The expression of Fas ligand (FasL) on the membrane of many kinds of leukemia or solid tumor cells played an important role in the immune escape of tumor cells. This study was aimed to know if the soluble Fas (sFas), expressed by adenovirus, could block the immune escape of tumor cells by FasL pathway. The two recombinant adenoviral vectors, AdsFas with murine soluble Fas gene and AdEGFP with enhanced GFP protein gene, were constructed by homologous recombination between two plasmids in Escherichia coli with the AdEasy adenovirus vector system. The viruses were propagated and purified by two times ultracentrifugation. Their titres were detected by plaque assays. The expressed protein was evaluated by Western blot analysis. Then the tumor EL4 cells were infected with AdsFas and AdEGFP respectively. The apoptosis ratio of the target cells-YAC-1 cells induced by EL4 cells was respectively detected by (3)H-thymidine ((3)H-TdR) labeling. The results showed that the recombinant adenoviral vectors AdsFas and AdEGFP were successfully obtained. The titres of viruses purified by two times ultracentrifugation were up to 10(11) pfu/ml by plaque assays. The sFas protein was highly expressed in the target cells by Western blot analysis. After the EL4 cells were transfected with the adenoviruses AdsFas, the apoptosis rate of YAC-1 cells in the sFas transfection group (respectively 6%, 7% and 9% when the effector:target (E:T) was 3:1, 10:1 and 30:1) was obviously lower than that in the control group (respectively 28%, 37% and 45%), P < 0.01. But when the EL4 cells were transfected with AdEGFP, the apoptosis rate of YAC-1 cells (respectively 30%, 36% and 48%) was similar to the control group, P > 0.05. In conclusion, the transfer of sFas by adenovirus could inhibit the apoptosis of Fas(+) cells-YAC-1 cells induced by tumor EL4 cells. It showed that the transduction of sFas could block the effect of the immune escape of EL4 cells through FasL in vitro. These results thus provide a new direction to find a way to treat tumors.
Adenoviridae ; genetics ; Animals ; Apoptosis ; Blotting, Western ; Fas Ligand Protein ; Leukemia, T-Cell ; immunology ; Membrane Glycoproteins ; genetics ; physiology ; Mice ; Mice, Inbred C57BL ; Transfection

To explore the new approach to prevent graft versus host disease (GVHD) by purging ex vivo T lymphocytes of bone marrow graft through Fas-FasL way, FasL-cDNA was transfected into BALB/c mouse bon e marrow cells by liposome ex vivo. The transfected cells were cultured together with BAC (BALB/c x C57BL/6) mouse bone marrow graft. The mixing bone marrow graft was infused into BALB/c mouse recipients after 60Co-gamma irradiation. The mortality, manifestation and pathologic change of GVHD in recipient mice were observed. The CFU-S and Y chromosome from donor mice were detected. The results showed that compared with control group, the mortality in 60 days of the recipients in the experimental group decreased (20% vs 70%, P < 0.01) and the morbidity of GVHD lowered (40% vs 100%, P < 0.01). The CFU-S counts for all groups were at normal level on 20 days after transplantation. The Y chromosome from donor mice was discovered in 70% bone marrow nucleated cells of recipient mice survived over 2 months in the experimental group. It is concluded that mFasL-cDNA transfected mouse bone marrow cells prevent GVHD after culturing together with bone marrow graft, and accelerate hematopoietic reconstitution in recipient mice.
Animals ; Bone Marrow Cells ; metabolism ; Bone Marrow Purging ; Bone Marrow Transplantation ; Fas Ligand Protein ; Female ; Genetic Therapy ; Graft vs Host Disease ; Male ; Membrane Glycoproteins ; genetics ; Mice ; Mice, Inbred BALB C ; Transfection

OBJECTIVEA new pattern recognition method suitable for traditional Chinese patent medicine was established in this paper, which is named as the Dual index grade sequence pattern recognition.

METHODIn this method the quality gradation was defined mathematically relying on normal distribution. By this way samples can be clustered and classified depending on which quality gradation is wanted, and the grading samples quantitatively relative to quality can be performed simultaneously. Especially, the redundant information with respect to pattern recognition hiding in dual index sequences of samples can be removed effectively by applying the good grade sequences, which make the pattern recognition results accurate excellently. This approach possesses the advantages of both supervised classification and unsupervised cluster methods. Samples can be clustered and classified at the same time without any standard samples, and the operation is accomplished based on the good grade similar sequences themselves being as the classifying marks. Moreover, the subclasses in each class can be identified more subtly.

