Explore of allogenic melanocytes transplantation on vitiligo
Keywords:transplantation，homologous;melanocytes;vitili-go
CLC number:R758.41
Abstract:AIM To study the feasibility of vitiligo treatment through allogenic transplantation of pure melanocytes.METHODS By negative pressure suction blisters were made at a female vitiligo volunteer's lesions.Suspension of pure cultured melanocytes from a healthy male donor was injected into the blisters.The effects in appearance were observed continuously.The outcome of the allogenic melanocytes was determined by pathological examination and specific fragment of Y chromosome detection.After color-recovery were ob-served at the transplanted areas，ten volunteers joined the test too.RESULTS The four transplanted spots'colors of the first female transplanter appeared some re-pigmentation1mo after transplantation.The transplant spots'colors were almost identical to those of adjacent normal skin2mo later.One hundred and sixty days later pathological examination of one repigmented spot displayed that there were melanocytes that could secrete melanin into adjacent keratinocytes.Y chromosome fragment was detected positive in the DNA ex-tracted from the skin of another repigmented spot.Two hun-dred days after second grafting one transplanted spot showed obvious color-recovery too.Similar results were observed in some of other transplanted patients.CONCLUSION Be-cause the allogenic melanocytes can survive in acceptor graft-ing of cultured allogenic melanocytes and can repiment vitiligo lesions and it is promising to develop a new way to treat vi-tiligo.
INTRODUCTION
Vitiligo，an acquired pigment loss of the skin，af-fects approximately1%～4%of the worldwide pop-ulation.It always causes psychological problems.The melanocytes are destroyed in the lesions[1] .The reason of vitiligo is inclined to an autoimmune dis-ease[2] which is difficult to treat now.Autologous transplantation methods have been applied to treat vitiligo and the results proved effective to some ex-tent[3] .But the patients have to suffer from a surgi-cal operation and the melanocytes got from it are fi-nite.So transplantation of allogenic melanocytes，which can solve the problem of the shortage of melanocyte，is an ideal way to treat vitiligo[4] .Eisenberg engraftment cultured human epidermal allograft successfully in a child with recessive dys-trophic epidermolysis bullosa.Allogenic melan-o-cytes were proved in the skin several months lat-er[5] .Recently studies show that cultured pure melanocytes do not express MHC-Ⅱantigen and are not likely to be rejected in vitiligo patient[6-8] .In order to know the real reaction after transplant allo-genic melanocytes and to investigate the feasibility of treated vitiligo by this method，we carried out a series of clinical trials without administerring any immunosuppressants.Before transplant the donors and the receptors had no genetic connection and the major histocompatibility complex were not matched[9，10] .
MATERIALS AND METHODS
Preparation of melanocytes suspension Melano-cytes were obtained from a healthy young man's foreskin.We modified the melanocytes culture method of the previous literatures[11] .The cells were established in1640medium containing100g L-1 calf serum，6nmol L-1 Cholera Toxin，2mg L-1 basic fibroblast growth factor(all GIBCO prod-ucts).The fourth generation of melanocyte suspen-sions were prepared with saline at different cell den-sities.
Subjects and transplantation The first volunteer was a27-year-old female.She had suffered from vi-tiligo for more than20years.Her lesions did not expand in recent three years.There were other10patients(7female，3male)joined this test.Except one male patient was in active stage，all patients were in stable stage.
In the abdomen lesion of the first female patient(Fig1)four blisters were formed in about40min-utes by epidermis separate machine which could hold the transplanted sites in compressive stress of negative48kPa and temperature of43℃.Most of exudates in the blisters were drawn out by a new in-jector.The melanocyte suspensions were injected into the cavity of the blisters(about0.1mL sus-pension per blister).Two blisters marked A and B were injected both20，000melanocytes.The melanocytes number of the other two blisters C and D was10，000and1，000respectively(Fig2).The blisters were kept wholly and carefully for a few days.
Follow up and examination concerned If the trans-planted spots were color-recovered，the skin of one spot would be biopsied for histopathological exami-nation half year later.One of the other re-pigment-ed spot was made blister.DNA was got from the roof of the blister(Shanghai Biomedicine LTD)and sent for examing of Y chromosome peculiar frag-ment by high sensitive detects KIT(Fuhua Biomedicine corporation LTD.Shanghai).
