BACKGROUND: Umbilical cord blood (UCB) with limited karyocytes is mainly used in child patients. Recently, physicians have tried to mix two units of cord blood in the treatment of adults with hematological system diseases.OBJECTIVE: To monitor quantitatively the dynamic changes and the development rules of engraftment, chimera types and relative amount after allogeneic transplantation of mixed UCB from two units in adults with leukemia.DESIGN: Donors and the recipient were regarded as observational subjects in umbilical cord blood transplantation (UCBT). DNA extracted from blood samples of donors and the recipient before and after transplantation was considered as detecting samples. Short tandem repeat (STR) loci were as observational measures.SETTING: Key Laboratory of Immunology and Genetics of Institute of Transfusion Medicine of Shenzhen Blood Center.PARTICIPANT: A 43-year male patient with acute myeloid leukemia (AML), 75 kg, who was hospitalized at Shenzhen Hospital of Peking University, was enrolled in June 2005. The patient received two units of human leucocyte antigen (HLA), one locus mismatched unrelated UCBT (2.5×107 kg-1 karyocytes in UCB 1, and 1.53×107 kg-1 karyocytes in UCB 2) at month 6 after complete remission from first chemotherapy. UCB was collected from Guangzhou umbilical cord blood bank. The patient signed the informed consent.METHODS: The adult with AML received two units of HLA, one locus mismatched unrelated UCBT (2.5×107 kg-1 karyocytes in UCB 1, and 1.53×107 kg-1 karyocytes in UCB 2). Nine STR loci of the blood sample were determined before and after transplantation by quantitative technique of fluorescence labeling with multiplex polymerase chain reaction (MPCR), while the engraftment and chimera types were qualitatively evaluated by comparing differential loci between the recipient and the donors. The relative amount of two units of UCB was calculated in the patient after transplantation according to the differential gene peak areas of two donors with 377XL DNA sequencer after fluorescence scanning. The engraftment level and the development rules of donors' cells were analyzed quantitatively. In addition, the results were also compared with that of HLA loci distinct analysis for engraftment.MAIN OUTCOME MEASURES: After UCBT, transition process of nine STR loci of the recipient and two donors was observed, and engraftment was quantitatively and qualitatively described.RESULTS: Two units of UCB at day 15 after transplantation were engrafted simultaneously and revealed a complete chimera of the two. The relative amounts of UCB 1 and UCB 2 were 51.3% and 48.7%, respectively. Subsequently, UCB 1 went up to 70.0% and UCB 2 declined to 30.0% at day 30. However, only the genotype of UCB 1 was detected at day 52, and engraftment turned to a complete chimera of a single donor. The one with fewer karyocytes was rejected and the one with more karyocytes was engrafted for a long term.CONCLUSION: To detect quantitatively STR chimera with fluorescence labeling and MPCR can show precisely the engraftment level and the change of two units of UCB. It provides an accurate and reliable experimental basis for clinical UCB application and donor selection. It is proved that adult transplantation at the same time with mixed UCB from two units HLA one locus mismatched unrelated donors is feasible.

