8-甲氧补骨脂素脂质体凝胶的浓度筛选及初步药效学研究
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摘要
目的：考察8-甲氧补骨脂素脂质体凝胶对实验性白癜风的疗效，并确定其最佳浓度。
方法：将8-甲氧补骨脂素脂质体(LMOP)悬液作用于体外培养的小鼠B16F1黑素瘤细胞，并与等浓度的8-甲氧补骨脂素(8-MOP)溶液作比较，用四甲基偶氮唑蓝(MTT)比色法测定细胞增殖情况；NaOH裂解法测定黑素合成；多巴氧化法测定酪氨酸酶活性。从细胞增殖率、黑素含量、酪氨酸酶活性等方面进行比较，初步作出浓度筛选。用化学脱色法制备豚鼠实验性白癜风动物模型，以市售敏柏宁酊的推荐使用浓度(0.15％)为最高浓度，设定LMOP凝胶低、中、高三个浓度组(0.03％，0.075％，0.15％)，进行药效学比较性研究，以8-MOP酊剂(敏柏宁酊稀释至含8-MOP 0.15％)为对照，考察其对皮肤黑素分布、DOPA阳性细胞数、含黑素毛囊数、胆碱酯酶(ChE)及丙二醛(MDA)的影响。
结果：体外初步浓度筛选实验表明，与等浓度8-MOP溶液相比，LMOP悬液低浓度(3，7.5mg·L~(-1))时即显著促进B16F1细胞增殖，提高酪氨酸酶活性和黑素含量(P＜0.05)，但高浓度(15～30mg·L~(-1))时表现为明显的抑制作用(P＜0.05)，30mg·L~(-1)中细胞几乎不能生长。LMOP凝胶对实验性白癜风模型动物的作用中，发现与8-MOP酊剂组相比，0.03％LMOP凝胶组调节黑素生成的能力及血清ChE活力均较低，但差别无显著性(P＞0.05)，受试处皮肤MDA生成最少，皮肤刺激性也最小；0.075％LMOP凝胶组黑素及血清ChE活力明显增高(P＜0.05)，受试处皮肤MDA含量较8-MOP酊剂组显著降低(P＜0.05)；0.15％LMOP凝胶组黑素生成、ChE活力及MDA生成均明显增多，表明其疗效提高的同时，皮肤刺激性也增加。
结论：本研究表明，LMOP凝胶的最佳浓度为0.075％，可以明显刺激黑素生成，提高血清ChE活力，降低皮肤MDA含量，减轻皮肤刺激性。
Objectives: To study the curative effect of experimental vitiligo to the 8-MOP liposomal gels, and to ensure the optimum concentration of 8-MOP liposomal gels.
Methods: Different concentration levels of LMOP suspension were used to B16F1 murine melanoma cells in vitro, and comparing with 8-MOP liquors on the same concentration levels. MTT was used to study the proliferation of B16F1 cells. The melamin content was mesured by NaOH-assay, and the tyrosinase activity was detected by DOPA-oxidation. Comparing the proliferation rate, melanin content and tyrosinase activity of B16F1 cells, so as to preliminarily select the concentration levels of LMOP suspension. The experimental vitiligo models were prepared by chemical decolour method in Guinea Pigs. And we choose the Meladinine's recommeded level (0.15%) as high concentration, use the high, middle and low concentration levels (0.15%, 0.075 % , 0.03 % ) of LMOP gels to progress the pharmacodynamics comparative study, 8-MOP tincture (the dilution of Meladinine) was used as controll group, observing the melanin distribution, the number of DOPA active cells, the number of hair follicles contented melanin, the levels of ChE
and MDA of the experimental vitiligo models.
Results: Compared with 8-MOP liquor groups on the same concentration, in cultured B16F1 cells in vitro, LMOP groups in low concentrations (3, 7.5mg L-1 ) could significantly promote the proliferation, tyrosinase activity and melanin content (P<0.05) while significantly inhibit the proliferation and melanogenesis in high concentrations (15~ 30mg L-1, P<0.05) , furthermore B16F1 cells almost cannot exist in 30mg L-1 LMOP. In the treatment of experimental vitiligo, compared with 8-MOP tincture group, the regulation ability of 0.03% LMOP gel group was small, but MDA level of 0.03% group was the lowest of all; 0.075% LMOP gel group had significant differences regulation ability with 8-MOP
tincture group (P<0.05) while MDA level lower than 8-MOP tincture group, but had no significance (P>0.05) . In 0.15% LMOP gel group, the regulation ability and MDA level were all highest, it suggest that it increase not only the curative effect but also the skin irritation at the same time.
Conclusions: In clinical use, the optimum concentration of LMOP gel is 0.075%.