Second transplantation 160days after the first transplantation，the patient accepted another trans-plantation and the melanocytes transplanted came from another male donor.The melanocytes sus-pends were grafted to the spots E，F，G at the adja-cent site(Fig4)and spots H of her right sciatic site(Fig7).The spot H was injected suspend without melanocytes.The spot I didn't injected anything.Other transplanted patients From then on，10oth-er patients(7female，3male)received allogenic melanocytes transplantation.All patients were in stable phase except a male patient.Results were ob-served continuously.Three months later color re-covered skin was sent to examine Y chromosome pe-culiar fragment.
RESULTS
Clinical effects First volunteer didn't feel any dis-comfort after transplantation.The spot began to turn red，and slight re-pigment appeared thirty days later.The re-pigmentations were getting more and more obviously.The colors of the four sites were similar to the normal adjacent skin75days after transplantation(Fig2)and existed stably.
After second transplantation，she didn't feel any discomfort yet.The photo of two hundred days later(three hundred and sixty days after the first transplantation)showed obviously color-recover at her right sciatic site H(Fig3).There were no col-or-recover at the spot H and I that didn't graft melanocytes.The re-pigmentations of the first transplantation were not influenced(Fig4).
Examination results Fontana dying of the spot A displayed a lot of melanin existing in the ker-atinocytes at the basal layer.There were some melanocytes existing around the keratinocytes(Fig5).The DNA of the re-pigmented spot C's roof was positive of chromosome peculiar fragment(Fig6).Results of other patients Among the other ten pa-tients，two males and one female were lost in fol-low-up survey.Six females'lesions showed slight re-pigment three months later.The colors of some transplanted spots in two cases were similar to those of normal skin.Some graft spots didn't change in evidence.We detected Y chromosome peculiar frag-ment in DNA of one female acceptor's color-recov-ered skin(Fig7).The male patient with active dis-ease got re-pigment for several months but the re-pigmentation disappeared six months later.
Fig1 The depigmented area at the abdomen of the first patient before transplantation(略)
Fig2 The four spots at the abdomen of the first patient75days after transplantation(略)
Fig3 Obvious color-recover at the first patient's right sciatic spot K200days after the second transplantation(略)
Fig4 360days after the first transplantation and200days after the second transplantation at the abdomen(略)
Fig5 A lot of melanin demonstrated in the keratinocytes around melanocytes at the re-pigment area A with Fantana dyeing(略)
DISCUSSION
In above transplant test，MHC was not matched be-fore transplantation，donors and receptors had no genetic connection，and immunosuppressants were not administered.We found color-recovered in some patients'transplant sites.Reestablishment color could existed stably，without spreading into sur-rounding areas.The first volunteer got certain ef-fects in secondary allogenic transplantation.Histopathological examination of the color-recov-ered spot A showed that there were melanocytes se-creting melanin.It is the melanocytes that worked normally lead to re-pigmentation of the lesions.
Fig6 Y chromosome peculiar fragments was positive in the DNA of the blister C's roofH2(略)
The melanocytes that existed at the basal layer were concluded to be the allogenic melanocytes.Firstly，in the hair follicle of spot A where melanocytes of oneself could exist had no melanin dying.It proved that there were not melanin that was secreted by herself and the melanin at the basal layer could only come from the allogenic melanocytes.Secondly，the DNAs of the re-pig-ment spots'skins of two female receptors was veri-fied to contain Y chromosome peculiar fragment that only exists in man(Fig3).In addition，no re-pig-mentation was observed in the sites where melanocytes were not injected.
According to transplantation immunology，this phenomenon belongs to donor specific transplanta-tion tolerance[12] .Mechanism of the survival of allo-genic melanocytes may be related to the special im-munological condition of vitiligo and the properties of purely cultured melanocyte.It is well known that in immunologic rejection caused by allograft trans-plantation，presentation of heterogeneous MHC antigens and T cell recognition goes first，and then proliferation and differentiation of T cell occurs with lymphokines production，leading to the rejection of transplants.The following factors may contribute to the survival of the allogenic melanocytes:①De-layed allergy is poor in most vitiligo patients.The number of CD4+ cells and the ratio of CD4+ to CD8+ cells are significantly decrease in vilitigo pa-tients[13] .This immunodeficiency is helpful for the transplantation of allogenic melanocytes.②Melanocytes do not express or express very few MHC-Ⅱmolecules that play an important role in transplantation rejection.MHC-Ⅱantigens begin to express when melanocytes were cultured in vitro and affected by some growth factors for many genera-tions[6，7] .So the pure cultured melanocytes of few generations are not easy to be recognized and reject-ed.③Vitiligo is highly related to autoimmunity[2] .Autoantibodies produced by the body and sensitized lymphocytes can invade self-cell components and damage self-melanocytes.In stationary and restora-tion stages，areas of lesions decrease or do not change，and immunological reaction to melanocyte stops.Thus，allogenic melanocytes perhaps are easy to survive.
