血友病FⅧ/FⅨ抑制物是一种特异性中和抗体,加重出血、死亡和致残,是血友病的严重并发症和新的挑战.抑制物产生的高危因素与遗传和环境等因素有关,为预防提供了方向.目前认为血友病抑制物的有效治疗方法为旁路药物止血、免疫耐受诱导治疗清除抑制物和新型止血药预防治疗,但仍不能满足需要.最近研发的多种新型止血药,其半衰期长,可4周皮下注射1次进行预防治疗,年化出血率0,无抑制物产生,治疗取得了突破性进展.

OBJECTIVE:To evaluate the efficacy and safety of Shengxue mixture combined with intraosseous blood infusion for treatment of aplastic anemia patients.METHODS:From 2011 to 2015, Institute of blood diseases of Shaanxi Medical University admitted 53 patients with aplastic anemia. The patients were treated with shengxue mixture 200 ml, orally, twice a day. Stanozolol tablets, Adult 2 mg, three times a day, mycophenolate mofetil 1.0 g, twice a day. Intraosseous infusion of the following medicine were administered in patients: recombinant human EPO 10000 U, recombinant human G-CSF 450 µg, recombinant human IL-11 4.5 mg, dexmethasone 20 mg, once a week, a total of four times. One month later, the blood cell counts and bone marrow biopsy were performed. Consolidation treatment continued for 3 to 6 months after discharge, and therapeutic effect was observed and followed-up for more than a year.RESULTS:After one month of treatment, 40 patients were basically cured (75.47%), 8 patients were remitted(15.09%), Hemoglobin level, white blood cell count and platelet count were significantly improved after treatment (P<0.01). The overall response rate was 90.57%(48 patients). Patients with bone marrow hyperplasia was 46 (86.79%), versus 9(16.98%) before treatment. There was a difference (P<0.05). After 3 to 6 months of treatment, 40 patients were cured (75.47%); 8 patients were remitted(15.09%); 3 patients were obviously improved(5.66%); 2 patients were ineffective(3.77%). The overall response rate was 96.23%(51 cases). No obvious side effects were observed. No patients were relapsed after one year.CONCLUSION:Shengxue mixture combined with Intraosseous infusion is a fast, efficient, safe method for the treatment of aplastic anemia.

Objective: Research on the efficacy and safety of non-factor drugs applied the etiological classification for treatment of hemophilia.

Objective To study the etiological classification method in hemophillia.Methods From February 2005 to May 2010,500 hemophillia patients were included in this study in Shaanxi Medical University Hematology Institute.The activity of coagulation factor was tested by one stage assay.The antibodies of FⅧ and FIX were tested by Bethesda assay.According to the diagnostic criteria of thrombosis and homeostasis conference in 2000,the etiology was classified as four types,as flowers:Congential hereditary hemophilia,congential non-hereditary hemophilia,acquired allogeneic hemophilia and acquired autogeneic henophillia.Results The congential hemophillia were 490(98％)cases,of which 376(75.2％)cases were hereditary,114(27.8％)cases were non-hereditary,411(82.2％)cases were hemophillia A and 79(15.8％)cases were Hemophillia B.Hemophillia A associated with FⅧ antibody were 151 cases (30.2％),hemophillia B combined with FIX antibody were 18 cases(3.6％).There were 10 cases(2.0％)of acquired autogeneic hemophilia.All of them were multiple coagulation factors deficient.Conclusion This study recommended an important basis for the etiologic classification of hemophillia.

