[Transplantation of human umbilical cord stem cells improves neurological function recovery after spinal cord injury in rats].

OBJECTIVE: To study whether intraspinally transplanted human cord blood CD34+ cells can survive, differentiate, and improve neurological functional recovery after spinal cord injury in rats. METHODS: Rats were randomly divided into two groups. One group of rats was subjected to spinal cord left-hemisection and transplanted with human cord blood CD34+ cells labeled by bromodeoxyuridine (BrdU); The other group was carried by left-hemisection with injection of PBS (control group). The neurological function was determined before and 24 h, 1, 2, 3 and 4 weeks after spinal cord injury and cell transplantation using the modified Tarlov score. The distribution and differentiation of transplanted human cord blood cells in vivo in rat spinal cord were evaluated by histological and immnuhistochemical analysis. RESULTS: Functional recovery determined by modified Tarlov score was significantly improved in the group receiving human cord blood CD34+ cells compared with the control group (P < 0.05). Moreover, human cord blood CD34+ cells were found to survive in rat spinal cord microenvironment, with the expression of the neural nuclear specific protein (NeuN) in 2% BrdU-reactive human cells and of the astrocytic specific protein glial fibrillary acidic protein (GFAP) in 7% BrdU-reactive human cells. CONCLUSIONS: Intraspinally administered human cord blood CD34+ cells can survive, differentiate, and improve functional recovery after spinal cord injury in rats. Transplantation of human cord blood cells may provide a novel strategy for the treatment of neural injury.

[Reversal of multi-drug resistance in K562/A02 cells by small interference RNA of mdr1 gene].

OBJECTIVE: To investigate the effect of small interference RNA (siRNA) on mdr1 and P-glyco-protein (P-gp) expression of multi-drug resistance (MDR) human leukemia cell line K562/A02. METHODS: Three si RNAs (si-mdr1-1, si-mdr1-2, si-mdr1-3) which were specifically targeted mdr1 gene were synthesized and transfected into K562/A02 cells. Expression of mdr1 mRNA was assayed by RT-PCR. P-gp expression and intracellular daunorubicin (DNR) concentration were determined by flow cytometry. 50% inhibition concentration (IC(50)) of doxorubicin (ADM) on K562/A02 was determined by MTT method. RESULTS: Treatment of K562/A02 cell with the 3 kinds of siRNAs resulted in a reversal of MDR of a different extent. The third siRNA was more effective in the suppression of mdr1 with a significant reduction of (58.0 +/- 1.54)% of the mdr1 mRNA expression. Positive expression rate of p170 decreased from (76.0 +/- 1.0)% to (19.6 +/- 1.9)%, and the relative efficiency of K562/A02 to ADM was 70.4%. The intracellular accumulation of DNR increased after siRNA treatment. CONCLUSION: The siRNA could effectively restore the sensitivity of K562/A02 cells to conventional chemotherapeutic agents.

[Inhibition of tumor angiogenesis in nude mice by adenovirus-mediated PF4 p17-70 cDNA transfection].

OBJECTIVE: To investigate the in vivo effect of modified platelet factor 4 (PF4)-p17-70 cDNA on tumor angiogenesis in nude mice. METHODS: The p17-70 cDNA was cloned into the AdEasy system to transfect packing cell line 293 and produce viral particles encoding p17-70cDNA (Ad p17-70). The integration of p17-70 cDNA was confirmed by RT-PCR and the P17-40 peptide Western blot. The biological activity of purified recombinant adenovirus was determined by umbilical veinal endothelial cell proliferation assay in vitro and in vivo tumor angiogenesis suppression of nude mice bearing human head and neck carcinoma. RESULTS: p17-70 significantly inhibited in vitro proliferation of endothelial cells being 58% lower than that of empty vector and reduced tumor volume in vivo. The tumor mass was (0.086 +/- 0.054) g, (0.171 +/- 0.076) g and (0.195 +/- 0.067) g, the tumor volume was (16.7 +/- 5.2) mm(3), (36.5 +/- 23.7) mm(3) and (41.5 +/- 12.2) mm(3) in p17-70 cDNA transfected group, empty vector group and PBS group, respectively. Immunohistochemical staining demonstrated a decreased number of blood vessels in the tumors. CONCLUSION: P17-70 peptide mediated by adenoviral vector could inhibit the endothelial proliferation in vitro and the tumor growth in vivo.

[The preliminary study on in vitro differentiation of human umbilical cord blood cells into neural cells].

