BACKGROUND: There are no reports about adult degenerative disc cell culture model currently. This establishment of disc cell culture is the cellular basis of intervertebral disc tissue engineering. OBJECTIVE: To establish cell culture models from human degenerative intervertebral disc, and to settle the cytology base of disc tissue engineering. DESIGN AND SETTING: The controlled observational experiment with regard to cytology was performed in Shandong Institute of Orthopaedics and Traumatology. MATERIALS: Five degenerative intervertebral disc patients were recruited from Department of Orthopaedics, Affiliated Hospital of Qingdao University Medical College, including one case in L2-3, three cases in L4-5 and one case in L5-S1. METHODS: Five samples were taken from the operational disectomy for lumbar disc herniation, and these samples were cultured using enzymatic digestion cell culture and tissue cell culture method differently. All the samples were cultured with HAMF12 medium with the addition of 10% and 20% fetal bovine serum. The morphology of the cultured cell was observed with Giernsa staining and transmission electron microscopy respectively. MAIN OUTCOME MEASURES: ①Cellular growth; ②Morphology of the cultured cells;③Ultrastructure Of the cultured cells. RESULTS: By means of enzymatic digestion cell culture and tissue cell culture method, numerous degenerative lumbar disc cells were obtained and successfully subcultured. The cell secretions around the cultured cells may influence the growth of these cells. Destroying the secretions, the cultured cells could be highly proliferated. The degenerative intervertebral disc cells were proliferated vigorously in HAMF12 medium with 20% fetal bovine serum compared with that with 10% fetal bovine serum. The notochordal cell was observed in all the specimens. CONCLUSION: The adult degenerative disc can be used to obtain the required cell for tissue engineering.

Objective To compare the effects of fresh amniotic membrane (FAM), glycerol-preserved amniotic membrane (GPAM), alcohol-preserved amniotic membrane (APAM) and amniotic membrane preserved under vacuum drying condition (VXAM) on rabbit corneal alkali burn and to observe the outcomes of the grafted amniotic membrane. Methods Thirty-six New Zealand rabbits were used to establish corneal alkali burn models and then divided into 4 groups according different preserved amniotic membrane they received (FAM, GPAM, VXAM and APAM), with 9 rabbits in each group. After transplantation, we observed the changes of amniotic membrane graft, corneal opacity, corneal neovascularization (CNV) with slit lamp microscopy. The amniotic membrane graft was examined by HE staining 10 d, 14 d and 1 month after transplantation. Results One month later, FAM graft, GPAM graft and VXAM graft showed better curative effect than APAM graft. Ten days after transplantation, the amniotic membrane graft began to dissolve and desquamate, and disappeared completely till 14 d after transplantation. Amniotic membrane tissues were not found under light microscope in any rabbit. FAM graft dissolved most quickly, and APAM graft was the most slowly. The dissolution of GPAM graft and VXAM graft had no significant difference. Conclusion After amniotic membrane transplantation, it dissolves and sheds instead of fusing with recipient tissues. Corneal epithelium grows under amniotic membrane graft. Amniotic membrane graft transplantation should be called as amniotic membrane patching.

AIM:Recently,amnion transplantation has been one of effective methods for curing corneal alkali burn,but its biomechanism is unclear.This study was to discuss the efficiency of different kinds of amnion on rabbit corneal alkali burn and to find the bioactive functions of the amnion.METHODS:The experiment was carried out in the Central Laboratory of Experimental Animals at Chongqing University of Medical Sciences between January and July in 2004.All the disposals were in accordance with the ethical standard of animals.①Placenta from healthy puerperants underwent uterine-incision delivery were offered by Department of Obstetrics in the First Affiliated Hospital of Chongqing University of Medical Sciences.A total of 45 New Zealand rabbits were used for corneal alkali burn models and divided into fresh amniotic membrane(FAM),glycerin preserved amniotic membrane(GPAM),vacuum xeransis amniotic membrane(VXAM),alcolhol preserved amniotic membrane(APAM) and control groups,every group had 9 rabbits.②The FAM was prepared within 24 hours,and the preserved amnion were transplanted after 1 month.③The corneal opacity and corneal neovascularization were observed everyday during days 2-21 after operation and once every three days after days 21;corneal fluorescein staining was conducted to observe the repair of corneal epithelium on days 14,30,60 after operation;2 weeks,1 month and 2 months after operation,we did histopathological examination and scanning electron microscope observation.RESULTS:Forty-three rabbits were involved in the result analysis.①There were no significant differences in the corneal opacity and repair velocity of corneal epithelium among FAM,GPAM and VXAM,which were remarkably superior to APAM and controls.APAM group and control group had not distinct difference.②FAM had better effects on inhibiting corneal neovascularization than GPAM and VXAM.CONCLUSION:After transplantation,VXAM,GPAM and FAM can all accelerate corneal epithelial healing and lighten inflammation.FAM containing more bioactive materials,plays best.

