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<p><b>OBJECTIVE</b>To establish a culture system for transgenic Salvia miltiorrhiza hairy roots.</p><p><b>METHOD</b>Investigated the success rate of different explants, different infection time and different co-culture time to induce hairy roots of S. miltiorrhiza. Co-cultured explants were sterilizated with 400 g x L(-1) Cef water for 5 min, inoculated on MS solid medium supplied with 400 mg x mL(-1) cef and 2.5 g x L(-1) Hyg, and then transfered to the 67-V liquid medium with 2.5 g x L(-1) Hyg after complete sterilization. GFP fluorescence detection was performed to detect positive hairy root lines. PCR method to detect rolC gene which is the specific gene of hairy root. Biomass was determinated in different growth periods of root lines. HPLC was conducted to measure the content of dihydrotanshinone I of transgenic hairy roots.</p><p><b>RESULT</b>Leaf base of S. miltiorrhiza was used as a perfect explant to Induce hairy roots, the success rate can reach 93.3%. Inducing efficiency was up to 63.3% after Agrobacterium infection for 10 min. Co-culture for 2-3 d can reach the best induced effect. It is a high credibiliy to use PCR method combined with detection of GFP fluorescence to identified positive transformants. There is a close contact between biomass increases and secondary metabolite accumulation of transgenic hairy roots.</p><p><b>CONCLUSION</b>Successfully in vitro culture system has been established in transgenic S. miltiorrhiza, and this research can lay foundations for the further genetic engineering applications.</p>
Subject(s)
Cells, Cultured , Culture Media , Metabolism , Plant Proteins , Genetics , Metabolism , Plant Roots , Genetics , Metabolism , Salvia miltiorrhiza , Genetics , Metabolism , Tissue Culture Techniques , MethodsABSTRACT
<p><b>OBJECTIVE</b>A novel bHLH-like gene, designated SmbHLH1, was isolated from Salvia miltiorrhiza, in order to identify a bHLH gene in related to danshinone biosysnthesis.</p><p><b>METHOD</b>SmbHLH1 was isolated by RT-PCR,and Semi-quantitative RT-PCR was used to detect the gene expression level.</p><p><b>RESULT</b>The full length of SmbHLH1 cDNA has an open reading frame of 999 bp. The deduced amino acid sequence of SmbHLH1 has 332 amino acid residues which forms a 36 kDa polypeptide with a calculated pI of 5.4. SmbHLH1 gene was expressed at high level in root, but low level in stem, leaf and flower of S. miltiorrhiza. The transcripts of SmbHLH1 was suppressed when the plants were treated with exogenous MeJA, Yeast + Ag+. The transcripts of SmbHLH1 constitutively accumulated in response to exogenous ABA and low concentration of salicylic acid.</p><p><b>CONCLUSION</b>SmbHLH is a new member of the S. miltiorrhiza bHLH family, and its possible roles in brassinosteriods signaling responses.</p>
Subject(s)
Basic Helix-Loop-Helix Transcription Factors , Genetics , Physiology , Cloning, Molecular , Plant Proteins , Genetics , Physiology , Salvia miltiorrhiza , GeneticsABSTRACT
Objective To study the apoptosis of gallbladder carcinoma cell line GBC-SD induced by antisense oligodeoxynucleotide (ASODN) targeting survivin. Methods ASODN targeting survivin was transfected into GBC-SD cells mediated by lipofectin. Cultured cells were divided into 3 groups: control group,sense oligonucleotide (SODN) group and ASODN group. After transfected for 16 h, the cultured cells were harvested and the following texts were carried out. The expression of survivin mRNA was detected by RT-PCR. Flow cytometer were used to detect apoptosis. Morphological changes were observed by electron microscopy. Results The expression of survivin mRNA was decreased 47.83% in ASODN group while apoptosis was increased from (0.50?0.23)% to (26.28? 3.91)%. Abnormal morphological changes of cells were observed in ASODN group and apoptosis bodies were found in some gallbladder carcinoma cells. Conclusion The expression of survivin may be decreased in GBC-SD cells after ASODN transfection.ASODN targeting survivin could induce gallbladder carcinoma cells apoptosis effectively.
