【Objective】 To clone the squalene synthase gene of Artemisia annua L. for improving the quality and production of Artemisia annua L. by genetic engineering. 【Methods】 PCR amplification, RT-PCR amplification, ligation of the target fragment with a T-vector and sequence analysis of the interested gene were performed. 【Results】 An expected 3590 bp fragment was amplified by PCR and an expected 1257 bp fragment was amplified by RT-PCR. The two cloned fragments were identified by PCR and restriction enzyme digestion respectively. The preliminary sequence data indicated that the results obtained were similar to that from GenBank, and the difference was only found in several base pairs. 【Conclusion】 The squalene synthase gene and cDNA of Artemisia annua L. were successfully cloned and sequenced.

Objective To lay the foundation for studying the synthesis of artemisinin in microorganism,squalene synthase(SS) gene,a key enzyme gene from Saccharomyces cerevisiae,was cloned and a yeast expression vector was constructed.Methods After amplification of SS gene by polymerase chain reaction(PCR),ligation to T-vector and analysis of the cloned sequence,enzyme digestion and reconfirmation of the target gene,the antisense yeast expression vector was constructed by inverted insertion of the target gene into a yeast expression vector,pGAPZ?A,and digested with two restriction enzymes for vertifying the recombinant.Results The length of SS gene was 1335bp.The preliminary sequence data indicated that SS gene obtained from the experiment had a high sequence homology with that from GenBank,except for a few base pairs.The antisense yeast expression vector has been constructed and vertified by digesting with two enzymes.Conclusion SS gene from Saccharomyces cerevisiae has been successfully cloned and sequenced.An antisense yeast expression vector has been also constructed.

Objective To investigate the expression patterns of artemisinin biosynthetic genes in Artemisia annua L.during the development stage and in different tissues,and to explore the mechanism of spatial and temporal modulators for artmisinin production.Methods The transcriptional profiles of artemisinin biosynthetic genes in the capital and cooperative pathways were quantitatively assayed by real time fluorescence quantitative-polymerase chain reaction(RTFQ-PCR) in different tissues of roots,stems,leaves and flowers,and in leaf-flourishing,pre-floral,and post-floral periods.Results The expression levels of the tested genes were extremely low in June,raised in July,and reached their peak values in August(before flowering),but dropped gradually in September(after blooming).In August,the transcription levels of the tested genes increased by 3 to 15 times compared to the lowestlevels.In particular,ADS and CYP71AV1 mRNA levels had the great elevation,which were 12 and 15 times as much as those in June.During the flowering period,the artemisinin biosynthetic genes mRNA expression was detectable in roots,stems,leaves and flowers,and the expression levels had no obvious difference except that ADS mRNA level in leaves was 2 times higher than that in other tissues(P

This study was to explore a better three-dimensional (3-D) culture method of chondrocyte. The interpenetrating network (IPN) gel beads were developed through a photo-cross linking reaction with mixed barium ions and calcium ions at the ratio of 5:5 with the methacrylic alginate (MA), which was a chemically conjugated alginate with methacrylic groups. The second generation of primary cartilage cells was encapsulated in the MA gel beads for three weeks. In the designated timing, HE stain, Alamar blue method and Scanning electron microscopic were used to determine the cartilage cells growth, proliferation and the cell distribution in the scaffolds, respectively. The expression of type II collagen was investigated by an immunohistochemistry assay and the glycosaminoglycan content was quantitatively evaluated with the spectrophotometry of 1, 9 dimethylene blue assay. Compared to the alginate control group, the deposition of glycosaminoglycan was significantly upregulated in IPN-MA gel beads with higher cell proliferation. The secretion of extracellular matrix and proliferation of chondrocyte in methacrylic alginate gel beads were higher than that in Alginate beads. Cells were able to attach, to grow well on the scaffolds under scanning electron microscopy. The result of immunohistochemistry staining of collagen type II was positive, confirming the maintenance of chondrocyte phenotype in methacrylic alginate gel beads. This study shows a great potential for three-dimensional culture of cartilage.
Alginates ; chemistry ; Barium ; chemistry ; Calcium ; chemistry ; Cartilage ; cytology ; Cations ; Cell Culture Techniques ; instrumentation ; Cells, Cultured ; Chondrocytes ; cytology ; Collagen Type II ; chemistry ; Glucuronic Acid ; chemistry ; Glycosaminoglycans ; chemistry ; Hexuronic Acids ; chemistry ; Metals ; chemistry ; Microscopy, Electron, Scanning

