BACKGROUND:Recently,some overseas in vitro researches and animal models confirmed that donor leukomonocyte cultured by fludarabine can significantly reduce acute graft-versus-host disease,but the mechanism of action is not clear.OBJECTIVE:To explore immunophenotype changes of donor leukomonocyte cultured with fludarabine in vitro.DESIGN,TIME AND SETTING:The observational cytology experiment was performed at the Department of Hematology,First People's Hospital from July 2007 to January 2008.MATERIALS:Leukomonocytes were collected from four hour healthy persons as donors of allogenic peripheral blood hematopoietic stem cell transplantation.Fludarabine was offered by Schering AG,Germany.METHODS:Peripheral blood hematopoietic stem cells collected by COBO SPECTRA were washed with RPMI1640 twice.These cells were cultured in RPMI1640 containing 0.10 volume fraction of fetal bovine serum till cell density of 2?109 L-1.1 mL of hematopoietic stem cells were washed with phosphate-buffered saline,and added CD3,CD4,CD8,CD44mAb 10 ?L,and added Mouse Ig-G1-FITC/PE as control group.These cells were attenuated with phosphate-buffered saline after incubated for 30 minutes at a low temperature,then detected by flow cytometry.Other cells were put in 24-well cultivate plate,added 8 mmol /L fludarabine as a final concentration for 24 hours(5% CO2,37 ℃).MAIN OUTCOME MEASURES:Studying immunophenotypic changes of donor leukomonocytes by flow cytometry.RESULTS:There was no significant change in the ratio of CD4+CD44+T cells before or after donor leukomonocytes cultured with fludarabine for 24 hours,and proportion of CD8+CD44+T cells declined significantly(P

Objective To investigate the effect of mesenchymal stem cell(MSC) on the balance of Th1/ Th2 with different stimulations.Methods To inoculate MSC in 24-well tissue culture plates.after 3 days, added T lymphocytes co-stimulated by PMA and Ionomycin,or the MSC were added to the two-way mixed lymphocyte culture according to different proportion.The subsets and cytokines of T lymphocytes were analyzed by flow cytomety.Results Differentiation into Th1 of T lymphocytes activated by co-stimulation can be inhibited MSC;In MLC,CD8~+T cell subsets and Th1 were evidently decreased.But Th2 cells were slightly increased.Conclusion MSC can significantly suppress CD8~(+)T cell,Th1,increase Th2.MSC has potentialities of alleviating acute graft-versus-host disease(aGVHD) and maintaining graft versus leukemia(GVL).

Objective To investigate the immunophenotype characteristics,the clinical significance of CD28 and International staging system in multiple myeloma (MM).Methods BM aspirate samples from 49 newly diagnosed MM patients and 22 patients after treatment were assessed using 4 color flow cytometric analyses.These MM patients were classified according to International staging system and the consolidated risk staging system,and the survival rates and treatment efficiency of 2 staging system were compared.Results The higher stage,according to the international staging system,the lower treatment efficiency [the efficacy rates of Ⅰ,Ⅱ,Ⅲ stage were 83.3 ％ (4/6),38.5 ％ (5/13),38.5 ％ (5/13),36.5 ％ (19/52)](x2 =4.235,P =0.04),and the survival and progression-free survival time of high-risk group were significantly shortened (P ＜ 0.05).The rate of CD28 abnormal expression was no significant different between the initial and the treatment group [27 (55.1％),14 (63.4 ％)],also similar in the impact of survival and progression-free survival time (P ＞ 0.05).According to the consolidated risk staging system,the treatment efficiency,the survival and progression-free survival time of high-risk group were significantly shortened than the low ones (P =0.040).Conclusions The patients with higher ISS stage,abnormal expression of CD28,and high-risk group have poor prognosis.

