OBJECTIVETo construct self-assembly fibrocartilage model of goat temporomandibular joint disc and observe the biological characteristics of the self-assembled fibrocartilage constructs, further to provide a basis for tissue engineering of the temporomandibular joint disc and other fibrocartilage.

METHODSCells from temporomandibular joint discs of goats were harvested and cultured. 5.5 x 10(6) cells were seeded in each agarose well with diameter 5 mm x depth 10 mm, daily replace of medium, cultured for 2 weeks.

RESULTSOne day after seeding, goat temporomandibular joint disc cells in agarose wells were gathered and began to self-assemble into a disc-shaped base, then gradually turned into a round shape. When cultured for 2 weeks, hematoxylin-eosin staining was conducted and observed that cells were round and wrapped around by the matrix. Positive Safranin-O/fast green staining for glycosaminoglycans was observed throughout the entire constructs, and picro-sirius red staining was examined and distribution of numerous type I collagen was found. Immunohistochemistry staining demonstrated brown yellow particles in cytoplasm and around extracellular matrix, which showed self-assembly construct can produce type I collagen as native temporomandibular joint disc tissue.

CONCLUSIONProduction of extracellular matrix in self-assembly construct as native temporomandibular joint disc tissue indicates that the use of agarose wells to construct engineered temporomandibular joint disc will be possible and practicable.

Animals ; Cells, Cultured ; Collagen Type I ; Fibrocartilage ; Glycosaminoglycans ; Goats ; Temporomandibular Joint Disc ; Tissue Engineering

Background C57BL/6(B6) is a kind of routine mouse specie used in experimental autoimmune uveitis (EAU) research.Previous studies showed that the pathogenesis of uveitis related to inflammatory cytokines secreted by different helper T(Th) cells.However,the interaction of different Th cells in EAU is unclear.Objective This study was to investigate the dynamic changes of inflammatory factors in the spleen and serum after immunization in EAU mice.Methods Forty-four SPF B6 mice were immunized by injection of interphotoreceptor retinoid-binding protein (IRBP) and complete Freund adjuvant (CFA) emulsion via caudal vein and footpad.Indirect ophthalmoscope was used to examine the eyes 3 times per week and the inflammatory response was scored based on Thurau's criteria.In the thirty day after injection,20 model eyes were extracted and the sections of eye tissue were prepared for histopathological examination.The spleens of model mice were enucleated before injection and 2,5,10,15,20,25,30 days after injection,and reverse transcriptase PCR (RT-PCR) was used to detect the contents of interleukin-17 (IL-17) mRNA,interferon-γ (IFN-γ) mRNA,tumor necrosis factor-α (TNF-α) mRNA and IL-10 mRNA,and the contents of IL-17,IFN-γ,TNF-α and IL-10 in model serum were assayed by ELISA in 24 model mice.The experimental protocol and use of the animals were approved by Ethic Committee for Care and Use of Laboratory Animals of Shandong University of Traditional Chinese Medicine.Results Mild inflammatory response was seen in 12 days under the indirect ophthalmoscope with the scores of 0.5.The inflammatory scores peaked in 13-15 days with the scores of 1.0 and alleviated after that with the inflammatory scores of 0.5 in 30 days after injection.The histopathological score was consistent with the clinical score in the models on the 30 days.The serum IL-17 content of model mice was (0.98±0.05) ng/L before injection and increased to (51.85 ±2.42) ng/L on the fifth day,and decreased to (4.01±0.06)ng/L on the fifteen day.But,the serum IL-17 level increased to (25.00±0.94)ng/L again on the 25th day,and then lowed to (6.01 ±0.21)ng/L 30 days after injection,showing a significant elevation in comparison with that of before injection (P=0.000).The serum IFN-γ content of the model mice was (1.02±0.09)ng/L before injection and increased to (50.54±0.48) ng/L on the fifth day,and (73.21±0.12) ng/L on the tenth day,and then it declined gradually until (5.15±0.18)ng/L in the 30th day,which was still higher than that of before injection (P=0.000).After injection of IRBP+CFA,the serum TNF-α level upregulated from the second day to fifth day with the peak values (134.25±0.59)ng/L,and declined to valley on 15th day.A repeat elevation of serum TNF-α level was found on the 20th day with the values (60.54±0.62)ng/L and followed by decrease till the 30th day,which was higher than that of before injection (P=0.660).Serum IL-10 was detectable in the tenth day and peaked on the 15th day.Then a slight decrease was seen till the 30th day,compared with before injection(P =0.000).The contents of IL-10 mRNA,IL-17 mRNA,TNF-α mRNA,IFN-γmRNA in mice spleens followed the same pattern with serum levels of their proteins.Conclusions IL-17,IFN-γ,TNF-α and IL-10 are key inflammatory factors of Th1,Th2 and Th17,they present with specific changes during EAU,it confirming that IFN-γ probably play a pathogenic role in EAU,IL-17 and TNF-α levels probably associated with the chronic and recurrent procedure of uveitis,IL-10 plays an inhibit role in EAU.

