Expert Consensus on Rheumatic Immune Diseases Responding Specifically to Traditional Chinese Medicine / 中国实验方剂学杂志

In the clinical practice of rheumatic immune diseases in traditional Chinese medicine （TCM），it`s still unclear about the dominant diseases and breakthrough points. It`s urgent missions to formulate TCM diagnosis and treatment guidelines widely recognized and integrated by traditional Chinese medicine and Western medicine. In order to clarify the dominant diseases and breakthrough points in rheumatism，China association of Chinese medicine initiated a research group covering experts in the field of rheumatism of traditional Chinese medicine and Western medicine. Based on questionnaire survey and on-site discussion，experts had reached the following consensus. Evidence-based medicine research using modern medical methods and scientific methods should be carried out to provide objective clinical evidences. "Four mutuality" were put forward as the basis for the work of integrated traditional Chinese and Western medicine，that is the mutual communication using the exchangeable context，the mutual explanation using common theories，the mutual certification using common standards，and the mutual integration using common means. Key works should focus on solving refractory rheumatism in the future. In terms of dominant diseases and breakthrough points，this paper introduces 21 breakthrough points in 6 dominant diseases，including rheumatoid arthritis，ankylosing spondylitis，Sjogren's syndrome，hyperuricemia and gout，systemic lupus erythematosus and fibromyalgia syndrome. Advice on this discussion can provide valuable references for developing the treatment scheme of rheumatism with TCM and integrated Chinese and Western medicine and clinical practice and scientific research.

Influence of Rho kinase inhibitor combined furosemide and spironolactone on cardiac function , serum levels of AST , LDH and CK‐MB in patients with acute left heart failure／ / 心血管康复医学杂志

To explore application value of Rho kinase inhibitor (RKI) combined furosemide and spironolactone in patients with acute left heart failure (ALHF).Methods : A total of 94 ALHF patients were randomly and equally divided into diuretic group (received furosemide and spironolactone based on routine treatment ) and triple therapy group (received RKI‐‐fasudil hydrochloride based on diuretic group ) , both groups were continuously treated for 7d.LVESV , LVEDV , LVEF ,serum levels of aspartate transaminase (AST) , lactate dehydrogenase (LDH) and creatine kinase isoenzyme MB (CK‐MB) before and after treatment , therapeutic effects were observed and compared between two groups .Results : Total effective rate of triple therapy group was significantly higher than that of diuretic group (95.75% vs.82.98%) , P＝0.045. Compared with before treatment , there was significant rise in LVEF , and significant reductions in LVESV , LV‐EDV ,serum levels of AST , LDH and CK‐MB in two groups after treatment , P＝0.001 all ;compared with diuretic group after treatment , there was significant rise in LVEF [ (48.27 ± 5.95)% vs.(55.14 ± 6.74)%] , and significant reductions in LVESV [ (86.29 ± 10.41) ml vs.(65.96 ± 9.84) ml] , LVEDV [ (133.71 ± 13.42) ml vs.(120.35 ± 11.25) ml] , serum levels of AST [ (81.23 ± 10.44) U／L vs.(57.58 ± 8.42) U／L] , LDH [ (184.24 ± 13.51) U／Lvs.(124.65 ± 12.42) U／L] and CK‐MB [ (187.84 ± 13.45) U／L vs.(132.54 ± 11.69) U／L] in triple therapy group , P＝0.001 all. There was no significant difference in adverse reactions during treatment between two groups , P＞0.05 both .Conclusion :Rho kinase inhibitor combined furosemide and spironolactone can significantly improve cardiac function and reduce myocar ‐dial damage , and it＇s safe and reliable , which is worth extending .

Protective Effect of Norcantharidin on Collagen-Induced Arthritis Rats / 中国结合医学杂志

