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(3S)-Linalool synthase (LIS) is a key enzyme involved in the monoterpene biosynthetic pathway. Based on our previous transcriptome study, the expression level of LIS gene was exceedingly related to glycyrrhizic acid (GA) biosynthesis. Therefore, we used hairy root culturing to further investigate the effect of LIS on the GA biosynthesis. A LIS gene (GenBank accession number: MZ169552) was cloned from Glycyrrhiza uralensis. The plant binary overexpression vector pCA-LIS was constructed by gene fusion. G. uralensis hairy roots overexpressing LIS were induced by the Agrobacterium rhizogenes ATCC15834. The expression levels of LIS were analyzed by real-time quantitative PCR (RT-qPCR) and the contents of GA in hairy root lines were determined by UPLC. It was found that in the hairy root lines overexpressing LIS, the expression levels of LIS were significantly higher than that in the wild type, while the contents of GA were remarkably lower than those in the wild type and negative control. These findings indicate that the expression level of LIS is negatively correlated with the accumulation of GA. In this study, LIS was cloned from G. uralensis for the first time and the negative regulatory effect of LIS on GA biosynthesis was confirmed by reverse genetics. This work provides support for further improvement of the molecular regulatory network of GA biosynthesis in G. uralensis.
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1-Deoxy-D-xylulose-5-phosphate synthase (DXS) is a rate-limiting enzyme involved in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for terpenoid precursor biosynthesis. DXS plays an essential role in glycyrrhizic acid (GA) biosynthesis. Based on our previous transcriptome study, there was a negative correlation between DXS expression and GA content. Therefore, we explored the regulatory role of DXS in GA biosynthesis using both gene overexpression and gene knockout in a hairy root culture system. DXS was cloned from Glycyrrhiza glabra L. (GenBank Accession No. MN158121). A plant binary expression vector pCA-DXS was constructed by a gene fusion method. The sgRNA sequence was designed based on the first exon of DXS to construct the gene editing vector pHSE-DXS. Hairy roots overexpressing or knocking out DXS were generated through an Agrobacterium-mediated method with licorice hypocotyls as explants. Wild-type hairy roots and negative control hairy roots containing empty plasmids were also evaluated. UPLC was used to determine the GA content in each licorice hairy root line. Results showed that the content of GA in the hairy root group knocking out DXS was significantly higher than that in the wild-type and negative control groups, while in the hairy root group overexpressing DXS was significantly lower, suggesting that DXS plays a negative role in GA biosynthesis. This study provides a foundation for determining the function of DXS in terpenoid metabolism and for further establishment of a molecular regulatory network of GA biosynthesis.
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Chalcone isomerase (CHI) is the second rate-limiting enzyme involved in the biosynthetic pathway of flavonoids in Glycyrrhiza uralensis. Based on our previous studies, we selected the specific CHI haplotype (GenBank Accession No. KY115232) to maximize flavonoid accumulation. We constructed a plant binary expression vector for overexpression of this CHI gene by the gene fusion method and transfected the plasmid into Agrobacterium tumefaciens ACCC10060 by electroporation. The recombinant A. tumefaciens ACCC10060 subsequently was used to infect cotyledons and hypocotyls of G. uralensis to obtain transgenic hairy roots. A qRT-PCR method was used to determine the copy number of CHI and a UPLC method was used to assay the content of four flavonoids in different hairy root lines. The qRT-PCR results showed that the copy number of CHI in hairy roots was 1 or 5. UPLC results showed that the content of total flavonoids, liquiritin, liquiritigenin, and isoliquiritigenin in transgenic hairy root samples was significantly higher than that in wild-type samples. This study demonstrates that overexpression of CHI significantly increases the content of flavonoids in hairy roots of G. uralensis. This work provides a theoretical basis for clarifying the function of CHI. Three transgenic hairy root lines of G. uralensis were isolated which can be used to increase the accumulation of licorice flavonoids in vitro.