OBJECTIVE: To explore the feasibility of in vitro differentiation of human umbilical cord blood cells (HUCBC) into neural cells induced by receptor activator of NF-KappaB ligand (RANKL) and brain-derived neurotrophic factor (BDNF). METHODS: Normal fresh HUCBC were cultured as the following: (1) Control group cultured by differentiation medium only; (2) BDNF group, cultured by differentiation medium + BDNF; (3) RANKL group, cultured by differentiation medium + human soluble RANKL (sRANKL); (4) BDNF + RANKL group, cultured by differentiation medium + BDNF and sRANKL. Cultured cells were observed with invert microscope. After ten-days culture, the expression of glial fibrillary acidic protein (GFAP) and neuron-specific nuclear protein (NeuN) of the cultured cells were detected by immunocytochemical staining. RESULTS: After 10 day's culture, the NeuN positive cells were (97.0 +/- 13.5), (85.0 +/- 5.6), (167.0 +/- 19.7) in RANKL, BDNF and BDNF + RANKL groups, respectively, with 1.7, 1.5, 3.0 fold in crease than that of control (55.7 +/- 8.5), the GFAP positive cells were (114.7 +/- 18.0), (233.3 +/- 21.7), (289.0 +/- 24.7), respectively, with 1.4, 2.9, 3.6 fold increase compared with the control group. The differentiation ratio of neurons in RANKL group was similar to that of the BDNF group, but the differentiation ratio of glial cells was lower than that in the BDNF group. In the RANKL + BDNF group, the differentiation of HUCBC into neurons and glial cells were enhanced obviously, the differentiated neural cells were typical with longer axons and dendrites. CONCLUSION: RANKL and BDNF could induce HUCBC into neurons and glial cells, and they have synergistic effect on the induced differentiation. It is hopeful that HUCBC might be an source of stem cells for the treatment of central nervous system injury.

[Efficient generation of recombinant adenoviruses expressing antiangiogenic fragment of human thrombospondin1].

OBJECTIVE: To construct recombinant adenoviruses expressing antiangiogenic fragment of human thrombospondin1 (TSP1f). METHODS: TSP1f cDNA was amplified by RT-PCR from normal human peripheral blood mononuclear cells and was subcloned into a shuttle vector pShuttle-CMV. After sequence confirmation, the resultant plasmid was linearized by the restriction endonuclease Pme I and cotransformed with the supercoiled adenoviral vector pAdEasy-1 into Escherichia coli strain BJ5183. Recombinants were selected by Kanamycin resistance and screened by restriction endonuclease digestion. Then, the recombinant adenoviral construct was cleaved with Pac I and transfected into the packaging cell line 293. The adenoviral vector ADV-TSP1f was propagated in 293 cells and purified by cesium chloride (CsCl) density centrifugation. PCR and Western blot analysis were performed to confirm TSP1f expression. RESULTS: Of 43 Kanamycin-resistant colonies obtained from cotransformation, all of the 10 smallest ones were the correct recombinants. TSP-1f was expressed efficiently by ADV-TSP1f. The virus stock titer after CsCl banding was 1.0 x 10(11) pfu/mL. CONCLUSIONS: Generating recombinant adenoviruses using AdEasy System results in highly efficient viral production and significantly decrease the time required to construct usable viruses. ADV-TSP1f can be further used in in vivo gene therapy studies.

[Expression of vascular endothelial growth factor and its receptors KDR and Flt1 in acute myeloid leukemia].

OBJECTIVE: To evaluate the expression of vascular endothelial growth factor (VEGF) and its receptors KDR and Flt1 in patients with acute myeloid leukemia (AML). METHODS: The expression of VEGF and its receptors mRNA was assayed by RT-PCR, the plasma of VEGF by ELISA. RESULTS: In 13 AML cell lines, the expression of VEGF, KDR and Flt1 mRNA were found in 13 (100%), 7 (53.8%) and 12 (92.3%), respectively. There were 21 (65.6%), 1 (3.1%), and 17 (53.1%) of 31 (AML) patients bone marrow mononuclear cells (BMMNCs) expressing VEGF, KDR and Flt1 mRNA, respectively. None of BMMNCs from 3 normal donor and CD(34)(+) cells from 2 normal donor was found to express VEGF, KDR and Flt1 mRNA. The plasma level of VEGF of 39 patients (new diagnosed, relapsed and secondary-AML) before treatment was (135.3 +/- 87.9) ng/L which was significantly higher than that of 15 complete remission (CR) patients (80.6 +/- 36.9) ng/L and 12 normal donors (80.6 +/- 33.1) micro g/L (P = 0.028, 0.007). The plasma level of VEGF of 15 non-responsive patients was (188.2 +/- 118.6) ng/L after two cycles of chemotherapy which was higher than that of 20 CR patients [(104.2 +/- 30.9) ng/L] (P = 0.004). CONCLUSION: VEGF and its receptors KDR and Flt1 mRNAs were expressed in BMMNCs of AML patients. The plasma level of VEGF directly affected the response to chemotherapy in AML patients.

[Adenoviral vector expressing an antiangiogenic fragment of thrombospondin 1 inhibits the growth of K562 cell xenografts].

