Objective To explore the method of primary isolation, cultivation and identification of rat brain microvessel pericytes. Methods The brain tissue of 10 3 week-old Wistar rats was separated sterilely. The brain microvessel fragments were separated using two-step enzyme digestion and one-step gradient centrifugation and were seeded in 35-mm dishes for primary culture. The cell morphology was observed by phase contrast microscopy; the immunofluorescence assay was used to identify the associated antigns, such as the α-smooth muscle actin (α-SMA), neuron-glial antigen 2 (NG2), von Willebrand factor (vWF), and glial fibrillary acidic protein (GFAP). Methyl thiazolyl tetrazolium was used to determine the cell growth curve. Results Pericytes climbed out from the adherent brain microvascular fragments around,showing polygonal, and the cell fusion was 95％ after 12-14 days. Immunofluorescence staining revealed that the molecular markers of the pericytes α-SMA and NG2 related antigens showed double positive, while the vWF and GFAP related antigens showed double negative and the cultured cells were confirmed as brain microvascular pericytes. The growth rate of primary cells was slower. The passage cells entered into logarithmic growth phase after 36 to 60 hours and entered into plateau phase after 72 to 108 hours. Conclusions This method may successfully isolate rat brain microvascular pericytes with higher purity.

Chondrodysplasia Punctata ; congenital ; Humans ; Infant, Newborn ; Male

One of the causes of the high cost of pharmaceuticals and the major obstacles to rapidly assessing the bioavailability and risk of a chemical is the lack of experimental model systems. A new pre-treatment technology, in vitro bionic digestion was designed for metal analysis in Lianhua Qingwen capsule. The capsule was digested on 37 degrees C under the acidity of the stomach or intestine, and with the inorganic and organic compounds (including digestive enzymes) found in the stomach or intestine, and then the chyme was obtained. Being similar to the biomembrane between the gastrointestinal tract and blood vessels, monolayer liposome was used as biomembrane model Affinity-monolayer liposome metals (AMLMs) and water-soluble metals were used for metal speciation analysis in the capsule. Based on the concentration of AMLMs, the main absorption site of trace metals was proposed. The metal total contents or the concentration of AMLMs in the capsule were compared to the nutritional requirements, daily permissible dose and heavy metal total contents from the "import and export of medicinal plants and preparation of green industry state standards". The metal concentrations in the capsule were within the safety baseline levels for human consumption. After in vitro bionic digestion, most of trace metals were absorbed mainly in intestine. The concentration of As, Cd, Pb was 0.38, 0.07, 1.60 mg x kg(-1), respectively, far less than the permissible dose from the "import and export of medicinal plants and preparation of green industry state standards".
Biological Availability ; Capsules ; adverse effects ; pharmacokinetics ; Digestion ; Drugs, Chinese Herbal ; adverse effects ; pharmacokinetics ; Humans ; Metals, Heavy ; adverse effects ; pharmacokinetics ; Models, Biological ; Stomach ; metabolism ; Trace Elements ; adverse effects ; pharmacokinetics

Administration, Oral ; Anti-Bacterial Agents ; administration & dosage ; Cytomegalovirus Infections ; drug therapy ; immunology ; virology ; Erythromycin ; administration & dosage ; Female ; Humans ; Infant ; Male ; T-Lymphocyte Subsets ; immunology ; Virus Replication

Aged ; Alzheimer Disease ; drug therapy ; physiopathology ; Brain ; physiopathology ; Chitosan ; pharmacology ; therapeutic use ; Electroencephalography ; Female ; Humans ; Male ; Phospholipids ; pharmacology ; therapeutic use

BackgroundLimbal stem cell deficiency usually leads to blindness, and traditional therapy is limited. Recent research demonstrated that bone mesenchymal stem cells ( BMSCs ) and human amniotic epithelial cells(AECs) could differentiate into many kinds of cells including corneal epithelial cells, but the outcome and effect of these cells on corneal stem cell deficiency are still unclear. ObjectiveThis study aimed to observe and compare the effects of rabbit BMSCs and human AECs transplantation for rabbit limbal stem cell deficiency. Methods Eighteen clean New Zealand rabbits were randomly divided into the amniotic stroma(AS) group, rabbit BMSCs group and human AECs group with 6 rabbits for each group. Limbal stem cell deficiency models were established by putting a piece of filter paper that had been soaked in a NaOH solution at the corneal center. Rabbit BMSCs were isolated and purified by density gradient centrifugation combined with the attachment culture method, and human AECs were collected by a sequential trypsin digestion technique,and the third generation rabbit BMSCs and the first generation human AECs were identified with RT-PCR. After that,cells were inoculated onto the denuded AS and grafted to the corneal surface of the experimental animals. Twenty-eight days after cell transplantation, the therapeutic effects were evaluated based on the corneal neovascularization and opacity scores. Corneal histopathological examination and immunohistochemistry were performed to evaluate and compare the effectiveness among AS,rabbit BMSCs and human AECs on corneal stem cell deficiency. The procedure complied with the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. ResultsThe third generation of rabbit BMSCs grew well after 12 hours, and the first generation of human AECs formed a membrane-like monolayer after 48 hours of incubation on AS. Immunohistochemistry staining showed that, 28 days after transplantation, the surface cells of the cornea were positive for cytokeratin 3 in both the rabbit BMSCs group and human AECs group.Compared with the AS group,the corneal neovascularization and opacity grades were significantly decreased in the rabbit BMSCs group( Z=-2. 983, P =0. 003 ; Z =-2. 844, P =0. 004 ) and human AECs group ( Z =-2. 817, P =0. 005 ; Z =-2.041, P =0. 041 ). Histopathological analysis exhibited that stratified corneal epithelial-like cells formed on the corneal stroms 28 days after grafting and no signs of goblet cells and neovascularization were found. Less inflammatory cells and regular collagen fiber could be seen in the rabbit BMSCs group and human AECs group. In addition,clinical observation also revealed that the corneas were much clearer in the rabbit BMSCs group than the human A ECs group( Z =-2. 091 , P=0. 037 ), but the corneal neovascularization score was similar between them (Z = -0. 267,P=-0. 789). ConclusionsRabbit BMSCs and human AECs can differentiate into corneal epithelial-like cells onthedamagedcornealsurfaceandfurtherdemonstrateremarkableinhibitoryeffectsoncornealneovascularization and inflammatory cells. The more dominant and prominent effect is the role played by rabbit BMSCsin the improvement of corneal transparency.

