摘要
研究背景
糖尿病导致的下肢缺血是缺血性疾病中的常见症状,具有发生率高、致残率高及病死率高等特点,严重的影响人们的生活质量,成为社会经济发展的沉重负担。对于缺血性疾病,治疗性血管性新生是当前的研究热点。治疗性血管新生主要包括包括干细胞移植、直接注射重组成血管因子的蛋白疗法和基于传递编码成血管因子基因的基因疗法。这些方法在缺血性疾病中取得了一定的疗效,但是其价格昂贵,技术要求高,对具有庞大患病人群但医疗资源相对十分缺乏的我国难以普遍推广。随着脉冲电磁场的发展,在临床中广泛应用于促进骨愈合、治疗肿瘤和一些神经损伤性疾病,取得了较好的疗效,在实验研究方面,已发现脉冲电磁场可促进内皮细胞增殖、加快血管化的作用,因此,本实验研究脉冲电磁场对糖尿病大鼠急性缺血下肢血管再生的影响。
研究目的
建立糖尿病大鼠急性下肢缺血模型,对实验组大鼠给予脉冲电磁场处理,应用激光多普勒、免疫组化、免疫荧光、ELISA、Western-blot等多种方法研究脉冲电磁场对糖尿病大鼠急性下肢缺血模型血管再生的作用,并探讨其机制。
实验方法
1.糖尿病大鼠模型的建立:雄性SD大鼠禁食12小时,以60mg/kg的剂量腹腔注射链脲菌素(streptozotocin, STZ)(溶于0.1 mol/L新鲜配制柠檬酸缓冲液中)。1周后检测空腹血糖,采用鼠尾采血方法,空腹血糖>300mg/dl者为造模成功。
2.急性下肢缺血动物模型的建立选用符合糖尿病标准的SD雄性大鼠,术前常规禁食12小时,自由饮水,腹腔注射3%戊巴比妥进行麻醉,麻醉成功后在大鼠的右侧腹股沟韧带致膝关节间切一纵行切口,分离各层组织,暴露股动、静脉,分离股动脉,于近心端结扎,并切除下端及其分支。术后随机分为实验组和对照组(每组60只),实验组:术后即给予脉冲电磁场治疗(脉冲宽度4.5 ms、频率15 Hz、磁感应强度为12 Gs)。治疗时间为每天2小时,共28天。对照组除给予正常饮食外不给予任何处理。
3.在术后当天及术后7、14、28天运用激光多普勒技术检测脉冲电磁场治疗组及对照组下肢血流灌注情况。并于术后7、14、28天在实验组与对照组每组随机每次处死10只大鼠,分离缺血后肢的肌肉,采用免疫组织化染色法检测CD31、α-SMA的表达;免疫荧光方法检测RECA-1的表达,ElISA检测VEGF、FGF-2;采用Western-blot法检测VEGF、FGF-2、VEGFR2、FGFR1、ERK1/2、P-ERK1/2、P38及P-P38的表达。
实验结果
1.激光多普勒检测显示:在术后、术后7天、14天、28天,实验组缺血右下肢与对侧相应部位的血流比率分别为:0.15±0.013,0.32±0.017,0.64±0.020,0.85±0.021;而对照组分别为:0.15±0.009,0.27±0.014,0.48±0.023,0.61±0.021。在术后14天及28天,实验组血流恢复明显高于对照组(P<0.05),而术后当天及术后7天,实验组与对照组的血流恢复无明显差异(P>0.05)。
2.免疫组化染色结果显示:术后7、14、28天,实验组CD31的表达分别为430.2±18.15/mm~2,677.4±15.63/mm~2,837.2±25.60/mm~2;而对照组为357.8±26.64/mm~2,495.2±25.31/mm~2,619.4±19.24/ mm~2。实验组术后14、28天明显高于对照组(P<0.05),而术后7天两组无显著性差异(P>0.05)。实验组α-SMA的表达在7、14、28天分别为22.6±1.91/mm~2,35.8±2.08/mm~2和50.6±3.08/mm~2;对照组为18.2±1.07/mm~2,25.6±1.94/mm~2和32.4±1.72/mm~2,两组α-SMA的表达对比结果与CD31一致。
3.免疫荧光染色可见:术后7、14、28天,实验组RECA-1表达分别为:96.4±4.00/mm~2,179.8±6.95/mm~2,253.0±8.67/mm~2;对照组为89.6±4.51/mm~2, 150.4±5.35/mm~2,184.6±6.09/mm~2;实验组在14、28天时明显高于对照组(P<0.05);而7天时两者差异无统计学意义(P>0.05)。
4.VEGF、FGF-2及其受体检测结果:实验组7、14、28天等不同时间点VEGF的ELISA检测值分别为38.6±6.27pg/ml,91.4±10.51pg/ml,59.8±6.48 pg/ml;对照组为40.4±5.83 pg/ml,80.4±10.88pg/ml,51.6±2.90 pg/ml;两组相比差异无统计学意义(P >0.05),Western blot的结果与ELISA一致。实验组FGF-2的ELISA检测值在各个时间段内分别为13.4±1.91pg/ml,28.4±3.67 pg/ml,47.4±6.80 pg/ml;对照组分别为:7.6±1.21pg/ml,11.8±1.53 pg/ml,10.8±2.17 pg/ml,在各个时间点实验组明显高于对照组(P<0.05),Western的结果与之一致。同时Western对VEGF受体VEGFR2检测,未发现差异有统计学意义(P>0.05),而对FGF-2受体FGFR1检测,我们发现,实验组在各个阶段明显高于对照组(P<0.05)。
5.P-ERK1/2、T-ERK1/2、P-P38、T-P38检测结果通过Western blot方法检查,实验组P-ERK1/2与T-ERK1/2比值在各个时间段均高于对照组(P<0.05),而两组P-P38与T-P38比值无显著性差异(P >0.05)。
结论
一定频率、脉冲宽度、磁感应强度的PEMF可促进糖尿病大鼠急性下肢缺血的血管新生,在脉冲电磁场促血管新生作用过程中,FGF-2是重要的促血管新生因子,而MAPK/ERK1/2是FGF-2促进血管新生的一种重要信号转导途径。
Background
Ischemic diseases, particularly in diabetics, result in significant morbidity and mortality and have a profound economic impact. To ischemic disease, therapeutic angiogenesis is the focus of current research.Stem and progenitor cell therapy, protein of promoting angiogenesis factor therapy and gene therapy were the main methods to improve neovascularization and function of ischemic tissue. These methods in ischemic diseases have certain effect, but they were expensive, technically, especially to our country of lack of medical resources,it is still very difficult for the general clinical promotion. Therefore, we need an effective treatment..