To induce spontaneous donor specific trans-plantation tolerance is an ideal that people pusued hardly.In our study we find allogenic melanocytes can survive in vitiligo acceptors'body when MHC was not matched before transplantation，donors and receptors had no genetic connection，and no im-munosuppressants were administered.It seems that the ideal has come true.This phenomenon is very interesting.It is well known that the main methods that can induce donor specific transplantation toler-ance are bone morrow refer to acceptors，donors'blood transfusion and spleen cells transplantation to acceptors before transplantation and so on[14-16] .So the acceptor's immune system can be reconstructed.If the acceptors were suffered a kind of immune dis-ease and stepped from active stage to steady one，his or her immune system had been suffered self-re-construction and may be tolerant to the same anti-gen from outer.This special situation was worthy of more attention.
The results above revealed us that allogenic melanocytes might survive in acceptor and cultured allogenic melanocytes are possible to repigment vi-tiligo lesions especially large achromic areas[17] .This is a promising procedure to treat vitiligo.Transplantation of allogenic melanocytes has the following advantages:The patients need not to suf-fer from biopsy.Treating process is greatly simpli-fied with less pain and fewer risks;treatment can be conducted conveniently and quickly with little cost.The effects of melanocytes suspension should be better than those from transplantation of skin flap.Long-term study with large numbers of samples is necessary to verify the conclusion.At the same time，the mechanism of successful survival of allo-genic melanocytes has yet to be further studied and the transplantation method need to be modified.
REFERENCES:
[1]Le Poole IC，van den Wijingard RM，Westerhof W，Dutrieux RP，Das PK.Presence or absence of melanocytes in vitiligo lessons:An immunohistochemical investigation [J].J Invest Dermatol，1993;100(6):816-822.
[2]Park YK，Kim NS，Hann SK，Im S.Identification of autoanti-body to melanocytes and characterization of vitiligo antigen in vitiligo patients [J].Dermatol Sci，1996;11(2):111-120.
[3]Njoo MD，Westerhof W，Bos JD，Bossuyt PM.A systematic review of autologous transplantation methods in vitiligo [J].Arch Dermatol，1998;134(12):1543-1549.
[4]Liu YF.Culture of melanocyte，see:The disorders of pigmenta-tion [M]，Zhu Tie-Jun，Beijing:The Press of Beijing Medial University and Peking Union Medical College，1996:15-29.
[5]Eisenberg M，Llewellyn DM，Moran K，Kerr A.Successful en-graftment of cultured human epidermal allograft in a child with recessive dystrophic epidermolysis bullosa [J].Med J Aust，1987;147(10):520-521.
[6]Hedley SJ，Metcaalfe R，Gawkrodger DJ，Weetman AP，Mac Neil S.Vitiligo melanocytes in long-term culture show normal constitutive and cytokine-induced expression of intercellular ad-hesion molecule-1and major histocompatibility complex classⅠand classⅡmolecules [J].Br J Dermatol，1998;139(6):965-973.
[7]Xiang LH，Zheng ZH，Zhu LH，Fu WW，Lu HF.The HLA-DR antigen expression on human melanocytes in vitro and after growth in culture [J].Linchuang Pi Fuke Zazhi(J Clin Der- matol)，1999;28(5):275-277.
[8]Xiang lH，Zheng ZZ，Chen WH.The effects of melanocyte to the transformation and proliferation of allogenic lymphocyte [J].Zhongguo PifuxingbinxueZazhi(Chin J Dermatol Venere-ol)，2001;15(3):149-151.
[9]Lu T，Gao TW，Liu YF，Li CY，Sun LC.Explore of allogenic transplantation of melanocytes to treat vitiligo [J].Di-si Junyi Daxue Xuebao(J Fourth Mil Med Univ)，2001;22(12):1147.
