Establishment of Patient-Derived Xenograft (PDX) Zebrafish Model of Multiple Myeloma and Its Application in Drug Screening / 中国实验血液学杂志

OBJECTIVE@#To establish a MM patient-derived tumor xenograft model (MM-PDX) in zebrafish, and to evaluate the anti-myeloma activity of indirubin-3'-monoxime(I3MO) using this model.@*METHODS@#Zebrafish embryos 2 days after fertilization were transplanted with fluorescence labeled myeloma primary tumor cells, the survival of primary tumor cells in zebrafish was observed at 0,16 and 24 hours after cell injection. The zebrafish embryos after tumor cell transplantation were randomly divided into control group, BTZ treatment and I3MO treatment group. Before and 24 hours after treatment with BTZ and I3MO, the positive area with calcein or Dil in zebrafish were observed under fluorescence microscope to reflect the survival of tumor cells, and it was verified.@*RESULTS@#MM patient derived tumor cells survived in zebrafish. The construction of MM-PDX was successful. Compared with control group, the fluo- rescence area of the BTZ and I3MO treatment groups in zebrafish were significantly decreased(P<0.05), and BTZ and I3MO significantly inhibited the survival of MM cells in zebrafish.@*CONCLUSION@#MM-PDX model was successfully established. Zebrafish model derived from tumor cells of MM patients can be used as a tool for drug screening of MM.

Establishment and comparison of three human multiple myeloma cell line transplantation models in mice / 中华血液学杂志

Objective: To establish three types of xenotransplantation models using human myeloma cell lines ARP1, MM.1S, and NCI-H929 and to compare the proliferation, tumor load, and biological characteristics of the three types of cells after transplantation. Methods: Suspensions of human myeloma cell lines ARP1, MM.1S, and NCI-H929 were implanted into NOD/SCID mice by subcutaneous injection or tail vein injection. The survival of the mice was observed weekly, and the tumor load was measured. Flow cytometry was used to detect the proportion of CD138(+) cells in tumor tissue or the mouse bone marrow. CD138(+) cells and light chains were detected by immunofluorescence. Light chains in bone marow and peipheral blood were measured by ELISA, and bone disease was assessed by micro-CT. Results: Mice injected with ARP1, MM.1S, and NCI-H929 cells all formed tumors subcutaneously in about 2 weeks. Immunofluorescence detection supported plasma cell tumors. Kappa light chains were detected in the peripheral blood of ARP1 mice on day 20 after tail vein transplantation (8.2±1.0 ng/ml) . After 6 weeks of tail vein transplantation, mice in the ARP1 group showed signs of weight loss, mental depression, and dragging legs, and human CD138(+)CD38(+) cells were detected in the bone marrow (BM) . Furthermore, bortezomib (BTZ) treatment given once the tumor was established significantly reduced the tumor burden[ (5.7±0.2) % vs (21.3±2.1) %, P<0.01]. Human CD138(+)CD38(+) cells were not detected in the BM of the MM.1S or NCI-H929 groups. Conclusion: The results of this study suggest that the mouse models constructed by the three cell lines (ARP1, MM.1S, and NCI-H929) can be used as models for the pathogenesis and clinical research of MM.

Progress of TP53 Family Gene TP73 and Tumorigenesis / 肿瘤防治研究

P73 protein is one of the main members of p53 protein family, and its coding gene TP73 is highly homologous to TP53 gene. On one hand, similar to p53, p73 protein is involved in all aspects of cell life. On the other hand, unlike the p53, not only p73 protein plays the role of tumor suppressor in normal cell activities and tumor development, but also the function of p73 protein is no less complex and important than that of p53 protein. Tumorigenesis involves a variety of cellular biological processes, such as apoptosis, autophagy, cell migration and cell metabolism. These normal cellular biological processes are tightly regulated by multiple cellular signaling pathways to maintain homeostasis. Once a carcinogenic abnormality occurs, it will eventually lead to tumorigenesis. Therefore, this paper mainly focuses on the important research results of p73 and tumorigenesis and its unique function different from p53 protein in recent years.

