The effects of Gremlin1 on human umbilical cord blood hematopoietic progenitors.

Bone morphogenetic proteins (BMPs) support malignant hematopoiesis in CML. Conversely, the multi-functional BMP antagonist Gremlin1 supports self-renewing cancer stem cells of other malignancies. Inhibition of BMP signaling in CML, or of Gremlin1 in solid tumors, may therefore have therapeutic potential. However, since BMPs regulate hematopoietic stem cell (HSC) decisions in the stem cell niche, it is necessary to determine how Gremlin1 influences normal HSC. We examined the effects of Gremlin1 on long-term culture-initiating cells (LTC-IC) and transplantable hematopoietic stem cells (SCID-repopulating cells: SRC) in human umbilical cord blood. Gremlin1 inhibited BMP signaling, downregulated BMP-6 and cyclin E2 expression and upregulated hairy and enhancer of split-1 (HES-1; a Notch transcriptional target) and Hedgehog interacting protein-1 (HHIP-1; an inhibitor of Hedgehog signaling). The functional effects of Gremlin1 on SRC, i.e. skewing of their myelopoietic:lymphopoietic potential towards B lymphopoiesis without affecting long-term engraftment potential, were entirely consistent with changes in gene expression induced by Gremlin1. Since both BMPs and Gremlin1 are secreted by osteoblasts in vivo, our studies provide potential insights into the molecular regulation of hematopoiesis in the stem cell niche. These results also suggest that Gremlin1 (and possibly its mimetics that may be developed for therapeutic use) may not adversely affect normal human hematopoietic stem cell survival, though they may reduce their myelopoietic potential.

Establishment of mouse model of orbital diffuse large B cell lymphoma / 中华实验眼科杂志

Background Recently, the morbidity of orbital lymphoma increased gradually, and we have made deeper research in pathology,therapy and pathogenesis of the disease.There were few reports of mice model of orbital lymphoma up to now for its lower morbidity and culture difficulty.Objective This study was to establish a mouse model of orbital diffuse large B cell lymphoma (DLBCL) by injection of systemic DLBCL cell line pfeiffer.Methods Ten SPF BALB/c mice and 5 nod-SCID mice were radiated firstly using 137Cs,with the absorption dose 3.5 Gy in the BALB/c mice and 2.6 Gy in the nod-SCID mice,and then pfeiffer cells were intraobitally injected in 4eyes of BALB/c mice (orbital injection group) and suncutaneously injectied in 4 eyes of BALB/c mice and 4 eyes of nod-SCID mice (subcutaneous injection group) at the concentration of 1.5×l08/ml.The developing status of tumors were examined once per day and the growth curve was drawn.The tumors and nearby lymph nodes were obtained 54 days after injection for the preparation of 4 μm thickness of serial sections.Hemotoxylin-eosin staining was used to examine the histopathology of the tumors, and immunochemistry was employed to detect the expressions of CD20,CD79α and CD45RO proteins.The tumors were typed based on the expressions of CD10, BCL-6 and mum-1 in the specimens,and the expressions of Ki-67 and survivin were assyed to assess the prognosis of the tumor.All the results were compared with 3 diagnosed human orbital DLBCL sections.The use and care of the mice complied with Chinese Administration Rule of Laboratory Animal.Results The tumor formation rates were 100％ in both the orbital injection group and subcutaneous injection group, and the tumors grew much faster in nod-SCID mice than BALB/c mice.Infiltration of tumor cells in lymph nodes were found in the subcutaneous injection group rather than the orbital injection group.The pathological features were accordant among the orbital injection group, subcutaneous injection group and human orbital DLBCL sections.The number of ＜50％ and ≥50％ CD20+ specimens was 3 and 5,CD79αwas 2 and 6,CD45RO was 8 and 0 in the BALB/c mice;while that in the nod-SCID mice was 1 and 3 in CD20,0 and 4 in CD79α,4 and 0 in CD45RO;the number of human orbital DLBCL specimens was 1 and 2 in CD20,1 and 2 in CD79α,2 and 1 in CD45RO,without significant differences among them (all at P=1.00).No significant differences were seen in Ki-67+ number and survivin+ number among the BALB/c mice, nod-SCID mice and human orbital DLBCL specimens (all at P=1.00).The detection of CD10,BCL-6 and mum-1 expressions indicated that the tumors of BALB/c mice,nod-SCID mice and human orbital DLBCL specimens all were the non-germinal center B cell-like types.Conclusions The orbital and subcutaneous DLBCL mouse models are successfully established by injection of pfeiffer cell line.There are the same findings and features in biological behavior, pathology and immunohistochemistry in orbital,subcutaneous models with human orbitl DLBCL.Nod-SCID mice appear to be more suitable for the growth of lymphoma cells.

