Astragalus polysaccharide has been widely used in food and medicinal industry owing to its health-promoting properties. In order to characterize better the relationship among molecular weight, structure-activity and activities, a simple method was used different concentration of ethanol including 30% (PW30), 50% (PW50), 70% (PW70), 75% (PW75), 80% (PW80) and 90% (PW90) to precipitate Astragalus polysaccharides into different molecular weight. As a result, PW90 showed smooth surface and the strongest antioxidant activity among these six fractions (P < 0.05). In conclusion, graded ethanol precipitation was a simple method to separate Astragalus polysaccharides into different molecular weight with different antioxidant activity fractions.
Antioxidants ; pharmacology ; Astragalus Plant ; chemistry ; Chemical Precipitation ; Ethanol ; chemistry ; Polysaccharides ; chemistry ; pharmacology

Tumor angiogenesis provides adequate oxygen and nutrition for tumor development and supports tumor growth and metastasis. Stromal cell derived factor 1 (SDF-1) and its receptor C-X-C motif chemokine receptor 4 (CXCR4) in pancreatic cancer microenvironment are involved in tumor growth such as promoting tumor cell proliferation, migration, and angiogenesis. In this study, anti-CXCR4 nanobody (CXCR4 Nb) and anti-programmed cell death ligand 1 (PD-L1) &CXCR4 bispecific nanobody (PX4 BsNb) were expressed in Escherichia coli system and purified by nickel column affinity chromatography. We investigated the anti-angiogenesis activity and mechanism of CXCR4 Nb by in vivo and in vitro experiments. Ethical approval was obtained for collection of human peripheral blood mononuclear cell (hPBMC) samples from the Local Ethics Committee of Shanghai Jiao Tong University. All animal experiments were approved by the Animal Ethic Committee of Shanghai Jiao Tong University. The results showed that CXCR4 Nb at 0.1 μmol·L^-1 could effectively inhibit the proliferation and migration of human umbilical vein endothelial cells (HUVEC) promoted by pancreatic stellate cells in vitro. CXCR4 Nb and PX4 BsNb at 0.3 mg·kg^-1 obviously decreased tumor angiogenesis and inhibited the tumor growth in NOD/SCID mice, the inhibitory rates were 28.8% and 36.1%, respectively. CXCR4 Nb significantly inhibited tumor growth and angiogenesis with great safety, which provides support for application of CXCR4 Nb and anti-angiogenesis therapy of pancreatic cancer.

OBJECTIVETo identify SNP in flos Lonicerae, and authenticate Lonicera japonica from its adulterants and the mixture by using bidirectional PCR amplification of specific alleles (Bi-PASA).

METHODSNP of L. japonica and its adulterants was identified by using ClustulW to align trnL-trnF sequences of the Lonicera genus from GenBank database. Bi-PASA primer was designed and the PCR reaction systems including annealing temperature optimized. Optimized result was performed in 84 samples to authenticate L. japonica, its adulterants and the mixture.

RESULTWhen the annealing temperature was 61 degrees C, DNA from L. japonica would be amplified 468 bp whereas PCR products from all of the 9 adulterants were 324 bp. The established method also can detect 5% of intentional adulteration DNA into L. japonica.

CONCLUSIONThe Bi-SPASA could authenticate L. japonica from its adulterants and the mixture.

Alleles ; DNA Barcoding, Taxonomic ; methods ; DNA, Plant ; analysis ; genetics ; Flowers ; genetics ; Lonicera ; classification ; genetics ; Plants, Medicinal ; classification ; genetics ; Polymerase Chain Reaction ; methods ; Polymorphism, Single Nucleotide ; RNA, Transfer, Leu ; genetics ; RNA, Transfer, Phe ; genetics ; Reproducibility of Results ; Species Specificity

