2.Construction of subtracted cDNA libraries of gastrocarcinoma and normal tissue with suppression subtractive hybridization and their quality analysis
Lan-jun, WU ; Bing-zhi, MAO ; Sheng-qi, WANG
Bulletin of The Academy of Military Medical Sciences 2001;25(1):9-12,53
Objective: To construct subtracted cDNA libraries of stomach tumors and normal stomach tissue using suppression subtractive hybridization(SSH).Methods: cDNA Library subtraction was performed using the protocol described in the Clontech PCR-Select cDNA Subtraction Kit. cDNA was synthesized from 2 μg of poly A+RNA from the tumor and normal tissues using AMV reverse transcriptase. The tester and driver cDNAs were digested with RsaⅠ, a four-base-cutting restriction enzyme that yields blunt ends. The tester cDNA was then subdivided into two portions, and each was ligated with different cDNA adaptor. Two hybridizations were performed. In the first, an excess of driver was added to each sample of tester. Hybridization kinetics led to equalization and enrichment of differentially expressed sequences. During the second hybridization, the two primary hybridization samples were mixed together without denaturing and thus the templates were generated from differentially expressed sequences for PCR amplification. Using suppression PCR, only differentially expressed sequences were amplified exponentially and after second PCR amplification the background was reduced and differentially expressed sequences were further enriched. The cDNAs were then directly inserted into a T/A cloning vector to generate a stomach tumor-specific subtracted cDNA library. Results: The amplified library contained 800 positive clones. Plasmid inserts were PCR amplified and showed 250-700 bp inserts. Conclusions: The successfully constructed subtracted cDNA library of gastrocarcinoma and normal tissue enables us to compare two populations of mRNA and obtain clones of genes that expressed in one population but not in the other.The characterization of these genes will allow them to be exploited for their diagnostic and therapeutic potentials.
3.The mechanism of Er-xian Decoction in regulating lipid metabolism disorders on bilateral ovariectomized rats based on metabolomics study
Ning SHENG ; Cai-hong WANG ; Zhi-xin JIA ; Zhe WANG ; Cai-sheng WU ; Jin-lan ZHANG
Acta Pharmaceutica Sinica 2021;56(9):2403-2409
Studies have shown that women's menopause caused by permanent cessation of ovarian function is closely related to lipid metabolism disorders. Er-xian Decoction has been used in the clinical treatment for gynecological diseases and has a good effect on diseases related to reduced sex hormone function. In this study, metabolomics was performed on bilateral ovariectomized model rats within 12 weeks after modeling to mimic the physiological state of menopausal women in different menopausal stages and Er-xian Decoction dosed model rats. The results of liver oil red O staining sections showed lipid metabolic disorder of bilateral ovariectomized model rats and the regulating effects of Er-xian Decoction. 46 potential biomarkers (6 steroid hormones, 3 sphingolipids, 11 phospholipids and 26 glycerides) in plasma and 32 potential biomarkers (1 steroid hormones, 20 phospholipids and 11 glycerides) in liver were obtained based on lipidomics analysis. Then, we analyzed the differential metabolic pathways and construct the lipid metabolism network significantly regulated by Er-xian Decoction. The results provided valuable information for in-depth understanding of the gradual changes on lipid metabolism disorders under menopausal conditions and the characteristics and mechanisms of compound Er-xian Decoction's regulatory effects. The study complied with the procedures established by the Animal Experiment Ethics Committee of the Institute of Materia Medica, Chinese Academy of Medical Sciences and passed the animal experiment ethics examine (No. 00000918).
4.Histomorphological study on folk medicine Lysimachia fortunei.
Zhi-gui WU ; Xiao-mei FU ; Sheng-fu HU ; Jian-guo PEI ; Fei GE ; Xiao-lan CHU ; Cui-sheng FAN
China Journal of Chinese Materia Medica 2015;40(4):639-642
To set standards for histomorphological studies on Lysimachia fortunei, an efficacious and widely applied folk medicine in this study, in order to develop its resources. Its species were identified by observing plant morphology and herbs appearance characters, preparing slices with routine methods and defining structural characters. According to the results of morphologic observation, leaves, stamen and pistil of this plant were different from the descriptions in Flora of China. The whole herb can be used in medicines, mainly including rhizomes, stems and leaves. According to the findings in the first study on microscopic structures, its rhizomes, stems and leaves were characteristic and worth identifying. The transaction tissue structures of rhizomes and stems were under developed and contained endodermis, secretory structures; Stems had sclerenchymata of different shapes of sclereids; Leaves were bifacial and had vascular bundles under midribs, which were surrounded by parenchymal sheathes. On the surface of leaves, stomata, glandular hairs and keratin lines were morphologically different in upper and lower epidermis. The herbal power had glandular hairs, sclereids and vessels. In conclusion, herbs of L. fortunei can be identified by the above histomorphological characteristics, which lays a foundation for further development and application of L. fortunei.
Medicine, Traditional
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Plant Leaves
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anatomy & histology
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growth & development
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Plant Stems
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anatomy & histology
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growth & development
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Plants, Medicinal
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anatomy & histology
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growth & development
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Primulaceae
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anatomy & histology
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growth & development
5.Auricular acupuncture for primary insomnia: a systematic review based on GRADE system.
