNA制备与转染
各对siRNA序列如下TSP-1sence5'-UACACUUGGCACCAGCAAAGCAGGG-3', LacZ引物序列上游引物
5'-ACCAGAAGCGGRGCCGGAAA-3'下游引物
5'-CCACAGCGGATGGTTCGGAT-3',上述siRNA由英維捷基(上海)贸易有限公司合成。上述细胞准备后,进行转染,而后轻轻振荡混匀,5%二氧化碳、37℃湿化空气中常规培养48小时。
2.3扫描电镜观察
上述细胞培养48小时后,吸干铺有小圆玻片48孔培养板中的培养液,4℃生理盐水洗1次,2.5%戊二醛固定1h,0.01M PBS洗2次,每次15min,1%锇酸固定1h,0.1M PBS溶液漂洗2次,每次15分钟。梯度酒精脱水,100%丙酮脱水20min。用50%醋酸异戊酯置换15min,100%醋酸异戊酯置换15min,临界点干燥,粘台,镀金,上机观察。
2.4免疫组化检测及Western Blot检测
上述细胞培养48小时后,0.01MPBS含0.3%的TRITON X-100(pH值7.4,PBST)分别洗净,然后沉浸含2%正常山羊血清的PBS2小时,TSP-1或TGF-β1抗体(1:100)和含有1%的牛血清白蛋白的PBS中4℃过夜,PBS(3×5min)洗净,生物素标记的羊抗兔IgG(1:200)孵育4h,PBS(3×5min)洗净,免疫标记diaminobenzdine0.05%,加0.3%H202的PBS,染色,镜下控制反应时间,然后用乙醇和二甲苯梯度脱水。其中PBS被用来代替一抗作为阴性对照。
等量提取的蛋白装载到SDS/PAGE胶上并被分离后,再转移到PVDF膜上,然后用含有5%脱脂奶粉的TBST液封闭1小时,再分别用TSP-1或TGF-β1抗体(1:100)在常温下孵育,膜用TBST液洗涤4次,然后用抗鼠IgG-HRP(DAKO)在常温下孵育1小时,最后蛋白用ECL体系进行检测。蛋白相对表达量=待测蛋白的光密度值／内参的光密度值。
2.5统计学分析
所有数值以均数士标准差(mean±SD)表示,多组之间采用SPSS16.0统计软件进行单因素方差分析,两组间比较采用t检验。
3结果
3.1扫描电镜观察
各组培养细胞扫描电镜可见,正常组神经元表面细胞微绒毛结构完整,神经元间联系广泛而紧密,高糖培养组神经元细胞表面微绒毛结构缺失,伴脱落,细胞膜可见轻微破损,神经元间联系稀疏,而RNA转染组神经元上述病变较轻,同时阴性转染组神经元显示和高糖培养组程度类似的损伤。
3.2免疫组化检测及Western Blot检测结果
通过检测神经元TSP-1和TGF-β1蛋白表达来评估TSP-1siRNA对神经元上述蛋白表达的影响,结果发现TSP-1siRNA转染组TSP-1和TGF-β1蛋白的表达与高糖培养组相比显著地减少(P<0.01)。
4结论
通过siRNA靶向干扰TSP-1能有效改善高血糖状态下神经元形态改变和TSP-1和TGF-β1蛋白的表达,同时保护神经元之间的连接和形态。
Background
Diabetes (Diabetes Mellitus, DM) has become the one of highest incidence and the most serious disease to human health. One important mechanism of type2diabetes was insulin resistance and insulin dysfunction, then leading to islet failure. Disorder of glucose metabolism, that leading to microvascular disease, and toxic effects of high blood sugar on tissue cells, could give rise to a series of complications, including diabetic erectile dysfunction, diabetic nephropathy, diabetic cardiomyopathy, and diabetic dementia, etc. The research data indicated that about half of patients diagnosed with diabetes after10years with a possible merger erectile dysfunction (ED). ED was the first manifestation in about12%of ED patients with diabetes. Hyperglycemia itself has toxic effects on cardiac, epidemiological studies have found that more than70%of patients with diabetes died of cardiovascular diseases. Diabetic cardiomyopathy was a major cardiac complications in patients with diabetes, and closely related to high incidence and high mortality of cardiovascular disease. It is well established, diabetic cardiomyopathy was an independent primary disease, and was independent on hypertension, coronary artery disease and other known heart disease. Diabetes was also closely related to the cognitive impairment and dementia, as in Europe, the incidence rate of dementia in diabetes patients older than65is6.4%. Leibson et al reported1455cases of adult onset diabetes,981patients were observed after one year later,101had dementia, of which77cases of senile dementia. The mechanism of multi-organ damage in diabetes has still unclear. The non-enzymatic formation of advanced glycation end products, protein kinase C signaling pathway, oxidative stress, polyol pathway activation and increased hexosamine pathway activity were the some roles involved in. With further research, we found that the multiple organ injury in diabetes, that had interstitial fibrosis, apoptosis-related or pathological changes in synaptic plasticity. The high glucose activiated TSP-1and TGF-β1signaling pathway, then caused the multiple organ damage in patients with diabetes, brought to our concern. Thrombospondin (Thrombospondin, TSP), especially TSP-1, is an important physiological activator. TSP-1is an extracellular glycoprotein molecules, initially in platelets stimulated by thrombin product of a-granule release was found, after study has confirmed the TSP is not a platelet-specific, can be mesangial cells, fibroblasts and other cells, many organizations such as the kidney, heart, cartilage and brain are the product of the TSP gene expression, many different organizations, an important component of the extracellular matrix. Study