Increasing evidence has revealed that maternal cytomegalovirus (CMV) infection may be associated with neurodevelopmental disorders in offspring.Potential relevance between the placental inflammation and CMV-related autism has been reported by clinical observation.Meanwhile,abnormal expression of Toll-like receptor 2 (TLR2) and TLR4 in placenta of patients with chorioamnionitis was observed in multiple studies.IL-6 and IL-10 are two important maternal inflammatory mediators involved in neurodevelopmental disorders.To investigate whether murine CMV (MCMV) infection causes alterations in placental IL-6/10 and TLR2/4 levels,we analyzed the dynamic changes in gene expression of TLR2/4 and IL-6/10 in placentas following acute MCMV infection.Mouse model of acute MCMV infection during pregnancy was created,and pre-pregnant MCMV infected,lipopolysaccharide (LPS)-treated and uninfected mice were used as controls.At E13.5,E14.5 and E18.5,placentas and fetal brains were harvested and mRNA expression levels of placental TLR2/4 and IL-6/10 were analyzed.The results showed that after acute MCMV infection,the expression levels of placental TLR2/4 and IL-6 were elevated at E13.5,accompanied by obvious placental inflammation and reduction of placenta and fetal brain weights.However,LPS 50 μg/kg could decrease the IL-6 expression at E13.5 and E14.5.This suggests that acute MCMV infection during pregnancy could up-regulate the gene expression of TLR2/4 in placental trophoblasts and activate them to produce more proinflammatory cytokine IL-6.High dose of LPS stimulation (50 tg/kg) during pregnancy can lead to down-regulation of IL-6 levels in the late stage.Imbalance ofIL-6 expression in placenta might be associated with the neurodevelopmental disorders in progeny.

OBJECTIVETo probe into effective methods for treatment of ischemic cerebral infarction and specificity of acupoints in the acupuncture treatment.

METHODSThe rat model of middle cerebral artery occlusion (MCAO) was established with thread ligation according to Zea-Longa's method. They were divided into normal group, non-operation group, model control group, non-acupuncture group and acupuncture group, and the acupuncture group was divided into sham-acupuncture group and four Xingnao Kaiqiao groups (contain Shuigou group, Neiguan group, Chize group, Sanyinjiao group and Weizhong group). Then they were treated by acupuncture at "Shuigou" (GV 26), "Neiguan" (PC 6), "Chize" (LU 5), "Sanyinjiao" (SP 6), "Weizhong" (BL 40) and non-acupoints, 3 times/second, for 5 seconds. Cerebral blood blow (CBF) was used for assessment of the effect.

RESULTSAs compared with the model control group, in the non-acupuncture group CBF did not significantly change (P>0.05); compared with the non-acupuncture group, after acupuncture CBF was significantly increased in the Shuigou group and the Neiguan groups (all P<0.05), but did not significantly increased in the Chize group, Sanyinjiao group and Weizhong group (all P>0.05).

CONCLUSIONCBF has a tendency of spontaneous cure within 72 h after cerebral infarction in the MCAO rat; acupuncture can significantly improve CBF in the MCAO rat, so it is an effective method for treatment of ischemic stroke; among the acupoints in the "Xingnao Kaiqiao" needling method, "Shuigou" (GV 26) and "Neiguan" (PC 6) have obvious effect in improvement of CBF, indicating acupoint specificity of "Shuigou" (GV 26) and "Neiguan" (PC 6) in treatment of ischemic stroke.

Acupuncture Points ; Acupuncture Therapy ; Animals ; Brain ; blood supply ; Brain Ischemia ; therapy ; Cerebral Infarction ; therapy ; Disease Models, Animal ; Humans ; Male ; Random Allocation ; Rats ; Rats, Wistar

OBJECTIVETo investigate the effect of acupoint Shuigou (GV 26) and non-acupoint on inhibiting the neuronal necrosis induced by the middle cerebral artery obstruction (MCAO) in rats through the morphological observation.

METHODSForty two healthy adult male Wistar rats were randomly divided into a normal group, false-ope ration group, model control group, non-acupuncture group, Shuigou-acupuncture group and non-acupoint acupuncture group, 7 rats in each group. Besides the normal and false-operation groups, the experimental modal of MCAO was established in the other groups by using the Zea-Longa thread method. Acupoint Shuigou (GV 26) and non-acupoint (below the costal region) were selected respectively in the Shuigou-acupuncture group and non-acupoint acupuncture group for puncturing at 180 times/min for 5 seconds. Its effect was estimated by measuring the rate of the neuronal necrosis at the level of the light microscope and the degree of the neuronal necrosis at the level of the electron microscope.