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Ferulate 5-hydroxylase (F5H) is a key enzyme involved in the phenylpropane metabolism pathway. Based on our previous transcriptome sequencing study, F5H played a negative regulatory role in glycyrrhizic acid (GA) biosynthesis. Therefore, in this study we cloned the F5H gene and investigated its regulatory effect on GA accumulation through gene overexpression and knockout. F5H was cloned from Glycyrrhiza glabra L. (GenBank Accession No. MK882511). A plant binary expression vector pCA-F5H was constructed by inserting F5H into pCAMBIA1305.1 at Spe I and Bgl II sites. The sgRNA sequences were designed based on the first exon of F5H. The CRISPR/Cas9 gene editing vector pHSE-F5H was constructed by inserting F5H sgRNA into pHSE401 at two Bsa Ⅰ sites. PCA-F5H and pHSE-F5H were transfected into Agrobacterium tumefaciens ATCC15834, which was used to induce hairy root overexpressing or knocking out F5H with licorice hypocotyl as explants. At the same time, wild type and negative control hairy roots were also generated. UPLC was used to assay the GA content in different hairy root lines, and results showed that the GA content in hairy root lines knocking out F5H was significantly higher, whereas in hairy root lines overexpressing F5H GA content was lower than that in the wild-type and negative control. In this work, through a reverse genetics strategy, the negative regulatory effect of F5H on GA biosynthesis was confirmed through gene overexpression and knockout. This work will lay a foundation for further elucidation of the molecular regulatory network of GA biosynthesis.
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Objective::To clone the complementary deoxyribonucleic acid (cDNA) of auxin/indole acetic acid protein (Aux/IAA) from Glycyrrhiza glabra (GgARPI) and analyze its sequence by bioinformatics. Method::RNA was extracted from fresh root of G. glabra, the cDNA sequence of GgARPI gene was cloned by reverse transcription polymerase chain reaction (RT-PCR), then sequencing and bioinformatic analysis were performed. Result::The GgARPI cDNA sequence with the full length of 686 bp was obtained from G. glabra. The full open reading frame (ORF) was 585 bp, encoding 194 amino acid residues. The bioinformatic analysis showed that the protein coded by GgARPI was a stable hydrophilic protein, with a relative molecular weight of 21.95 kDa and isoelectric point of 6.85.It contained no signal peptides or transmembrane domain. Its secondary structure mainly consisted of random coil. An Aux/IAA superfamily was included in the conversed domain. Homology analysis indicated that it had a close evolutionary relationship with leguminous plants, and a distant evolutionary relationship with monocotyledon, such as Setaria italica. Conclusion::GgARPI cDNA sequence is successfully cloned from G. glabra for the first time, which will lay a foundation for studying the function of GgARPI and explaining the molecular regulatory mechanism of biosynthesis of glycyrrhizic acid in G. glabra.
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Objective::To clone the cDNA sequence of UDP-glucose 4-epimerase (UGE) in Glycyrrhiza glabra and analyze its sequence, so as to explore the potential relationship between the UGE gene and the molecular regulatory mechanisms of glycyrrhizic acid biosynthesis. Method::The cDNA sequence of UGE was cloned from the root of G. glabra by reverse transcription polymerase chain reaction (RT-PCR), then sequenced and analyzed by bioinformatics software. Results::A GgUGE cDNA sequence with the full length of 1 121 bp was obtained. The open reading fame (ORF) of GgUGE was 1 053 bp, encoding 350 amino acid residues. The GgUGE cDNA sequence was submitted to GenBank, and the accession No. was MK638908. Sequence analysis showed that GgUGE was an unstable hydrophilic protein, its average relative molecular weight was 39.02 kDa, and isoelectric point was 6.13. It contained no signal peptides or transmembrane domains. Its secondary structure mainly constituted of α-helix and had a conversed domain of UDP-glucose 4-epimerase superfamily. The homologoue analysis showed that the cDNA and amino acid sequences of GgUGE had the closest evolutionary relationship to Leguminosae and relatively distant evolutionary relationship to Salicaceae. Conclusion::In this study, GgUGE cDNA sequence is successfully cloned from G. glabra for the very first time, which will provide reference for studying the function of GgUGE and explaining the molecular regulatory mechanisms of glycyrrhizic acid biosynthesis in G. glabra.