OBJECTIVE To study the effect and safety of haemostatic apozem combined with haemostatic mixture on hemophilia hemorrhage. METHODS Five hundred hemophilia patients were randomly recruited from Shaanxi Yida Hematology Institute from February 2005 to July 2010. Under the condition of using no blood products such as platelet cofactors VIII and IX, oral administration of haemostatic apozem combined with intravenous dripping of haemostatic mixture were given to 332 hemorrhagic patients and 451 patients in need of surgery for hemorrhagic prevention. The treatment was lasted for three successive weeks. The hemostatic time, hemorrhage absorption (recovery) time, and their safety were observed. RESULTS The hemostatic time for open bleeding and closed bleeding was (0.85 +/- 0.83) h and (2.69 +/- 0.65) h respectively. The average hemostatic time was (2.00 +/- 0.69) h. The recovery time for different portions was as follows respectively: intra-cranial hemorrhage (14.13 +/- 6.01) days; muscular hemorrhage (18.18 +/- 7.34) days; hematuria (8.25 +/- 4.69) days; arthrorrhagia(3.27 +/- 1.31) days; ecchymoma (7.16 +/- 2.32) days; bleeding of oral and nasal cavities (4.26 +/- 1.35) days; intramedullary hemorrhage (19.15 +/- 1.36) days; hematoma ulceration (50.01 +/- 20.91) days. The hemorrhage recovery ratio was 99.10% (329/332). The success rate of preventing from surgery hemorrhage was 100% (451/451). No severe adverse reaction occurred during the therapeutic course. CONCLUSIONS Haemostatic apozem combined with haemostatic mixture was effective and fast in preventing and treating hemophilia hemorrhage, with no complications or adverse reactions. It could be taken as the first choice for prevention and treatment of hemophilia hemorrhage.

Realgar has been used in Western medicine and Chinese traditional medicine since ancient times, and its promising anticancer activity has attracted much attention in recent years, especially for acute promyelocytic leukemia (APL). However, the therapeutic action of realgar treatment for APL remains to be fully elucidated. Cellular cytotoxicity, proliferation, apoptosis and differentiation were comprehensively investigated in realgar-treated cell lines derived from PML-RARα+ APL patient, including the all-trans retinoic acid (ATRA)-sensitive NB4 and ATRA-resistant MR2 cell lines. For analysis of key regulators of apoptosis and differentiation, gene expression profiles were performed in NB4 cells. Realgar was found to induce apoptosis and differentiation in both cell lines, and these effects were exerted simultaneously. Gene expression profiles indicated that genes influenced by realgar treatment were involved in the modulation of signal transduction, translation, transcription, metabolism and the immune response. Given its low toxicity, realgar is a promising alternative reagent for the therapy of APL. Our data contribute to an understanding of the underlying mechanism responsible for the therapeutic effects of realgar in the clinical treatment of APL.

Prostate apoptosis response-4 (Par-4) is a tumor-suppressor protein that induces apoptosis in cancer cells, but not in normal cells. The cancer-specific pro-apoptotic action of Par-4 is encoded in its centrally located SAC domain. In this study, to further enhance the anti-cancer effect of Par-4 in order to overcome the limitations of peptide therapy, a recombinant adeno-associated virus was constructed using the following strategies: the secretory expression of therapeutic peptide, a HA2TAT-mediated cytosolic delivery technique, and an adeno-associated virus gene transfer system. To test the hypothesis that Par-4 has an additive bystander effect as an anti-cancer therapy, we designed a secretory protein by adding a secretory signal peptide NT4(Si) to the Par-4 SAC-HA2TAT peptide gene sequence [NT4(Si)-Par-4 SAC-HA2TAT]. The results indicated that, compared to the normal NIH3T3 cell line, AAV-NT4(Si)-Par-4 SAC-HA2TAT significantly suppressed cell growth and induced rapid cell death in HepG2 cells in a time-dependent manner through successful gene transfer and secretory expression of therapeutic peptide at 48 h post-transfection. In addition, the secretory properties of Par-4 may greatly increase its effectiveness in cancer therapy when delivered in vivo.

