【作者】 陈彬；

【导师】 柳大烈；

【作者基本信息】 南方医科大学 ， 整形外科学， 2014， 博士

【摘要】 第一章：促渗剂二甲基亚砜在小鼠透皮实验中的最适浓度研究研究目的：检测二甲基亚砜(dimethylsulfoxide, DMSO)的不同浓度是否影响报告基因pORF-LacZ透过小鼠皮肤的转染效率以及探讨DMSO在促进报告基因pORF-LacZ经皮渗透效率过程中的最适浓度,为二期实验中应用最适浓度的DMSO制剂促进pORF-LacZ基因透过皮肤(皮瓣)作进一步研究奠定理论基础。研究方法：选取8周龄清洁级健康雄性Balb/c小鼠(购买于南方医科大学动物实验中心)20只,体重20—-22g,实验室环境：环境温度为22.0±2.0℃,相对湿度55.6%±4%。按浓度不同随机分为0%DMSO组,5%DMSO组,10%DMSO组,30%DMSO组4组,每组5只小鼠。DMSO配方的浓度主要以在总体积150μl中DMSO所占百分比为基础,各配方中DMSO的浓度分别为30%(formula1),10%(formula2),5%(formula3),0%(formula4),每个配方中均含有150ug的报告基因pORF-LacZ质粒。按如下步骤操作：每只小鼠以1%戊巴比妥钠溶液进行腹腔注射麻醉后固定,于小鼠背部中央设计一个蒂在头侧的模拟随意型皮瓣(蒂宽1cm、长3cm),在转染皮肤之前,脱毛膏背部脱毛,3-5min后,用0.9%氯化钠溶液(生理盐水)处理15min,随后于小鼠背部皮瓣相应区域涂抹20u1含0.05%维甲酸(retinoic acid, RA)(维甲酸溶于0.9%氯化钠溶液中)、20u1的浓度为1%Toll样受体2(Toll like receptor2,TLR2)抑制剂--脂质羊毛硫氨酸肽(lipolanthionine peptides,LP)及DMSO混合制剂,这个过程每隔一天进行一次,连续1周。到第7天,当涂抹最后一次DMSO、LP和RA混合制剂4小时后,用移液器吸取10ul的混合物(150ug的pORF-LacZ和15u1的不同浓度DMSO溶于水中,总体积为150u1)均匀地涂抹于小鼠背部皮瓣上,这个过程进行3天,每天1次。第4天于皮瓣下部收集透过的质粒,样本分成两份,实时荧光定量PCR检测pORF-lacz基因透过量。用水合氯醛麻醉实验小鼠,然后进行处死,并收集它们的背部皮肤样本。样本先用PBS溶液处理,而后被固定在带有一定扩散面积的扩散池中,此处包括有测定位点隔间和受体隔间,且两种隔间分别被安装在扩散池的两侧。皮肤表面朝向测定位点隔间的一侧,受体隔间中充满RPMI1640培养基,此实验过程中,pEGFP-N1质粒(5ug)被加入到培养基中作为内参。已经准备好的基于不同浓度的DMSO等混合制剂被加到测定位点隔间。受体隔间中的培养基在整个实验当中以45转/min的转速进行搅拌。整个装置被置于37.0±0.5℃的环境条件下。取样后,背部皮肤在2%的甲醛和0.2%的戊二醛的混合液当中浸泡2-4小时,溶液的温度保持在4℃的环境下,然后在室温下先后用三种不同浓度的PBS(PH7.4)洗涤60min,接着所有的样本在37.0℃的环境下被置于1mg/ml X-gal当中浸泡过夜。第2天,所有的样本在室温条件下先后用三种不同浓度的PBS(PH7.4)洗涤60min,接着在室温条件下被放置于10%福尔马林溶液当中浸泡24小时,然后用水冲洗过夜。研究结果：在不同浓度DMSO混合制剂的作用下,报告基因pORF-lacz透皮表达量分别为(F=337.718)：a.当DMSO浓度为10%时,pORF-lacz基因透皮表达量最高(5.378±0.346μg/cm2),显著性高于另外三组,P=0.000,差异具有统计学意义；b. DMSO浓度为0时,pORF-lacz基因透皮表达量最低(0.940±0.095μg/cm2),显著性低于另外三组,P=0.000,差异具有统计学意义；c.而DMSO浓度为5%和30%时,pORF-lacz基因透皮表达量居中(分别为2.376±0.190μg/cm2和2.516±0.199μg/cm2),两者之间差异并无统计学意义。结论：当DMSO浓度为10%时,报告基因pORF-lacz透皮表达量最高,可确定此为促进报告基因透过小鼠皮肤的最适使用浓度,因此在本课题进一步研究中,将选用浓度为10%的DMSO作为关键促渗剂。第二章：RA和LP对小鼠皮肤claudin-4和ZO-1影响的实验研究研究目的：探讨经10%DMSO作为促渗剂,维甲酸(retinoic acid, RA)和Toll样受体2(Toll like receptor2,TLR2)抑制剂--脂质羊毛硫氨酸肽(lipolanthionine peptides,LP)按照分组施加干预后,小鼠皮肤连接蛋白claudin-4和ZO蛋白-1(ZO-1)表达的改变情况和皮肤相应透过功能的变化规律。研究方法：选取8周龄清洁级健康雄性Balb/c小鼠(购买于南方医科大学动物实验中心)30只,体重20-22g。实验室环境：环境温度为22.0±2.0℃,相对湿度55.6%±4%。将小鼠随机分为5组：RA+LP+DMSO+pORF-LacZ; DMSO+pORF-LacZ; LP+DMSO+pORF-LacZ; RA+DMSO+pORF-LacZ;生理盐水对照组,每组6只。按如下步骤操作：选取8周龄小鼠背部一定面积(1cm×1cm)的皮肤,用脱毛膏脱出此选定皮肤区域的毛发,3-5min后,用0.9%氯化钠溶液处理,15mmin后,按照分组,小鼠背部皮肤分别被涂抹20u1含0.05%维甲酸(RA)(维甲酸溶于0.9%氯化钠溶液中)+10%DMSO制剂和20u1的浓度为1%TLR2抑制剂LP+10%DMSO制剂,或者涂抹两者的联合制剂+10%DMSO制剂,或者10%DMSO制剂,这个过程每隔一天进行一次,连续1周,对照组作同样处理。第7天,最后用上述相应制剂处理后,施用最适浓度10%DMSO配置的pORF-LacZ质粒,处理过程进行3天,每天1次,第4天处死小鼠,收集皮肤标本,然后运用皮肤免疫组化和western blot检测紧密连接蛋白claudin-4和ZO蛋白-1(ZO-1)的表达情况。背部皮肤在2%的甲醛和0.2%的戊二醛的混合液当中浸泡2-4小时,溶液的温度保持在4℃C的环境下,然后在室温下先后用三种不同浓度的PBS(PH7.4)洗涤60mmin,接着所有的样本在37.0℃C的环境下被置于1mg/ml X-gal当中浸泡过夜。