The N-butyl alcohol extract from Hibiscus rosa-sinensis L. flowers enhances healing potential on rat excisional wounds.

ETHNO-PHARMACOLOGICAL RELEVANCE: Hibiscus rosa-sinensis L. (HRS), a folk medicine named Zhujin in China, possess anti-tumor, antioxidant, antibacterial, low density lipoprotein oxidation prevention and macrophage death prevention effects. The leaves and red flowers of HRS have been traditionally used to treat with furuncle and ulceration. AIM OF THE STUDY: To investigate the efficacy and possible mechanism of the N-butyl alcohol extract of HRS (NHRS) red flowers in wound healing by analyzing the collagen fiber deposition, angiogenic activity and macrophages action of the NHRS. MATERIALS AND METHODS: In an excisional wound healing model in rats, different concentrations of NHRS, or recombinant bovine basic fibroblast growth factor (rbFGF), were respectively applied twice daily for 9 days. Histopathology was assessed on day 9 via hematoxylin and eosin (HE) and Masson's trichrome (MT) staining, and immunohistochemistry for vascular endothelial growth factor (VEGF), transforming growth factor-ß1 (TGF-ß1) and CD68. Immunomodulation by NHRS was evaluated by a carbon clearance test in mice. RESULTS: Wound healing post-surgery was greater in the rbFGF-control, NHRS-M and MHRS-H groups than in the model and 5% dimethylsulfoxide (DMSO)-control groups after the third day. By the sixth day the wound contraction of NHRS-M and MHRS-H groups was much higher than the rbFGF-control group. HE and MT staining revealed that epithelialization, fibroblast distribution, collagen deposition of NHRS-M- and NHRS-H-control groups were significantly higher than the model group. Moreover, immunohistochemistry showed more intense staining of VEGF, TGF-ß1 and CD68 in the rbFGF- and NHRS-control groups, compared to that in model and 5% DMSO-control groups. The clearance and phagocytic indices of NHRS-M- and NHRS-H-control groups were significantly higher than that of the carboxyl methyl cellulose (CMC) group in mice. CONCLUSION: NHRS accelerates wound repair via enhancing the macrophages activity, accelerating angiogenesis and collagen fiber deposition response mediated by VEGF and TGF-ß1.

Bone marrow stromal cell line co-transfected with IL-2 and IL-3 genes can accelerate restoration of T-cell immunity in allo-BMT mice.

BACKGROUND: After T-cell depleted allogeneic bone marrow transplantation, impaired immune reconstitution is a major cause of morbidity and mortality in the recipient. The purpose of this study was to observe the effects of the gene-engineered bone marrow stromal cell line QXMSC1-IL-2 + IL-3 on the reconstitution of T-cell immunity in allo-BMT mice. METHODS: The bone marrow stromal cell line QXMSC1 was co-transfected with IL-2 and IL-3 genes using a Tet-on gene expression system. T lymphocyte subset counts per spleen were analyzed by flow cytometry. Lymphocyte proliferation response to ConA was examined to evaluate T-cell function. CDR3 spectratyping techniques were performed to evaluate TCR repertoire diversity at various time points post-transplantation. RESULTS: Gene engineered bone marrow stromal cell line QXMSC1-IL-2 + IL-3 could express IL-2 and IL-3 [1,300 ng.day(-1).10(-6) cells and 1100 ng.day(-1).10(-6) cells, respectively] under the control of doxycycline. QXMSC1-IL-2 + IL-3 in combination with allogeneic bone marrow could significantly increase the counts of CD(4)(+) and CD(8)(+) T cell, 1.72 and 1.27-fold respectively at week 3 compared with TCD-BMT group (P < 0.01); make CD(4)(+)/CD(8)(+) ratio return to normal level at week 4; enhance splenocytes mitotic response to ConA (P < 0.01), and accelerate restoration of TCR repertoire diversity in the lethally irradiated mice (P < 0.05). CONCLUSION: The gene transduced stromal cell line QXMSC1-IL-2 + IL-3 is able to accelerate T-cell immunity in allo-BMT mice.

[The enhancing effects of IL-3 gene transfected murine bone marrow stromal cells on hematopoiesis under control of doxycycline].