Objective To analyze the karyotypes of 11 cases of Turner syndrome with marker chromosome,and study the phenotypic effects resulting from the abnormal karyotype.Methods Eleven Turner syndrome patients had a mosaic karyotype and carried a marker chromosome,and 6 marker chromosomes were ring chromosomes.Their karyotypes were showed as mos.45,X/46,X,+mar or mos. 45,X/46,X,+r.Fluorescence in situ hybridization(FISH)technique with X/Y centromere probes was performed to determine the origin of the marker chromosome.Reverse chromosome painting technique was used to identify the breakpoints of two largest markers.Phenotype effects with different chromosome breakpoints were compared.Results All the 11 marker chromosomes were ring X chromosomes.The breakpoints of the r(X)were involved in Xp22,Xq22,Xq24 and Xq26,etc.Conclusions The marker chromosomes in Turner syndrome mainly originate from X chromosome and form ring chromosome X.Each r (X)in our patients was mosaic,indicating it was originated from mitosis error during early embryo development.To analyze the origin of the marker chromosome and the breakpoint of r(X)will provide guidance for the therapy and prognosis of the Turner syndrome patient.

Objective To evaluate the potential relationship between isoforms of BRCA1 associated RING domain 1 ( BARD1 ) and the pathophysiologic markers of sporadic breast carcinoma of female Han ethnic group. Methods The expression of BARD1 isoforms in 39 breast carcinomatous tissue, 12paracancerous-normal breast tissue and 7 controlled normal breast tissue was detected by reverse transcription polymerase chain reaction (RT-PCR) and then cloned and sequenced.The difference of isoforms expression and their clinical significance were analyzed. Results There were four transcriptive products of BARD1 found in all these candidates,named full lenth,isoform γ,isoform δ and isoform e.The positive rate of isoform γ and δ was higher in carcinomatous tissues than in paracancerous-normal tissues and normal breast tissue in healthy women ( P ＜ 0.05 ). Carcinomatous tissue expressed more kinds of isoforms.There was significant difference between carcinomatous tissue and paranormal/nomal tissue ( P =0.0075 ).There was significant correlation between isoform ε positive and poor prognosis factors such as poorly differentiation,HER2 positive,poor pathologic type and larger breast cancer lumps(P ＜ 0.05 ). Conclusions There are significant differences in the expression of BARD1 isoforms among different kinds breast tissues in the female Han ethnic group.Positive isoform ε may predict poor prognosis of breast carcinoma in the female Han ethnic group.

Antineoplastic Agents ; administration & dosage ; therapeutic use ; Child ; Female ; Humans ; Injections, Intralesional ; Interferon-alpha ; administration & dosage ; therapeutic use ; Male ; Neoplasms ; drug therapy ; Recombinant Proteins ; Treatment Outcome

Objective To evaluate the value of a CT ribs unfolding algorithm in the diagnosis of rib fracture. Methods Retrospective analysis of 180 patients who suffered chest trauma to do the chest or/and abdominal CT examination, and they also had the surgical or CT referral information. The images of these patients were postprocessed by software(Bone Reading software)and hand-drawn method(multi-point hand-painted CPR method). The rib fracture was observed and the time of reading was record. The diagnosis of fractures was confirmed by follow-up review or surgery. The fractures diagnosis sensitivity of the two post-treatment methods were measured, and the McNemar test was used to compare the difference between the software method and the hand-drawn method. Results Eight patients were excluded due to program failure, 172 cases were included in the study. Of the 172 patients, 63 patients suffered 259 fractures(178 ribs). The sensitivity of the software group was 91.7％(475/518), which was higher than that of the hand-painted group(86.3％, 447/518), the difference was statistically significant(P=0.005). The time of reading were (30.3 ± 3.3)and(173.2 ± 4.5)s, respectively, and the difference had statistically significant(P=0.001). Conclusion Compared to the traditional CPR method, the bone reading technique was used in patients with rib fractures during thoracic CT postprocessing can shorten the reading time and increase the sensitivity of the diagnosis.