第2天,所有的样本在室温条件下先后用三种不同浓度的PBS(PH7.4)洗涤60mmin,接着在室温条件下被放置于10%福尔马林溶液当中浸泡24小时,然后用水冲洗过夜。研究结果：1.根据HE染色图片所示：DMSO、RA和LP干预后的实验组小鼠皮肤角质层细胞连接间隙增大,细胞连接疏松,生理盐水对照组小鼠的皮肤并未出现上述现象；所有的实验组小鼠皮肤并未出现病理性的破坏。在各实验组中,DMSO组和RA+DMSO组小鼠皮肤略微出现了皮肤乳头状突起、皮肤水肿、中性粒细胞聚集和毛细管扩张等现象,与之相比,LP+DMSO组和RA+LP+DMSO组的小鼠皮肤水肿现象减少,毛细血管扩张现象有所缓解。2.免疫组化实验结果：(1)claudin-4的表达情况(F=16.174,P=0.000)：a.与盐水对照组相比,RA+LP+DMSO+pORF-LacZ、LP+DMSO+pORF-LacZ两组目的蛋白-claudin-4的表达量(分别为29.827±2.849、32.932±4.890)显著低于前者的蛋白表达(42.830±3.865)(p<0.01),差异具有显著统计学意义；b.与RA+DMSO+pORF-LacZ相比,RA+LP+DMSO+pORF-LacZ目的蛋白-claudin-4的表达量(29.827±2.849)显著低于前者的蛋白表达(39.388±2.053)(p<0.01),差异具有显著统计学意义；c.LP+DMSO+pORF-LacZ目的蛋白-claudin-4的表达量(32.932±4.890)低于RA+DMSO+pORF-LacZ组的蛋白表达(39.388±2.053)(p<0.05),差异具有统计学意义；d.与DMSO+pORF-LacZ组相比,RA+LP+DMSO+pORF-LacZ> LP+DMSO+pORF-LacZ两组目的蛋白-claudin-4的表达量(分别为分别为29.827±2.849、32.932±4.890)显著低于前者的蛋白表达(40.202±1.872)(p<0.01),差异具有统计学意义。(2)zo-1蛋白表达情况(F=97.827,P=0.000)：a.与盐水对照组相比,RA+LP+DMSO+pORF-LacZ、LP+DMSO+pORF-LacZ、 RA+DMSO+pORF-LacZ、DMSO+pORF-LacZ四组目的蛋白—zo-1的表达量(分别为21.367±1.892、31.613±0.911、32.047±1.976、34.697±1.748)显著低于盐水对照组的蛋白表达量(41.473±2.174)(p<0.01),差异具有显著统计学意义；b.RA+LP+DMSO+pORF-LacZ目的蛋白——zo-1的表达量(21.367±1.892)显著低于RA+DMSO+pORF-LacZ组的蛋白表达量(32.047±1.976)(p<0.01),盐水对照组目的蛋白表达量(41.473±2.174)显著高于RA+DMSO+pORF-LacZ组的蛋白表达量(32.047±1.976)(p<0.01),差异具有显著统计学意义；c.DMSO+pORF-LacZ目的蛋白—-zo-1的表达量(34.697±1.748)高于RA+DMSO+pORF-LacZ组的蛋白表达(32.047±1.976)(p<0.05),差异具有统计学意义；d.与DMSO+pORF-LacZ组相比,RA+LP+DMSO+pORF-LacZ、 LP+DMSO+pORF-LacZ组目的蛋白—zo-1的表达量(分别为21.367±1.892、31.613±0.911)显著低于前者(34.697±1.748)(p<0.01),差异具有显著统计学意义。e.RA+LP+DMSO+pORF-Lac组目的蛋白—zo-1的表达量显著低于LP+DMSO+pORF-LacZ组表达量(分别为21.367±1.892、31.613±0.911)(p<0.01),差异具有显著统计学意义。3.Western blot实验结果：(1)claudin-4的表达情况(F=124.557,P=0.000)：a.与盐水对照组相比,RA+LP+DMSO+pORF-LacZ、LP+DMSO+pORF-LacZ、 RA+DMSO+pORF-LacZ三组目的蛋白-claudin-4的表达量(分别为0.208±0.018、0.241±0.020、0.285±0.017)显著低于前者的蛋白表达(0.458±0.031)(p<0.01),差异具有统计学意义；b.与DMSO+pORF-LacZ组相比,RA+LP+DMSO+pORF-LacZ、 LP+DMSO+pORF-LacZ、RA+DMSO+pORF-LacZ三组目的蛋白-claudin-4的表达量(分别为0.208±0.018、0.241±0.020、0.285±0.017)显著低于前者的蛋白表达(0.410±0.030)(p<0.01),差异具有统计学意义。c.与RA+DMSO+pORF-LacZ组相比,RA+LP+DMSO+pORF-LacZ、 LP+DMSO+pORF-LacZ组目的蛋白-claudin-4的表达量(分别为0.208±0.018、0.241±0.020)显著低于前者的蛋白表达(0.285±0.017)(p<0.01),差异具有统计学意义；d. RA+LP+DMSO+pORF-LacZ组目的蛋白-claudin-4的表达量低于LP+DMSO+pORF-LacZ组(分别为0.208±0.018、0.241±0.020)(p<0.05),差异具有统计学意义；(2)zo-1蛋白表达情况(F=64.080,P=0.000)：a.与盐水对照组相比,RA+LP+DMSO+pORF-LacZ、LP+DMSO+pORF-LacZ、 RA+DMSO+pORF-LacZ三组目的蛋白—zo-1的表达量(分别为0.541±0.054、0.789±0.076、0.955±0.064)显著低于前者的蛋白表达(1.129±0.093)(p<0.01),差异具有统计学意义；b.与DMSO+pORF-LacZ相比,RA+LP+DMSO+pORF-LacZ、 LP+DMSO+pORF-LacZ两组目的蛋—zo-1的表达量(分别为0.541±0.054、0.789±0.076)显著低于前者的蛋白表达(1.067±0.069)(p<0.01), RA+DMSO+pORF-LacZ目的蛋白—zo-1的表达量(0.955±0.064)低于DMSO+pORF-LacZ组蛋白表达(1.067±0.069)(p<0.05),差异具有统计学意义；c.