RESULTS(1) At the level of the light microscope, comparing with the model control group (0.66 +/- 0.18), of the neuronal necrosis there were no significant differences in the rate of neuronal necrosis in the non-acupuncture group (0.67 +/- 0.34) and non-acupoint acupuncture group (0.59 +/- 0.11) (both P > 0.05), while it was significantly decreased in the Shuigou-acupuncture group (0.200 +/- 12) (P < 0.05). It indicates that no intervention and wrong (non-acupoint) intervention could light the damage of the neurons, however, only right intervention (Shuigou, GV 26) can obviously decrease the rate of the neuronal necrosis, showing with the specificity of acupoint. (2) At the level of the electron microscope, the ultrastructure of the involved neurons in the non-acupuncture group and non-acupoint acupuncture group is similar to that of the model control group, showing with the serious edema and structure damage. In contrast, the ultrastructure of the involved neurons in the Shuigou-acupuncture group is similar to that of the normal group and false-operation group, showing with light neuronal damage.

CONCLUSIONAcupuncturing Shuigou (GV 26) could protect the involved neurons and inhibit the neuronal necrosis induced by the MCAO in rats, but acupuncturing the non-acupoint couldn't, which imply that there is the specificity of acupoint in the acupuncture.

Acupuncture Points ; Acupuncture Therapy ; Animals ; Brain Ischemia ; pathology ; physiopathology ; therapy ; Disease Models, Animal ; Humans ; Male ; Necrosis ; Neurons ; pathology ; ultrastructure ; Random Allocation ; Rats ; Rats, Wistar

OBJECTIVESTo establish DHPLC method in detecting mutations of mismatch repair genes, hMLH1 and hMSH2, and to identify germline mutations of hMLH1 and hMSH2 in HNPCC kindreds fulfilling Chinese HNPCC criteria.

METHODSFourteen peripheral blood DNA samples from 14 unrelated HNPCC probands fulfilling Chinese HNPCC criteria were obtained respectively. PCR amplified 35 exons of two main mismatch repair genes, hMLH1 and hMSH2. DHPLC followed by DNA sequencing was used to detect and confirm mutations.

RESULTSa total of 41 colorectal cancers and 19 extracolonic tumors were developed in 14 HNPCC kindreds, and gastric cancer was the most common extracolonic tumor type. Twelve single nucleotide changes were identified by DHPLC in 14 probands. Among them, three were missense mutations, one was a nonsense mutation. Other single nucleotide changes included five single nucleotide polymorphisms, two intron single nucleotide changes, one synonymous mutation. hMLH1 EXON19 CODON749 TAC-->TAG (Tyr-->X), hMSH2 EXON12 CODON629 CAA-->CGA (Gln-->Arg) and hMSH2 EXON15 CODON839 CAT-->CGT (His-->Arg) were new discovered mutations.

CONCLUSIONS(1) DHPLC was considered to be highly effective, convenient technique with consistent results for the mutation detection of hMLH1 and hMSH2 genes. (2) Valid mutations of hMLH1 and hMSH2 genes were identified in about one-third HNPCC kindreds fulfilling Chinese HNPCC criteria and missense mutation was the most common mutational types in this cohort of families.

Adaptor Proteins, Signal Transducing ; Asian Continental Ancestry Group ; genetics ; Base Pair Mismatch ; Carrier Proteins ; genetics ; Chromatography, High Pressure Liquid ; Codon, Nonsense ; Colorectal Neoplasms, Hereditary Nonpolyposis ; genetics ; DNA Mutational Analysis ; Female ; Genetic Testing ; Germ-Line Mutation ; Humans ; Male ; MutL Protein Homolog 1 ; MutS Homolog 2 Protein ; genetics ; Mutation, Missense ; Nuclear Proteins ; genetics ; Pedigree