Objective　To study chemotherapy sensitivity and apoptosis in tumor cell line.Methods　Two different human colon carcinoma cell lines, LoVo and Ls-174-t were incubated with DDP,MMC,5-FU，EPI at various peak plasma concentrations (PPC), 1/10PPC，1/5PPC，5PPC and 10 PPC. Apoptosis was detected by FACScan and TUNEL technique after 24 hours and 48 hous. Cell growth inhibition rates were assessed by MTT, DNA ladder were examined by agarose electrophoresis.Results　At 48 hours, the highest cellular apoptosis rates were observed with PPC which was assessed by FACScan technique. There was a positive linear correlation between apoptosis assessed by TUNEL technique and growth inhibition rates of cells determined by MMT(P＜0.05).Conclusion　Using Annexin-V-Fluos staining FACScan technique to assess apoptosis of tumor cells is an exploring and useful method which can be used to choose both kinds and doses of chemotherapeutic drugs.

Objective To study the effect of expansion ex vivo on the hematopoietic reconstitution of umbilical cord blood (UCB) hematopoietic cells, and to find a probable way of UCB transplantation (UCBT) to be used on adults. Method Three groups of UCB hematopoietic cells were transplanted to lethally irradiated BALB/C mice: fresh cells, a half part of fresh cells and a half part of cells expanded for a week, cells expanded for a week. Results All the three groups of cells could reconstitute hematopoiesis. The cells in the latter two groups could more rapidly accelerate the recovery of early-period hematopoiesis. The death rate of the mice in second group was the lowest among three groups. Conclusion The way transplanted with a half part of fresh UCB hematopoietic cells and a half part of UCB hematopoietic cells ex vivo expanded for a week may be a probable one for adult UBCT.

Objective The loss of heterozygosity (LOH) on tumor suppressor genes is believed to play a key role in the carcinogenesis of colorectal cancer. When LOH occurs at a tumor suppressor gene locus where one of the alleles is already abnormal, it can result in neoplastic transformation. In this study, we analyzed the LOH on the chromosome 22 in sporadic colorectal cancer to identify additional loci involved in colorectal tumorigenesis. Methods Six polymorphic microsatellite markers were analyzed in 83 cases of colorectal cancer and normal tissue DNA by PCR. PCR products were eletrophoresed on an ABI Prism 377 DNA sequencer; Genescan 3.1 and Genotype 2.1 software were used for LOH scanning and analysis. Comparison between LOH frequency and clinicopathological data were performed by ? 2 test. Results The average LOH frequency on chromosome 22 was 27.27%. The region between markers D22S280 and D22S274 (22q12.2-q13.33) exhibited relatively high LOH frequency. The two highest LOH loci with frequencies of 35.09% and 34.04% was identified on D22S280 (22q12.2-q12.3) and D22S274 (22q13.32-q13.33). On D22S274 locus, LOH frequency of rectal cancer was 50% (9/18), which was higher than that of proximal colon cancer (12%, 2/17) ( P =0.018). The frequency of distal colon cancer was 42% (5/12), also higher than that of proximal colon cancer. But there was no statistical significance. Putting both the tumors in distal colon and rectum together into consideration, the frequency, 47% (14/30), was higher than that of proximal colon cancer ( P = 0.015 ),suggested the mechanism of carcinogenesis was different in both groups.Conclusion This study provided evidence for the involvement of putative tumor suppressor genes related to the sporadic colorectal carcinoma on chromosome 22q. The tumor suppressor gene(s) might locate on the 22q12.2-q12.3 and/or 22q13.32 -q13.33.

Purpose:To study the relationship between Fas/FasL expression and apoptosis of colon adenocarcinoma cell lines induced by chemotherapeutic drugs and to screen out hypersensitive drugs. Methods:Colon adenocarcinoma cell lines LS174T and LoVo were incubated respectively in cultural media with DDP,MMC,5 FU and EPI at concentrations of PPC(plasma peak concentration),1/10 PPC,1/5 PPC,5 PPC and 10 PPC. Fas/FasL expression and apoptosis were assessed by FACScan, and DNA ladder was examined by agarose gel electrophoresis. Results:In LS174T cells, Fas expression and apoptosis of DDP group, MMC group and EPI group reached their highest peaks at 1/5 PPC or PPC,and Fas expression and apoptosis were positively correlated by relativity analysis. There was no significant correlation between Fas expression and apoptosis in LoVo cells. Conclusions:We could find out hypersensitive drugs and their appropriate dosage according to apoptosis, partly according to Fas expression. The different apoptosis paths between LS174T cells and LoVo cells demonstrate the importance of individualized selection of chemotherapeutic drugs.

