Detection of auto antibodies and transplantation of cultured autologous melanocytes for the treatment of vitiligo.

The aim of the present study was to establish an immunofluorescence method of antibody detection to identify melanocytes in the serum of vitiligo patients. Furthermore, we aimed to establish a method for the culture and proliferation of autologous pure melanocytes and to observe the effect of their transplantation for the treatment of vitiligo. Suspension of epidermal cells with melanocytes was performed using trypsin digestion of normal epiderm from eyelid operation and melanocytes were selectively cultured and proliferated in serum-free M2 medium. FITC-labeled rabbit anti-human antibody was used to detect the relative fluorescence intensity of the melanocytes. After identification with immunological and biological examinations, the melanocytes were transplanted to depigmented areas of vitiligo. Repigmentation was observed continuously. The results indicated that melanocytes could be selectively proliferated in the medium. Subsequently, pure melanocytes without contamination of fibroblast and keratinocyte were harvested. A total of 34 patients suffering vitiligo for between 3 months and 20 years with depigmented area (between 4 cm2 and 70% of body surface) were divided into 19 cases of developing stage and 15 cases of stable stage, according to the change of depigmentation. A total of 15 developing cases were positive for the antibody against melanocytes, with the positive rate of 79%. The titers of serum was >1:50 in 10 patients at the developing stage, and 5 developing patients were 1:10. Among the 15 stable cases, four were positive, with a positive rate of 27%. Fluorescence of antibody was localized in the cytoplasm of the melanocytes. Autologous melanocytes of vitiligo patients could be selectively proliferated in the medium. Next, pure melanocytes without contamination with fibroblasts and keratinocytes were harvested. A total of 16 vitiligo patients with 28 depigmented areas (2-200 cm2) were treated with transplantation of melanocytes. Repigmentation of the transplanted areas appeared as red coloration after one month. All the vitiligous areas received transplantation were repigmented significantly with hypo- or hyper-pigmentation after 3-5 months. After 6-8 months, 87.5% of lesions showed repigmentation of >50% of the lesion area. No scarring or other side-effects occurred. After follow-up of 5 years, no relapse was observed in transplantation area. Thus, an immunofluorescence method for the test of antibody to melanocytes in the serum of vitiligo patients was established. Transplantation of cultured autologous melanocytes was an effective and safe measure for treatment of vitiligo, particularly for patients with a large depigmented area.

Hydroxysafflor yellow A enhances survival of vascular endothelial cells under hypoxia via upregulation of the HIF-1 alpha-VEGF pathway and regulation of Bcl-2/Bax.

Hydroxysafflor yellow A (HSYA) is a component of the flower Carthamus tinctorius L. The present investigation determines whether HSYA can modify the effects of hypoxia on vascular endothelial cells (EC) and its mechanisms. Human EC line (EAhy926) viability was determined using the MTT assay. EC cycle phase distribution was done with PI staining and flow cytometric analysis, and EC apoptosis was done by AnnexinV-FITC detection and the TUNEL assay. The protein levels of VEGF, Bcl-2, Bax, and HIF-1 alpha were determined by ELISA or Western blot analysis, and the mRNA expression of these genes by RT-PCR analysis. HIF-1 alpha transcriptional activity was measured using a reporter gene assay. HSYA improved cell viability under hypoxia in a concentration-dependent manner by attenuating its cycle arrest and inhibiting its apoptosis. HSYA upregulated the bcl-2/bax ratio, which is downregulated under hypoxia, increased VEGF protein concentration and VEGF mRNA expression and enhanced HIF-1 alpha protein accumulation and its transcriptional activity. In conclusion, HSAY could enhance the survival of ECs under hypoxia, which may be correlated with its effect of upregulating the bcl-2/bax ratio and promoting HIF-1 alpha protein accumulation, which increases VEGF. These findings provide evidence for the mechanisms by which HSYA maintains EC survival under hypoxia.

[Expression and activity of tissue-type plasminogen activator mutant reteplase with deletion of PAI-1 binding sites].

