Objective:To develop a simple culture and purifying method for rat dental follicle cells.Methods:The upper and lower first and second intact molar germs of SD rat were separated. Then, dental follicle and enamel organ were stripped together, minced into little pieces, digested with collagenase and cultured. Dental follicular cells were purified by differential passage and indentified by immunohistochemical staining of vimentin and cytokeratin. Results:The primary cells were mixed, consisting of dental follicle cells and enamel organ cells. After differential passage, the cells of fourth passage became purified dental follicle cells. Purified dental follicle cells were elongated spindle or triangle in shape, positive for vimentin and negative for cytokeratin.Conclusion:Dental follicle cells can be purified by several differential passages from the mixed primarily cultured cells.

Objective:To investigate the temporal and spatial distrib ut ions of collagenⅠ and Ⅲ during mouse tooth germ development and their function s during tooth mineralization.Methods:Immunohistochemistry stain ing technique was used to test the expressions of collagen Ⅰ and Ⅲ during mous e tooth germ development. Results:collagen Ⅲ was positive in or al epithelial cells in bud stage,in oral epithelial cells and in stellate reticu lum cells in cap stage. During bell and differentiation stage,collagen Ⅲ was positive in oral epithelial cells, stellate reticulum cells, dental papilla cell s and dental sac cells. During P2-10 d(crown development stage), collagen Ⅲ was expressed possitively in ameloblasts,enamel matrix,odontoblasts,predentin, dental papilla cells,dental sac cells and pulp tissues. During P10-30 d(toot h root development stage),collagen Ⅲ was strongly positive in Hertwig's epithe lial root sheath, cementum, alveolar bone and periodontal ligament cells apart f rom above mentioned cell types. CollagenⅠ was not expressed in bud stage and wa s positive in oral epithelial cells,stellate reticulum cells in cap stage. Durin g bell and differentiation stage,collagen Ⅰ was positive in oral epithelial ce lls, stellate reticulum cells, dental papilla cells and dental sac cells.After P2 d (crown and root development stage), the distribution and expression of co llagen Ⅰ were similar to those of collagen Ⅲ.Conclusions:Coll agenⅠand Ⅲ are involved in tooth germ and tooth tissue development. But the fu nction of collagenⅢ is more extensive than that of collagenⅠ.

Objective:To study the multiple alternative sp l iceosome of nfic gene in postnatal rat molar tissues. Methods: 3 d postnatal SD rat molar tissues were removed and mRNA was obtained by magne tic beads coupled with oligo-dT18. Two pairs of primers specific to the nfic gene were designed, and nfic gene in the rat molar tissue was amplified b y RT-PCR method.The fragments were inserted into competent DH5? bacteria.Posit ive clones were selected randomly and evaluated by enzyme digestion and sequenci ng.Results:Three alternative spliceosomes of nfic gene with the size of 1.5 kb,900 bp and 650 bp respectively were obtained. The spliceosome s were named rNFIC-1、 rNFIC-2 and rNFIC-3 respectively.Conclusion s:The nfic gene is expressed in postnatal rat molar, and there is a multiple alternative splicing way for the nfic gene to play function.

Objective:To prepare and identify a polyclonal antibody againstadam28 gene product.Methods:Theprotein coding region of ADAM28 was amplified by RT-PCR and cloned into pMD18-T Vector to produce the newconstruct, pMD18-T-adam28. The cloned ADAM28 segment was cut with two restriction enzymes and theadam28fregment was directed into the prokaryotic expression vector, pGEX-4T-1,to produce the expression vector pGEX-4T-adam28. The recombinant plasmid was transformed intoE. coliDH5?and GST-ADAM28 fusion protein was ob-tained after the inducement by IPTG. The fusion protein was extracted and purified by SDS-PAGE,and the newpro-tein band of 35 300 was isolated as antigen, the antigen was injected into rabbits to produce polyclonal antibody a-gainst ADAM28 product.Results:The expression vector pGEX-4T-adam28 was constructed successfully,and GST-ADAM28 fusion protein was obtained. The rabbit serum containing polyclonal antibody against ADAM28 productwas obtained and the antibody was purified by salting out method. Western blot analysis displayed that the antibodyhad high specificity. ELISA analysis confirmed that the titer for the antibody reached 1∶16 000.Conclusion:Thepolyclonal antibody against ADAM28 product with high titer is successfully prepared,it may be used for further studyof the role and expression of ADAM28 during tooth development.