<p><b>OBJECTIVE</b>To observe the effect of norcantharidin (NCTD) on collagen-induced arthritis (CIA) rats.</p><p><b>METHODS</b>Sixty Sprague-Dawley(SD) rats were randomly divided into 6 groups (n=10): normal group, CIA model group(model group), NCTD low-dose group [1.35 mg/(kg•d)], NCTD middle-dose group [2.7 mg/(kg•d)], NCTD high-dose group [5.4 mg/(kg•d)] and methotrexate (MTX) group [1.8 mg/(kg/w)]. Anesthetized rats were sacrificed by luxation of cervical vertebra after 4 weeks of administration. The arthritis scores were evaluated twice a week. The pathological changes in the ankle joints of rats were observed by hematoxylin-eosin (H&E) staining. The serum levels of interleukin (IL) 1β, IL-6, tumor necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF), IL-17 and transform growth factor (TGF) β were detected by enzyme linked immunosorbent assay (ELISA). The mRNA expression of retinoid-related orphan nuclear receptorγt (RORγt) and forkhead box P3 (Foxp3) in peripheral blood lymphocytes were confirmed by real-time polymerase chain reaction.</p><p><b>RESULTS</b>MTX and high-dose NCTD not only decreased the arthritis scores but also alleviated the pathological changes in CIA rats' ankle joints compared with the model group (P<0.05 or P<0.01). All doses of NCTD significantly inhibited the serum levels of IL-6, IL-17 and TNF-α in CIA rats (P<0.05). Only middle- and high-dose of NCTD prominently decreased serum IL-1β and TGF-β levels of CIA rats (P<0.05). However, NCTD has no effect on vascular endothelial growth factor (VEGF) level in CIA rats. The Foxp3 mRNA expression in all NCTD groups were increased significantly than in the model group (P<0.05). The mRNA expression of RORγt in NCTD high-dose group was decreased apparently in comparison with the model group (P<0.05).</p><p><b>CONCLUSIONS</b>NCTD showed therapeutic effect on CIA rats by inhibition of cytokines and regulation of Th17/Treg cells.</p>

Role of miR137-MITF in Prognostic Analysis of Multiple Myeloma / 中国实验血液学杂志

<p><b>UNLABELLED</b>Objectiive：To explore the effect of miR137 target gene MITF on the prognosis of multiple myeloma (MM).</p><p><b>METHODS</b>The target genes of miR137 were predicted by software, the GFP analysis was carried out for detecting MITF as the prognosis of multiple myeloma. The cell line overexpressing miR137 in MM cell line was constructed. Real-time qPCR and Western blot were used to detect the expression of MITF in this cell line.</p><p><b>RESULTS</b>The target genes of miR137 were MITF, BUE2H, SH3BP5 and KLF12. High expression of MITF in MM patients showed a good prognosis according to GFP analysis, but no significant difference was detected between the different subgroups. MITF expression was higher in MM cell line that over expressed miR137.</p><p><b>CONCLUSION</b>The miR137-MITF is an important index in judging the prognosis of multiple myeloma.</p>

Expression of B7-H1 gene in leukemia cells and its clinical significance / 中国实验血液学杂志

This study was purposed to investigate the expression of B7-H1 gene in leukemia cells and its clinical significance. The expression of B7-H1 mRNA was detected by SYBR Green I real-time quantitative PCR in a panel of 9 leukemia cell lines, 4 leukemia cell lines induced with IFN-γ, the bone marrow mononuclear cells (BMMNC) from 59 initial leukemia patients and 10 dendritic cells (DC) derived from BMMNC of initial leukemia patients, 2 solid tumour cell lines and BMMNC from 10 normal persons. The correlation between the clinical features of 59 acute leukemia patients and the expression level of B7-H1 mRNA in leukemia cells was analyzed. The results showed that the lower level of B7-H1 mRNA expression was found in leukemia cell lines and primary acute leukemia cells, but the expression level of B7-H1 mRNA was up-regulated significantly in the leukemia cell lines induced by IFN-γ and DC derived from BMMNC of leukemia patients. The expression level of B7-H1 mRNA in non complete remission (CR) patients after therapy was significantly higher than that in CR patients. It is concluded that the expression level of B7-H1 mRNA in leukemia cells is lower, but is up-regulated when affected by some factors. A correlation exists between the expression level of B7-H1 gene in leukemia cells and response of patients to therapy.