AIM: To construct a mutant of human tissue plasminogen activator (t-PA) with activity not inhibited by plasminogen activator inhibitor-1 (PAI-1). METHODS: The finger, epidermal growth factor and kringle-1 domains were removed from native t-PA to obtain a t-PA mutant with the PAI-1 binding sites. A pUC18 plasmid containing the human t-PA full-length cDNA sequence was used as template, and the DNA sequences encoding 1-3 and 176-527 amino acids were amplified by PCR. The nucleotides AAG CAC AGG AGG (from 373 to 384) in the PAI-1 binding site changed to GCG GCC GCG GCG, so amino acid KHRR was replaced by AAAA correspondently. RESULTS: Sequencing result showed that the above t-PA mutant DNA sequence was consistent with the expected. Then it was cloned in an E.coli expression vector and was highly expressed. The expressed protein occupied about 30% of total bacterial proteins as inclusion body. After denaturation and renaturation, the active t-PA mutant was obtained,activity of which was not inhibited by PAI-1. CONCLUSION: This t-PA mutant may be developed to a new gene-engineering drug with higher therapeutic activity and lower dose requirement in the treatment of acute myocardial infarction and cerebrovascular thrombosis diseases.

[Prokaryotic expression of DnaJ-homologous chaperon PBP and preparation of rabbit antibody against PBP].

AIM: To express DnaJ-homologous chaperon peripherin-binding protein(PBP) gene in E.coli and prepare the rabbit antibody against PBP. METHODS: The PBP cDNA was amplified from the human fetal brain tissue by RT-PCR. After confirmed by DNA sequencing, the PBP-cDNA was cloned into expression vector pET28a and then the PBP gene was expressed in E.coli under the IPTG induction. The expressed protein was purified through Ni-NTA affinity chromatography column. The rabbit antibody against PBP was prepared by immunizing two New Zealand white rabbits using the purified PBP as immunogen. The titer and specificity of the antisera were determined by Western blot. RESULTS: The 720 bp PBP gene was amplified, cloned, and expressed in E.coli. The expressed product existed in the bacterial inclusion body and the supernatant of the bacteria lysate. The purified PBP reached electrophoretic purity. The rabbit antibody against PBP was prepared and its titer was about 1:1,600. Western blot analysis showed that the antibody could bind to the expressed PBP protein specifically. CONCLUSION: The PBP protein was expressed in E.coli and rabbit antibody against PBP was prepared successfully, which lays the foundation for further study on the structure and biological function of PBP.

[Cloning of a DnaJ homolog chaperon PBP and its subcellular localization].

AIM: To isolate and identify a human DnaJ homolog chaperon, PBP, from a human skeleton cDNA library, and to analyze its expression and distribution in transfected mammalian cells. METHODS: (32)p-dCTP labeled probe hybridization was used to screen the human skeleton cDNA library and sequence of the positive clones were analyzed. Then PBP gene was transfected into COS-7 cells using lipofectamin. PBP expressed in the cells were detected by Western-blot and indirect immunofluorescence staining. RESULTS: A full-length(1.5 kb) cDNA of peripherin-binding protein (PBP) was identified, which is identical with that of mrj. Full length PBP was mainly localized to cytoplasms of COS-7 cells in interphase, and to nuclei in mitosis. CONCLUSION: The results indicate that besides cooperating with DnaK (HSP70), PBP itself plays an important role as a member of DnaJ family. PBP may also be involved in the regulation of cell cycle.

[Construction of a cDNA subtractive library of rat brain after repeated +Gz exposure].

Objective. To construct a cDNA subtractive library of rat brain after repeated + Gz exposures with suppression subtractive hybridization (SSH). Method. Wistar [correction of Wister] rats were randomly divided into control group and repeated +Gz exposure group. Using an animal centrifuge, control rats were exposed to +1 Gz and exposure rats were exposed to +10 Gz for three times, each for 1 min with 30 min interval in between. Brains were taken 6 h after the last centrifuge run and Poly (A) + RNA were isolated. Moreover, single-strand cDNAs and double-strand cDNAs were synthesized in turn. After Rsa I enzyme restriction, +Gz exposure rat brain cDNAs were divided into two groups and ligated to the specific adaptor 1 and adaptor 2R, respectively. Then +Gz exposure rat brain cDNAs were hybridized with the control rat brain cDNA twice and underwent nested PCR twice. The PCR product was ligated with T/A plasmid vectors to set up the subtractive library. Result. The cDNA subtractive library of rat brain after repeated +Gz exposures with high subtractive efficiency was set up successfully. Conclusion. The highly efficient cDNA subtractive library may provide a solid foundation for screening and cloning differentially expressed genes in rat brain after repeated exposures to +Gz.