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载N-(4-羟基苯基)维甲酰胺脂质微泡联合超声对人瘢痕疙瘩成纤维细胞的影响

Effects of N-(4-hydroxyphenyl) retinamide lipid microbubble combined with ultrasound on human keloid fibroblasts

摘要:

目的 探讨载N-(4-羟基苯基)维甲酰胺(4HPR)及4HPR脂质体(4HPR-L)与4HPR脂质微泡(4HPR-LM)联合超声对人瘢痕疙瘩成纤维细胞(Fb)增殖、凋亡及细胞周期的影响. 方法 (1)采用水化超声法制备4HPR-L及4HPR-LM,采用高效液相色谱法、动态光散射法、透射电镜对4HPR-L外观形态、粒径分布、Zeta电势、载药浓度、包封率、载药量进行考察.(2)取人瘢痕疙瘩Fb,采用随机数字表法(分组方法下同)分为13组,每组6孔.对照组细胞不给予任何处理,0.5 W30s组、0.5 W60s组、0.5 W 120s组、0.7W30s组、0.7 W60s组、0.7W 120s组、1.0W30s组、1.0W60 s组、1.0W120s组、1.5W30s组、1.5W60s组、1.5 W 120s组12个超声组细胞分别给予对应参数超声处理.常规培养24 h后,酶标仪测定细胞活力.另取人瘢痕疙瘩Fb,分为5组,每组6孔.对照组细胞不给予任何处理,1、10、20、50 μg/mL空白脂质微泡组细胞给予对应质量浓度的空白脂质微泡处理,常规培养24 h后同前测定细胞活力.另取人瘢痕疙瘩Fb,分为6组,每组12孔.对照组细胞不给予任何处理,1、10、20、50、100 μg/mL 4HPR-L组细胞分别加入含对应质量浓度4HPR的4HPR-L处理,常规培养24、48 h后每组各取6孔同前测定细胞活力.另取人瘢痕疙瘩Fb,分为4组,每组6孔.对照组细胞不给予任何处理,4HPR组、4HPR-L组和4HPR-LM+超声组细胞分别加入4HPR、4HPR-L、4HPR-LM(4HPR质量浓度均为20 μg/mL)处理,4HPR-LM+超声组细胞给药后立即给予0.5 W60s超声处理.常规培养24 h后同前测定细胞活力.(3)取人瘢痕疙瘩Fb,分为对照组、4HPR组、4HPR-L组和4HPR-LM+超声组,每组3孔,各组细胞处理同前.常规培养24 h后流式细胞仪检测细胞凋亡情况.(4)取人瘢痕疙瘩Fb,同(3)分组及处理,常规培养24 h后流式细胞仪检测细胞周期分布情况.对数据行单因素方差分析和t检验. 结果 (1)4HPR-L颗粒呈大小均匀的球形或类球形结构,粒径为(100.1±1.3)nm,Zeta电势为(-34.3 ±2.3)mV.4HPR-L溶液中4HPR质量浓度在1400 μg/mL左右,包封率为(95.8±1.2)%,载药量为(8.3±0.4)%.(2)12个超声组细胞活力均高于93.0%.1、10、20、50 μg/mL空白脂质微泡组细胞活力均高于95.0%.1μg/mL4HPR-L组细胞给药24、48 h细胞活力相近(t=0.393,P>0.05),给药24 h时10、20、50、100 μg/mL4HPR-L组细胞活力明显高于给药48 h(t=44.593、22.961、32.224、35.337,P<0.01).4HPR组、4HPR-L组与4HPR-LM+超声组给药24 h细胞活力分别为(47.3±0.7)%、(42.3±1.7)%、(38.6±0.8)%.4HPR组细胞活力明显高于4HPR-L组和4HPR-LM+超声组(t=4.551、15.895,P<0.05或P <0.01),4HPR-L组细胞活力明显高于4HPR-LM+超声组(t=-3.360,P<0.05).(3) 4HPR组、4HPR-L组与4HPR-LM+超声组总凋亡细胞百分比分别为(32.8±2.4)%、(42.5±2.4)%、(58.5±6.3)%,明显高于对照组的(14.9±1.6)%(t=8.748、13.637、9.500,P<0.01);4HPR-L组与4HPR-LM+超声组总凋亡细胞百分比明显高于4HPR组(t=4.049、5.393,P<0.05或P <0.01);4HPR-LM+超声组总凋亡细胞百分比明显高于4HPR-L组(t=3.371,P<0.01).(4)4HPR组G2/M期细胞百分比高于对照组,但差异无统计学意义(t =2.107,P>0.05);4HPR-L组G2/M期细胞百分比明显高于4HPR组和对照组(t=18.169、30.026,P<0.01);4HPR-LM+超声组G2/M期细胞百分比明显高于4HPR-L组、4HPR组和对照组(t=4.932、25.854、66.231,P<0.01).结论 4HPR可抑制人瘢痕疙瘩Fb增殖、诱导细胞凋亡及发生G2/M期阻滞,且4HPR-LM联合超声的作用效果优于4HPR-L和游离4HPR.