Hui-Juan TAN ; Ying LAN ; Fu-Sheng WU ; Han-Dan ZHANG ; Li WU ; Xi WU ; Fan-Rong LIANG
Chinese Acupuncture & Moxibustion 2014;34(7):726-730
The clinical efficacy and safety of auricular acupuncture (AA) for treatment of primary insomnia was evaluated. After a comprehensive retrieval in domestic and foreign databases, literatures were strictly screened and Revman 5.2 software was applied to perform a Meta-analysis on eligible randomized controlled trials (RCTs). The evidence quality was assessed with GRADE profiler 3.6 software. As a result, 8 articles were included involving 894 patients. Compared among AA and sham AA, placebo AA, blank control, there was significant difference in Pittsburgh sleep quality index (PSQI) [WMD = -3.48, 95% CI (-3.96, -3.00)], sleep latency LWMD = -10.14, 95% CI (-17.16, -3.12)] and sleep awakening times [WMD = -9.98, 95% CI (-1.10,-0.48)]. Compared between AA and western medication, there was significant difference in PSQI [WMD = -3.62, 95% CI (-4.59, -2.65)]. The evidence quality was moderate in AA vs. sham AA, placebo AA or blank control, while that of the rest was extremely low. No reports of adverse events were described in all studies. In conclusion, for the treatment of primary insomnia, AA could effectively improve sleep quality, but due to the low evidence quality, cautious attitude should be taken on this conclusion, and clinical trials with large sample and high quality were needed in the further.
Acupuncture, Ear
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Humans
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Randomized Controlled Trials as Topic
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Sleep
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Sleep Initiation and Maintenance Disorders
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physiopathology
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therapy
6.Protective effect of jiedu tongluo injection on cerebral edema in rats with lesion of cerebral ischemia/reperfusion.
Li-Fei WU ; Yue XING ; Ya-Lan GUAN ; Zhen-Quan LIU ; Wen-Sheng ZHANG
China Journal of Chinese Materia Medica 2014;39(6):1088-1092
OBJECTIVETo investigate the protective effects of Jiedu Tongluo injection on cerebral edema induced by focal lesion of cerebral ischemia/reperfusion, the hydrous content of brain and the expressions of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin and MMP-9 in rats.
METHODThe model of brain middle cerebral artery ischemia/reperfusion was established by the thread approach. After 24 hours of reperfusion, cerebral edema formation was determined by the hydrous content of brain. The permeability of blood brain barrier was evaluated based on the leakage of Evans blue. Enzyme-linked immunoadsordent assay (ELISA)was used to examine the expression of ICAM-1, VCAM-1, E-selectin. The expression of MMP-9 was measured by immunohistochemistry.
RESULTJDTL, in the dose of 2 mL x kg(-1) and 4 mL x kg(-1), relieved cerebral edema (P < 0.05, P < 0.01), reduced the expressions of ICAM-1, VCAM-land E-selectin and decreased MMP-9 activity (P < 0. 05, P < 0.01) in model rats.
CONCLUSIONJiedu Tongluo injection has a protective effect on rat brain from cerebral edema induced by the injury of focal cerebral ischemia/reperfusion. The mechanism is related to that Jiedu Tongluo injection can reduce the expressions of ICAM-1, VCAM-1 and E-selectin and inhibit of MMP-9 activation in rat brain.
Animals ; Blood-Brain Barrier ; drug effects ; metabolism ; Brain Edema ; etiology ; metabolism ; prevention & control ; Brain Ischemia ; complications ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; E-Selectin ; metabolism ; Evans Blue ; metabolism ; Gene Expression Regulation, Enzymologic ; drug effects ; Injections ; Intercellular Adhesion Molecule-1 ; metabolism ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Permeability ; drug effects ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; complications ; Vascular Cell Adhesion Molecule-1 ; metabolism
7.Optimization of Fermentation Medium for S-adenosylmethionine Production by Candida sp.YQ5
Fang-Lan GE ; Sheng YE ; Gui-Ying CHEN ; Wei LI ; Ke WU ; Liang-Jun DU ;
Microbiology 1992;0(05):-
The optimized cultural medium of fermentation for Candida sp.mutant strain YQ5 to produce S-adenosylmethionine was studied.The results of single factor experiment showed that the most favorable pH value,carbon source,nitrogen source organic nutrient is 6.0,8% sucrose,1.5% tryptone and 2% yeast extract,respectively.As to inorganic salts,MgSO4?7H2O,CaCl2,FeSO4?7H2O,CoCl2,CuSO4?5H2O,H3BO3 could improve accumulation of the intercellular SAM.The ingredients of the culture medium are also opti-mized by the orthogonal experiment.Fermentation for 48 h under the optimal condition resulted in AdoMet production at 1740.0 mg/L.