It is well documented that electromagnetic fields play a role in the repair of human issues.Electrical stimulation by means of capacitive coupling, combined magnetic fields and pulsed electromagnetic fields has been used for over 30 years to augment healing. and subsequently demonstrated electromagnetic fields increase angiogenesis in animal experiments . Based on the above findings, we hypothesized that pulsed electromagnetic fields might be beneficial in diabetic limb ischemia . In this study, we discuss pulsed electromagnetic fields on lower limb ischemia in diabetic rats the impact of angiogenesis, and explore its mechanism.
AIM
To observe the effects of pulsed electromagnetic fields (PEMF) on acute hindlimb ischemia diabetic rats in microcirculation angiogenesis. Establishing diabetic rats acute hindlimb ischemia model, the experimental group was given pulsed electromagnetic field treatment, analysis effects of pulsed electromagnetic fields on hindlimb ischemia diabetic rat microcirculation angiogenesis, and explore its mechanism.
Methods
Diabetes were induced by intraperitoneal injection of streptozotocin(60μg/g body with in 50 mmol/L citric acid buffer, pH 4.5) once to Sprague-Dawley rats. Then, 1 week later, blood was obtained periorbitally after 8 hours of fasting. Only rats with blood glucose values >300 mg/dl were kept in the protocol and randomized for experiment.
Diabetic rats were anesthetized intraperitoneally with 3% pentobarbital. An incision was made along the inner right hind limb along the line of the femoral artery and vein. The proximal end of the femoral artery was tied with 4-0 silk suture .The femoral artery was dissected free from the limb and its peripheral branches. The distal end was severed, the artery was removed, and the skin was sutured closed. After surgery, rats were randomly divided into experimental group and control group(n=60).After operation, rats in experiment group were exposed to pulsed electromagnetic fields for 2 hours every day while those in control group were not given any treatment. Laser-Doppler perfusion measurements was used to determine the blood flow of ischemia hindlimbs respectively on the day 0, 7, 14 and 28 after operation, and immunohistochemistry analysis of CD31 andα-SMA were used to evaluate the changes in angiogenesis,immunofluorescence to detect expression of RECA-1. ELISA and Western blot were analyzed for the protein levels of VEGF,VEGFR2,FGF-2,FGFR1, P-ERK1/2,ERK1/2, P-P38 and P38.
Results
The perfusion ratios were significantly higher in pulsed electromagnetic field-treated diabetic rats at day 14 and 28 compared with those in controls (0.64±0.02、0.85±0.021vs0.48±0.023、0.61±0.021,P<0.05)。CD31 density in tissues measured by immunohistochemistry significantly increased in pulsed electromagnetic field-treated groups on 14d and 28d(677.4±15.63/mm~2、837.2±25.60/mm~2vs495.2±25.31/mm~2、619.4±19.24/mm~2 , P<0.05 ) . Immunohistochemical analysis revealed significantly higher numbers ofα-SMA in animals exposed to PEMF at day 14 and 28(35.8±2.08/mm~2、50.6±3.08/mm~2vs 25.6±1.94/mm~2、32.4±1.72/mm~2,P<0.05), . Immunofluorescence analysis of RECA-1 density in tissues were significantly increased in pulsed electromagnetic field–treated groups at day 14 and 28 ( 179.8±6.95vs150.4±5.35/mm~2,253.0±8.67vs184.6±6.09/ mm~2,P<0.05).The levels of FGF-2 and FGFR1 in ischemic hindlimbs significantly increased in PEMF group at all time points and the same were p-ERK1/2 /total ERK1/2. No significant differences were observed in VEGF, VEGFR2 and p-p38/total p38 between the two groups.
Conclusion
Pulsed electromagnetic fields can promote angiogenesis on acute hindlimb ischemia diabetic rat by up-regulation of FGF-2. As an indispensable signal pathway for FGF2-induced angiogenesis, MAPK/ERK1/2 is responsible for coupling FGFR1 to multiple downstream pathways to mediate blood vessel formation.
引文
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