found that TSP-1peptide by the three chains of the same homologous matrix glycoprotein trimer, each peptide chain from the N-terminal, C-terminal globular domain, procollagen homology region, typel repeats, type2repeats, type3repeat sequences. One typel repeats (thrombospondin typel repeats, TSRs) prepared by the three solution-like motif (TSR1, TSR2, TSR3) composition. Studies have shown that, TSP-1molecule KRFK sequence with L-TGF-β1of the LAP area, thereby changing the spatial configuration of the LAP, and exposure TGF-β1receptor binding site, then become activated TSP-1/L-TGF-β1. The research also showed that thrombospondin in hippocampus are closely related to the establishment of synaptic activation, plasticity the morphology and function. Therefore, we propose the following hypothesis:the diabetic state (such as high sugar stimulation) conditions caused by various types of body cells TSP-1ang TGF-β1activation, thus promoting proliferation of these cells and collagen fibers, leading to fibrosis, and changes of synaptic plasticity, which leads to diabetic multiple organ injury. Recent studies have revealed that interference in the mammalian small RNA (siRNA) through the conservative structure of biological RNA interference to inhibit the expression of the corresponding target genes. RNA interference is highly sequence-specific, can shut down the expression of target proteins. Many studies have confirmed that RNA interference can block many types of mammalian virus replication, inhibition of cancer growth. RNA interference has been developed as a powerful reverse genetics tool and has shown broad prospects for therapeutic application on.
In this study, we investigated the expression of TSP-1and TGF-β1changes under high glucose on hippocampal neurons, design of siRNA on silencing expression of TSP-1gene, and to assess the reduction of TSP-1expression on neuronal morphology and synapse formation. It would provide the in vitro experimental evidence on a target gene therapy of TSP-1in diabetic learning and memory ability injury.
PART Ⅰ TSP-1and TGF-β1expression in penis, myocardium, and hippocampus of diabetic rat
1Objective
Investgating the TSP-1and TGF-(31express in penis, myocardium, and hippocampus of diabetic rat, proved TSP-1and TGF-β1was involved in diabetic above-mentioned organ injury under high glucose.
2Materials and Methods
2.1Preparation of diabetic rats
SD rats were randomly divided into two groups, diabetes rat's model were induced by intraperitoneal injection of streptozotocin (STZ)65mg/kg, regular testing urine, blood glucose and body weight in order to ensure the maintenance of high blood sugar status. While the normal control group rats were injected with normal saline, with same feeding conditions for six weeks.
2.2The organ function tests
2.2.1Behavioral testing: The learning and memory changes in each group of rats were to detect by Morris water maze test (recording the escape latency and search strategy).
2.2.2Erectile function testing: Using subcutaneous injection of apomorphine hydrochloride (APO) solution (150ug/kg) about1ml, observation30min, recording its erection situation.
2.2.3Heart function testing: The rats were killed after full anesthesia thoracotomy, the heart quickly removed, placed in4°C KH solution to remove blood, then quickly transferred and fixed on the Langendorff perfusion device with a modified KH solution by conventional retrograde perfusion constant (76mmHg), observed the end of perfusion balance the amount of cardiac work changes (rate-pressure product, RPP), that is left ventricular developed pressure*heart rate (HR*LVDP).
2.3Pathological changes of organs observation
The rats were killed after anesthesia, penis, heart and brain were taken out and making paraffin slice. Pathological changes of these organs were observed by HE staining, while heart and hippocampus tissue slices were produced embedding and using electron microscope to observe the ultra-micro-structure changes.