Objective To clone and construct the plasmid containing human thyroid stimulating hormone receptor(TSHR) gene ectodomain,and then identify the immunoreactivity of the purified recombinant protein.Methods TSHR total RNA was extracted from human thyroid,and cDNA was obtained with RT-PCR technique.Human TSHR ectodomain gene(hETSHR) was cloned into pcDNA3.1(+) vector.The recombined construct was transfected into CHO cells by Lipofectin 2000.The transcript mRNA was detected by RT-PCR,and protein immunoreactivity was assayed by TSHR antibody with immunocytochemistry staining and Western blot.Results DNA sequencing results showed that the recombinant of human thyrotropin receptor ectodomain had confirmed sequence reported in GenBank.The fusion protein had the immunoreactivity.Conclusion Human thyroid stimulating hormone receptor was successfully cloned in eukaryotic expression vector and the constructor was expressed in eukaryotic cells very well.

Anginex is a novel artificial peptide that can inhibit angiogenesis. AdNT4-anginex was constructed by inserting the artificial anginex gene into a recombinant adenoviral vector. We demonstrated that AdNT4-anginex inhibited migration of human endothelial cells, angiogenesis and tumor growth in in vitro and in vivo studies. Tumor growth of human H22 hepatoma in mice was inhibited after AdNT4-anginex treatment for 4 weeks, and a significant decrease in tumor size was observed as compared with the control group. Overall, these studies indicate that AdNT4-anginex is an effective anti-tumor agent, and deserves more attention and research.

Objective:To use gene engineering technique to clone and sequence the par-4 SAC(prostate apoptosis response-4 gene SAC domain).Methods:By means of asymmetrical primer/template,double stranded cDNA of par-4 SAC was constructed,which had restriction enzymes sites on the two extremes.Then the cDNA was subcloned into the plasmid pGEM-T Easy,which was then transformed into E.coli DH5α and identified by restriction endonuclease digestion and sequence analysis.Results:Evidences of DNA sequence analysis and restriction enzymes digestion showed that the inserted fragment was par-4 SAC cDNA,consistent with the fragment we designed.Conclusion:The par-4 SAC cDNA was cloned successfully.

AIM: To investigate the inhibitory effect of As2O3 on gastric tumor growth and angiogenesis and on the expression of Flt-1.METHODS: Solid tumor models were established in 30 nude mice with gastric cancer cell line SGC-7901.The mice were divided randomly into two treatment groups(5 mg/kg As2O3 and 2.5 mg/kg As2O3) and control group,with 10 in each group.As2O3 was injected to the arsenic-treatment groups(2.5 mg/kg and 5 mg/kg) and the same volume of saline solution was injected to the control group.Tumor growth inhibition(TGI) was used to assess the inhibitory efficacy of As2O3 against xenografts.Microvessel density(MVD) and Flt-1 were detected by immunofluorescence laser confocal microscopy.RESULTS: In 5 mg As2O3 and 2.5 mg As2O3 treatment groups,the tumor growth inhibition rates were 29.08% and 52.17% respectively.Decrease of MVD was observed in As2O3 treatment tumors compared with that in control group(P<0.01).MVD in tumors was significantly lower in 5mg group than that in 2.5 mg group(P<0.01).The expression of Flt-1 was less in As2O3 treatment mice compared with that in controls,and less in 5mg As2O3 treatment group compared with that in 2.5mg As2O3 treatment group(P<0.01).CONCLUSION: As2O3 inhibits solid tumor growth and decreases MVD in tumors,possibly by inhibiting the expression of Flt-1.

AIM:To study if the effect of arsenic sulfide combined with IFN-alpha can be increased on K562 cells.METHODS:Telomerase activity was determined by PCR-ELISA. Flow cytometry was used to analyze the cell apoptosis. The final concentration of IFN-alpha and arsenic sulfide was 10,000 U/mL and 0.6 mg/L.RESULTS:The rates of apoptosis was 37.8% and 37% in K562 cells treated with IFN-alpha or arsenic sulfide alone for 8 days; The rates of apoptosis and inhibition of telomerase activity was 59.9% and 81.2% in K562 cells treated with IFN-alpha and arsenic sulfide simultaneously for 8 days, or 60.37% and 78.8% in K562 cells was treated with arsenic sulfide for 5 days after affected by IFN-alpha for 3 days. 71.3% telomerase activity was inhibited in K562 cells by arsenic sulfide alone for 8 days.CONCLUSION:Combination of arsenic sulfide and IFN-alpha can increase the apoptosis and inhibit the telomerase activity of K562 cells obviously comparing with the two drugs used alone. IFN-alpha maybe promote arsenic sulfide inducing apoptosis of K562 cells.