方法：将8-甲氧补骨脂素脂质体(LMOP)悬液作用于体外培养的小鼠B16F1黑素瘤细胞，并与等浓度的8-甲氧补骨脂素(8-MOP)溶液作比较，用四甲基偶氮唑蓝(MTT)比色法测定细胞增殖情况；NaOH裂解法测定黑素合成；多巴氧化法测定酪氨酸酶活性。从细胞增殖率、黑素含量、酪氨酸酶活性等方面进行比较，初步作出浓度筛选。用化学脱色法制备豚鼠实验性白癜风动物模型，以市售敏柏宁酊的推荐使用浓度(0.15％)为最高浓度，设定LMOP凝胶低、中、高三个浓度组(0.03％，0.075％，0.15％)，进行药效学比较性研究，以8-MOP酊剂(敏柏宁酊稀释至含8-MOP 0.15％)为对照，考察其对皮肤黑素分布、DOPA阳性细胞数、含黑素毛囊数、胆碱酯酶(ChE)及丙二醛(MDA)的影响。
结果：体外初步浓度筛选实验表明，与等浓度8-MOP溶液相比，LMOP悬液低浓度(3，7.5mg·L~(-1))时即显著促进B16F1细胞增殖，提高酪氨酸酶活性和黑素含量(P＜0.05)，但高浓度(15～30mg·L~(-1))时表现为明显的抑制作用(P＜0.05)，30mg·L~(-1)中细胞几乎不能生长。LMOP凝胶对实验性白癜风模型动物的作用中，发现与8-MOP酊剂组相比，0.03％LMOP凝胶组调节黑素生成的能力及血清ChE活力均较低，但差别无显著性(P＞0.05)，受试处皮肤MDA生成最少，皮肤刺激性也最小；0.075％LMOP凝胶组黑素及血清ChE活力明显增高(P＜0.05)，受试处皮肤MDA含量较8-MOP酊剂组显著降低(P＜0.05)；0.15％LMOP凝胶组黑素生成、ChE活力及MDA生成均明显增多，表明其疗效提高的同时，皮肤刺激性也增加。
结论：本研究表明，LMOP凝胶的最佳浓度为0.075％，可以明显刺激黑素生成，提高血清ChE活力，降低皮肤MDA含量，减轻皮肤刺激性。
Objectives: To study the curative effect of experimental vitiligo to the 8-MOP liposomal gels, and to ensure the optimum concentration of 8-MOP liposomal gels.
Methods: Different concentration levels of LMOP suspension were used to B16F1 murine melanoma cells in vitro, and comparing with 8-MOP liquors on the same concentration levels. MTT was used to study the proliferation of B16F1 cells. The melamin content was mesured by NaOH-assay, and the tyrosinase activity was detected by DOPA-oxidation. Comparing the proliferation rate, melanin content and tyrosinase activity of B16F1 cells, so as to preliminarily select the concentration levels of LMOP suspension. The experimental vitiligo models were prepared by chemical decolour method in Guinea Pigs. And we choose the Meladinine's recommeded level (0.15%) as high concentration, use the high, middle and low concentration levels (0.15%, 0.075 % , 0.03 % ) of LMOP gels to progress the pharmacodynamics comparative study, 8-MOP tincture (the dilution of Meladinine) was used as controll group, observing the melanin distribution, the number of DOPA active cells, the number of hair follicles contented melanin, the levels of ChE
and MDA of the experimental vitiligo models.
Results: Compared with 8-MOP liquor groups on the same concentration, in cultured B16F1 cells in vitro, LMOP groups in low concentrations (3, 7.5mg L-1 ) could significantly promote the proliferation, tyrosinase activity and melanin content (P<0.05) while significantly inhibit the proliferation and melanogenesis in high concentrations (15~ 30mg L-1, P<0.05) , furthermore B16F1 cells almost cannot exist in 30mg L-1 LMOP. In the treatment of experimental vitiligo, compared with 8-MOP tincture group, the regulation ability of 0.03% LMOP gel group was small, but MDA level of 0.03% group was the lowest of all; 0.075% LMOP gel group had significant differences regulation ability with 8-MOP
tincture group (P<0.05) while MDA level lower than 8-MOP tincture group, but had no significance (P>0.05) . In 0.15% LMOP gel group, the regulation ability and MDA level were all highest, it suggest that it increase not only the curative effect but also the skin irritation at the same time.
Conclusions: In clinical use, the optimum concentration of LMOP gel is 0.075%.
