[Establishment of multivariate diagnosis and treatment system of modern gynecology of traditional Chinese medicine].

Gynecology of traditional Chinese medicine (TCM) is a clinical subject concentrating on women's specific diseases. With the advance of the time, clinical gynecology of TCM has to be developed from clinical practice and establish an open mode of multivariate diagnosis and treatment. The author of this article expatiates on the relationships between syndrome differentiation and disease identification, constitution differentiation and syndrome differentiation, stage differentiation and syndrome differentiation, time differentiation and syndrome differentiation, macroscopic differentiation of syndrome and microcosmic differentiation of syndrome, and integral syndrome differentiation and local syndrome differentiation, and then states that it is necessary for us to establish a multivariate diagnosis system to adapt to the complex clinical practice.

Screening of aplastic anaemia-related genes in bone marrow CD4+ T cells by suppressive subtractive hybridization.

BACKGROUND: CD4(+) T cells play a crucial role in the pathogenesis of aplastic anaemia. However, the mechanisms of over-proliferation, activation, infiltration of bone marrow and damage to haematopoietic cells of CD4(+) T cells in aplastic anaemia are unclear. Therefore, we screened differentially expressed genes of bone marrow CD4(+) T cells of aplastic anaemia patients and normal donors by suppressive subtractive hybridization to investigate the pathogenesis of aplastic anaemia. METHODS: The bone marrow mononuclear cells of a first visit aplastic anaemia patient and a healthy donor of the same age and sex were isolated using lymphocyte separating medium by density gradient centrifugation. With the patients as "tester" and donor as "driver", their CD4(+) T cells were separated with magnetic bead sorting and a cDNA library established by suppressive subtractive hybridization. Then 15 of the resulting subtracted cDNA clones were randomly selected for DNA sequencing and homological analysis. With semiquantitative RT-PCR, bone marrow samples from 20 patients with aplastic anaemia and 20 healthy donors assessed the expression levels of differentially expressed genes from SSH library. RESULTS: PCR detected 89 clones in the library containing an inserted fragment of 100 bp to 700 bp. Among 15 sequenced clones, 12 were known genes including 3 repeated genes. Compared with normal donors, there were 9/12 genes over-expressed in bone marrow CD4(+) T cells of patients with aplastic anaemia. The effects of these genes included protein synthesis, biology oxidation, signal transduction, proliferative regulation and cell migration. Not all these genes had been reported in the mechanisms of haematopoietic damage mediated by CD4(+) T cells in aplastic anaemia. CONCLUSIONS: Screening and cloning genes, which regulate functions of CD4(+) T cells, are helpful in elucidating the mechanisms of over proliferation, activation, infiltrating bone marrow and damaging haematopoietic cells of CD4(+) T cells in aplastic anaemia.

[Mechanism of radiation induced premature senescence of bone marrow stromal cells: experiment with murine bone marrow stromal cells].

OBJECTIVE: To investigate the mechanism of radiation induced premature senescence of bone marrow stromal cells induced by gamma-radiation. METHODS: Bone marrow stromal cells were isolated from the bones of mice, cultured, and divided into 2 groups: control group and irradiation group. The cells of the irradiation group were exposed to 60Co gamma-rays of the dose of 8.0 Gy. 24 h, 72 h, 1 week, and 2 weeks after irradiation MTT method was used to test the proliferation of the cells, Giemsa staining was used to observe the morphology, the percentage of cells positive in beta-galactosidase (SA-beta-Gal), a senescence associated biomarker, was detected by cytochemistry, and RT-PCR was used to detect the mRNA expression of p21(Cip1/Waf1) and p53 gene, both associated with senescence. One week after the irradiation flow cytometry was used to analyze the cell cycle distribution. RESULTS: Characteristics of mature senescence were seen in the cells of the irradiation group. One week after the irradiation the percentage of G0/G1 of the irradiation group was 89.83% +/- 0.05%, significantly higher than that of the control group (66.95% +/- 0.36%, P < 0.01). 24 h, 72 h, 1 week, and 2 weeks after irradiation the percentages of SA-beta-gal positive cells in the irradiation group were 20.33% +/- 0.03%, 34.33% +/- 0.03%, 86.33% +/- 0.02%, and 95.67% +/- 0.02% respectively, significantly higher than those of the control group (2.33% +/- 0.006%, 14.33% +/- 0.02%, 24.67% +/- 0.02%, and 45.00% +/- 0.02% respectively, all P < 0.01). The mRNA expression of p53 and p21(Cip1/Waf1) mRNA expressions in the irradiation group increased 24 h after the irradiation and lasted for two weeks. CONCLUSION: Gamma-radiation induces changes of premature senescence in bone marrow stromal cells in which p53-p21(Cip1/Waf1) manner pathways may play an important role.