Objective To suppress the proliferation of lens epithelial cells (LECs) is a primary goal in prevention of after cataract. Recent study demonstrated an effective inhibition of elemene(Ele) on tumor cells. Present study was to investigate the effects of Ele on proliferation and cell cycle of human LECs B3 (HLE-B3). Methods Recombinant human basic fibroblast growth factor(rhbFGF) was utilized to induce proliferation of HLE-B3. Proliferative HLE-B3 was incubated with 80 mg/L Ele in CO_2 incubator for 24 hours. Then the inhibitory effects of Ele on proliferation of HLE-B3 was evaluated by methyl thiazolyl tetrazolium(MTT). The effect of Ele on HLE-B3 morphology was observed under the optical microscope. The effect of Ele on HLE-B3 cell cycle was analyzed by flow cytometer(FCM). Results MTT test showed that the optical density (OD) value of rhbFGF group was remarkably higher than that of control group(0. 599 0 ± 0. 053 1 versus 0. 409 1 ± 0. 042 2) (P ＜ 0. 01), and that of Ele group(0. 450 0 ±0. 061 4) was obviously declined in comparison to rhbFGF group(P ＜0. 01) . The inhibitory rate of Ele was 24.90 %. In proliferation group, the number of HLE-B3 was increased with the normal cell structure and abundant cytoplasm under the optical microscope. However, in Ele group, the number of HLE-B3 was evidently decreased with less cytoplasm, undistinguished cell structure, condensed and aggregated nucleuses. The result of flow cytometer showed that the percentage of HLE-B3 in G_1 phase in rhbFGF group was 42. 062% ± 1. 270% and that in control group was 46. 422% ±3. 765% with a significant difference(P ＜ 0. 05). HLE-B3 in G_1 phase in Ele group (60. 665% ±2.069%) was evidently increased in comparison with rhbFGF group (P ＜ 0. 01). HLE-B3 in S phase in rhbFGF group compared with control group was increased (51.647% ±1.123% versus 31.842% ± 2. 798%) (P ＜ 0. 01), but that in S phase in Ele group(30. 222% ±3.429%) was lower than rhbFGF group (P ＜ 0. 01). HLE-B3 in G_2 phase in rhbFGF group was decreased in comparison with control group (6. 288% ± 0. 966% versus 21. 735% ± 3. 806%, P ＜ 0. 01), and that in G_2 phase in Ele group (9. 112% ± 1. 659%) compared with proliferation group was increased (P ＜ 0. 01). Conclusion Ele could alter the cell cycle of HLE-B3 and effectively inhibit the HLE-B3 proliferation induced by rhbFGF. Ele may be a reliable and effective drug for prevention and treatment of after cataract.