与RA+DMSO+pORF-LacZ组相比,RA+LP+DMSO+pORF-LacZ、 LP+DMSO+pORF-LacZ两组目的蛋白—zo-1的表达量(分别为0.541±0.054、0.789±0.076)显著低于前者蛋白表达(0.955±0.064)(p<0.01),DMSO+pORF-LacZ目的蛋白—-zo-1的表达量(1.067±0.069)高于RA+DMSO+pORF-LacZ组的蛋白表达(0.955±0.064)(p<0.05),差异具有统计学意义。d. RA+LP+DMSO+pORF-LacZ组目的蛋白—zo-1的表达量显著低于LP+DMSO+pORF-LacZ组(分别为0.541±0.054、0.789±0.076)(p<0.05),差异具有统计学意义。结论：LP/RA/DMSO单独或者联合干预均能不同程度地降低小鼠皮肤claudin-4、zo-1蛋白表达量,其中,LP+RA+DMSO干预的效果最为明显,LP+DMSO次之,RA+DMSO再次,说明通过TLR2抑制剂LP抑制TLR2信号通路可以下调小鼠皮肤连接蛋白claudin-4和ZO-1的表达,降低皮肤紧致度,改变紧密连接(tight junction,TJ)的固有结构,从而为增加报告基因pORF-LacZ对小鼠皮肤的透过效率创造条件。但是此过程中claudin-4、ZO-1的表达降低和TJ皮肤屏障作用改变的程度和时限需要进一步研究和评估,以保障皮肤正常的防御能力不受影响。TLR2抑制剂LP不仅能够增强小鼠皮肤对药物或者基因的透过效率,同时也能减轻DMSO所引起的微小皮肤损害,可以作为一种有效而安全的药物或者基因透过皮肤的增强剂。本实验为今后将治疗基因或者药物通过透皮传输发挥治疗作用奠定研究基础。第三章：RA和LP对pORF-lacz基因透皮效率影响的实验研究研究目的：经10%DMSO作为促渗剂,维甲酸(RA)和TLR2抑制剂--脂质羊毛硫氨酸肽(LP)按照分组施加干预后,测定报告基因pORF-lacz透过小鼠皮肤进入血液中数量的情况,以此探讨LP/RA/DMSO对基因透皮效率的影响。研究方法：选取8周龄清洁级健康雄性Balb/c小鼠(购买于南方医科大学动物实验中心)30只,体重20-22g。实验室环境：环境温度为22.0±2.0℃,相对湿度55.6%±4%。将小鼠随机分为5组：RA+LP+DMSO+pORF-LacZ; DMSO+pORF-LacZ; LP+DMSO+pORF-LacZ; RA+DMSO+pORF-LacZ;生理盐水对照组,每组6只。按如下步骤进行操作：选取8周龄小鼠背部一定面积(1cm×1cm)的皮肤,用脱毛膏脱出此选定皮肤区域的毛发,3-5min后,用0.9%氯化钠溶液处理,15min后,按照分组,小鼠背部皮肤分别被涂抹20u1含0.05%维甲酸(RA)(维甲酸溶于0.9%氯化钠溶液中)+10%DMSO制剂和20u1的浓度为1%TLR2抑制剂LP+10%DMSO制剂,或者涂抹两者的联合制剂+10%DMSO制剂,或者10%DMSO制剂,这个过程每隔一天进行一次,连续1周,对照组作同样处理。第7天,最后用上述相应制剂处理后,施用最适浓度10%DMSO配置的pORF-LacZ质粒,处理过程进行3天,每天1次,第4天处死小鼠,收集血液标本,随后应用RT-PCR检测血液中的pORF-LacZ质粒表达量。研究结果：a.盐水对照组的pORF-LacZ基因透皮表达量最低(0.897±0.228μg/cm2), RA+LP+DMSO组的pORF-LacZ基因透皮表达量则最高(5.310±0.273μg/cm2),差异具有统计学意义(p<0.01)；b. LP+DMSO组(3.632±0.219μg/cm2)、RA+DMSO组(2.948±0.289μg/cm2)和DMSO组(2.070±0.23μg/cm2)的基因透皮表达量都低于RA+LP+DMSO组(5.310±0.273μg/cm2)(p<0.05)、显著高于盐水对照组(0.897±0.228μg/cm2)(p<0.01),差异具有统计学意义。c. LP+DMSO组、RA+DMSO组和DMSO组三组之间比较：LP+DMSO组的基因透皮表达量(3.632±0.219μg/cm2)最高,DMSO组基因透皮表达量(2.070±0.231μg/cm2)则最低,差异具有统计学意义(p<0.05)。F=265.903,P=0.000结论：LP/RA/DMSO单独或者联合干预均能不同程度地提高报告基因pORF-LacZ的透皮表达量,其中,LP+RA+DMSO干预的效果最为明显,LP+DMSO次之,RA+DMSO再次,与干预后小鼠皮肤claudin-4、zo-1蛋白表达量下降程度相一致,说明通过LP抑制TLR2信号通路可以增加报告基因pORF-LacZ对小鼠皮肤的透过效率,其机制可能是通过下调小鼠皮肤连接蛋白claudin-4和ZO-1的表达,降低皮肤紧致度,暂时打破紧密连接(tight junction,TJ)所起的皮肤屏障作用。今后将进一步研究如何改进LP/RA/DMSO的配比效率,并对某些具体药物或者治疗基因的透皮表达量进行详细研究,观察LP/RA/DMSO制剂对药物和基因透皮吸收并改善皮瓣缺血损伤、提高皮瓣成活率的作用。更多还原

【Abstract】 The first part:The Research of optimum concentration of dimethyl sulfoxide in the transdermal experimentsObjectives:To detect Whether the effects of DMSO concentration affect the gene transfection efficiency and promote the efficiency of percutaneous penetration optimum concentration, identify the best option which concentration is the best one.Methods:24male Balb/c mice which are8weeks old (which are bought from southern medical university),weight:20-22g.We randomly divided them into5groups, fed with ambient temperature of22±2℃and