RESULTThe infrared fingerprint spectra of extracts of 27 kinds of Mingmu Dihuangwan pills and Zhibo Dihuangwan pills samples extracted with ethanol were analyzed with the method proposed in this paper. The results showed that these pills can be classified in their subclasses clearly, respectively.

CONCLUSIONThe Dual index grade sequence pattern recognition is a new and effective one for identifying complex biological products made from complex herbal medicines.

Cluster Analysis ; Drugs, Chinese Herbal ; analysis ; chemistry ; classification ; Ethanol ; chemistry ; Pattern Recognition, Automated ; methods ; Quality Control ; Reproducibility of Results ; Spectrophotometry, Infrared

OBJECTIVETo investigate whether murine soluble Fas gene transfected marrow graft could block the immune escape of leukemia cells, so as to eliminate the residual leukemia cells and reduce relapse after bone marrow transplantation (BMT).

METHODSThe murine leukemia/lymphoma models were established by inoculating female C57BL/6 mice (H-2b) with 10(5) EL4 cells/mouse through caudal vein. Donors of BM grafts were C57BL/6 male mice. Bone marrow mononuclear cells (BMMCs) were transfected with sFas or EGFP by adenovirus (adsFas or adEGFP) 24 hours before BMT (group D or E). The following three groups were set simultaneously: group A, no BMMCs transplanted; group B, BMMCs transplanted with no adenoviruses transfection; group C, EL4 cells transfusion only. Hematopoietic reconstitution, generation of leukemia/lymphoma and the survival rate were observed in all the groups after BMT.

RESULTSThe spleen indices examined 11 days after BMT were not obviously different among group B, D and E (P > 0.05), but in group A were significantly lower than those in the groups B, D, E (P < 0.01). The leukocyte and platelet counts on day 30 after BMT were recovered in group B and D, but were very low in group C and E. The Y-chromosomes appeared 2 months after BMT. Bone marrow pictures in group B and D were almost normal, but in group C and E had plenty of lymphoblast-like tumor cells. Tumors were obviously revealed in the mice of group C and E by histopathology examination, but did not in group B and D. The survival rate was 0 in group A, 100% in group B and D, 12.5% in group C and 6.25% in group E. Compared with that in group E, the survival was significantly increased in the sFas group (P < 0.01).

CONCLUSIONSGraft transfected with sFas gene prolonged the post-BMT survival of leukemia/lymphoma mice. The transfection of sFas might block the effect of the immune escape of EL4 cells through FasL.

Animals ; Bone Marrow Transplantation ; immunology ; Combined Modality Therapy ; Female ; Genetic Therapy ; methods ; Leukemia, Experimental ; immunology ; therapy ; Male ; Mice ; Mice, Inbred C57BL ; Transduction, Genetic ; Transfection ; Transplantation, Homologous ; Tumor Escape ; fas Receptor ; genetics

To explore the absorption mechanism of paeonol-beta-CD from various intestinal segments and offer biopharmaceutics data for paeonol new dosage form. The absorption kinetics and permeability rate consatants were investigated by the in situ perfusing method in rats. The absorption of the drug conforms to the firt-order kinetics and passive transport mechanism . The results indicate that paeonol-beta-CD absorption mechanism wasn't change.
Acetophenones ; pharmacokinetics ; Animals ; Intestinal Absorption ; physiology ; Intestines ; metabolism ; Kinetics ; Male ; Rats ; Rats, Sprague-Dawley

This study was aimed to investigate the effect of small interfering RNA (siRNA) on the expression of mll-af9 oncogene and the proliferation of human acute monocytic leukemia cell line THP-1. One group of siRNA was designed targeting mll-af9 mRNA and finally obtained by chemosynthesis. Then the obtained siRNA was transfected into cultured human acute monocytic leukemia cell line THP-1 by lipofectamine. Flow cytometry was used to detect siRNA transfection efficiency. The level of mll-af9 mRNA expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR). The cell proliferation rate was assayed by MTT. The change of cell cycles and apoptosis rate was detected by flow cytometry. The results showed that the siRNA transfection efficiency was 69.1%+/-1.8%. The level of mll-af9 mRNA expression was significantly inhibited in siRNA-transfected cells as compared with the controls. mll-af9-targeted siRNA inhibited the proliferation of THP-1 cells and induced cell apoptosis effectively after transfection. The percentage of G0/G1 phase cells significantly increased in siRNA-transfected cells in comparion with the control cells, but the percentage of S phase cells significantly decreased. It is concluded that the mll-af9-targeted siRNA can effectively inhibit the proliferation of human acute monocytic leukemia cell line THP-1.
Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; Humans ; Leukemia, Monocytic, Acute ; genetics ; pathology ; Myeloid-Lymphoid Leukemia Protein ; genetics ; metabolism ; Oncogene Proteins, Fusion ; genetics ; metabolism ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Transfection

