ISOLATION AND INDUCTION OF RABBIT BONE MARROW MESENCHYMAL STEM CELLS TO EXPRESS CHONDROCYTIC PHENOTYPE

Objective To isolate rabbit bone marrow mesenchymal stem cells (MSCs), and observe the inducing effect of growth factors on MSCs to express chondrocytic phenotype. Methods MSCs were seperated from bone marrow of New Zealand rabbit. TGF-β 1, IGF-I, Vitamin C and dexamethasone were added into culture medium to induce proliferation and differention of MSCs. Procollagen α1(Ⅱ) mRNA in cells was detected by RT-PCR to observe the chondrogenous effect of inducing factors. ALP in culture medium was detected by automatic biochemical analyser, and lipid droplet in cells was stained by Sudan Ⅲ to clarify whether these factors given had osteogenic and adipogenic potential. Results Expression of articular cartilage specific procollagen α1 (Ⅱ)mRNA was promoted by inducing factors-TGF-β 1, IGF-I, Vitamine C and dexamethasone; elevated level of ALP in culture medium and lipid droplet in cells were also detected. Whereas ALP level was decreased and lipid stain were negative in groups without dexamethasone. Conclusion ① Expression of chondrocytic phenotype by MSCs could be induced by the synergistic action of TGF-β 1, IGF-I and Vitamine C. ② Dexmathasone had osteogenic and adipogenic potential, it should not be chosen to induce chondrogenic differention of MSCs.

Transdifferentiation of Fetal Liver-delivered Mesenchymal Stem Cells into Cardiomyocyte-like Cells

Objectives To explore the possibility to induce mesenchymal stem cells from human fetal livers （FMSCs） to differentiate along cardiac lineage and the way to obtain high rate of differentiation. Methods Cells from passage 6-9 were plated at the density of 1.5 × 10^4/cm^2 and were treated with the combination of 5-azacytine（5-aza）, retinoitic acid（RA） and Dimethylsulfoxide （DMSO） in different doses when near confluence. 24 hours later, the treatment was removed by changing into normal medium without inducers. Different culture conditions were tried, including temperature, oxygen content and medium. Results When FMSCs were treated with highdose combination （ 5-aza 50 μM ＋RA 10-1 μM ＋ DMSO 1%） and modified combination（5-aza 50 μM ＋RA 10-3 μM ＋ DMSO 0.8 %） in cardiac differentiation medium （CDM）, at 37℃ and 20% 02, the cardiac differentiation was induced. When near confluence, cells became round and tended to gather together to form ball-like structures. 3 weeks after treatment, the cells were harvested and stained with anti-desmin and cardiac troponin I antibodies, and about 40% of the cells were positively stained. No beating cells observed during observation. Conclusions FMSCs cardiac have lineage the potential to differentiate along , and the stimulus for the cardiac differentiation is different from those for MSCs from different species.

Expressions of Tissue Factor and Tissue Factor Pathway Inhibitor in Patients with Acute Graft-versus-host Disease after Allogeneic Hematopoietic Stem Cell Transplantation

This study examined the expressions of human serum tissue factor （TF） and tissue factor pathway inhibitor （TFPI） in patients with acute graft-versus-host disease （aGVHD） after allogeneic hematopoietic stem cell transplantation （allo-HSCT） and their clinical significance. The serum TF and TFPI levels were detected by ELISA in 28 allo-HSCT recipients before and after the transplanta-tion and the changes of TF and TFPI levels were dynamically monitored at different phases of the disease. No significant differences in the serum TF and TFPI levels were found in allo-HSCT recipi-ents in the absence of aGVHD or with gradeⅠaGVHD before and after the transplantation. The lev-els of serum TF and TFPI were substantially increased in the patients with gradeⅡ aGVHD at the peak of aGVHD （P〈0.05） and they were even higher in the patients with grade Ⅲ–Ⅳ aGVHD （P〈0.01）. When the conditions became stable after treatment with immunosuppressive agents, the serum TFPI level was decreased to the baseline level （P〉0.05） and the TF level was lowered but still higher than the baseline level （P〈0.05）. It was concluded that the levels of serum TF and TFPI were increased significantly in the patients with grade Ⅱ–Ⅳ aGVHD after allo-HSCT and decreased markedly after the treatment. Monitoring the levels of serum TF and TFPI in the patients with allo-HSCT is important to predict the occurrence, outcome and prognosis of aGVHD.