Expression of SOX11 mRNA in mantle cell lymphoma and its clinical significance / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression level of SOX11 mRNA in mantle cell lymphoma (MCL) and other B-cell non-Hodgkin lymphoma (B-NHL) and its prognostic value in MCL.</p><p><b>METHODS</b>The expression level of SOX11 mRNA in 80 B-NHL patients were determined by real-time quantitative RT-PCR, GAPDH was used as internal control. The dispersion of SOX11 expression ratio of groups with different prognostic factors was described by Mann-Whitney U test.</p><p><b>RESULTS</b>The SOX11 mRNA expression level was 2.90 (0.75 - 4.63) in 80 B-NHL patients, and the expression level was significantly higher in MCL than that in other B-NHL (P = 0.014). The SOX11 expression level was statistically lower in the group of MCL with hyperleukocytosis, 12 trisomy, MYC amplification and therapeutic effect < PR (P = 0.042, 0.013, 0.028, 0.009) than that of MCL in other group. But SOX11 expression was not associated with MCL international prognostic index (MIPI) (P = 0.333), lactate dehydrogenase (LDH) (P = 0.790), ATM mutation (P = 0.865) and P53 deletion (P = 0.116). The progression free survival (PFS) and overall survival (OS) were significantly longer in the MCL patients with high level of SOX11 than that of other MCL patients.</p><p><b>CONCLUSION</b>There was statistically significant differences in SOX11 mRNA expression between MCL with other B-NHL. SOX11 maybe a good prognostic factor in MCL.</p>

Overrepresentation of specific gene segments of expressed immunoglobulin heavy chain variable region among unmutated and mutated patients with chronic lymphocytic leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the overrepresentation of specific gene segments of immunoglobulin heavy chain variable region (IgVH) among unmutated and mutated chronic lymphocytic leukemia (CLL) patients and its prognostic implication.</p><p><b>METHODS</b>Multiplex PCR was used to identify the expression of IgVH segment and its mutation status in CLL.</p><p><b>RESULTS</b>Analyses were successfully performed in 80 of 85 samples. Marked skewed IgVH families were disclosed. The most commonly used VH was VH3 (40.0%), followed by VH4 (30.0%), VHI (13.8%), VH2 (10.0%) and VH5, VH7 (2.5%). Fifty-six patients (70.0%) had mutated VH, 24 (30.0%) unmutated VH. Nine cases (11.3%) were with 100% germline sequence. Fifteen cases (15/24, 62.5%) in VH4, 29 (29/32, 90.7%) in VH3, and 4 (4/11, 36.3%) in VH1 had mutated VH. The most frequently used IgVH gene was VH4-39 (13.8%), and VH4-34 (8.8%). J4 (36/66, 54.5%) and D3 (25/66, 37.8%) were the most frequently used in J and D genes. The progression-free survival (PFS) was 82 and 17 months (P = 0.000), and the overall survival (OS) was 90 and 41 months (P = 0.009), respectively, for mutated and unmutated cases. Recurrent CDR3 sequences were found in our patients and 2 patients with VH1-69 had CDR3 sequences highly similar to those reported in literature.</p><p><b>CONCLUSION</b>There is difference in IgVH gene segment usage and mutational status in different area CLL patients. Recurrent CDR3 sequences were found in specific IgVH gene segments, which highlights the importance of immunoglobulin mediated stimulation in the development of CLL.</p>

Influence of siRNA interfering β-catenin on tumorigenicity in vivo of K562 cell line / 中国实验血液学杂志

The aim of this study was to investigate the effects of β-catenin on the tumorigenicity of K562 cells in vivo. The β-catenin expression in K562 cells was down-regulated through sequence-specific siRNA, and the treated K562 cells were implanted into BALB/c nude mouse subcutaneously. And the tumor-forming rate and tumor-forming curve (interference group) were observed. Experiments were divided into 3 group: interference group (implanted K562 cells transfected with β-catenin interfering plasmid DNA), control group (implanted K562 cells transfected with unrelated sequence plasmid DNA) and untreated group (implanted K562 cells transfected without plasmid DNA). The results indicated that the tumor-forming rates of untreated group (n = 9), control group (n = 8) and interference group (n = 9) were 100%, 87.5% and 0% respectively. The tumor-forming rate of interference group was significantly lower than those of the other 2 groups (p < 0.001). Comparison of the tumor-forming curve between 3 groups, showed that in first 2 groups existed tumor-forming and their final tumor volumes were almost the same, but the tumor growth of untreated group was faster than that in control group; while in the interference group there was not tumor-forming. It is concluded that the β-catenin expression level in K562 cells is down-regulated through the interference of sequence-specific siRNA, thus affecting their tumor-forming potential in vivo.

