[Treatment of chronic refractory wound with autologous platelet-rich plasma in a hemophilia A patient after knee joint surgery].

On December 22, 2017, a 35-year-old male hemophilia A patient with a secondary chronic refractory wound after left knee joint surgery was transferred from the Department of Hematology of Maoming People's Hospital to the Department of Burns and Plastic Surgery in the same hospital. The physical examination revealed that the patient's left knee joint was swollen, with a full-thickness skin defect wound of 4 cm×4 cm on the lateral side of the joint and a large number of dark red blood clots at the bottom of the wound. The wound bleeding was controlled by intravenous infusion of plasma, cryoprecipitate, and human coagulation factor Ⅷ. After con- ventional debridement and dressing changes until the wound infection was controlled and necrotic tissue was removed, a subcutaneous cavity wound of 2 cm×2 cm in area and 3 cm in depth remained in the left knee joint and was difficult to heal. Nineteen days after transfer, the patient received autologous platelet-rich plasma (PRP) treatment, and 32 days after PRP treatment, the wound in left knee joint was healed with epithelialization. This case suggests that autologous PRP therapy would be a good option for hemophilia complicated chronic refractory wounds when they could not be repaired by surgery.

[Ultrasonic and pathological comparative analysis of different subtypes of breast encapsulated papillary carcinoma].

Objective: To investigate the clinicopathological and ultrasonic characteristics of patients with breast encapsulated papillary carcinoma (EPC) and the comparative analysis of different subtypes. Methods: A total of 57 patients with pathological diagnosis of breast EPC in the First Affiliated Hospital of Nanjing Medical University from September 2014 to August 2020 were retrospectively collected. Based on pathological diagnosis, patients were divided into 3 subtypes, and their clinical, pathological and ultrasonic manifestations were compared and analyzed. Results: Among the enrolled patients, there were 2 males and 55 females, aged 41-88 (63±11) years. The lesion diameter of EPC was 7.0-7.5 (2.9±1.9) cm. There were 16 cases of simple EPC, 9 cases of EPC with ductal carcinoma in situ (DCIS), and 32 cases of EPC with infiltration. The molecular classification was mainly luminal (55/57, 96.6%), of which 38 cases were Luminal A type and 17 cases were Luminal B type. The majority ultrasound images of EPC showed nodules with size greater than 2 cm (68.4%), regular morphology (64.9%), edge finishing (54.4%), no burr forming angles (82.5%), and no calcification (93%). There were differences between different subtypes. The simple EPC was closer to the nipple than the EPC with infiltration, with regular shape and smooth edge (P<0.05). Compared with the EPC with DCIS, the EPC with infiltration had richer blood flow and was farther away from the nipple (P<0.05). There was no significant difference between the simple EPC and the EPC with DCIS (P>0.05). Conclusion: The simple EPC often showed benign signs on ultrasound, such as regular morphology, smooth edge, and cystic-solid internal structure. If the distance between the lesion and the nipple was greater than 3 cm, the shape was irregular, and the edge was blurred, the possibility of EPC with infiltration was high. Multi-modality image fusion was helpful for accurate diagnosis and treatment of EPC.

TM4SF1 facilitates non-small cell lung cancer progression through regulating YAP-TEAD pathway.

OBJECTIVE: Transmembrane-4-L- Six-Family-1 (TM4SF1) has been found involved in the development and progression of tumor. This study aims to investigate the effect of TM4SF1 on the proliferation, migration, and invasion of non-small cell lung cancer (NSCLC) and reveal its underlying mechanisms. MATERIALS AND METHODS: qRT-PCR, immunohistochemical analysis, and Western blot were used to evaluate the expression of TM4SF1 in human NSCLC tissues and cells. Cell proliferation was measured by CCK-8 and colony formation assay. Cell apoptosis was evaluated by flow cytometry assay. Cell migration and invasion were detected by wound healing and transwell assays. Co-immunoprecipitation (Co-IP) assay was used to examine the interactions between proteins. Expression levels of related proteins were determined by Western blot. For in vivo experiment, xenograft tumor models were used. RESULTS: TM4SF1 was upregulated in NSCLC tissues and cell lines and closely correlated to survival time, tumor size, lymph node metastasis, distant metastasis, and clinical stage. Gain-of function and loss-of function experiments demonstrated the oncogenic effect of TM4SF1 on NSCLC cell proliferation, apoptosis, migration, and invasion. Notably, mechanism studies showed that TM4SF1 regulated the interaction between YAP and TEAD and the level of downstream target genes. Besides, sh-YAP or Peptide 17 treatment (YAP-TEAD protein-protein interaction inhibitor) reversed the effect of TM4SF1 on NSCLC cells. The in vivo research suggested that the knockdown of TM4SF1 inhibited the growth of xenograft tumor of NSCLC. CONCLUSIONS: This is the first evidence demonstrating that TM4SF1 could promote proliferation, migration, and invasion in NSCLC, at least partially through a potential YAP-TEAD signaling pathway-dependent mechanism. This study might provide a potential therapeutic target for the treatment of NSCLC.