Objective:To investigate spatiotemporal expression of ADAM28 in mouse tooth germ development.Methods:Immunohistochemistry and image analysis technique were used to observe the expressions of ADAM28 at mouse tooth germ development stages.Results:Different expression levels of ADAM28 at tooth germ development stages were observed.At cap stage,ADAM28 was found strongly positive in oral epithelial,stellate reticulum cells of enamel organ,basement membrane,dental papilla cells and dental sac cells.At late bell stage,positive staining was found in ameloblasts,enamel matrix,epithelial root sheath and dental papilla cells.At crown and root development stage,positive staining for ADAM28 was detected in ameloblasts,odontoblasts,cementoblasts,epithelial root sheath,dental papilla cells and dental sac cells.Conclusion:ADAM28 participates in crown and root morphogenesis process ranging from bud stage to late bell stage and from matrix secretion to sclerous tissue formation.It might play an important role in early formation,proliferation and differentiation of odontogenic mesenchymal cells.

Objective:To observe the dynamic process of the development of dentin and enamel during their mineralization period. Methods: Dental germs in maxillae and mandibulae were obtained from spontaneously aborted 21 weeks and 32 weeks fetus. The samples were prepared and stained with modified Mallory's trichrome staining and observed under light microscope. Results: Different mineralized layers in different period of development appeared different color variation in the dental germs. Conciusion: Modified Mallory's trichrome staining method may be used to study the mineralization of dental germ.

Objective:To detect the expression of upstream stimulatory factor 1(USF1) and its tempo-spatial distribution during mice teeth development. Methods: Total protein was extracted from P1 and P11 d mice first molar teeth germ, and Western blot for USF1 was undertaken. Paraffin sections of first molar teeth germs from E13, 16, 19, P1, 5, 8, 11, 21 d and 6-month-old adult mice were prepared respectively and immunohistochemical staining was carried out. Results: Western blot analysis identified one Mr 43 000 protein from P11 d mice teeth germs, but none from P1d mice. Immunohistochemically, evidently positive staining for USF1 in mice teeth germs began from P5 d, and extended to P11 d, which was mainly confined to the cytoplasm of secreting ameloblasts and odontoblasts, but no staining in bud, cap and early bell stages of tooth germ. However, after tooth eruption on P21 d, USF1 became negative again, although it was still positive in the adjacent muscles, and the same result was observed in adult mice tooth. Conclusion: USF1 is expressed in tooth germ, which localizes solely in secreting ameloblasts and odontoblasts, and its expression was quite dynamic during mice tooth development.

Objective:To evaluate the physiological roles of bmp3,bmp4 and bmp7 during the formation of permanent tooth roots in dog. Methods:The expression of bmp3,bmp4 and bmp7 mRNA at different stages of the development of permanent tooth roots was examined by in situ hybridization in 3 dogs aged 12-18 weeks. Results:bmp3 was found in dental sac surrounding the germs at the early stage of tooth root development, and in cementoblasts and periodontal cells at the later stage. bmp4 was found in odontoblasts, dental papilla and osteoblasts. bmp7 positive signals was found only in epithelial cells of root sheath around cervical circulus at early stage, then located in cementoblasts and odontoblasts at later stage. Conclusion:The spatiotemporal expressions of bmp3,bmp4 and bmp7 are widely diverse, indicating that they participate in the regulation of tooth root development.

