Preliminary experimental research on gene therapy for hemophilia A / 中华血液学杂志

<p><b>OBJECTIVE</b>To accomplish a kind of therapeutic gene for hemophilia A, and observe the expression of human factor VIII (hF VIII) in vivo.</p><p><b>METHODS</b>Human clotting factor VIII cDNA with B-domain deleted (Delta760aa approximately 1639aa) was inserted into vector pRC/RSV to form pRC/RSV-hF VIII BD, which conjugated with in vivo liposome transfection reagent (DOTAP-Cholesterol) to accomplish a kind of therapeutic gene, pRC/RSV-hF VIII BD-DOTAP-Cholesterol. Mice were injected with pRC/RSV-hF VIII BD-DOTAP-Cholesterol i.m. and sacrificed 48 hours, 10 days, 20 days, 30 days, 40 days and 50 days later, respectively. Tissues such as heart, liver, spleen, lung, kidney and muscle were harvested, the distribution and transcription as well as expression of hF VIII BD cDNA were detected by means of PCR, RT-PCR and immunohistochemistry techniques. In addition, the antigen and antibody of hF VIII in plasma were measured.</p><p><b>RESULTS</b>There was high expression of hF VIII in plasma and tissues at the 48(th) hour after injection. On day 10, antigen level of hF VIII in plasma reached its peak, 17.55 ng/ml, and gradually reduced later. The antibody of hF VIII in plasma emerged on day 10 after injection, and increased and gradually reached 37.06 U/ml on day 50 after injection. PCR, RT-PCR and immunohistochemistry showed that hF VIII BD cDNA and its transcription as well as expression existed in all kinds of tissues, and lasted longer in spleen, lungs and kidneys than in heart, liver and muscle.</p><p><b>CONCLUSION</b>Therapeutic gene, pRC/RSV-hF VIII BD-DOTAP-Cholesterol, produced by combination of pRC/RSV-hF VIII BD and DOTAP-Cholesterol liposome can express human F VIII successfully in vivo, which lays an experimental foundation for curing hemophilia A by gene-drug in clinic.</p>

In vitro expression of human factor VIII gene induced by sodium butyrate / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the effect and mechanism of sodium butyrate on expression of human clotting factor VIII in vitro.</p><p><b>METHODS</b>Mouse NIH/3T3 cell line was transfected with recombinant plasmid vector pRC/RSV-BDD-hFVIII, which enclosed B-domain deleted (760aa approximately 1 639aa) human factor VIII cDNA (BDD-hFVIII cDNA). Then cells were incubated in Dulbecco's modification of Eagle's medium (DMEM) containing sodium butyrate for 24 hours, hFVIII: C and hFVIII: Ag in the cell culture medium were measured by ELISA assay and one-stage method, respectively. In addition, the effect of sodium butyrate on transcription of cDNA encoding the whole hFVIII, heavy and light chain of hFVIII was also investigated by means of run-on assay.</p><p><b>RESULTS</b>After stimulation of sodium butyrate, the levels of hFVIII: C and hFVIII: Ag increased 70% than those of control. Run-on assay showed that sodium butyrate enhanced the transcription of cDNA which encoded heavy chain of hFVIII.</p><p><b>CONCLUSION</b>Sodium butyrate can improve the expression of hFVIII through enhancing the transcription of hFVIII heavy chain encoding cDNA. It demonstrated that sodium butyrate had potential utility in inducing the expression of hFVIII in vitro.</p>

STUDIES ON SKIN PIGMENT IN CADAVER / 解剖学报

Cadaver preserved in formalin may develop pigmentation in the epidermis astime goes on.What is the nature of the colour substance?Are they degradationproduct of hemoglobin,melanin-like substance or others?In this paper,we reportthe results of observation on the pigmented substance from cadaver skin and charac-terize it by chemical and physical means.The epidermal layer of pigmented skin of cadaver was hydrolyzed by aceticacid,the hydrolysate was extracted with water and organic solvent in steps,thusthree portions were obtained:acidic water soluble,organic solvent soluble and inso-luble black precipitate.The later was identified as melanin by infrared spectrumanalysis,and chromatographic behavior after alkali fuse.The pigmented substancein acidic water and organic solvent are not identified yet.We also tried to find out the degradation products of hemoglobin,but wecouldn't detect them either by spectrum analysis or by crystallization of haem chlo-ride derivative.Hence,we intend that the deep colour of the cadaver skin after formalintreatment might be closely related to melanin,the mechanism of its formation isdiscussed.