OBJECTIVE:To establish a method for the contents of gallic acid,catechin,sennosides B,aloe-emodin,rhein, emodin,chrysophanol,physcion,chrysophanol-1-O- glucoside and emodin-8-O- glucoside in Rhei Radix et Rhizoma,Jiu Rhei Radix et Rhizoma,Shu Rhei Radix et Rhizoma,Rhei Radix et Rhizoma tan,Cu Rhei Radix et Rhizoma,and analyze the differ-ences. METHODS:HPLC was performed on the column was Hypersil C18 with mobile phase of methanol- 0.2% acetic acid(gradi-ent elution)at a flow rate of 1.0 ml/min,the detection wavelength was 260 nm,column temperature was 25 ℃,injection volume was 10 μl. RESULTS:The linear range was 0.252 5-4.040 0 μg for gallic acid(r=0.999 6),0.600 0-9.600 0 μg for catechin(r=0.999 6),0.297 4-4.758 4 μg for sennosides B(r=0.999 9),0.001 8-0.028 8 μg for aloe-emodin(r=0.999 9),0.005 0-0.080 0 μg for rhein(r=0.999 9),0.019 0-0.304 0μg for emodin(r=0.999 8),0.380 2-6.083 2μg for chrysophanol(r=0.999 7),0.008 2-0.131 2μg for physcion(r=0.999 8),0.126 0-2.016 0 μg for chrysophanol-1-O-glucoside(r=0.999 6)and 0.111 3-1.780 8 μg for emo-din-8-O-glucoside (r=0.999 8);RSDs of precision,stability and reproducibility tests were lower than 3.0%;recoveries were 96.17%-97.21%(RSD=1.67%,n=6),97.60%-100.54%(RSD=2.55%,n=6),99.45%-101.32%(RSD=1.63%,n=6), 95.31%-98.19%(RSD=2.42%,n=6),98.99%-100.35%(RSD=1.86%,n=6),98.95%-101.21%(RSD=2.17%,n=6), 99.81%-100.62%(RSD=1.66%,n=6),96.78%-98.52%(RSD=1.99%,n=6),97.80%-100.14%(RSD=3.32%,n=6) and 97.40%-101.24%(RSD=2.89%,n=6). Compared with Sheng Rhei Radix et Rhizoma,the contents of gallic acid,catechin,sen-nosides B and anthraquinones in Cu Rhei Radix et Rhizoma,Jiu Rhei Radix et Rhizoma and Rhei Radix et Rhizoma tan decreased. The contents of catechin,sennosides B and anthraquinones in Shu Rhei Radix et Rhizoma. Catechin,sennosides B,chrysopha-nol-1-O- glucoside,aloe-emodin and rhein were not detected in Dahuang tan. CONCLUSIONS:The method is simple with good precision,stability and reroducibility,and can be used for the simultaneous determination of 10 chemical components in processed products of Rhei Radix et Rhizoma;there were significant differences in contents of 10 chemical components in processed prod-ucts of Rhei Radix et Rhizoma.

Objective To investigate the possibility of survivin inhibition by lentiviral vector-mediated RNA interference and the influence on cell apoptosis in pancreatic cancer cell line.Methods The lentiviral vector of SiRNA targeted against survivin(LV-shRNA-survivin-1,LV-shRNA-survivin-2,LV-shRNA-survivin-3) was constructed and transfected into the packaging cells 293T,and then the supernatant with virus was collected to transfect SW1990 cells.Quantitative real-time fluorescent PCR and Western-blot were used to detect the expression of survivin.DAPI staining and detection of enzymatic activity of caspase 3/7 were employed to examine cell apoptosis.Results Three lentiviral vector-survivin-shRNA were constructed successfully.In the LV-shRNA-survivin-1 group,the survivin mRNA and protein expression inhibitory rate was 73.50% and 87.64% respectively;when compared to control group,the activity of caspase-3/7 increased significantly,which showed a 14.5-fold increase,and apoptosis increased 11.95%.Conclusions Lentiviral vector-mediad RNA interference targeted against survivin can effectively inhibit survivin expression and increase cell apoptosis significantly.

Objective To explore the effect of beraprost sodium (BPS) on hypoxia-induced pulmonary artery hypertension (HPH) in rats and the expression of oxygen-sensitive Kv channels in pulmonary artery smooth muscle (PASM).Methods The HPH model of rats was established by exposing rats to low-pressure and low-oxygen cabin which was auto-modulated for 8h every day.Rats in the BPS group were given an intragastric administration of BPS [300 μg/(kg · d)],while those in the control group and HPH group were given an intragastric administration of 3 ml/kg of 0.9％ saline.After 4 weeks,the mean pulmonary artery pressure (mPAP) was measured and right heart ventricle hypertrophy index (RVHI) was calculated;pulmonary artery remodeling was evaluated by HE staining;the expressions of Kv 1.2,Kv 1.5 and Kv2.1 in the pulmonary artery were detected by Real-time PCR and Western blot.Results The HPH model was successfully established in rats exposed to chronic hypoxia for 4 weeks.Compared with those in HPH group,mPAP,RVHI and pulmonary artery remodeling were decreased in BPS group [mPAP:(13.48±2.18)mmHg vs.(23.87±2.23)mmHg vs.(17.09±1.20)mmHg;RVHI:0.28±0.02 vs.0.46±0.03 vs.0.36±0.04;％ area of medial smooth muscle:35.72±6.58 vs.68.52±5.64 vs.46.58±8.43;P＜0.05],and the mRNA and protein expressions of Kv 1.2,Kv 1.5 and Kv 2.1 were increased (relative protein expression level:Kv1.2,0.78±0.10 vs.0.15±0.03 vs.0.57±0.13;Kv1.5,0.61±0.10 vs.0.31±0.05 vs.0.59±0.13;Kv2.1,0.29±0.05 vs.0.10±0.02 vs.0.28±0.07;P＜0.05).Conclusion BPS can improve pulmonary arterial hypertension induced by hypoxia,and upregulate the decreased mRNA and protein expressions of Kv 1.2,Kv 1.5 and Kv 2.1 in pulmonary artery.