In view of the anatomical and physiological barrier of the ocular surface and the intraocular structure, the conventional ophthalmic agents cannot efficiently reach the lesion site.Currently, the different types of nanomaterials possess great advantages in delivering drugs due to their characteristics of small size, easy preparation, degradability, strong targeting and less irritation to biological tissue.As drug delivery carriers, nanomaterials have been widely used in ocular drug delivery so as to treat different types of eye diseases.In this paper, the applications of nanomaterials as drug delivery carriers in treating eye diseases are briefly reviewed.

Objective To establish a method for studying molecular mechanism of Rhubarb inhibiting anti-Yersinia pesti based on DNA microarray.Methods A whole genome DN A microarray containing 4005 annotated genes of Yersiniapesti Was used.The minimal inhibitory concentration(MIC)of Rhubarb to Yersiniapestiwas determined by liquid dilution method.The gene expression profile of Yersinia pesti was performed after the exposure to Rhubarb at a concentration of 10×MIC for 30 minutes.The total RNA extracted and purified from Yersinia pesti Was reversely transfected to cDNA and labeled by Cy3-Cy5 dye.The labeled probes were hybridized to the microarray anti the results were obtained by a laser scanner and the microarray data was confirmed by real-time quantitative RT-PCR.Results The platform of the DNA microarray-based bacteria transcriptional profile was established.A total of 498 genes of Yersinia pesti changed significantly in response to Rhubarb.Among them.358 genes were up-regulated,140 down-reguated.Conclusions The whole genome DNA microarray can be used in the studying of molecular anti-Yersinia pesti mechanism of Rhubarb.

Objective To explore the biological exposure limit of liver dysfunction induced by arsenic-coal burning, and screen sensitive biornarkers for its' liver dysfunction monitoring. Methods One hundred and eighteen subjects from the exposed area and 50 control from non-pollution area were studied. Their urinary and hair contents of arsenic were tested as exposure biomarkers by Ag-DDC assay. Total bile acid(TBA, detected by enzymatic cycling method), glutathione S-transferase (GSTs, detected by chemical colorimetry) and γ-glutamyl transpeptidase (γ-GT, detected by colorimetry of diazotization reagent) were used as biomarkers indicating liver cell damage. were used as liver fibrosis biomarkers. The benchmark dose (BMD) and the lower confidence limit of benchmark dose(BMDL) of urinary and hair arsenic were calculated. Sensitivity of each biomarker was estimated according to the BMD and BMDL value. Results The geometric mean of urinary and hair arsenic(98.50 mg/kg Cr, 7.42 mg/kg) μg/L) in the exposed group were significantly higher than urinary and hair arsenic (22.98 mg/kg Cr, 1.28 mg/kg) and each biomarker in the control group(4.63 μmol/L, 13.76 U/L,36.45 U/L,54.62 μg/L,74.45 μg/L,54.81 μg/L, P<0.01). Significant dose-effect relationship existed between urinary and hair arsenic contents and each biomarker. BMD and BMDL value of urinary arsenic was 49.53-101.96 mg/kg Cr and 39.02-70.15 mg/kg Cr, respectively. Those of hair arsenic were 3.04-5.02 mg/kg and 2.36-3.25 mg/kg, respectively. According to BMD and BMDL value of urinary and hair arsenic, the sensitivity of biomarkers decreased in the order of GSTs, TBA and Conclusions According to the lowest BMD and BMDL of urinary and hair arsenic, averaged reference value of urinary and hair arsenic in the local normal population, we suggest urinary 35.0 mg/kg Cr and hair 2.5 mg/kg as their biological exposure limits for those with liver dysfunction induced by arsenic-coal burning. GSTs, TBA, γ-GT and HA, Ⅳ. C, PC-Ⅲ can respectively reflect liver cell damage and liver fibrosis caused by arsenic-coal burning in different degrees, among which, GSTs and HA are the most sensitive biomarkers respectively for liver cell damage and liver fibrosis.