8.Study on risk factors of brain metastases of locally advanced non-small-cell lung cancer
Xia CA0 ; An-lan NG WA ; Pei YANG ; Yuan YUAN ; Sheng-qi WU ; Rong-xi LUO
Cancer Research and Clinic 2012;24(1):24-27
Objective To evaluate the prognostic factors in locally advanced non-small-cell lung cancer (LA-NSCLC) for selectively carrying out prophylactic cranial irradiation (PCI).Methods 114 patients with LA-NSCLC between Jun 2006 and Oct 2010 were retrospectively analyzed. Related risk factors and features about brain metastases were analyzed.Results The 2-year incidence rate of brain metastases was 31.58 % (36/114),the first brain metastases was 20.18 % (23/114),and sole brain metastases was 9.65 %(11/114),respectively.Variables involved in the equation of binary logistic regression analysis were pathology (OR =5.892) and treatment mode(OR =2.888).The incidence rate of brain metastases in patients of non-squamous carcinoma and single treatment mode was higher than others (P < 0.01) Model fitting is better (P > 0.05).Overall accuracy rate of predicting brain metastases is 67.7 %.The increased rate of lactate dehydrogenase in the patients with brain metastases or death was 17.54 %, which was higher than that in the survival patients without brain metastases (P < 0.01).At the same time,the station number and the number of mediastinal lymph node metastases were positively correlated (r =0.716, P < 0.01).The incidence rate of brain metastases or mortality rate was higher in the adenocarcinoma cases than that in the squamous carcinoma cases (P < 0.01,P < 0.05),with more frequent occurrence of mediastinal metastases.The mean diameter of squamous carcinoma and adenocarcinoma were 5.8 cm and 3.9 cm, respectively (P < 0.01).Conclusions The incidence rate of brain metastases was higher in patients with single treatment.Large primary tumors, high lactate dehydrogenase, non-squamous carcinoma, multiple stations, and multiple mediastinal lymph nodes metastases can be regarded as risk factors of brain metastases to perform PCI.
9.A comparison of four methods for extraction of human fecal DNA by using real time PCR
Zhong-Wen WU ; Ying HAN ; Hai-Feng LU ; Lan-Juan LI ; Ji-Fang SHENG ; Jian ZUO ;
Chinese Journal of Laboratory Medicine 2001;0(01):-
Objective To compare the relative efficacy and quality of extraction of human fecal DNA using four methods.Methods Real-time PCR were utilized for analysis both quantification and quality of the fecal targeted bacteria(including gut all eubaeterium,Bacteriodes-PrevoteUa group,Bifidobacterium spp Enterobacteriaceae and Enterococcus spp)by using 16s rRNA gene-targeted genus or group-specific primer sets.Results The negative rat of PCR product from method 3(phenol-chloroform plus bead-beating) was about 40%(4/10)by using universal primers,the PCR inhibition disappeared after fecal DNA purified with column.The total fecal 16s rRNA gene copy numbers(per gram of wet weight of feces)as well as the numbers of Bacteriodes-Prevotella group from method 1(QIAamp~DNA stool mini kit)and 4(QIAamp~ DNA stool mini kit combined with bead-beating)was higher significantly than that from method 2(FastDNA ~Kit,Biol01)and 3(P
10.Application of 11C-choline PET/CT for the hepatic space-occupying lesions with an indeterminate diagnosis by 18F-FDG PET/CT
Hu-bing, WU ; Quan-shi, WANG ; Ming-fang, WANG ; Wen-lan, ZHOU ; Hong-sheng, LI ; Li-juan, WANG
Chinese Journal of Nuclear Medicine 2010;30(4):217-221
Objective To explore the value of 11C-choline PET/CT in patients with hepatic spaceoccupying lesions that have an indeterminate diagnosis by 18F-fluorodeoxyglucose (FDG) PET/CT. Methods A total of 25 liver masses in 20 patients with an indeterminate diagnosis based on 18F-FDG PET/CT were enrolled. Regional 11C-choline PET/CT scan was performed in all of the patients. Lesions with intense 11C-choline uptake were considered as positive. The semiquantitative maximum standardized uptake value(SUVmax) was measured and the tumor-to-liver (T/L) radioactivity ratio was calculated. The Mann-Whitney test,Kruskal-Wallis test and crosstabs x2-test were performed by using SPSS version 11.5. Results Of the 25 lesions,21 were proven to be hepatocellular carcinomas (HCC),3 hemangiomas,and 1 parasitic granuloma. The sensitivity of 11C-choline PET/CT for the detection of HCC was 66.7% (14/21). 11C-choline PET/CT had a higher sensitivity for well differentiated HCC than moderately and poorly differentiated HCC on a patient basis (8/9 vs 2/5,respectively). There were significant differences of 11C-choline T/L ratios between the HCC positive group,HCC negative group and benign lesion group ( 1.70 ± 0.35,0.86 ± 0.15,and 0.36 ± 0.18,x2 = 19.00,P <0.01 ). The lesion size and alphafetoprotein (AFP) level between the HCC positive and negative groups had no significant difference respectively ( Mann Whitney U = 39.00,P >0.05,and U=16.00,P>0.05,respectively). Conclusions 11C-choline is complementary to 18F-FDG PET/CT for the detection of HCC,especially for well differentiated HCC.