relative humidity of55.6%±4%.Four groups in experiment:0%DMSO,5%DMSO,10%DMSO,30%DMSO,there are5mice in every group.According to a total volume of150ul,Different concentrations of DMSO formulations primarily are the basis of the percentage share of DMSO.Each formulation contains150ug of pORP-LacZ plasmid.The concentration of DMSO in each formulation is30%(formula1),10%(formula2),5%(formula3),0%(formula4).The experimental procedure was operated as follows:after the mice fixed,anesthesia by intraperitoneal injection of1%sodium pentobarbital solution,hair removal cream removed the back hair,3to5minutes later, the mice were treated by concentration of0.09%saline water.In the central,design a pedicle flap random simulation cephalic (pedicle width of lcm and length3cm).Before the transfection of skin, mice’s skin was treated with hair removal cream.15minutes later,after then, It dealt the back skin with0.05%of the Retinoic acid concentration and1%of LP.The procedure was operated once every other day, the process was operated7days.when it was the seventh day,the retinoic acid mixture was painted,4hours later,We pipetted lOul mixture (150ug of pORF-LacZ and15ul of different concentration of DMSO in water, the overall volume is150ul) to paint on the back skin of mice,The process was carried out3days,1time every day.Collect the pORF-LacZ through the lower flap plasmid on the fourth day,Sample was divided into the two parts.RT-PCR detected the transdermal expression of pORF-lacz.After depilated3-5min, it dealt the skin with0.09%concentration of saline for15min. After then, it dealt the back skin with0.05%of the Retinoic acid concentration and1%of LP.The procedure was operated once every other day, the process was7days.On the seventh day,chloral hydrate anesthetized mice,then killed them,and collected their back skin.Firstly, samples were treated with PBS solution.And then the samples were fixed in the diffusion cell proliferation with a certain area.There spotting receptor compartment and the other compartment and a compartment colors were mounted on both sides of the diffusion cell.Determination of skin epithelial side faces sited compartment side, the receptor compartment was filled with RPMI1640medium,In the experiment, pEGFP-Nl plasmid (5ug) was added to the medium as an internal reference.The prepared different concentrations of DMSO was added to a mixture of the compartment-side positioning point.The medium in the receptor compartment stirred45r/m.The whole apparatus was placed under37±0.5±C.The back skin soaked for2-4hours in a mixture of2%formaldehyde and0.2%glutaraldehyde in which the temperature of the solution was maintained at4±C environment, and then at room temperature successively with three different concentrations