Treatment and Prognosis of Adult T Cell Acute Lymphoblastic Leukemia / 中国医学科学院学报

To analyze the treatment and prognosis of T cell acute lymphoblastic leukemia(T-ALL)in adults. Method The clinicobiogical and survival data of 68 adult patients with newly diagnosis T-ALL were retrospectively analzyed. Results The median age of these 68 patients was 23 years(14-60 years).T-ALL was more common in men(81%).After the first cycle of treatment,complete remission was achieved in 50 patients(73%).The highest complete remission(CR) rate was in patients with cortex T-ALL(100%),followed by other T-ALL(73%)and early T-cell precursor lymphoblastic leukemia(54%),(=5.712,=0.058).The CR rate for adults aged >35 years was significantly lower than that of patients aged ≤ 35 years(40% 79%,=6.364,=0.012).The overall CR rate after the second treatment course was 93%.For patients treated with chemotherapy,autograft hematopoietic stem cell transplantation(auto-SCT),and allogeneic SCT,the median relapse free survival was 10 months,24 months,and not reached,respectively(=0.002).The 5-year overall survival rate was 25% for all patients;for patients treated with chemotherapy,auto-SCT and allogeneic SCT,the median overall survival was 24 months,34 months,and 30 months,respectively(=0.007),and the 5-year overall survival rate was 9%,33%,and 38%(=0.037).Multivariate analysis showed leukocyte count ≥100×10 /L was a risk factor for decreased relapse free survival(risk ratio 2.540,95%=1.058-6.099,=0.037). Conclusion Adult T-ALL patients have poor prognosis,which may be improved by SCT.

Progress of the relationship between aberrant p53 protein and tumorigenesis / 肿瘤研究与临床

TP53 gene has been found to have the highest correlation with human tumors, and its mutations occurr in about 50% malignant tumors. Its encoded p53 protein is a well-known tumor-suppressor factor in vivo, which is closely related to tumorigenesis. It is found that tumorigenesis has a close relationship with various abnormal biological processes, including cell cycle regulation, apoptosis, DNA damage repair, cell senescence, autophagy, metabolic regulation. This paper reviews the complex network relationship between p53 protein and tumorigenesis from biological processes affecting the tumorigenesis.

The prognostic significance of minimal residual disease detection after first induction treatment in adult acute lymphoblastic leukemia patients treated with autologous stem cell transplantation / 中华血液学杂志

Objective@#To investigate the prognostic significance of detection of minimal residual disease after first induction treatment (MRD1) in adult acute lymphoblastic leukemia (ALL) patients treated with autologous stem cell transplantation (auto-HSCT).@*Methods@#The clinical data of 87 ALL patients who underwent auto-HSCT during February 2006 to April 2017 with MRD1 detection data by flow cytometry were analyzed retrospectively. The relationship between MRD1 and relapse and survival of ALL patients after auto-HSCT was studied.@*Results@#Of 87 patients, 26 (29.9%) were MRD1 positive. The proportion of high-risk immunophenotype (pro-B, pro-T, pre-T, mature T) was significantly higher in MRD1-positive patients than that in MRD1 negative patients (34.6% vs 14.5%, P=0.038). There was no significant difference between positive and negative MRD1 patients at age, sex, lineage (T/B), immunophenotype (standard risk/high risk), high white blood cell count (B-ALL>30×109/L or T-ALL>100×109/L), high-risk chromosome/gene ratio, the time from first complete remission to transplantation and pre-treatment regimen. The 5-year overall survival (OS) and leukemia-free survival (LFS) in MRD1 negative and positive patients were 72.7% vs 47.3% (P=0.004) and 75.7% vs 29.6% (P<0.001), respectively. Multivariate analysis showed that positive MRD1 was an independent risk factor for OS (HR=3.007, 95% CI 1.256-7.200, P=0.013) , and positive MRD1 and high-risk immunophenotype were risk factors for LFS (HR=3.986, 95% CI 1.813-8.764, P=0.001; HR=2.981, 95% CI 1.373-6.473, P=0.006) .@*Conclusions@#Auto-HSCT could not reverse the poor prognosis of MRD1 positive patients. Auto-HSCT treatment is optional for patients with MRD1 negative and maintaining MRD1 negative status during intensive therapy.