To study enhancing effect of IL-3 gene transfected bone marrow stromal cell which can be induced by doxycycline (Dox) to express IL-3 cytokine on the proliferation and differentiation of hematopoietic stem cell, retrovirus vector system contained mIL-3 cDNA was established and bone marrow stromal cell line was transfected, and obtained QXMSC1Tet-on-IL-3, in which expression level of IL-3 can be modulated by Dox. The activities of IL-3 were measured under different Dox concentrations. The numbers of hematopoietic progenitors (CFU-GM, CFU-E, CFU-GEMM and LTC-IC) were measured and the capacity of QXMSC1Tet-on-IL-3 sustaining hematopoietic progenitor cell growth was evaluated. The results showed that IL-3 gene transfected stromal cell line QXMSC1-Tet-on + IL-3 expressed high concentration of IL-3 in vitro under control of Dox. The supernatant of QXMSC1-Tet-on + IL-3 was able to increase the number of CFU-GM, CFU-E and CFU-GEMM. The total numbers of nucleated cells and long-term cultured colonies increased in LTC-IC assay. It is concluded that in the culture of QXMSC1-Tet-on + IL-3 cells, Dox actually enhanced IL-3 expression, and thus augmented the proliferation and differentiation of hematopoietic stem/progenitor cells in vitro.

[The improving effect of bone marrow stromal cell transfected with IL-3 gene on hematopoietic reconstitution in bone marrow transplantation of mice].

To study the improving effect of regulatable gene of IL-3 engineered bone marrow stromal cell on the hematopoietic reconstitution in allogeneic bone marrow transplantation, an inducible gene expression system was established in a bone marrow stromal cell line which expressed IL-3 gene induced by doxycycline (Dox). The lethally irradiated mice C57BL/6 (H-2(d)) were co-transplanted with allogeneic bone marrow (BALB/c, H-2(d), 1 x 10(7)/mice) in which T cell were depleted by monoclonal antibody anti-Thy1.2 added with complement and the gene engineered stromal cell QXMSC1tet-on + IL-3 (5 x 10(5)/mice) at the same time. Dox was administrated continuously for 15 days to induce the expression of IL-3. The hematopoiesis in the bone marrow transplanted mice were observed at 30, 60 days post-transplantation, respectively. The numbers of RBC and WBC in peripheral blood were counted, and nucleated cells, CFU-S, CFU-GM, CFU-E and CFU-GEMM were measured in recipient bone marrow. The results showed that the engineered stromal cell line achieved high-level and controllable IL-3 expression. Co-graft with QXMSC1tet-on + IL-3 significantly increased the number of RBC, WBC in recipient peripheral blood, and the nucleated cells, CFU-S, CFU-GM, CFU-E, CFU-GEMM in bone marrow, compared with those coinfused with QXMSC1 or QXMSC1tet-on-TRE as control. In conclusion, regulatable gene IL-3 engineered bone marrow stromal cells accelerates hematopoietic reconstitution after allogeneic bone marrow transplantation.

[Accelerating hematopoietic recovery in allogeneic bone marrow transplantation mice by co-infusion of marrow stromal cells transduced with dual genes of IL-3/IL-6].

To observe whether bone marrow stromal cell line QXMSC1 (H-2(d)) engineered to secrete IL-3 and IL-6 can improved the hematopoiesis in allogeneic bone marrow transplantation (allo-BMT) mice, the stromal cell line QXMSC1IL-3/IL-6 was established by QXMSC1 cells transduced with the recombined retrovirus vector pL3SN containing mouse IL-3 cDNA and pL6SN containing human IL-6 cDNA. The lethally irradiated C57BL/6 (H-2(b)) mice were engrafted with bone marrow cells (1 x 10(7) cells/mouse BALB/c mice, H-2(d)) in which T cells were depleted by anti-Thy1.2 monoclonal antibody adding complement, and QXMSC1IL-3/IL-6 cells (5 x 10(5)/mouse) were co-infused at same time. The hematopoiesis of recipient mice was observed in 20 and 40 days post-transplantation. Blood RBC and WBC were counted, and nucleated cells, CFU-S, CFU-GM, CFU-E and CFU-GEMM were assayed in recipient bone marrow. Results showed that IL-3 and IL-6 were stably expressed in QXMSCQIL-3/IL-6 cells. Compared with BMT and co-infusion with QXMSC1 or QXMSC1 pLXSN cell groups, co-graft with QXMSC1IL-3/IL-6 cells increased the number of blood RBC and WBC in the recipients, and also significantly increased nucleated cells, CFU-S, CFU-GM, CFU-E and CFU-GEMM in recipient bone marrow. It is concluded that the marrow stromal cells transduced with IL-3/IL-6 cDNA improve the hematopoiesis in allo-BMT mice. Co-graft with QXMSC1IL-3/IL-6 cells has synergistic effect in accelerating hematopoietic reconstitution.