Objective To study the expression of Fas associated phosphatase-1(FAP-1)in cerebral cortex and hippocampus after cerebral ischemia in rats.Methods The distribution and quantity of FAP-1 immunological positive cells in the brain were observed with immunohistochemistry method after focal ischemia.Results The number of FAP-1 positive cells in cerebral cortex and hippocampal gyrus of cerebral ischemia rats were 10.76?1.23 and 9.24?0.87,obviously higher than sham operation group(4.12?0.36,3.83?0.43)(all P

Objective: To further study the effect of apoptin in inducing cancer cell specific apoptosis and the possible applications in cancer therapy. Methods: Apoptin gene was amplified by PCR and inserted into pcDNA3.1(+) with a FLAG tag in front of the multi-cloning-site. Apoptin gene with the FLAG tag was sub-cloned into an adenovirus vector. Several cancer cell lines were transfected with pcDNA3. 1/FLAG/apoptin or infected with apoptin containing recombinant adenoviruses to study the morphologic changes. Ad/apoptin infected cells were also analyzed by flowcytometry after staining with PI. Results: Expressed apoptin was localized in the nucleus of cancer cells. Chromatin condensation occurred 2 or 3 days after Ad/apoptin(+) infection. Cell number in G 2-M phase increased dramatically after Ad/apoptin(+) infection. Conclusion: Apoptin can induce cell cycle G 2-M arrest and chromatin condensation in cancer cells.

OBJECTIVETo explore the effect of p21(WAF1) on the proliferation and the sensitivity to Vp16 of leukemia cell line K562.

METHODSA p21(WAF1) retroviral expression vector pLXSN-p21(WAF1) was constructed by FuGENE 6, pLXSN-p21(WAF1) and pLXSN-neo, and transfected into p21(WAF1) defect leukemia cell line K562. After selected with G418, K562-p21(WAF1) cell clones that stably expressed p21(WAF1) were isolated. The ectopic expressions of p21(WAF1) mRNA and protein in K562-p21(WAF1) were identified by RT-PCR and Western b1ot. The cell growth rate was tested by trypan blue dye, the cell cycle by FCM and the sensitivity to Vpl6 by cell count and MTT assay.

RESULTSThe expression of p21(WAF1) protein and mRNA could be detected in K562-p21(WAF1) cells. A strong inhibition of cell proliferation was observed in K562-p21(WAF1) cell as compared with that of the control. The cell number in G(0)/G(1) phase was remarkably increased. The sensitivity to Vpl6 decreased, the IC (50) of K562-neo cells was (56.4 +/- 6.5) microgram/ml, and that of K562-p21(WAF1) cells was (131.0 +/- 8.7) microgram/ml (P < 0.01).

CONCLUSIONp21(WAF1) can inhibit the proliferation of leukemia cell and decrease its sensitivity to Vp16.

Antineoplastic Agents, Phytogenic ; pharmacology ; Blotting, Western ; Cell Division ; drug effects ; Cell Survival ; drug effects ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; genetics ; metabolism ; physiology ; Dose-Response Relationship, Drug ; Etoposide ; pharmacology ; G1 Phase ; drug effects ; Gene Expression ; Humans ; K562 Cells ; cytology ; drug effects ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Resting Phase, Cell Cycle ; drug effects ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors

Varied TCM constitutions in the patient of acute cerebral infarction were classifyed by using HLA - DQAl allehc gene to analyze hereditary susceptibility of constitution types and relation among constitution, syndrome and treatment. PCR-SSP technique was used for classification of Yin - deficiency Yang - deficiency, Qi - deficiency, phlegm - dampness and blood stasis constitution in 103 cases of acute cerebral infarction and HLA - DQAl allelic gene was used for gent classification in 99 cases of nomal constituion. Results indicated that HLA -DQA * 0501 gene type in Yin -deficiency constitution was significant higher than that of normal constitution CP

Objective　To observe the effect of aloe-emodin on the expression of proliferating cell nuclear antigen (PCNA) in smooth muscle cells (SMCs) after arterial injury and study the molecular mechanism of inhibition of aloe-emodin on SMC proliferation. Methods　Deendothelialization was performed at the abdominal aorta in Japanese white rabbits using a 3F Fogarty arterial embolectomy catheter. 48 hours later, the medium of abdominal aorta was isolated and primary SMCs culture was performed. Cells were synchronized to G0 by serum starvation, then aloe-emodin at a concentration of 20?μg/ml was added to the culture medium containing 10%［v/v］ fetal calf serum. Vehicle was also added to the medium as a control. After 18 hours, the expression of PCNA at the level of mRNA and protein were examined using techniques of RT/PCR, Western blotting and inmmunocytochemistry respectively. Results　Compared with the control group, the expression of PCNA mRNA and protein was prominently decreased after addition of aloe-emodin. Conclusion　The inhibition of aloe-emodin on SMCs proliferation may be caused by inhibiting the expression of the PCNA gene.