Leukemic cells from patients expressed high level FasL cause apoptosis of autologous activated T cells via the Fas/FasL pathway. To investigate the role of soluble Fas (sFas) in reversing this process, a retroviral-mediated expression vector pLXIN-sFas was established. A retroviral-mediated expression system of human sFas was established in vitro and the biological activity of the expression product sFas was observed. To obtain the soluble Fas cDNA, the specific part of the full-length Fas cDNA was deleted by multiple PCR. After pLXIN-sFas packaged by PA317 cells, it was transferred into the target cell COS-7. The quantity of the sFas was (2.2 +/- 0.7) micro g/ml in supernatant of cultured COS-7 cells, and it could greatly inhibit apoptosis of Jurket cells induced by anti-Fas antibody. Our results suggested that the recombinant is able to express the target proteins in vitro and it has the perfect biological activity.
3T3 Cells ; Animals ; Apoptosis ; drug effects ; COS Cells ; Cell Line ; Cell Survival ; drug effects ; Culture Media, Conditioned ; pharmacology ; Dose-Response Relationship, Drug ; Gene Expression ; Genetic Vectors ; genetics ; Humans ; Jurkat Cells ; Mice ; Retroviridae ; genetics ; Solubility ; fas Receptor ; genetics ; metabolism ; pharmacology

OBJECTIVETo observe the effect of electroacupuncture on recovery of urinary bladder function after radical hysterectomy.

METHODSOne hundred and ten cases were randomly divided into an electroacupuncture (EA) group and a control group, 55 cases in each group. In the control group, the urinary tube was placed and kept with routine method and the urinary bladder was rinsed, and from the eighth day the abdomen was radiated with TDP, 30 min each day, for 5 days. In the EA group, on the basis of treatment in the control group EA was given at Sanyinjiao (SP 6), Zusanli (ST 36), Waiguan (TE 5), Shuidao (ST 28), Guilai (ST 29), etc. from the eighth day to twelfth day after operation. The recovery time of urinary bladder function after radical hysterectomy, urine dynamic indexes and hospitalization days were compared between the two groups.

RESULTSThe cases of the bladder function recovery, retention of urine, urinary incontinence were 51(51/55), 4(4/55), 0 on the 14 th day after operation and 53(53/55), 2(2/55), 0 on the 28 th day in the EA group, and 27(27/55), 25(25/55), 3(3/55) on the 14 th day and 43(43/55), 11(11/55), 1(1/55) on the 28th day in the control group, respectively, with a very significant difference between the two groups (P < 0.01); the EA group in residual urine volume, bladder volume, mean urinary flowing rate was better than the control group on the 14 th day after operation (P < 0.01 or P < 0.05); the hospitalization days after operation was (21.1 +/- 3.3) days in the EA group and (25.5 +/- 3.5) days in the control group, the former being shorter than the later (P < 0.01).

CONCLUSIONEA can promote recovery of bladder function, shorten the keeping time of urinary tube after radical hysterectomy, which is benefit to decreasing incidence rate of urinary system infection and shortening hospitalization days.

Adult ; Aged ; Electroacupuncture ; Female ; Humans ; Hysterectomy ; adverse effects ; Medicine, Chinese Traditional ; Middle Aged ; Postoperative Complications ; physiopathology ; therapy ; Urinary Bladder ; physiopathology ; Urinary Bladder Diseases ; physiopathology ; therapy

OBJECTIVETo investigate the application of serum glutathione S-transferase (GST) and urinary 8-hydroxy-2-deoxyguanosine (8-OHdG) as the monitoring biomarkers for coke oven workers exposed to polycyclic aromatic hydrocarbons (PAHs).

METHODS47 male coke oven workers and 31 male control workers were investigated. Urinary 8-OHdG and serum GST were analyzed using high performance liquid chromatography (HPLC) with electrochemical detection and test kit. Urinary 1-hydroxypyrene (1-OHP) as internal exposure of PAHs was also determined simultaneously by alkaline hydrolysis and HPLC.