By integration of traditional and Western medicine under particular historical conditions in China, some doctors of Western medicine are learning traditional Chinese medicine (TCM)and try to interconnect the two theories.To promote medical progress, they provide TCM services in clinic, such as acupuncture, herbal medicine, etc.But as we know that the laws are lacking for the doctors of Western medicine who want to carry out traditional Chinese medicine service.For the reason, the paper holds that at present there are three fundamental problems that should be solved.First of all, it must be clear that doctors should have qualifications and conditions to provide TCM service.Secondly, it must be clear that the doctors should have practicing scope to guarantee medical security and medical quality.Thirdly, it is imperative to strengthen TCM education and training for doctors.Now it is necessary to further strengthen the standardization of health supervision and law enforcement.

OBJECTIVETo develop a rapid and reproducible method for the culture of human fetal hair follicle bulge cells, and observe the plasticity of its differentiation into sebaceous gland in vitro.

METHODSThe bulge cells isolated from fetal human hair follicles by enzymatic digestion (digestion method) and manual microdissection (conventional method) were cultured and passaged respectively, the efficiency and biological features of cells were investigated , the clone forming efficiency was assayed by MTT, and the expression of K19 was further compared by immunocytochemistry (ABC). The morphological change and the expression of EMA of bulge cells were also observed after induction.

RESULTSBy conventional method, 8-10 bulges were harvested in one hour, 40%-50% of their cells were found to adhere to the culture plate after culturing for 48h, and they became confluent after 14 days. In comparison, about 100 bulges were harvested in one hour by digestion method, the adherence efficiency of their cells was 30% after cultivation for 12h and became confluent after 7 days. The cells grew larger with time, with irregular shape and droplets of lipid around the nucleus. The clone forming efficiency of bulge cells cultured by digestion method was (18.2 +/- 2.1) %, which was much higher than that of cells obtained by conventional method[ (12.7 +/- 3.4) %, P < 0.05]. Immunocytochemistry staining showed that positive staining of K19 was observed in most of the bulge cells, with a large amount of brown granules in the cytoplasm.

CONCLUSIONHuman hair follicle bulge cells can be efficiently cultured and multiplied in vitro, and they retained the characteristics of stem cells. And they have the potential to differentiate into sebaceous glands by induction in vitro.

Cell Culture Techniques ; Cell Differentiation ; Cells, Cultured ; Fetal Stem Cells ; cytology ; Hair Follicle ; cytology ; Humans ; Sebaceous Glands ; cytology

OBJECTIVETo examine the effects of high and low concentrations of transforming growth factor (TGF) β(1) and insulin-like growth factor-I (IGF-I) on the extracelluar matrix synthesis of the self-assembled constructs of temporomandibular joint (TMJ) disc.

METHODSThe experimental groups of self-assembled constructs were exposed to IGF-I (10, 100 µg/L) and TGF-β(1) (5, 50 µg/L), the control groups were not added with any growth factors. All groups were examined at 3 and 6 weeks for gross morphological, histological, and biochemical changes. Safranin-O/fast green staining was used to examine glycosaminoglycan (GAG) distribution, picrosirius red and immunohistochemical staining to observe type I collagen distribution. Type I collagen contents were tested by ELISA assay kit, GAG contents were measured by Blyscan GAG assay kit, and the cell numbers were quantified with a Picogreen reagent kit.

RESULTSThe growth factor groups all upregulated the matrix synthesis of the self-assembled constructs compared with control groups. TGF-β(1) (5 µg/L) and IGF-I (10 µg/L) were the two most potent concentration in increasing type I collagen and GAG synthesis and cells proliferation. IGF-I group (10 µg/L) produced nearly 2 times (109.16 ± 5.12 µg) as much type I collagen as the control group (69.13 ± 5.94 µg) at 3 weeks. The matrix contents and the number of the proliferated cells in control group and all GF groups at 6 weeks were more than those at 3 weeks.

CONCLUSIONSIGF-I (10 µg/L) is the most beneficial growth factor and can be applied in tissue-engineering stratigies of the temporomandibular joint disc. At the same time, the exposure time of growth factors is another key factor that affects matrix synthesis of TMJ disc constructs.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen Type I ; biosynthesis ; Extracellular Matrix ; metabolism ; Glycosaminoglycans ; biosynthesis ; Goats ; Insulin-Like Growth Factor I ; pharmacology ; Temporomandibular Joint Disc ; cytology ; metabolism ; Tissue Engineering ; methods ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo investigate the feasibility of fabricating tissue engineering skin with human hair follicle bulge cells (HFBCs) to repair full-thickness skin wound.