[Study on commutability evaluation of reference materials for fibrinogen].

Objective: To discuss the commutability evaluation method of reference materials for fibrinogen measurement and evaluate the commutability of the Third WHO International Standard Fibrinogen Plasma (WHO 09/264), SSC/ISTH Secondary Coagulation Standard (SSC LOT4) and homemade reference materials (RM01, RM02) in order to provide suggestions on how to determine the suitable method of commutability evaluation and reliable traceability standard. Methods: The comparability of fibrinogen among different measurement systems were evaluated and WHO 09/264 was used to calibrate each system to improve the comparability if the comparability among different systems couldn't be accepted. Forty clinical samples and the reference materials randomly interspersed among the clinical samples were measured on Stago STA-R Evolution, Sysmex CS 5100, IL ACL TOP 700 simultaneously. Measurement results were pairwise analyzed by Deming regression and difference in bias approach according to the Clinical and Laboratory Standards Institute (CLSI) EP14-A3 protocol and the recommendations of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) working group on commutability, respectively. Results: The comparability of fibrinogen measurement among common systems could not meet the criterion. WHO 09/264 could improve the agreement among different measurement systems. The prediction interval of Deming regression was affected by the comparability of measurement systems, resulting in unreliable results. The difference in bias approach was more suitable because its criterion was related to the medical requirements. WHO 09/264 was commutable between Stago and Sysmex, inconclusive between Stago and IL, Sysmex and IL (The calibration effectiveness of WHO 09/264 showed that it was commutable among the three measurement systems). SSC LOT4 was commutable between Stago and Sysmex, inconclusive between Stago and IL, Sysmex and IL. RM01 and RM02 were commutable between all systems pairs assessed by difference in bias approach. Conclusions: There are differences in the results of two commutability evaluation approaches. The difference in bias approach is recommended for commutability evaluation. WHO International Standard and homemade reference materials can be used as traceability standard for fibrinogen measurement.

TUG1 promotes diabetic atherosclerosis by regulating proliferation of endothelial cells via Wnt pathway.

OBJECTIVE: To explore the specific role of TUG1 in regulating the occurrence and progression of diabetic atherosclerosis and its underlying mechanism. PATIENTS AND METHODS: TUG1 expressions in coronary artery disease (CAD) tissues, normal arterial tissues, endothelial cells induced by high-dose glucose and tumor necrosis factor-α (TNF-α) were detected by quantitative Real-time polymerase chain reaction (qRT-PCR). The effects of TUG1 on proliferation, migration and cell cycle of human umbilical vein endothelial cells (HUVECs) were detected by cell counting kit-8 (CCK-8), transwell assay and flow cytometry, respectively. Subsequently, protein expressions of proliferation-related genes, cell cycle-related genes and Wnt pathway-related genes were detected by Western blot after altering TUG1 expression in HUVECs. Further rescue experiments were carried out to explore whether TUG1 could regulate diabetic atherosclerosis via Wnt pathway. RESULTS: Overexpressed TUG1 was found in CAD tissues and endothelial cells induced by high-dose glucose and TNF-α compared with those of controls. TUG1 overexpression remarkably promoted proliferation, migration and cell cycle of HUVECs. Protein expressions of ß-catenin and c-Myc were upregulated by overexpression of TUG1. Rescue experiments indicated that XAV-939, the inhibitor of Wnt pathway, could partially reverse the increased proliferative and migratory changes in HUVECs induced by TUG1 overexpression. CONCLUSIONS: We found that overexpressed TUG1 stimulates proliferation and migration of endothelial cells via Wnt pathway, thereby promoting the occurrence and progression of diabetic atherosclerosis.