Expansive effects of aorta-gonad-mesonephros-derived stromal cells on hematopoietic stem cells from embryonic stem cells.

BACKGROUND: Hematopoietic stem cells (HSCs) give rise to all blood and immune cells and are used in clinical transplantation protocols to treat a wide variety of refractory diseases, but the amplification of HSCs has been difficult to achieve in vitro. In the present study, the expansive effects of aorta-gonad-mesonephros (AGM) region derived stromal cells on HSCs were explored, attempting to improve the efficiency of HSC transplantation in clinical practice. METHODS: The murine stromal cells were isolated from the AGM region of 12 days postcoitum (dpc) murine embryos and bone marrow (BM) of 6 weeks old mice, respectively. After identification with flow cytometry and immunocytochemistry, the stromal cells were co-cultured with ESCs-derived, cytokines-induced HSCs. The maintenance and expansion of ESCs-derived HSCs were evaluated by detecting the population of CD34+ and CD34+Sca-1+ cells with flow cytometry and the blast colony-forming cells (BL-CFCs), high proliferative potential colony-forming cells (HPP-CFCs) by using semi-solid medium colonial culture. Finally, the homing and hematopoietic reconstruction abilities of HSCs were evaluated using a murine model of HSC transplantation in vivo. RESULTS: AGM and BM-derived stromal cells were morphologically and phenotypically similar, and had the features of stromal cells. When co-cultured with AGM or BM stromal cells, more primitive progenitor cells (HPP-CFCs) could be detected in ESCs derived hematopoietic precursor cells, but BL-CFC's expansion could be detected only when co-cultured with AGM-derived stromal cells. The population of CD34+ hematopoietic stem/progenitor cells were expanded 3 times, but no significant expansion in the population of CD34+Sca-1+ cells was noted when co-cultured with BM stromal cells. While both CD34+ hematopoietic stem/progenitor cells and CD34+Sca-1+ cells were expanded 4 to 5 times respectively when co-cultured with AGM stromal cells. AGM region-derived stromal cells, like BM-derived stromal cells, could promote hematopoietic reconstruction and HSCs' homing to BM in vivo. CONCLUSIONS: AGM-derived stromal cells in comparison with the BM-derived stromal cells could not only support the expansion of HSCs but also maintain the self-renewal and multi-lineage differentiation more effectively. They are promising in HSC transplantation.

An experimental study on astrocytes promoting production of neural stem cells derived from mouse embryonic stem cells.

BACKGROUND: The production of neural stem cells (NSCs) derived from embryonic stem (ES) cells was usually very low according to previous studies, which was a major obstacle for meeting the needs of clinical application. This study aimed at investigating whether astrocytes could promote production of NSCs derived from ES cells in vitro. METHODS: Mouse ES cells line-D3 was used to differentiate into NSCs with astrocytes as inducing stromal cells by means of three-stage differentiation procedure. Another group without astrocytes served as control. The totipotency of ES cells was identified by observation of cells' morphology and formation of teratoma in severe combined immunodeficiency disease (SCID) mice. The quantity and purity of NSCs derived from ES cells were analyzed using clonogenic assay, immunohistochemical staining and flow cytometry assay. The plasticity of NSCs was detected by differentiating test. Octamer-binding transcription factor 4 (Oct-4) and nestin, the specific marker genes of ES cells and NSCs respectively, were detected continuously using reverse transcription-polymerase chain reaction (RT-PCR) method to monitor the process of cell differentiation. RESULTS: The ES cells of D3 line could maintain the ability of differentiating into cellular derivations of all three primary germ layers after continuous passage culture. At the end of two-stage of inducing process, 23.2 +/- 3.5 neurospheres per plate formed in astrocyte-induced group and only 0.8 +/- 0.3 per plate in the control group (clonogenic assay, P < 0.01), and the ratio of nestin positive cells was (50.2 +/- 2.8)% in astrocyte-induced group and only (1.4 +/- 0.5)% in the control group (flow cytometry, P < 0.01). With the induction undergoing, the expression of Oct-4 gradually decreased and then disappeared, while the expression of nestin was increased step by step, and the ratio of nestin positive cells was up to 91.4% by the three-stage differentiation. The nestin positive cells could be further induced into neurons, astrocytes, and oligodendrocytes in differentiating medium supplemented with fetal calf serum. The results of differentiating test showed that the ratio of NF-200 and NSE positive cells was (42.7 +/- 2.6)% in astrocyte-induced group and only (11.2 +/- 1.8)% in the control group (P < 0.01). CONCLUSIONS: Astrocytes can not only increase the production of NSCs derived from ES cells but also promote the differentiation of NSCs toward neuronal lineage.