引文
1. Kopera D. Historical aspects and definition of vitiligo[J].Clinics in Dermatology, 1997, 15: 841-843.
2. Helm TN, Dijkstra JW, Ferrara RJ Jr, et al. PUVA therapy[J]. Am Fam Physician. 1991, 43(3): 908-12.
3．李德爱，邵友梅，裴晓冬．国家基本药物临床手册[M]．北京：化学工业出版社，1999：421．
4．赵红，郑俊明．新型的皮肤给药系统——脂质体[J]．沈阳药科大学学报，1997：14(2)：148-153．
5. Egbaria K, Ramachandran C, Weiner N. Topical delivery of ciclosporin: evaluation of various formulation using in vitro diffusion studies in hairless mouse skin [J]. Skin Pharmacol, 1990,3(1): 21-811.
6．何文，吴宏霞，蔡鸿生，等．8-甲氧补骨脂素脂质体凝胶的研制[J]．中国医院药学杂志，2004，24(1)：3-5．
7．吴宏霞，蔡鸿生，罗顺德，等．反相高效液相色谱法测定白癜风治疗药脂质体凝胶中8-甲氧补骨脂素的含量[J]．中国医院药学杂志，2003，23(3)：148-150．
8．吴宏霞，何文，蔡鸿生，等．8-甲氧补骨脂素脂质体凝胶的皮肤渗透性研究[J]．中国药房，2003，14(11)：655-657．
9．徐叔云，卞如濂，陈修．药理学实验方法学[M]．2002，第三版，北京：人民卫生出版社：1849，1855．
10．陈啸梅，周文郁，彭俊云，等．组织化学手册[M]．1982，第一版，北京：人民卫生出版社：192，193．
11．雷铁池，朱文元，夏明玉，等．甘草酸、熊果苷及氢醌对小鼠黑素瘤细胞黑素生成影响的比较研究[J]．临床皮肤科杂志，2000，29(2)：69-72．
12. Nakajima M, Shinoda I, Fukuwatari Y, et al. Arbutin Increases the Pigmentation of Cultured Human Melanocytes Through Mechanisms Other Than the Induction of
Tyrosinase Activity[J]. Pigment Cell Res, 1998,11 : 12-17.
13. Ando H, Itoh A, Mishima Y, et al. Correlation between the number of melanosomes,tyrosinase mRNA levels, and tyrosinase activity in cultured murine melanoma cells in response to various melanogenesis regulatory agents[J]. J Cell Physiol, 1995,163 : 608-614.
14．龙子江，白玫，樊彦，等．化学脱色法制备白癜风动物模型[J]．安徽中医学院学报，1997，16(6)：60-61．
15．谢勇，盛国荣．自制祛白酊对白癜风动物模型的实验研究[J]．皮肤病与性病，2002，24(3)：2-3．
16. Imokawa G, Kawai M, Mishima Y, et al. Differential analysis of experimental hypermelanosis induced by UVB, PUVA, and allergic contact dermatitis using a brownish guinea pig model[J]. Arch Dermatol Res, 1986, 278: 352-362.
17．全国中西医结合皮肤性病学会色素病学组．白癜风临床分型及疗效标准(草案)[J]．中华皮肤科杂志，1995，28(4)：212-213．
18．刘介眉，严庆汉，路英杰，等．病理染色技术[M]．1983，第一版，北京：人民卫生出版社：119-120．
19. Ben-Hur E, Moor ACE, Margolis-Nunno H, et al. The photodecontamination of cellular blood components: mechanisms and use of photosensitization in transfusion medicine[J]. Transfusion medicine reviews, 1996, 10(1): 15-22.
20. Danno K. PUVA therapy:current concerns in Japan[J]. J of Dermatol Sci, 1999, 19: 89-105.
21. Brown DA. Skin Pigmentation Enhancers[J]. J Photochem Photobiol B, 2001, 63:148-161.
22. Anthony FA, Laboda HM, Costlow ME, et al. Costlow, Psoralen-fatty acid adclucts activate melanocyte protein kinase C: a proposed mechanism for melanogenesis induced by 8-methoxypsoralen and ultraviolet A light[J]. Photodermatol Photoimmunol Photomed, 1997, 13:9-16.
23. P.A.Riley. Melanin[J]. Int J Biochem Cell Biol, 1997, 29, 11: 1235-1239.
24. Grimes PE. Melasma: etiologic and therapeutic considerations[J]. Arch Dermatol, 1995, 131 (12): 1453-1457.
25．朱光斗．白癜风病因初步探讨[J]．临床皮肤科杂志，1990，19(1)：9-11．
26．李雯，刘贞富．皮肤病、精神因素与神经—内分泌—免疫调节[J]．国外医学皮肤性病学分册，2002，28(2)：102-105．
27．陈瑗，周玟．脂质过氧化作用与疾病[J]．中华医学杂志，1985，65(11)：704-706．
2. Helm TN, Dijkstra JW, Ferrara RJ Jr, et al. PUVA therapy[J]. Am Fam Physician. 1991, 43(3): 908-12.
3．李德爱，邵友梅，裴晓冬．国家基本药物临床手册[M]．北京：化学工业出版社，1999：421．
4．赵红，郑俊明．新型的皮肤给药系统——脂质体[J]．沈阳药科大学学报，1997：14(2)：148-153．
5. Egbaria K, Ramachandran C, Weiner N. Topical delivery of ciclosporin: evaluation of various formulation using in vitro diffusion studies in hairless mouse skin [J]. Skin Pharmacol, 1990,3(1): 21-811.