Objective To explore the feasibility of utilizing the cytosine deaminase A (CodA) gene as an effective negative selectable marker in Artemisia annua for gene targeting.Methods The PCR procedure was employed to amplify CodA gene from Escherichia coli.After being cloned and sequenced, the gene was inserted into a plant expression vector, pROKⅡ, and then introduced into Agrobacterium tumefaciens LBA4404 (pAL4404).The leaf disks of A.annua were transformed by the co-cultivation protocol, after which the transformed calli were selected and green shoots of A. annua were regenerated on N6 medium supplemented with 25 ?g/mL Kanamycin (Kan).Then the Kan-resistant transgenic shoots were transplanted onto the MS medium containing 500 ?g/mL 5-fluocytosine (5-FC) plus 25 ?g/mL Kan and continuously cultured for up to two weeks.Results The transgenic shoots have totally died while untransformed shoots still kept normal growth, indicating that A.annua cells introduced into the CodA gene had conferred an expected negative selection phenotype.When detected by RT-PCR, the transgenic shoots displayed a CodA-positive amplified band, but untransformed shoots gave no such CodA-specific amplified pattern.This result suggested that CodA gene had transcribed into corresponding mRNA in A.annua cells with furtherly verifying the result of phenotypic assay.Conclusion The CodA gene can be utilized as an effective negative selectable marker in A.annua for gene targeting.

Objective To increase artemisinin yield in transgenic Artemisia annua plants by regulating metabolic affluxion through metabolic pathway engineering. Methods The gene targeting vector was constructed by squalene synthase (SS) gene of A. annua, green fluorescent protein (GFP) gene, and cytosine deaminase (CodA) gene, and the vector was introduced into Agrobacterium tumefaciens by freeze-thawing procedure. A. annua was transformed through Leaf Disk method and regenerated transgenic plants were screened by the “Step-by-Step Selection”. Results Among the transgenic A. annua plants emitting green fluorescence after expression of GFP gene, the exogenous GFP gene rather than endogenous SS gene was detected in one transgenic plant by PCR as well as hybridization of PCR products. The preliminary data showed that the wild-type SS gene was replaced by mutated SS gene in the transgenic A. annua plant. Conclusion Gene targeting of squalene synthases of A. annua is successful.

Objective To investigate the effect of hepatocyte growth factor (HGF) on mitral regurgitation caused by acute myocardial infarction in canines.Methods The acute myocardial infarction model was established by ligating proximal left anterior descending coronary artery (LAD) with 13 hybrid canines. The myocardial infarction model was successfuly established in 12 animals and those were randomly divided into the HGF-group and control group with 6 animals in each group. Canines of the HGF-group were injected with pc-DNA3-HGF 1 ml (about 300 μg) at the margin of infarcted myocardial and animals of the control group were injected with equal volume saline. The data were measured through echocardiography in the 1st, 4th and 8th week after ligation as following parameters: left ventricular ejection fraction (LVEF), left ventricular end-systolic volume (LVESV), left atrial area, mitral regurgitation area and the ratio of left atrial area to mitral regurgitation area. Left ventricular myocardium specimens were obtained in the 8th week after ligation and stained with hematoxylin and eosin for histological examination or with picrosirius red staining to assess the collagen content.Results Compared with the control group, LVEF in the HGF-group increased in the 4th week after ligation; LVEF significantly improved and LVESV decreased in the 8th week after ligation ( P<0.05). In the 8th week after ligation, left atrial area, mitral regurgitation area and the ratio of left atrial area to mitral regurgitation area in the HGF-group were lower than that in the control group. In the HGF-group, neovascularization and fewer scars were observed histologically. Compared with the control group, the HGF-group showed higher capillary density in margin of infarcted area by factor Ⅷ-related immunohistochemistry staining ( P<0.01).Conclusion HGF gene can improve cardiac function and relieve mitral regurgitation after acute myocardial infarction by stimulating angiogenesis, reducing fibrosis, diminishing myocardiolysis and scarring.