Objective To study the effect of antivirus therapy of HBV reinfection and YMDD mutation after liver transplantation. Methods Fifteen of 317 patients with HBV-related end-stage liver diseases received lamivudine ( LAM ) monothereapy, others received combination low-dose hepatitis B immune globulin( HBIG) and LAM (or adefovir dipivoxil, ADV) therapy, as prophylaxis against HBV reinfection after OLT. Hepatitis serum markers, HBsAg, HBeAg, HBcAb-IgM, and HBcAg were detected every 2 weeks by immunohistochemistry. Serum HBV DNA was examined by PCR every 2 weeks. HBsAg and HBcAg in the liver specimens were examined by immunohistochemistry. YMDD mutation was detected by PCR in those patients with recurrence of positive HBV DNA posttransplantation. Results In LAM monotherapy group, 4 developed HBV reinfection out of 15 patients with pretreatment positve HBV DNA. Sixteen of 302 patients with combination HBIG and LAM therapy suffered from posttransplant HBV reinfection, the difference between the two groups was significant (26.7% vs. 5. 30% ,P

Objective To investigate molecular cytogenetic abnormalities in chronic lymphocytic leukemia and clinic prognostic significance. Methods Conventional cytogenetics (CC) examination was performed in 17 cases with CLL by I-FISH with five probes [DI3S25(13q14.3), ATM(11q22.3), RB1(13q14), p53(17p13.1) and CSP12(12p11.1-12q11.1)]to detect molecular cytogenetic abnormalities in CLL. Results Among 17 cases of CLL, by CC examination, only 18.75 % patient were found to have chromosomal abnormalities;whereas on I-FISH, 70.6 % patient were found to have molecular cytogenetic abnormalities including 13q-(47.1%) del(RB1) (23.5 %), del(13q13.4)(29.4 %), trisomy 12 (29.4%), del(17p13.1)(11.8 %), del (ATM)(5.6 %), the frequency of complex abnormalities were 11.8 %. No correlation of molecular cytogenetic abnormalities with sex, age, Binet stage, LDH and β_2-MG were found. Conclusion I-FISH is a more rapid, accurate and sensitive technique for detection of molecular cytogenetic abnormalities in CLL than CC, There was no statistically significant difference between molecular cytogenetic abnormalities and clinic characteristics, but its prognostic significance in CLL needs to be further investigated.

Objective To investigate the characters of morphology,immunology and cytogenetics of adult acute leukemia (AL) in different ages. Methods 172 cases of newly diagnosed adult AL were divided into two groups:the non-aged group (age＜60 years) and the aged group ( age≥60 years). Morphology,immunology and cytogenetics between the two groups were compared. Results The incidence of M3 in aged AL was significantly lower than that in non-aged AL[6.0 %(3/50) vs 18.9 %(23/122),P ＜0.05]. The incidence of hypo-or extremely hypo-cellular AL in aged AL was significantly higher than that in non-aged AL[14.0 %(7/50) vs 4.1 %(5/122),P ＜0.05],but the incidence of hyper-or extremely hyper-cellular was significantly lower than that in non-aged AL[52.0 %(26/50) vs 73.8 %(90/122),P ＜0.05]. Among aged acute myeloid leukemia (AML),the incidence of lymphoid antigen positive AML (Ly+AML) was significantly higher than that in non-aged AML[63.4 %(26/41) vs 41.3 %(38/92),P ＜0.05]. The incidence of adverse karyotypes in aged AML was significantly higher than that in non-aged AML[33.3 %(11/33) vs 14.1 %(10/71),P ＜0.05].Conclusion Age is an important prognostic factor in AL. Generally,aged AL has poorer prognosis than nonaged AL.