Objective To observe the characteristics of folate binding proteins (FBP) in myelodysplastic syndromes (MDS) and leukemia and to study the clinical significance of reduced folate carrier (RFC) present in MDS and its relationship with multidrug resistance (MDR).Methods The features of FBP on bone marrow cells were analyzed using radiolabeled 3 H-folic acid (3 H-FA) binding membrane proteins and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). In the same time, P-glucoprotein and mRNA of MDR gene were detected using immunocytochemistry and reverse transcription ploymerase chain reaction (RT-PCR) respectively in patients with MDS and leukemia.Results Two kinds of FBP, folate receptor (FR) and reduced folate carrier (RFC), were found on the leukemic cells. The same results were presented on mononuclear cells of bone marrow in 5 out of 14 MDS patients, and MDR positive was seen in 4 patiens of them. In normal control and other 9 cases of MDS FRs were only found on the mononuclear cells of bone marrow.Conclusion Reduced folate carrier, which is present in the leukemic cell, is a product of neoplastic cell. It might reveal preleukmic state and have the same significance with MDR that RFC is found in MDS patients.

For the purpose of thrombocytopenia gene therapy, recombinant RevTet-On and pRevTRE/TPO retrovirus were packaged and transfected to NIH3T3 after selected with G418 and hygromycin, the two inserting recombinant retrovirus cell strain RevTet-On3T3/TPO were established. TPO expression was controlled and regulated by doxycydine (Dox). After using Dox to control the expression of TPO in RevTet-On3T3/TPO cells, the result showed that when Dox is added to the RevTet-On3T3/TPO cells, cell populations expressed TPO highly in the presence of 2 mg/L of Dox, and lowly in the absence of Dox. By using the RevTet-On gene expression system (the retrovirus vector RevTet-On regulation system to control the expression gene by Dox), it could modulate the expression of multiple genes by tetracyline and its derivatives. This system maybe provides a safe and efficacient way for the thrombocy to penia gene therapy.

The aim of this work was to test the effect of p16 on the proliferation of leukemic cells and its potential in gene therapy for leukemia. The full-length p16 cDNA was transfered by recombinant retrovirus vector into leukemia cell line K562, which is homozygous p16 deletion and retains functional retinoblastoma (RB) protein. The cell proliferation was tested in liquid and in soft agar culture after transduction of p16 retrovirus. The results showed a strong inhibition of cell proliferation. Phosphorylation of RB protein was also inhibited. The findings demonstrated that p16 (MTS/CDKN2) inactivation is a significant factor in the genesis and progression of leukemia and p16 could be a candidate gene for gene therapy in leukemia.

To study the significance of calcium in the apoptosis of HL-60 cells induced by VP-16, the technology of flow cytometry, confocal laser scanning microscopy and Western blot were used. The results showed that VP-16 could induced the apoptosis of HL-60 cells and transient increase of intracellular calcium concentration; EGTA [ethylene glycol-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid], that could combine the extracellular calcium, did not prevent the apoptosis of HL-60 cells. BAPTA-AM [1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxy-methyl) ester], however, a chelating agent of intracellular calcium ions, could prevent apoptosis and the release of cytochrome C from HL-60 cells. It was concluded that the calcium plays an important role in apoptosis and the release of cytochrome C.

Abstract:Objective To detect the mutation frequency of the bcl 10 gene in the early and advanced stages of hepatocellular carcinoma (HCC).Methods Genome DNA samples were extracted from 46 cases of fresh HCC tumor tissues and their non-tumor adjacent tissues. Polymerase chain reaction-single strand conformation polymorphism method was used to detect point mutations of the three exons of the bcl 10 gene. For each individual exon, six random samples from those showing abnormal DNA bands were sequenced to verify those mutations. The relationship between serum alpha-fetoprotein (AFP) level and bcl 10 mutation, between the tumor size and bcl 10 mutation was also analyzed.Results Among the 46 samples, 26 cases (56.5%) were found to have mutations in exon 1, 5 out of the 6 cases were shown to have 5744 C→G mutation by sequencing; 25 cases (54.3%) were found to have mutations in exon 2, 4 out of the 6 cases were shown to have 11?311 T deletion mutation by sequencing. Twenty-one cases (45.7%) were found to have mutations in exon 3, all of the 6 cases selected for sequencing were shown to have 14?116 C→T mutation. Statistical analysis showed that neither serum alpha-fetoprotein level nor the size of hepatocellular carcinoma has a significant relationship with bcl 10 mutation.Conclusion The bcl 10 gene has a high mutation frequency in liver cancer.