Background Our previous study showed that curcumin suppresses the proliferation of human lens epithelial cells (LECs) in vitro and has an influence on the signal transduction of cyclic adenosine monophosphate (cAMP) and cyclic guanosinc monophosphate (cGMP).Actually,the regulation for biological behavior of LECs in vivo is complex.Objective This study was to investigate the changes of signal transduction of protein kinase (PK) in inhibition of curcumin on human LECs proliferation.Methods HLE-B3 was cultured and then divided into the blank control group,recombinant human basic fibroblast growth factor (rhbFGF) group and rhbFGF+ curcumin group.rhbFGF of 10 ng/ml was added in the medium in the rhbFGF group,and 20 mg/L curcumin was added into rhbFGF-induced cell medium for 24 hours in the rhbFGF+ curcumin group.The expression rates of PKA,PKC,PKG and calmodulin (CaM) in the cells were assayed using flow cytometry.Results The expression rates of PKA protein were (46.847± 1.673) ％,(33.250 ± 2.242) ％ and (71.645 ±2.432) ％ in the blank control group,rhbFGF group and the rhbFGF+ curcumin group,respectively,and the expression rate of PKA protein was significantly reduced in the rhbFGF group compared with the blank control group (t =11.904,P＜0.01),but the expression rate of PKA protein in the rhbFGF+ curcumin group was significnatly higher than that in the rhbFGF group (t=28.430,P＜0.01).In the blank control group,rhbFGF group and the rhbFGF+ curcumin group,the expression rates of PKC protein in the cells were (35.575± 1.937) ％,(50.652±2.068) ％ and (27.662t4.481) ％,those of PKG protein were (63.838±0.486) ％,(86.562 ± 1.325) ％ and (40.733 ± 2.175) ％,while those of CaM protein were (67.408± 1.391)％,(83.887±3.499)％ and (53.785 ± 1.942)％,the expression rates of PKC,PKG and CaM were significantly lower in the rhbFGF group in comparison with the blank control group (all at P＜0.01),and those in the rhbFGF+ curcumin group were significantly declined in comparison with the rhbFGF group (all at P＜0.01).Conclusions Suppression of curcumin on HLE-B3 proliferation probably is associated with the up-regulation of PKA expression and down-regulation of PKC,PKG and CaM expression in the cells.

Objective To assess polymerase chain reaction(PCR)combined with restriction fragment length polymorphism(RFLP)and gene sequencing technologies in the detection and typing of HPV DNA.Methods Tissue specimens were collected from skin diseases and venereal disease in perianal or genitals.PCR was performed with HPV DNA general primers(MY09/11)in tissue samples. Positive fragments of HPV DNA were purified and digested by restriction enzymes.The digested fragments were typed by po]yacrylamide gel electrophoresis(PAGE).The Resultswere verified by direct sequencing.Results In 50 clinical samples there were 35 HPV DNA positive,including 26 from patients with condyloma acuminatum,8 from patients with bowenoid papulosis,and 1 from patients with squamous cell carcinoma.In HPV DNA positive samples,19 were HPV6,3 were HPV11,8 were HPV16,4 were HPV6 and HPV 11,and I was HPV62.Sequencing Resultswere in accordance with the PCR-RFLP Results .Conclusion PCRRFLP method is effective in the detection and typing of HPV DNA.

This study was intended to look for anti-HIV chemical constituents of aerial parts of Caragana rosea Turcz. Column chromatographic technique was used for the isolation and purification of constituents of Caragana rosea under the guide of anti-HIV assay. The structures were established on the basis of physical and chemical properties and spectroscopic data. Five compounds were obtained from the EtOAc fraction of aerial parts of Caragana rosea and identified as myricetin (1), mearnsetin (2), p-hydroxy cinnamic acid (3), cararosinol A (4) and cararosinol B (5). At the same time, one possible transformation route between cararosinol B and kobophenol A, another resveratrol tetramer isolated from this plant previously, was proposed. Compounds 4, 5 are new resveratrol tetramers, compounds 1 -3 were isolated from this plant for the first time. All compounds showed no activities in an in vitro assay against HIV-1.
Anti-HIV Agents ; chemistry ; isolation & purification ; pharmacology ; Benzofurans ; chemistry ; isolation & purification ; pharmacology ; Caragana ; chemistry ; Coumaric Acids ; chemistry ; isolation & purification ; pharmacology ; Flavonoids ; chemistry ; isolation & purification ; pharmacology ; HIV-1 ; drug effects ; Molecular Structure ; Plant Components, Aerial ; chemistry ; Plants, Medicinal ; chemistry ; Propionates ; Stilbenes ; chemistry ; isolation & purification ; pharmacology

OBJECTIVETo explore the main points of clinical differentiation between hemangioma and vascular malformation in infant.