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abstracts:

Objective To explore the effects of N-(4-hydroxyphenyl) retinamide (4HPR),4HPR liposome (4HPR-L),and 4HPR lipid microbubble (4HPR-LM) combined with ultrasound on proliferation,apoptosis,and cell cycle of human keloid fibroblasts (Fbs).Methods (1) 4HPR-L and 4HPR-LM were prepared by hydration ultrasonic method.The appearance morphology,particle size distribution,Zeta potential,loading drug concentration,encapsulation efficiency,and drug loading rate of 4HPR-L were investigated by high performance liquid chromatography,dynamic light scattering,and transmission electron microscope.(2) Human keloid Fbs were cultured and divided into 13 groups by random number table (the same grouping method below),with 6 wells in each group.Cells in control group were given no treatment,while cells in 12 ultrasound groups including 0.5 W 30 s group,0.5 W 60 s group,0.5 W 120 s group,0.7 W 30 s group,0.7 W 60 s group,0.7 W 120 s group,1.0 W 30 s group,1.0 W 60 s group,1.0 W 120 s group,1.5 W 30 s group,1.5 W 60 s group,and 1.5 W 120 s group were treated by ultrasound with corresponding parameters.The cells viability was measured by a microplate reader after 24 hours of routine culture.Another batch of human keloid Fbs were divided into 5 groups,with 6 wells in each group.Cells in control group were given no treatment,while cells in 1,10,20,and 50 μg/mL blank lipid microbubble groups were treated with blank lipid microbubbles in corresponding mass concentration.The cells viability was measured as before after 24 hours of routine culture.Another batch of human keloid Fbs were divided into 6 groups,with 12 wells in each group.Cells in control group were given no treatment,while cells in 1,10,20,50,and 100 μg/mL 4HPR-L groups were added with 4HPR-L carrying corresponding mass concentration of 4HPR.The cells viability in 6 wells of each group was detected after 24 and 48 hours of routine culture,respectively.Another batch of human keloid Fbs were divided into 4 groups,with 6 wells in each group.Cells in control group were given no treatment,while cells in 4HPR,4HPR-L,and 4HPR-LM + ultrasound groups were treated with 4HPR,4HPR-L,and 4HPR-LM (all the mass concentration of 4HPR was 20 μg/mL),respectively,and cells in 4HPR-LM + ultrasound group were given 0.5 W 60 s ultrasound treatment immediately after drug administration.The cells viability was measured as before after 24 hours of routine culture.(3) Another batch of human keloid Fbs were divided into control group,4HPR group,4HPR-L group and 4HPR-LM + ultrasound group,with 3 wells in each group,and the cells in each group were treated as before.Apoptosis of the cells was detected by flow cytometer after 24 hours of routine culture.(4) Another batch of human keloid Fbs were grouped and treated as in (3),and then the cell cycle distribution was detected by flow cytometer after 24 hours of routine culture.Data were processed with oneway analysis of variance and t test.Results (1) 4HPR-L particles had a spherical or spheroidal structure and were uniform in size,with particle size of (100.1 ± 1.3) nm and Zeta potential of (-34.3 ±2.3) mY.The mass concentration of 4HPR in 4HPR-L solution was about 1 400 μg/mL,with the encapsulation efficiency of (95.8 ± 1.2) % and drug loading rate of (8.3 ± 0.4) %.(2) The viability of cells in the 12 ultrasound groups was higher than 93.0%,and the viability of cells in 1,10,20,and 50 μg/mL blank lipid microbubble groups was higher than 95.0%.The viability of cells in 1 μg/mL 4HPR-L group at administration hour 24 was similar to that at 48 (t =0.393,P >0.05).The viability of cells in 10,20,50,and 100 μg/mL 4HPR-L groups at administration hour 24 was significantly higher than that at administration hour 48 (t =44.593,22.961,32.224,35.337,P < 0.01).The viability of cells in 4HPR group,4HPR-L group,and 4HPR-LM + ultrasound group was (47.3 ± 0.7) %,(42.3 ± 1.7) %,and (38.6 ± 0.8) %,respectively.The viability of cells in 4HPR group was significantly higher than that in 4HPR-L group and 4HPR-LM + ultrasound group (t =4.551,15.895,P < 0.05 or P < 0.01).The viability of cells in 4HPR-L group was significantly higher than that in 4HPR-LM + ultrasound group (t =-3.360,P <0.05).(3) The percentages of total apoptotic cells in 4HPR group,4HPR-L group,and 4HPR-LM + ultrasound group were (32.8 ± 2.4) %,(42.5 ± 2.4) %,and (58.5 ± 6.3) %,respectively,which were significantly higher than the percentage of control group [(14.9 ± 1.6) %,t =8.748,13.637,9.500,P <0.0l].The percentages of total apoptotic cells in 4HPR-L group and 4HPR-LM + ultrasound group were significantly higher than the percentage in 4HPR group (t =4.049,5.393,P <0.05 orP <0.01),and the percentage of total apoptotic cells in 4HPR-LM + ultrasound group was significantly higher than that in 4HPR-L group (t =3.371,P < 0.0l).(4) The percentage of G2/M phase cells in 4HPR group was high-er than that in control group,but there was no statistically significant difference (t =2.107,P > 0.05).The percentage of G2/M phase cells in 4HPR-L group was significantly higher than that in 4HPR group or control group (t =18.169,30.026,P <0.01).The percentage of G2/M phase cells in 4HPR-LM + ultrasound group was significantly higher than that in 4HPR-L group,4HPR group,and control group (t =4.932,25.854,66.231,P < 0.01).Conclusions 4HPR can inhibit proliferation,induce apoptosis,and arrest G2/M phase of human keloid Fbs,and the effects of 4HPR-LM combined with ultrasound are better than those of 4HPR-L and free 4HPR.

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作者: 王梦蛟 [1] 方宇辉 [1] 金承龙 [1] 金哲虎 [1]
期刊: 《中华烧伤杂志》2018年34卷10期 683-689页 MEDLINEISTICPKUCSCD
栏目名称: 论著·瘢痕治疗与机制研究
DOI: 10.3760/cma.j.issn.1009-2587.2018.10.007
发布时间: 2018-11-13
基金项目:
吉林省科技发展计划 Science and Technology Development Plan of Jilin Province of China
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