of PBS (PH7.4) and washed1h. Then all the samples at37℃ambient was placed1mg/ml X-gal them soak overnight. The second day, all samples was washed for60minutes with three different concentrations of PBS (PH7.4) at room temperature successively,then at room temperature followed by tissue was placed in10%formalin solution were immersed for24hours, then washed with water overnight.Results:Different concentrations of DMSO affect transdermal expression of pORF-lacz gene:while it is10%DMSO, the transdermal expression of pORF-lacz gene is significantly higher than the other three groups;while it is0%DMSO, the transdermal expression of pORF-lacz gene is significantly lower than the other three groups;while they are5%DMSO and30%DMSO, the transdermal expression of pORF-lacz gene is in middle,there is no significant difference between the two groups.Conclusions:10%DMSO group promotes the penetration effect of the skin of the mice best. So,we choose the10%DMSO to be the optional one.The second part:The research of RA and LP affecting claudin-4and ZO-1of mice’s skinObjectives:Through treating with retinoic acid (RA) and TLR2inhibitors-LP,to see the skin connexin (claudin-4) and ZO protein (ZO-1) in mice, by this to test the change of skin penetration rate.Methods:30male Balb/c mice(which are bought from southern medical university),weight:20-22g.We randomly divided them into5groups, fed with ambient temperature of22±2℃and relative humidity of55.6%±4%.Five groups in experiment:RA+LP+DMSO+pORF-LacZ;LA+DMSO+pORF-LacZ; DMSO+pORF-LacZ; RA+DMSO+pORF-LacZ; the salt water control group. We chose the back skin(1×1cm) of mice,and used depilatory creams to remove hair in selected place for all mice.Then treating with retinoic acid (RA) and TLR2inhibitors-LP for1week. From the Seventh day, use inhibitors treat and the mice were treated with optimal concentration of plasmid pORF-LacZ configuration in once a day. Our mice were sacrificed on the fourth day,while we collected the skin and blood samples.Finally we used Immunohistochemical skill for skins,and western blot to test connexin (claudin-4) and ZO protein (ZO-1).Intervention Process:we chose8weeks mice’s back skin (1×1cm), and used depilatory creams to remove hair in selected place for all mice.After3-5minutes, we used physiological saline (0.09%) to clean,then through15minutes,they were put into several groups:one group were blotted with20ul contain0.05%(RA)(RA dissolved in NS) on where treated;20ul of1%TLR2inhibitor (Lipolanthionine Peptides,LP) concentration; applied two joint preparation;10%DMSO.This process was carried out once every other day,for one week, also treated as such. From the Seventh day, used inhibitors treat and the mice were treated with optimal concentration