[10]Lu T，Gao TW，Liu YF，Li CY，Sun LC.Explore of allogenic transplantation of melanocytes to treat vitiligo [J].Zhongguo Pifuxingbingxue Zazhi(Clin J Dermatol Venereol)，2001;15(4):240-245.
[11]Gao TW，Ye QX，Liu RQ.The culture of melanocyte in nevus cell and epidermal melanocyte [J].Zhongguo Pifuxingbingxue Zazhi(Clin J Dermatol Venereol)，1993;26(1):51.
[12]Chen S.Transplant immunology [M].Shijiazhuang:The press of Hubei Science and Technology，1998:300-301.
[13]Wang XY，Zhang，FR，Shi ZX，DU WL，Shi BQ，Pan FT.Study on T-lymphocyte sub-populations and levels of soluble in-terleukin-2receptor in the peripheral blood of patients with vi-tiligo [J].Linchuang PifukeZazhi(J Clin Dermatol)，2000;29(5):258-260.
[14]Zhang LY，Chimerism and transplantation [J].Guowaiyixue Mianyixue Fence(Foreign Med Sci Sect Immunol)，2000;23(1):39-42.
[15]Yang G，Cai ZJ，Zhang TZ，Wang ZW.Does fluid nitrogen cry-opreservation act on MHC antigenicity of aorta of rats [J]?Di-si Junyi DaxueXuebao(J Fourth Mil Med Univ)，2001;22(5):410-413.
[16]Yang YL，Li KZ，Dou KF.Can anti-idiotypi antibodies induce transplantation immunoreaction of mice [J]?Di-si Junyi Daxue Xuebao(J Fourth Mil Med Univ)，2001;22(1):1569-1571.[17]Chen YF，Chang JS，Yang PY，Hung CM，Huang MH，Hu DN.Transplant of cultured autologous pure melanocytes after laser abrasion for the treatment of segmental vitiligo [J].J Dermatol，2000;27(7):434-439.
Center of Dermatology&Venereology of Chinese PLA，Xijing Hospital，Fourth Military Medical University，Xi'an710033，China
Editor XU Fu-Ming, http://www.100md.com(LU Tao，GAO Tian-Wen，LI Chun-Ying，SUN Lin)
CLC number:R758.41
Abstract:AIM To study the feasibility of vitiligo treatment through allogenic transplantation of pure melanocytes.METHODS By negative pressure suction blisters were made at a female vitiligo volunteer's lesions.Suspension of pure cultured melanocytes from a healthy male donor was injected into the blisters.The effects in appearance were observed continuously.The outcome of the allogenic melanocytes was determined by pathological examination and specific fragment of Y chromosome detection.After color-recovery were ob-served at the transplanted areas，ten volunteers joined the test too.RESULTS The four transplanted spots'colors of the first female transplanter appeared some re-pigmentation1mo after transplantation.The transplant spots'colors were almost identical to those of adjacent normal skin2mo later.One hundred and sixty days later pathological examination of one repigmented spot displayed that there were melanocytes that could secrete melanin into adjacent keratinocytes.Y chromosome fragment was detected positive in the DNA ex-tracted from the skin of another repigmented spot.Two hun-dred days after second grafting one transplanted spot showed obvious color-recovery too.Similar results were observed in some of other transplanted patients.CONCLUSION Be-cause the allogenic melanocytes can survive in acceptor graft-ing of cultured allogenic melanocytes and can repiment vitiligo lesions and it is promising to develop a new way to treat vi-tiligo.
INTRODUCTION
Vitiligo，an acquired pigment loss of the skin，af-fects approximately1%～4%of the worldwide pop-ulation.It always causes psychological problems.The melanocytes are destroyed in the lesions[1] .The reason of vitiligo is inclined to an autoimmune dis-ease[2] which is difficult to treat now.Autologous transplantation methods have been applied to treat vitiligo and the results proved effective to some ex-tent[3] .But the patients have to suffer from a surgi-cal operation and the melanocytes got from it are fi-nite.So transplantation of allogenic melanocytes，which can solve the problem of the shortage of melanocyte，is an ideal way to treat vitiligo[4] .Eisenberg engraftment cultured human epidermal allograft successfully in a child with recessive dys-trophic epidermolysis bullosa.Allogenic melan-o-cytes were proved in the skin several months lat-er[5] .Recently studies show that cultured pure melanocytes do not express MHC-Ⅱantigen and are not likely to be rejected in vitiligo patient[6-8] .In order to know the real reaction after transplant allo-genic melanocytes and to investigate the feasibility of treated vitiligo by this method，we carried out a series of clinical trials without administerring any immunosuppressants.Before transplant the donors and the receptors had no genetic connection and the major histocompatibility complex were not matched[9，10] .