METHODSBased on Mulliken and Waner's classification, from March, 1997 to February, 1999, 81 baby patients with hemangioma were included in this study. Thirty-eight cases, 43 cases received medical treatment of steroids.

RESULTSAll the patients were followed up from 5 to 7 years. Thirty-eight cases of red strawberry-like lesions limited in the skin began to involute within two years old. Of the 30 patients with strawberry-like lesions and subcutaneous mass, 20 cases involuted in varying degree; 10 cases' subcutaneous mass grew gradually and didn't involute, in 4 cases biopsy was performed, 3 cases were confirmed as hemangioma accompanied with venous malformation by pathology, 1 case was hemangioma accompanied with arteriovenous malformation. Of 13 cases with light blue or normal skin and subcutaneous mass, 7 cases involuted in varying degree; 6 cases grow gradually and didn't disappear, 2 cases were confirmed as venous malformation by biopsy.

CONCLUSIONSHemangioma in infant begins to involute within two years old. Vascular malformation or hemangioma with deep vascular malformation grows persistently and does not disappear. Skin temperature of lesion surface and dilative veins on the skin artery pulsation, are indexes compressibility, for differentiation between hemangioma and vascular malformation in clinical diagnosis.

Child, Preschool ; Diagnosis, Differential ; Female ; Follow-Up Studies ; Hemangioma ; diagnosis ; Humans ; Infant ; Male ; Vascular Malformations ; diagnosis

HLA genes constitute a highly polymorphic multigene system. In the present study, HLA-B oligonucleotide chips were manufactured by using a set of sequence-specific oligonucleotide probes derived from polymorphic regions in exon 2 and exon 3 of HLA-B gene spotted by microarrayer onto the aldehyde modified glass slides. In addition, the sequenced HLA-B gene clones used as standard samples were amplified from exon 2 and exon 3 by PCR. Together with the correct hybridization and wash conditions, the PCR products were bound with the array probes on the chip, and the hybridization patterns were transformed to HLA-B genotypes. The results showed that the genotypes of standard samples by the HLA-B oligonucleotide chips were completely identical with the sequenced clones. In conclusion, the oligonucleotide chip method presented here for HLA-B genotyping is a rapid, accurate, sensitive and attractive high throughput biochemical way.
Genotype ; HLA-B Antigens ; classification ; genetics ; Humans ; Oligonucleotide Array Sequence Analysis ; Sequence Analysis, DNA

OBJECTIVETo explore the clinical classification and ideal therapy for maxillofacial AVMs.

METHODSAccording to the clinical characteristics, 106 patients with maxillofacial AVMs were divided into the 4 types Of them, 38 cases were cystic dilatation lesions, 22 cases were limited thicken lesions, 42 case were diffuse thicken lesions, 4 cases were central maxillary hemangioma. 106 patients with maxillofacial AVMs were treated in our hospital, of them, 8 cases received operation (group 1); 23 cases received embolization of supplying artery alone (group 2); 37 cases received embolization of supplying artery plus hardener intra-tumorous injection (group 3); 38 cases received embolization of supplying artery plus tumor resection (group 4).

RESULTSOf all the patients were followed up 1 - 11 years, In group 1, 2, 3, and 4, the cure rates is 62.50%, 17.39%, 89.19%, and 97.37% respectively. one patient died of embolization of abnormal communication branches between external carotid and intra-cranical arteries.

CONCLUSIONS(1) This new clinical classification is beneficial for selecting method of treatment. (2) It is necessary that a good digital subtraction angiography for maxillofacial AVMs. (3) The embolization of tumor supplying artery alone could cure the small AVM with single branch terminal blood supply. (4) The embolization of supplying artery plus hardener intratumorous injection or the embolization of supplying artery plus tumor resection is an effective method for maxillofacial AVMs.

Adolescent ; Adult ; Angiography, Digital Subtraction ; Arteriovenous Malformations ; diagnosis ; therapy ; Carotid Artery, External ; diagnostic imaging ; Child ; Child, Preschool ; Embolization, Therapeutic ; methods ; Female ; Follow-Up Studies ; Humans ; Jaw ; blood supply ; Male ; Middle Aged ; Mouth ; blood supply ; Sclerotherapy