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Sudden unexpected death means a "healthy" person died suddenly of unknown diseases,usually within 6 hours after onset. It is reported that sudden unexpected death occurred from neonates(sudden infant death syndrome)to adults(sudden adult death syndrome). The patients suddenly died during daily activities,sleep or exercise. Underlying genetic diseases are main cause of sudden death. The etiological studies are performed in the patients of sudden death. Heart attack and encephalopathy due to varied genetic disorders are the two major causes. Sudden cardiac death accounts for more than half. It is known that some inherited metabolic diseases associated with sudden death sometimes. Mitochondrial diseases are a group of inherited metabolic diseases. Some patients of mitochondrial diseases suddenly died of acute heart failure,malignant arrhythmia or encephalopathy. With the advancement of genetic technology,post-mortem genetic diagnosis became available in some cases. The definite genetic diagnosis is the key for the genetic counseling of the families and prenatal diagnosis of their fetuses.
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Objective To investigate the internal quality control(IQC) of hemoglobin A2 (HbA2) and hemoglobin F (HbF) from 2014 to 2017 in China.Methods The results of IQC were collected from the laboratories which participated in external quality assessment (EQA) of National Center for Clinical Laboratories (NCCL) from 2014 to 2017,then the coefficient of variation (CV) was compared with 1/3TEa (6.67 ％),1/4TEa (5 ％).The proportion of laboratories meeting criteria were calculated to analyze IQC of HbA2 and HbF in China.The data were grouped based on the instruments used in laboratories,the acceptable rates of CVs of HbA2 and HbF in each group under two criteria in 2017 were calculated,respectively.Results In HbA2,more than 84％ of participant laboratories met 1/3TEa criteria and 70.83％ ～84.47％ of laboratories met 1/ 4TEa criteria.In HbF except for 2015,the more than 80％ laboratories whose month and cumulative CVs met 1/3TEa and 1/4TEa criteria accounted for 68.42 ％ ～ 85.07 ％,respectively.Under 1/3TEa and 1/4TEa criteria,sebia capillarys 2 instru ment and fully automatic hemoglobin analyzer bole Variant Ⅱ instrument group the acceptable rates of CVs above 85％,showed good precision for HbA2 and HbF detection.Conclusion At present,the precision level of HbA2 and HbF need to be further improved in laboratories of China,especially HbF.Laboratory should continue to strengthen the internal quality control,establish strict internal quality system to improve detection capacity.
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Objective To investigate the current status of the coefficients variations (CVs) of internal quality control (IQC) data for serum procalcitonin in China.Methods Data had been collected by Web based submission system,the laboratories which enrolled in 2017 serum procalcitonin external quality assessment (EQA) program had attended.The data had includ ed:the CVs of two levels of IQC materials (level 1 and 2) in March of 2017 and long-term cumulative in control data.Mi crosoft Office Excel 2007 was used to analyze and process the data,the acceptable rates of CVs were calculated based on the 1/3TEa and 1/4TEa standards.The instruments which was used in laboratory internal quality control system of EQA,were grouped and counted,the acceptable rates of each group was calculated according to two evaluation standards.According to the laboratory detecting system was matched or not,to calculate the proportion of laboratories,and to adapt to the two standards.Results The acceptable rates of the same standards were close and the acceptable rates of level 2 were relatively higher.After grouping according to the instruments,the acceptable rates of each group were uneven.According to the labo ratory detecting system was matched or not,the acceptable rates of the matching system were much more higher.Conclusion To strengthen internal quality control system,and to improve the detection quality level much further.Laboratory should pay more attention to the mission of internal quality control,in order to ensure the reliability of test results.