[Analysis of internal quality control data of complete blood count from laboratories participating in national external quality assessment].

Objective: To investigate current status and problems of internal quality control (IQC) of complete blood count in China so as to perform IQC normally. Methods: The IQC data of complete blood count for five parameters were collected from laboratories participating in national external quality assessment during 2012-2017 (totally 12 times), including WBC, RBC, Hb, Hct and PLT. After confirmation of all data, data for the 12 times were analyzed as follows.The proportions of using different levels of quality control materials were calculated.The 25th, 50th, 75th, 90th percentiles CV of data collected for the 12 times were calculated respectively and the trends of CV were observed over time.The difference of CV among laboratories running three control levels was compared.The CV of each parameter in 2017 was compared with precision requirements based on biological variation, health standards and German Medical Association Directive; The proportions of laboratories using different control rules were calculated. Results: After invalid data was excluded from those IQC data of laboratories for the 12 times external quality assessment (up to 2 402, as low as 1 449) from 2012 to 2017, the residual data (up to 2 332, as low as 1 431, accounting for 96.0%-99.2%) was used for analysis. 61.9%-66.1%, 18.2%-23.6% and 14.3%-17.3% of laboratories ran one, two and three control levels respectively, and the proportions of laboratories running more than two control levels increased from 33.9% to 38.1%. The decrease trend of the 75th, 90th percentiles CV of WBC, RBC, Hb, Hct for three levels, PLT for normal level and the 90th percentiles CV of PLT low level had statistically significance over time (P<0.05); the decrease trend of the 75th percentiles CV of PLT low level and 75th, 90th percentiles CV of PLT high level had no statistically significance over time. The CV had significant difference between low and normal, low and high control level for WBC and PLT, while there were no difference between normal and high control levels. There were no significant difference of CV among three control levels for RBC, Hb, and Hct. Except for the CV of Hct low, normal level and PLT low level, 85% of laboratories for the other parameters could meet the minimum precision requirements based on biological variation; more than 85% laboratories met the requirements of health standards; except for the CV of PLT low level, more than 80% laboratories met the requirements of German Medical Association Directive. The proportion of laboratories using 1(3s)/2(2s) quality control rules increased from 59.2% to 76.0%. Conclusions: During the past 6 years, the CV for IQC has shown a decrease trend over time. However, the control level and quality control rules used by some laboratories do not meet management requirements. The CV of Hct and PLT in a few laboratories do not meet the minimum requirements of the health standards, and need to implement quality improvements fatherly.

[Study and analysis of national current status and problems of anticoagulant proteins assay].

Objective: To investigate current status and problems of anticoagulant proteins assay in domestic laboratories so as to provide suggestions for implementing the standardization and quality improvement. Methods: Two hundred and seventy-four laboratories those had developed or prepared to do anticoagulant proteins assay were selected from one thousand and five hundred participants in the national coagulation screening External Quality Assessment(EQA) program by an internet survey and then a questionnaire and quality control materials were sent to them to carry out a further survey. The questionnaire information was analyzed statistically. The results of quality control materials were grouped by the reagents and the average, median, standard deviation(s), coefficient variation(CV) of each group were calculated. The deviations or percentage deviations were determined by comparing the results of each laboratory to the target defined as the peer-group median after exclusion of outliers, and then the pass rates were calculated based on the criterion of RCPA, DGKL and the allowable total error based on biological variation. Results: Two hundred and thirty-five questionnaires were collected. The number of laboratories testing antithrombin(AT), protein C(PC) and protein S(PS) activity were 194, 63 and 50 respectively. The instruments and reagents were mainly from abroad (more than 96%), the matching rate of which were above 94%. For AT, PC and PS activity testing, there were 30.4%, 33.3%, 34.0% of laboratories did not perform verification assays respectively, and 8.8%, 7.9%, 14.0% of laboratories did not renew calibration curve when the reagent lots were changed. 11.3%, 17.5%, 16.0% of laboratories didn't run internal quality control, and 34.9%, 26.9%, 21.4% of laboratories only performed a single level of quality control. 4.1% of laboratories set the reference intervals of AT activity according to different age groups, and the percentage of that of PC and PS activity were 1.6% and 2.0%. 16.0% of laboratories set the reference interval of PS activity by sex. For normal control materials, the CV of AT, PC and PS activity results were 5.7%-12.9%, 4.2%-7.7% and 18.4%-33.1% while the CV for abnormal level were 13.3%-38.3%, 6.1%-14.4% and 31.5%-34.5% respectively. The pass rate was different when it was judged by different criteria. A suitable criterion for each item should be selected according to the concentration level of quality control materials. Conclusion: The comparability between laboratory results are not satisfactory and in order to promote quality improvement, it is necessary to develop guidelines, organize trainings and establish a national EQA scheme.