Effect of human umbilical cord mesenchymal stem cells on the CD34+ cells transplantation in NOD/SCID mice / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the effect of human umbilical blood (UB) mesenchymal stem cells (MSC) on the CD34(+) cells transplantation in NOD/SCID Mice.</p><p><b>METHODS</b>Umbilical blood CD34(+) cells (3.5 x 10(5) cells) alone or combined with umbilical cord MSC cells were transplanted into NOD/SCID mice that had been irradiated with (137)Cs (3.0 Gy) before transplantation. Changes in peripheral blood cells within 6 post-transplantation weeks were detected. The mice were sacrificed 6 weeks after transplantation. The human hematopoietic cells (hCD45(+)) and multi-lineage engraftment cells (CD3/CD19, CD33, CD14, CD61, and CD235a) in NOD/SCID recipients bone marrow, spleen, and peripheral blood were analyzed by flow cytometry.</p><p><b>RESULTS</b>In the 3rd post-transplantation week, white blood cells (WBC), platelets (PLT), and red blood cells (RBC) began to increase in both two groups. In the 6th post-transplantation week, WBC and PLT counts in CD34(+) + MSC group reached peak levels and were significantly higher than CD34(+) alone group (P < 0.05), while RBC level was not significantly different between these two groups P > 0.05). hCD45(+) cell levels in bone marrow and peripheral blood were (42.66 +/- 2.57) % and (4.74 +/- 1.02) % in CD34(+) + MSC group, which were significantly higher than those in CD34(+) alone group [(25.27 +/- 1.67) % and (1.19 +/- 0.54) %, respectively, P = 0.006]. Also in the 6th post-transplantation week, the proportions of CD19(+), CD33(+), CD14(+), CD61(+), and CD235a(+) in CD34(+) + MSC group were significantly higher than those in CD34(+) alone group (P < 0.05), while the proportion of CD3(+) T lymphocyte in CD34(+) + MSC group was significantly lower than that in CD34(+) alone group (P = 0.003). The amplification of CD19(+) B lymphocyte was significantly higher than other blood cell lineages (P < 0.05).</p><p><b>CONCLUSION</b>The co-transplantation of MSC cells and CD34(+) cells can promote hematopoietic stem cell transplantation and hematopoietic recovery in vivo.</p>

Comparative study of genetic aberrations in human multiple myeloma cell lines and newly diagnosed MM by fluorescence in situ hybridization / 中国实验血液学杂志

Multiple myeloma (MM) is a neoplasm of a terminally differentiated B-cell. Human myeloma cell lines were shown to be suitable model systems for use in various fields of the biological sciences. This study was aimed to investigate the genetic aberrations in human multiple myeloma cell lines. Interphase fluorescence in situ hybridization (FISH) with probes for the regions containing 13q14 (RB-1), 13q14.3 (D13S19), 14q32 (IGHC/IGHV) , 1q12 (CEP1), 17p13 (TP53) were was used to detect 7 HMCL and 85 cases of newly diagnosed MM. FISH with LSI IGH/CCND1 , LSI IGH/FGFR3 and LSI IGH/MAF probes were used to detect t(11;14) (q13;q32) , t(4;14) (p16;q32) and t(14;16) (q32;q23) in HMCL and MM with 14q32 rearrangement. The results showed that molecular cytogenetic aberrations were found in all 7 HMCL, six (85.7%) HMCL simultaneously had 13q14, 13q14.3 deletion. Chromosome 1q21 abnormality was found in six (33.3%) HMCL with at least 3 copies amplifications. Illegitimate 14q32 rearrangement was found in five (71.4%) HMCL, including one with t(11;14), two with t(4;14) and three with t(14;16). 17p13 deletion was detected in 5 HMCL. Chromosomal changes were observed in 85.9% of the 85 cases of newly diagnosed MM. The del(13), 1q12 amplification, del(17p), 14q32 rearrangement, t(11;14), t(4;14), t(14;16) were present in 44.7%, 52.9%, 20%, 62.4%, 27.1%, 24.7% and 3.5% of the patients respectively. There was no significant difference in the prevalence of genetic abnormalities of del(13q), 14q32 rearrangement, 1q12 amplification, t(11;14), t(4;14) except del(17p) and t(14;16). It is concluded that HMCL representative of the most aggressive phase of plasma cell neoplasms accumulated a large amount of genetic aberrations. Loss of p53 are strikingly common in HMCL suggesting that the impairment of the P53 tumor suppressor pathway is an important contributor to extramedullary tumor expansion.