of plasmid pORF-LacZ configuration in once a day. Our mice were sacrificed on the fourth day,while we collected the skin and blood samples.Finally we used Immunohistochemical skill for skins,and western blot to test connexin (claudin-4) and ZO protein (ZO-1)Back skin soaked for2-4hours in a mixture of2%formaldehyde and0.2%glutaraldehyde in which the temperature of the solution was maintained at4℃environment, and then at room temperature successively with three different concentrations of PBS (PH7.4) and washed1h. Then all the samples at37℃ambient is placed1mg/ml X-gal them soak overnight. The second day, all samples at room temperature successively with three different concentrations of PBS (PH7.4) was washed for60minutes at room temperature followed by tissue was placed in 10%formalin solution were immersed for24hours, then washed with water overnight.Results:1.According to HE staining picture,we could find that:after DMSO,RA and LP Intervention,experimental mice stratum corneum cells connection gap increased, cells loose connection,but control group of mice skin above phenomenon did not appear;and all the experimental mice’s skin didn’t show pathological skin damage.However,DMSO group and RA+DMSO group appeared slightly mouse skin skin papillae, skin edema, neutrophil aggregation and capillary dilation phenomenon;LP+DMSO group and RA+LP+DMSO group mouse skin edema reduction, telangiectasia phenomenon eased. Thus, we can speculate:LP not only enhances the transdermal skin of mice, but will also reduce the slight skin damage caused by DMSO, LP is an effective and safe skin penetration enhancer.2.mmunohistochemical results:(1)compare to the control group:the claudin-4protein’s expression of the groups of RA+LP+DMSO+pORF-LacZ, LP+DMSO+pORF-LacZ, was significantly lower than the amount of protein expression (p <0.01) in the saline control group, with statistical significance; Compared with RA+DMSO+pORF-LacZ, the expression of the RA+LP+DMSO+pORF-LacZ group was significantly lower than the RA+DMSO+pORF-LacZ group (p<0.01), with statistical significance, the expression of the RA+LP+DMSO+pORF-LacZ group was significantly lower than the RA+DMSO+protein pORF-LacZ group (p <0.05), with statistical significance.Compared with DMSO+pORF-LacZ, the expression of the RA+LP+DMSO+pORF-LacZ group and LP+DMSO+pORF-LacZ group was significantly lower than the DMSO+pORF-LacZ group (p <0.01).