METHODSHFBCs and dermal papilla cells (DPCs) isolated from human fetal hair follicles by collagenase digestion were cultured, purified and passaged. PGA-collagen scaffolds as bioengineered dermis were randomly divided into A and B groups. The HFBCs and DPCs (1 : 2) were seeded in scaffolds of group A and the equal amount of DPCs was seeded in scaffolds of group B as control. Then the keratinocyte sheets were seeded onto the surfaces of the scaffolds as bioengineered epidermis. The tissue engineering skins were then transplanted to repair the full-thickness wound on the back of nude mice. The wound healing process was observed and the plant histological changes of the transplanted engineered skin was observed with light microscope on 2, 4, 6 post-operation weeks (POW).

RESULTSThe full-thickness defect of nude mice in A and B groups could be effectively repaired by bioengineered skins. On 2 POW, integral epidermal and dermal structures were observed in the wounds in A and B groups, with thin epithelial layer and basement membrane. On 4 POW, epithelial layer became thickening and rete pegs formation was observed in basement membrane in A group, but only thickening of epithelial layer was observed in B group. On 6 POW, rete pegs structure was seen to descend and hair-follicle-like structure was formed, while only thickened epithelial layer with flat basement membrane were formed in B group.

CONCLUSIONFrom the composite skin engineered with PGA-collagen hybrid scaffolds and keratinocytes, HFBCs and DPCs could effectively repair the full-thickness skin defect of nude mice. The hair follicle stem cells participate in the process of anatomic repair of wound, and might be able to induce the repair of skin structure and function.

Animals ; Cell Culture Techniques ; Cells, Cultured ; Dermis ; cytology ; Fetus ; cytology ; Hair Follicle ; cytology ; Humans ; Male ; Mice ; Mice, Nude ; Skin Transplantation ; Skin, Artificial ; Tissue Engineering ; methods

15-hydroxyeicosatetraenoic acid (15-HETE) plays an important role in hypoxia-induced pulmonary vasoconstriction. Release of nitric oxide (NO) is apparently decreased and activity of endothelial nitric oxide synthase (eNOS) is impaired in chronic hypoxia. However, little is known whether 15-HETE contributes to eNOS/NO pathway in the constriction induced by 15-HETE. We examined the response of rat pulmonary artery (PA) rings to 15-HETE, the production of NO, total eNOS expression and the phosphorylation of eNOS in bovine pulmonary artery endothelial cells (BPAECs) stimulated by 15-HETE. Rat PA rings were divided into three groups: endothelium intact group, endothelium denuded group, and nitro-L-arginine methyl ester (L-NAME, 0.1 mmol/L, an inhibitor of eNOS) group. Constrictions to 15-HETE were significantly enhanced in endothelium denuded group and L-NAME group (both P< 0.05 vs endothelium intact group, n= 9); BPAECs were incubated in different conditions to test nitrite production by Greiss method. Nitrite production was significantly reduced by 1 mumol/L 15-HETE (P<0.05), and increased by the lipoxygenase inhibitors, 10 mumol/L cinnamyl 3,4- dihydroxy-[alpha] -cyanocinnamate (CDC, P< 0.05) and 0.1 mmol/L nordihydroguiairetic acid (NDGA, P< 0.01 ); Western blot analysis of extracts from BPAECs incubated with 15-HETE in different time was carried out to test total eNOS expression, and the expression was changed unobviously. Immunoprecipitation (IP) and Western blot analysis of cell extracts from BPAECs treated with 2 mumol/L 15-HETE in different length of time were accomplished, using phospo-eNOS-threonine 495 (Thr495, an inhibitory site) antibody for IP, and eNOS or 15-lipoxygenase (15-LO) antibodies for Western blot. 15-HETE depressed eNOS activity by increasing the levels of phospho-eNOS-Thr 495. The data suggest that eNOS/NO pathway is involved in PA constrictions induced by 15-HETE and that 15-HETE depresses eNOS activity by phosphorylation in Thr495 site. The protein interaction between phospho-eNOS (Thr495) and 15-LO is discovered for the first time.
Animals ; Cattle ; Down-Regulation ; drug effects ; Endothelium, Vascular ; cytology ; drug effects ; enzymology ; Hydroxyeicosatetraenoic Acids ; pharmacology ; In Vitro Techniques ; Male ; Nitric Oxide Synthase Type III ; metabolism ; Pulmonary Artery ; cytology ; enzymology ; physiology ; Rats ; Rats, Wistar