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Pharmacometabolomics is a new and rapidly growing field in life science, which use metabolomics for studying drug effects and variation in drug response. Recently, it has been widely used in individualized medicine research. The research process of pharmacometabolomic can be divided into three parts: metabolomic study of baseline samples, drug response analysis after drug administration and statistical analysis. By combining the baseline information on metabotype of an individual with the drug response phenotype after drug exposure, pharmacometabolomic method can be used to predict the efficacy and toxicity of drugs, which providing the theoretical basis for individualized medical treatment. In this paper, we give an overview of present studies in the application of pharmacometabolomics for predicting the individualized drug response. Besides, we also summarized the specific research processes and pharmacometabolomic methods.
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Objective To study the mechanism of anti-atherosclerosis of astragaloside IV by observing the regulation of PI3K/Akt/mTOR signaling pathway. Methods In the in vitro experiments, macrophages were randomly divided into control group, astragaloside IV group, Kangshide group, rapamycin group and siRNA group. The changes of autophagy of macrophages were observed by transmission electron microscopy (TEM) after 48 h of mouse RAW264.7 macrophages in vitro treated by astragaloside IV-containing serum, Akt inhibitor Kangshide, mTOR inhibitor rapamycin and mTOR-siRNA. The expression of Akt, mTOR and autophagy-associated protein Beclin 1 was detected by real-time quantitative RT-PCR and Western blotting. The expression of Beclin 1 was detected by immunofluorescence and Western blotting. IL-4, IL-10, IL-2, and IFN-γ in RAW264.7 cells were detected by ELISA. In the in vivo experiments, the pathological changes of aorta were detected by HE staining. The expression of Akt, mTOR mRNA and protein in aorta of mice was detected by qRT-PCR and Western blotting, respectively. Results In vitro experimental results showed that compared with control group, the autophagus of astragaloside IV-containing serum group and each inhibitor group was significantly increased under the TEM (P < 0.05). The expression levels of LC3-II and Beclin1 were significantly up-regulated (P < 0.05). The expression of Akt and mTOR mRNA and protein was significantly decreased (P < 0.05). The secretion of IL-10 was significantly decreased and the secretion of IFN-γ was significantly increased (P < 0.05). HE staining results showed that, compared with the model group, the blood vessels of astragalus membranaceus group were normal, arranged neatly, with small focal calcification of the granules. The lesions were mild, the patches were small, the foam cells and the lipids were reduced, the elastic plates were basically complete, The degree of lesion was significantly lighter and lighter than that of the inhibitor groups. The expression of Akt, mTOR mRNA and protein were significantly lower in the aorta of astragaloside IV group (P < 0.05). Conclusion Astragaloside IV inhibition of atherosclerotic plaque formation mechanism and regulation of PI3K/Akt/mTOR signaling pathway, inhibition of inflammatory response.
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Diagnostic ions filter method was used to rapidly detect and identify the phenolic compounds in Rheum palmatum based on ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MSE). The representative authentic standards of phenolic compounds, including gallic acid, (+)-catechin, (－)-epicatechin, (－)-epicatechin-3-O-gallate and procyanidin B2, were subjected to analysis by UPLC-Q-TOF/MSE system with negative ion mode. Fragmentation patterns of each standard were summarized based on assigned fragment ions. The prominent product ions were selected as diagnostic ions. Subsequently, diagnostic ions filter was employed to rapidly recognize analogous skeletons. Combined with retention time, accurate mass, characteristic fragments and previous literature data, the structures of the filtered compounds were identified or tentatively characterized. A total 63 phenolic compounds (36 phenolic acid derivatives, 8 flavonoid derivatives and 19 tennis derivatives) in R. palmatum were identified, including 6 potential new compounds. The method of diagnostic ions filter could rapidly detect and identify phenolic compounds in R. palmatum This study provides a method for rapid detection of phenolic compounds in R. palmatum and is expected to complete the material basis of rhubarb.