Effect of mesenchymal stem cells on multiple myeloma cells growth and inhibition of bortezomib induced cell apoptosis / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the role of mesenchymal stem cells (BMSCs) in multiple myeloma (MM) bone marrow (BM) microenrivonment and their effect on myeloma cells survival and bortezomib induced apoptosis.</p><p><b>METHODS</b>BMSCs were derived from BM of untreated myeloma patients (MM-BMSCs) and healthy donors (HD-BMSCs), respectively. The phenotype, proliferation time and cytokine secretion of MM-BMSCs were detected and compared with HD-BMSCs. Then BMSCs were co-cultured with myeloma cell line NCI-H929 and bortezomib in vitro. The NCI-H929 cells proliferation and bortezomib induced cell apoptosis were investigated.</p><p><b>RESULTS</b>MM-BMSCs and HD-BMSCs were isolated successfully. The phenotype of MM-BMSCs was similar to that of HD-BMSCs. Expressions of CD73, CD105, CD44 and CD29 were positive, but those of CD31, CD34, CD45 and HLA-DR (< 1%) negative. The proliferation time of MM-BMSCs was longer than that of HD-BMSCs (82 h vs 62 h, P < 0.05). Moreover, over-expressions of IL-6 and VEGF in MM-BMSCs culture supernatant were detected as compared with that in HD-BMSCs [(188.8 ± 9.4) pg/ml vs (115.0 ± 15.1) pg/ml and (1497.2 ± 39.7) pg/ml vs (1329.0 ± 21.1) pg/ml, respectively]. MM- BMSCs supported survival of the myeloma cells NCI-H929 and protected them from bortezomib induced cell apoptosis.</p><p><b>CONCLUSIONS</b>MM-BMSCs is benefit for myeloma cells proliferation and against cell apoptosis induced by bortezomib. Over-expression of IL-6 and VEGF maybe play a critical role in these effects.</p>

Study of influence of umbilical cord mesenchymal stem cells on CD34+ cells in vivo homing in NOD/SCID / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect and the potential mechanism of umbilical cord (UC) derived mesenchymal stem cells (MSCs) on umbilical cord blood (UCB) derived CD34+ cells in vivo homing in xenotransplanted NOD/SCID mice model.</p><p><b>METHODS</b>CD34+ cells and MSCs were derived from fresh UCB and UC, respectively. CD34+ cells (5 x 10(5) per mice) and MSC cells (5 x 10(6) per mice) were co-transplanted into irradiated NOD/SCID mice intravenously. CD34+ cells (5 x 10(5) per mice) alone were transplanted into the mice as control group. CD34+ cells home in bone marrow and spleen of recipient mice were detected 20 hours after transplant by FACS and RT-PCR, and the homing efficiencies were calculated. The effect of MSCs on CD34+ cells chemotactic function was investigated after co-cultured UCB CD34+ cells with UC MSCs in vitro. After 4 and 7 days coculture, the homing related adhesion molecules (the CD49e, CD31, CD62L, CD11a) expressed on CD34+ cells were detected by FACS.</p><p><b>RESULTS</b>1) The homing efficiencies in bone marrow in experimental and control group were (7.2 +/- 1.1)% and (5.4 +/- 0.9)%, respectively (P < 0.05). 2) Human GAPDH gene was detected in bone marrow in experimental group and in spleen in both groups. 3) The migration efficiency of CD34+ cells was significantly higher in experimental group (35.7 +/- 5.8)% than in control group (3.5 +/- 0.6)% (P < 0.05). 4) The expression of CD49e, CD31, CD62L on CD34+ cells kept higher level in MSCs cocultured group than in CD34+ cells alone group.</p><p><b>CONCLUSIONS</b>MSCs can efficiently increase homing of CD34+ cells to bone marrow and spleen in vivo by keeping a high level of homing adhesion molecules expression and improving migration efficiency of UCB CD34+ cells.</p>

Analysis of relationship between bcr-abl transcription level detected by real-time quantitative polymerase chain reaction and clinical status of CML patients / 中国实验血液学杂志