Experimental study of the appropriate time to block adrenal androgens in endocrine therapy of prostate cancer / 中华泌尿外科杂志

Objective To explore the appropriate time to block adrenal androgens in endocrine therapy of prostate cancer.Methods An human androgen-dependent prostate carcinoma xenograft model in SCID mice was established with LNCaP cells.Firstly,the serum PSA and tumor volume of 2 groups (castrated and not castrated) were measured on the 1,4,7,10,14,17,21,28,35,42,49 and 56 day to determine the recurrent time of prostate cancer after castration.Secondly,3 different groups of castration and adrenalectomy at the same time,adrenalectomy in recurrence after castration and sham adrenalectomy in recurrent after castration,were set to measure the serum PSA and tumor volume on the 1,4,7,10,14,17,21,28,35,42,49 and 56 day.The tumor tissues of 5 groups were harvested to measure testosterone concentration,and tumor progress in these groups was compared.Results The recurrence time was 14 days in castrated group,21 days in group with castration and adrenalectomy at the same time and 35 days in group with adrenalectomy in recurrence after castration.The testosterone concentration in tumor tissues was (2.69± 0.21) pmol/g in the group with castration and adrenalectomy at the same time,and (2.16±0.13) pmol/g in the group with adrenalectomy in recurrence after castration,and the difference was significant (P＜0.05).Conclusion Compared with the therapy of castration and adrenalectomy at the same time,the therapy of adrenalectomy in recurrence after castration may have slower progress course in prostate cancer.

Study on the novel models with human rheumatoid arthritis synovial fibroblasts-cartilage-SCID mice for rheumatoid arthritis / 中华风湿病学杂志

Objective To establish the models with human rheumatoid arthritis (RA) synovial fibroblasts (RASFs)-cartilage-severe combined immune deficient (SCID) mice for the study of the RASFs in the pathogenesis of RA.Methods The 4th passage RASFs were marked with 5-bromodexyuridine (5-Brdu)and injected into a cavity of inert sterile gel sponge,then with the normal human cartilage co-implanted in the back subcutaneously of SCID mice to set up a novel model of RA.Osteoarthritis synovial fibroblasts (OASFs)were injected as control group.Thirty days after the surgery,the mice were killed,the grafts and the knee joints were proceed with histological study and immunohistochemistry to detect the expression of 5-Brdu and Vimentin in synoviocytes.The serum level of interleukin (IL)-6 and matrix metalloproteinase (MMP)-3 were detected with enzyme-linked immunosorbent assay (ELISA).Results Twenty-three mice survived except for one mouse in the RASFs group died of anesthesia.① Only in one case in the RASFs group,the IL-6 was detected,the others were unable to be detected.The MMP-3 in the OASFs group was (40±17) pg/ml,but in RASFs group only one case was detected.② There were 4 and 3 implanted cartilages loss in the RASFs group and OASFs group respectively.The histological scores of cartilage invasion by synoviocytes and cartilage degradation in grafts were higher in RASFs groups than in OASFs groups (0.6±0.7 vs 0.3±0.5,2.3±0.8 vs 1.7±1.0 respectively).③ The histological scores of synovial hyperplasia and cartilage invasion in the knee joints was significantly higher in the RASFs group than in the OASFs group (3.1±0.8 vs 1.7±1.0,P＜0.01,1.6±1.7 vs 0.6±1.4,P＜0.05 respectively).④ During the grafts,a lot of 5-Brdu and Vimentin markers positive synovio-cytes were found in the mice subcutaneous tissue,but manipulus positive synoviocytes were found on the area of cartilage invasion in both groups.In knee joints,single positive synoviocytes could be detected in bone marrow and hyperplasic area of the synovial tissue in both groups.Conclusion The isolated RASFs can survival and have the ability to invade and degrade the cartilage in vivo without the limitation of immunity induced inflammations,and can also migrate to the synovial joints in distance and induce arthritis.