OBJECTIVE: To explore the effect of adenovirus-mediated delivery of an antiangiogenic fragment of human thrombospondin 1 (TSP1(f)) on K562 cell growth in nude mice. METHODS: TSP1(f) cDNA was amplified by RT-PCR from normal human peripheral blood mononuclear cells and was used as transgene to construct a adenoviral vector (ADV-TSP1(f)). Human leukemia K562 cells were cultured and infected with ADV-TSP1(f) or ADV-LacZ. PBS was used as control. TSP1(f) expression/secretion by these infected K562 cells was demonstrated using Western blot analysis. MTT assay was performed to determine the effect of ADV-TSP1(f) infection on K562 cell growth kinetics. Eighteen female Balb/c nude mice were inoculated subcutaneously with human leukemia K562 cells. When the diameter of the tumor reached 5 approximately 7 mm the rats were randomly divided into 3 groups of 6 rats injected intratumorally with ADV-TSP1(f), ADV-LacZ, and PBS respectively. The volume of K562 xenografts was measured every three days during the 3-week treatment. By the end of the 21st day the mice were killed and the tumors were taken to undergo histological examination. The intratumoral microvessel density (MVD) was determined by immunohistochemical staining. RESULTS: TSP-1f was expressed and secreted efficiently by ADV-TSP1(f)-infected K562 cells. Three weeks after the initial treatment, the volume of K562 xenografts in the mice treated with ADV-TSP1(f), ADV-LacZ, and PBS was (1,108 +/- 179) mm(3), (4,518 +/- 452) mm(3), and (4,666 +/- 458) mm(3) respectively (P < 0.01). The number of CD31+ microvessels counted per x200 field was 34 +/- 9, 36 +/- 7, and 14 +/- 4 in the tumors treated with PBS, ADV.LacZ, and ADV.TSP-1f respectively (P < 0.001). CONCLUSION: Adenovirus-mediated TSP-1f gene transfer greatly inhibits K562-derived tumor growth and angiogenesis in mouse xenograft model, and may serve as a new therapy for hematological malignancies.

Adenovirus-mediated gene therapy with an antiangiogenic fragment of thrombospondin-1 inhibits human leukemia xenograft growth in nude mice.

Recent investigations support the idea that angiogenesis is involved in the pathophysiology of leukemia. Within a given microenvironment, the angiogenic response is regulated by a delicate balance of angiogenesis inducers and inhibitors. Thrombospondin-1 (TSP-1) is a multifunctional extracellular glycoprotein showing angiostatic properties in multiple in vitro and in vivo assays. Interestingly, there is also proangiogenic domain in this complex molecule. Development of TSP-1 as an antiangiogenic drug has been hindered by multiplicity of its functional effects, difficulties in its production and its poor pharmacokinetics. The aim of the present study was to establish a recombinant adenovirus (ADV.TSP-1(f)) expressing antiangiogenic fragment of TSP-1 (TSP-1(f)), and to determine the feasibility for use of the adenovirally expressed TSP-1(f) in leukemia gene therapy. The results of this investigation showed that TSP-1(f) was expressed efficiently in adenovirus-transduced human myelogenous leukemia K562 cells. Compared to the controls, although there was almost no effect on proliferation of K562 cells in vitro, adenovirus-mediated TSP-1(f) transduction inhibited the growth of K562 xenografts dramatically. Furthermore, the microvessel density (MVD) was much lower in the ADV.TSP-1(f)-treated tumors compared to the controls. These data support the use of in vivo gene delivery approach to produce antiangiogenic fragment of TSP-1 for leukemia therapy.

[The CXC-chemokine platelet factor 4 (PF4) increases KG1a cells adherence and modulates actin polymerization of KG1a cells].

OBJECTIVE: To study the effects on adherence of hematopoietic stem/progenitor cells, PF4 was assessed alone or in combination with IL-3 for effects on the total adherence and various kinds of adhesion molecules of KG1a cells as well as actin polymerization in KG1a cells. METHODS: The total adherence was assayed by crystal violet dye staining. The adhesion molecule expression was determined by FACS analysis. These adhesion molecule monoclonal antibodies individually blocked total adherence by MTT. F-actin content was monitored by fluorospectrophotometry. RESULTS: 100 ng/ml PF4 could increase the total adherence of KG1a cells by 80%. 20 ng/ml IL-3 could increase the total adherence of KG1a cells by 96%. When PF4 and IL-3 were combined, the total adherence could be promoted by 97%. Exposure of 1 x 10(6) cells/ml of KG1a cells to 100 ng/ml PF4 the increased total adherence of KG1a cells was mediated by PECAM-1 (CD31), CD44, LFA-1 (CD11a) and Mac-1 (CD11b) but not by P-selectin (CD62P) and E-selectin (CD62E). These adhesion molecule monoclonal antibodies could individually block total adherence for 34%-43%. Similar phenomenon was observed when IL-3 was added onto KG1a cells. Further study found that PF4 induced actin polymerization of KG1a cells. CONCLUSIONS: Our study indicated that PF4 promoted total adherence, as well as several adhesion molecule expression and actin polymerization of KG1a cells. The results suggest that PF4 may have therapeutic utility along with other cytokines by enhancing the total adhesion of hematopoietic stem/progenitor cells to promote the homing.