MATERIALS AND METHODS
Preparation of melanocytes suspension Melano-cytes were obtained from a healthy young man's foreskin.We modified the melanocytes culture method of the previous literatures[11] .The cells were established in1640medium containing100g L-1 calf serum，6nmol L-1 Cholera Toxin，2mg L-1 basic fibroblast growth factor(all GIBCO prod-ucts).The fourth generation of melanocyte suspen-sions were prepared with saline at different cell den-sities.
Subjects and transplantation The first volunteer was a27-year-old female.She had suffered from vi-tiligo for more than20years.Her lesions did not expand in recent three years.There were other10patients(7female，3male)joined this test.Except one male patient was in active stage，all patients were in stable stage.
In the abdomen lesion of the first female patient(Fig1)four blisters were formed in about40min-utes by epidermis separate machine which could hold the transplanted sites in compressive stress of negative48kPa and temperature of43℃.Most of exudates in the blisters were drawn out by a new in-jector.The melanocyte suspensions were injected into the cavity of the blisters(about0.1mL sus-pension per blister).Two blisters marked A and B were injected both20，000melanocytes.The melanocytes number of the other two blisters C and D was10，000and1，000respectively(Fig2).The blisters were kept wholly and carefully for a few days.
Follow up and examination concerned If the trans-planted spots were color-recovered，the skin of one spot would be biopsied for histopathological exami-nation half year later.One of the other re-pigment-ed spot was made blister.DNA was got from the roof of the blister(Shanghai Biomedicine LTD)and sent for examing of Y chromosome peculiar frag-ment by high sensitive detects KIT(Fuhua Biomedicine corporation LTD.Shanghai).
Second transplantation 160days after the first transplantation，the patient accepted another trans-plantation and the melanocytes transplanted came from another male donor.The melanocytes sus-pends were grafted to the spots E，F，G at the adja-cent site(Fig4)and spots H of her right sciatic site(Fig7).The spot H was injected suspend without melanocytes.The spot I didn't injected anything.Other transplanted patients From then on，10oth-er patients(7female，3male)received allogenic melanocytes transplantation.All patients were in stable phase except a male patient.Results were ob-served continuously.Three months later color re-covered skin was sent to examine Y chromosome pe-culiar fragment.
RESULTS
Clinical effects First volunteer didn't feel any dis-comfort after transplantation.The spot began to turn red，and slight re-pigment appeared thirty days later.The re-pigmentations were getting more and more obviously.The colors of the four sites were similar to the normal adjacent skin75days after transplantation(Fig2)and existed stably.
After second transplantation，she didn't feel any discomfort yet.The photo of two hundred days later(three hundred and sixty days after the first transplantation)showed obviously color-recover at her right sciatic site H(Fig3).There were no col-or-recover at the spot H and I that didn't graft melanocytes.The re-pigmentations of the first transplantation were not influenced(Fig4).
Examination results Fontana dying of the spot A displayed a lot of melanin existing in the ker-atinocytes at the basal layer.There were some melanocytes existing around the keratinocytes(Fig5).The DNA of the re-pigmented spot C's roof was positive of chromosome peculiar fragment(Fig6).Results of other patients Among the other ten pa-tients，two males and one female were lost in fol-low-up survey.Six females'lesions showed slight re-pigment three months later.The colors of some transplanted spots in two cases were similar to those of normal skin.Some graft spots didn't change in evidence.We detected Y chromosome peculiar frag-ment in DNA of one female acceptor's color-recov-ered skin(Fig7).The male patient with active dis-ease got re-pigment for several months but the re-pigmentation disappeared six months later.