Objective:To investigate the changes of monocytic myeloid-derived suppressor cells (M-MDSC) in children with acute Kawasaki disease (KD) and its roles in the immunological pathogenesis of KD.Methods:A total of 38 children with acute KD were enrolled in the present study and 32 age-matched healthy children were selected as control group. The proportions of HLA-DR -CD11b + CD33 + CD15 -CD14 + M-MDSC and CD4 + CD25 + CD127 - regulatory T cells (Treg) in peripheral blood, concentrations of reactive oxygen species (ROS) and expression of arginase-1 (Arg-1), CD39, CD73, CD40, CD40L and CCR5 at protein levels were detected by flow cytometry. Quantitative real-time PCR was used to evaluate the transcription levels of inducible nitric oxide synthase (iNOS) in M-MDSC and the transcription levels of cytotoxic T-lymphocyte associated antigen 4 (CTLA4) and lymphocyte-activation gene 3 (LAG3) in Treg. Concentrations of NO, CCL3, CCL4, CCL5, IL-10 and TGF-β in the supernatants of cell culture were measured by ELISA. Results:(1) The proportion of HLA-DR -CD11b + CD33 + CD15 -CD14 + M-MDSC, the concentration of intracellular ROS and the expression of iNOS, CD39 and CD73 in M-MDSC decreased significantly in patients with acute KD as compared with those in the control group ( P<0.05), and the concentrations of NO, IL-10 and TGF-β in culture supernatant of M-MDSC were lower than those in the control group upon lipopolysaccharide (LPS) stimulation for 48 h ( P<0.05). All of the aforementioned indexes restored to some extent after intravenous immunoglobulin (IVIG) therapy ( P<0.05). No statistical differences were found in Arg-1 expression between healthy controls and patients with KD before or after IVIG therapy ( P<0.05). (2) CD40 expression on M-MDSC was significantly lower in the acute KD group than in the control group ( P<0.05). The concentrations of CCL3, CCL4 and CCL5 in the culture supernatants of M-MDSC were lower in the acute KD group than in the control group after LPS stimulation ( P<0.05). With IVIG treatment, all of the indexes were up-regulated significantly ( P<0.05), although CD40 expression was still lower in the acute KD group than in the control group ( P<0.05). (3) The proportion of CD4 + CD25 + CD127 -Treg and the expression of CTLA4, LAG3, CD40L and CCR5 reduced significantly in patients with acute KD as compared those in healthy controls ( P<0.05), and all increased remarkably after IVIG therapy ( P<0.05). Pearson correlation analysis showed a positive correlation between the proportions of M-MDSC and Treg in patients with acute KD ( r=0.58, P<0.05). Conclusions:Insufficiency and impaired function of M-MDSC might be a major cause of immune dysfunction in patients with acute KD.

OBJECTIVESTo investigate the risk factors for cervical cancer in the areas of high incidence, and provide evidence for current intervention of cervical cancer.

METHODSIn the areas of Xiangyuan County, Shanxi Provicne with high incidence of cervical cancer, 1 997 women were interviewed using a questionnaire, including baseline information, menstrual, marital and pregnancy histories, sexual behavior, health habits, contraception, medical history and family history of cancer, etc., after its screening with six kinds of methods. All subjects, including 84 cases with pathological diagnosis of greater than cINI, and 1 784 cases with pathological diagnosis of normal, were tested for high-risk HPV.

RESULTSThe overall rates of HPV infection were 20.8% (415/1 997) in high-risk subjects, 97.7% and 14.2% in the cases and control groups, respectively. Univariate analysis showed that risk factors with statistical significance included high-risk HPV infection, age at first sexual intercourse, history of pregnancy and abortion, the number of sexual partners and family history of cancer. Analysis with non-conditional logistic regression model revealed high-risk HPV infection, multiple sexual partners and family history of cancer associated obviously with occurrence of cervical cancer. In addition, there was significantly positive relationship between HPV infection, which increased with the number of sexual partners, and extramarital sexual activity both in males or females.

CONCLUSIONSThe main risk factor for cervical cancer in this region was high-risk HPV infection, which related to sexual behavior, hygienic habits during menstruation and puerperium. It was particularly important to detect and treat precancerous lesions and to implement behavior modification. In addition, further research on genetic susceptibility was suggested.

Abortion, Induced ; Adult ; Analysis of Variance ; China ; epidemiology ; Female ; Humans ; Interviews as Topic ; Multivariate Analysis ; Papillomaviridae ; Papillomavirus Infections ; epidemiology ; Risk Factors ; Sexual Behavior ; Sexual Partners ; Surveys and Questionnaires ; Tumor Virus Infections ; epidemiology ; Uterine Cervical Neoplasms ; epidemiology ; virology