RT-PCR amplification of ginseng ?-amyrin synthase gene was successfully performed based on the total RNAs extracted from ginseng hairy roots induced by Agrobacterium rhizogenes A4 using a modified guanidine isothiocyanate-method. Sequence analysis of this gene revealed that its sequence was consistent with the sequence of a previously reported ginseng ?-amyrin synthase gene (GenBank No. AB009030). This gene was inserted into pMD-119T simple vector and transformed into E.coli DH5?. Furthermore antisense plant expression vector of this gene was constructed using the pBI121 vector, laying foundation for studies on antisense regulation of ginseng ?-amyrin synthase gene.

Genetic engineering is a powerful tool in Panax ginseng breeding.Genetic transformation and plant regeneration are the premise and foundation involved in genetic engineering of Panax ginseng.Ginseng can be regenerated through organogenesis or somatic embryogenesis and indirect somatic embryogenesis is mainly used for its regeneration.Summurized the factors influencing plant regeneration such as different explants,different carbohydrates,somatic embryo optimization and hormone-free approach.Ginseng transformation has been achieved by Agrobacterium tumefaciens and Agrobacterium rhizogenes and transgenic ginseng with good characters was obtained by introducing genes associated with biosynthesis of ginsenosides or herbicide gene.Hairy root culture system can supply large scale of ginsenosides,thus effect of rolC genes on ginseng hairy root induction,regeneration and bioreactor culture of hairy root were discussed.Additionally,problems that are present in genetic engineering of Panax ginseng were also discussed in this review.

Aim To establish a new model of skin damage by u-sing vincristine to transgenic zebrafish (krt4:NTR-hKikGR).Methods Skin fluorescent transgenic zebrafish embryos after 24 h fertilization were treated with the 0.01~0.04 mmol·L-1 vincristine,and zebrafish skin cell ablation was investigated un-der fluorescence microscope after 24 h,at same time skin death cells were detected with TUNEL assay and image processed, then the protein expressions of KRT4, caspase-3 and p53 were assessed with Western blot. Results 0.02 mmol·L-1and 0.04 mmol·L-1vincristine could obviously induce zebrafish skin cell apoptosis(P<0.01) with statistically significant differ-ence compared with the control, and the same result could be accomplished in TUNEL assay. Results of Western bolt showed that vincristine could increase the embryos caspase-3 and p53 expression(P <0.01), while vincristine in high concentration might decrease KRT4 markedly(P<0.01). Conclusion Vin-cristine can induce damage on zebrafish skin with suppression KTR4,which can be used as a new skin damage model.

OBJECTIVETo observe the change of nitric oxide (NO) and expression of nuclear factor-kappa B (NF-kappaB) in the cortex of cerebral infarction rat induced by photochemical reaction, and study the effect of extract from Cornus officinalis (whose main ingredient is iridoid glycoside) in the course of disease.

METHODAfter rats were fed with experimental drugs for 7 days, the model of cerebral infarction was induced. Spectrophotography and immunohistochemistry were used to detect the change of the content of NO, NOS and the expression of NF-kappaB in the cortex.

RESULTCompared with control group, distinct infarction was visible in the model group, and the content of NO, the activity of NOS and the positive cell number of NF-kappaB were increased obviously. Compared with model group, the extract of C. officeinalis decreased the area of infarction, the content of NO, the activity of NOS and the positive cell number of NF-kappaB.

CONCLUSIONThe iridoid glycoside of C. officinalis may have therapeutical effect on cerebral infarction through regulating the content of NO and NF-kappaB.

Animals ; Cerebral Cortex ; metabolism ; pathology ; Cerebral Infarction ; metabolism ; pathology ; Cornus ; chemistry ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Male ; NF-kappa B ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; Plants, Medicinal ; chemistry ; Random Allocation ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo observe the hair follicle regeneration after subcutaneous implantation of hair follicle cell mixture in nude mice.

METHODSThe hair papilla cells, dermal sheath cells, outer root sheath and fibroblasts of human scalp were mixed with the hair follicle epithelial cells and implanted subcutaneously in nude mice to observe the regeneration of the hair follicle.

RESULTS AND CONCLUSIONFormation of intact hair follicle-like structures was observed in the skin sections of the recipient nude mice, suggesting the feasibility of this approach for hair follicle regeneration in vivo.