AIM:To explore the different effect and mechanism of arsenic sulfide on telomerase activity and hTERT-mRNA expression in CML cell lines-K562 and APL cell lines-NB4.METHODS:Telomerase activity was determined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). The expression of hTERT-mRNA was analyzed by semi-quantitative RT-PCR. Flow cytometry was used to analyze the cell cycle and apoptosis.RESULTS:0.15-0.6 mg/L arsenic sulfide (72 h)can induce apoptosis and inhibit telomerase activity and hTERT-mRNA expression in NB4 cell. The concentration of arsenic sulfide with the same effect on K562 cell was 0.3-3 mg/L. 0.3 mg/L arsenic sulfide (72 h) can cause the proportion of the NB4 cell in G2/M phase increased, but for K562 cell, The concentration of arsenic sulfide was 1.5 mg/L.CONCLUSION:Telomerase system may be one of the pathway for arsenic sulfide inducing apoptosis of NB4 and K562 cell; G2/M phrase arrest may have correlation with decrease of telomerase activity; The sensitivity of NB4 and K562 cell for arsenic sulfide is different, the mechanism of it need to study more.

Objective To investigate the effect of arsenic trioxide(As2O3)on expression of angiopoietin-2 and vascular endothelial growth factor(VEGF)in cancer.Methods The solid tumor model was formed in nude mice with gastric cancer cell line SGC-7901.The 30 nude mice were randomly divided into the two arsenic-treated groups(2.5 mg/kg and 5 mg/kg)and control group.As2O3 was injected to mice in the treated groups for 10 days;the same volume of saline solution was injected to the control group.The volume of gastric tumor xenografts was measured and counted for tumor growth inhibition(TGI).Microvessel density(MVD)labeled by CD31 was measured with immunofluorescence.Expression of angiopoietin-2 was detected by immunohistochemistry,and expression of VEGF was detected by immunofluorescence laser confocal technology.Results Tumor growth inhibitions were 30.33% and 50.85% in 2.5 mg/kg and 5 mg/kg As2O3,respectively.Decrease of MVD appeared in As2O3-treated tumors compared with that in the control group.The fluorescence intensity level of VEGF in tumor cells was lower significantly than that in the arsenic-treated groups.Angiopoietin-2 was mostly expressed in endothelial cells in tumors.There was no difference in expression level of angiopoeitin-2 between arsenic-treated groups and the control group.Conclusion As2O3 does not affect the expression of angiopoietin-2.Angiopoietin-2 may induce vessel regression,and VEGF expression is decreased by As2O3 in tumor.Angiopoietin-2 may become a new indicator in study on angiogenesis in tumor.

This study was aimed to investigate on effect of As(2)O(3) on expressions of COX-2, MMP-2 and MMP-9 in SGC7901 and K562 cells. SGC7901 and K562 cells were cultured in RPMI 1640 medium and were inoculated in culture medium with different concentrations of As(2)O(3) and at different times. Expressions of COX-2, MMP-2 and MMP-9 in SGC7901 and K562 cells were measured by using Western blot, while the levels of COX-2 mRNA and MMP-2 mRNA were measured with fluorescence quantitative RT-PCR. The results showed that the expression of COX-2, MMP-2 and MMP-9 decreased in dose- and time-dependent manners after treating with As(2)O(3). The levels of COX-2 mRNA and MMP-2 mRNA reduced in groups treated with As(2)O(3). In conclusion, As(2)O(3) inhibits expressions of COX-2, MMP-2 and MMP-9 in K562 and SGC7901 cells, suggesting that As(2)O(3) inhibits tumor development through its effect on angiogenesis involved in solid and hematologic malignancies.