This study was aimed to investigate the bcr-abl transcription level and its relationship with the clinical status of patients so as to provide some bases for predicting patient status according to absolute value of bcr-abl transcript. The bcr-abl/abl values (%) of bone marrow samples from 30 newly diagnosed CML patients at the baseline bcr-abl/abl value obtained in CML patients with bcr-abl positive were defined, then 161 bone marrow samples from 82 patients were detected at virions time points, and the bcr-abl/abl value of each sample was compared with baseline value and its relationship with clinical status of patient at same time point was investigated. The results showed that bcr-abl/abl values (%) of 30 patients showed positive skew distribution and a large variation with mean 13.5631 (1.0206 - 98.3159) and mathematical mean of 21.1491 (95% CI: 12.3532 - 29.9450). For strict standard, the baseline value of bcr-abl/abl (%) was set as 1, the lower limit of these values. In the detected results of 161 samples, there were 33 samples' values above the baseline value, in which resistance/relapse/progression (R/R/P) 13 (39.4%, 13/33), no remission (NR) 17 (51.5%, 17/33) and complete hematologic remission (CHR) 3 (9.1%, 3/33) were observed. the values of 26 samples decreased by 0 - 1 order of magnitude (0.1 < or = bcr-abl/abl % < 1), in which R/R/P 6 (23.1%, 6/26), NR 7 (26.9%, 7/26), CHR 7 (26.9%, 7/26) and cytogenetic remission (CyR) 6 (23.1%, 6/26) were observed, the values of 19 samples decreased by 1 - 2 order of magnitude (0.01 < or = bcr-abl/abl % < 0.1), in which NR 2 (10.5%, 2/19), CHR 3 (15.8%, 3/19) and CyR 14 (73.7%, 14/19) were determined. 7 samples decreased by 2 - 3 order of magnitude (0.001 < or = bcr-abl/abl % < 0.01) in which major CyR (MCyR) 2 (28.6%, 2/7) and complete CyR (CCyR) 5 (71.4%, 5/7) were determined, the values of 76 samples decreased by 3 or more order of magnitude (bcr-abl/abl % < 0.001), and all these were CCyR. In conclusion, the using decrease degree of one time point-detected value compared to the baseline could well assess the patient clinical status. The bcr-abl/abl % < 0.01 can reliably reflect CyR obtained by patients at the time point, and bcr-abl/abl % < 0.001 can reflect CCyR obtained by patients. However, exact judgments of patient status relies on dynamic and serial monitoring.

Influence of mesenchymal stem cells on UCB CD34+ cell expansion and characteristics / 中国实验血液学杂志

The aim of this study was to investigate the support effects of mesenchymal stem cells (MSCs) on umbilical cord blood (UCB) CD34+ cell (HSPC) expansion in vitro and its influence on cell characteristics including the surface marker of CD34+ cells, homing adhesion molecules and colony-forming ability. The mononucleated cells (MNCs) were isolated from UCB, then the CD34+ cells were isolated from freshly obtained MNCs by immunomagnetic beads, the MSC feeder cells exposed to gamma-ray of 137Cs were prepared by MSC feeder. The CD34+ cells were inoculated in different culture media. Experiment was divided into 3 groups: HSPC+CK group in which cytokines were added to medium (SCF, FL and TPO); HSPC+MSC group in which CD34+ cells were inoculated on MSC feeder; HSPC+MSC+CK group in which cytokines and MSC feeder cells were added to medium. After culture for 4, 7, 10, 14 days the MNC amount was counted and expansion ability of CD34+ cells was evaluated. The immunotypes of CD34+ cells and subsets, homing adhesion molecules and colony-forming ability in different groups detected by flow cytometry. The results showed that the amount of MNCs and CD34+ cells all obviously increased during culture for 14 days, the expansion levels of MNCs in 3 groups were HSPC+MSC+CK group>HSPC+CK group>HSPC+MSC group in proper order. Within 10 days of expansion in vitro amount of MNCs obtained significant expansion, meantime the expansion of CD34+ cells was higher also. The CD34+ count in 3 groups at day 4 of culture decreased significantly as compared with 0 day of culture (p<0.01). The CD34+ cells ratios in 3 groups after expansion were HSPC+MSC group>HSPC+MSC+CK group>HSPC+CK group in proper order (p<0.01), while CD34+ subset levels in 3 groups were different, the CD34+CD38- cells in HSPC+CK group at 4 days of culture increased transiently (62.71%), then quickly decreased, the CD34+CD38- cell ratio at day 7 was 0.05%, while the CD34+CD38- cell ratio in HSPC+MSC group at day 7 was 18.92%, difference was significant as compared with HSPC+CK group (p<0.05). The analysis of colony-forming units showed that the colony-forming ability at various time points after expansion all sustained in high level. It is concluded that in short-time (<7 day) culture of UCB CD34+ cells the combination of MSCs with cytokines can significantly expand the CD34+ cells and make the HSPCs to maintain original biologic characteristics.