To produce specific monoclonal antibody (McAb) against human thrombomodulin (hTM), the full-length hTM cDNA-expressing plasmid pThr402 was transfected into CHO cells by Lipofectamine 2000 reagent. The hTM-expressing CHO cells, which was confirmed by flow cytometry and Western blot, were obtained by G418 selection. Then the McAb against hTM was prepared with classic hybridoma technique. A cell line of CHO-TM5 with high level of hTM was used to immunize female Balb/c mice 3 times at an interval of 4 weeks. On the third day after the third immunization, mice were sacrificed and spleen cells were harvested to prepare hybridoma cells with SP2/0 cells at the ratio of 10 to 1. Hybridoma cells were then cultured at 96-well plates for screening. Cellular enzyme-linked immunoabsorbent assay (CELISA) was applied twice. The first CELISA was done with polythene ELISA plate with a monolayer of CHO-TM5 cells. The positive clones from the first screen were then selected by reacting with similar screening ELISA plate but with CHO cell monolayer instead. Only clones that were positive for the first screening and negative for the second screening were kept, and called as CHO-TM5(+)CHO(-) hybridoma cells. Balb/c mice were intraperitoneally injected with the selected hybridoma cells. Ascites were collected and monoclonal antibodies were purified using FPLC, and its Ig class, subclass, and titer were then determined respectively. The specificity of the yielded McAb was identified with CELISA, flow cytometry, ABC immunohistochemistry and immunoblotting. One line of hybridoma cells with high expression of specific McAb against hTM, NH-1, was obtained. The Ig subclass of the McAb was IgG1 and the titer of ascitic McAb was 1x10(-6). Flow cytometry, CELISA and Western blot assays demonstrated that McAb NH-1 could specifically recognize hTM expressed in CHO-TM5 cells and human umbilical vascular endothelial cells. Meanwhile, the tissue specificity of antigen recognized by McAb NH-1 was identified by immunohistochemical ABC staining. NH-1 can specifically recognize the natural hTM expressed mainly in vascular endothelial cells, which will potentially be useful for investigation of the functions and clinic values of hTM.
Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibody Specificity ; CHO Cells ; Cricetulus ; Female ; Humans ; Hybridomas ; secretion ; Mice ; Mice, Inbred BALB C ; Thrombomodulin ; immunology ; Transfection

OBJECTIVETo study on needling safe depth of Fengfu (GV 16) with CT, so as to provide reference for safe needling depth of Fengfu (GV 16) in clinical acupuncture treatment.

METHODSForty-one adult volunteers were divided into 3 groups, a thin person group, a moderate person group and a fat person group according to Luo's indexes, and computer-aided tomography (CT) was used to measure the needling depth of Fengfu (GV 16).

RESULTSThe safe depths of perpendicular needling of Fengfu (GV 16) were different for persons of different somatotypes. The safe needling depth was (27.73 +/- 3.45) mm for the thin person group, (30.78 +/- 2.90) mm for the moderate person group, and (33.39 +/- 4.27) mm for the fat person group.

CONCLUSIONThe safe needling depth < or = the dangerous depth x 75% can be used for reference for the safe needling depth of Fengfu (GV 16) for different somatotypes persons.

Acupuncture Points ; Acupuncture Therapy ; Female ; Humans ; Male ; Neck ; Somatotypes ; Tomography, X-Ray Computed

OBJECTIVETo evaluate the test-retest reliability of Nordic Musculoskeletal Questionnaire in nurses and to provide supplementary data for evaluating the reliability of the questionnaire in different occupational populations.

METHODSNordic Musculoskeletal Questionnaire was translated into Chinese according to the Guidelines for the process of cross-cultural adaptation of self-report measures. We carried out a study to examine the reliability of Chinese-version Nordic Questionnaire among Chinese nurses. This study was conducted in 120 nurses recruited from our hospital, who underwent questionnaire survey twice within one week. The test-retest reliability of questionnaire was analyzed.

RESULTSThe Chinese-version Nordic Questionnaire showed a high test-retest reliability, with a Kappa coefficient of 0.72∼1.00.

CONCLUSIONThe Chinese-version Nordic Questionnaire has a high test-retest reliability in nurses, so it can be used for the screening and epidemiological investigation of musculoskeletal disorders in this population.

Humans ; Musculoskeletal Diseases ; diagnosis ; Nurses ; Reproducibility of Results ; Surveys and Questionnaires