2.4Immunohistochemistry and real-time quantitative PCR
The rats were killed after anesthesia, penis, heart and brain were taken out. TSP-1and TGF-β1protein expression were examed by immunohistochemical ABC method, while TSP-1and TGF-β1mRNA expression was detected by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR).
2.5Statistical analysis
Values are mean±SD. The differences in multiple groups were analysised by statistical software SPSS16.0one-way analysis of variance, while the differences between the two groups were analysised by/t test.
3Results
3.1The body weight, blood and urine glucose
Before diabetes model induction, the body weight, blood and urine glucose were no significant differences between the two groups (P>0.05). After6weeks, the diabetic group body weight251.6±27.4g, blood glucose were22.6±3.6mmol/L, urine for the++-+++. Normal rats weighing397.4±21.3g, blood glucose5.1±0.5mmol/L, urine glucose (-). There are significant differences between the two groups (P<0.01).
3.2The organs function
The erectile function was detected in rats with diabetes and erectile erections per unit time and rate were1.4±0.5times/30min and40%. It was significantly lower than the normal control group (2.4±0.8times/30min and100%)(P<0.01).
Heart function tests also showed that diabetic rats RPP was significantly lower than normal rats,(P<0.01).
Morris water maze behavioral experiments showed that normal rats on day1of the escape latency of (40.1±26.0s);2days (24.8±14.2s);3days (13.4±4.8s); through platform number is (6.1±3.8); diabetic rats on day1of the escape latency of (71.6±33.7s);2days (43.4±13.8s);3days (26.9±19.1s), through platform number is (3.2±1.3), the difference was statistically significant (P<0.01).
3.3Pathological changes in penis, myocardium and hippocampus
HE staining of the tissues showed that the thickness of the albuginea penis increased, penis collagen fiber damaged, and lost its characteristic undulating appearance, but also degradation of endothelial and smooth muscle cells in diabetic rats. Diabetic rats heart have varying degrees of interstitial fibrosis and myocardial hypertrophy, myocardial fiber structural appeared sparse. Micro-structure of diabetic rat heart revealed that myocardial myofibril necrosis, dissolution, loss of normal sarcomere structure, and even myocardial apoptosis under electron microscope. Diabetic rat hippocampus shows that mitochondria within neurons disappear, transformed the synaptic structure between neurons, while the number of synapses per unit area of the hippocampus was significantly less than normal rats.
3.4Immunohistochemistry and real-time quantitative PCR assay
TSP-1and TGF-β1protein expression in penis, heart and hippocampus of diabetes rats were significantly up-regulated, in which the penis, heart and hippocampus of TSP-1and TGF-β1in unit area of positive cells were respectively higher than the control group rats (P<0.01). The real-time quantitative PCR showed that TSP-1mRNA expression in penis, myocardium and hippocampus were2.8folds,1.7folds,6.6folds and TGF-β1mRNA expression were6.9folds,4.3folds,1.4folds while compared with the comtrol group rats.
4Conclusion
STZ-induced diabetic rats had increased blood sugar, urine sugar, and decreased the weight, sexual function, cardiac function and learning and memory ability, with the penis, the heart and hippocampus of different degrees of pathological changes. In addition, TSP-1and TGF-β1protein and mRNA expression in dibetes rats were significantly increased while comparing with the normal control rats.
PART Ⅱ The protection of siRNA against TSP-1on high glucose cultured neurons form TSP-1expression changes
1Objective
To explore the siRNA targeting TSP-1can effectively improve the high glucose cultured neuronal morphology and expression of TSP-1, and the protection of neurons and synapses of the contact normal.
2Materials and Methods
2.1Cell culture
SD rats pregnant16days,0.5%sodium pentobarbital anesthesia, opening the uterus, remove the fetus, and isolated hippocampal brain to take. Organization and the purpose of grinding through200mesh, cells were collected, centrifuged and resuspended. Inoculated at1×Polylysine-coated6-well plates and a small round glass slides in48-well culture plates at37℃with5%CO2and95%air.
2.2siRNA preparation and transfection
Each of the siRNA sequence is as follows. TSP-1sence5'-UACACUUGGCACCAGCAAAGCAGGG-3', LacZ primers upstream primer5'-ACCAGAAGCGGRGCCGGAAA-3'primer5'-CCACAGCGGATGGTTCGGAT-3', cell preparation, transfection, and then gently Shakers,5%carbon dioxide,37℃humidified air in the conventional culture for48hours. After transfected for48h, cells were harvested for the next experiments.