6．何文，吴宏霞，蔡鸿生，等．8-甲氧补骨脂素脂质体凝胶的研制[J]．中国医院药学杂志，2004，24(1)：3-5．
7．吴宏霞，蔡鸿生，罗顺德，等．反相高效液相色谱法测定白癜风治疗药脂质体凝胶中8-甲氧补骨脂素的含量[J]．中国医院药学杂志，2003，23(3)：148-150．
8．吴宏霞，何文，蔡鸿生，等．8-甲氧补骨脂素脂质体凝胶的皮肤渗透性研究[J]．中国药房，2003，14(11)：655-657．
9．徐叔云，卞如濂，陈修．药理学实验方法学[M]．2002，第三版，北京：人民卫生出版社：1849，1855．
10．陈啸梅，周文郁，彭俊云，等．组织化学手册[M]．1982，第一版，北京：人民卫生出版社：192，193．
11．雷铁池，朱文元，夏明玉，等．甘草酸、熊果苷及氢醌对小鼠黑素瘤细胞黑素生成影响的比较研究[J]．临床皮肤科杂志，2000，29(2)：69-72．
12. Nakajima M, Shinoda I, Fukuwatari Y, et al. Arbutin Increases the Pigmentation of Cultured Human Melanocytes Through Mechanisms Other Than the Induction of
Tyrosinase Activity[J]. Pigment Cell Res, 1998,11 : 12-17.
13. Ando H, Itoh A, Mishima Y, et al. Correlation between the number of melanosomes,tyrosinase mRNA levels, and tyrosinase activity in cultured murine melanoma cells in response to various melanogenesis regulatory agents[J]. J Cell Physiol, 1995,163 : 608-614.
14．龙子江，白玫，樊彦，等．化学脱色法制备白癜风动物模型[J]．安徽中医学院学报，1997，16(6)：60-61．
15．谢勇，盛国荣．自制祛白酊对白癜风动物模型的实验研究[J]．皮肤病与性病，2002，24(3)：2-3．
16. Imokawa G, Kawai M, Mishima Y, et al. Differential analysis of experimental hypermelanosis induced by UVB, PUVA, and allergic contact dermatitis using a brownish guinea pig model[J]. Arch Dermatol Res, 1986, 278: 352-362.
17．全国中西医结合皮肤性病学会色素病学组．白癜风临床分型及疗效标准(草案)[J]．中华皮肤科杂志，1995，28(4)：212-213．
18．刘介眉，严庆汉，路英杰，等．病理染色技术[M]．1983，第一版，北京：人民卫生出版社：119-120．
19. Ben-Hur E, Moor ACE, Margolis-Nunno H, et al. The photodecontamination of cellular blood components: mechanisms and use of photosensitization in transfusion medicine[J]. Transfusion medicine reviews, 1996, 10(1): 15-22.
20. Danno K. PUVA therapy:current concerns in Japan[J]. J of Dermatol Sci, 1999, 19: 89-105.
21. Brown DA. Skin Pigmentation Enhancers[J]. J Photochem Photobiol B, 2001, 63:148-161.
22. Anthony FA, Laboda HM, Costlow ME, et al. Costlow, Psoralen-fatty acid adclucts activate melanocyte protein kinase C: a proposed mechanism for melanogenesis induced by 8-methoxypsoralen and ultraviolet A light[J]. Photodermatol Photoimmunol Photomed, 1997, 13:9-16.
23. P.A.Riley. Melanin[J]. Int J Biochem Cell Biol, 1997, 29, 11: 1235-1239.
24. Grimes PE. Melasma: etiologic and therapeutic considerations[J]. Arch Dermatol, 1995, 131 (12): 1453-1457.
25．朱光斗．白癜风病因初步探讨[J]．临床皮肤科杂志，1990，19(1)：9-11．
26．李雯，刘贞富．皮肤病、精神因素与神经—内分泌—免疫调节[J]．国外医学皮肤性病学分册，2002，28(2)：102-105．
27．陈瑗，周玟．脂质过氧化作用与疾病[J]．中华医学杂志，1985，65(11)：704-706．