[Advances in the research of role of integrin-linked kinase in angiogenesis].

Integrin-linked kinase (ILK) has been found for twenty years, and its biological characteristics have been extensively studied by multi-discipline. At present, studies of ILK are mainly focused on its roles in angiogenesis, tumor formation, and tissue fibrosis, etc. In recent years, the regulation effect of ILK in angiogenesis attracts attention of researchers. The studies showed that ILK can stimulate the secretion of angiogenic factor, promote the proliferation and migration of endothelial cells and inhibit their apoptosis, and therefore play an important role in the regulation of angiogenesis. Further research on molecular mechanism about the role of ILK playing in angiogenesis may provide an effective method for the treatment of some diseases.

[Advances in effects of integrin-linked kinase on cutaneous wound healing and the relative mechanism].

Previous studies have demonstrated that integrin-linked kinases (ILKs) are abundantly expressed in extracellular matrix (ECM) riche dermis, hair follicles, and basal cells of epidermis. ILKs are not only essential for the maintenance of skin structure, but also play important roles in wound healing. ILKs can promote the formation of granulation tissue by stimulating the proliferation of fibroblasts and secretion of ECM, accelerate wound contraction by inducing the differentiation of fibroblasts to myofibroblasts, and boost reepithelization by promoting proliferation, migration, and differentiation of keratinocytes and follicle epidermal stem cells.

Borax-induced apoptosis in HepG2 cells involves p53, Bcl-2, and Bax.

Borax, a boron compound and a salt of boric acid, is known to inhibit the growth of tumor cells. HepG2 cells have been shown to be clearly susceptible to the anti-proliferative effects of borax. However, the specific mechanisms regulating this effect are poorly understood. This study aimed to investigate the pathways underlying the growth inhibition induced by borax in HepG2 cells. The effects of borax on HepG2 cell viability were characterized using MTT. Apoptosis was also verified by annexin V/propidium iodide staining. JC-1 dye and western blotting techniques were used to measure mitochondrial membrane potential and p53, Bax, and Bcl-2 protein expression, respectively. Relevant mRNA levels were measured by qRT-PCR. Borax inhibited the proliferation of HepG2 cells in a time- and dose-dependent manner in vitro. The apoptotic process triggered by borax involved the upregulation of p53 and Bax and the downregulation of Bcl-2, which was confirmed by a change in the mitochondrial membrane potential. These results elucidate a borax-induced apoptotic pathway in HepG2 cells that involves the upregulation of p53 and Bax and the downregulation of Bcl-2.

[Evaluation and characterization of the certified reference materials for coagulation factor â § and â ¨ activity testing].

OBJECTIVE: To evaluate and characterize the certified reference materials for coagulation factor Ⅷ (FⅧ) and factor Ⅸ (FⅨ) activity testing. METHODS: The homogeneity and stability of three lots of certified reference materials (F01-F03) with different factor concentrations were evaluated according to guidelines"Reference materials-general and statistical principles for certification","Guidance on evaluating the homogeneity and stability of samples used for proficiency testing"and"Technical Norm of Primary Reference Material". The certified reference materials were characterized by eight laboratories using one-stage method, which were calibrated by the coagulation standard provided by the National Institute for Biological Standards and Control (NIBSC) in UK. RESULTS: The Coefficient of Variation (CV) of homogeneity test of FⅧ activity of three lots of certified reference materials were 3.9%, 3.3% and 3.4%, respectively. While that of FⅨ activity were 3.7%, 3.0% and 1.8%, respectively. The results of one-way ANOVA showed that all certified reference materials had good homogeneity (P>0.05), and the between-bottle homogeneity uncertainties (ubb) of FⅧ and FⅨ activity were 0.5%-2.9% and 0.1%-3.9%, respectively. All certified reference materials stored in -80 ℃ remained stable in 9 months by trend analysis, and the long-term stability uncertainties(ults) of FⅧ and FⅨ activity were 0.5%-5.1% and 1.3%-4.4%, respectively. The characterization uncertainties (uchar) of FⅧ and FⅨ activity testing were 0.9%-2.4% and 1.1%-2.4%, respectively. The combined uncertainties and extended uncertainties (coverage factor k=2) were calculated. The assigned values of each lot of certified reference materials for FⅧ activity were (85±13)%, (36.0±3.4)% and (20.5±2.3)%, and that were (102±13)%, (47.8±6.9)% and (29.3±3.8)% for FⅨ activity, respectively. CONCLUSIONS: The certified reference materials for FⅧ and FⅨ activity testing have good homogeneity and stability. The results of the characterization are accurate and reliable.