TEMPORAL TRENDS IN ETIOLOGY AND IN-HOSPITAL OUTCOME IN CHINESE PATIENTS WITH PERICARDIAL EFFUSION:10-YEAR EXPERIENCE OF A SINGLE CENTER

Objective To evaluate the evolution of etiology, clinical characteristics, and in-hospital outcomes of pericardial effusions in the recent decade. Methods All patients with a diagnosis of pericardial effusion during hospitalization were recruited from the Hospital Inpatient System between January 1996 and December 2005. Demographic and clinical characteristics, laboratory measurements, echocardiographic and treatment features, and in-hospital outcomes were retrospectively reviewed by using a standardized data collection form. Results One hundred and fifry-three consecutive patients were recruited. Mild, moderate and large pericardial effusion occurred in 61 （40%）, 52 （34%） and 40 （26%） patients, respectively. The most frequent etiologic diagnoses were tuberculous pericarditis （ n = 50, 33% ） , malignancy （ n = 36, 24% ） and idiopathic pericarditis （n = 35, 23% ）. Large effusions were more likely＇ associated with malignancy （P 〈 0. 01 ）. Compared to the initial 5 years （from 1996 to 2000） , the incidence of tuberculous effusion was decreased but neoplastic effusion increased significantly in the recent 5 ）,ears （from 2001 to 2005 ）. Forty-four patients underwent percardiocentesis （tuberculous in 23, neoplastic in 16, and others in 5） and 28 patients required pericardectomy （tuberculous in 11 and neoplastic in 17）. One patient with tuberculous and 3 patients with neoplastic pericardial effusion died during hospitalization. Conclusion Tuberculosis remains the major cause of pericardial effusion, but neoplastic pericardial effusions are on the rise. Pericardial drainage or pericardectomy are often required for symptomatic relief in those with malignancy-caused pericardial effusion.

Hemostatic mechanism of Jianpi Yiqi Shexue decoction(健脾益气摄血方) in treatment of immune thrombocytopenia

OBJECTIVE: To explore the early hemostatic mechanism of Jianpi Yiqi Shexue decoction(健脾益气摄血方, JYSD) in treating immune thrombocytopathy(ITP), based on the functional homeostasis of brain-intestine axis and blood neurotransmitter METHODS: Non-drug treatment cases: Healthy volunteers were selected as normal control group and compared with patients with dysfunctional uterine bleeding, gastrointestinal tumors with bleeding and ITP, to detect the changes of blood 5-hydroxytryptamine(5-HT), β-endorphin(β-EP), vasoactive intestinal peptide(VIP) and compare the changes of blood neurotransmitters in patients with different disease symptoms. Drug treatment cases: According to the randomized controlled multicenter clinical trial, 272 ITP patients were randomly divided into three groups: treatment group(JYSD) combined group(JYSD + Prednisone) control group(Prednisone). The changes of blood neurotransmitter(5-HT, β-EP, VIP) before and after treatment were detected on the basis of peripheral blood platelet(PLT) and grade score. RESULTS: Non-drug treatment cases: compared with the normal control group, the 5-HT level was higher, and the VIP and β-EP levels were both lower in the ITP group(P < 0.001), and the 5-HT, VIP and β-EP levels in the Gastrointestinal tumors with bleeding group were also lower compared with the normal control group(P < 0.05, 0.001). Drug treatment cases: The PLT grading scores of the combination group and the control group after treatment were lower than that before treatment(P < 0.05, 0.001). The PLT grading score of the 3 groups were compared in pairs after treatment: the combination group was the lowest among the 3 groups, which was better than the treatment group, but no better than the control group(vs the treatment group, P = 0.005, vs the control group, P = 0.709). The statistical results of full analysis set(FAS) and per protocol set(PPS) were consistent. The bleeding symptom scores of the treatment and combination groups began to drop 7 d after treatment, and kept dropping 14 d after treatment until the end of the study(P < 0.05). On the other hand, the control group started to show favorable results 14 d after treatment(P < 0.05). The FAS and PPS analysis results were consistent. In the control group, the 5-HT level was higher and VIP level was lower after treatment, compared with those before treatment(P < 0.05, 0.001). The β-EP levels were both increased in the treatment and combination group after treatment, compared with those before treatment(P < 0.05). After treatment, the β-EP levels in the treatment and control groups were significantly lower compared with the combination groups(P < 0.05). After treatment, compared with the control group, the VIP levels in the treatment and combination groups were upregulated, and the differences were statistically significant by rank sum test(P < 0.01), and by t-test(P = 0.0002, 0.0001). CONCLUSIONS: The prednisone tablet is better than the JYSD in increasing the level of PLT, while prednisone tablet combined with JYSD has more advantages in improving patients' peripheral blood PLT levels. However, in improving the bleeding time of ITP patients, the combination of the two drugs was significantly delayed compared with the single usage, showing the characteristics and advantages of traditional Chinese medicine. JYSD can regulate the neurotransmitter level of ITP patients through the function of the brain-gut axis, mobilize 5-HT in the blood of ITP patients to promote the contraction of blood vessels and smooth muscles, and activate the coagulation mechanism are the early hemostatic mechanisms of JYSD. Up-regulate the levels of β-EP and balancing VIP levels may be an important part of the immune mechanism of JYSD for regulating ITP patients.