RESULTSThe values of urinary 1-OHP, serum GST and urinary 8-OHdG were reported as median with interquartile range (P(25)-P(75)). Urinary 1-OHP [5.7 (1.4-12.0) micromol/mol Cr], serum GST [22.1 (14.9-31.2) U/ml], and urinary 8-OHdG [1.9 (1.4-15.4) micromol/mol Cr] in coke oven workers were significantly higher than in control workers [3.0 (0.5-6.4) micromol/mol Cr (P < 0.05), 13.1 (9.5-16.7) U/ml (P < 0.01), and 1.3 (1.0-4.0) micromol/mol Cr (P < 0.05) respectively]. Categorizing by smoking status, significant differences in urinary 1-OHP and serum GST were found only in smokers among coke oven workers compared to control workers (P < 0.01), and 8-OHdG levels only in non-smokers (P < 0.01). Additionally, there was significant correlation between urinary 1-OHP and serum GST activity (r(s) = 0.31, P < 0.01, n = 78). The multiple logistic regression analysis showed that coke oven workers were at the higher risk of having GST activities above 16.7 U/ml (OR = 13.2) and 8-OHdG levels above 1.8 micromol/mol creatinine (OR = 4.4). High body mass index was an independent factor to affect urinary 8-OHdG levels.

CONCLUSIONSThe elevated serum GST activities and increased oxidative DNA damage were found in the coke oven workers. Occupational exposure and smoking interact on each other. Serum GST may be used as a biomarker for assessing the exposure of PAHs. Assay of urinary 8-OHdG may be useful for evaluating the risk of lung cancer in coke oven workers.

Adult ; Case-Control Studies ; Coke ; Deoxyguanosine ; analogs & derivatives ; urine ; Glutathione Transferase ; blood ; Humans ; Male ; Occupational Exposure

BACKGROUNDTo investigate if bone marrow transplantation (BMT) with bone marrow mononuclear cells (BMMCs) transducted with murine soluble Fas gene (sFas) using adenovirus vector could block the immune escape of leukemia cells eliminate the residual leukemia cells and reduce their relapse.

METHODSThe recombinant adenovirus vector with murine sFas, adsFas, and the control vector adEGFP were constructed using homologous recombination between two plasmids in Escherichia coli. BMT was carried out after the BMMCs were infected with Adenoviruses. The mice models of leukemia/lymphoma were constructed by inoculating female C57BL/6 mice (H-2b) with 10(5) EL4 cells/mouse through caudal vein. Donors of bone marrow grafts were syngeneic male mice. BMMCs were infected with AdsFas or AdEGFP 24 hours before (Group D or E). The following three groups were simultaneously used: Group A, no BMMCs transplanted; Group B, transplanted with BMMCs not infected with adenoviruses; Group C, only transfusing EL4 cells, neither irradiation nor BMT. The hematopoietic reconstitution, generation of leukemia/lymphoma and the survival rate were observed in all groups after BMT.

RESULTSThe adenovirus vectors were successfully constructed. The titre of virus after purification was up to 2.5 x 10(11) pfu/ml. Spleen indices examined 11 days after BMT were not obviously different among Group B, D and E (P > 0.05), but indices in Group A were significantly lower than those in the latter three groups (P < 0.01). Counts of leukocytes and platelets on +30 day showed mice were reconstituted satisfactorily in Group B and D, but very low in Group C and E. The Y-chromosomes existed 2 months after BMT and examination of bone marrow cytology showed that Group B and D were almost normal, but Group C and E had plenty of lymphoblast-like tumor cells. Tumors were obviously observed in the mice of Group C and E by histopathological examination, but the mice in Group B and D were normal. The survival rates were 0 (0/4) in Group A, 100% in Group B (6/6) and D (16/16), 12.5% (2/16) in Group C and 6.25% (1/16) in Group E respectively. It is demonstrated that, in contrast with the control (Group EGFP), survival rate was significantly increased in the sFas Group (P < 0.01).

CONCLUSIONSThe transfer of sFas gene by adenovirus changed the prognosis state of leukemia/lymphoma mice after auto-BMT. The transduction of sFas might block the effect of the immune escape of EL4 cells through FasL. These results could thus provide a new direction to find a way to treat the leukemia and its recurrence after BMT.

Adenoviridae ; Animals ; Bone Marrow Transplantation ; Fas Ligand Protein ; Female ; Genetic Vectors ; Leukemia, Experimental ; Leukocytes, Mononuclear ; Male ; Membrane Glycoproteins ; genetics ; Mice ; Mice, Inbred C57BL ; Recombination, Genetic ; Transduction, Genetic ; Transfection ; Tumor Escape ; physiology