MicroRNA-mediated regulation of melanoma.

Melanoma is one of the most aggressive and deadly skin cancers, and, in its advanced stages, accounts for > 80% mortality. The incidence of melanoma is increasing worldwide; however, beyond surgical removal of the tumour, there is currently no curative therapy available, especially for its advanced stages. This may, in part, be owing to incomplete understanding of the molecular mechanisms that regulate the initiation and/or progression of melanoma to metastasis. The molecular mechanisms leading to the development and progression of melanoma are the focus of intense investigation, and many genetic/epigenetic alterations affecting melanoma progression and development have been identified. microRNAs (miRNAs) are emerging as important causal modulators in the development and progression of melanoma. The understanding of miRNA-mediated regulation of tumours has grown immensely over the last few years, as it has been understood to regulate most biological processes. Here, we review the currently available data on miRNAs associated with melanoma, highlighting those deregulated miRNAs that target important genes and pathways involved in the progression of melanocytes to primary and metastatic melanoma. We also review their potential clinical utility as biomarkers and potential use in targeted therapy.

Delivery of an miR155 inhibitor by anti-CD20 single-chain antibody into B cells reduces the acetylcholine receptor-specific autoantibodies and ameliorates experimental autoimmune myasthenia gravis.

MicroRNA-155 (miR155) is required for antibody production after vaccination with attenuated Salmonella. miR155-deficient B cells generated reduced germinal centre responses and failed to produce high-affinity immunoglobulin (Ig)G1 antibodies. In this study, we observed up-regulation of miR155 in the peripheral blood mononuclear cells (PBMCs) of patients with myasthenia gravis (MG), and miR155 was also up-regulated in torpedo acetylcholine receptor (T-AChR)-stimulated B cells. We used an inhibitor of miR155 conjugated to anti-CD20 single-chain antibody to treat both the cultured B cells and the experimental autoimmune MG (EAMG) mice. Our results demonstrated that silencing of miR155 by its inhibitor impaired the B cell-activating factor (BAFF)-R-related signalling pathway and reduced the translocation of nuclear factor (NF)-κB into the nucleus. Additionally, AChR-specific autoantibodies were reduced, which may be related to the altered amounts of marginal zone B cells and memory B cells in the spleens of EAMG mice. Our study suggests that miR155 may be a promising target for the clinical therapy of MG.

Adipophilin affects the expression of TNF-alpha, MCP-1, and IL-6 in THP-1 macrophages.

Macrophages accumulated in the arterial intima play an important role in the development of atherosclerosis by producing a large number of proinflammatory cytokines which accelerate the disease. Recent studies show that adipophilin might be involved in inflammatory processes in macrophages. In this study, we observe the effect of adipophilin on proinflammatory cytokine expression and secretion in THP-1 macrophages. SiRNA and adipophilin gene overexpression mediated by an pEGFP-C3 vector were used to observe the effect of adipophilin on proinflammatory cytokines in THP-1 macrophages in vitro. Realtime PCR and enzyme-linked immunosorbent assay (ELISA) were applied to detect the production of tumor necrosis factor alpha (TNF-alpha), monocyte chemoattractant protein 1 (MCP-1), and interleukin-6 (IL-6). It was found that acetylated low-density lipoprotein (AcLDL), pioglitazone [a peroxisome proliferator-activated receptor gamma (PPARgamma) agonist] increased adipophilin expression in macrophages, while glucose had no such affect. It was also shown that adipophilin augments TNF-alpha, MCP-1, and IL-6 expression in AcLDL induced macrophages. Our results suggest that adipophilin augment inflammation in macrophages, which might be one role of adipophilin in atherosclerosis.

Solid-phase photocatalytic degradation of polyethylene-goethite composite film under UV-light irradiation.