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Most protocols for in vitro producing red blood cells (RBC) use the CD34(+) cells or embryonic stem cells from cord blood, bone marrow or peripheral blood as the start materials. This study was purposed to produce the mature RBC in vitro by using peripheral blood mononuclear cells as start material. The peripheral blood mononuclear cells (PBMNC) were isolated from buffy coat after blood leukapheresis, the mature red blood cells (RBC) were prepared by a 4-step culture protocol. The results showed that after culture by inducing with the different sets of cytokines and supporting by mouse MS-5 cell line, the expansion of PBMNC reached about 1000 folds at the end of the culture. About 90% of cultured RBC were enucleated mature cells which had the comparable morphological characteristics with normal RBC. Colony-forming assays showed that this culture system could stimulate the proliferation of progenitors in PBMNC and differentiate into erythroid cells. The structure and function analysis indicated that the mean cell volume of in vitro cultured RBC was 118 ± 4 fl, which was slight larger than that of normal RBC (80-100 fl); the mean cell hemoglobin was 36 ± 1.2 pg, which was slight higher than that of normal RBC (27-31 pg); the maximal deformation index was 0.46, which approachs level of normal RBC; the glucose-6-phosphate dehydrogenase and pyrurvate kinase levels was consistant with young RBC. It is concluded that PBMNC are feasble, convenient and low-cost source for producing cultured RBC and this culture system is suitable to generate the RBC from PBMNC.
Subject(s)
Animals , Mice , Bone Marrow , Cell Differentiation , Cell Line , Cytokines , Erythrocytes , Cell Biology , Erythroid Cells , Leukocytes, Mononuclear , Cell BiologyABSTRACT
It has been showed that there were obvious obstacle effects of Panax notoginseng replanting. Crop rotation was the main effective technique to overcome the obstacle. To find a reasonable crop rotation system for P. notoginseng, aqueous extracts from root, stem and leaf of P. notoginseng were analyzed for allelopathic effect on three maize varieties (which are often grown in regions where P. notoginseng grown). The main results were as follows: (1) Allelopathic effect of P. notoginseng stem and leaf extracts on the three other tested plants was stronger than that of root extracts; (2) Corn was more vulnerable to the effects of allelochemicals at seedling stage than at germination stage, and the corn root was more sensitive than aerial part to allelochemicals; (3) Lusan No. 3 and Yunrui No. 1 showed resistance to P. notoginseng allelopathy, with respective comprehensive sensitivity indexes (M3) of - 0.089 3 and -0.159 2, while Bainuo No. 1 is sensitive at M3 = -0.261 0. It then can be concluded that Lusan No. 3 and Yunrui No. 1 may be an alternative rotation plants for overcoming P. notoginseng continuous cropping obstacle.
Subject(s)
Allelopathy , Panax notoginseng , Chemistry , Pheromones , Pharmacology , Plant Extracts , Pharmacology , Plant Leaves , Chemistry , Plant Roots , Chemistry , Plant Stems , Chemistry , Zea maysABSTRACT
<p><b>OBJECTIVE</b>To explore the mechanism and effect of maternal high-fat diet before and during pregnancy on bone growth of neonatal offspring rats.</p><p><b>METHODS</b>Forty female Sprague-Dawley rats were divided into high-fat diet and control groups (n=20) that were fed with 35% high-fat diet and standard chow, respectively. After 8 weeks, 8 female rats from each group were sacrificed for liver pathological examinations and the other female rats were mated with male rats and fed continuously with 35% high-fat diet and standard chow throughout gestation, respectively. The body lengths (from apex nasi to end of tail) of the offspring rats from both groups were measured within 24 hours after birth. Enzyme-linked immunosorbent assay was used to detect serum insulin-like growth factor (IFG-I) levels. Liver pathological changes were observed under a light microscope. The expression of insulin receptor substrate 1 (IRS-1) and phosphorylation IRS-1 (Phospho-IRS-1) in tibia and femur samples were detected by immunohistochemistry. The expression of mitogen-activated protein kinase (MAPK) and phosphorylation MAPK (Phospho-MAPK), phosphatidylinositol 3-kinase (PI3K) and phosphorylation PI3K (Phospho-PI3K), protein kinase B (AKT1) and phosphorylation AKT1 (Phospho-AKT1) in tibia and femur samples were detected by Western blot.