Animals ; Cell Transplantation ; Hair Follicle ; cytology ; metabolism ; physiology ; transplantation ; Humans ; Injections, Subcutaneous ; Mice ; Mice, Nude ; Regeneration ; Skin Pigmentation ; Time Factors

The hematopoietic system of the mouse arises from extraembryonic mesoderm that migrate through primitive streak to the presumptive yolk sac at day 7.0 of gestation. However, the mechanisms regulating mesoderm commitment to hematopoietic lineages remain poorly understood. Previous studies demonstrated that the development kinetics and growth factor responsiveness of hematopoietic precursors derived from embryonic stem cells (ES cells) is similar to that found in the yolk sac, indicating that the onset of hematopoiesis within the embryoid bodies (EBs) parallels that found in the embryo. Furthermore, in vitro differentiation of ES cells to hematopoietic cells is valuable for establishment of therapeutic clone against a variety of hematological disorders. Despite the identification of multipotential hematopoietic progenitors in EBs, a subset of more primitive progenitors, identical to the high proliferative potential colony-forming cells (HPP-CFC) derived from human and murine hematopoietic tissues, have not been clearly identified regarding particular their replating potential in vitro. HPP-CFC is among the most primitive hematopoietic multipotent precursors cultured in vitro. In this study, our aim was to investigate the in vitro and in vivo hematopoietic capacity of HPP-CFC within the day 12 EBs, rather than the expansion of more committed progenitors. In this study the HPP-CFC could be detected within EBs differentiated for 5 to 14 days of murine ES cells, but the development dynamics of the HPP-CFC differed greatly among distinct serum lots. Qualitatively HPP-CFC is capable of forming secondary colonies. As to our expectation the ES cells-derived HPP-CFC demonstrated similar regeneration capacity to those from yolk sac, giving rise to secondary granulocyte, erythrocyte, macrophage and mast cells, however largely differed from the counterparts of adult bone marrow. In addition, by RT-PCR ES cells-derived HPP-CFC were found to express transcription factors associated closely with stem cell proliferation including SCL, GATA-2 and AML1 as well as various receptors of hematopoietic growth factors such as c-kit, GM-CSF receptor and interleukin 3 receptor et al. Finally, in order to understand the in vivo hematopoietic capacity of the ES cells-derived HPP-CFC, spleen colony-forming unit (CFU-S) assay was performed. Nevertheless, typical CFU-S was not observed after transplantation of the day 12 EB cells or HPP-CFC colonies into lethally irradiated adult murine. In conclusion the HPP-CFC differentiated from murine ES cells displayed robust hematopoietic activity in vitro, however their in vivo reconstitution ability was not detected. The difference between in vitro and in vivo hematopoietic activities of ES cells-derived primitive hematopoietic precursors deserves further investigation.
Animals ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; Cell Differentiation ; genetics ; physiology ; Colony-Forming Units Assay ; Core Binding Factor Alpha 2 Subunit ; genetics ; Embryonic Stem Cells ; cytology ; GATA2 Transcription Factor ; genetics ; Hematopoietic Stem Cells ; cytology ; metabolism ; Humans ; Mice ; Proto-Oncogene Proteins ; genetics ; Proto-Oncogene Proteins c-kit ; genetics ; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; Receptors, Interleukin-3 ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; T-Cell Acute Lymphocytic Leukemia Protein 1

OBJECTIVETo investigate the relationship between the clinical features, pathogenesis, immunophenotype, different classification models and prognosis in Chinese patients with diffuse large B-cell lymphoma (DLBCL).

METHODSA total of 147 patients with DLBCL who were treated with CHOP-like or R-CHOP were subjected to analysis. Standard two-step Envision method of immunohistochemical staining was used to assess the expression of CD10, Bcl-6, MUM1, FOXP1, GCET1, CD5, Bcl-2, Ki-67, then according to Hans algorithm, Choi algorithm and Molecular markers, we compared the differences of their prognoses.

RESULTS(1) Kaplan-Meier univariate analysis of the clinical data of 147 DLBCL patients found that the 3-year overall survival (OS) rates were better in early stage (P=0.032), low IPI score (P=0.001), less than one extranodal involvement (P=0.014), and complete remission (P<0.01). The prognoses had no significant difference in terms of gender, age, LDH, B symptoms and treatment options (P value> 0.05). (2 )For Hans model, GCB group had 42 cases, the ABC group 85 cases; GCB were 47 cases, ABC 80 cases (according to Choi model). Choi model suggested GCB subtype showed much better prognosis than ABC subtype (P=0.047), while Hans model shed no statistically significant difference (P=0.285). (3) Ki-67 of 75% was found to significantly discriminate patients with good or bad prognosis. In R-CHOP group at the same time, low Ki-67 (P=0.017) and CD5-negative groups (P=0.012) were better. Cox proportional hazards regression model showed that IPI score (P=0.002) and Ki-67 (P=0.019) were independent adverse prognostic factors.

CONCLUSIONThe Ann Arbor stage, IPI score, extranodal involvement status and Ki-67 were significantly associated with prognosis .Compared to Hans algorithm, Choi had an advantage to predict the different prognosis between subtypes, and ABC group had poor outcome. Finally, both Ki-67 and IPI score were independent adverse prognostic factors.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Immunophenotyping ; Lymphoma, Large B-Cell, Diffuse ; diagnosis ; pathology ; therapy ; Male ; Middle Aged ; Prognosis ; Young Adult