Bilateral Masculine Mastoplasia Associated with Imatinib Mesylate:A Case Report and Literature Review

1 CASE REPORT In June 2009, a 29-year-old Chinese male was diagnosed as having Philadelphia chromosome-positive chronic myeloid leukemia （chronic phase）; other than a high white blood cell count in peripheral blood （WBC, 254.00×10^9/L） and splenomegaly, the patient exhibited no abnormal physical signs in mammary glands. He was given hydroxyurea for several days before he received treatment with 400 mg of imatinib mesylate daily.

Effect of transplanted mesenchymal stem cells from rats of different ages on the improvement of heart function after acute myocardial infarction

Background Mesenchymal stem cells （MSCs） transplantation is of therapeutic potential after ischemic injury in both experimental and clinical studies. Clinically, elderly patients are more vulnerable to acute myocardial infarction （AMI）. But little is known about the characteristics of young donor-derived MSCs transplanted to old patients with AMI. The present study was designed to investigate the effect of transplanted MSCs from rats of different ages on the improvement of heart function after AMI. Methods MSCs from Sprague-Dawley （SD） rats were isolated and cultured in vitro. The apoptosis characteristics of MSCs were observed under conditions of ischemia and anoxia. SD rats underwent MI received intramyocardial injection of MSCs from young donor rats （n=-8）, old donor rats （n=-8）, respectively. AMI control group received equal volume physiological saline. Immunofluorescence was used to observe the differentiation of the grafted cells into cardiomyocytes Four weeks after cell transplantation, reverse transcriptase-polymerase chain reaction （RT-PCR） and immunohistochemistry for vascular endothelial growth factor （VEGF）, VIII-factor immunohistochemistry for vessel density, TUNEL, caspase-3 for cardiomyocyte apoptosis, echocardiography and hemodynamic detection for heart function were performed. Results The apoptosis rate of the old donor-derived MSCs group was significantly higher than that of the young donor-derived MSCs group under conditions of ischemia and anoxia （P 〈0.05）. Engrafted MSCs survived, proliferated and differentiated into myocardium-like cells. VEGF gene expression and capillary density in the old donor-derived group were lower than those in the young donor-derived group but higher than those in the control group （P 〈0.05）. The transplantation of old donor-derived MSCs attenuated apoptosis of cadiomyocytes in the peri-infract region compared with the control group and the effect was elevated in young donor-derived MSCs （P 〈0.05）. The heart functions （left ventricle ejection fraction （LVEF）, left ventricle fractional shortening （LVFS）） were improved more significantly in the old donor-derived MSCs group than in the control group and the heart function in the young donor-derived MSCs group further improved （P 〈0.05）. Conclusions Young donor-derived MSCs can improve heart function significantly through angiogenesis and decreasing cardiomyocyte apoptosis when transplanted to the infarcted area.