</p><p><b>RESULTS</b>The offspring rats from the high-fat diet group showed a significant shorter body length compared with those from the control group (P<0.05). The level of serum IGF-I in offspring rats from the high-fat diet group decreased by 20.1% in comparison to those from the control group, but there was no significant difference between the two groups (P>0.05). Fatty degeneration was found in livers of both high-fat diet-fed maternal rats and their offspring rats under a light microscope. There were no significant differences in IRS-1 and Phospho-IRS-1 expression in chondrocytes of tibia and femur samples between the offspring rats of the two groups (P>0.05). The protein expression of MAPK in chondrocytes of tibia and femur samples of offspring rats from the high-fat diet group was higher than that from the control group (P<0.05). There were no significant differences of PI3K and AKT1/Phospho-AKT1 between the offspring rats of the two groups (P>0.05).</p><p><b>CONCLUSIONS</b>A maternal high-fat diet before and during pregnancy may affect the bone growth of offspring rats in utero, which is possibly associated with the decreased IGF-I level. However, further study on the exact mechanism of IGF-I on the bone growth is needed.</p>
Subject(s)
Animals , Female , Pregnancy , Rats , Bone Development , Diet, High-Fat , Insulin-Like Growth Factor I , Liver , Pathology , MAP Kinase Signaling System , Maternal Nutritional Physiological Phenomena , Phosphatidylinositol 3-Kinases , Physiology , Proto-Oncogene Proteins c-akt , Physiology , Rats, Sprague-DawleyABSTRACT
<p><b>OBJECTIVE</b>To explore the clinical application of high-frequency ultrasound in the diagnosis and treatment of epididymal stasis after vasectomy.</p><p><b>METHODS</b>We retrospectively studied the sonographic characteristics of 23 cases of epididymal stasis treated by vasectomy, which were divided into a mild (n = 5), a moderate (n = 11) and a severe group (n = 7) according to the results of color Doppler flow imaging. We analyzed the significance of high-frequency ultrasonography in the treatment of epididymal stasis after vasectomy.</p><p><b>RESULTS</b>High-frequency ultrasonography revealed 14 cases of increased bilateral epididymal volume, 6 cases of left epididymal thickening and 3 cases of right epididymal thickening, mainly the thickening of the epididymal body and tail. After conservative treatment, 18 of the epididymal stasis cases (5 mild, 11 moderate and 2 severe) were improved, and the other 5 severe cases significantly relieved and discharged from hospital following conservative treatment combined with vasectomy reversal.</p><p><b>CONCLUSION</b>Post-vasectomy epididymal stasis has typical sonographic characteristics, and high-frequency ultrasonography has an extremely important application value in the clinical classification, diagnosis and treatment of the disease.</p>
Subject(s)
Adult , Aged , Humans , Male , Middle Aged , Epididymis , Diagnostic Imaging , Genital Diseases, Male , Diagnostic Imaging , Retrospective Studies , Ultrasonography , Methods , VasectomyABSTRACT