(2) zo-1protein:Compared with the saline control group, RA+LP+DMSO+pORF-LacZ, LP+DMSO+pORF-LacZ, RA+DMSO+pORF-LacZ, DMSO+ pORF-LacZ,the four groups’target protein-zo-1protein expression level was significantly lower than the saline control group protein (p<0.01), with statistical significance, Compared with RA+DMSO+pORF-LacZ group, RA+LP+DMSO+pORF-LacZ group’ target protein-zo-1protein expression level was significantly lower than RA+DMSO+pORF-LacZ expression group (p<0.01), the saline control group was significantly higher than RA+DMSO+pORF-LacZ expression group (p <0.01), DMSO+pORF-LacZ group’ protein expression level is lower than the RA+DMSO+protein expression (p<0.05).Compared with DMSO+pORF-LacZ group, RA+LP+DMSO+pORF-LacZ, LP+DMSO+pORF-LacZ,the two groups ’target protein-zo-1protein expression level was significantly lower than the DMSO+pORF-LacZ group (p<0.01),the saline control group is significantly higher than the RA+DMSO+protein expression (p<0.01);RA+DMSO+pORF-LacZ group’protein expression level is lower than the DMSO+protein expression (p<0.05)3.Western blot results:(1)compared to the control group:RA+LP+DMSO+pORF-LacZ, LP+DMSO+pORF-LacZ, RA+DMSO+pORF-LacZ, claudin-4protein’s expression of these three groups was significantly lower than the amount of protein expression (p<0.01) in the saline control group, with statistical significance; Compared with DMSO+pORF-LacZ, RA+LP+DMSO+pORF-LacZ, LP+DMSO+pORF-LacZ, RA+DMSO+pORF-LacZ expression of three purpose-claudin-4protein was significantly lower than the DMSO+protein pORF-LacZ group (p<0.01), with statistical significance.(2) zo-1protein:Compared with the saline control group, RA+LP+DMSO+pORF-LacZ, LP+DMSO+pORF-LacZ, RA+DMSO+pORF-LacZ three groups target protein-zo-1protein expression level was significantly lower than the saline control group protein (p<0.01), with statistical significance, Compared with DMSO+pORF-LacZ, RA+LP+DMSO+pORF-LacZ, LP+DMSO+pORF-LacZ two groups target protein-zo-1protein expression level was significantly lower than DMSO+pORF-LacZ expression group (p<0.01), RA+DMSO+pORF-LacZ-zo-1protein expression level is lower than the DMSO+protein expression (p<0.05) pORF-LacZ group, with statistical significance; with RA+DMSO+pORF-compared LacZ group, RA+LP+DMSO+pORP-LacZ, LP+DMSO+pORF-LacZ,two groups target protein,-zo-1protein expression level was significantly lower than RA+DMSO.Conclusions:Based on the experimental results, we can find that after the intervention, the mouse skin claudin-4, zo-1protein in varying degrees to reduce, reduce connexin mouse skin, mouse skin penetration increases.The third part:The research of RA and LP affecting the transdermal efficiency of pORF-lacz of mice’s skinObjective:After the RA and LP dealed with the mice,we measured the Transdermal rate of the mice(the change of pORP-lacz which entered into blood across the skin of the mice).Methods:30male Balb/c mice(which are bought from southern medical university),weight about20g.We randomly divided them into8groups, fed with ambient temperature of22±2°C and relative humidity of55.6%±4%.Five