| 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. |
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