Effects of beta-catenin-specific siRNA interference on Jurkat and K562 cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To inhibit the expression of beta-catenin and investigate the effect of the beta-catenin gene on Jurkat and K562 cells.</p><p><b>METHODS</b>siRNA specifically knocking down the expression of beta-catenin was used to testify the function of beta-catenin in Jurkat and K562 cells. Real time polymerase chain reaction and Western blot were performed respectively to testify the mRNA level and protein level of beta-catenin. Growth curve was determined by counting viable cells using trypan blue refusal-dyed method. The proliferation of cells was assayed by clonogenic counting and MTT method. The apoptotic cells were measured by Annexin V/PI staining. The cell cycle analysis was performed based on propidium iodide staining.</p><p><b>RESULTS</b>Compared with the control group (transfected with siRNA directed against scramble gene), the survival, colonogenicity, and proliferation of the Jurkat and K562 cells were significantly decreased in experimental group transfected with beta-catenin siRNA. The colonogenicity was decreased from 31.9 +/- 5.55 (siRNA) to 25.0 +/- 5.13 (control) in Jurkat cells, and from 47.33 +/- 8.52 (siRNA) to 39.33 +/- 6.26 (control) in K562 cells (both P <0.05). The inhibition rate was (49.3 +/- 9.86)% (siRNA) and (15.1 +/- 6.55)% (control) respectively in Jurkat cells, and (39.4 +/- 7.56)% (siRNA) and (10.1 +/- 6.89)% (control) in K562 cells (both P <0.05). In addition, the apoptotic rate increased from (23.5 +/- 2.82)% (control group) to (55.9 +/- 2.22)% (experiment group) in Jurkat cells and from (14.9 +/- 8.54)% (control group) to (27.9 +/- 15.3)% (experiment group) in K562 cells. However, cell cycle analysis revealed no obvious phases change both in Jurkat and in K562 cells.</p><p><b>CONCLUSION</b>Knock-down of beta-catenin gene may decrease the proliferation, survival, and clonogenicity in Jurkat cells and K562 cells.</p>

Effect of shRNA targeted to beta-catenin on K562 cell growth / 中国实验血液学杂志

In order to investigate the effect of shRNA targeted to beta-catenin on the growth of K562 cells, plasmid containing beta-catenin specific shRNA sequence was transfected into K562 cells by lipofectamine 2000, and G418 was added to screen the positive cells. Real-time PCR and Western blot were used to detect the expression of beta-catenin. Cell growth curve, MTT and colony forming cell assays were used to evaluate the proliferation potential of cells. The results showed that the mRNA level of beta-catenin was reduced significantly in K562 cells transfected into interfering plasmid as compared with control plasmid, while the protein level failed to demonstrate difference by the time of 72 hours after transfection. After long-term culture with G418, the count of positive cells enhanced in control group while no positive cells survived in the interfering group. Colony-forming cell assays revealed that the K562 cells in interfering group formed colonies with very small size and low forming rate, compared with the control group, though the growth curve and MTT failed to illustrate differences. It is concluded that the beta-catenin-specific shRNA mediated by plasmid can effectively knockdown the expression of beta-catenin gene and inhibit the colony-forming ability in K562 cells, it is a potential target for the therapy of CML, even in blast crisis.

The expression of beta-catenin and its significance in leukemia cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression of beta-catenin in patients with leukemia and explore its significance in leukemias.</p><p><b>METHODS</b>RT-PCR was used to detect the expression of beta-catenin in bone marrow mononuclear cells (BMMNCs) from patients with leukemia. Immunocytochemistry was in some of patients to detect the distribution of beta-catenin at the same time. The clinical significance of beta-catenin was analyzed in combination with patients' clinical information.</p><p><b>RESULTS</b>Expression of beta-catenin was statistically higher in acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) samples than in normal donors (P = 0.001 and 0.016 respectively) and chronic phase chronic myeloid leukemia (CML) patients (P = 0.001 and P = 0.008 respectively), while there was no statistic difference between AML and ALL patients (P = 0.58). In addition, beta-catenin expression in chronic phase CML patients was like that in normal donors (P = 0.49), but increased significantly in blast crisis and accelerated phase. Immunocytochemical analysis revealed that BMMNCs from normal donors expressed beta-catenin on the plasma membrane and cytoplasma, while those from acute leukemia expressed beta-catenin to varying degrees in the nucleus as well. The expression of beta-catenin gene statistically showed the highest level in M5 (n = 15) and the lowest level in M3 (n = 18). No clinical features, such as, age, initial WBC count, therapy response rate, blast cell numbers or cytogenetic risk was found to be correlated with the expression of beta-catenin excepting for CD34+ positive rate (P = 0.004) in AML.</p><p><b>CONCLUSION</b>As a key mediator of Wnt signal transduction way, overexpression of beta-catenin in leukemia cells indicates that it might be aberrantly activated in acute leukemia, accelerated or blastic phase of CML.</p>