groups in experiment,and there are6mice in every group:RA+LP+DMSO+pORF-LacZ; LP+DMSO+pORP-LacZ; DMSO+pORP-LacZ; RA+DMSO+pORP-LacZ; the salt water control group.The progress was operated as follows:we chose8weeks mice’s back skin (lxlcm), and used depilatory creams to remove hair in selected place for all mice.3-5minutes later, we used physiological saline (0.09%) to clean them.And then,15minutes later,according to the groups:one group were blotted with20ul contain0.05%(RA)(RA dissolved in NS) on where treated;20ul of1%TLR2inhibitor (Lipolanthionine Peptides,LP) concentration; apply two joint preparation; 10%DMSO alone,or jointed,and so on. In a word,the experiment groups are RA+LP+DMSO+pORF-LacZ group; LP+DMSO+pORF-LacZ group; DMSO+pORF-LacZ group; RA+DMSO+pORF-LacZ group;This process is carried out once every other day,for one week,the control group was also treated as such. On the Seventh day, we treated them with inhibitors and the mice were treated with optimal concentration of plasmid pORF-LacZ configuration in once a day,it last3days. Our mice were sacrificed on the fourth day,while we collected the blood sample.Finally we measured the pORF-LacZ in the blood by RT-PCR..Results:Compared with the control group, DMSO+pORF-LacZ group, LP+DMSO+pORF-LacZ group, RA+DMSO+pORF-LacZ group, RA+LP+DMSO+pORF-LacZ group is significantly higher than them in the percutaneous penetration(p<0.01).Compared with the control group, DMSO+pORF-LacZ group, RA+DMSO+pORF-LacZ group, LP+DMSO+pORF-LacZ group is significantly higher than them in the percutaneous penetration(p<0.01).Compared with the control group, DMSO+pORF-LacZ group, RA+DMSO+pORF-LacZ group is significantly higher than them in the percutaneous penetration(p<0.01).Compared with the control group, Conclusions:According to the experimental result,we know that after the intervention,the number of the change of pORF-lacz which entered into blood across the skin of the mice increases in different degree,the Transdermal rate of the mice increases,and the effect of LP+RA+DMSO is best of the five groups.DMSO+pORF-LacZ group is significantly higher than it in the percutaneous penetration(p <0.01),compered with RA+DMSO+pORF-LacZ group,DMSO+pORF-LacZ group is significantly lower than it in the percutaneous penetration(p<0.01).Compared with DMSO+pORF-LacZ group, RA+DMSO+pORF-LacZ group,the control group is significantly lower than it in the percutaneous penetration(p<0.01).Conclusion:According to the experimental result,we know that after the intervention,the number of the change of pORF-lacz which entered into blood across the skin of the mice increases in different degree,the Transdermal rate of the mice increases,and the effect of LP+RA+DMSO is best of the five groups.更多还原