BACKGROUND:A number of studies have shown that bone marrow mesenchymal stem cells can survive in the infarcted myocardium and improve cardiac function. OBJECTIVE:To investigate the effects of al ogeneic rat bone marrow mesenchymal stem cells on heart failure in acute myocardial infarction models of rats and possible mechanisms. METHODS:Rat bone marrow mesenchymal stem cells were isolated from the bone marrow of 39 male Wistar rats by density gradient centrifugation with Percol . After ligating anterior descending coronary artery, 39 female Wistar rats were randomly divided into three groups:control group (Dulbecco’s modified Eagle’s medium, n=12), mesenchyma stem cells group (n=15) and mononuclear cells group (n=12). Eight weeks later, hemodynamics and left ventricular function were measured. Histopathological and immunohistochemical examinations were performed. RESULTS AND CONCLUSION:Compared with the control group, left ventricular end diastolic pressure, left ventricular relative weight, the col agen volume fraction of type I and type III in the infarction zone of the left ventricle were al significantly decreased, in contrast to ±dp/dtmax,-dp/dtmax/left ventricular systolic pressure, body weight and vascular density in infarction zone were al significantly increased both in mesenchymal stem cells group and mononuclear cells group. There were no significant differences between two treatment groups except for interventricular septal thickness and vascular density in non-infarction zone. 5-Bromo-2'-deoxyuridine positive cells were observed in the infarction area of mesenchyma stem cells group but no positive cells in mononuclear cells group. Some bal-like cellmasses were found positively stained with desmin and cardiac troponin T. Results have suggested that embedded bone marrow mesenchymal stem cells survived in exogenous host hearts. The therapy of mononuclear cells and mesenchymal stem cells could limit the left ventricular remodeling after acute myocardial infarction and improve left ventricular function through angiogenesis inducing and col agen deposition decreasing.

Object To explore the feasibility of breeding genetic modified (GM) medicine by expressing human cytokine in transgenic Chinese materia medica Methods Human interferon ? gene and RANTES gene available from the amplification in vitro were enzymatically excised, recoveried, and inserted into intermediate vectors The recombinants were identified by double enzyme digestion of EcoRⅠand HindⅢ The plasmids were extracted from Escherichia coli and introduced into A tumefaciens, and the transformants harboring binary vectors were screened by addition of antibiotics of kanamycin (Km) and rifampicin (Rif), and the explants of M charantia and P vulgaris were transformed by co cultivation of leaf disks with A tumefaciens strain Results RT PCR was applied to detect the transient expression of human interferon ? gene and RANTES gene in transformed medicinal herbal calli Conclusion The expression of recombinant human interferon ? gene and RANTES gene in transgenic M charantia and P vulgaris cells was firstly reported, which opens an alternative road to antivirus, especially anti AIDS virus, by using transgenic Chinese materia medica

Objective To explore the value of MRI-R2 * and to compare clinical effect of two iron chelators(deferasirox and deferoxamine) in iron-overloaded patients.Methods By completely randomized balanced design,24 iron-overloaded patients were randomly divided into 2 groups,which consisted of 12 patients treated with deferasirox and 12 patients treated with deferoxamine.The planned deferasirox dose was 40 mg· kg-1 · d-1,and the deferoxamine dose was no less than 50 mg · kg-1 · d-1 All patients underwent quantitative MRI at the time points of the primary screening,6 months and 12 months.Pair Wilcoxon rank sum test was used to compare the differences of liver R2 * values of the 2 groups at various time points respectively.Wilcoxon rank sum test was used to compare the differences of change rate of liver R2 * values between the two groups at the time point of 6 months,12 months,respectively.Results Deferasirox group's liver R2 * values of primary screening,6 months and 12 months were 1081,889 and 712 Hz,while deferoxamine group's liver R2 * values were 1042,838 and 488 Hz.There was no statistically significant difference between liver R2 * values of two groups at primary screening (Z =-0.029,P ＞ 0.05).The change rate of liver R2 * of deferasirox group at 12 month was-32％,while it was-58％ for the deferoxamine group,and there was statistically significant difference between the two groups (Z =-3.060,P ＜0.01).The change rate of serum ferritin of deferasirox group at 12 month was-15％,while it was -55％ for the deferoxamine group,and there was statistically significant difference between the two groups (Z =-2.945,P ＜ 0.01).Conclusion By using MRI-R2*,it suggest that both deferasirox and deferoxamine can effectively remove liver iron and deferoxamine is superior to deferasirox.