The prognostic significance of minimal residual disease detection after first induction treatment in adult acute lymphoblastic leukemia patients treated with autologous stem cell transplantation / 中华血液学杂志

Objective: To investigate the prognostic significance of detection of minimal residual disease after first induction treatment (MRD(1)) in adult acute lymphoblastic leukemia (ALL) patients treated with autologous stem cell transplantation (auto-HSCT). Methods: The clinical data of 87 ALL patients who underwent auto-HSCT during February 2006 to April 2017 with MRD(1) detection data by flow cytometry were analyzed retrospectively. The relationship between MRD(1) and relapse and survival of ALL patients after auto-HSCT was studied. Results: Of 87 patients, 26 (29.9%) were MRD(1) positive. The proportion of high-risk immunophenotype (pro-B, pro-T, pre-T, mature T) was significantly higher in MRD(1)-positive patients than that in MRD(1) negative patients (34.6% vs 14.5%, P=0.038). There was no significant difference between positive and negative MRD(1) patients at age, sex, lineage (T/B), immunophenotype (standard risk/high risk), high white blood cell count (B-ALL>30×10(9)/L or T-ALL>100×10(9)/L), high-risk chromosome/gene ratio, the time from first complete remission to transplantation and pre-treatment regimen. The 5-year overall survival (OS) and leukemia-free survival (LFS) in MRD(1) negative and positive patients were 72.7% vs 47.3% (P=0.004) and 75.7% vs 29.6% (P<0.001), respectively. Multivariate analysis showed that positive MRD(1) was an independent risk factor for OS (HR=3.007, 95% CI 1.256-7.200, P=0.013) , and positive MRD(1) and high-risk immunophenotype were risk factors for LFS (HR=3.986, 95% CI 1.813-8.764, P=0.001; HR=2.981, 95% CI 1.373-6.473, P=0.006) . Conclusions: Auto-HSCT could not reverse the poor prognosis of MRD(1) positive patients. Auto-HSCT treatment is optional for patients with MRD(1) negative and maintaining MRD(1) negative status during intensive therapy.

Progress of the relationship between aberrant p53 protein and tumorigenesis / 肿瘤研究与临床

TP53 gene has been found to have the highest correlation with human tumors, and its mutations occurr in about 50％ malignant tumors. Its encoded p53 protein is a well-known tumor-suppressor factor in vivo, which is closely related to tumorigenesis. It is found that tumorigenesis has a close relationship with various abnormal biological processes, including cell cycle regulation, apoptosis, DNA damage repair, cell senescence, autophagy, metabolic regulation. This paper reviews the complex network relationship between p53 protein and tumorigenesis from biological processes affecting the tumorigenesis.

Plasma cell malignancies with t(11;14): clinical analysis of 380 cases / 白血病·淋巴瘤

Objective To investigate the clinical characteristics of plasma cell malignancies with t(11;14) and the effect of t(11;14) on prognosis. Methods A cohort of 380 newly diagnosed patients with plasma cell malignancies were analyzed,including 146 females and 234 males.There were 370 cases of newly diagnosed multiple myeloma (NDMM), as well as 10 cases of primary plasma cell leukemia (PCL). The relationship between the categorical variables was evaluated by using the bilateral Fisher exact probability test, with 95 % confidence interval. Results Of 370 NDMM cases, t(11;14) was detected in 101 cases (27.3 %). Of 10 PCL cases, 8 cases displayed t(11;14). The detection rate of t(11;14) was significantly higher in IgD, IgM and non-secreting MM [50.9 % (27/53)] than that in IgA MM [21.6 % (16/78)] and IgG [28.4 % (52/183)] (both P= 0.002). The rate of CD56+in t(11;14) positive group was lower than that in t(11;14) negative group [51.6 % (48/93) vs. 72.0 % (167/232), P= 0.001], and the rate of CD117+was also significantly decreased [23.7 % (22/93) vs. 37.7 % (87/231), P= 0.019]. There were 86 cases of non-t(11;14) IgH rearrangement in 269 cases of NDMM without t(11;14), which mainly were t(4;14) or t(14;16). The detection rate of high risk MM was only 11.9 %(12/101)in t(11;14)positive group,while that rate was 27.5 % (74/269) in t(11;14) negative group, the difference was statistically significant (P = 0.001). Conclusion MM with t(11;14)displays distinct biological,clinical and laboratory features,it is a heterogeneous disease.