Screening and cloning of multi-drug resistant genes in HL-60/MDR cells.

OBJECTIVE: To screen and clone multi-drug resistance (MDR) related genes in MDR acute myeloid leukemia cells (HL-60/MDR). METHODS: HL-60/MDR was established using All-Trans Retinoic Acid. With the HL-60 cells as "tester" and HL60/MDR as "driver", the cDNA library of HL-60/MDR was established by suppression subtractive hybridization. Then 12 of the resulting subtracted cDNA clones were selected for DNA sequencing and homology analysis. The obtained expressed sequence tags (ESTs) were analyzed with the GenBank BLASTN program to identify sequence homologies to known genes. RESULTS: The HL-60/MDR cells had different multi-drug resistance to six kinds of chemotherapeutic drugs. The 211 positive gene clones in differential cDNA library of HL-60/MDR cells were amplified with PCR and 46 gene clones exhibited differential expression (ratio >3). Twelve gene clones with significant differential expression (ratio >5) were screened out to homology analysis. Of these, 11 matched known genes and the rest 1 showed no significant homology to human or non-human known sequences. It was named as gene clone HA117. CONCLUSIONS: This effort provides the partial list of genes differential expressed in HL-60/MDR cells and a novel gene HA117 was found to be related to MDR. Identification of these genes contributes to our understanding of MDR development, and potentially provides candidate target genes to overcome MDR.

[Role of the sonic hedgehog pathway in regulation of the proliferation, migration and differentiation of hemangioblast derived from AGM].

OBJECTIVE: To explore the role of sonic hedgehog (Shh) pathway in regulating the proliferation, migration and differentiation of hemangioblasts derived from aorta-gonad-mesonephros (AGM). METHODS: The hemangioblasts were isolated from AGM region of 11-day postcoitum (dpc) murine embryos by using the immuno-magnetic with CD34 and Flk1 monoclonal antibodies. The phenotypic analysis of hemangioblasts and AGM-derived stromal cells were detected by flow cytometry. The secretion of Shh was examined by immunohistochemical staining. The roles of Shh in regulating the proliferation, migration and differentiation of hemangioblasts in the transwell non-contact coculture system with AGM-derived stromal cells were observed by adding exogenous Shh N-Terminus and its antibody. RESULTS: The protein of Shh was highly expressed on AGM-derived stromal cells. The proliferation of hemangioblasts was promoted when co-cultured with AGM-derived stromal cells, and the effects of the latter could be blocked by antibody of Shh. The proliferation of hemangioblasts was strengthened further and kept for a long time without differentiation and apoptosis when exogenous Shh N-Terminus was added into the transwell non-contact co-culture system with AGM-derived stromal cells. When exogenous Shh N-Terminus was added into the cultural supernatant of hemangioblasts without AGM-derived stromal cells, the hemangioblasts were observed to be induced to apoptosis or differentiation after a short time of proliferation. Furthermore, the ability of migration could be promoted in the co-cultured hemangioblasts by adding exogenous Shh N-Terminus. CONCLUSION: Shh pathway probably involves in the regulation of the proliferation, differentiation, apoptosis and migration of hemangioblasts, and is regulated by the AGM microenvironment.

[Screening and cloning the genes differentially expressed in human embryonic AGM-derived stromal cells].