Purpose To observe the pathologic types of glomerular diseases which have high renal interstitial foam cells infiltration and to evaluate the relationship between infiltration of foam cells (FC) in renal interstitial tissue and pathologic parameters.Methods A total of 2 862 patients who had received renal biopsy were enrolled in this study. Patients with Alport syndrome (AS,n=5), membranous proliferative glomerular nephritis (MPGN,n=28), focal segmental glomerulosclerosis (FSGS,n=144), idiopathic membranous nephropathy (IMN,n=132), IgA nephropathy (IgAN,n=893) were divided into two groups:FC+ group with foam cells and FC- group without foam cells.Results Infiltration of foam cells in renal interstitial tissue was commonly seen in AS.The frequency of interstitial foam cells was 46.43% in MPGN, 20.14% in FSGS, 13.64% in IMN, and 6.27% in IgAN. It was found that the segmental glomerular sclerosis and interstitial fibrosis were more severe in FC+ group than that in FC- group.Conclusions The renal interstitial foam cells are most common in patients with AS, but also seen in patients with MPGN, FSGS, IMN and IgAN. There might be a relationship between glomerular sclerosis, interstitial fibrosis and infiltration of foam cells. The present of foam cells in the renal interstitial tissue may contribute to the progression of renal diseases.

Objective To observe the change of regulatory T cells expression in severely burned rats gut ,To investigate the effects of regulatory T cells on CD3+CD4+ /CD3+CD8+lymphocytes and its relation with gut-origin endotoxin translocation .Meth-ods Fifty SD male rats were randomly divided into normal control group(n=10)and burn model groups(n=40) .Rats were burned to achieve Ⅲ degree scalding ,and a 30% total body surface area(TBSA) burn model was made .Rats were sacrificed before(normal control group) and after 0 .5 ,1 ,2 ,4 burn hour(PBH groups) .Flow cytometry techniques were used for the detection of the expres-sions of regulatory T cells and CD3+CD4+ /CD3+CD8+ lymphocyte in intestinal lymph nodes which were separated .The dynamic turbidity method was used for detection of endotoxin levels in portal vein blood .Results The expression of regulatory T cells was negativelycorrelatedwithCD3+CD4+ /CD3+CD8+ lymphocyteratio(r= -0.827,P<0.01)inintestinallymphnodeofrats,while the regulatory T cells was positively correlated with ET levels in portal vein blood plasma .(r=0 .782 ,P<0 .01) .Conclusion The expression of the regulatory T cells in intestinal lymph node in severely burned rats was increased compared to that in normal con-trol group .Regulatory T cells suppressed the expression of intestinal T lymphocytes ,leading to gut immune inhibition .The translo-cation of intestinal endotoxin has a close relationship with regulatory T cells in severely burned rats .Regulatory T cells could have portal effects on intestinal immunity barrier .

Berberine is the main active ingredients in Coptis.The content determination of berberine can provide the basis for its quality evaluation.Relevant provessinal literatures about determination methods of berberine were summarized,and the main methods included TLC,UV, HPLC,HPEC,fluorescence,and so on.HPLC was the most widely used method because of its high separation efficiency,specificity and sensitivity.