Fig1 The depigmented area at the abdomen of the first patient before transplantation(略)
Fig2 The four spots at the abdomen of the first patient75days after transplantation(略)
Fig3 Obvious color-recover at the first patient's right sciatic spot K200days after the second transplantation(略)
Fig4 360days after the first transplantation and200days after the second transplantation at the abdomen(略)
Fig5 A lot of melanin demonstrated in the keratinocytes around melanocytes at the re-pigment area A with Fantana dyeing(略)
DISCUSSION
In above transplant test，MHC was not matched be-fore transplantation，donors and receptors had no genetic connection，and immunosuppressants were not administered.We found color-recovered in some patients'transplant sites.Reestablishment color could existed stably，without spreading into sur-rounding areas.The first volunteer got certain ef-fects in secondary allogenic transplantation.Histopathological examination of the color-recov-ered spot A showed that there were melanocytes se-creting melanin.It is the melanocytes that worked normally lead to re-pigmentation of the lesions.
Fig6 Y chromosome peculiar fragments was positive in the DNA of the blister C's roofH2(略)
The melanocytes that existed at the basal layer were concluded to be the allogenic melanocytes.Firstly，in the hair follicle of spot A where melanocytes of oneself could exist had no melanin dying.It proved that there were not melanin that was secreted by herself and the melanin at the basal layer could only come from the allogenic melanocytes.Secondly，the DNAs of the re-pig-ment spots'skins of two female receptors was veri-fied to contain Y chromosome peculiar fragment that only exists in man(Fig3).In addition，no re-pig-mentation was observed in the sites where melanocytes were not injected.
According to transplantation immunology，this phenomenon belongs to donor specific transplanta-tion tolerance[12] .Mechanism of the survival of allo-genic melanocytes may be related to the special im-munological condition of vitiligo and the properties of purely cultured melanocyte.It is well known that in immunologic rejection caused by allograft trans-plantation，presentation of heterogeneous MHC antigens and T cell recognition goes first，and then proliferation and differentiation of T cell occurs with lymphokines production，leading to the rejection of transplants.The following factors may contribute to the survival of the allogenic melanocytes:①De-layed allergy is poor in most vitiligo patients.The number of CD4+ cells and the ratio of CD4+ to CD8+ cells are significantly decrease in vilitigo pa-tients[13] .This immunodeficiency is helpful for the transplantation of allogenic melanocytes.②Melanocytes do not express or express very few MHC-Ⅱmolecules that play an important role in transplantation rejection.MHC-Ⅱantigens begin to express when melanocytes were cultured in vitro and affected by some growth factors for many genera-tions[6，7] .So the pure cultured melanocytes of few generations are not easy to be recognized and reject-ed.③Vitiligo is highly related to autoimmunity[2] .Autoantibodies produced by the body and sensitized lymphocytes can invade self-cell components and damage self-melanocytes.In stationary and restora-tion stages，areas of lesions decrease or do not change，and immunological reaction to melanocyte stops.Thus，allogenic melanocytes perhaps are easy to survive.
To induce spontaneous donor specific trans-plantation tolerance is an ideal that people pusued hardly.In our study we find allogenic melanocytes can survive in vitiligo acceptors'body when MHC was not matched before transplantation，donors and receptors had no genetic connection，and no im-munosuppressants were administered.It seems that the ideal has come true.This phenomenon is very interesting.It is well known that the main methods that can induce donor specific transplantation toler-ance are bone morrow refer to acceptors，donors'blood transfusion and spleen cells transplantation to acceptors before transplantation and so on[14-16] .So the acceptor's immune system can be reconstructed.If the acceptors were suffered a kind of immune dis-ease and stepped from active stage to steady one，his or her immune system had been suffered self-re-construction and may be tolerant to the same anti-gen from outer.This special situation was worthy of more attention.
The results above revealed us that allogenic melanocytes might survive in acceptor and cultured allogenic melanocytes are possible to repigment vi-tiligo lesions especially large achromic areas[17] .This is a promising procedure to treat vitiligo.Transplantation of allogenic melanocytes has the following advantages:The patients need not to suf-fer from biopsy.Treating process is greatly simpli-fied with less pain and fewer risks;treatment can be conducted conveniently and quickly with little cost.The effects of melanocytes suspension should be better than those from transplantation of skin flap.Long-term study with large numbers of samples is necessary to verify the conclusion.At the same time，the mechanism of successful survival of allo-genic melanocytes has yet to be further studied and the transplantation method need to be modified.
REFERENCES:
[1]Le Poole IC，van den Wijingard RM，Westerhof W，Dutrieux RP，Das PK.Presence or absence of melanocytes in vitiligo lessons:An immunohistochemical investigation [J].J Invest Dermatol，1993;100(6):816-822.