Expression of beta-Catenin Gene in CML and its relationship with bcr/abl / 中国实验血液学杂志

This study was aimed to quantitatively detect the expression level of beta-catenin and bcr/abl in different phases of chronic myeloid leukemia (CML) and to analyze their potential relationship and significance in the progression of CML. First, the total RNA isolated from BMMNC of patients with CML and donors was reversely transcribed into cDNA. The real-time quantitative PCR method was used to analyze the expression level of beta-catenin and bcr/abl. The expression level of beta-catenin and bcr/abl in different phases of CML was compared and the correlation was analyzed between the two genes. The results showed that the beta-catenin gene in BMMNC of blast crisis of CML patients was expressed significantly higher than that in chronic phase (p < 0.001) and accelerated phase (p = 0.016) of CML patients and in normal donors (p = 0.004). The expression of bcr/abl in blast crisis of CML was statistically higher than that in chronic phase of CML (p = 0.001). The expression levels of beta-catenin and bcr/abl were correlated with each other in CML patients (r = 0.620, p < 0.001). It is concluded that the beta-catenin gene in blast crisis of CML patients express higher than that in chronic phase and accelerated phase of CML, and its expression level is correlated with the level of bcr/abl expression. The increased expression of beta-catenin may be account partly for the blast crisis of CML.

Related factors affecting the isolation of multipotent non-hematopoietic adult stem cells from umbilical cord blood / 中国实验血液学杂志

To investigate the related factors affecting the isolation of multipotent non-hematopoietic adult stem cells (MNASCs) from human umbilical cord blood in low serum (2%) condition, the isolation conditions were optimized and the yield of MNASCs was improved. MNASCs from human umbilical cord blood samples were isolated, and the effects of medium component, medium exchange time and initial plating density for isolation of MNASCs were studied. Then, the MNASCs were isolated and cultured in optimal condition, the surface antigen expression and differentiation potential of MNASCs were detected. The result showed that the medium of DMEM/F12 was better than IMDM and DMEM-LG for MNASCs culture in low serum condition. The optimal yield of MNASCs was obtained when mononuclear cells were cultured at a initial plating density of 1 x 10(6) cells/cm2 and the medium was exchanged to remove the nonadherent cells after 72 hours of inoculation. MNASCs isolated and cultured under the above-mentioned conditions maintained a homogenous morphology, high potential ability of expansion and differentiation. It is concluded that culture conditions with low serum defined in this study is optimal for the successful isolation and expansion of umbilical cord blood MNASCs with high numbers for subsequent cellular therapeutic approaches.

Transforming growth factor-beta1-loaded fibrin sealant promote bone marrow Mesenchymal stem cells to contract injectable tissue engineering cartilage in vivo / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the feasibility that transforming growth factor-beta1 (TGF-beta1) -loaded fibrin sealant (FS) promotes bone marrow mesenchymal stem cells (BMSCs) to create tissue engineering cartilage in vivo.</p><p><b>METHODS</b>The BMSCs were isolated from healthy human and amplified in vitro, and then induced by defined medium containing TGF-beta1 and dexamethasone. After 7 days the induced BMSCs were collected and mixed with TGF-beta1-loaded FS or FS as BMSCs+ FS-TGF-beta1 group and BMSCs+ FS experimental group. Then the mixture was injected by a needle into the dorsum of nude mice. In control group, only FS or BMSCs were injected. The tissue engineering specimens were harvested from nude mice 12 weeks later. Gross observation, average wet weight measurement, glycosaminoglycan (GAG) quantification, histology and immunohistochemistry were used to evaluate the results.</p><p><b>RESULTS</b>The BMSCs have possessed the shape and functional characters of chondrocyte when transferred to a defined medium. After injection of the mixture, the cartilage-like tissue were formed in two experimental groups. Compared with BMSC+ FS group, the specimens of BMSCs +FS-TGF-beta1 group were larger and firmer. Alcian staining showed better metachromatic matrix formation. The GAG contents were significantly higher. Immunohistochemical staining of collagen type II was stronger. However, no cartilage-like tissue was formed in two control groups.</p><p><b>CONCLUSION</b>TGF-beta1-loaded FS can promote BMSCs to contract injectable tissue engineering cartilage in vivo.</p>