OBJECTIVE:To study the influential factors related to efficacy of Peach kernel-Rheum palmatum couplet medi-cines in TCM formula,and to reveal the general regularity of compatibility environment,common ratio,processing variety and dosage forms of P. kernel-R. palmatum couplet medicines. METHODS:Using Chinese Medical Prescription Selected Dictionary ed-ited by Peng Huairen as data source,142 formulas of P. kernel-R. palmatum couplet medicines were collected. By establishing data-base,compatibility types of P. kernel-R. palmatum couplet medicines,as well as common ratio,processed prodact,dosage form were classified statistically. The influential factors related to efficacy of P. kernel-R. palmatum couplet medicines with different pro-portions were summarized. RESULTS:The efficacy of P. kernel-R. palmatum couplet medicines could be divided into 6 aspects and 11 roles,including activating blood circulation to dissipate blood stasis(activating blood to relieve pain,promoting blood circula-tion to eliminate disease,activating blood to promote menstruation,breaking stagnant and eliminating blood stasis),eliminating carbuncle and detoxicating(cleaning intestine and clearing away the pathogenic heat of lung,eliminating carbuncle and expelling pus,eliminating sore and detoxicating),expelling the pathogenic heat to loosen the bowels,warming yang for dispelling cold,forti-fying the spleen and nourishing the stomach,relaxing tendon and activating blood. The compatibility environment of P. kernel-R. pal-matum couplet medicines were mainly compatible with TCM for activating qi to eliminate stasis,activating blood to promote menstru-ation,breaking stagnant and eliminating blood stasis,expelling the pathogenic heat to expel stasis. The ratio of P. kernel to R. palma-tum ranged 1 : 8-4 : 1,and the ratio ranged 1 : 8-3 : 1 when performing the role of actirating blood circalation to dissipate blood stasis. Common processed products were crude P. kernel and prepared R. palmatum. Common dosage forms were mainly decoction,pill and powder. CONCLUSIONS:Compatibility environment,ratio,processing varieties,dosage forms influence the effects of P. kernel-R. palmatum couplet medicines,especially compatibility environment.

Objective To investigate levels of IL-17 and IL-22 mRNA in the blood of type 2 diabetes(T2DM)patients with the different stages of kidney injury and explore the relationship between the gene expression levels of IL-17,IL-22 and renal lesions in patients with diabetic nephropathy(DM).Methods Subjects included 60 T2DM patients with or without kidney injury and 20 normal controls(NC,n=20).Diabetes patients were divided into 3 groups by level of urinary albumin to creati-nine ratio (ACR):no proteinuria group (NA,ACR<30 mg/g,n=22),microalb uminuria group (MA,30 mg/g>ACR>300 mg/g,n=18)and diabetic nephropathy group (DN,ACR>300 mg/g,n=20).Quantitative Real-Time RT-PCR was used to detect IL-17 and IL-22 mRNA levels.Analysis differences of IL-17 and IL-22 mRNA levels among NA,MA,DN and NC groups.Results The levels of IL-17 and IL-22 mRNA were significantly higher in DN group than that in MA,NA and NC group (P <0.01,respectively),However,there were not significant difference among MA,NA and NC group (P >0.05,re-spectively).Conclusion Levels IL-17 and IL-22 mRNA were increased in blood of T2DM patients with nephropathy.IL-17 and IL-22 may play role in the pathogenicity of diabetic nephropathy.

Objective To compare the effect of 5-fluorouridine(5-FUR) prodrug liposome on the cell proliferation and apoptosis in HEP-2 cells.Methods MTT assay was used to investigate the cell proliferation after 5-FU or 5-FUR prodrug liposome at the dose of 3.0,0.6,0.12 or 0.024 ?g/ml was added into the culture medium of HEP-2 cells for 72 h.Flow cytometry(FCM) was used to measure the cell cycle when the cells were treated with 0.17 ?g/ml 5-FU or 0.05 ?g/ml 5-FUR prodrug liposome for 72 h.The apoptosis of cells treated with 0.05 ?g/ml 5-FU or 5-FUR for 48 h were assayed with FCM.Results The IC50 of 5-FU on HEP-2 cells was 300% higher when compared with 5-FUR prodrug liposome.Treated with 5-FU and 5-FUR prodrug liposome respectively,HEP-2 cell lines were both arrested at S stage.the apoptosis rate of HEP-2 were(10.35?1.33)% and(21.57?0.11)% respectively.Conclusion Compared with 5-FU,5-FUR prodrug liposome inhibits the cell proliferation of HEP-2 cells more significantly,arrests more cells at S phase,and improves cell apoptosis.