2.3Scanning electron microscopy observation
The cell culture, the dry glass slides covered with small round48-well culture plates in culture medium,4℃saline wash1,2.5%glutaraldehyde1h,0.01M PBS washed2timesx15min,1%osmium tetroxide fixed1h,0.1M PBS solution, rinsed twice for15minutes. Gradient alcohol dehydration,100%acetone dehydration20min. With50%isoamyl acetate replacement of15min,100%amyl acetate replacement of15min, the critical point drying, sticky table, gold-plated, observed on the machine.
2.4Immunohistochemistry and Western Blot assay
Immunohistochemistry:0.01M PBS containing0.3%of the rest were washed TRITON X-100(pH value7.4, PBS-T), then immersed in PBS2%normal goat serum for2hours at37℃4℃overnight, TSP-1antibody (1:100) and PBS containing1%bovine serum albumin, washed in PBS (3×5minutes), biotinylated goat anti-rabbit IgG (1:200) were incubated in PBS wash, PBS-T (3x5minutes), at room temperature for2hours, the immune markers diaminobenzdine0.05%, plus0.3%H2O2in PBS, staining, endoscopic control of reaction time, and then dehydrated with graded ethanol and xylene. Which PBS was used instead of primary antibody as negative control.
Western Blot asay:the same amount of protein extract loaded onto SDS/PAGE gel and was separated, then transferred to PVDF membrane, and then with5%skim milk TBST solution closed1hour, then using the antibody at room temperature incubated for2hours, then TBST membranes were washed4times, then use the anti-mouse IgG-HRP (DAKO) incubated for1hour at room temperature, the final protein with the ECL detection system. Protein relative expression=optical density of test protein/internal reference optical density value.
2.5Statistical analysis
Values are mean±SD. The differences in multiple groups were analysised by statistical software SPSS16.0one-way analysis of variance, while the differences between the two groups were analysised by ttest.
3Results
3.1SEM observation
The cultured cells by scanning electron microscopy showed normal group of neurons surface microvilli structural integrity, neuronal links between broad and close, high glucose group of neurons in cell surface microvilli structure loss, the membrane slightly injury, and neurons contact sparse, while the RNA interference group of neurons showed less severe injury.
3.2Immunohistochemistry and Western Blot assay
Detection of neuronal morphology and TSP-1and TGF-β1protein expression to assess the impact of siRNA on neurons. TSP-1and TGF-β1protein expression was significantly reduced in the TSP-1siRNA group while compare with the normal control group (P<0.01). The TSP-1and TGF-β1protein expression of negative transfection group and control group, the difference was not statistically significant (P>0.05).
4Conclusion
Our study implied that interference by siRNA targeting TSP-1can effectively improve the high glucose neuronal morphological and synapses changes and reduce TSP-1and TGF-β1protein expression.
引文
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2. Szendroedi J, Phielix E, Roden M. The role of mitochondria in insulin resistance and type 2 diabetes mellitus. Nat Rev Endocrinol.2011 Sep 13. [Epub ahead of print]
3. Gallagher EJ, Leroith D, Karnieli E. The metabolic syndrome--from insulin resistance to obesity and diabetes. Med Clin North Am.2011;95(5):855-73..
4. Viljoen A, Sinclair AJ. Diabetes and insulin resistance in older people. Med Clin North Am.2011 May;95(3):615-29.
5. Aziz A, Wheatcroft S. Insulin resistance in Type 2 diabetes and obesity:implications for endothelial function. Expert Rev Cardiovasc Ther.2011;9(4):403-7.
6. Kempler P, Amarenco G, Freeman R, et al. on behalf of the Toronto Consensus Panel on Diabetic Neuropathy*. Gastrointestinal autonomic neuropathy, erectile-, bladder-and sudomotor dysfunction in patients with diabetes mellitus:clinical impact, assessment, diagnosis, and management. Diabetes Metab Res Rev.2011 Jul 11. [Epub ahead of print].
7. Wada T, Shimizu M, Toyama T, et al. Clinical impact of albuminuria in diabetic nephropathy. Clin Exp Nephrol.2011 Aug 10. [Epub aheadof print]
8. Thandavarayan RA, Giridharan VV, Watanabe K, et al. Cardiomyopathy and Oxidative Stress:Role of Antioxidants. Cardiovasc Hematol Agents Med Chem. 2011 Sep 9. [Epub ahead of print]
9. Murarka S, Movahed MR. Diabetic cardiomyopathy. J Card Fail. 2010;16(12):971-9.
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