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Objective To study the expression of nuclear factor-?Bp65 (NF-?Bp65) in gastric carcinoma and its relationship with vascular endothelial growth factor (VEGF). Methods The expression of NF-?Bp65 and VEGF in 56 gastric carcinomas was detected with immunohistochemistry and compared with benign tissues. Results The positive rates of NF-?Bp65 and VEGF in 56 gastric carcinomas were 62.5% and 76.8% respectively,and which were higher than those of gastric mucosal atypical hyperplasia (33.3% and 44.4%) and the normal gastric mucosa(0 and 8.3%) (P0.05). There was positive correlation between NF-?Bp65 and VEGF expression (r=0.36,P
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Objective:To investigate the expression of lysozyme in ampullary carcinoma, and its relation with invasion, metastasis and prognosis. Methods: 36 cases were analyzed with immunohistochemistry of lysozyme. Results: The lysozyme-positive rate in patients with lymph node metastasis (30%) was significantly lower than that in patients without metastasis (71%), while that in patients with advanced cancer invading the pancreas (35%), was significantly lower than that in patients with cancer without invasion of the pancreas. The Survival rate of lysozyme-positive patients (66.7%) was significantly higher than that of lysozyme-negative patients (14.3%). Conclusion: The expression of lysozyme in carcinoma of ampulla is associated with invasion, metastasis and prognosis. Lysozyme is important in typing of cancer of ampulla.
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Objective :To investigate expression of E-Cadherin(E-CD) in breast cancer cells and its relationship to clinical and pathological character and to the incidence of tumor cells shed from the surgical field. In an attempt to study more deeply about the mechanisms of invasion and metastasis of breast cancer and give some reference to the diagnosis and treatment of breast cancer. Methods: The expressions of breast carcinoma cells were studied using immunohistochemical methods(ABC methods).Semi-quantitative methods were used to judge the results. For statistical analysis, Chi-square test and the exact probability in 2 ?2 table were used. Results : E-CD positive was located on cell membranes and cytoplasms, mainly on membranes, showing different intensity of brown pellet. Of 52 cases, twenty nine percent retained normal E-CD expression, seventy one percent have reduced or even none expression. E-CD expression has significant correlation with histological grade. Reduced and lost expression rate in Grade 1(36.4%,4/11) were much lower than grade 2(77.8%,28/36)and grade 3(75%,6/8) (p
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Objective : The patients with inguinal hernia were treated with plug mesh hernia repair. Methods:The hernia sacs were isolated and dissected back to the internal ring. The unoperated sacs were allowed to drop back through the internal ring into the abdominal cavity. A cone-shaped mesh hernia plug is inserted tapered end through ring and placed into position just beneath the crura. All our repairs were reinforced a second piece of flat was placed from the pubic tubercle, overlying the direct space. Results : Com-paired with conventional suture surgical techniques, a plug repair uses less disscection and ensures tenssion free hernioplusty. Conclusion : We believe that the two factors are the most important reseasons for greater patients confort, rapit rehabilitation, decreased recurrence and lessened overall complication rates with the mesh hernia plug techniques.
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Objective:To study the expression of NF-?Bp65 and bcl-x L gene in pancreatic carcinoma and their clinical significance.Methods:The expressions of NF-?Bp65 and bcl-x L protein in 52 pancreatic carcinoma were studied by immunohistochemistry,and their relationship to clinical pathology were analyzed.Results:The positive rates of NF-?Bp65 protein and bcl-x L protein in pancreatic carcinoma were 63.5% and 73.1% respectively,and which were higher than those of normal pancreatic tissues.The expression of NF-?Bp65 was related to tumor diameter,TNM stage and lymph node metastasis,and had no relation with the extent of differentiation and type of pathology.There was positive correlation between NF-? Bp65 and bcl-x L expression.Conclusion:NF-? Bp65 may be an important oncoprotein,and may have positive regulation on bcl-x L,then promote tumorgenesis.