Cotransplanted Bone Marrow Derived Mesenchymal Stem Cells (MSC) Enhanced Engraftment of Hematopoietic Stem Cells in a MSC-dose Dependent Manner in NOD/SCID Mice

Transplantation of marrow-derived mesenchymal stem cells (MSCs), expanded by culture in addition to whole bone marrow, has been shown to enhance engraftment of human hematopoietic stem cells (HSCs). Our hypothesis was that there might be an optimum ratio range that could enhance engraftment. We examined the percent donor chimerism according to the ratio of HSCs to MSCs in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. We tested a series of ratios of co-transplanted CD34+-selected bone marrow cells, and marrow-derived MSCs into sublethally irradiated NOD/SCID mice. In all experiments, 1 x 10(5) bone marrow derived human CD34+ cells were administered to each mouse and human MSCs from different donors were infused concomitantly. We repeated the procedure three times and evaluated engraftment with flow cytometry four weeks after each transplantation. Serial ratios of HSCs to MSCs were 1:0, 1:1, 1:2 and 1:4, in the first experiment, 1:0, 1:1, 1:2, 1:4 and 1:8 in the second and 1:0, 1:1, 1:4, 1:8 and 1:16 in the third. Cotransplantation of HSCs and MSCs enhanced engraftment as the dose of MSCs increased. Our results suggest that the optimal ratio of HSCs and MSCs for cotransplantation might be in the range of 1:8-1:16; whereas, an excessive dose of MSCs might decrease engraftment efficiency.

Cotransplanted Bone Marrow Derived Mesenchymal Stem Cells (MSC) Enhanced Engraftment of Hematopoietic Stem Cells in a MSC-dose Dependent Manner in NOD/SCID Mice

Transplantation of marrow-derived mesenchymal stem cells (MSCs), expanded by culture in addition to whole bone marrow, has been shown to enhance engraftment of human hematopoietic stem cells (HSCs). Our hypothesis was that there might be an optimum ratio range that could enhance engraftment. We examined the percent donor chimerism according to the ratio of HSCs to MSCs in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. We tested a series of ratios of co-transplanted CD34+-selected bone marrow cells, and marrow-derived MSCs into sublethally irradiated NOD/SCID mice. In all experiments, 1 x 10(5) bone marrow derived human CD34+ cells were administered to each mouse and human MSCs from different donors were infused concomitantly. We repeated the procedure three times and evaluated engraftment with flow cytometry four weeks after each transplantation. Serial ratios of HSCs to MSCs were 1:0, 1:1, 1:2 and 1:4, in the first experiment, 1:0, 1:1, 1:2, 1:4 and 1:8 in the second and 1:0, 1:1, 1:4, 1:8 and 1:16 in the third. Cotransplantation of HSCs and MSCs enhanced engraftment as the dose of MSCs increased. Our results suggest that the optimal ratio of HSCs and MSCs for cotransplantation might be in the range of 1:8-1:16; whereas, an excessive dose of MSCs might decrease engraftment efficiency.

Antitumor Activity of TRAIL Recombinant Adenovirus in Human Malignant Glioma Cells

Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) has been reported to specifically kill malignant cells but to be relatively nontoxic to normal cells. One of disadvantages to previous in vivo protocols was the need for large quantities of TRAIL recombinant protein to suppress tumor growth. To evaluate the antitumor activity and therapeutic value of the TRAIL gene, we constructed adenoviral vectors expressing the human TRAIL gene (Ad.hTRAIL) and transferred them into malignant glioma cells in vitro and tumors in vivo, as an alternative to recombinant soluble TRAIL protein. The results show that TRAIL-sensitive glioma cells infected Ad.hTRAIL undergo apoptosis through the production and expression of TRAIL protein. The in vitro transfer elicited apoptosis, as demonstrated by the quantification of viable or apoptotic cells and by the analysis of cleavage of poly (ADP-ribose) polymerase. Furthermore, in vivo administration of Ad.hTRAIL at the site of tumor implantation suppressed the outgrowth of human glioma xenografts in SCID mice. These results further define Ad.hTRAIL as an anti-tumor therapeutic and demonstrate its potential use as an alternative approach to treatment for malignant glioma.