[2]Park YK，Kim NS，Hann SK，Im S.Identification of autoanti-body to melanocytes and characterization of vitiligo antigen in vitiligo patients [J].Dermatol Sci，1996;11(2):111-120.
[3]Njoo MD，Westerhof W，Bos JD，Bossuyt PM.A systematic review of autologous transplantation methods in vitiligo [J].Arch Dermatol，1998;134(12):1543-1549.
[4]Liu YF.Culture of melanocyte，see:The disorders of pigmenta-tion [M]，Zhu Tie-Jun，Beijing:The Press of Beijing Medial University and Peking Union Medical College，1996:15-29.
[5]Eisenberg M，Llewellyn DM，Moran K，Kerr A.Successful en-graftment of cultured human epidermal allograft in a child with recessive dystrophic epidermolysis bullosa [J].Med J Aust，1987;147(10):520-521.
[6]Hedley SJ，Metcaalfe R，Gawkrodger DJ，Weetman AP，Mac Neil S.Vitiligo melanocytes in long-term culture show normal constitutive and cytokine-induced expression of intercellular ad-hesion molecule-1and major histocompatibility complex classⅠand classⅡmolecules [J].Br J Dermatol，1998;139(6):965-973.
[7]Xiang LH，Zheng ZH，Zhu LH，Fu WW，Lu HF.The HLA-DR antigen expression on human melanocytes in vitro and after growth in culture [J].Linchuang Pi Fuke Zazhi(J Clin Der- matol)，1999;28(5):275-277.
[8]Xiang lH，Zheng ZZ，Chen WH.The effects of melanocyte to the transformation and proliferation of allogenic lymphocyte [J].Zhongguo PifuxingbinxueZazhi(Chin J Dermatol Venere-ol)，2001;15(3):149-151.
[9]Lu T，Gao TW，Liu YF，Li CY，Sun LC.Explore of allogenic transplantation of melanocytes to treat vitiligo [J].Di-si Junyi Daxue Xuebao(J Fourth Mil Med Univ)，2001;22(12):1147.
[10]Lu T，Gao TW，Liu YF，Li CY，Sun LC.Explore of allogenic transplantation of melanocytes to treat vitiligo [J].Zhongguo Pifuxingbingxue Zazhi(Clin J Dermatol Venereol)，2001;15(4):240-245.
[11]Gao TW，Ye QX，Liu RQ.The culture of melanocyte in nevus cell and epidermal melanocyte [J].Zhongguo Pifuxingbingxue Zazhi(Clin J Dermatol Venereol)，1993;26(1):51.
[12]Chen S.Transplant immunology [M].Shijiazhuang:The press of Hubei Science and Technology，1998:300-301.
[13]Wang XY，Zhang，FR，Shi ZX，DU WL，Shi BQ，Pan FT.Study on T-lymphocyte sub-populations and levels of soluble in-terleukin-2receptor in the peripheral blood of patients with vi-tiligo [J].Linchuang PifukeZazhi(J Clin Dermatol)，2000;29(5):258-260.
[14]Zhang LY，Chimerism and transplantation [J].Guowaiyixue Mianyixue Fence(Foreign Med Sci Sect Immunol)，2000;23(1):39-42.
[15]Yang G，Cai ZJ，Zhang TZ，Wang ZW.Does fluid nitrogen cry-opreservation act on MHC antigenicity of aorta of rats [J]?Di-si Junyi DaxueXuebao(J Fourth Mil Med Univ)，2001;22(5):410-413.
[16]Yang YL，Li KZ，Dou KF.Can anti-idiotypi antibodies induce transplantation immunoreaction of mice [J]?Di-si Junyi Daxue Xuebao(J Fourth Mil Med Univ)，2001;22(1):1569-1571.[17]Chen YF，Chang JS，Yang PY，Hung CM，Huang MH，Hu DN.Transplant of cultured autologous pure melanocytes after laser abrasion for the treatment of segmental vitiligo [J].J Dermatol，2000;27(7):434-439.
Center of Dermatology&Venereology of Chinese PLA，Xijing Hospital，Fourth Military Medical University，Xi'an710033，China
Editor XU Fu-Ming, http://www.100md.com(LU Tao，GAO Tian-Wen，LI Chun-Ying，SUN Lin)