A novel photodegradable polyethylene-goethite (PE-goethite) composite film was prepared by embedding the goethite into the commercial polyethylene. The degradation of PE-goethite composite films was investigated under ultraviolet light irradiation. The photodegradation activity of the PE plastic was determined by monitoring its weight loss, scanning electron microscopic (SEM) analysis and FT-IR spectroscopy. The weight of PE-goethite (1 wt%) sample steadily decreased and led to the total 16% reduction in 300 h under UV-light intensity for 1 mW/cm(2). Through SEM observation there were some cavities around the goethite powder in the composite films, but there were few changes except some surface chalking phenomenon in pure PE film. The degradation rate could be controlled by changing the concentration of goethite particles in PE plastic. The degradation of composite plastic initiated on PE-goethite interface and then extended into polymer matrix induced by the diffusion of the reactive oxygen species generated on goethite particle surface. The photocatalytic degradation mechanism of the composite films was briefly discussed.

Diagnostic value of vascular endothelial growth factor and endostatin in malignant pleural effusions.

OBJECTIVE: To assess the value of vascular endothelial growth factor (VEGF) and endostatin in the differential diagnosis of malignant and tuberculous pleural effusions (PE). METHODS: Effusion samples were collected from 62 patients with malignant PE caused by lung cancer and from 64 patients with tuberculous pleurisy. Concentrations of pleural fluid VEGF and endostatin were measured simultaneously using enzyme-linked immunosorbent assay (ELISA) and enzyme immunoassay, respectively. The sensitivity, specificity and accuracy were calculated. RESULTS: PE levels of VEGF and endostatin were found to be significantly elevated in effusions with malignancy rather than in those of tuberculous origin (both P < 0.01). For VEGF, the sensitivity, specificity and accuracy in diagnosing malignant pleural effusions were respectively 71%, 61% and 66%, while for endostatin the values were respectively 69%, 83% and 76%. A combination of VEGF and endostatin can increase these to respectively 81%, 97% and 89%. CONCLUSIONS: Elevated levels of VEGF and endostatin in pleural effusions may be helpful in diagnosing malignant pleural effusions, while a combination of VEGF and endostatin can increase the sensitivity, specificity and accuracy of differentiating malignant from tuberculous pleural effusions.

Berberine inhibits the expression of TNFalpha, MCP-1, and IL-6 in AcLDL-stimulated macrophages through PPARgamma pathway.

Macrophages are the main source of cytokines in atherosclerotic plaques. Modified low-density lipoproteins may stimulate macrophages to produce large quantities of proinflammatory cytokines that promote atherosclerosis. Berberine is the main component of the traditional Chinese medicine umbellatine, which has a widespread effect and was used to treat many diseases clinically. Our previous study found that berberine could increase adipophilin expression in macrophages, which is a target gene of PPARgamma. PPARgamma agonist could decrease proinflammatory cytokines in macrophage. In this study, we investigated the effects and the mechanism of action of berberine on the expression and secretion of TNFalpha, MCP-1, and IL-6 in vitro to identify new pharmacological actions of berberine. The results of RT-PCR and ELISA shows that berberine may inhibit the expression and secretion of the tumor necrosis factor alpha (TNFalpha), monocyte chemoattractant protein 1 (MCP-1), and interleukin-6 (IL-6) in macrophages stimulated by acetylated low-density lipoprotein (AcLDL), whereas the peroxisome proliferator-activated receptor gamma (PPARgamma) inhibitor GW9662 could attenuate this effect of berberine. This study demonstrates that berberine may inhibit the expression and production of TNF-alpha, MCP-1, and IL-6 in AcLDL-stimulated macrophages. This effect might be partially mediated through PPARgamma activity.

[Decolorization and isolation of recombinant hirudin expressed in the methylotrophic yeast Pichia pastoris].

Schemes for decolorization of broth and isolation of recombinant hirudin from the fermentation supernatants of recombinant yeast Pichia pastoris were developed to remove a great deal of non-desired proteins and coloring matters. Based on the stability of hirudin, heating is desirable to pre-treat the fermentation broth. Several different column chromatographies, including anion-exchange, cation-exchange, hydrokyapatite adsorption, hydrophobic interaction, gel filtration and their combination were investigated on the efficiencies of decolorization and isolation. Among these methods, the combination of cation-exchange and anion-exchange was found to be a success in both decolorization and isolation of hirudin.