To screen and separate the genes differentially expressed in human embryonic aorta-gonad-mesonephros (AGM)-derived stromal cells, a subtracted library was generated through the suppression subtractive hybridization using the cDNA of human embryonic AGM-derived stromal cells as target and human fetal liver (FL)-derived stromal cells as drivers. Then a high though screening technique, gene chip, was used to screen the differentially expressed genes in the established subtractive library. Approximately 18 of the resulting subtracted cDNA clones were partially sequenced and analyzed by blastn in the GenBank database. The results showed that 211 Clones were selected and identified from the established subtractive library, the positive ratio was amount to 76.4%. 18 over-expressed genes were screened by gene chip with more than a 5-fold difference expression levels between AGM and FL-derived stromal cells, and were selected to sequence, results of sequencing indicated that the 18 sequences was compared to known sequences in the GenBank database, and among the sequenced clones, 14 sequences were considered as part of the known genes, and 4 sequences representing previously unknown genes. The known genes were reported to involve the regulation of cell migration, cell differentiation, cell proliferation, cell cycle, signal transduction, and angiogenesis. Most of these genes have not been reported to relate to the haematogenesis in ontogeny. It is concluded that many genes both known and unknown are differentially expressed in human embryonic aorta-gonad-mesonephros-derived stromal cells. Discovery of these genes provides a solid foundation to elucidate the mechanism of haematogenesis in ontogeny.

Sonic hedgehog protein promotes bone marrow-derived endothelial progenitor cell proliferation, migration and VEGF production via PI 3-kinase/Akt signaling pathways.

AIM: To investigate the effects of Sonic hedgehog (shh) protein on bone marrow- derived endothelial progenitor cells (BM-EPC) proliferation, migration and vascular endothelial growth factor (VEGF) production, and the potential signaling pathways involved in these effects. METHODS: Bone marrow-derived Flk-1(+) cells were enriched using the MACS system from adult Kunming mice and then BM-EPC was cultured in gelatin-coated culture dishes. The effects of shh N-terminal peptide on BM-EPC proliferation were evaluated using the MTT colorimetric assay. Cell migration was assayed using a modified Boyden chamber technique. The production of VEGF was determined by ELISA and immunofluorescence analysis. The potential involvement of PKC and PI3K signaling pathways was explored using selective inhibitor or Western blot. RESULTS: The proliferation, migration and VEGF production in BM-EPC could be promoted by endogenous shh N-terminal peptide at concentrations of 0.1 microg/mL to 10 microg/mL, and could be inhibited by anti-shh antibodies. Shh-mediated proliferation and migration in BM-EPC could be partly attenuated by anti-VEGF. Phospho-PI3-kinase expression in newly separated BM-EPC was low, and it increased significantly when exogenous shh N-terminal peptide was added, but could be attenuated by anti-human/mouse shh N-terminal peptide antibody. Moreover, the inhibitor of the PI3-kinase, but not the inhibitor of the PKC, significantly inhibited the shh-mediated proliferation, migration and VEGF production. CONCLUSION: Shh protein can stimulate bone marrow-derived BM-EPC proliferation, migration and VEGF production, which may promote neovascularization to ischemic tissues. This results also suggests that the PI3-kinase/Akt signaling pathways are involved in the angiogenic effects of shh.

[The effects of Mcl-1 gene on ATRA-resistant HL-60 cell].

OBJECTIVE: To investigate the role of Mcl-1 gene in resistance of all-trans retinoic acid (ATRA) of leukemia cells. METHODS: Long-term, intermittent and repetitive exposure of HL-60 cells to ATRA was used to establish a multidrug-resistance cell line (HL-60/ATRA). HL-60/ATRA cells were transfected with Mcl-1 small interference RNA (siRNA) by Lipofectamine 2000. Western blot was used to detect the expression of Mcl-1. The proliferation, apoptosis and differentiation were evaluated by MTT assay, in situ nick end-labeling (TUNEL) and NBT assay, respectively. RESULTS: The HL-60/ATRA could keep its undifferentiated and proliferative status to a high concentration of ATRA (100 nmol/L) with highly expressed Mcl-1 protein (relative grey scale 0.624 +/- 0.127). Mcl-1 gene knockdown by siRNA (relative grey scale 0.267 +/- 0.086) could reverse the resistance of ATRA of HL-60/ATRA by inhibiting proliferation, and inducing differentiation and apoptosis [apoptosis rate (18.5 +/- 4.5)%]. CONCLUSION: Mcl-1 gene might be involved in ATRA resistance in HL-60 cells and inhibiting its expression could be a new approach to ATRA resistance reversion.