Aim To investigate the protective effect of Qi ZhiXiaoke granules ( QZXK ) on nerve injury using zebrafish and nerve cell injury models. Methods The nerve injury model was established using wild zebrafish AB line, 72 hours after fertilization treated with 1-methyl-4-phenyl-1 , 2 , 3 , 6-four pyridine ( MPTP ) .Then QZXK of different doses were administered for three days,and the trajectory of the zebrafish behavior was recorded and analyzed. Neuroblastoma PC12 cells were incubated with different concentrations of QZXK and MPTP,and the cell viability of PC12 cells was de-tected by MTT. The mitochondrial membrane potential and expression of apoptosis related protein Caspase3 were measured by kits. Results Compared with con-trol group,MPTP reduced the movement distance of ze-brafish,and with the increase of concentration, QZXK promoted the movement distance and reversed the swimming behavior abnormality of zebrafish. Compared with control group, QZXK could inhibit the apoptosis induced by MPTP and promote the cell viability of PC12 cells with MPTP. QZXK improved the membranepotential and decreased the expression of Caspase3 . Conclusions QZXK exerts neuroprotective effect in the process of nerve injury induced by MPTP. The mechanism may be related with inhibiting apoptosis of neural cells. These experiment provides experimental and theoretical foundation for QZXK promoting cogni-tive function.

OBJECTIVETo evaluate the efficacy and safety of dose-reduced intravenous busulfan, cyclophosphamide and etoposide (BCV) as conditioning for autologous stem cell transplantation (ASCT) in multiple myeloma (MM).

METHODSFrom September 2007 to September 2010, thirty-two ASCT-eligible patients with MM received high dose melphalan (HDM) as conditioning in our center. Median age was 53.5 (30-63) years. From October 2010 to October 2012, thirty-eight patients conditioned by BCV regimen (intravenous busulfan, total doses 9.6 mg/kg), whose median age was 54(35-64) years.

RESULTSThere were no statistical differences in clinical characteristics between the two groups, including myeloma isotype, Durie-Salmon staging, international staging system(ISS), and patients received the first line, second line or more than third line therapy. The median time to neutrophil and platelet engraftment were 10.5 vs 11 days (P=0.057) and 11 vs 12 days (P=0.100) in the BCV and HDM groups, respectively. The toxicity of two conditioning regimens had no significant difference. None of hepatic veno-occlusive disease and early transplant related mortality was observed. Although overall response rates showed no significant difference between two groups (P>0.05), the CR rates increased from 44.74% pre-ASCT to 63.18% post-ASCT in the BCV group, while 37.50% to 59.38% in the HDM group. During the median follow-up of 16 months (range 2-27) in BCV group, ten patients (26.32%) developed progressive disease and PFS at 12 months were 71.37%.

CONCLUSIONSIn this study, the dose-reduced intravenous busulfan, cyclophosphamide and etoposide (BCV) conditioning was demonstrated an effective and safety regimen for ASCT-eligible patients with MM. However, the long term observation is needed.

Adult ; Busulfan ; therapeutic use ; Cyclophosphamide ; therapeutic use ; Etoposide ; therapeutic use ; Female ; Follow-Up Studies ; Hematopoietic Stem Cell Transplantation ; Humans ; Male ; Melphalan ; therapeutic use ; Middle Aged ; Multiple Myeloma ; therapy ; Transplantation Conditioning ; Transplantation, Autologous ; Treatment Outcome

BACKGROUNDTumstatin is a novel endogenous angiogenesis inhibitor which is widely studied using purified protein. The current study evaluates the antiangiogenic effects of tumstatin-overexpression plasmid in vitro, reveals the mechanism underlying the vascular endothelial cell growth inhibition and searches for a novel method administering tumstatin persistently.

METHODSThe eukaryotic expression plasmid pcDNA-tumstatin encoding tumstatin gene was constructed and transfected to human umbilical vein endothelial cell ECV304 and human renal carcinoma cell ACHN. Expression of tumstatin in the two cell lines was determined by RT-PCR and Western blotting. Vascular endothelial cell proliferation was assessed by CCK-8 assay and cell cycle was analyzed by flow cytometry. To investigate the mechanism by which pcDNA-tumstatin inhibited vascular endothelial cell proliferation in vitro, cyclin D1 protein was detected by Western blotting.

RESULTSDNA sequence confirmed that pcDNA-tumstatin was successfully constructed. RT-PCR and Western blotting indicated that tumstatin could express in the two cell lines effectively. After tumstatin gene transfer, ECV304 cell growth was significantly inhibited and the cell cycle was arrested in G1 phase. And Western blotting showed that pcDNA-tumstatin decreased the level of cyclin D1 protein.

CONCLUSIONSOverexpression of tumstatin mediated by pcDNA 3.1 (+) specially inhibited vascular endothelial cells by arresting vascular endothelial cell in G1 phase resulting from downregulation of cyclin D1 and administration of tumstatin using a gene therapy might be a novel strategy for cancer therapy.

Autoantigens ; genetics ; metabolism ; Blotting, Western ; Cell Cycle ; genetics ; physiology ; Cell Line ; Cell Line, Tumor ; Cell Proliferation ; Collagen Type IV ; genetics ; metabolism ; Endothelial Cells ; cytology ; metabolism ; Flow Cytometry ; Humans ; Plasmids ; genetics ; Reverse Transcriptase Polymerase Chain Reaction