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Objective:To observe the expression of Th1 versus Th2 type cytokines in primary hepatic cancer(PHC)and its adjacent liver tissues.Methods:The gene expression of Th1/Th2 cytokines was detected by RT-PCR using IFN-?and IL-2 as Th1 type cytokine genes,IL-4 and IL-10 as Th2 type cytokine genes.Results:Thl type cytokines were expressed in 7 and 9 cases,while Th0 type cytokines in 4 and 2 among 11 PHC and their adjacent liver tissues,respectively.Conclusion:Th1 type cytokines are expressed predominantly in primary hepatic cancer and its adjacent liver tissue.
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Objective: To set up the animal model of precancerous lesion of rats' pancreatic acinar cellular cancer,and to learn estrogen's effect on this lesion's biological feature and K-ras gene mutation. Methods: male Wistar rats were injected Azaser-ine 30mg/kg(ip). Some rats received Estradiol-17 P Valerate(0.18mg/100g/2weeks, im) .4 months later, rats'pancreas were autopsied and K-ras gene mutation were detected by PCR-SSCP method. Results : The incidence of AACN were very high. The incidence of K-ras gene mutation was 42.1% in rats without estrogen and 25.6% with estrogen. Conclusion: Azaserine could induce precancerous lesion of rats' pancreatic acinar cancer successfully, K-ras gene mutation was early change during this process Estrogen could inhibit this precancerous lesion's happen and development to some degree.
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ObjectiveTo evaluate the efficacy and feasibility of extraperitoneal approach (EPA) for the resection of primary retroperitoneal tumors (PRT). MethodsForty six cases undergoing resection of PRT were analyzed retrospectively, of which, 26 cases were through transabdominal approach (TAA group) and 20 through EPA. ResultsThe postoperative complications in EPA group was lower than in TAA group (2/20 vs. 11/26, P
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Objective: To investigate the effect of survivin and bcl-2 antisense oligodeoxynucleotides ( AsODN) combined transfection on the human gallbladder carcinoma cell line GBC-SD in vitro. Methods: Survivin and Bcl-2 protein expressions were detected by immunohistochemical method; Cultured cells were divided into 4 groups: Nomal control group, survivin antisense observed group, bcl-2 antisense observed group and combined group. After transfected for 24 h, the expression of survivin mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). Cell morphological changes were observed under electron microscopy. Apoptosis index (AI) was examined by flow cytometry; Inhibitory rate (IR) was determined by the colorimetri MTT cell viability and proliferation assay. Results: Survivin and Bcl-2 protein were highly expressed in gall bladder carcinoma cells; The expression of survivin mRNA was decreased 47. 8%. Abnormal morphological changes of cells were observed in the three AsODN transfection groups; The AI in survivin antisense observed group,bcl-2 antisense observed group,and combined group was 11.38%?3.91% , 9.26%?4.15% , 28.45%?6.34% respectively and significantly higher than the nomal control group (P
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Objective To study the effect of ribozyme mediated inhibition on telomerase activity and cell(proliferation) of GBC-SD cells.Methods According to the hTR template two wings region sequence,the hammerhead ribozyme gene sequence was synthesized in vitro.Then,it was engineered into the eukaryon(expression) vector pTriEx-4.Lipofectamine was used to transfect the carcinoma of gallbladder.The inhibition of telomerase activity and cell proliferation in GBC-SD cell was observed,and compared with control group.(Results) Compared to control group,ribozyme can significantly inhibit telomerase activity and cell proliferation of GBC-SD cell.After ribozyme action,the cell ratio of G_0/G_1 markedly increased,and the cell ratio of S phase markedly decreased.Cellular cleavage and proliferation was markedly inhibited.The apoptosis rate was 17.10% and 31.01% on day10 and day13 after ribozyme action.Conclusions Ribozyme can inhibit(telomerase) activity of GBC-SD cells effectively and induce cell apoptosis.