<p><b>OBJECTIVE</b>To investigate the expression of IGF-1 and IGFBP-3 in nonalcoholic fatty steatosis hepatocyte models induced by oleic acid.</p><p><b>METHOD</b>Nonalcoholic fatty steatosis hepatocyte models induced by oleic acid on immortalized human hepatocyte, Oil red O staining and intracellular triglycerides were detected for observing the situation of IHH cells fatty degeneration. IHH cells were divided into control group, NAFLD group, which the control group cultured in DMEM/F12 medium, NAFLD group were treated with oleic acid, 0.5 mmol/L treatment for 72 h. The expression of mRNA and protein of IGF-1 and IGFBP3 were measured by immunofluorescent staining, Western blot and RT-PCR methods. Between the two groups were compared using the t- test.</p><p><b>RESULTS</b>The steatosis models of the hepatocytes were established successfully with 0.5 mmol/L oleic acid. Lipid droplets were observed through Oil red O staining. The level of hepatocyte TG was increased (275.7+/-27.2) mug/mg from (150.2+/-15.6) mug/mg (t = 21.67, P less than 0.01). Compared with the control group, the mRNA of IGF-1 (0.76+/-0.04 vs 4.82+/-1.51, t = 17.915, P less than 0.01), IGFBP-3 (1.58+/-0.93 vs 5.41+/-1.37, t = 12.893, P less than 0.01) and protein expression of IGF-1 (1.00+/-0.29 vs 2.56+/-0.71, t = 29.17, P less than 0.01), IGFBP-3 (0.65+/-0.36 vs 1.23+/-0.91, t = 32. 12, P less than 0.01) significantly decreased in oleic acid-treated group. The results of immunofluorescence staining also confirm the significantly decreased protein expression of IGF-1 and IGFBP-3 in NAFLD group compared with control group.</p><p><b>CONCLUSION</b>The expression of IGF-1 and IGFBP-3 decreased in nonalcoholic fatty steatosis hepatocyte models, which will provide the experimental basis for the further study of the mechanism of the limited height of some children with non-alcoholic fatty liver disease in clinical.</p>
Subject(s)
Humans , Cell Line , Fatty Liver , Metabolism , Pathology , Hepatocytes , Metabolism , Pathology , Insulin-Like Growth Factor Binding Protein 3 , Genetics , Metabolism , Insulin-Like Growth Factor I , Genetics , Metabolism , Non-alcoholic Fatty Liver DiseaseABSTRACT
<p><b>OBJECTIVE</b>To study the effect of growth hormone (GH) combined with Radix Dipsaci on the body growth and the bone mineral content (BMC) of hypophysectomized rats.</p><p><b>METHODS</b>The GH deficiency rats model was established using the hypophysectomized operation through the skull and the throat. Qualified rats were divided into the sham-operation group (n = 15), the negative control group (n = 13), the GH intervention group (n = 13), and the GH combined with Radix Dipsaci group(n = 12). GH (0.25 mg/kg) was subcutaneously injected from the cervical part in the GH intervention group and the GH combined with Radix Dipsaci group at the same time, while equal volume of normal saline was injected to the rest groups. 0.7 mL/100 kg Radix Dipsaci was given by gastrogavage to the GH combined with Radix Dipsaci group at the same time, while equal volume of normal saline was given by gastrogave to the rest groups. The body weight, the tail length, and the body length were measured during the intervention period. Blood was withdrawn after 14-day intervention. The femoral bone and the tibial bone were taken out. The levels of GH, insulin-like growth factor 1 (IGF-1), alkaline phosphatase (ALP), and osteocalcin (OC) were measured. The width of the tibial epiphyseal plate was measured. The bilateral femur bone mineral density (BMD) and BMC were measured using the dual energy X-ray absorptiometry.</p><p><b>RESULTS</b>The body weight, the body length, the length of the femoral bone, the length of the tibial bone, the width of the epiphyseal plate, the levels of the GH, IGF-1, ALP, and OC increased in the GH intervention group and the GH combined with Radix Dipsaci group after 2-week intervention, showing statistical difference when compared with the model group (P < 0.01). But there was no statistical difference in the tail length though it also increased (P > 0.05). There was insignificant difference in the aforesaid indices between the two groups (P > 0.05). Compared with the model group, the BMD of the GH combined with Radix Dipsaci group increased with statistical difference (P < 0.01). Compared with the model group, the BMC of the GH intervention group and the GH combined with Radix Dipsaci group increased with statistical difference (P < 0.01). It was highest in the GH combined with Radix Dipsaci group (P < 0.01).</p><p><b>CONCLUSIONS</b>GH combined with Radix Dipsaci showed unobvious effect on promoting the growth. But it could elevate BMD and BMC, and improve the bone metabolism.</p>