T2 mapping in the quantitative evaluation of articular cartilage changes in children with hemophilia: A pilot study

Importance: Joint disease affects more than 90% of severe hemophiliacs. Early diagnosis is critical in preventing hemophilic arthritis. Magnetic resonance imaging (MRI) enables visualization of early arthropathic changes and plays an important role in treatment. Objective: To evaluate the role of T2 mapping in detecting early cartilage lesions in the knee and ankle joints of children with hemophilic arthropathy. Methods: Target joints of 15 male patients with clinically confirmed moderate or severe hemophilia were evaluated with MRI. In addition to routine MRI protocols (T1WI, T2_FFE, T2_SPAIR, PDW_TSE), T2 mapping was used to evaluate the articular cartilage of target joints. results: The mean T2 value of the distal femoral cartilage was 46.72 ± 10.94 ms, which is higher than the reported age-matched normal value (40.27 ± 3.50 ms). The mean T2 value of the proximal tibial cartilage was 45.60 ± 8.82 ms, which is higher than the reported normal value (31.15 ± 1.86 ms). Four examined joints (two ankles, two knees) showed normal morphology with no abnormal signal on routine MR sequences. However, T2 mapping showed locally increased T2 values in the cartilage, along with uneven color scales. Interpretation: The quantitative assessment method of T2 mapping might be helpful to early diagnosis for articular cartilage lesions. It might be a potential tool for early assessment of cartilage changes and quantification of lesion's severity for hemophilia joint.

Cytokine-induced killer cells showing multidrug resistance and remaining cytotoxic activity to tumor cells after transfected with mdr1 cDNA

Background Routine treatment of cancer such as surgery, radiation or chemotherapy is sometimes unable to erdiacate metastatic malignant cells. So we tried a new method and increased the adoptive immunotherapy of Cytokine-induced killer (CIK) cells in tumor patients and the multidrug resistance (mdr1) cDNA was transfected into CIK cells. Methods CIK cells were obtained from peripheral blood and induced by IFN-γ, anti-CD3 monoclonal antibody, IL-2 and IL-1. CIK cells were transfected with plasmid PHaMDR containing human mdr1 cDNA by electroporation. RT-PCR was used to detect mdr1 mRNA in transfected CIK cells. P-glycoprotein (P-gp) expressed on surface of CIK cells was assayed by FITC-conjugated anti-P-gp monoclonal antibody and flow cytometry. Multidrug resistance to doxorubicin and colchicine and cytotoxic activity to human breast cancer cell line MCF7 were performed using MTT method. Results mdr1 mRNA was detected in transfected CIK cells. P-gp was expressed on the surface of the transfected CIK cells, and the P-gp positive cells reached 21%-37% of the total CIK cells after transfection. The IC50 to doxorubicin increased to 22.3-45.8 times, and that to colchicines to 6.7-11.35 times, as compared to those of untransfected CIK cells. However, the cytotoxic activity to MCF7 cell line remained unaltered.Conclusions CIK cells were successfully transfected with mdr1 cDNA by using electroporation. The transfected CIK cells had the characteristics of multidrug resistance without change in their cytotoxic activity to tumor cells.

Relationship between Cytokines and Leukocytosis in Patients with APL Induced by All-Trans Retinoic Acid or Arsenic Trioxide

Leukocytosis or hyperleukocytosis occurs during ATRA or arsenic trioxide differentiation therapy, which is related to the RA syndrome. The number of WBC often increased by ten or more times as high as that of pretreatment, around 7 to 20 days after treatment with ATRA or arsenic trioxide. Usually, when number of WBC tended to peak, there was concomitance with down-regulation of promyelocytes, up-regulation of myelocytes and more mature neutrophils. The same trend of classification of BM was observed in most of the patients with APL to whom leukocytosis happened. Although the mechanism of leukocytosis has not been demonstrated clearly, so far the proliferation hypothesis by cytokines and rheological hypothesis by adhesion molecules were taken into consideration. Otherwise, hypothesis about more divisions of differentiated myelocytes induced by ATRA or arsenic trioxide remains unclear. Usually, this kind of leukocytosis or hyperleukocytosis itself requires no additional cytotoxic treatment.