This study was aimed to investigate the effect of arsenic trioxide (As2O3) on expression of vascular endothelial growth factor-C (VEGF-C) and its receptor VEGFR-3 in gastric cancer in order to clarify the role of As2O3 in lymphangiogenesis and metastasis of tumor. The gastric cancer model was established in nude mice by using gastric cancer cell line SGC-7901. As2O3 was injected to the two treatment groups (2.5 mg/kg and 5 mg/kg) and the same volume of saline solution was injected to the control group. Expression of VEGF-C and VEGFR-3 were detected by immunohistochemistry and were analyzed with QWin550cW image Acquiring & Analysis System. The results showed that the expression of VEGF-C and VEGFR-3 in cancer cells significantly reduced in the arsenic -treated groups. The expression of VEGF-C and VEGFR-3 in 5 mg/kg group was significantly less than that in 2.5 mg/kg group. The gray ratio analysis confirmed that there were significant difference between control group and two treated group, as well as between 2.5 mg/kg-treated group and 5 mg/kg-treated group. It is concluded that As2O3 can inhibit expression of VEGF-C and VEGFR-3 of human gastric cancer xenografts in nude mice, which suggests that As2O3 may inhibit the lymphangiogenesis by suppressing the expression of VEGF-C and VEGFR-3.

Objective:To study the effecs on effect of arsenic trioxide on proliferation and cell cycle of vascular endothelial cells.Methods:ECV304 cells were treated with As2O3.Cell viability of ECV304 cells was measured by MTT assay.Cell cycle was analyzed by using flow cytometry with PI staining;apoptosis was estimated by flow cytometry by Annexin V-FITC conjugated with PI staining.Results:FCM assays showed that the percent of the cells treated with As2O3 were higher in sub-G1,lower in sub-S and lower in G2/G1 than that of the control,and 14.84% and 18.9% of the cells treated with As2O3 at 2.5μmol/L and 5μmol/L respectively were in apoptosis.Early apoptosis in As2O3 treated groups were 2.88~5.1 times higher than that of the control,late apoptosis in As2O3 treated groups were 1.17~1.67 times higher than that of the control.Conclusions:As2O3 may directly inhibit proliferation of vascular endothelial cells and induce apoptosis.

Objective Study on effect of As2O3 on expressions of Vascular Endothelial Growth Factor (VEGF). Methods K562 and HL-60 cells were routinely grown in RPMI 1640 and were inoculated in culture medium no added (control group) and added As2O3 according to different concentration and different time. Expression of VEGF protein in K562 and HL-60 cells were measured with Western bloting. VEGF mRNA was measured with fluorescence quantitative RT-PCR in K562 cells. Results Expression of VEGF protein decreased in dose- and time-dependent manner after the treatment with As2O3, but in VEGF mRNA no significant difference between control group and treated groups was found. Conclusion As2O3 may inhibit VEGF protein expression in K562 and HL-60 cells.

Par-4 gene(prostate apoptosis response-4 gene) is first identified for proapoptotic genes in prostate cancer cells.Par-4 is a widely expressed protein that sensitizes both prostatic and non-prostatic cells to apoptosis.Par-4 SAC domain shows selective action against cancer cells but not normal cells.It has both academic and therapeutic(target therapy) applications.

anginex是利用碱性折叠原理和掺入来自PF-4、IL-8和BP1β-片层域短序列相结合的方法设计的一种新的小分子肽。体内和体外研究证明anginex通过诱导激活的内皮细胞(EC)凋亡,具有较强的抑制EC增殖、移行作用、抑制肿瘤血管生成作用并有与化疗协同和放疗增敏作用。因此,它是一个有可能应用于临床靶向治疗的抗血管生成的小分子肽。