Subject(s)
Animals , Male , Rats , Bone Development , Bone and Bones , Metabolism , Dipsacaceae , Chemistry , Drugs, Chinese Herbal , Pharmacology , Growth Hormone , Pharmacology , Hypophysectomy , Rats, Sprague-DawleyABSTRACT
In order to investigate the influence of cytokine combinations on proliferation and differentiation of human umbilical cord blood CD34(+) cells into megakaryocytes/platelets in vitro, the CD34(+) cells from human umbilical cord blood were amplified in serum-free medium StemSpan(SFEM) supplemented with several cytokine combinations by three-phase culture system. The effects of the cytokine combinations were compared. The results showed that at day 14 of the first culture phase, the CD34(+) cells cultured with cytokine combinations SCF + TPO + FL + IL-3 were amplified (11 000 ± 1 000) times, which were significantly higher than that of cells cultured with SCF + TPO + FL, but were not significantly different from that of cells cultured with SCF + TPO + IL-3 or SCF + TPO + FL + IL-3+ hydroxyl-corticosteroids. At day 7 of the second culture phase, the CD34(+) cells cultured with cytokine combination SCF + TPO + FL + IL-11 were amplified by (204666.7 ± 11718.9) times, which were significantly higher than that of cells cultured with SCF + TPO + FL + IL-3, but were not significantly different from that of cells cultured with SCF + TPO + FL + IL-11 + BMP4 + VEGF. At day 3 and day 6, the CD34(+) platelet-like cells accounted for about (39.8 ± 1.9)%, (39.7 ± 2.6)% and (25.5 ± 1.4)%, (23.1 ± 3.5)% cultured with SCF + TPO + FL + IL-11 and SCF + TPO + FL + IL-11 + BMP4 + VEGF, and significantly higher than that of the cells cultured with SCF + TPO + FL + IL-3. It is concluded that the cytokine combination of SCF + TPO + FL + IL-3 is most suitable cytokines combination for the amplification of CD34(+) hematopoietic progenitor cells. The cytokine combination of SCF + TPO + FL + IL-11 is preferred for the proliferation and differentiation of megakaryocytes, this study lays an experimental basis for investigating the proliferation and differentiation of CD34(+) into megakaryocytes/platelets in vitro.
Subject(s)
Humans , Antigens, CD34 , Allergy and Immunology , Blood Platelets , Cell Biology , Cell Differentiation , Fetal Blood , Cell Biology , Allergy and Immunology , Interleukin-11 , Pharmacology , Interleukin-3 , Pharmacology , Megakaryocytes , Cell Biology , Stem Cell Factor , Pharmacology , Thrombopoietin , PharmacologyABSTRACT
<p><b>OBJECTIVE</b>To investigate the feasibility and therapeutic effect of lateral thoracic flaps pedicled with subscapular vessels for defects in the upper extremities.</p><p><b>METHODS</b>From June 2003 to September 2009, 5 cases with large soft tissue defects in the upper extremities were treated with lateral thoracic flaps pedicled with subscapular vessels. The flap size ranged from 23 cm x 8 cm to 40 cm x 20 cm. The subscapular vessels, the thoracodorsal vessels, the lateral branch and the cutaneous perforators of thoracodorsal vessels were all included in the flap. A muscular sleeve of 2-3 cm in width was preserved to protect the musculocutaneous perforator. The defects in the donor area were closed directly or covered by skin graft.</p><p><b>RESULTS</b>The lateral thoracic flaps were used in four cases. A combination of lateral thoracic flap and paraumbilical flap was used in one case. Partial necrosis happened at the distal portion of the flap in one case. All the other flaps survived completely. 4 cases were followed up for two to fourteen months with satisfactory cosmetic and functional results. The color, texture and thickness of the flaps were also satisfactory.</p><p><b>CONCLUSIONS</b>The lateral thoracic flap pedicled with the subscapular vessel is flexible for repairing defects in the upper extremities with a reliable blood supply, leaving less morbidity to donor site.</p>