Mito-cpYFP cDNA转基因小鼠的建立及其自由基分布规律的初步探讨

目的:利用可实时动态测量超氧阴离子自由基的环状排列黄色荧光蛋白(circularly permuted yellow fluorescent protein,cpYFP)构建可实时动态监测活体动物体内自由基的转基因动物模型。方法:应用转基因技术将载有可定位于线粒体内的cpYFP(mitochondria-targeted cpYFP,mito-cpYFP)的载体pRP(Exp)-CAGG>mitocpYFP注射到C57BL/6小鼠的受精卵中,采用PCR及RT-PCR的方法鉴定阳性小鼠,通过荧光观察自由基的分布并辅助鉴定阳性小鼠。结果:PCR、RT-PCR及荧光观察结果表明mito-cpYFP cDNA转基因小鼠构建成功。通过与野生型C57BL/6小鼠交配,得到F1、F2和F3代的新生幼鼠共计494只,其中,阳性幼鼠为255只,阳性率为51.6%。转基因阳性鼠体内出现了绿色荧光,其解剖伤口、肠道、脑部及口腔黏膜等荧光明显,而肾脏、肝脏及胸腺等荧光则较弱,耻骨联合部和膀胱出现绿色荧光,但约0.5 h后消失。结论:我们成功制备了可稳定表达并遗传mito-cpYFP的转基因小鼠品系。在活体动物体内,自由基在黏膜层含量较多。此模型可研究自由基在生物体内的含量及分布规律,为后续生物自由基作为信号分子在机体内传导途径的研究提供有效的工具。

铁皮石斛的抗氧化性与其脏腑归经作用差异性的研究

目的研究铁皮石斛(Dendrobium offcinale Kimura et Migo)的体外抗氧化活性及通过测量经络电压探索铁皮石斛的归经作用。方法测定铁皮石斛汤剂的氧化还原电位值、pH值、总抗氧化能力。召集20名健康志愿者按随机数字表法分为2组,试验组(服用铁皮石斛汤剂,8 g铁皮石斛粉冲泡于80 mL水中),对照组(饮用等量水),每组10名。服用药物前后进行人体经络电压的测量,选取12条经络上(肺经、肝经、脾经、肾经、胃经、心经、大肠经、胆经、心包经、三焦经、小肠经、膀胱经)的合穴和原穴作为经络电压的测量点,均以合穴为正极,原穴为负极,记录经络电压的变化。结果通过pH的测定结果可以看出铁皮石斛呈现弱酸性。与蒸馏水比较,铁皮石斛氧化还原电位降低,抗氧化能力及总抗氧化能力升高(P<0.01)。在经络电压测定中,与对照组比较,试验组大肠经、胆经、心包经、三焦经、小肠经和膀胱经的电压升高,肺经、肝经、脾经、肾经、胃经和心经的电压则无显著变化。结论铁皮石斛对可以显著增加大肠经、胆经、心包经、三焦经、小肠经和膀胱经所属的六腑经络的电压,对肺经、肝经、脾经、肾经、胃经和心经所属的五脏经络的电压则无显著作用。经络电压的变化规律与铁皮石斛的抗氧化性具有一定的关联。

Effects of TAT-SOD at Acupoints on Essential Hypertension by Monitoring Meridians Electrical Potential

Objectives:To investigate the effect on essential hypertension of the topical application of TAT-Cu,Zn-superoxide dismutase(TAT-SOD)at left acupoint Zusanli(ST 36),and to observe whether the change of electrical potential difference(EPD)can be related to the change of blood pressure.Methods:Sixteen patients with essential hypertension and 16 healthy subjects were included in the study.EPD between the left acupoints of Yanglingquan(GB 34)and Qiuxu(GB 40)was firstly screened out for the EPD detection.An intracellular superoxide quenching enzyme,TAT-SOD,was topically applied to the acupoint ST 36 within an area of 1 cm^2 once a day,and the influence on EPD was investigated.The dosage applied to TAT-SOD group(n=8)was 0.2 mL of 3000 U/mL TAT-SOD cream prepared by adding purified TAT-SOD to a vehicle cream,while placebo group(n=8)used the vehicle cream instead.The left acupoints of Yanglingquan(GB 34)and Qiuxu(GB 40)were selected for EPD measurement after comparing EPD readings between 5 acupoints on each of all 12 meridians.Results:EPDs between the left acupoints of GB 34 and GB 40 for 16 patients of essential hypertension and 16 healthy subjects were 44.9±6.4 and 5.6±0.9 mV,respectively.Daily application of TAT-SOD for 15 days at ST 36 of essential hypertension patients significantly decreased systolic blood pressure(SBP)and diastolic blood pressure(DBP)of 179.6 and 81.5 mm Hg to 153.1 and 74.1 mm Hg,respectively.Responding to the change in blood pressure,EPD between the left acupoints of GB 34 and GB 40 also declined from 44.4 to 22.8 mV with the same trend.No change was observed with SBP,DBP and EPD between the left acupoints of GB 34 and GB 40 with the daily application of the placebo cream.Conclusion:Enzymatic scavenging of the intracellular superoxide at ST 36 proved to be effective in decreasing SBP and DBP.The results reconfirm the involvement of superoxide anions and its transportation along the meridians,and demonstrate that EPD between acupoints may be an indicator to reflect its functioning status.Moreover,prellmlnary results suggest a close correlatlon between EPD and blood pressure readings,implying a possibility of using EPD as a sensitive parameter for blood pressure and to monitor the effect of antihypertensive treatment.