Plasma cell malignancies with t(11;14): clinical analysis of 380 cases / 白血病·淋巴瘤

Objective To investigate the clinical characteristics of plasma cell malignancies with t(11;14) and the effect of t(11;14) on prognosis. Methods A cohort of 380 newly diagnosed patients with plasma cell malignancies were analyzed,including 146 females and 234 males.There were 370 cases of newly diagnosed multiple myeloma (NDMM), as well as 10 cases of primary plasma cell leukemia (PCL). The relationship between the categorical variables was evaluated by using the bilateral Fisher exact probability test, with 95 % confidence interval. Results Of 370 NDMM cases, t(11;14) was detected in 101 cases (27.3 %). Of 10 PCL cases, 8 cases displayed t(11;14). The detection rate of t(11;14) was significantly higher in IgD, IgM and non-secreting MM [50.9 % (27/53)] than that in IgA MM [21.6 % (16/78)] and IgG [28.4 % (52/183)] (both P= 0.002). The rate of CD56+in t(11;14) positive group was lower than that in t(11;14) negative group [51.6 % (48/93) vs. 72.0 % (167/232), P= 0.001], and the rate of CD117+was also significantly decreased [23.7 % (22/93) vs. 37.7 % (87/231), P= 0.019]. There were 86 cases of non-t(11;14) IgH rearrangement in 269 cases of NDMM without t(11;14), which mainly were t(4;14) or t(14;16). The detection rate of high risk MM was only 11.9 %(12/101)in t(11;14)positive group,while that rate was 27.5 % (74/269) in t(11;14) negative group, the difference was statistically significant (P = 0.001). Conclusion MM with t(11;14)displays distinct biological,clinical and laboratory features,it is a heterogeneous disease.

Clinical feature and efficacy of patients with multiple myeloma and renal impairment treated with bortezomib based chemotherapy / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the efficacy and safety of bortezomib (btz) based chemotherapy in multiple myeloma (MM) patients with renal-function impairment (RI).</p><p><b>METHODS</b>Fifty-six MM patients with impaired renal function treated with bortazomib based regimens in our single center were retrospectively analyzed.</p><p><b>RESULTS</b>The median age was 59 (ranged 30-77) years. 39.3% were κ-restricted MM, while 57.1% were λ-restricted MM. Nine patients were IgD-MM, and 14 were light chain MM. Median creatinine clearance (CrCl) was 25.33 (7.23-59.55) ml/min. The number of patients with mild, moderate and severe RI was 6, 35 and 15, respectively. Overall response rate of MM was 82.4% (≥MR), including 32.4% complete response (CR), 17.6% very good partial response (VGPR) and 26.5% partial response (PR). The rate of renal response was 89.3%, including 62.5% CR, 14.3% PR and 12.5% minor response (MR). A median time of optimal response was 25.5 (ranged 5-240) days. There was no significant difference in the median overall survival and the time to progress in different RI groups. Adverse events observed were similar to those patients with normal renal function previously reported. Most adverse events were manageable, 55.6% patients developed peripheral neuropathy and 10 patients discontinued bortezomib.</p><p><b>CONCLUSION</b>The incidence of RI is higher in patients with IgD-MM and λ restricted MM. Bortezomib based treatment is a highly effective and safe option in MM patients with impaired renal function. In this analysis, renal function was improved in a substantial proportion of patients. Peripheral neuropathy is the major adverse events which limit its use in MM patients.</p>

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>

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>

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>

Clinical application of avidin-biotin ELISA to detect serum hepatoma-specific gamma-glutamyltransferase in patients with primary hepatic cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To detect serum hepatoma-specific datura stramonium lectin-tightly binding gamma-glutamyl transferase (DSA-GGT) in patients with primary hepatic cancer (PHC) by avidin-biotin ELISA method which was established in our laboratory, and carry on a study of its clinical application.</p><p><b>METHODS</b>To detect serum DSA-GGT in 45 healthy control subjects, 58 PHC patients and 203 non-PHC patients (including 36 patients with other tumors and 167 patients with benign liver diseases) with the method was established; meanwhile, AFP was detected by ELISA method.</p><p><b>RESULTS</b>38 individuals were DSA-GGT positive in 58 PHC patients, the sensitivity was 65.5%. 18 individuals were DSA-GGT positive in 203 patients without PHC, the specificity was 91.1%. The sensitivity and specificity of AFP in diagnosis of PHC patients was 69.0% and 90.6%, respectively. The sensitivity and specificity of combination of DSA-GGT and AFP was 93.1% and 85.7%, respectively. The average intra-CV and inter-CV of DSA-GGT ELISA was 8.9% and 11.5%, respectively.</p><p><b>CONCLUSION</b>The sensitivity and specificity of DSA-GGT ELISA method established in our lab is similar with that of AFP assay and the accuracy is good. Combination of DSA-GGT and AFP may improve the diagnostic sensitivity. The method should be potentially as a new way to improve diagnosis of PHC.</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>

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.